The world’s premier stem cell 9th Annual Meeting research event

Thursday Poster Abstracts

June 15 – 18, 2011 Metro Toronto Convention Centre Toronto, Ontario Canada www.isscr.org

Co-sponsored by

Mouse neural crest stem cells image ©Deepak Srivastava The world’s premier stem cell 9th Annual Meeting research event

Thursday Poster Abstracts

June 15 – 18, 2011 Metro Toronto Convention Centre Toronto, Ontario Canada www.isscr.org

Co-sponsored by

Mouse neural crest stem cells image ©Deepak Srivastava 1 ISSCR 9th Annual Meeting www.isscr.org

Poster Floor Plan

Totipotency/Early Embryo Cells Mesenchymal Mesenchymal Pre-Clinical and Clinical Germline Technologies for Tissue Hematopoietic Cell Lineage Stem Cell Applications of Neural Cell Cancer Cells Cells Stem Cell Research Engineering Stem Cells Analysis Di erentiation Mesenchymal Cells 2350 2250 2251 2270 2271 2290 2291 2310 2311 2011 2030 2031 2050 2051 2070 2071 2090 2091 2110 2111 2130 2131 2150 2151 2170 2171 2190 2191 2210 2211 2230 2351 2370 2371 2390 2391 2410 2411 2430 2431 2010 2450 2451 2470 2471 2490 2491 2510 2511 2530 2531 2550 2551 2570 2571 2590 2591 2330 2331 2231

745 844 845 944

743 842 843 942

741 840 841 940 2241 2260 2261 2280 2281 2300 2301 2320 2240 2020 2021 2040 2041 2060 2061 2080 2081 2100 2101 2120 2121 2140 2141 2160 2161 2180 2181 2200 2201 2220 2221 2001 2321 2340 2341 2440 2360 2361 2380 2381 2400 2401 2420 2421 739 838 839 938 2441 2460 2461 2480 2481 2500 2501 2520 2521 2540 2541 2560 2561 2580 2581 2600

1291 1300 3001 3020 1290 1281 Other 3040 3021 1271 1280 433 532 533 632 832 833 932 Neural Cell 20' Chromatin in 3041 3060 1270 1261 Stem Cells 3080 3061 1251 1260 331 430 431 530 531 630 63 733 831 930 931 1030 20' 3081 3100 1250 1241 3120 3101 329 428 629 728 729 830 829 928 929 1028 1231 1240 Muscle Cells 20' 20' 20' 20' 3121 3140 1230 1221 iPS Cells 3160 3141 427 526 527 626 1211 1220 Ethics 325 724 824 825 924 925 1024 3161 3180 20' 20' 20' 1210 1201 50' 3200 3181 Regeneration Epithelial Cells 1191 1200 323 422 623 722 723 823 922 923 1022 3201 3220 1190 1181 Eye or Retinal Cells 20' (not skin) Mechanisms 3240 3221 1171 1180 820 3241 20' 20' 20' 720 721 821 920 20' 1020 3260 1170 1161 Intestinal/Gut Cells Reprogramming 3280 3261 1151 1160 319 418 619 718 719 818 819 918 919 1018 3300 Intern 3281 1150 1141 Epidermal Cells 60' Embryonic Stem Cells 3320 3301 Endothelial Cells/ Clinical Application 1131 1140 715 814 815 914 3321 3340 1130 1121 Hemangioblasts 3360 3341 1111 1120 313 413 712 713 812 813 912 1012 3361 3380 1110 1101 20' 30' 30' 3400 3381 311 410 710 711 811 910 1091 1100 20' 20' 3401 3420 1090 1081 Cardiac Cells 20' 3440 3421 309 408 611 708 709 810 809 908 909 1008 1071 1080 20' 20' 20' Embryonic Stem Cell 3441 3460 1070 1061 Di erentiation 3480 3461 407 506 507 EXH Lung Cells SER 1051 1060 305 404 504 605 704 705 804 805 1004 3481 3500 1050 1041 20' 20' 20' 3520 3501 1031 1040 30Liver3 Cells 403 502 503 603 702 703 802 803 Embryonic902 90 3Stem1002 Cell 20' 20' 3521 3540 1030 1021 Pluripotency 3560 3541 1011 1020 30Pancreatic1 400 Cells401 500 501 600 601 700 701 800 801 900 901 1000 3561 3580 1010 1001 3600 3581 Main Entrance Poster Session Schedule Wednesday, June 15 Thursday, June 16 Friday, June 17 Saturday, June 18 3:30 – 4:00 p.m. 11:00 a.m. – 8:00 p.m. 11:00 a.m. – 8:00 p.m. 11:00 a.m. – 4:00 p.m. All Posters Put On Display Posters Open for Viewing Posters Open for Viewing Posters Open for Viewing

4:00 – 8:00 p.m. 6:00 – 8:00 p.m. 6:00 – 8:00 p.m. 4:00 p.m. MAIN Posters Open for Viewing Poster Presentation Reception Poster Presentation Reception PostersE DismantleNTRANCE ALL ODD NUMBERED ALL EVEN NUMBERED POSTERS PRESENTED POSTERS PRESENTED ii www.isscr.org Thursday Poster Abstracts

Poster Floor Plan

Totipotency/Early Poster Boards by Topic Embryo Cells Mesenchymal Mesenchymal Pre-Clinical and Clinical Posters 1001 – 1300 Germline Technologies for Tissue Hematopoietic Cell Lineage Stem Cell Applications of Pancreatic Cells 1001 – 1020 Cancer Cells Cells Stem Cells Analysis Di erentiation Neural Cell Stem Cell Research Engineering Mesenchymal Cells Liver Cells 1021 – 1048 Lung Cells 1049 – 1060 2350 2250 2251 2270 2271 2290 2291 2310 2311 2351 2370 2371 2390 2391 2410 2411 2430 2431 2011 2030 2031 2050 2051 2070 2071 2090 2091 2110 2111 2130 2131 2150 2151 2170 2171 2190 2191 2210 2211 2230 2010 2450 2451 2470 2471 2490 2491 2510 2511 2530 2531 2550 2551 2570 2571 2590 2591 2330 2331 2231 Cardiac Cells 1061 – 1106 Endothelial Cells/ Hemangioblasts 1107 – 1140

745 844 845 944 Epidermal Cells 1141 – 1155

743 842 843 942 Intestinal/Gut Cells 1156 – 1165 Epithelial Cells (not skin) 1166 – 1177 741 840 841 940 Eye or Retinal Cells 1178 – 1204 2241 2260 2261 2280 2281 2300 2301 2320 2240 2020 2021 2040 2041 2060 2061 2080 2081 2100 2101 2120 2121 2140 2141 2160 2161 2180 2181 2200 2201 2220 2221 2001 2341 2321 2340 2440 2360 2361 2380 2381 2400 2401 2420 2421 739 838 839 938 2441 2460 2461 2480 2481 2500 2501 2520 2521 2540 2541 2560 2561 2580 2581 2600 Ethics and Policy 1205 – 1220 Muscle Cells 1221 – 1240 1291 1300 3001 3020 Neural Cell 1241 – 1300 1290 1281 Other 3040 3021 1271 1280 433 532 533 632 832 833 932 Neural Cell 20' Chromatin in 3041 3060 1270 1261 Stem Cells 3080 3061 Posters 2001 – 2600 1251 1260 331 430 431 530 531 630 63 733 831 930 931 1030 20' 3081 3100 1250 1241 3120 3101 Neural Cells (cont.) 2001 – 2064 1240 329 428 629 728 729 830 829 928 929 1028 1231 Muscle Cells 20' 20' 20' 20' 3121 3140 Cancer Cells 2065 – 2150 1230 1221 iPS Cells 3160 3141 427 526 527 626 Germline Cells 2151 – 2170 1211 1220 Ethics 325 724 824 825 924 925 1024 3161 3180 Technologies for 20' 20' 20' 1210 1201 50' 3200 3181 Stem Cell Research 2171 – 2274 Regeneration Epithelial Cells 1191 1200 323 422 623 722 723 823 922 923 1022 3201 3220 1190 1181 Eye or Retinal Cells 20' Tissue Engineering 2275 – 2320 (not skin) Mechanisms 3240 3221 1171 1180 820 3241 20' 20' 20' 720 721 821 920 20' 1020 3260 Totipotency/Early 1170 1161 Intestinal/Gut Cells Reprogramming 3280 3261 Embryo Cells 2321 – 2330 1151 1160 319 418 619 718 719 818 819 918 919 1018 3281 3300 Intern Hematopoietic Stem Cells 2331 – 2416 1150 1141 Epidermal Cells 60' Embryonic Stem Cells 3320 3301 Clinical Application Mesenchymal Cell Endothelial Cells/ Lineage Analysis 2417 – 2443 1131 1140 715 814 815 914 3321 3340 1130 Hemangioblasts 1121 3360 3341 Mesenchymal Stem 1111 1120 313 413 712 713 812 813 912 1012 3361 3380 1110 1101 20' Cell Differentiation 2444 – 2544 30' 30' 3400 3381 311 410 710 711 811 910 1091 1100 20' 20' 3401 3420 Pre-Clinical and Clinical 1090 1081 Cardiac Cells 20' 3440 3421 Applications of 309 408 611 708 709 810 809 908 909 1008 1071 1080 20' 20' 20' Embryonic Stem Cell 3441 3460 Mesenchymal Cells 2545 – 2600 1070 1061 Di erentiation 3480 3461 407 506 507 EXH Lung Cells SER Posters 3001 – 3600 1051 1060 305 404 504 605 704 705 804 805 1004 3481 3500 20' 20' 20' 1050 1041 3520 3501 Other 3001 – 3048 1031 1040 30Liver3 Cells 403 502 503 603 702 703 802 803 Embryonic902 90 3Stem1002 Cell 20' 20' 3521 3540 1030 1021 Pluripotency 3560 3541 Chromatin in Stem Cells 3050 – 3072 1011 1020 30Pancreatic1 400 Cells401 500 501 600 601 700 701 800 801 900 901 1000 3561 3580 iPS Cells 3073 – 3222 1010 1001 3600 3581 Main Entrance Regeneration Mechanisms 3223 – 3264 Reprogramming 3265 – 3314 Embryonic Stem Cells Clinical Application 3315 – 3340 Embryonic Stem Cell Differentiation 3341 – 3508 Embryonic Stem Cell Pluripotency 3509 – 3600 MAIN ENTRANCE NO PHOTOGRAPHY OR RECORDINGS PERMITTED IN THE POSTER AREAS iii ISSCR 9th Annual Meeting www.isscr.org

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PANCREATIC CELLS...... 1. Poster Board Number: 1023 DIFFERENTIAL METHYLATION PATTERNS THAT Poster Board Number: 1001 MODULATE STAGE-SPECIFIC DEVELOPMENT OF LIVER EFFECT OF ENDOTHELIAL CELL CO-CULTURE ON LINEAGES...... 4 MATURATION OF HUMAN EMBRYONIC STEM CELL Poster Board Number: 1025 (HESC) TO PANCREATIC ISLET-LIKE CELLS ...... 1 HUMAN ES CELLS ENGINEERED WITH A REPORTER IN Poster Board Number: 1003 THE CYP-3A4 GENE FOR HEPATOCYTE-BASED DRUG EXPANSION OF ADULT HUMAN PANCREATIC STEM CELLS METABOLISM AND TOXICITY SCREENING...... 4 WITH β-CELL AND α-CELL DIFFERENTIATION POTENTIAL Poster Board Number: 1027 BY SUPPRESSION OF ASYMMETRIC CELL KINETICS (SACK)...... 1 DERIVATION OF HEPATOPANCREATIC STEM/PROGENITOR CELL LINES FROM HESC AND HIPSC ...... 5 Poster Board Number: 1005 Poster Board Number: 1029 INDUCTIVE MOLECULES DERIVED FROM HUMAN FETAL LIVER STROMAL CELLS ENHANCE THE GROWTH AND ISOLATION AND CLONOGENIC EXPANSION OF CD34+ DIFFERENTIATION OF HUMAN PANCREATIC PROGENITOR STEM CELLS CAPABLE OF PRODUCING HUMAN CELLS...... 1 HEPATOCELLULAR CARCINOMA...... 5 Poster Board Number: 1007 Poster Board Number: 1031 CHARACTERIZATION OF MURINE LIVER, PANCREAS AND HUMAN ADULT HEPATIC PROGENITOR CELLS, BONE KIDNEY PROGENITORS...... 2 MORPHOGENIC PROTEIN AND GREMLIN IN THE PATHOGENESIS OF HEPATOCELLULAR CARCINOMA ON Poster Board Number: 1009 TOP OF CHRONIC HCV INFECTION...... 6 ISOLATING FETAL MOUSE ISLET PROGENITORS WITH A Poster Board Number: 1033 NOVEL MONOCLONAL ANTIBODY...... 2 THE PLOIDY-CONVEYOR OF MATURE HUMAN Poster Board Number: 1011 HEPATOCYTES AS A SOURCE OF EXTENSIVE GENETIC COMPARISON BETWEEN VARIOUS METHODS IN DIVERSITY ...... 6 EVALUATION OF B-ISLET CELL TRANSFORMATION . . . . 2 Poster Board Number: 1035 Poster Board Number: 1013 DISEASE-CORRECTED HEPATOCYTES FROM HUMAN REPROGRAMMING PANCREATIC EXOCRINE CELLS FAMILIAL HYPERCHOLESTEROLEMIA INDUCED TOWARDS BETA CELLS USING PTD-TRANSCRIPTION PLURIPOTENT STEM CELLS ...... 7 FACTORS ...... 2 Poster Board Number: 1037 Poster Board Number: 1015 ROUTINE PRODUCTION AND APPLICATIONS OF HUMAN THE HISTONE DEMETHYLASE LSD1 IS REQUIRED FOR PLURIPOTENT STEM CELL-DERIVED HEPATOCYTE-LIKE ENDOCRINE CELL FORMATION IN THE DEVELOPING CELLS...... 7 PANCREAS...... 3 Poster Board Number: 1039 Poster Board Number: 1017 MICROENVIRONMENT CHANGES BUT NOT DIRECT THE ESTABLISHMENT OF A SCREENING SYSTEM FOR DIFFERENTIATION ASSOCIATE WITH THE PREVENTION LOW MOLECULAR COMPOUNDS FOR β CELL INDUCING OF NON-ALCOHOLIC STEATOHEPATITIS ONSET ACTIVITY...... 3 OBSERVED AFTER SYSTEMIC ADMINISTRATION OF MULTIPOTENT MESENCHYMAL STROMAL CELLS INTO Poster Board Number: 1019 OBESE MICE WITH METABOLIC SYNDROME...... 7 RETINOIC ACID AMELIORATES TYPE I DIABETES Poster Board Number: 1041 MELLITUS BY UPREGULATING REGULATORY T CELLS AND BY PROMOTING BETA-CELL REGENERATION ...... 3 INTRAHEPATIC TRANSPLANT OF ALLOGENEIC MOUSE LIVER DERIVED-MESENCHYMAL STEM CELLS PRODUCING HUMAN FACTOR IX CORRECTS THE LIVER CELLS...... 4. HEMOPHILIA B PHENOTYPE...... 8 Poster Board Number: 1021 Poster Board Number: 1043 EFFICIENT DIFFERENTIATION OF PLURIPOTENT HESC TO MOLECULAR ANALYSIS OF THE ROLE OF CASPASE-8 GENERATE HEPATOCTYES CAPABLE OF CELL THERAPY- IN ACTIVATION AND IN VIVO SELECTION OF THE BASED RESCUE IN ACUTE LIVER FAILURE ...... 4 HEPATIC STEM CELL COMPARTMENT AND LIVER REGENERATION ...... 8 iv www.isscr.org Table of Contents

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Poster Board Number: 1045 Poster Board Number: 1067 NITRIC OXIDE ACTS VIA TWO INDEPENDENT MATRIX-PROMOTED EPITHELIAL-TO-MESENCHYMAL MECHANISMS TO REGULATE LIVER DEVELOPMENT AND TRANSITION LEADS TO EFFICIENT MESODERM REGENERATION IN ZEBRAFISH...... 9 FORMATION AND CARDIOGENESIS OF HUMAN Poster Board Number: 1047 PLURIPOTENT STEM CELLS ...... 13 Poster Board Number: 1069 KERATIN 7 AND PROMININ-1 EXPRESSION IS ASSOCIATED WITH LIVER DAMAGE AND PREDICTS GENERATION AND CARDIOMYOCYTES DIFFERENTIATION SHORT-TERM MORTALITY IN PATIENTS WITH ALCOHOL OF HUMAN INDUCED PLURIPOTENT STEM CELLS FROM HEPATITIS...... 9 POST-MYOCARDUAL INFARCTION HEAET FAILURE PATIENTS ...... 13 LUNG CELLS...... 10. Poster Board Number: 1071 Poster Board Number: 1049 VOLTAGE DEPENDENCE AND KINETICS OF ENDOGENOUS IKS CHANNELS IN HUMAN EMBRYONIC PEDIGREE TRACKING REVEALS REGIOSPECIFIC STEM STEM CELL DERIVED CARDIOMYOCYTES: IMPLICATIONS CELLS OF THE HUMAN AIRWAYS...... 10 FOR SUBUNIT STOICHIOMETRY IN THE ASSEMBLED Poster Board Number: 1051 CHANNEL...... 13 PNEUMACULT™-ALI: AN IMPROVED MEDIA Poster Board Number: 1073 FORMULATION FOR THE DIFFERENTIATION OF PRIMARY UNIQUE IMMUNOLOGICAL PROPERTIES OF HUMAN BRONCHIAL EPITHELIAL CELLS AT THE AIR- HUMAN EMBRYONIC STEM CELL-DERIVED LIQUID INTERFACE...... 10 CARDIOMYOCYTES...... 14 Poster Board Number: 1053 Poster Board Number: 1075 CANCER STEM CELLS IN A MOUSE MODEL OF LUNG GENE TARGETING IN HUMAN EMBRYONIC STEM CELLS ADENOCARCINOMA...... 10 UNDERPINNING THE STUDY OF PRIMARY HEART FIELD Poster Board Number: 1055 IN VITRO ...... 14 TRACKING MOUSE LUNG EPITHELIAL PROGENITORS Poster Board Number: 1077 DURING EMBRYOGENESIS AND ADULTHOOD ...... 11 HUMAN CARDIAC STEM CELLS ISOLATED FROM ATRIAL Poster Board Number: 1057 APPENDAGES STABLY EXPRESS C-KIT WITHOUT LINEAGE TARGETED MURINE BONE MARROW CELL THERAPY FOR DEPLETION ...... 14 CFTR RESTORATION IN THE LUNG ...... 11 Poster Board Number: 1079 Poster Board Number: 1059 ASSEMBLY OF AN ELECTROMECHANICALLY FUNCTIONAL THE STUDY FOR THE ROLE OF PULMONARY STEM/ 3D BIOSYNTHETIC TISSUE USING MOUSE EMBRYONIC OR PROGENITOR CELLS IN HYPEROXIA INDUCED LUNG INDUCED PLURIPOTENT STELL CELL-DERIVED CARDIAC INJURY...... 11 PROGENITOR CELLS...... 15 Poster Board Number: 1081 CARDIAC CELLS...... 12. A NOVEL CELL-LINE MODEL SYSTEM FOR MOUSE Poster Board Number: 1061 CARDIAC PROGENITOR CELLS...... 15 FUNCTIONAL COMPARISON OF LONG QT-ASSOCIATED Poster Board Number: 1083 CARDIAC ION CHANNELS (INA, IKR, IKS) IN HUMAN MITOCHONDRIAL DNA MUTATIONS AS USEFUL EMBRYONIC STEM CELLS AND HUMAN INDUCED MARKERS FOR FUNCTIONAL COMPETENCE OF MOUSE PLURIPOTENT STEM CELLS ...... 12 ADULT STEM CELLS...... 15 Poster Board Number: 1063 Poster Board Number: 1085 LINEAGE SPECIFICATION OF MOUSE AND MICRORNA PROFILING PREDICTS A VARIANCE IN THE HUMAN EMBRYONIC STEM CELL DERIVED CARDIAC PROLIFERATIVE POTENTIAL OF CARDIAC PROGENITOR MESODERM...... 12 CELLS DERIVED FROM NEONATAL AND ADULT MOUSE Poster Board Number: 1065 HEARTS ...... 16 CHARACTERIZATION OF NOVEL CIRCULATING Poster Board Number: 1087 MULTIPOTENT STEM CELLS DERIVED FROM HUMAN GCSF AND SITAGLIPTIN ENHANCE STEM CELL PERIPHERAL BLOOD ...... 12 HOMING, CARDIAC FUNCTION AND SURVIVAL AFTER MI IN MICE...... 16

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Poster Board Number: 1089 Poster Board Number: 1111 NOVEL SMALL MOLECULES FACILITATE HIGH DO HUMAN ENDOTHELIAL PROGENITOR CELLS EFFICIENT CARDIAC DIFFERENTIATION FROM ES AND PARTICIPATE IN VASCULOGENESIS DURING FETAL IPS CELLS...... 17 DEVELOPMENT? ...... 20 Poster Board Number: 1091 Poster Board Number: 1113 IN VITRO MODELING OF ARRHYTHMOGENIC RIGHT HUMAN EMBRYONIC STEM CELLS AND HUMAN VENTRICULAR CARDIOMYOPATHY...... 17 INDUCED PLURIPOTENT STEM CELLS FOR Poster Board Number: 1093 STUDYING THE CARDIOVASCULAR DEFECT OF MARFAN SYNDROME ...... 21 A MODEL FOR LQT3 USING DISEASE-SPECIFIC IPS CELL- Poster Board Number: 1115 DERIVED CARDIOMYOCYTES...... 17 Poster Board Number: 1095 DIFFERENTIATION OF HUMAN INDUCED PLURIPOTENT STEM CELLS INTO FUNCTIONAL CD34+ CELLS BY EPIGENETIC REGULATION OF CARDIAC MODULATING MEK/ERK AND BMP4 SIGNALING DIFFERENTIATION ...... 18 PATHWAYS...... 21 Poster Board Number: 1097 Poster Board Number: 1117 SCREENING OF NOVEL CHEMICALS EFFICIENTLY MAJOR ROLE FOR P-SELECTIN DURING HOMING INDUCING CARDIOMYOCYTE DIFFERENTIATION FROM OF TRANSPLANTED MOUSE BONE MARROW PLURIPOTENT STEM CELLS ...... 18 MONONUCLEAR CELLS TO ISCHEMIC HINDLIMB. . . 22 Poster Board Number: 1099 Poster Board Number: 1119 IN VIVO DIFFERENTIATION OF EPIGENETICALLY INDUCTION OF LYVE-1/STABILIN-2-POSITIVE LIVER REPROGRAMMED ENDOTHELIAL PROGENITOR SINUSOIDAL ENDOTHELIAL CELLS FROM MOUSE CELLS INTO CARDIOMYOCYTES ENHANCES EMBRYONIC STEM CELL-DERIVED EMBRYOID BODIES FUNCTIONAL AND ANATOMICAL POST-INFARCT BY MODULATION OF ADRENOMEDULLIN-RAMP2 MYOCARDIAL REPAIR...... 18 SIGNALING...... 22 Poster Board Number: 1101 Poster Board Number: 1121 RGS4-LACZ EXPRESSION IS ASSOCIATED WITH DEVELOPMENT OF A HIGH-THROUGHPUT AND ROBUST SPONTANEOUSLY BEATING CARDIOMYOCYTES DURING VASCULAR DIFFERENTIATION ASSAY USING MOUSE SINOATRIAL NODE DEVELOPMENT...... 18 EMBRYONIC STEM CELLS...... 22 Poster Board Number: 1103 Poster Board Number: 1123 THE EFFECT OF ELECTRICAL FIELD STIMULATION ON IDENTIFICATION OF THE EMERGENCE OF CLONALLY CARDIOMYOCYTE GROWTH ON COLLAGEN GEL AND MULTIPOTENT HEMATOPOIETIC STEM AND PROGENITOR 2D-COATING...... 19 POPULATIONS IN THE MOUSE EMBRYO...... 23 Poster Board Number: 1105 Poster Board Number: 1125 VESSEL-DERIVED STEM CELLS FOR THERAPY OF HEMATOGENIC ENDOTHELIUM DERIVED FROM SKELETAL AND CARDIAC MUSCLE IN DUCHENNE EMBRYONIC STEM CELLS IN RHESUS MONKEY ...... 23 MUSCULAR DYSTROPHY...... 19 Poster Board Number: 1127 ENDOTHELIAL CELLS/HEMANGIOBLASTS. . . .20 SCL/TAL1 IS REQUIRED TO INHIBIT CARDIOGENESIS FROM ENDOTHELIUM IN HEMATOPOIETIC TISSUES. . 23 Poster Board Number: 1107 Poster Board Number: 1129 OPTIMIZATION OF A DERIVATION NICHE TO ELUCIDATE THE LINK BETWEEN HUMAN OVER EXPRESSION OF DOMINANT NEGATIVE RETINOIC PLURIPOTENT STEM CELL ORIGIN AND ENDOTHELIAL ACID RECEPTOR-ALPHA (DNRARΑ) IMMORTALIZES DIFFERENTIATION CAPACITY ...... 20 EMBRYONIC HEMANGIOBLAST-LIKE PRECURSORS WITH HEMATOPOIETIC AND ENDOTHELIAL CELL Poster Board Number: 1109 DEVELOPMENTAL POTENTIAL...... 24 SYNTHETIC PEPTIDE-ACRYLATE SURFACE FOR LONG Poster Board Number: 1131 TERM EXPANSION AND ENDOTHELIAL DIFFERENTIATION OF HUMAN INDUCED PLURIPOTENT STEM CELLS . . .20 ADULT UTERINE HEMANGIOBLASTS: EXTRAMEDULLARY SELF-RENEWING AND BILINEAGE ...... 24

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Poster Board Number: 1133 INTESTINAL/GUT CELLS...... 28. ANGIOGENIN-1 IS NECESSARY FOR THE GENERATION OF ENDOTHELIAL PROGENITOR CELL COLONIES ...... 24 Poster Board Number: 1157 Poster Board Number: 1135 THE COLORECTAL CANCER STEM CELL MARKER, CD166/ ALCAM PLAYS AN ESSENTIAL ROLE IN MAINTAINING VEGFR-3 SIRNA NANOPARTICLES INHIBIT THE NORMAL MOUSE AND HUMAN INTESTINAL STEM LYMPHANGIOGENESIS OF LYMPHATIC ENDOTHELIAL CELL NICHE ...... 28 PROGENITOR CELLS...... 25 Poster Board Number: 1159 Poster Board Number: 1137 CRITICAL ROLE OF THE MACROPHAGE COLONY- CYTOPROTECTIVE EFFECTS OF AUTOPHAGY ON STIMULATING FACTOR RECEPTOR (C-FMS) IN MOUSE SURVIVAL OF HYPOXIA-INDUCED ENDOTHELIAL INTESTINAL STEM CELL MAINTENANCE THROUGH PROGENITOR CELLS...... 25 PANETH CELLS...... 29 Poster Board Number: 1139 Poster Board Number: 1161 TISSUE PLASMINOGEN ACTIVATOR IMPAIRS STEM CELL TELOMERASE-EXPRESSING MOUSE INTESTINAL STEM FUNCTION, THUS LIMITING RECOVERY FOLLOWING CELLS ARE ACTIVATED IN RESPONSE TO FASTING. . .29 STROKE ...... 25 Poster Board Number: 1163 EPIDERMAL CELLS ...... 26 MYB CONTROLS INTESTINAL STEM CELL GENES AND SELF RENEWAL ...... 29 Poster Board Number: 1141 Poster Board Number: 1165 EMBRYONIC STEM CELL LIKE CELLS FROM HUMAN FORESKIN FIBROBLASTS ...... 26 IN VIVO GENERATION OF FUNCTIONAL INSULIN-PRODUCING CELLS IN THE GUT BY FOXO1 Poster Board Number: 1143 KNOCKOUT...... 30 ROLE OF AGE IN MOUSE STEM CELL MEDIATED WOUND HEALING ...... 26 EPITHELIAL CELLS (NOT SKIN)...... 30. Poster Board Number: 1145 Poster Board Number: 1167 FINE-TUNING OF MOUSE EPIDERMAL STEM CELL THERAPEUTIC POTENTIAL OF HUMAN AMNION BEHAVIOR BY THE MOLECULAR CLOCK PROTEIN EPITHELIAL CELLS FOR AUTOIMMUNE-MEDIATED BMAL1...... 26 DEMYELINATION ...... 30 Poster Board Number: 1147 Poster Board Number: 1169 IGF1 SIGNALING CONTRIBUTES TO BULGE EPIDERMAL HUMAN PLACENTA DERIVED MESENCHYMAL STEM STEM CELL HOMEOSTASIS AND IS REQUIRED FOR CELLS MODULATE ALLOGENEIC IMMUNE CELL THEIR ACTIVATION IN AGED MICE IN RESPONSE TO RESPONSE...... 30 WOUNDING...... 27 Poster Board Number: 1171 Poster Board Number: 1149 THE ROLE OF ΔNP63Α IN HUMAN CYTOTROPHOBLAST REGULATING HAIR FOLLICLE STEM CELL MAINTENANCE, STEM CELL PROLIFERATION AND LINEAGE ACTIVATION AND SELF-RENEWAL...... 27 SPECIFICATION...... 30 Poster Board Number: 1151 Poster Board Number: 1173 IMPACT OF CRYOPRESERVATION ON EPITHELIAL STEM EPITHELIAL TO MESENCHYMAL TRANSITION OF HUMAN CELLS GROWN IN MONOLAYER CULTURE AND IN TISSUE AMNION EPITHELIAL CELLS...... 31 ENGINEERED SKIN...... 27 Poster Board Number: 1175 Poster Board Number: 1153 TENDON HEALING INDUCED BY ALLOTRANSPLANTED ADULT SALIVARY GLAND DERIVED PROGENITOR CELLS OVINE EPITHELIAL AMNIOTIC CELLS...... 31 AND THEIR POTENTIALITY TO DIFFERENTIATE INTO ENDODERM ORIGINATED CELL TYPES...... 27 Poster Board Number: 1177 Poster Board Number: 1155 LACRIMAL GLAND REGENERATION PTENTIAL BY THEIR PROGENITOR CELLS...... 32 ROLE OF RAC1 IN INTERCELLULAR COMMUNICATION WITHIN THE EPIDERMIS...... 28

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EYE OR RETINAL CELLS...... 32 Poster Board Number: 1203 TRANSFER OF MRNA AND MICRO-RNA BY EMBRYONIC Poster Board Number: 1179 STEM CELL-DERIVED MICROVESICLES INDUCES GENE DIFFERENTIATION OF RETINAL CELLS FROM HUMAN EXPRESSION CHANGES IN RETINAL PROGENITOR INDUCED PLURIPOTENT STEM CELLS UNDER XENO-FREE MüLLER CELLS...... 36 CULTURE CONDITIONS...... 32 Poster Board Number: 1181 ETHICS AND POLICY...... 37. EX VIVO EXPANSION OF AUTOLOGOUS HUMAN Poster Board Number: 1205 CONJUNCTIVAL EPITHELIAL CELL GRAFT FOR HOPEFUL JOURNEYS: EXPERIENCES OF AUSTRALIANS TREATMENT OF RECURRENT PTERYGIUM: A MULTI- TRAVELLING OVERSEAS FOR STEM CELL TREATMENT CENTRIC CLINICAL STUDY REPORT...... 32 AND IMPLICATIONS FOR STEM CELL SCIENCE AND Poster Board Number: 1183 REGENERATIVE MEDICINE...... 37 MICRORNA-145 REGULATES THE DIFFERENTIATION OF Poster Board Number: 1207 HUMAN CORNEAL STEM CELLS...... 33 TAKING STEM CELLS INTO THE CLASSROOM - TAILORED Poster Board Number: 1185 EDUCATIONAL MATERIAL TO ASSIST AUSTRALIAN TEACHERS ...... 37 SUBRETINAL TRANSPLANTATION OF NEURAL PROGENITORS DERIVED FROM HUMAN EMBRYONIC Poster Board Number: 1209 STEM CELLS INTO RETINAL DEGENERATION RAT INFORMING THE PUBLIC - STEM CELL THERAPIES, NOW MODEL ...... 33 AND IN THE FUTURE...... 37 Poster Board Number: 1187 Poster Board Number: 1211 A SIMPLE AND EFFICIENT PROTOCOL FOR EXPERIMENTAL TREATMENT, STEM CELLS AND SPINAL DIFFERENTIATION OF RETINAL PIGMENTED EPITHELIAL CORD INJURIES: IS THERE A BALANCED RIGHT BETWEEN CELLS FROM HUMAN INDUCED PLURIPOTENT STEM RISK AND LUXURY? ...... 38 CELLS...... 33 Poster Board Number: 1213 Poster Board Number: 1189 TRACKING DONORS IN STEM CELL RESEARCH AND INDUCTION OF HUMAN AND MOUSE STEM CELLS BANKING: POLICY CONSIDERATIONS...... 38 TOWARDS A PHOTORECEPTOR-LIKE FATE...... 34 Poster Board Number: 1215 Poster Board Number: 1191 REGULATION OF STEM CELL-BASED THERAPIES EXOGENOUS FACTORS ENRICH THE PROPORTION OF IN CANADA: A SURVEY OF CURRENT ISSUES AND NEURAL RETINAL PROGENITORS DERIVED FROM ADULT CONCERNS...... 38 MOUSE RETINAL STEM CELLS...... 34 Poster Board Number: 1217 Poster Board Number: 1193 GERMANY’S SEARCH FOR NEW STEM CELL RESEARCH A BIOENGINEERED DELIVERY SYSTEM FOR THE RULES BETWEEN THE POLES OF THE RECENT TRANSPLANTATION OF MOUSE RETINAL STEM PREIMPLANTATION GENETIC DIAGNOSIS RULING OF CELL-DERIVED ROD PHOTORECEPTORS DIRECTLY THE FEDERAL SUPREME COURT AND THE IMPACT OF ENCOURAGES CELL SURVIVAL...... 35 REPROGRAMMED STEM CELLS...... 39 Poster Board Number: 1195 Poster Board Number: 1219 LOSS OF P-CADHERIN NEGATIVELY AFFECTS MOUSE A HISTORY OF THE GUIDELINES FOR CLINICAL RETINAL STEM CELLS IN VITRO, BUT NOT IN VIVO.. . 35 TRANSLATION OF REGENERATIVE MEDICINE Poster Board Number: 1197 RELEASED BY MINISTRY OF HEALTH, LABOR AND WELFARE, JAPAN...... 39 GENERATION OF RETINAL PIGMENT EPITHELIUM FROM ADIPOSE-DERIVED STEM CELLS ...... 35 Poster Board Number: 1199 MUSCLE CELLS...... 39. Poster Board Number: 1221 LIMBAL STEM CELL CULTURE’S JOURNEY TO WIDESPREAD ADOPTION...... 36 PAX7 EXPRESSING CELLS CONTRIBUTE TO DERMAL WOUND REPAIR; REGULATING SCAR SIZE THROUGH A Poster Board Number: 1201 β-CATENIN MEDIATED PROCESS ...... 39 RESVERATROL PROTECTS RETINAL STEM CELLS FROM OXIDATIVE STRESS VIA EXPRESSION OF SIRT1. . . . 36 viii www.isscr.org Table of Contents

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Poster Board Number: 1223 Poster Board Number: 1253 ORIGIN OF MUSCLE SATELLITE CELLS IN THE XENOPUS DIFFERENTIATION AND CHARACTERIZATION OF RETT EMBRYO...... 40 SYNDROME PATIENT-SPECIFIC NEURAL PROGENITOR Poster Board Number: 1225 CELLS...... 43 Poster Board Number: 1255 CLONED MURINE SATELLITE CELLS DISPLAY HETEROGENEITY AND DISTINCT DIFFERENTIATION DISEASE RELEVANT PHENOTYPE IN MOTOR NEURONS POTENTIAL BOTH IN VITRO AND IN VIVO ...... 40 DERIVED FROM AMYOTROPHIC LATERAL SCLEROSIS Poster Board Number: 1227 (ALS) PATIENT IPS CELLS...... 44 Poster Board Number: 1257 A MICRORNA CONTROLS THE SKELETAL MUSCLE / BROWN FAT SWITCH ...... 40 GENERATION AND MAINTENANCE OF NEURAL Poster Board Number: 1229 PROGENITOR CELLS FROM HUMAN INDUCED PLURIPOTENT STEM CELLS ...... 44 SMALL MOLECULE SCREENING FOR MOUSE SATELLITE Poster Board Number: 1259 CELL PROLIFERATION...... 41 Poster Board Number: 1233 DERIVATION OF HUMAN NEURAL PROGENITORS FROM FRAGILE X SYNDROME INDUCED PLURIPOTENT A MUSCLE PRECURSOR DEFECT ASSOCIATED WITH LOW STEM CELL LINES WITH VARIATION IN CGG REPEAT, LEVELS OF SURVIVAL OF MOTOR NEURON PROTEIN.. 41 DNA METHYLATION, AND EXPRESSION OF THE FMR1 Poster Board Number: 1235 LOCUS...... 44 A LONG-TERM SELF-RENEWABLE POPULATION IN Poster Board Number: 1261 ACTIVATED SATELLITE CELLS IN MOUSE SKELETAL CHARACTERIZATION OF WNT/GSK-3 SIGNALING IN MUSCLE. . . Poster. . . .Withdrawn ...... 41 HUMAN NEURAL PROGENITOR CELL MODELS OF Poster Board Number: 1239 FRAGILE X SYNDROME DERIVED FROM INDUCED PLURIPOTENT STEM CELLS ...... 45 TRANSPLANTATION OF MESENCHYMAL STEM CELLS FROM MOUSE ES CELLS AFTER MUSCLE INJURIES . . . 41 Poster Board Number: 1263 DEVELOPMENT OF A NOVEL HTS AMENABLE ASSAY NEURAL CELLS...... 42. PLATFORM FOR VISUAL ASSESSMENT OF HUMAN EMBRYONIC STEM CELL DERIVED NEURAL PROGENITOR Poster Board Number: 1243 MIGRATION ...... 45 REPROGRAMMING OF HUMAN RETINAL PROGENITOR Poster Board Number: 1265 CELLS TO PLURIPOTENCY VIA LENTIVIRAL INDUCTION OF SOX2...... 42 MMP-9 ASSOCIATED WITH THE TETRASPANIN CD82 AND REGULATED THE MIGRATION TO HUMAN NEURONAL Poster Board Number: 1245 STEM CELL...... 45 USING HUMAN IPS AND INDUCED NEURONAL CELLS Poster Board Number: 1267 (IN CELLS) TO INVESTIGATE PSYCHIATRIC DISEASE RISK GENES ...... 42 DOUBLECORTIN EXPRESSION PROMOTES THE MIGRATION OF HUMAN EMBRYONIC STEM CELL- Poster Board Number: 1247 DERIVED NEURONS AND STIMULATES THE DEVELOPMENT OF AN ASSAY TO IDENTIFY ACTIVATORS CONSTRUCTION OF NEURAL PROGENITOR SCAFFOLDS OF TRKB SIGNALING USING HUMAN INDUCED THAT GUIDE MIGRATION ...... 46 PLURIPOTENT STEM CELL DERIVED NEURONS ...... 42 Poster Board Number: 1269 Poster Board Number: 1249 USING HUMAN IPSC DERIVED NEURAL CELLS FROM OLIGODENDROCYTE PROGENITORS DERIVED FROM GENETICALLY RELATED INDIVIDUALS TO UNDERSTAND HUMAN INDUCED PLURIPOTENT STEM CELLS IMPROVE BIPOLAR DISORDER...... 46 RECOVERY OF RAT MODEL OF OPTIC CHIASM Poster Board Number: 1271 DEMYELINATION ...... 43 AZACYTIDINE INDUCED DNA DEMETHYLATION Poster Board Number: 1251 PROMOTES ASTROCYTIC DIFFERENTIATION OF NEURAL IPS CELL-DERIVED MODELS FOR HUMAN PROGENITORS DERIVED FROM HUMAN EMBRYONIC MITOCHONDRIAL DISORDERS...... 43 STEM CELLS...... 46

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Poster Board Number: 1273 Poster Board Number: 1297 SCALABLE HUMAN NEURAL STEM CELLS PRODUCE MOLECULAR REGULATION OF NEURAL STEM CELL ROBUST NEURAL MODEL...... 47 QUIESCENCE IN THE AGING MURINE BRAIN. . . . . 51 Poster Board Number: 1275 Poster Board Number: 1299 ACCELERATED DIFFERENTIATION OF HUMAN JMJD3-MEDIATED EPIGENETIC REGULATION OF NEURAL STEM CELLS BY A CELL-CONTACT MEDIATED NEUROGENESIS FROM ADULT MOUSE NEURAL STEM MECHANISM ...... 47 CELLS...... 51 Poster Board Number: 1277 Poster Board Number: 2001 ELECTROPHYSIOLOGICAL PROPERTIES AND LIF-DEPENDENT PRIMITIVE NEURAL STEM CELLS EXPRESSION OF VOLTAGE-GATED ION CHANNELS IN IN THE ADULT MOUSE SUBEPENDYMA COMPRISE A HUMAN EMBRYONIC STEM CELLS DURING NEURAL DISTINCT POPULATION THAT CAN BE MANIPULATED DIFFERENTIATION ...... 47 INDEPENDENT OF ADULT DEFINITIVE NEURAL STEM Poster Board Number: 1279 CELLS...... 52 Poster Board Number: 2003 EXPANSION AND DIFFERENTIATION OF PSA-NCAM POSITIVE NEURAL PRECURSORS DERIVED FROM INSULIN RECEPTOR ACTIVATION PROMOTES MURINE HUMAN PLURIPOTENT STEM CELLS...... 48 NEURAL PRECURSOR STEMNESS...... 52 Poster Board Number: 1281 Poster Board Number: 2005 IDENTIFYING BIOMARKERS OF FETAL ALCOHOL VENTRAL HIPPOCAMPUS AS THE ORIGIN FOR THE EXPOSURE USING METABOLOMICS AND DERIVATIVES MOUSE SUBGRANULAR NEURAL STEM CELLS AT ALL OF HUMAN EMBRYONIC STEM CELLS ...... 48 LEVELS...... 52 Poster Board Number: 1283 Poster Board Number: 2007 METABOLOMICS OF HUMAN EMBRYONIC AND MASH1 IS A NOVEL TARGET OF GLI2 DURING GLI2- INDUCED PLURIPOTENT STEM CELLS TO PREDICT INDUCED NEUROGENESIS IN MURINE P19 EMBRYONAL DEVELOPMENTAL TOXICITY: A COMPARISON ...... 49 CARCINOMA STEM CELLS ...... 52 Poster Board Number: 1285 Poster Board Number: 2009 METFORMIN ACTIVATES ATYPICAL PKCS TO PROMOTE TRANSGENIC ENRICHMENT OF MOUSE EMBRYONIC RODENT AND HUMAN NEUROGENESIS AND ENHANCE STEM CELL-DERIVED PROGENITOR MOTOR NEURON HIPPOCAMPUS-DEPENDENT LEARNING...... 49 CELLS...... 53 Poster Board Number: 1287 Poster Board Number: 2011 NEUROTROPHIN SIGNALING IN NEURONAL CHEMOTAXIS OF MOUSE NEURAL STEM CELL-DERIVED DIFFERENTIATION OFMOUSE AND HUMAN EMBRYONIC NEUROBLASTS IS STIMULATED BY CXCL12 N-TERMINAL STEM CELLS IN 2D AND 3D MODELS ...... 49 PEPTIDES...... 53 Poster Board Number: 1289 Poster Board Number: 2013 MAMMALIAN GLIAL CELLS MISSING GENES INDUCE FOXP2 REGULATES NEUROGENESIS IN THE EMBRYONIC HES5 EXPRESSION BY ACTIVE DNA DEMETHYLATION IN MURINE CORTEX ...... 53 EARLY MOUSE EMBRYOS...... 50 Poster Board Number: 2015 Poster Board Number: 1291 STEM CELL TRANSPLANTATION IMPROVES AXONAL HUMAN NEURAL STEM CELLS PROMOTE RECOVERY OF CONDUCTION IN THE SPINAL CORD BY ALTERING BK FUNCTION IN A MOUSE UNILATERAL CERVICAL SPINAL CHANNEL EXPRESSIONS IN THE RAT ...... 54 CORD INJURY MODEL ...... 50 Poster Board Number: 2017 Poster Board Number: 1293 TRACING NEURONAL PROGENY OF ADULT RAT ADULT NEUROGENESIS AFTER ACUTE SGN EPENDYMAL CELLS USING ELECTROPORATION. . . .54 DEGENERATION IN THE MOUSE INNER EAR. . . . . 50 Poster Board Number: 2019 Poster Board Number: 1295 AMYGDALA REGULATION OF ADULT NEUROGENESIS IS ALTERED LIPID REGULATION INVOLVED IN AGE AND FEAR-RELATED MEMORY IN RATS...... 54 RELATED DEFICITS IN ADULT NEUROGENESIS IN THE Poster Board Number: 2021 MURINE SUBVENTRICULAR ZONE?...... 51 VASCULAR NICHE FACTORS REGULATE RODENT ADULT CAROTID BODY STEM CELL ACTIVITY...... 54 x www.isscr.org Table of Contents

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Poster Board Number: 2023 Poster Board Number: 2047 OPTIMIZATION AND VALIDATION OF A SELECTION IN VIVO VISUALISATION OF MIR-124 IN NEURAL STEM STRATEGY FOR ES-DERIVED MIDBRAIN DOPAMINE CELLS USING LENTIVIRAL TRANSGENESIS...... 59 NEURONS FOR TRANSPLANTATION AND DISEASE Poster Board Number: 2049 MODELING IN PARKINSON DISEASE ...... 55 DAMAGED PROTEIN SEGREGATION DIFFERS Poster Board Number: 2025 BETWEEN ASYMMETRICALLY DIVIDING STEM CELL HIGH-THROUGHPUT SCREENING OF SMALL MOLECULES POPULATIONS ...... 59 THAT DIRECT DOPAMINERGIC NEUROGENESIS IN Poster Board Number: 2051 NEURAL PROGENITOR CELLS...... 55 THE AVIAN TRUNK NEURAL CREST COMPRISES Poster Board Number: 2027 MULTIPOTENT PROGENITORS WITH NEUROGENIC, NEURAL PRECURSOR CELLS IN NEUROSPHERES SHOW OSTEOGENIC AND ADIPOGENIC CAPACITIES...... 59 GLIAL COMMITMENT IN EMBRYONIC NEUROGENIC Poster Board Number: 2053 ENVIRONMENTS...... 55 THE EFFECT OF POST-MI TIMING ON THE Poster Board Number: 2029 ENGRAFTMENT AND COLONIZATION OF MYOCARDIAL STAUFEN 2 REGULATES THE MAINTENANCE AND SCAR TISSUE BY A PRIMITIVE NEURAL CREST-LIKE DIFFERENTIATION OF NEURAL PRECURSORS DURING STEM CELL POPULATION DERIVED FROM ADULT ORAL MAMMALIAN CORTICAL DEVELOPMENT...... 56 MUCOSA ...... 60 Poster Board Number: 2031 Poster Board Number: 2055 DIRECT CONVERSION OF FIBROBLASTS TO A INTEGRATED PLATFORM FOR CREATION OF ASSAY- MULTIPOTENT NEURAL PRECURSOR POPULATION WITH READY NEURAL CELLS...... 60 DEFINED FACTORS...... 56 Poster Board Number: 2057 Poster Board Number: 2033 EFFICIENT INDUCTION AND PURIFICATION OF NEURAL EVALUATING STEM CELL-BASED PLASTICITY IN A PROGENITORS DERIVED FROM LINEAGE SPECIFIC MODEL OF CERVICAL SPINAL CORD INJURY...... 56 KNOCKIN HPSC REPORTERS...... 60 Poster Board Number: 2035 Poster Board Number: 2059 SKIN-DERIVED PRECURSORS DIFFERENTIATED INTO DIFFERENTIATION OF INDUCED PLURIPOTENT STEM SCHWANN CELLS (SKP-SCS) TRANSPLANTED EIGHT CELLS TO NEURAL - LIKE CELLS WITHOUT GROWTH WEEKS POST SPINAL CORD CONTUSION IMPROVE FACTORS ...... 61 RECOVERY OF MOTOR AND BLADDER FUNCTION AFTER Poster Board Number: 2061 INJURY...... 57 PRECISE TEMPORAL CONTROL OF THE Poster Board Number: 2037 MICROENVIRONMENT TO STEER NEURAL STEM CELL PPARS ROLES IN MOUSE ADULT NEURAL PRECURSORS DIFFERENTIATION ...... 61 CELLS...... 57 Poster Board Number: 2063 Poster Board Number: 2039 CELLULAR ORGANIZATION OF THE FOREBRAIN FGF-2 CAN RECRUIT VEGF SIGNALING THROUGH NEUROGENIC NICHE IN ADULT ZEBRAFISH...... 61 ACTIVATION OF FLK-1 IN VIVO TO PROMOTE NEUROGENESIS...... 57 CANCER CELLS...... 62. Poster Board Number: 2041 Poster Board Number: 2067 THE P53 FAMILY MEMBER P63 IS REQUIRED FOR THE CHARACTERIZATION OF HUMAN ACUTE MYELOID MAINTENANCE OF ADULT NEURAL PRECURSORS AND LEUKEMIA STEM CELLS...... 62 COGNITIVE FUNCTION...... 58 Poster Board Number: 2069 Poster Board Number: 2043 IDENTIFICATION AND CHARACTERIZATION OF HUMAN PROPERTIES OF ADULT NEURAL STEM CELLS ARE LEUKEMIA STEM CELL FUNCTIONAL REGULATORS. . . 62 REVEALED BY ALTERATIONS IN DISC1...... 58 Poster Board Number: 2071 Poster Board Number: 2045 miR-126 BIOACTIVITY MARKS THE PRIMITIVE THE EFFECTS OF PHYSICAL VS. COGNITIVE ACTIVITY COMPARTMENT IN AML AND REGULATES HUMAN ON ADULT NEUROGENESIS: UNRAVELLING THEIR LEUKEMIA STEM CELL NUMBERS ...... 62 MECHANISMS OF ACTION...... 58

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Poster Board Number: 2073 Poster Board Number: 2097 IDENTIFICATION OF HUMAN LEUKEMIA STEM CELL PARATHYROID HORMONE-RELATED PROTEIN (PTHRP) GENES BY IN VIVO RETROVIRAL INSERTIONAL PROMOTES THE ACQUISITION OF CANCER STEM CELL- MUTAGENESIS...... 63 LIKE PROPERTIES IN PROSTATE CANCER ...... 66 Poster Board Number: 2075 Poster Board Number: 2099 TRANSCRIPTION PROGRAM OF LEUKEMIA STEM CELL NOVEL MURINE CELL LINES ISOLATED FROM SELF- DEVELOPMENT IN MLL-ENL-INDUCED LEUKEMIA IN RENEWING PTEN-/-TP53-/- PROTOSPHERES HAVE MICE ...... 63 MULTIPOTENT PROGENITOR ACTIVITY AND FORM Poster Board Number: 2077 ADENOCARCINOMA AND METASTASES...... 67 Poster Board Number: 2101 A2B5-DEFINED TUMOR PROGENITORS DERIVED FROM HUMAN GLIOMAS EXHIBIT A CORE SET OF ATTACKING NEUROBLASTOMA CANCER STEM CELLS DYSREGULATED GENES AT ALL STAGES OF TUMOR WHERE THEY RESIDE: NICHE-SPECIFIC APPROACHES PROGRESSION, IN ADDITION TO GRADE-ASSOCIATED FOR RELAPSE METASTATIC DISEASE ...... 67 TRANSCRIPTIONAL CHANGES CONSISTENT WITH Poster Board Number: 2103 EPITHELIAL-TO-MESENCHYMAL TRANSITION ...... 63 IN SEARCH OF THE ELUSIVE NEUROBLASTOMA (NBL) Poster Board Number: 2079 TUMOR INITIATING CELLS (TIC) IN PERIPHERAL BLOOD ADAPTATION OF HUMAN GLIOMA STEM-LIKE CELLS TO HEMATOPOIETIC STEM CELLS (PBHSC) COLLECTIONS SEVERE HYPOXIA...... 64 AFTER G-CSF MOBILIZATION IN PATIENTS WITH NBL IN Poster Board Number: 2081 REMISSION...... 67 Poster Board Number: 2105 HOX GENES ARE ESSENTIAL FOR GLIOMA STEM CELL SURVIVAL AND TUMORIGENICITY ...... 64 KRUPPEL-LIKE FACTOR 4 SUPPRESSES Poster Board Number: 2083 NEUROBLASTOMA GROWTH BY PROMOTING SMOOTH- MUSCLE DIFFERENTIATION ...... 68 OVEREXPRESSION OF CD133 PROTMOTES DRUGS Poster Board Number: 2107 RESISTANCE IN U87 GLIOMA CELLS...... 64 Poster Board Number: 2085 MODELING THE NEUROBLASTOMA TUMOR INITIATING CELL MICROENVIRONMENTPoster Withdrawn IN 3D CULTURE...... 68 HOXA10 IS A POSTERIORIZING SIGNAL THAT Poster Board Number: 2109 CONTRIBUTES TO THE TUMORIGENIC PROPERTIES OF GLIOMA-INITIATING CELLS ...... 64 THE PROGNOSTIC ROLE OF HUMAN ADULT CANCER Poster Board Number: 2087 STEM CELL MARKER PROFILES IN AGGRESSIVE METASTATIC COLORECTAL CANCER...... 68 DEVELOPMENT OF HUMAN PROSTATE BENIGN AND Poster Board Number: 2111 CANCER STEM CELL SPECIFIC GENE EXPRESSION PROFILES...... 65 EXPRESSION OF SSEA-1/CD-15 IN DIFFERENT COLON Poster Board Number: 2089 CANCER CELL LINES...... 69 Poster Board Number: 2113 CONSTITUTIVE NF-KB SIGNALING MARKS A SUBSET OF STEM-LIKE HUMAN PROSTATE TUMOR-INITIATING INTESTINAL STEM CELL-LIKE CELLS IN COLORECTAL CELLS...... 65 CANCER RELAPSE...... 69 Poster Board Number: 2091 Poster Board Number: 2115 AMENDMENT OF THE HUMAN PROSTATE STEM CELL HUMAN CANCER STEM CELLS FROM MODEL: ACTIVE ANDROGEN RECEPTOR IS EXPRESSED CHEMORESISTANT OVARIAN EPITHELIAL CANCER: AT ALL STAGES OF PROSTATE DIFFERENTIATION. . . 66 A PERSONALIZED MODEL FOR OVARIAN CANCER Poster Board Number: 2093 THERAPY SCREENING...... 69 Poster Board Number: 2117 CELL DIVISION IN MOUSE AND HUMAN PROSTATE STEM CELLS...... 66 ALDEHYDE DEHYDROGENASE, A WIDELY-USED MARKER Poster Board Number: 2095 TO ISOLATE CANCER STEM CELLS IN VARIOUS TUMORS, SEEMS NOT TO ENRICH THE CSC-FRACTION IN OVARIAN ANDROGEN ABLATION MITIGATES DEFECT OF B CELLS CANCER...... 70 TO A PROSTRATE CANCER AND INCREASE SURVIVAL Poster Board Number: 2119 RATE OF TRAMP MICE...... 66 THE ROLE OF PERICYTES AS CANCER ASSOCIATED FIBROBLASTS...... 70 xii www.isscr.org Table of Contents

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Poster Board Number: 2121 Poster Board Number: 2145 ONCOLYTIC VIRUSES FOR ERADICATION OF CANCER MURINE HEMATOPOIETIC STEM CELLS ARE ACTIVELY STEM CELLS...... 70 CYCLING...... 75 Poster Board Number: 2123 Poster Board Number: 2147 CHARACTERIZATION AND TARGETING OF CANCER STEM BMP2 AND dkk1 INHIBITOR INDUCE OSTEOGENIC CELLS IN HUMAN MPNST...... 71 DIFFERENTIATION OF OSTEOSARCOMA CELLS ...... 75 Poster Board Number: 2125 Poster Board Number: 2149 AMAZING CANCER STEM CELLS AND THE MOST TOXICITY OF HEAVY METALS IN STEM CELLS. . . . .75 DESIRABLE CANCER IMMUNOTHERAPY...... 71 Poster Board Number: 2127 GERMLINE CELLS...... 76. NATURAL KILLER CELL LINES PREFERENTIALLY TARGET Poster Board Number: 2153 CLONOGENIC MULTIPLE MYELOMA CELLS ...... 71 THE TRANSDIFFERENTIATION OF THE HUMAN FORESKIN Poster Board Number: 2129 FIBROBLASTS TO FORM GERM CELLS USING RETINOIC ACID...... 76 A NOVEL FORKHEAD-ASSOCIATED SIGNALING DOMAIN CONTAINING PROTEIN ENHANCES SURVIVAL OF HUMAN Poster Board Number: 2155 EMBRYONIC STEM CELLS VIA CD30-DEPENDANT MEIOTIC GERM CELLS ENRICHED FROM ACTIVATION OF CANONICAL NFκB SIGNALING. . . . 72 DIFFERENTIATING HUMAN EMBRYONIC STEM CELLS Poster Board Number: 2131 VIA TRANSGENIC MANIPULATION, SURFACE MARKER SELECTION AND GROWTH FACTOR STIMULATION. . .76 PHENOTYPIC HETEROGENEITY OF TUMOR-INITIATING CELLS IN PRIMARY HUMAN HEPATOCELLULAR Poster Board Number: 2157 CARCINOMA...... 72 GERMLINE DIFFERENTIATION OF HUMAN EMBRYONIC Poster Board Number: 2133 STEM CELLS...... 76 INHIBITION OF AKT/PI3K PATHWAY INCREASES Poster Board Number: 2159 LOW RHODAMINE 123 RETENTION CELLS WITH CHARACTERIZATION OF A NOVEL LIF-RESPONSIVE, CHARACTERISTICS OF CANCER STEM CELLS IN HUMAN EMBRYONIC-LIKE, PRIMITIVE NEURAL STEM CELL IN MELANOMA...... 72 THE ADULT MOUSE BRAIN REVEALED FOLLOWING THE Poster Board Number: 2135 TARGETED ABLATION OF GFAP EXPRESSING ADULT NEURAL STEM CELLS...... 77 DISCOVERY OF NEW CELL SURFACE MARKERS TO IDENTIFY TUMOR INITIATING CELLS IN HUMAN Poster Board Number: 2161 MESENCHYMAL TUMORS...... 73 INVESTIGATION OF STEM CELLS IN ADULT AND PRE- Poster Board Number: 2137 ADULT MOUSE OVARIES ...... 77 RESVERATROL INHIBITS PANCREATIC CANCER STEM Poster Board Number: 2163 CELL CHARACTERISTICS IN HUMAN AND KRASG12D CROSSTALK OF HYPOXIA AND INSULIN-LIKE GROWTH TRANSGENIC MICE BY INHIBITING PLURIPOTENCY FACTOR-1 RECEPTOR SIGNALING IN SELF-RENEWAL MAINTAINING FACTORS AND EPITHELIAL- PROLIFERATION AND MIGRATION OF PLURIPOTENT MESENCHYMAL TRANSITION...... 73 MOUSE GERMLINE STEM CELLS ...... 77 Poster Board Number: 2139 Poster Board Number: 2165 REGULATION OF MOUSE ADULT STEM TO PROGENITOR IMPACT OF A SINGLE DOSE OF 12 GY IRRADIATION ON CELL TRANSITION BY MICRORNAS...... 73 BOVINE SPERMATOGONIAL STEM CELL SURVIVAL AND Poster Board Number: 2141 RECOVERY...... 78 POLYCOMB-MEDIATED EPIGENETIC REPRESSION OF Poster Board Number: 2167 KIT EXPRESSION UNDERLIES THERAPY RESISTANCE OF FUNCTION OF BAZOOKA/PAR3 IN ASYMMETRIC STEM MOUSE STEM CELLS FOR GASTROINTESTINAL STROMAL CELL DIVISION AND THE CENTROSOME ORIENTATION TUMORS...... 74 CHECKPOINT IN DROSOPHILA MALE GERMLINE STEM Poster Board Number: 2143 CELLS...... 78 TUMORIGENIC CELLS ARE ABUNDANT IN MOUSE MALIGNANT PERIPHERAL NERVE SHEATH TUMORS BUT THEIR FREQUENCY DEPENDS UPON TUMOR GENOTYPE AND EXPOSURE TO EXTRACELLULARMATRIX. . . . .74

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Poster Board Number: 2169 Poster Board Number: 2193 THE TRIM-NHL PROTEIN MEI-P26 REGULATES BOTH DEVELOPMENT OF REPORTER GENE PET IMAGING GERMLINE STEM CELL SELF-RENEWAL TECHNIQUES FOR LONG-TERM ASSESSMENT OF AND DIFFERENTIATION THROUGH DISTINCT TRANSPLANTED HUMAN CIRCULATING PROGENITOR MECHANISMS ...... 78 CELLS...... 82 Poster Board Number: 2195 TECHNOLOGIES FOR STEM CELL RESEARCH. . .79 ANTIBODY-DIRECTED LENTIVIRAL GENE TRANSDUCTION Poster Board Number: 2171 FOR LIVE-CELL MONITORING AND SELECTION OF A MULTI-STEP APPROACH FOR TARGETED CORRECTION HUMAN IPS AND HES CELLS ...... 83 OF THE ENDOGENOUS MUTATED GENE IN SCID-X1 Poster Board Number: 2197 FIBROBLASTS AND GENERATION OF REPROGRAMMING ADDRESSING THE SAFETY BOTTLENECK FOR USE FACTOR-FREE IPS CELLS...... 79 OF HUMAN PLURIPOTENT STEM CELLS IN CLINICAL Poster Board Number: 2173 APPLICATIONS: DEVELOPMENT OF ANTIBODIES TO DIAGNOSTIC MICROBIOREACTOR ARRAYS FOR NOVEL EPITOPES ON LIVE HUMAN PLURIPOTENT STEM MULTIPLEXED MICROENVIRONMENTAL SCREENING OF CELLS...... 83 PLURIPOTENT STEM CELL EXPANSION, MAINTENANCE Poster Board Number: 2199 AND DIFFERENTIATION ...... 79 TRANSIENTLY TRANSFECTED HUMAN MESENCHYMAL Poster Board Number: 2175 STEM CELLS PROVIDE A READY-TO-USE PLATFORM FOR A NEW EXTRACELLULAR MATRIX FOR FEEDER-FREE CELL-BASED ASSAYS...... 83 GROWTH OF PLURIPOTENT STEM CELLS...... 80 Poster Board Number: 2201 Poster Board Number: 2177 HIGHLY EFFICIENT EXPANSION OF HUMAN NOVEL SYNTHETIC, XENO-FREE SURFACE FOR MESENCHYMAL STEM CELLS IN A NOVEL SERUM FREE, EXPANSION AND DIFFERENTIATION OF HUMAN NEURAL ANIMAL COMPONENTS FREE AND CHEMICALLY DEFINE STEM CELLS IN SERUM-FREE MEDIUM...... 80 MEDIUM ...... 84 Poster Board Number: 2179 Poster Board Number: 2203 SYNTHETIC SURFACES FOR THE CULTURE OF HUMAN DEVELOPMENT OF SERUM FREE DEFINED MEDIUM FOR EMBRYONIC AND ADULT STEM CELLS...... 80 HUMAN MESENCHYMAL STEM CELL EXPANSION . . . . 84 Poster Board Number: 2181 Poster Board Number: 2205 APPLICATION OF SYNTHETIC MEDIUM AND GMP SYNTHETIC SURFACE FOR LONG-TERM CULTURE OF COMPLIANT HUMAN FEEDER CELLS IN THE HUMAN MESENCHYMAL STEM CELLS IN SERUM-FREE, MAINTENANCE, EXPANSION AND CLONAL RECOVERY OF XENO-FREE CONDITIONS...... 84 HUMAN EMBRYONIC STEM CELLS...... 80 Poster Board Number: 2207 Poster Board Number: 2183 ENHANCEMENT OF MESENCHYMAL STEM CELL YIELDS THE CORNING® SYNTHEMAX™ SURFACE: FROM HUMAN UMBILICAL CORD TISSUE...... 85 A COMPLETE SOLUTION FOR RECOVERY, EXPANSION, Poster Board Number: 2209 AND DIFFERENTIATION OF HUMAN EMBRYONIC STEM SCALING UP HUMAN MESENCHYMAL STEM CELLS CELLS...... 81 ON MICROCARRIERS IN SUSPENSION FOR CULTURE IN Poster Board Number: 2185 SINGLE-USE BIOREACTORS ...... 85 SINGLE-CELL BASED XENO-FREE CULTURE OF HUMAN Poster Board Number: 2211 EMBRYONIC STEM CELLS...... 81 EFFICIENT PRODUCTION OF HUMAN Poster Board Number: 2187 OLIGODENDROCYTE-LINEAGE CELLS FROM A NEW TYPE GENERATION OF NEW MONOCLONAL ANTIBODIES OF HUMAN NEURAL STEM CELLS ...... 85 ELICITED AGAINST HUMAN PLURIPOTENT STEM CELL Poster Board Number: 2213 DERIVATIVES AS MARKERS FOR EARLY STAGES OF A 3D CULTURE SYSTEM FOR MEDIUM-SCALE HUMAN DEVELOPMENT...... 81 PRODUCTION OF HUMAN EMBRYONIC STEM CELLS. . 86 Poster Board Number: 2189 Poster Board Number: 2215 BAR-CODING HUMAN EMBRYONIC PROGENITOR CELL NON-VIRAL STRATEGIES FOR ENHANCEMENT LINES USING PHAGE DISPLAY ...... 82 OF HUMAN INDUCED PLURIPOTENT STEM CELL GENERATION ...... 86 xiv www.isscr.org Table of Contents

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Poster Board Number: 2217 Poster Board Number: 2241 COMPARATIVE PROTEOMIC AND PHOSPHOPROTEOMIC EXPANSION AND CHARACTERIZATION OF CORD BLOOD- PROFILING OF HUMAN EMBRYONIC STEM CELLS DERIVED CD34+ CELLS ON 3-D NANEX NANOFIBER AND THEIR PURE PAX6+ NEUROECTODERMAL SCAFFOLD...... 90 DERIVATIVES ...... 86 Poster Board Number: 2243 Poster Board Number: 2219 SUR8 INVOLVES BOTH INHIBITION OF DIFFERENTIATION ANALYSIS OF THE HUMAN EMBRYONIC STEM CELL AND MAINTENANCE OF SELF-RENEWAL OF NEURAL DEPOSITOME...... 87 STEM CELLS VIA MODULATION OF ERK SIGNALING . . 91 Poster Board Number: 2221 Poster Board Number: 2245 DEFINING THE ADHESION CHARACTERISTICS OF FIBROBLAST-DERIVED EXTRACELLULAR MATRIX HUMAN EMBRYONIC STEM CELLS TO RECOMBINANT CONTROLS MATURATION OF ESC-DERIVED ECM FRAGMENTS USING SURFACE PLASMON CARDIOMYOCYTES...... 91 RESONANCE...... 87 Poster Board Number: 2247 Poster Board Number: 2223 LARGE PARTICLE FLOW CYTOMETRY FOR CELL LATE PASSAGE HUMAN FORESKIN FIBROBLASTS CLUSTERS, MICROCARRIERS AND OTHER 3-D CELL SUPPORT HUMAN EMBRYONIC STEM CELLS IN CULTURE ENTITIES...... 91 CULTURE...... 87 Poster Board Number: 2249 Poster Board Number: 2225 IRON IN NEURAL STEM CELLS CAN BE TRACKED WITH S-SHIP PROMOTER EXPRESSION IDENTIFIES A SYNCHROTRON X-RAY FLUORESCENCE MICROPROBE.. 92 SUBPOPULATION OF MOUSE PROSTATE BASAL CELLS Poster Board Number: 2251 WITH STEM CELL CHARACTERISTICS...... 88 HIGH CONTENT ANALYSIS OF NEURAL STEM CELL Poster Board Number: 2227 EXPANSION AND DIFFERENTIATION...... 92 NANOG EXPRESSION ON MOUSE PARAFFIN TISSUE. . 88 Poster Board Number: 2253 Poster Board Number: 2229 CHARACTERIZATION OF TRANSGENIC HUNTINGTON ESTABLISHMENT OF MOUSE EMBRYONIC STEM CELLS MONKEY NEURAL PROGENITOR CELLS DERIVED CULTURE IN 96 WELL PLATE FOR HIGH-THROUGHPUT FROM INDUCED PLURIPOTENT DENTAL PULP STROMAL SCREENING ...... 88 CELLS...... 92 Poster Board Number: 2231 Poster Board Number: 2255 MULTIPLEX SINGLE CELL REAL TIME PCR FOR STEMNESS ISOLATION OF MESENCHYMAL STEM CELL-LIKE CELL MARKERS IN MOUSE EMBRYONIC STEM CELLS. . . .89 POPULATION FROM BLASTAMA OF RABBIT PINNA . . . 93 Poster Board Number: 2233 Poster Board Number: 2257 SORTING LIVE MOUSE EMBRYONIC AND MOUSE ADULT ULTRASENSITIVE IMAGE-BASED PROFILING TO STEM CELLS BASED ON MRNA EXPRESSION...... 89 FORECAST STEM CELL FATE AND IDENTIFY CELLULAR Poster Board Number: 2235 HETEROGENEITY...... 93 Poster Board Number: 2259 A TECHNOLOGY PLATFORM FOR THE ISOLATION AND EXPRESSION PROFILING OF RARE CELL POPULATIONS MICRO FLUIDIC BASED CELL MANIPULATION CHIP FOR FROM DEVELOPING MOUSE EMBRYOS...... 89 STEM CELL SCREENING...... 93 Poster Board Number: 2237 Poster Board Number: 2261 ISOLATION OF MOUSE HEMATOPOIETIC STEM CELL AN IMPROVED WORKFLOW FOR qRT-PCR GENE POPULATIONS USING SPKLS IN COMBINATION WITH EXPRESSION STUDIES OF SINGLE CULTURED CELLS . . 94 HIGH-THROUGHPUT SINGLE CELL GENE ARRAYS Poster Board Number: 2263 SHOW DISTINCT DIFFERENCES IN STEM CELL MARKER EXPRESSION INVOLVED IN HEMATOPOIETIC UNCOVERING THE DIVERSITY OF INDIVIDUAL CELLS: DEVELOPMENTAL PATHWAYS ...... 90 GENE EXPRESSION PROFILING WITH THE BIOMARK SYSTEM ...... 94 Poster Board Number: 2239 Poster Board Number: 2265 PIVOTAL ROLE FOR GSK3 IN HEMATOPOIETIC STEM CELL HOMEOSTASIS...... 90 NANOTECHNOLOGY APPROACHES FOR INVESTIGATING MICROENVIRONMENTAL CUES REGULATING STEM CELL FATE...... 94

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Poster Board Number: 2267 Poster Board Number: 2291 BIODISTRIBUTION OF TRANSPLANTED BONE EX VIVO EXPANSION OF MURINE AND HUMAN MARROW MONONUCLEAR CELLS IN SUBACUTE HEMATOPOIETIC STEM CELLS WITH NON-CELL STROKE PATIENTS ...... 95 AUTONOMOUS FACTORS...... 99 Poster Board Number: 2269 Poster Board Number: 2293 MODULAR MICROFLUIDIC BIOREACTOR PLATFORM CAN BETA-BOSWELLIC ACID SYNERGIZE DERIVATION OF FOR THE AUTOMATED CULTIVATION AND TIME-LAPSE DOPAMINERGIC NEURON FROM MOUSE EMBRYONIC ES IMAGING OF EMBRYONIC STEM CELLS ...... 95 CELLS CULTURED ON MATRIGEL COATED ELECTROSPUN Poster Board Number: 2271 PLGA/PCL NANOFIBROUS SCAFFOLD...... 99 Poster Board Number: 2295 A NEW PHOTOPOLYMERIZABLE HYALURONAN- BASED HYDROGEL FOR STEM CELL CULTURE AND DO MATRIGEL-EMBEDDED TRANSGENIC MOUSE TRANSPLANTATION ...... 95 EMBRYONIC ES CELLS CULTURED ON ELECTROSPUN Poster Board Number: 2273 PLGA-PCL SCAFFOLDS EFFICIENTLY DIFFERENTIATE TO DOPAMINERGIC NEURONS?...... 100 IN VIVO CLONAL ANALYSIS OF ADULT RADIAL GLIA- Poster Board Number: 2297 LIKE NEURAL PRECURSORS REVEALS SELF-RENEW AND MULTIPOTENT CHARACTERISTICS...... 96 IMPROVEMENT OF ADULT MOUSE NEURAL STEM CELL DELIVERY TO THE BRAIN USING A BIOMATERIAL TISSUE ENGINEERING...... 96 COMPOSED OF HYALURONAN AND METHYLCELLULOSE (HAMC) ...... 100 Poster Board Number: 2275 Poster Board Number: 2299 EFFICIENT SKELETAL MUSCLE REGENERATION FROM HUMAN ES AND IPS CELLS ...... 96 ACCELERATED RECONSTRUCTION OF MOUSE CALVARIAL DEFECT BY THE ADIPOSE-DERIVED STEM CELL SEEDED Poster Board Number: 2277 ONTO POLYLACTIC ACID SCAFFOLD ...... 100 TISSUE ENGINEERING OF THREE-DIMENSIONAL Poster Board Number: 2301 CONTRACTILE SKELETAL MUSCLES FROM HUMAN PLURIPOTENT STEM CELLS ...... 96 INDUCTION OF CORNEAL ENDOTHELIAL CELLS FROM MOUSE ADULT NEURAL CREST STEM CELLS ...... 101 Poster Board Number: 2279 Poster Board Number: 2303 DETECTION OF PRO-ARRHYTHMIC EFFECTS WITH HUMAN ENGINEERED HEART TISSUE...... 97 TRANSPLANTATION OF AUTOLOGOUS BONE MARROW DERIVED MONONUCLEAR CELLS COMBINED WITH Poster Board Number: 2281 XENOGRAFTS IN ADVANCED MAXILLARY AND INFECTION OF HUMAN ENDOTHELIAL PROGENITOR MANDIBULAR ATROPHY...... 101 CELLS WITH BARTONELLA HENSELAE INDUCES VESSEL Poster Board Number: 2305 GROWTH IN VITRO...... 97 DECELLULARIZATION OF KIDNEYS TO BE USED AS A Poster Board Number: 2283 TISSUE SCAFFOLD FOR IN VITRO CULTURE OF WHOLE THE ROLE OF FOXO3A IN THE REGULATION OF HUMAN ORGANS...... 101 MESENCHYMAL STEM CELL SENESCENCE UNDER Poster Board Number: 2307 HYPOXIA...... 97 INFANTILE HEMANGIOMA A TUMOR DERIVED FROM Poster Board Number: 2285 TROPHOBLASTS...... 101 BIOPROCESSING HUMAN PLURIPOTENT STEM CELLS Poster Board Number: 2311 FOR CARDIAC CELL THERAPY...... 98 FUNCTIONAL VALIDATION OF DECELLULARIZED Poster Board Number: 2287 EXTRACELLULAR MATRIX PREPARATIONS FOR EXTRACELLULAR MATRIX DEPOSITED BY HUMAN EX-VIVO CULTURE OF HEMATOPOIETIC STEM/ BONE MARROW STROMAL CELLS FACILITATES STEM PROGENITOR CELLS...... 102 CELL EXPANSION AND TISSUE-SPECIFIC LINEAGE Poster Board Number: 2313 DIFFERENTIATION POTENTIAL...... 98 INTERVERTEBRAL DISC-DERIVED STEM CELLS: Poster Board Number: 2289 IMPLICATIONS FOR REGENERATIVE MEDICINE AND TISSUE ENGINEERING OF A HUMAN VESICAL NEURAL REPAIR...... 102 EQUIVALENT USING ADIPOSE-DERIVED STEM CELLS...... 98 xvi www.isscr.org Table of Contents

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Poster Board Number: 2315 Poster Board Number: 2335 TISSUE ENGINEERED BIPHASIC CERAMICS FOR MODULATION OF NOTCH SIGNALLING IS REQUIRED OSTEOCHONDRAL DEFECT SHORT STUDY...... 102 DURING IN VITRO T CELL DIFFERENTIATION OF HUMAN Poster Board Number: 2317 ADULT AND CORD BLOOD-DERIVED HAEMATOPOIETIC STEM CELLS...... 106 IN VITRO AND IN VIVO INVESTIGATIONS ON Poster Board Number: 2337 RADIOOPAQUE STRONTIUM CALCIUM PHOSPHATE CELL SEEDED SCAFFOLDS FOR EASY CLINICAL CHEMOTACTIC AND ADHESION PROPERTIES OF HUMAN EVALUATION ...... 103 POSTNATAL LYMPHOID PROGENITORS SEEDING THE Poster Board Number: 2319 THYMUS...... 107 Poster Board Number: 2339 REGENERATION OF ELASTIC CARTILAGE FROM ELASTIC CARTILAGE DERIVED CHONDROCYTES...... 103 INDUCTION OF HUMAN T-CELL DEVELOPMENT FROM CD34+ PROGENITORS BY EXPOSURE TO IMMOBILIZED TOTIPOTENCY/EARLY EMBRYO CELLS. . . . 104. NOTCH LIGAND DELTA-LIKE-4...... 107 Poster Board Number: 2321 Poster Board Number: 2341 IDENTIFICATION OF MOUSE STEM CELL-SURFACE TRACKING THE SCID REPOPULATING ACTIVITY OF EX VIVO PROTEINS ENABLES ISOLATION OF LINEAGE HUMAN HEMATOPOIETIC CELLS DURING CULTURE...... 107 PROGENITOR CELLS DIRECTLY FROM THE MOUSE BLASTOCYST USING FLOW CYTOMETRY...... 104 Poster Board Number: 2343 Poster Board Number: 2323 THE ROLE OF THE PKC ISOFORM δ IN LINEAGE COMMITMENT OF HUMAN HEMATOPOIETIC STEM SUCCESSIVE ROLES OF PDGF SIGNALING IN THE CELLS...... 108 EXTRAEMBRYONIC ENDODERM OF THE MOUSE EMBRYO...... 104 Poster Board Number: 2345 Poster Board Number: 2325 PREDICTIVE CONTROL OF HUMAN HEMATOPOIETIC STEM CELL SELF-RENEWAL BY MANIPULATING CRITICAL REQUIREMENT OF FGF SIGNALING TO NON-STEM CELL ASSOCIATED ENDOGENOUS PRIMITIVE ENDODERM LINEAGE COMMITMENT IN SIGNALLING ENABLES A GREATER THAN 10-FOLD PREIMPLANTATION EMBRYOS...... 104 EXPANSION OF LONG TERM NOD/SCID REPOPULATING Poster Board Number: 2327 CELLS IN A CLINICALLY RELEVANT CLOSED SYSTEM CELL POLARITY AND TROPHECTODERM SEGREGATION BIOREACTOR ...... 108 IN EARLY MOUSE EMBRYOS...... 105 Poster Board Number: 2347 Poster Board Number: 2329 EXPRESSION OF INHIBITORY RECEPTOR ILT3 BY HUMAN DISSECTING EARLY LINEAGE SPECIFICATION BY ADULT HEMATOPOIETIC STEM CELLS AND MYELOID CONVERSION OF MURINE EXTRAEMBRYONIC PROGENITORS...... 108 TROPHOBLAST STEM CELLS TO PLURIPOTENT STEM Poster Board Number: 2349 CELLS...... 105 MODELLING TEL/AML1+ ACUTE LYMPHOBLASTIC LEUKEMIA USING HUMAN EMBRYONIC STEM HEMATOPOIETIC STEM CELLS...... 105 CELLS...... 109 Poster Board Number: 2331 Poster Board Number: 2351 CONDITIONING WITH BUSULFAN CONFERS ENFORCED EXPRESSION OF THE MIR-144~451 CLUSTER ENGRAFTMENT POTENTIAL ON HUMAN HEMATOPOIETIC DECREASED HUMAN ERYTHROID DIFFERENTIATION. 109 STEM CELLS COMPARABLE TO HOXB4 TRANSDUCTION Poster Board Number: 2353 IN SHEEP IN UTERO TRANSPLANTATION...... 105 Poster Board Number: 2333 L4, AN IMPORTANT REGULATOR FOR HEMATOPOIESIS DURING MOUSE EMBRYONIC DEVELOPMENT. . . .109 GENERATION OF MYELOID PROGENITORS AND Poster Board Number: 2355 EVALUATION OF THEIR OSTEOCLASTOGENIC POTENTIAL FROM HUMAN PLURIPOTENT STEM CELLS ...... 106 ENGRAFTMENT OF EMBRYONIC MURINE HEMATOPOIETIC STEM CELLS IN THE MORE PERMISSIVE NEONATAL HEMATOPOIETIC MICROENVIRONMENT...... 109

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Poster Board Number: 2357 Poster Board Number: 2377 HEDGEHOG SIGNALING SPECIFIES THE STRESS BCR-ABL IMPAIRS T CELL DEVELOPMENT ERYTHROID PROGENITOR CELL FATE AND PROMOTES FROM MURINE INDUCED PLURIPOTENT STEM CELLS; A THE EXPANSION OF SELF RENEWING STRESS ERYTHROID POSSIBLE EXPLANATION FOR T CELL ESCAPE PROGENITORS IN THE MURINE SPLEEN...... 110 FROM LEUKEMIC CLONE IN CHRONIC MYELOID Poster Board Number: 2359 LEUKEMIA...... 114 Poster Board Number: 2379 ENDOTHELIAL AND PERIVASCULAR STROMAL CELLS PROMOTE MURINE HEMATOPOIETIC STEM CELL THE ROLE OF ALCAM MEDIATED CELLULAR MAINTENANCE IN THE BONE MARROW BY SECRETING INTERACTION IN THE REGULATION OF STEM CELL FACTOR (SCF) ...... 110 HEMATOPOIESIS ...... 114 Poster Board Number: 2361 Poster Board Number: 2381 CHRONIC KIDNEY DISEASE IMPAIRS THE BONE THE SHELTERIN PROTEIN ACD/TPP1 PLAYS A CRITICAL MARROW CELLS CAPACITY TO RECONSTITUTE A ROLE IN THE MAINTENANCE OF HEMATOPOIETIC STEM COMPLETE HEMATOPOIETIC SYSTEM IN MICE. . . .110 CELLS INDEPENDENT OF TELOMERE LENGTH...... 114 Poster Board Number: 2363 Poster Board Number: 2383 IN VIVO IMAGING OF REGULATORY T CELLS PROVIDING MECHANISMS OF BONE MARROW DERIVED CELLS IMMUNE PRIVILEGE TO ADULT MOUSE HEMATOPOIETIC ENTRY TO THE BRAIN...... 115 STEM CELL NICHE ...... 111 Poster Board Number: 2385 Poster Board Number: 2365 VASOPRESSIN AND OXYTOCIN REGULATE BONE MARROW-DERIVED STEM CELL HEMATOPOIESIS THROUGH THE WNT AND AKT TRANSPLANTATION IN A MOUSE MODEL OF RETINAL PATHWAYS...... 115 DEGENERATION...... 111 Poster Board Number: 2387 Poster Board Number: 2367 DIRECT INVOLVEMENT OF BMP4 IN ADULT SINGLE LINEAGE TRANSCRIPTOME ANALYSIS HEMATOPOIETIC STEM CELL HOMING...... 116 REVEALS KEY MOLECULAR CHANGES IN ERYTHROID Poster Board Number: 2389 PROGENITORS IN LATE GASTRULATION MOUSE EMBRYOS...... 112 ERYTHROID DIFFERENTIATION OF CD133 STEM CELLS OF UMBILICAL CORD BLOOD BY UP REGULATION OF Poster Board Number: 2369 MIR 451 AND DOWN REGULATION OF MIR 150 . . . . 116 A FETAL LIVER-DERIVED CONDITIONED MEDIUM Poster Board Number: 2391 ENHANCES THE GROWTH FACTOR-DEPENDENT PROLIFERATION AND SURVIVAL OF ADULT MOUSE THE BASIC PROTEIN G0S2 ACTIVATES QUIESCENCE IN BONE MARROW ERYTHROID PROGENITORS BY HEMATOPOIETIC STEM CELLS BY INTERACTING WITH MODULATING THE DYNAMICS OF ERK SIGNALING . . 112 NUCLEOLIN ...... 116 Poster Board Number: 2371 Poster Board Number: 2393 SCL GENE DOSAGE SUSTAINS THE QUIESCENCE AND EPOXYEICOSATRIENOIC ACIDS REGULATE MAINTENANCE OF MOUSE ADULT HEMATOPOIETIC HEMATOPOIETIC STEM CELL SELF-RENEWAL STEM CELLS...... 113 DURING STRESS RESPONSE AND EMBRYONIC HEMATOPOIESIS ...... 117 Poster Board Number: 2373 Poster Board Number: 2395 THE ADAPTOR PROTEIN SHB REGULATES CELL CYCLE PROGRESSION IN ADULT MOUSE HEMATOPOIETIC STEM GENE CORRECTION BY DIRECT PROTEIN DELIVERY. . 117 CELLS...... 113 Poster Board Number: 2397 Poster Board Number: 2375 DELETION OF TET2 IN MICE LEADS TO DYSREGULATED WNT/BETA-CATENIN ACTIVITY IS REQUIRED FOR HEMATOPOIETIC STEM CELLS AND SUBSEQUENT HEMATOPOIETIC STEM CELL DEVELOPMENT IN THE DEVELOPMENT OF MYELOID MALIGNANCIES. . . .117 MOUSE EMBRYO ...... 113 Poster Board Number: 2399 LONG-TERM RECONSTITUTION ABILITIES AND DIFFERENTIATION OF HUMAN HEMATOPOIETIC STEM CELLS REQUIRE HYPOXIA-INDUCIBLE FACTOR-1 ALPHA AND -2 ALPHA EXPRESSIONS...... 118 xviii www.isscr.org Table of Contents

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Poster Board Number: 2401 Poster Board Number: 2427 THE HIPPO SIGNALING PATHWAY REGULATES HETEROTOPIC OSSIFICATION IS MEDIATED BY BMP- HEMATOPOIETIC STEM-LIKE PROGENITORS IN RESPONSIVE PROGENITORS IN THE SKELETAL MUSCLE DROSOPHILA MELANOGASTER...... 118 INTERSTITIUM ...... 123 Poster Board Number: 2403 Poster Board Number: 2429 DISEASE SPECIFIC LENTIVIRAL TARGET SITE SELECTION AGRIN IS A NOVEL NON-REDUNDANT PLAYER OF THE IN PROGENITORS OF FANCONI ANEMIA PATIENTS. . 118 MOUSE HEMATOPOIETIC STEM CELL NICHE ...... 123 Poster Board Number: 2405 Poster Board Number: 2431 DIRECT DIFFERENTIATION OF HEMATOPOIETIC STEM MESENCHYMAL STROMAL CELLS ISOLATED FROM CELLS INTO MONOCYTIC LINEAGE BY INDUCTION OF MULTIPLE SCLEROSIS PATIENTS INHIBIT LYMPHOCYTE MIR-424 EXPRESSION ...... 119 PROLIFERATION AND INDUCE REGULATORY T CELLS Poster Board Number: 2407 GENERATION ...... 123 Poster Board Number: 2433 AUTOLOGOUS TRANSPLANTATION OF BONE MARROW MONONUCLEAR CELLS IN A COMPARTMENT SYNDROME MESENCHYMAL STROMAL CELLS SHOW ENHANCED SALINE MODEL IN SINCLAIR MINI-SWINE: A PILOT MIGRATION TO HEART AFTER PRE-TREATMENT WITH STUDY ...... 119 TNF-α AFTER ACUTE MYOCARDIAL INFARCT ...... 124 Poster Board Number: 2409 Poster Board Number: 2435 LIGHT REGULATE HEMATOPOIETIC STEM CELL SPECIFIC MIGRATION OF MESENCHYMAL STROMAL DIFFERENTIATION ...... 120 CELLS TO CHEMOKINES IN VIVO...... 124 Poster Board Number: 2411 Poster Board Number: 2437 DYNAMIC AND DEMAND-ADAPTED CELL CYCLE EXPANSION OF MESENCHYMAL STEM CELLS CAUSES REGULATION IN HEMATOPOIETIC STEM CELLS . . . . . 120 DNA-METHYLATION CHANGES WHICH CORRELATE WITH Poster Board Number: 2413 REPRESSIVE HISTONE MARKS...... 124 Poster Board Number: 2439 SHEEP YOLK SAC AS A SOURCE OF HEMANGIOBLASTIC CELLS...... 120 A MSC POPULATION WITH ENHANCED RESPONSE Poster Board Number: 2415 TOWARDS TUMOR STROMA ...... 125 Poster Board Number: 2441 CHARACTERIZATION OF SHEEP MESENCHYMAL STEM CELLS FROM HEMATOPOIETIC PLACENTAL TISSUES AND OPTIMIZING GFP TRANSFECTION EFFICIENCY OF EMBRYONIC ORGANS...... 121 WHARTON’S JELLY- MESENCHYMAL STROMAL CELLS...... 125 MESENCHYMAL CELL LINEAGE ANALYSIS. . . .121 Poster Board Number: 2443 Poster Board Number: 2419 ISOLATION AND IDENTIFICATION OF STEM CELLS FROM FGFR2 MUTATION CONFERS LESS FUNCTIONAL DAMAGE DOG ADIPOSE TISSUE ...... 125 IN ADULT HUMAN MESENCHYMAL STEM CELLS AS COMPARED TO FIBROBLASTS ...... 121 MESENCHYMAL STEM CELL Poster Board Number: 2421 DIFFERENTIATION...... 125. CHARACTERISTICS OF MESENCHYMAL STROMAL CELLS Poster Board Number: 2445 IN SYNOVIAL FLUID OF CHILDREN WITH JUVENILE IDIOPATHIC ARTHRITIS AND CHILDREN WITHOUT JOINT HUMAN UMBILICAL CORD-DERIVED MESENCHYMAL INFLAMMATION...... 121 STROMAL CELLS AS A SOURCE OF TREATMENT FOR Poster Board Number: 2423 NEUROPATHIC PAIN AFTER SPINAL CORD INJURY . . . 125 Poster Board Number: 2447 HETEROGENEITY OF HUMAN MULTIPOTENT MESENCHYMAL STROMAL CELLS ...... 122 TRANSPLANTED NEURALLY MODIFIED HUMAN BONE Poster Board Number: 2425 MARROW DERIVED MESENCHYMAL STEM CELLS REDUCE THE VOLUME OF CAVITY, PROMOTE WHITE ACCELERATING THE CELL CYCLE OF HUMAN ADIPOSE MATTER SPARING AND SIGNIFICANTLY IMPROVE TISSUE STEM CELLS ELEVATES THEIR DIFFERENTIATION LOCOMOTOR RECOVERY IN SPINAL CORD INJURED PLASTICITY TOWARDS THE MESODERM AND ECTODERM RATS...... 126 LINEAGES...... 122

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Poster Board Number: 2449 Poster Board Number: 2469 ENHANCED DIFFERENTIATION OF HUMAN BONE DIFFERENTIATION OF HUMAN ADIPOSE MESENCHYMAL MARROW-DERIVED MESENCHYMAL STEM CELLS INTO STEM CELLS INTO SMOOTH MUSCLE-LIKE CELLS: FUNCTIONAL NEURAL CELLS BY DTOPV-MODIFIED A POTENTIAL CELL SOURCE FOR VASCULAR TISSUE SURFACES ...... 126 ENGINEERING...... 130 Poster Board Number: 2451 Poster Board Number: 2471 NEURAL GENE EXPRESION OF HUMAN UMBILICAL COMPARISON OF ADIPOGENIC DIFFERENTIATION OF CORD BLOOD DERIVED MULTI-LINEAGE PROGENITOR HUMAN MESENCHYMAL STEM CELL LINES FROM CELL (MLPC) AFTER BEEN CULTURED IN NEURAL DIFFERENT TISSUES OF ORIGIN...... 130 DIFFERENTIATION MEDIA ...... 127 Poster Board Number: 2473 Poster Board Number: 2453 HUMAN ADIPOSE TISSUE-DERIVED SSEA-4 STAT3 ACTIVITY IS CORRELATED WITH THE NEUROGENIC SUBPOPULATION CAN BE DIFFERENTIATED TOWARDS POTENTIAL OF HUMAN UMBILICAL CORD-DERIVED THE ENDOTHELIAL AND OSTEOGENIC LINEAGES . . . 131 MESENCHYMAL STEM CELLS...... 127 Poster Board Number: 2475 Poster Board Number: 2455 NON-ADHERENT MESENCHYMAL PROGENITORS ARE GLYCOSAMINOGLYCAN-RICH MATRIX PRODUCTION PRESENT IN THE STROMAL VASCULAR FRACTION OF AT CHONDROGENIC CULTURE OF HUMAN MARROW- FRESHLY ISOLATED HUMAN ADIPOSE TISSUE AND ARE DERIVED MESENCHYMAL STEM CELLS TREATED WITH ABLE TO SELF-RENEW IN SUSPENSION WHEN CULTURED GSK3 INHIBITORS...... 127 ON THEIR NICHE...... 131 Poster Board Number: 2457 Poster Board Number: 2477 A MOLECULAR MARKER FOR SELECTION OF UMBILICAL SYSTEMIC HUMAN ORBITAL FAT-DERIVED CORD BLOOD-DERIVED HUMAN MESENCHYMAL STEM STEM CELL TRANSPLANTATION AMELIORATES CELLS (HUCB-MSCS) WITH HIGH CHONDROGENIC MACROPHAGE-MEDIATED ACUTE INFLAMMATION IN CAPACITY...... 128 LIPOPOLYSACCHARIDE-INDUCED ACUTE LUNG Poster Board Number: 2459 INJURY...... 132 Poster Board Number: 2479 DECELLULARIZED MATRICES MODULATE HUMAN SYNOVIUM-DERIVED STEM CELL HUMAN ORBITAL FAT-DERIVED STEM CELLS PROMOTE CHONDROGENESIS...... 128 CORNEAL RE-EPITHELIALIZATION IN VIVO. . . . . 132 Poster Board Number: 2461 Poster Board Number: 2481 HUMAN ADIPOSE-DERIVED STEM CELL CONTROLLING HUMAN ADULT STEM CELL FATE BY CHARACTERIZATION AND COMPARATIVE GENE COMBINATORIAL BIOPHYSICAL STIMULI...... 132 EXPRESSION PROFILING...... 128 Poster Board Number: 2483 Poster Board Number: 2463 IN VITRO POTENTIAL OF HUMAN BONE MARROW CD10 POSITIVE CELLS FROM HUMAN ADIPOSE TISSUE STROMAL CELLS TO DIFFERENTIATE INTO INSULIN AS A POTENTIAL SOURCE OF LUMINAL MAMMARY PRODUCING CELLS IN CO-CULTURE WITH PANCREATIC EPITHELIAL CELLS...... 129 STROMAL CELLS ...... 133 Poster Board Number: 2465 Poster Board Number: 2485 FLUORESCENCE MICROSCOPY TO ASSESS CHARACTERIZATION OF DISTINCT HUMAN BONE DIFFERENTIATION OF HUMAN ADIPOSE-DERIVED STEM MARROW DERIVED MESENCHYMAL STEM/STROMAL CELLS AND SKELETAL MUSCLE PRECURSORS TOWARDS CELL SUBSETS ...... 133 BROWN ADIPOCYTES...... 129 Poster Board Number: 2487 Poster Board Number: 2467 HUMAN STEM CELLS FROM ELEVATOR PALATE MUSCLE MICROARRAY ANALYSIS OF IN-VITRO OSTEOGENESIS TO BONE TISSUE ENGINEERING FOR CRANIOFACIAL IN HUMAN ADIPOSE DERIVED MESENCHYMAL STEM DISEASES...... 133 CELLS...... 129 Poster Board Number: 2489 IN VITRO TRANSFORMATION OF HUMAN MESENCHYMAL STEM CELL TO HEPATOCELLULAR CARCINOMA DURING OSTEOGENIC DIFFERENTIATION ...... 134 xx www.isscr.org Table of Contents

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Poster Board Number: 2491 Poster Board Number: 2513 IDENTIFICATION OF EARLY LINEAGE SPECIFIC MARKER TRACKING OF MOUSE MESENCHYMAL STEM CELLS GENES OF DIFFERENTIATING HUMAN MESENCHYMAL USING SPECIFIC REPORTER GENES...... 138 STROMAL CELLS ...... 134 Poster Board Number: 2515 Poster Board Number: 2493 TRANSGENIC ADULT MURINE MESENCHYMAL STEM NANOSTRUCTURES INHIBIT MESENCHYMAL CELLS CONDITIONALLY OVEREXPRESSING SDF-1β DIFFERENTIATION OF HUMAN INDUCED PLURIPOTENT ENHANCE NEW BONE FORMATION IN BOTH IN VITRO STEM CELLS...... 134 AND IN VIVO MODEL SYSTEMS...... 138 Poster Board Number: 2495 Poster Board Number: 2517 OSTEOGENIC DIFFERENTIATION OF HUMAN HIGH-THROUGHPUT AND SYSTEMATIC INVESTIGATION INDUCED PLURIPOTENT STEM CELLS: IN VITRO AND OF MATRIX MECHANICAL AND BIOCHEMICAL IN VIVO ...... 135 CONTROL OF MOUSE BONE MARROW STEM CELL Poster Board Number: 2497 DIFFERENTIATION ...... 139 Poster Board Number: 2519 CHARACTERIZATION OF CARTILAGE GENERATED FROM HUMAN MESENCHYMAL STEM CELLS ON NANOFIBER MURINE BONE MARROW DERIVED VERY SMALL SCAFFOLDS: A COMPARATIVE STUDY USING HUMAN EMBRYONIC-LIKE (VSEL) STEM CELLS GIVE RISE TO CARTILAGE SAMPLES AS THE BENCHMARK. . . . .135 MESENCHYMAL STROMAL CELLS ...... 139 Poster Board Number: 2499 Poster Board Number: 2521 SHEAR IS A REQUIREMENT FOR MECHANICALLY MURINE VERY SMALL EMBRYONIC LIKE STEM CELLS INDUCED CHONDROGENESIS OF HUMAN BONE ARE THE PRIMARY SOURCE OF BONE MARROW MARROW DERIVED STEM CELLS...... 135 DERIVED LUNG EPITHELIAL CELLS...... 140 Poster Board Number: 2501 Poster Board Number: 2523 SMALL MOLECULE MESENGENIC INDUCTION EFFICIENT GENERATION OF MESENCHYMAL STEM CELLS OF HUMAN IPS FOR MESENCHYMAL STEM CELL FROM IPSCS AND THEIR USE IN DRUG DISCOVERY. . 140 GENERATION ...... 136 Poster Board Number: 2525 Poster Board Number: 2503 MORE EFFECTIVE REPAIR OF ISCHEMIC REGIONS BY DIFFERENTIATION OF HUMAN PLURIPOTENT STEM ADIPOSE TISSUE-DERIVED MSCS IS ASSOCIATED WITH CELLS INTO MESENCHYMAL STEM CELLS STRONGER EXPRESSION OF CCL5 ...... 140 BY MODULATING INTRACELLULAR SIGNALING Poster Board Number: 2527 PATHWAYS...... 136 OSTEOPONTIN PLAYS A CRITICAL ROLE IN DIRECTING Poster Board Number: 2505 OSTEOBLASTOGENESIS AND ADIPOGENESIS OF RESVERATROL PROMOTES OSTEOGENESIS OF HUMAN MESENCHYMAL STEM CELLS...... 141 MESENCHYMAL STEM CELLS BY UP-REGULATING RUNX2 Poster Board Number: 2529 GENE EXPRESSION VIA SIRT1/FOXO3A AXIS ...... 136 TWIST-1 INTERACTIONS INVOLVED IN MEDIATING Poster Board Number: 2507 MESENCHYMAL STROMAL/CELL PROLIFERATION, HUMAN UMBILICAL CORD PERIVASCULAR CELLS DIFFERENTIATION AND LINEAGE COMMITMENT. . .141 (HUCPVCS) VERSUS BONE MARROW-DERIVED Poster Board Number: 2531 MESENCHYMAL STROMAL CELLS (BM-MSCS): POTENTIAL ROLE OF EPIGENETICS ON MULTIPOTENT IMPROVED CALCIFICATION OF ARTIFICIAL BONE TISSUE CELL DIFFERENTIATION CAPACITY...... 137 IN 3D ALGINATE-COLLAGEN CAPSULES MADE FROM ULTRA-HIGH-VISCOSITY (UHV) ALGINATE...... 141 Poster Board Number: 2509 Poster Board Number: 2533 NANOPARTICLE LABELING OF BONE MARROW-DERIVED RAT MESENCHYMAL STEM CELLS AND THEIR USE IN HOMING OF ALLOGENIC OVINE MESENCHYMAL STEM DIFFERENTIATION AND TRACKING...... 137 CELLS IN COLLAGENASE INDUCED TENDONITIS OF THE ACHILLE’S TENDON...... 142 Poster Board Number: 2511 Poster Board Number: 2535 EFFECT OF ESTROGEN ON THE DIFFERENTIATION OF BONE MARROW-DERIVED RAT MESENCHYMAL STEM ISOLATION, IDENTIFICATION AND ADIPOCYTE CELLS INTO ADIPOCYTES AND OSTEOCYTES...... 137 DIFFERENTIATION OF HYPERBARIC OXYGEN-TREATED EQUINE PERIPHERAL BLOOD-DERIVED MESENCHYMAL STEM CELLS...... 142

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Poster Board Number: 2537 Poster Board Number: 2561 SIRNA-MEDIATED KNOCKDOWN OF VINCULIN TARGETING HUMAN MESENCHYMAL STEM CELLS MODULATES MAPK1 ACTIVATION AND STIFFNESS- TO MURINE KIDNEYS USING PULSED FOCUSED BASED STEM CELL DIFFERENTIATION ...... 142 ULTRASOUND: IMPLICATIONS FOR STEM CELL Poster Board Number: 2539 THERAPY ...... 146 Poster Board Number: 2563 MOBILIZATION OF BONE MARROW MONONUCLEAR CELLS BY ADULT NEURAL STEM PROGENITORS CELLS ELUCIDATION OF THE HUMAN MESENCHYMAL STEM INVOLVES A SDF1-DEPENDENT MECHANISM ...... 143 CELL HOMING MECHANISM...... 146 Poster Board Number: 2541 Poster Board Number: 2565 DIFFERENTIATION OF UMBILICAL CORD DERIVED STEM CLINICAL GRADE EXPANSION OF HUMAN CELLS TO HEPATOCYTE-LIKE-CELLS IN VITRO ...... 143 MESENCHYMAL STEM CELLS USING A MICROCARRIER- Poster Board Number: 2543 BASED SYSTEM UNDER SERUM-FREE AND XENO-FREE CONDITIONS ...... 147 EFFECT OF HAZARDOUS CHEMICALS ON THE CORD Poster Board Number: 2567 BLOOD CELLS ...... 143 THE ASSESSMENT OF CONTROLLED CRYOPRESERVATION PRE-CLINICAL AND CLINICAL APPLICATIONS OF CONDITIONS OF HUMAN UMBILICAL CORD STROMAL TISSUE FOR THE POTENTIAL USAGE OF MSCs FOR STEM MESENCHYMAL STEM CELLS...... 144 CELL TRANSPLANTATIONS AND BANKING...... 147 Poster Board Number: 2569 Poster Board Number: 2547 MRI TRACKING OF IRON-LABELED CD105+-MESENCHYMAL STEM CELLS MIGRATES FROM THERAPEUTIC HUMAN NEURAL STEM CELLS IN A PERIPHERICAL BLOOD INTO OSTEOARTHRITIS JOINT: AN MURINE BRAIN TUMOR MODEL: IMPLICATIONS FOR ANIMAL MODEL ...... 144 CLINICAL USE...... 148 Poster Board Number: 2549 Poster Board Number: 2571 A CHEMICALLY DEFINED, SERUM FREE GROWTH IMMUNOSUPPRESSION BY IDO AND B7-H1 IN MEDIUM FOR HUMAN MESENCHYMAL STEM CELLS MESENCHYMAL STEM CELLS DERIVED FROM ADULT FROM VARIOUS TISSUES ...... 144 HUMAN TISSUES ...... 148 Poster Board Number: 2551 Poster Board Number: 2573 EFFICIENT CLINICAL-SCALE PRODUCTION OF CARDIOMYOBLAST-LIKE CELLS DIFFERENTIATED FROM HUMAN MESENCHYMAL STEM CELLS IN COMPUTER- HUMAN ADIPOSE TISSUE-DERIVED MULTILINEAGE CONTROLLED MICROCARRIER BIOREACTORS IN A PROGENITOR CELLS IMPROVE LEFT VENTRICULAR DEFINED MEDIUM...... 144 DYSFUNCTION AND SURVIVAL IN A SWINE MYOCARDIAL Poster Board Number: 2553 INFARCTION MODEL ...... 148 IN VITRO EVALUATION OF ADIPOGENESIS FROM Poster Board Number: 2575 HUMAN ADIPOSE DERIVED STEM CELLS...... 145 NON CLINICAL STUDIES (GLP) FOR CLINICAL Poster Board Number: 2555 APPLICATION OF HUMAN ADIPOSE TISSUE THERAPEUTIC POTENTIAL OF HUMAN ADIPOSE DERIVED MULTILINEAGE PROGENITOR CELLS TISSUE-DERIVED MESENCHYMAL STEM CELLS FOR FOR THE PATIENTS WITH SEVERE FAMILIAL ALZHEIMER’S DISEASE...... 145 HYPERCHOLESTEROLEMIA...... 149 Poster Board Number: 2557 Poster Board Number: 2577 THE CONVENTIONAL DENSITY GRADIENT SEPARATION NON CLINICAL STUDIES (GLP) FOR CLINICAL TECHNIQUE IS NOT AN EFFECTIVE METHOD FOR THE APPLICATION OF CARDIOMYOBLAST LIKE CELLS ISOLATION OF HUMAN BONE MARROW MESENCHYMAL DIFFERENTIATED FROM HUMAN ADIPOSE TISSUE STEM CELLS...... 145 DERIVED MULTILINEAGE PROGENITOR CELLS. . . .149 Poster Board Number: 2559 Poster Board Number: 2579 A STRATEGY FOR ENHANCING THE ENGRAFTMENT OF BIODISTRIBUTION AND EFFECTS OF IV-INJECTED HUMAN HEMATOPOIETIC STEM CELLS IN NOD/SCID MESENCHYMAL BONE MARROW CELLS IN A MOUSE MICE ...... 146 MODEL OF CHAGAS DISEASE ...... 150

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Poster Board Number: 2581 Poster Board Number: 3005 MSC STIMULATE ENDOGENOUS NEUROGENESIS RAPID BEDSIDE PROCESSING AND CHARACTERIZATION THROUGH CCL2 IN NP-C DISEASE MICE...... 150 OF HUMAN AUTOLOGOUS BONE MARROW STEM CELL Poster Board Number: 2583 CONCENTRATE FOR CELL THERAPY...... 154 Poster Board Number: 3007 TRANSPLANTATION OF BONE MARROW STROMAL CELLS PRODUCES LONG-TERM ATTENUATION OF PAIN HUMAN MESENCHYMAL STEM/STROMAL CELLS INHIBIT HYPERSENSITIVITY IN RATS ...... 150 THE PROLIFERATION AND PROMOTE THE CELL CYCLE Poster Board Number: 2585 ARREST OF HUMAN LEUKEMIC CELLS...... 154 Poster Board Number: 3009 PERI-OCULAR AND INTRA-ARTICULAR INJECTION OF CANINE MSCS: AN IN VIVO SAFETY, IMAGING, MONITORING HUMAN STEM CELL DIFFERENTIATION MIGRATION, AND IMMUNOMODULATION STUDY. . . 151 USING MULTI-PARAMETER FLOW CYTOMETRY . . . . . 155 Poster Board Number: 2587 Poster Board Number: 3011 THE EFFECT OF HUMAN ADIPOSE TISSUE-DERIVED CARBON NANOTUBES AND HUMAN EMBRYONIC STEM MESENCHYMAL STEM CELLS ON AVASCULAR NECROSIS CELLS: INHIBITION OF PROLIFERATION AND INDUCTION OF THE FEMORAL HEAD IN BEAGLE DOGS ...... 151 OF DIFFERENTIATION...... 155 Poster Board Number: 2591 Poster Board Number: 3013 COMPLETE PULP REGENERATION BY TRANSPLANTATION COXSACKIE VIRUS B INFECTION INDUCES APOPTOSIS IN OF DENTAL PULP STEM CELLS...... 151 HUMAN EMBRYONIC STEM CELLS...... 155 Poster Board Number: 2593 Poster Board Number: 3015 ROLE OF UMBILICAL CORD DERIVED MESENCHYMAL HUMAN ADULT DENTAL PULP STEM CELLS: SURVIVAL, STEM CELLS IN THE REMISSION OF TUBULAR DEFECTS MIGRATION AND DIFFERENTIATION FOLLOWING IN A PRECLINICAL MODEL...... 152 TRANSPLANTATION INTO STROKE RAT BRAIN ...... 156 Poster Board Number: 2595 Poster Board Number: 3017 UMBILICAL CORD WHARTON’S JELLY: A NEW POTENTIAL BOTH ION CHANNELS AND CALCIUM SIGNALS CELL SOURCE OF MESENCHYMAL STEM CELLS FOR REGULATE PROLIFERATION IN HUMAN ADULT WOUND HEALING ...... 152 MESENCHYMAL STEM CELLS FROM BONE Poster Board Number: 2597 MARROW...... 156 Poster Board Number: 3019 DIFFERENTIATION POTENTIAL AND IMMUNOGENIC CHARACTERIZATION FOR PLACENTAL MESENCHYMAL ROLES OF PEROXISOME PROLIFERATOR-ACTIVATED STEM CELLS OF MATERNAL ORIGIN...... 152 RECEPTOR GAMMA ON PROLIFERATION STATE OF Poster Board Number: 2599 MOUSE EMBRYONIC STEM CELLS DUE TO THE ABSENCE OR PRESENCE OF LEUKEMIA INHIBITORY FACTOR. . 156 CHARACTERIZATION OF MESENCHYMAL STEM CELL Poster Board Number: 3023 TRANSMIGRATION...... 153 PREPARATION AND CHARACTERIZATION OF PITUITARY OTHER...... 153. STEM PROGENITOR CELLS IN MOUSE...... 157 Poster Board Number: 3001 Poster Board Number: 3025 REGULATION OF HUMAN LEUKOCYTE ANTIGEN-G MESENCHYMAL PRECURSOR CELLS RESTORE SALIVARY GLAND FUNCTION IN NON-OBESE DIABETIC MICE WITH EXPRESSION BY INTERFERON-γ-MEDIATED JAK/STAT-1 SIGNALING PATHWAY IN HUMAN PLACENTA-DERIVED SJöGREN’S SYNDROME ...... 157 MULTIPOTENT CELLS...... 153 Poster Board Number: 3027 Poster Board Number: 3003 MISIDENTIFICATION AND CROSS CULTURE HUMAN FIRST TRIMESTER CHORIONIC STEM CONTAMINATION OF STEM CELL LINES: A CONTINUING CELLS HAVE TRANSLATIONALLY ADVANTAGEOUS PROBLEM...... 157 CHARACTERISTICS OVER THEIR TERM Poster Board Number: 3029 COUNTERPARTS...... 153 SCHWANN CELLS DIFFERENTIATED FROM ADULT RAT SKIN STEM CELLS (SKPS) ARE HIGHLY SIMILAR TO SCHWANN CELLS ISOLATED FROM PERIPHERAL NERVE AND ARE DIFFERENT FROM SKPS, THEIR CELL OF ORIGIN...... 158

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Poster Board Number: 3031 Poster Board Number: 3055 EFFECTS OF CRYOPRESERVATION ON FUNCTION OF CONTROL OF GENE EXPRESSION AND CHROMOSOME THE OOPLASM AND SPINDLE OF THE UNFERTILIZED MORPHOLOGY IN MOUSE ESCS BY COHESIN AND OOCYTES ANALYZED BY THE “METAPHASE II SPINDLE CONDENSIN...... 161 INJECTION (MESI)” METHOD...... 158 Poster Board Number: 3057 Poster Board Number: 3033 GENOME-WIDE INTERACTIONS OF THE NANOG LOCUS TGFB SIGNALLING INHIBITS DLK1 EXPRESSION DURING IN MOUSE PLURIPOTENT STEM CELLS...... 161 CHONDROGENESIS IN VITRO...... 158 Poster Board Number: 3059 Poster Board Number: 3035 COMPARATIVE EPIGENETIC ANALYSIS OF A NUCLEAR AGO2 REGULATES ATSCS SURVIVAL FEW HISTONE MARKS IN HUMAN ESC, IPS AND THROUGH DIRECT CONTROL OF MIR10B AND SEPN1 DIFFERENTIATED CELLS...... 162 EXPRESSION...... 158 Poster Board Number: 3061 Poster Board Number: 3037 OVERCOMING EPIGENETIC SILENCING OF LENTIVIRAL NUCLEAR AGO2 AND HSP60 COOPERATIVELY VECTORS DURING MYELOID DIFFERENTIATION: NOVEL CONTROL ATSC SELF-RENEWAL AND DIFFERENTIATION ROLE OF INSULATOR AND SCAFFOLD ATTACHMENT VIA OCT4...... 159 REGIONS ...... 162 Poster Board Number: 3039 Poster Board Number: 3063 DNA METHYLTRANSFERASE CONTROLS STEM CELL RETT SYNDROME; UNDERSTANDING THE NEURON-GLIA AGING BY REGULATING BMI1 AND EZH2 THROUGH CONNECTION IN DIFFERENTIATED NEURAL STEM MICRORNAS...... 159 CELLS...... 162 Poster Board Number: 3041 Poster Board Number: 3065 SISTEMQCTM: A NOVEL, BROADLY-APPLICABLE VARIATION IN MYOD BINDING MOTIFS IS ESSENTIAL MICRORNA-BASED MONITORING TOOL FOR STEM FOR DEVELOPMENTAL REGULATION OF TARGET CELL QUALITY CONTROL AND DIFFERENTIATION GENES ...... 163 MONITORING...... 159 Poster Board Number: 3067 Poster Board Number: 3043 INTEGRATIVE GENOMIC ANALYSIS OF DIRECT ISOLATION AND CHARACTERIZATION OF PROGENITOR CONVERSION OF FIBROBLASTS TO NEURONS...... 163 CELLS DERIVED FROM EQUINE YOLK SAC. . . . . 160 Poster Board Number: 3069 Poster Board Number: 3045 DISSECTING EPIGENETIC REGULATORY NETWORKS ABSENCE OF T AND B LYMPHOCYTES ENHANCES DURING DEFINED STAGES OF CARDIOMYOCYTE SKELETAL MUSCLE REGENERATION AND AMELIORATES DIFFERENTIATION ...... 163 DYSTROPHIC PATHOLOGY IN DYSFERLIN DEFICIENT Poster Board Number: 3071 ANIMAL MODEL ...... 160 SWI/SNF-MEDIATED EPIGENETIC CONTROL OF Poster Board Number: 3047 MULTIPOTENT CARDIAC PROGENITOR CELLS ...... 163 DIRECTIONAL AND NONDIRECTIONAL RNA-SEQ EXPRESSION ANALYSIS ON THE ILLUMINA iPS CELLS...... 164. PLATFORM...... 160 Poster Board Number: 3075 CHROMATIN IN STEM CELLS...... 160. HUMAN INDUCED PLURIPOTENT STEM CELLS FROM PATIENTS WITH ATAXIA TELANGIECTASIA...... 164 Poster Board Number: 3051 Poster Board Number: 3077 TRANSCRIPTIONAL PULSING OF RETROVIRAL TRANSGENES IN MOUSE EMBRYONIC STEM CELLS. . 160 MISREPAIR OF RADIATION-INDUCED DNA DOUBLE- STRAND BREAKS IN HUMAN PLURIPOTENT CELLS. . 164 Poster Board Number: 3053 Poster Board Number: 3079 GLOBAL EPIGENETIC LANDSCAPE OF MOUSE INDUCED PLURIPOTENT STEM CELLS ...... 161 EFFICIENT HUMAN IPSC DERIVATION USING FLUORESCENCE ACTIVATED CELL SORTING TECHNOLOGY...... 164

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Poster Board Number: 3081 Poster Board Number: 3103 GENERATION OF DOWN SYNDROME IPSC MODEL TO ANALYSIS OF THE HETEROGENITY OF X STUDY TRISOMY 21 ALTERATIONS OF HEMATOPOIETIC CHROMOSOME INACTIVATION IN HUMAN PLURIPOTENT CELL DEVELOPMENT...... 164 STEM CELLS...... 168 Poster Board Number: 3083 Poster Board Number: 3105 INDUCED PLURIPOTENT STEM CELLS DERIVED ISOLATION OF MECP2-NULL RETT SYNDROME PATIENT FROM HUMAN PANCREATIC ISLET BETA CELLS HIPS CELLS AND ISOGENIC CONTROLS THROUGH SHOW EPIGENETIC MEMORY AND PREFERRED X-CHROMOSOME INACTIVATION...... 168 DIFFERENTIATION INTO INSULIN-PRODUCING Poster Board Number: 3107 CELLS...... 165 NEURONAL MATURATION DEFECT IN INDUCED Poster Board Number: 3085 PLURIPOTENT STEM CELLS FROM RETT SYNDROME RESTORATION OF FMR1 EXPRESSION IN FRAGILE-X PATIENTS ...... 168 INDUCED PLURIPOTENT STEM CELLS AND NEURONAL Poster Board Number: 3109 CELLS BY A PHARMACOLOGICAL TREATMENT. . . .165 EPIGENETIC ABNORMALITIES IN RETT SYNDROME Poster Board Number: 3087 FIBROBLASTS ARE PARTIALLY CORRECTED BY FULLY-PLURIPOTENT HUMAN IPS CELLS AND REPROGRAMMING...... 169 TRANSMITOCHONDRIAL DERIVATIVES: TOWARD Poster Board Number: 3111 PATIENT-SPECIFIC STEM CELL THERAPY FOR MITOCHONDRIAL DNA DISEASE ...... 165 IN VITRO OSTEOGENIC DIFFERENTIATION OF NOVEL HUMAN EMBRYONIC STEM CELL DERIVATIVES. . . . . 169 Poster Board Number: 3089 Poster Board Number: 3113 INITIAL CULTURE CONDITIONS OF HUMAN DERMAL FIBROBLASTS INCREASE EFFICIENCY AND PACE OF ENDOGENOUS PROTEIN TAGGING IN HUMAN THEIR REPROGRAMMING...... 166 PLURIPOTENT STEM CELL-DERIVED NEURAL STEM CELLS...... 169 Poster Board Number: 3091 Poster Board Number: 3115 MULTIPLE TARGETS OF ESCC MIRNAS PROMOTE HUMAN INDUCED PLURIPOTENCY...... 166 THE HUMAN EMBRYONIC STEM CELL INDUCED PLURIPOTENT STEM CELL SHARED RESOURCE FACILITY Poster Board Number: 3093 AT MOUNT SINAI, NEW YORK ...... 170 VALIDATING HUMAN IPS CELL-DERIVED MOTOR Poster Board Number: 3117 NEURONS FOR ALS MODELING...... 166 IINDUCTION OF HEMATOPOIESIS FROM HUMAN Poster Board Number: 3095 INDUCED PLURIPOTENT STEM CELLS (HIPSCS) IN HUMAN INDUCED PLURIPOTENT STEM CELLS COCULTURE WITH AUTOLOGUS HIPSC-DERIVED DERIVED FROM A SPORADIC ALZHEIMER’S DISEASE MESENCHYMAL STEM CELLS UNDER ANIMAL SERUM- PATIENT...... 167 FREE CONDITIONS...... 170 Poster Board Number: 3097 Poster Board Number: 3119 AN IPSC MODEL OF SORL1 VARIANTS IN SPORADIC MONOCLONAL ANTIBODIES RAISED SPECIFICALLY ALZHEIMER’S DISEASE...... 167 AGAINST UNDIFFERENTIATED HUMAN EMBRYONIC Poster Board Number: 3099 STEM (HES) CELLS PROVIDE A NOVEL TOOL TO DETECT AND ISOLATE EARLY REPROGRAMMED COLONIES UNDERSTANDING SPINAL MUSCULAR ATROPHY DURING HUMAN INDUCED PLURIPOTENT STEM (HIPS) MECHANISMS THROUGH HUMAN IPSC MODELS CELL LINE GENERATION...... 171 DERIVED FROM HAPLOIDENTICAL SIBLINGS WITH A Poster Board Number: 3121 DISCORDANT PHENOTYPE OF THE DISEASE. . . . .167 Poster Board Number: 3101 PARKIN MUTATIONS INCREASE OXIDATIVE STRESS IN HUMAN MIDBRAIN DOPAMINERGIC NEURONS DERIVED HIGH CONTENT-IMAGING CHARACTERIZATION OF IPS FROM PATIENT-SPECIFIC IPS CELLS...... 171 CLONES FOR SPINAL MUSCULAR ATROPHY (SMA) Poster Board Number: 3123 PROVIDES A CONVENIENT DRUG SCREENING PLATFORM...... 167 LRRK2 AND ALPHA-SYNUCLEIN MUTANT PARKINSON’S DISEASE IPSC-DERIVED DOPAMINERGIC NEURONS DEMONSTRATE DISEASE RELATED PHENOTYPES. . .171

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Poster Board Number: 3125 Poster Board Number: 3149 INDUCED PLURIPOTENT STEM CELLS DERIVED FROM WHERE THE TRANSCRIPTION FACTORS INTEGRATE IN PARKINSON’S DISEASE PATIENT DIFFERENTIATED INTO THE HUMAN GENOME DURING THE REPROGRAMMING MIDBRAIN DOPAMINERGIC NEURONS...... 172 IN THE GENERATION OF HUMAN INDUCED PLURIPOTENT Poster Board Number: 3127 STEM (iPS) CELLS?...... 176 Poster Board Number: 3151 USING HUMAN INDUCED PLURIPOTENT STEM CELL TECHNOLOGY TO STUDY NEURONAL PHENOTYPIC REPROGRAMMING OF HUMAN DENTAL PULP DIFFERENCES IN SCHIZOPHRENIA...... 172 STEM CELLS TO STUDYING THE BIOLOGICAL Poster Board Number: 3129 MECHANISM INVOLVED IN AUTISM SPECTRUM DISORDER (ASD)...... 176 XENO-TRANSPLANTATION OF IPSC DERIVED NEURAL Poster Board Number: 3153 STEM CELLS FROM A PATIENT WITH LYSOSOMAL STORAGE DISEASE...... 172 EPITHELIAL CELLS FROM HUMAN ORAL MUCOSA Poster Board Number: 3131 AS A CELLULAR MODEL FOR STUDYING GENETIC DISEASES...... 176 CYTOGENETIC ABERRATIONS IN HUMAN ESC AND Poster Board Number: 3155 IPSC LINES SHOW SIGNIFICANT DIFFERENCES: A COMPARISON OF CHROMOSOME CHANGES IN 1500 IPS EXTRAVILLOUS TROPHOBLAST STEM CELLS (EVT) FROM AND 1500 ES CELL LINES...... 173 HUMAN TERM PLACENTA ARE NOT GOOD CANDIDATES Poster Board Number: 3133 FOR USING IN CELL THERAPY PROTOCOLS ...... 177 Poster Board Number: 3157 GENERATION OF HUMAN INDUCED PLURIPOTENT STEM CELLS USING THE EXTRACELLULAR MATRIX OF HUMAN A LARGE NUMBER OF POINT MUTATIONS IN MOUSE IPS DECIDUA-DERIVED MESENCHYMAL CELLS...... 173 CELLS...... 177 Poster Board Number: 3135 Poster Board Number: 3159 EEFFICIENT GENERATION OF INTEGRATION FREE CONSTITUTIVE HETEROCHROMATIN PATIENT-SPECIFIC PLURIPOTENT STEM CELLS USING REORGANIZATION DURING MOUSE SOMATIC CELL TEMPERATURE SENSITIVE SENDAI VIRUS VECTORS. . 174 REPROGRAMMING...... 177 Poster Board Number: 3137 Poster Board Number: 3161 ROBUST GENERATION OF INDUCED PLURIPOTENT STEM CARDIOMYOCYTES OBTAINED FROM MURINE INDUCED CELLS FROM HUMAN AMNIOTIC MESENCHYMAL STEM PLURIPOTENT STEM CELLS WITH LONG QT SYNDROME CELLS...... 174 3 RECAPITULATE TYPICAL DISEASE-SPECIFIC FEATURES Poster Board Number: 3139 IN VITRO ...... 178 Poster Board Number: 3163 IPS CELL BASED IN VITRO MODELLING OF METACHROMATIC LEUKODYSTROPHY...... 174 REPROGRAMMING EFFICIENCIES OF PEI TRANSFECTION Poster Board Number: 3141 IN MURINE INDUCED PLURIPOTENT STEM CELL GENERATION ...... 178 CHARACTERIZATION OF IPSC-BASED DISEASE MODELS Poster Board Number: 3165 DERIVED FROM PATIENTS WITH TWO MAJOR TYPES OF X-LINKED ADRENOLEUKODYSTROPHY...... 174 RETROVIRAL SILENCING AND PLURIPOTENCY IN MOUSE Poster Board Number: 3143 IPS CLONES...... 178 Poster Board Number: 3167 IMMUNOHISTOCHEMICAL ANALYSIS OF THE HUMAN IPS CELL DERIVED TERATOMAS...... 175 AN EX VIVO GENE THERAPY APPROACH TO Poster Board Number: 3145 TREAT MUSCULAR DYSTROPHY USING MOUSE IPS CELLS...... 179 COMPARATIVE ANALYSIS OF CHARACTERISTICS Poster Board Number: 3169 AMONG HUMAN IPS, ES AND NEUROBLASTOMA CELL LINES...... 175 PATTERNED DIFFERENTIATION IN 3D FORMAT USING Poster Board Number: 3147 MOUSE IPS CELLS IN A MICROFLUIDICS DEVICE. . .179 Poster Board Number: 3171 EXPANSION OF HUMAN IPS CELLS ON XENO-FREE, SYNTHETIC SURFACE IN DEFINED MEDIUM. . . . .175 PRODUCTION OF IPS CELL-CHIMERAS FROM AGED MICE ...... 180

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Poster Board Number: 3173 Poster Board Number: 3197 TREATMENT OF ACUTE HEPATIC FAILURE IN MICE MICRORNAS AS INDICATORS OF PLURIPOTENCY BY TRANSPLANTATION OF HUMAN INDUCED STATUS...... 184 PELURIPOTENT STEM CELLS-DERIVED MESENCHYMAL Poster Board Number: 3199 PROGENITORS...... 180 ESTABLISHMENT OF AN INDUCED PLURIPOTENT STEM Poster Board Number: 3175 CELL BIOBANK AT CORIELL INSTITUTE FOR MEDICAL MICRORNA MIR15A/16 DEFECT REMAINS IN NEW RESEARCH...... 185 ZEALAND BLACK DERIVED MOUSE “IPS-LIKE” CELLS: Poster Board Number: 3201 POTENTIAL USEFULNESS FOR IDENTIFICATION OF CLL CANCER STEM CELLS...... 180 CELLULAR REPROGRAMMING OF ENDANGERED SPECIES FOR CONSERVATION AND RESEARCH ...... 185 Poster Board Number: 3177 Poster Board Number: 3203 CHIMERIC PIGS PRODUCED FROM INDUCED PLURIPOTENT STEM CELLS DEMONSTRATE NORMAL DIRECT REPROGRAMMING OF FIBROBLASTS INTO DEVELOPMENT AND LACK TUMOR FORMATION. . .181 EPIBLAST STEM CELLS...... 185 Poster Board Number: 3179 Poster Board Number: 3205 INCREASING THE EFFICIENCY OF GENERATING ZFX ENHANCES THE GENERATION OF INDUCED PORCINE INDUCED PLURIPOTENT STEM CELLS WITH PLURIPOTENT STEM CELLS ...... 186 MICRORNA...... 181 Poster Board Number: 3207 Poster Board Number: 3181 FEASIBILITY OF HYDROGEL-BASED CREATING BOVINE IPSCS: LENTIVIRAL INFECTION MICROENCAPSULATION FOR PROPAGATION AND EFFICIENCY OF FETAL BOVINE CELLS...... 182 DIFFERENTIATION OF INDUCED PLURIPOTENT STEM CELLS IN SCALABLE SUSPENSION CULTURE. . . . .186 Poster Board Number: 3183 Poster Board Number: 3209 ISOLATION AND INITIAL CHARACTERIZATION OF BABOON INDUCED PLURIPOTENT STEM CELLS. . . . . 182 GENETIC EVOLUTION AND MOSAICISM DURING REPROGRAMMING TO PLURIPOTENCY...... 186 Poster Board Number: 3185 Poster Board Number: 3211 INTRACEREBRAL TRANSPLANTATION OF NEURONS DIFFERENTIATED FROM INDUCED PLURIPOTENT STEM SINGLE CELL ANALYSIS OF GENETIC MOSAICISM IN CELLS INTO A RODENT MODEL OF MIDDLE CEREBRAL STEM CELL-DERIVED GENOMES ...... 187 ARTERY OCCLUSION...... 182 Poster Board Number: 3213 Poster Board Number: 3187 COMPARATIVE ANALYSIS OF REPROGRAMMING BY IPS, GENERATION OF INDUCED PLURIPOTENT STEM FMR AND NORMAL REPRODUCTION...... 187 CELLS FROM ADULT LIVER CELLS IN COMMON Poster Board Number: 3215 MARMOSET...... 183 PROBING EARLY GENE REGULATORY EVENTS IN Poster Board Number: 3189 REPROGRAMMING BY COMBINATORIAL BAYESIAN QUAIL INDUCED PLURIPOTENT STEM CELLS DERIVED MODELLING...... 187 USING HUMAN REPROGRAMMING FACTORS. . . . 183 Poster Board Number: 3217 Poster Board Number: 3191 INVESTIGATING THE ROLE OF ASTROCYTES IN NUCLEAR DELIVERY OF IPS TRANSCRIPTION FACTORS PLASTICITY AND DISEASE ...... 187 USING PROTEIN NANOCAPSULES...... 183 Poster Board Number: 3219 Poster Board Number: 3193 PROTEIN-BASED REPROGRAMMING: VIRUS VERSUS STEM CELL DETECTION AND ISOLATION USING PROTEIN APPROACH ...... 188 DIVERSITY ORIENTED FLUORESCENCE LIBRARY Poster Board Number: 3221 APPROACH (DOFLA) ...... 184 CHILDREN’S HOSPITAL OF PHILADELPHIA INDUCED Poster Board Number: 3195 PLURIPOTENT STEM CELL CORE FACILITY ...... 188 MIR-34A MICRO RNA PROVIDES A BARRIER FOR SOMATIC CELL REPROGRAMMING...... 184

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REGENERATION MECHANISMS...... 189. Poster Board Number: 3245 THE MECHANICAL COUPLING OF RAT ADULT Poster Board Number: 3223 MARROW STROMAL STEM CELLS DURING CARDIAC COMBINATORIAL HUMAN PROGENITOR CELL REGENERATION EXPLORED IN A 2-D CO-CULTURE TRANSPLANTATION OPTIMIZES ENDOGENOUS BETA MODEL ...... 193 CELL REGENERATION THROUGH SECRETION OF Poster Board Number: 3247 REGENERATIVE FACTORS...... 189 ORGAN ASSOCIATED DEVELOPMENTAL MARKERS IN Poster Board Number: 3225 PERI-ORGAN ADIPOSE: KIDNEY...... 193 SECRETOME OF HUMAN EMBRYONIC AND Poster Board Number: 3249 MESENCHYMAL STEM CELLS INCREASES MATRIX METALLPPROTEINASE-2 EXPRESSION AND PROMOTES A SMALL MOLECULE WNT INHIBITOR INCREASES HOST TISSUE REGENERATION IN A CHRONIC MODEL OF ENGAFTMENT AND INHIBITS LINEAGE COMMITTMENT LIVER INJURY...... 189 OF MESENCHYMAL STEM CELLS...... 193 Poster Board Number: 3227 Poster Board Number: 3251 CELL THERAPY IN ACUTE LUNG INJURY...... 189 FIBRO OF THE FIBRE - THE CRITICAL ROLE OF FIBRO- ADIPOGENIC PROGENITORS IN MUSCLE REGENERATION Poster Board Number: 3229 AND MYOPATHIC FIBROSIS ...... 194 HUMAN UMBILICAL CORD BLOOD-DERIVED Poster Board Number: 3253 MESENCHYMAL STEM CELLS MIGRATE IN RESPONSE TO COMPLEMENT...... 190 REGENERATION OF THE ADULT ZEBRAFISH BRAIN AFTER TRAUMATIC LESION: NEUROGENIC RADIAL GLIA-TYPE Poster Board Number: 3231 PROGENITORS MAKE NEW NEURONS...... 194 REGENERATION THERAPY IN ALZHEIMER’S Poster Board Number: 3255 DISEASE...... 190 THE ROLE OF THE RHOA/RHO KINASE SIGNALING Poster Board Number: 3233 PATHWAY IN GLIAL DIFFERENTIATION OF DOES HUMAN MOTOR NEURON STEM CELL-DERIVED MESOANGIOBLASTS...... 194 NEUROTROPHIC SUPPORT ENHANCE THE DEVELOPMENT Poster Board Number: 3257 OF THE NMJ IN SPINAL MUSCULAR ATROPHY? . . . . . 190 THE FLATWORM MACROSTOMUM LIGNANO: Poster Board Number: 3235 GENOMICS, TRANSGENICS AND NEOBLAST WHAT DETERMINES WHETHER MURINE EPENDYMAL MARKERS...... 195 STEM CELLS ARE ACTIVATED DURING PATHOLOGIES OF Poster Board Number: 3259 THE ADULT SPINAL CORD?...... 191 SUBSTANCE P STIMULATES TISSUE REPAIR ASSOCIATED Poster Board Number: 3237 WITH INDUCTION OF IL-10 AND ACTIVATION OF M2 ADULT MOUSE POST MORTEM NEURAL PRECURSORS MACROPHAGE AFTER SPINAL CORD INJURY...... 195 REGENERATE NEURONAL TISSUE AND PROMOTE Poster Board Number: 3261 FUNCTIONAL RECOVERY AFTER TRANSPLANTATION IN A SPINAL CORD INJURY MODEL...... 191 SIGNIFICANCE OF REMYELINATION BY GRAFTED NEURAL STEM/PROGENITOR CELLS INTO THE INJURED SPINAL Poster Board Number: 3239 CORD...... 195 ENHANCING NEUROREPAIR MECHANISMS: CLINICALLY Poster Board Number: 3263 RELEVANT ELECTRIC FIELDS PROMOTE ADULT MOUSE NEURAL PRECURSOR CELL MIGRATION...... 191 MECHANISTIC STUDY OF THE GLYCOME DURING VASCULAR REGENERATION ...... 196 Poster Board Number: 3241 THE INFLUENCE OF CHEMOKINES ON THE REPROGRAMMING...... 196. DIFFERENTIATION OF NEURAL PROGENITOR CELLS FROM THE YOUNG ADULT AND AGED RAT BRAIN. . . 192 Poster Board Number: 3265 Poster Board Number: 3243 AN INTEGRATION FREE REPROGRAMMING APPROACH USING HERPES SIMPLEX VIRUS (HSV)-1 AMPLICON SIGNALLING MECHANISMS UNDERLYING SYSTEM ...... 196 NEUROTROPHIC ACTIVITY OF RAT MESENCHYMAL STEM CELLS...... 192

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Poster Board Number: 3267 Poster Board Number: 3293 ENHANCED NEURONAL DIFFERENTIATION BY MECHANISTIC INSIGHTS INTO DIRECTED EXOGENOUS EXPRESSION OF SPECIFIC TRANSCRIPTION REPROGRAMMING USING CELL FUSION ...... 200 FACTORS ...... 196 Poster Board Number: 3295 Poster Board Number: 3269 NEUROGENIN3 PROMOTES PROLIFERATION OF DIRECT CONVERSION OF PERICYTE-LIKE CELLS OF TRANSDIFFERENTIATED BETA CELLS ...... 200 THE ADULT HUMAN BRAIN INTO FUNCTIONAL Poster Board Number: 3297 NEURONS...... 197 CONVERSION OF FIBROBLASTS INTO FUNCTIONAL Poster Board Number: 3271 SPINAL MOTOR NEURONS...... 201 EFFICIENT, HIGH-THROUGHPUT HUMAN IPS CELL Poster Board Number: 3299 DERIVATION...... 197 MYC DETERMINE THE PARTIAL REPROGRAMMED CELL Poster Board Number: 3273 FATE...... 201 PURIFICATION OF HUMAN IPSC-DERIVED Poster Board Number: 3301 ENDODERMAL AND RETINAL PROGENITORS AND SPECIALIZED CELLS...... 197 AKT RELATES TO C-MYC FOR PROMOTION OF REPROGRAMMING...... 201 Poster Board Number: 3275 Poster Board Number: 3303 IDENTIFICATION OF NANOG2, A NOVEL TRANSCRIPT IN PLURIPOTENT HUMAN STEM CELL POPULATIONS. . 198 GENE-TARGETED OSTEOGENESIS IMPERFECTA IPSCS EXPRESS NORMAL COLLAGEN AND PRODUCE BONE Poster Board Number: 3277 AFTER MESENCHYMAL DIFFERENTIATION. . . . . 201 INABILITY TO PROPERLY INITIATE EMBRYONIC Poster Board Number: 3305 TRANSCRIPTION PREVENTS DEVELOPMENT FOLLOWING HUMAN NUCLEAR TRANSFER...... 198 INVESTIGATING THE ROLE OF RB DURING CELLULAR REPROGRAMMING...... 202 Poster Board Number: 3279 Poster Board Number: 3307 A VALIDATED MRNA REPROGRAMMING PROTOCOL FOR THE REPRODUCIBLE GENERATION OF INTEGRATION- INDUCED PLURIPOTENT STEM CELLS AS A TOOL TO FREE HUMAN IPS CELLS...... 198 MODEL WILLIAMS-BEUREN SYNDROME...... 202 Poster Board Number: 3281 Poster Board Number: 3309 IN VIVO TRANSITION OF MOUSE HEPATOCYTES COMPONENTS THAT TRANSDUCE EXTRACELLULAR TOWARDS A BETA CELL PHENOTYPE BY DELIVERY OF MATRIX (ECM) SIGNALS ARE REQUIRED FOR BETA CELL TRNSCRIPTION FACTORS IN MOUSE. . . 198 DEVELOPMENTAL COMMITMENT OF C. ELEGANS EMBRYONIC PROGENITOR CELLS...... 202 Poster Board Number: 3283 Poster Board Number: 3311 THE CRITICAL ROLE OF PRDM14-KLF2 IN REPROGRAMMING MOUSE EPIBLAST STEM CELLS TO INSIGHTS INTO THE ROLE OF NANOG IN THE INDUCTION NAÏVE PLURIPOTENCY...... 199 OF NAIVE PLURIPOTENCY ...... 203 Poster Board Number: 3285 Poster Board Number: 3313 SYSTEMATIC ANALYSIS OF EPIGENETIC OVERCOMING BARRIERS THAT HINDER HIGH REPROGRAMMING PROCESS ACCOMPANIED BY THROUGHPUT SCREENING FOR FACTORS THAT MEDIATE GENERATION OF MOUSE INDUCED PLURIPOTENT STEM CELLULAR REPROGRAMMING...... 203 CELL...... 199 Poster Board Number: 3287 EMBRYONIC STEM CELLS CLINICAL EPIGENTIC REGULATION OF NEURAL GENE EXPRESSION APPLICATION...... 203. IN MOUSE MESENCHYMAL STEM CELLS...... 199 Poster Board Number: 3315 Poster Board Number: 3289 DERIVATION OF A GMP-GRADE HUMAN EMBRYONIC OPTIMIZING THE EFFICIENCY AND QUALITY OF IPS CELL STEM CELL LINE WITH THE DEFINED, LOW PROTEIN GENERATION ...... 199 AND XENO-FREE NUTRISTEMTM MEDIUM. . . . . 203 Poster Board Number: 3291 MODIFIED MRNA IS HIGHLY IMMUNOGENIC: IMPLICATIONS FOR DIRECTING CELL FATE. . . . . 200

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Poster Board Number: 3317 EMBRYONIC STEM CELL DIFFERENTIATION. . .208 DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS TO MATURE DENDRITIC CELLS IN A THERAPEUTIC Poster Board Number: 3341 MODEL FOR ALZHEIMER’S DISEASE...... 204 ANALYSIS OF HCN EXPRESSION IN HUMAN EMBRYONIC Poster Board Number: 3319 STEM CELL-DERIVED CARDIOMYOCYTES...... 208 DETECTION OF RARE HESC IMPURITY BY METHYLATION Poster Board Number: 3343 SPECIFIC CASTPCR...... 204 STEMDIFF™ APEL™ IS AN ANIMAL COMPONENT- Poster Board Number: 3321 FREE MEDIUM WHICH SUPPORTS MULTI-LINEAGE DIFFERENTIATION OF HUMAN PLURIPOTENT STEM CLINICAL STRATEGIES FOR CELL REPLACEMENT CELLS...... 208 THERAPY USING HUMAN EMBRYONIC STEM CELLS IN NEURODEGENERATIVE DISEASE...... 204 Poster Board Number: 3345 Poster Board Number: 3323 SUSPENSION CULTURE AND CARDIAC DIFFERENTIATION OF HUMAN INDUCED PLURIPOTENT STEM CELLS. . . 209 SORTING AND TRANSPLANTATION OF DOPAMINERGIC PROGENITORS DERIVED FROM HUMAN PLURIPOTENT Poster Board Number: 3347 STEM CELLS...... 205 PARTHENOGENETIC HUMAN ES CELLS FORM Poster Board Number: 3325 FUNCTIONAL NEURONS...... 209 CULTURE METHODS INFLUENCE THE GENETIC STABILITY Poster Board Number: 3349 OF HUMAN EMBRYONIC STEM CELLS ...... 205 EFFICIENT PRODUCTION OF NEURAL PROGENITOR Poster Board Number: 3327 CELLS FROM HUMAN PLURIPOTENT STEM CELLS USING STEMdiffTM NEURAL INDUCTION MEDIUM. . . . .209 THERAPEUTIC INTEGRIN-MEDIATED ECM REMODELING PATHWAY BY TRANSPLANTATION OF VASCULAR Poster Board Number: 3351 ANGIOGENIC PROGENITOR CELLS DERIVED FROM GENERATION OF PERIPHERAL SENSORY NEURONS FROM HESCS ON VASCULAR ISCHEMIC DISEASE...... 205 HUMAN EMBRYONIC STEM CELL-DERIVED NEURAL Poster Board Number: 3329 PROGENITORS...... 210 STABILITY OF CGMP HUMAN ES CELLS FOLLOWING Poster Board Number: 3353 EXTENDED CULTURE...... 206 NEURONAL DIFFERENTIATION OF HUMAN EMBRYONIC Poster Board Number: 3331 AND INDUCED PLURIPOTENT STEM CELLS ON 3D SCAFFOLDS: A KEY SOLUTION FOR TRANSPLANTATION DEFINED CULTURING AND DIFFERENTIATION OF THERAPY ...... 210 HUMAN INDUCED PLURIPOTENT STEM CELLS INTO ENDODERMAL LINEAGES USING A FULLY HUMAN Poster Board Number: 3355 RECOMBINANT VITRONECTIN PROTEIN...... 206 THE SPECIFICATION OF NOCICEPTIVE NEURONS FROM Poster Board Number: 3333 HUMAN EMBRYONIC STEM CELLS...... 211 ONE-YEAR OBSERVATION OF DOPAMINERGIC NEURONS Poster Board Number: 3357 DERIVED FROM HUMAN EMBRYONIC STEM CELLS IN IMMUNOPHENOTYPING CELL SUBPOPULATIONS PRIMATE MODELS OF PARKINSON’S DISEASE. . . .206 DEFINED BY EXPRESSION OF SOX1, SOX2, PAX6 Poster Board Number: 3335 AND DOUBLECORTIN IN NEURAL INDUCTION CULTURES OF HUMAN EMBRYONIC STEM CELLS BY FLOW TRANSPLANTATION OF EMBRYONIC STEM CELLS- CYTOMETRY...... 211 DERIVED BASAL FOREBRAIN CHOLINERGIC NEURONS IMPROVES THE COGNITIVE IMPAIRMENT OF Poster Board Number: 3359 ALZHEIMER’S DISEASE MICE...... 207 A NOVEL NODAL ANTAGONIST THAT IS NECESSARY Poster Board Number: 3337 AND SUFFICIENT FOR HUMAN NERVOUS SYSTEM INDUCTION ...... 211 INTERACTIONS BETWEEN MOUSE EMBRYONIC STEM CELL-DERIVED NEURAL PROGENITORS AND THE HOST Poster Board Number: 3361 BRAIN...... 207 DERIVATION OF FUNCTIONAL CRANIAL HUMAN Poster Board Number: 3339 PLACODE CELLS AND SENSORY NEURONS FROM HUMAN EMBRYONIC STEM CELLS...... 211 THE AUTISM IPSC BIOREPOSITORY: AN INTERNATIONAL RESOURCE FOR INVESTIGATORS STUDYING THE AUSTISM SPECTRUM DISORDERS ...... 207 xxx www.isscr.org Table of Contents

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Poster Board Number: 3363 Poster Board Number: 3385 WNT/β-CATENIN SIGNALING PROMOTES MIDBRAIN BRACHYURY AND CDX2 MEDIATE BMP-INDUCED SPECIFICATION OF NEURAL PRECURSOR CELLS DERIVED DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS FROM HUMAN EMBRYONIC STEM CELLS...... 212 INTO MESODERM, NOT TROPHOBLAST...... 215 Poster Board Number: 3365 Poster Board Number: 3387 OPTIMIZED METHOD FOR THE DERIVATION OF MOLECULAR FEATURES OF TROPHOBLAST MIDBRAIN DOPAMINE NEURONS FROM HUMAN DIFFERENTIATION FROM HUMAN EMBRYONIC AND EMBRYONIC STEM CELLS IMPROVES SURVIVAL INDUCED PLURIPOTENT STEM CELLS...... 216 IN VIVO ...... 212 Poster Board Number: 3389 Poster Board Number: 3367 DIFFERENCES BETWEEN 2D AND 3D PROTOCOL IN DIRECTED DIFFERENTIATION OF HUMAN EMBRYONIC HUMAN ESCS TOWARDS PANCREATIC ENDODERM STEM CELLS TO MIDBRAIN DOPAMINERGIC NEURONS ELUCIDATED...... 216 IN A 3D ENVIRONMENT...... 212 Poster Board Number: 3391 Poster Board Number: 3369 PRODUCTION OF MULTIPOTENT ANTERIOR FGF AND SMALL MOLECULES EFFICIENTLY INDUCE DEFINITIVE ENDODERM FROM HUMAN PLURIPOTENT NEURAL DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS...... 216 STEM CELLS...... 213 Poster Board Number: 3393 Poster Board Number: 3371 DEFINING THE HEPATIC CELL NICHE UPON HEPATIC NEURONS IN A SNAP: COMBINED SMALL MOLECULE SPECIFICATION OF HUMAN ES CELL-DERIVED INHIBITION EFFICIENTLY ACCELERATES HUMAN ENDODERM...... 217 PLURIPOTENT STEM CELL DIFFERENTIATION INTO Poster Board Number: 3395 NOCICEPTORS ...... 213 COLLAGEN THIN FILMS AS EXTRACELLULAR MATRICES Poster Board Number: 3373 FOR HUMAN PLURIPOTENT STEM CELLS...... 217 EXPLORING HUMAN PANCREATIC DEVELOPMENT USING Poster Board Number: 3397 CHEMICAL APPROACHES...... 213 LENGTHENED G1 PHASE INDICATES DIFFERENTIATION Poster Board Number: 3375 STATUS ACROSS MULTIPLE LINEAGES IN HUMAN EFFICIENT DIFFERENTIATION OF LIVER AND EMBRYONIC STEM CELLS...... 218 PANCREATIC PROGENITOR-LIKE CELLS FROM HUMAN Poster Board Number: 3399 EMBRYONIC STEM CELLS USING SMALL MOLECULE INHIBITORS ...... 214 EFFECTS OF FORCED ALIGNMENT ON HUMAN EMBRYONIC STEM CELLS...... 218 Poster Board Number: 3377 Poster Board Number: 3401 EXAMINATION OF CELL SIGNALING PATHWAYS DURING EARLY ENDODERM SPECIFICATION FROM HUMAN ADHERENT DIFFERENTIATION OF FUNCTIONAL EMBRYONIC STEM CELLS UTILIZING FLUORESCENCE NEURONS FROM HUMAN EMBRYONIC STEM CELL BARCODING AND INTRACELLULAR FLOW CELLS...... 218 CYTOMETRY...... 214 Poster Board Number: 3403 Poster Board Number: 3379 THE DERIVATION OF MELANOCYTES FROM IMPROVING THE FIDELITY OF HEPATIC DIFFERENTIATION HUMAN PLURIPOTENT STEM CELLS FOR DISEASE FROM HUMAN PLURIPOTENT STEM CELLS ...... 214 MODELING...... 219 Poster Board Number: 3381 Poster Board Number: 3405 PROMININ 1 (CD133) MARKS A POPULATION OF DEVELOPING A HUMAN DRUG DISCOVERY PLATFORM MESENDODERMAL CELLS IN HUMAN EMBRYONIC STEM FOR HUNTINGTON’S DISEASE ...... 219 CELLS...... 215 Poster Board Number: 3407 Poster Board Number: 3383 THE SIGNALING PROPERTIES OF INOSITOL PHOSPHATES PROMOTING MESODERM AND BLOCKING ENDODERM: AND PHOSPHOINOSITIDES IN HUMAN EMBRYONIC THE ROLE OF BRACHYURY IN HUMAN EMBRYONIC STEM STEM CELLS AND HUMAN EMBRYONAL CARCINOMA CELLS...... 215 CELLS...... 219

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Poster Board Number: 3409 Poster Board Number: 3431 CLONAL TRACKING OF THE GENESIS OF DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS HEMATOPOIETIC CELLS FROM HUMAN INTO CARDIOMYOCYTES DEPENDS ON EXPRESSION OF PLURIPOTENT STEM CELLS DIFFERENTIATING IN CONNEXINS...... 224 EMBRYOID BODIES ...... 220 Poster Board Number: 3433 Poster Board Number: 3411 LOW OXYGEN CULTURE AND SMALL MOLECULE DIRECTED DIFFERENTIATION OF HUMAN INHIBITION OF MITOGEN-ACTIVATED PROTEIN PLURIPOTENT STEM CELLS TO A DEFINITIVE KINASE AND GLYCOGEN SYNTHASE KINASE DURING HEMATOPOIETIC FATE...... 220 SELF-RENEWAL REVERSIBLY AFFECT SUBSEQUENT Poster Board Number: 3413 DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS TO CARDIOMYOCYTES...... 224 RUNX1C EXPRESSION IDENTIFIES HEMATOPOIETIC Poster Board Number: 3435 PROGENITORS IN CD34+ CELLS ISOLATED FROM DIFFERENTIATING RUNX1CGFP/W HUMAN EMBRYONIC DIFFERENTIAL EXPRESSION OF FORMINS DURING STEM CELLS...... 221 HEART DEVELOPMENT AND MOUSE EMBRYONIC STEM Poster Board Number: 3415 CELL DIFFERENTIATION TO CARDIOMYOCYTES. . . . . 224 Poster Board Number: 3437 NOVEL DIRECT DIFFERENTIATION METHOD FOR GETTING HIGHLY UNIFORM NEURAL PROGENITOR CELLS ROLE OF TRANSIENT RECEPTOR POTENTIAL CANONICAL AND DOPAMINE NEURONS FROM HUMAN ES CELLS CHANNELS IN DIFFERENTIATION OF MOUSE EMBRYONIC AND IPS CELLS...... 221 NEURAL STEM/PROGENITOR CELLS...... 225 Poster Board Number: 3417 Poster Board Number: 3439 PUTATIVE ROLE OF MICRORNA-21 IN HUMAN IN VITRO DIFFERENTIATION OF FUNCTIONAL INSULIN- EMBRYONIC STEM CELLS...... 221 PRODUCING CELLS FROM MOUSE ES CELLS. . . . 225 Poster Board Number: 3419 Poster Board Number: 3441 PROTEIN KINASE C INDUCES EPITHELIAL TO A NOVEL ROLE FOR P38 MAPK IN ENDODERM MESENCHYMAL TRANSITION IN HUMAN ES AND IPS FORMATION FROM MOUSE PLURIPOTENT CELLS . . . 225 CELLS...... 222 Poster Board Number: 3443 Poster Board Number: 3421 WNT-3A INDUCES THE SPECIFICATION OF MOUSE BALANCE OF PROLIFERATION AND DIFFERENTIATION EMBRYONIC STEM CELLS INTO THE VISCERAL POTENTIAL COULD BE MODULATED BY CULTURE ENDODERM LINEAGE ...... 226 MEDIUM IN HUMAN EMBRYONIC STEM CELLS. . . . . 222 Poster Board Number: 3445 Poster Board Number: 3423 FIBRILLAR FIBRONECTIN PROMOTES ENDODERM MECHANICAL STRETCHING ON OSTEOGENESIS OF SPECIFICATION IN MOUSE EMBRYONIC STEM HUMAN EMBRYONIC STEM CELLS...... 222 CELLS...... 226 Poster Board Number: 3425 Poster Board Number: 3447 THE EFFECT OF DISTINCT BMP LIGANDS ON SELF- EVALUATION OF NEUROTOXICITY OF ARSANILIC ACID, RENEWAL AND DIFFERENTIATION OF HUMAN DANOFLOXACIN AND MERCURY IN VITRO USING EMBRYONIC STEM CELLS AND CHORIONIC VILLI NEURAL PROGENITOR CELLS DERIVED FROM MOUSE CELL-DERIVED HUMAN INDUCED PLURIPOTENT STEM EMBRYONIC STEM CELLS...... 226 CELLS...... 222 Poster Board Number: 3449 Poster Board Number: 3427 RAPID AND ROBUST GENERATION OF FUNCTIONAL HYPOXIA MARKEDLY INFLUENCES DIFFERENTIATION OF OLIGODENDROCYTE PROGENITOR CELLS FROM MOUSE AND HUMAN PLURIPOTENT STEM CELLS. . . 223 PLURIPOTENT MOUSE EPIBLAST STEM CELLS...... 227 Poster Board Number: 3429 Poster Board Number: 3451 NON-INVASIVE CHARACTERIZATION OF MOUSE DIFFERENTIATING MOUSE EMBRYONIC STEM (ES) CELLS EMBRYONIC STEM CELL DIFFERENTIATION USING REVEAL A ROLE FOR FATTY ACIDS AND SEX STEROID RAMAN SPECTROSCOPY...... 223 MODIFYING MECHANISMS IN DETERMINING WHITE ADIPOCYTE LINEAGES...... 227

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Poster Board Number: 3453 Poster Board Number: 3477 GENERATION OF TRANSGENIC MURINE ES CELLS DISSECTING THE MECHANISMS OF PRIMARY GERM OVEREXPRESSING NURR1 AND PITX3 AS KEY LAYER INDUCTION IN EMBRYONIC STEM CELLS. . . 232 TRANSCRIPTION FACTORS IN DOPAMINERGIC NEURON Poster Board Number: 3479 DEVELOPMENT CONCOMITANTLY WITH GLUTATHIONE PEROXIDASE-1 AS AN ANTIOXIDANT...... 228 BENCHMARKING PLURIPOTENT STEM CELL MARKERS DURING DIFFERENTIATION INTO THE THREE GERM Poster Board Number: 3455 LAYERS UNVEILS A STRIKING HETEROGENEITY: ALL INFLUENCE OF DEFINED GROWTH FACTORS ON THE MARKERS ARE NOT EQUAL...... 232 DIFFERENTIATION OF MURINE EMBRYONIC STEM CELLS Poster Board Number: 3481 INTO ALVEOLAR TYPE II EPITHELIAL CELLS...... 228 MIRNA REGULATED SUICIDE GENE EXPRESSION TO Poster Board Number: 3457 AVOID TUMOR FORMATION FOLLOWING ES CELL THE ROLE OF L-PROLINE AND L-PROLINE TRANSPLANTATION ...... 232 TRANSPORTERS IN THE REGULATION OF MOUSE ES CELL Poster Board Number: 3483 DIFFERENTIATION IN CULTURE...... 229 EFFICIENT DERIVATION OF NEURAL STEM CELLS FROM Poster Board Number: 3459 COMMON MARMOSET ES CELLS AND iPS CELLS . . . 232 PROTEOMIC ASSESSMENT OF A CELL MODEL OF Poster Board Number: 3485 SPINAL MUSCULAR ATROPHY ...... 229 HETEROGENEITY OF P75(NGFR)+ CELLS ISOLATED Poster Board Number: 3461 DURING EARLY IN VITRO HESC DIFFERENTIATION: PROTEIN KINASE A ACCELERATES EARLY IMPLICATIONS ON NEURAL CREST-LIKE CELLS PROGRAMMING FROM MOUSE ES CELLS THROUGH PURIFICATION ...... 233 EPIGENETIC INHIBITION OF OCT3/4 AND NANOG Poster Board Number: 3487 EXPRESSION...... 229 MICRORNAS MIR17, MIR20A, AND MIR106B ACT IN Poster Board Number: 3463 CONCERT TO MODULATE E2F ACTIVITY ON CELL CYCLE THE SRC-FAMILY TYROSINE KINASE C-YES SUPPRESSES ARREST DURING NEURONAL LINEAGE DIFFERENTIATION MOUSE ES CELL DIFFERENTIATION...... 229 OF USSC...... 233 Poster Board Number: 3465 Poster Board Number: 3489 CALCINEURIN NFAT AND ERK MAPK PATHWAYS EPIGENETIC ALTERATION OF HOX GENES PROMOTE MOUSE EMBYRONIC STEM CELL DURING RETINOIC ACID-INDUCED NEURAL DIFFERENTIATION INTERDEPENDENTLY...... 230 DIFFERENTIATION ...... 234 Poster Board Number: 3467 Poster Board Number: 3491 CDX GENES RESTRICT CARDIOGENIC DEVELOPMENT BY PRIMITIVE ERYTHROPOIESIS IS REGULATED BY MIR-126 AFFECTING MESODERMAL PATTERNING ...... 230 VIA NON-HEMATOPOIETIC VCAM-1+ CELLS ...... 234 Poster Board Number: 3469 Poster Board Number: 3493 DERIVATION OF CANINE NEURAL PROGENITOR ENHANCER DECOMMISSIONING BY THE HISTONE CELLS FROM CANINE INDUCED PLURIPOTENT STEM DEMETHYLASE LSD1 DURING EMBRYONIC STEM CELL CELLS...... 230 DIFFERENTIATION ...... 234 Poster Board Number: 3471 Poster Board Number: 3495 CELLULAR DIVERSITY WITHIN EMBRYONIC STEM CELLS: ROLE OF HIF1α IN HESC DIFFERENTIATION...... 235 PLURIPOTENT CLONAL SUBLINES SHOW DISTINCT Poster Board Number: 3497 DIFFERENTIATION POTENTIAL...... 231 ISOLATION OF EMBRYONIC-LIKE STEM CELLS FROM RAT Poster Board Number: 3473 MARROW AND MYOCARDIAL REPAIRING WITH CELL SELF ASSEMBLING SCAFFOLDS FOR CULTURING AND TRANSPLANTATION ...... 235 DIFFERENTIATING STEM CELLS ...... 231 Poster Board Number: 3499 Poster Board Number: 3475 THE RNA-BINDING PROTEIN RBM24 IS REQUIRED FOR ECAT15-1 AND ECAT15-2 ARE DISPENSABLE FOR CARDIOGENESIS AND HEART FUNCTION...... 235 EARLY DEVELOPMENT BUT IMPORTANT FOR LATE Poster Board Number: 3501 EMBRYOGENESIS...... 231 GENERATION OF FUNCTIONAL MELANOCYTE FROM INDUCED PLURIPOTENT STEM CELLS...... 235

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Poster Board Number: 3503 Poster Board Number: 3527 DEFINITIVE ENDODERM SPECIFICATION IS A B-CELL RECEPTOR ASSOCIATED PROTEIN 31 IS CONSEQUENCE OF EXTRA-CELLULAR MATRIX EXPRESSED ON THE CELL SURFACE OF HUMAN REMODELING DRIVEN BY PI3K SIGNALLING...... 236 EMBRYONIC STEM CELLS AND DOWNREGULATED UPON Poster Board Number: 3505 DIFFERENTIATION ...... 239 Poster Board Number: 3529 PLURIPOTENT TRANSCRIPTION FACTORS REGULATE MESOENDODERM LINEAGE COMMITMENT DURING HUMAN ES CELL SPECIFIC MIR-302 CLUSTER ARE DIFFERENTIATION ...... 236 INTIMATELY INVOLVED IN COMPLETE NUCLEAR REPROGRAMMING OF IPS CELLS THROUGH EMBRYONIC STEM CELL PLURIPOTENCY. . . .236 SUPPRESSION OF MBD2 ...... 239 Poster Board Number: 3509 Poster Board Number: 3531 REGULATORY ROLE OF MICRORNAS IN RESPONSE TO ABCG2 MULTIDRUG TRANSPORTER EXPRESSION IN UVC-INDUCED DNA DAMAGE IN HUMAN EMBRYONIC HUMAN IPS CELLS DURING REPROGRAMMING AND STEM CELLS...... 236 DIFFERENTIATION ...... 240 Poster Board Number: 3511 Poster Board Number: 3533 COMPLEX CDK2/CYCLINB1 AND ITS ROLE IN HUMAN TERATOMA FORMATION BY FEEDER-DEPENDENT EMBRYONIC STEM CELLS...... 237 AND -INDEPENDENT HUMAN EMBRYONIC STEM CELLS...... 240 Poster Board Number: 3513 Poster Board Number: 3535 EMBRYONIC CHROMOSOMAL ANEUPLOIDY RECAPITULATED IN HUMAN EMBRYONIC STEM CELL PROTEOMIC COMPARISON BETWEEN HUMAN LINES...... 237 EMBRYONIC STEM CELLS AND THEIR DIFFERENTIATED FIBROBLASTS: IDENTIFICATION OF 206 GENES Poster Board Number: 3515 TARGETED BY HES CELL-SPECIFIC MICRORNAS. . . 240 A GENOME-WIDE RNAI SCREEN IDENTIFIES NOVEL Poster Board Number: 3537 REGULATORS IN HUMAN EMBRYONIC STEM CELLS. . 237 GENOME-WIDE IDENTIFICATION OF MICRORNA Poster Board Number: 3517 TARGETS IN HUMAN ES CELLS REVEALS A ROLE FOR THE FIDELITY OF DNA METHYLATION IN HESCS GROWN MIR-302 IN MODULATING BMP RESPONSE. . . . .241 IN DEFINED AND XENO-FREE CULTURE SYSTEMS . . . 237 Poster Board Number: 3539 Poster Board Number: 3519 HUMAN EMBRYONIC STEM CELLS EXPRESS A UNIQUE IDENTIFICATION OF NEW PLURIPOTENCY GENE REPERTOIRE OF BCL-2 FAMILY MEMBERS. . . . . 241 EXPRESSION PROFILES FOR HUMAN PLURIPOTENT STEM Poster Board Number: 3541 CELLS...... 238 PROTEOMIC ANALYSIS OF HUMAN EMBRYONIC STEM Poster Board Number: 3521 CELL INTEGRIN-ASSOCIATED COMPLEXES. . . . . 241 DISTINCT REGULATION OF FOXO ISOFORMS IN HUMAN Poster Board Number: 3543 EMBRYONIC AND ADULT MOUSE HEMATOPOIETIC STEM CELLS...... 238 FEMALE SEX BIAS IN HUMAN EMBRYONIC STEM CELL LINES...... 242 Poster Board Number: 3523 Poster Board Number: 3545 ADENOVIRUS EARLY REGION 1B-ASSOCIATED PROTEIN 5 REGULATES THE PROLIFERATION AND SELF-RENEWAL CHARACTERIZATION OF A NOVEL PLURIPOTENT STATE OF HUMAN EMBRYONIC STEM CELLS THROUGH AKT OF MOUSE ES CELLS EMERGING AFTER RETINOIDS AND ERK SIGNALING...... 239 TREATMENT...... 242 Poster Board Number: 3525 Poster Board Number: 3547 HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN LAMININ STIMULATES PROLIFERATION OF MOUSE A2/B1 IS EXPOSED AT THE CELL SURFACE OF HUMAN EMBRYONIC STEM CELLS VIA REDUCTION OF GAP EMBRYONIC STEM CELLS AND CANCER CELLS, AND JUNCTIONAL INTERCELLULAR COMMUNICATION DOWNREGULATED UPON DIFFERENTIATION. . . . 239 THROUGH CONNEXIN43 INACTIVATION...... 242 Poster Board Number: 3549 GENE REGULATION OF MOUSE EMBRYONIC STEM CELLS BY LOW NITRIC OXIDE...... 243 xxxiv www.isscr.org Table of Contents

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Poster Board Number: 3551 Poster Board Number: 3577 WNT PROTEINS ARE ESSENTIAL FOR SELF RENEWAL A PROTEOMIC APPROACH FOR HUNTING ELUSIVE OF MOUSE EMBRYONIC STEM CELLS BY PREVENTING STEMNESS PROTEINS...... 247 DIFFERENTIATION TO EPIBLAST STEM CELLS. . . . 243 Poster Board Number: 3579 Poster Board Number: 3553 INSULIN IN EMBRYO CULTURE MEDIUM FROM THE METABOLIC CHARACTERIZATION OF MOUSE CLEAVAGE STAGE; A SUCCESSFUL STRATEGY FOR EMBRYONIC STEM CELLS AND EPIBLAST DERIVED IMPROVING THE PLURIPOTENTIAL OF EMBRYOS. . .247 STEM CELLS...... 243 Poster Board Number: 3581 Poster Board Number: 3555 SUMOYLATION AND AKT-MEDIATED PHOSPHORYLATION MAINTENANCE OF GENOMIC INTEGRITY IN MOUSE OF OCT4REGULATE THE PRIMITIVE PLURIPOTENT EMBRYONIC STEM CELLS BY POST TRANSLATIONAL STATE...... 248 REGULATION OF PROTEINS INVOLVED IN DNA Poster Board Number: 3583 REPAIR...... 243 EPIGENETIC REGULATION OF STEM CELL SELF-RENEWAL Poster Board Number: 3557 AND DIFFERENTIATION ...... 248 ELUCIDATING FUNCTIONAL PLURIPOTENT STATES Poster Board Number: 3585 AMONG DIVERSE MOUSE STEM CELL TYPES. . . . 244 POST-TRANSCRIPTIONAL REGULATION OF PLURIPOTENT Poster Board Number: 3561 CELL FATE ...... 248 USING CONDITIONAL KNOCKOUT OF THE BLOOM’S Poster Board Number: 3587 SYNDROME GENE IN A RECESSIVE GENETIC SCREENING FOR NOVEL FACTORS INVOLVED IN DIFFERENTIATION THE PLURIPOTENT TRANSCRIPTION FACTOR ZIC3 OF MOUSE EMBRYONIC STEM CELLS...... 244 DIRECTLY ACTIVATES NANOG IN EMBRYONIC STEM CELLS...... 249 Poster Board Number: 3563 Poster Board Number: 3589 COMPARISON OF GENOME-WIDE SINGLE NUCLEOTIDE POLYMORPHISMS (SNP) IN MOUSE STEM CELL- MAKORIN-1 IS A NOVEL COMPONENT OF THE POST- LIKE, COLONY-FORMING FIBROBLASTS IN A SPECIFIC TRANSCRIPTIONAL GENE REGULATORY NETWORK IN CELLULAR ENVIRONMENT...... 244 EMBRYONIC STEM CELLS...... 249 Poster Board Number: 3565 Poster Board Number: 3593 INVOLVEMENT OF CONNEXIN43 PROTEIN EXPRESSION ALKBH1 IS A HISTONE H2A DEMETHYLASE INVOLVED IN LEVEL IN PROSTAGLANDIN E2-MEDIATED REGULATION OF PLURIPOTENCY AND SELF-RENEWAL IN MAINTENANCE OF MOUSE EMBRYONIC STEM CELL EMBRYONIC STEM CELLS...... 249 UNDIFFETENTIATED STATE...... 245 Poster Board Number: 3595 Poster Board Number: 3567 DISSECTING THE REGULATION OF ESC PROPERTIES BY OXYTOCIN STIMULATED CONNEXIN43 EXPRESSION MEMBERS OF THE CATENIN SUPERFAMILY ...... 250 THROUGH NF-κB/CREB/CBP COMPLEX VIA LIPID RAFT- Poster Board Number: 3597 INDEPENDENT OXYTOCIN RECEPTOR-MEDIATED CAMP/ PKA IN MOUSE EMBRYONIC STEM CELLS ...... 245 RETINOL/VITAMIN A SIGNALING AND REGULATION OF SELF RENEWAL OF STEM CELLS ...... 250 Poster Board Number: 3569 Poster Board Number: 3599 CHARACTERIZATION OF THE CNOT COMPLEX IN MOUSE EMBRYONIC STEM CELL SELF-RENEWAL...... 245 NOVEL LIVE ALKALINE PHOSPHATASE SUBSTRATE FOR IDENTIFICATION OF PLURIPOTENT STEM CELLS. . . . .250 Poster Board Number: 3571 CYCLIN E SUSTAINS SELF-RENEWAL OF EMBRYONIC STEM CELLS IN THE PLURIPOTENT STATE...... 246 Poster Board Number: 3573 SAF-A HAS A ROLE IN TRANSCRIPTIONAL REGULATION OF THE PLURIPOTENCY GENE OCT4...... 246 Poster Board Number: 3575 AN INTEGRATED OCT4 NETWORK FOR PLURIPOTENCY OF EMBRYONIC STEM CELLS...... 246

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(SACK) for general ex vivo expansion of tissue stem cells could be used to PANCREATIC CELLS expand adult human pancreatic stem cells with properties required for T1D Poster Board Number: 1001 cell transplantation therapy. In the SACK method, specific purine precursors are used to expand cellular guanine ribonucleotide (rGNP) pools. Increased EFFECT OF ENDOTHELIAL CELL CO-CULTURE levels of rGNP pools shift asymmetrically self-renewing tissue stem cells to symmetric self-renewal, which promotes their exponential expansion in ON MATURATION OF HUMAN EMBRYONIC culture. We evaluated the purine nucleoside xanthosine and the purine base STEM CELL (HESC) TO PANCREATIC ISLET-LIKE xanthine for their ability to promote the expansion of pancreatic stem cells CELLS from adult human cadaveric pancreases. Supplementation with these SACK agents increased the frequency of primary cell colony growth approximately Jaramillo, Maria, Banerjee, Ipsita 50%; and the clonogenicity of initial cell colonies was improved 3-fold. Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA, Chemical and Under SACK-supplemented conditions, the SACK-derived cells express the Petroleum Engineering, University of Pittsburgh, Pittsburgh, PA, USA endocrine progenitor marker Ngn3. When cultured in SACK-free medium under conditions known to induce islet cell differentiation, the cells form Directed differentiation protocol of embryonic stem cells tries to recapitulate islet-like clusters. Cells in the clusters co-express human glucagon and in-vivo environment of organogenesis in an in-vitro setting. The key factors human insulin, indicating the production of pancreatic precursor cells with affecting developmental process are soluble growth factors, extracellular potential to establish subsequent lineages with either α-cell or β-cell pheno- matrices and intercellular signaling. Likewise, soluble factors and ECM have type, respectively. Moreover, cluster cells also express human C-peptide, an been shown to have significant effect in the in-vitro differentiation of ESCs indicator of complete insulin production capability. In an initial cell transplan- to various lineages. The effects of intercellular signals in the context of pan- tation study with the SACK-derived cells, we detected human C-peptide in creatic differentiation of ESCs however remain less explored. In pancreatic the blood of injected immunodeficient mice at levels comparable to fasting embryogenesis, aortic endothelial cells are in close proximity to the dorsal human blood insulin levels (0.11 ng/ml). Based on these initial findings, we pancreas and it has been suggested that developing pancreatic cells secret are optimistic that the SACK method will provide a needed approach to pro- VEGF which attracts endothelial cells, which in turn secrete ECM compo- duction of human pancreatic stem cells for evaluation as a new transplanta- nents that promote maturation via integrin mediated pathways. Endothelial tion therapy for T1D. cells have also been demonstrated to induce Ptf1 expression and sustain Pdx1 expression. Poster Board Number: 1005 The current study explores the effect of endothelial cells in the differentia- tion of hESCs to pancreatic islet-like cells. A multi-stage directed differentia- INDUCTIVE MOLECULES DERIVED tion approach has been developed, involving definitive endoderm induc- FROM HUMAN FETAL LIVER STROMAL tion, followed by pancreatic progenitor specification and final maturation CELLS ENHANCE THE GROWTH AND into insulin producing cells. For the last step, the hESC-derived pancreatic progenitor cells have been co-cultured with rat micro-vascular endothelial DIFFERENTIATION OF HUMAN PANCREATIC cells, which results in high expression of insulin. Comparison with parallel PROGENITOR CELLS studies using previously reported protocol of notch inhibition elicits orders of magnitude lower expression of insulin than the co-culture. Furthermore, KA YAN, NG, PO SING, LEUNG the insulin expression elicits an increasing trend with increased co-culture The School of Biomedical Science, The Chinese University of Hong Kong, ratio. Different co-culture configurations have been tested in an attempt to Hong Kong, China understand the mechanism of differentiation. Both transwell co-culture and Studies of the stem cells differentiation are usually based on exogenous conditioned media shows high up-regulation of insulin, although contact addition of suitable growth factors and/or cytokines. However, the criteria co-culture has by far the best performance. to determine the suitable ones for development into pancreatic islet-like These preliminary studies establish the effect of endothelial cells in the cell clusters (ICCs) are controversial and variable among different stem cell in-vitro maturation of embryonic stems cells to pancreatic islets. It also indi- types. In this context, we selected a unique tissue microenvironment, i.e. the cates both soluble factors and extracellular matrices are responsible for co- fetal liver that shares many common signaling pathways with fetal pancreas culture mediated differentiation. Current research is underway in identifying during organogenesis, to co-culture with pancreatic progenitor cells (hPPCs) mechanistic details of the process. derived from human fetal pancreas. We thus hypothesize that human fetal Poster Board Number: 1003 liver stromal cells (hLSCs) enhance the differentiation of hPPCs into ICCs via its secretion of specific inductive factors. In order to achieve this, we co- EXPANSION OF ADULT HUMAN PANCREATIC cultured hPPC and hLSC so as to mimic the physical proximity between liver STEM CELLS WITH β-CELL AND α-CELL and pancreas development. hLSC and hPPC were separated by transwell thus being free of heterogeneous cell-cell contact. hLSC procured from DIFFERENTIATION POTENTIAL BY different gestational weeks were separated into 1st and 2nd trimester to fur- SUPPRESSION OF ASYMMETRIC CELL KINETICS ther mimic the temporal difference during development. To investigate the (SACK) effects of hLSC on hPPC growth, cell count and MTT were performed. In addition, some critical developmental gene expressions were examined for Pare, Jean-Francois, Sherley, James L. differentiation stages. Comparing with the established differentiation cock- Adult Stem Cell Technology Center, Boston Biomedical Research Institute, tail previously reported, growth and differentiation of hPPCs were enhanced Watertown, MA, USA in the hLSC co-culture system. hLSC derived from 2nd trimester exhibited a more prominent effect on ICC differentiation than that derived from 1st Type I diabetes (T1D) is a devastating disease that results from autoimmune trimester; this observation was in close agreement with islets development destruction of β-cells. Transplantation of functional islets is the most reliable occurred in 2nd trimester. Interestingly, we also found several growth factors way to restore normal glucose homeostasis in T1D patients, but the scarcity and cytokines to be expressed in hLSC and among them, IGF1 and IGF2 of donor islets limits this treatment strategy. In vitro expansion of transplant- showed a significant differential expression between 1st and 2nd trimesters, able pancreatic stem cells with the ability to produce glucose-responsive indicative of its differential effects on ICC differentiation. These data indi- β-cells could address the shortage of donor islets. We investigated whether cated that human fetal liver stroma promotes the development of pancreatic the previously described method of suppression of asymmetric cell kinetics

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progenitor via the mediation of inductive signaling molecules in a temporally WAM-21 followed by gene expression analysis revealed WAM-21 positive and spatially regulated manner. cells express pancreatic/islet precursor-specific cell markers, whilst WAM-21 negative cells did not. Both WAM-21 positive and negative populations ex- Poster Board Number: 1007 pressed a mixture of stem cell, ductal and mesenchymal markers. Cells from CHARACTERIZATION OF MURINE LIVER, e15.5 pancreas purified using WAM-21 were differentiated in vitro under defined cell culture conditions and WAM-21 positive cells gave rise to insulin PANCREAS AND KIDNEY PROGENITORS producing cells, whilst WAM-21 negative cells did not. These studies con- Dorrell, Craig, Canaday, Pamela S., Erker, Laura, Haft, Annelise, firm that our novel monoclonal antibody WAM-21 identifies islet progenitor cells in the developing mouse pancreas, and demonstrates the utility of this Streeter, Philip R., Grompe, Markus antibody for the study of the mechanisms underlying beta cell differentiation Oregon Health & Science University, Portland, OR, USA for the purpose of improving cell based therapies for Type 1 Diabetes. The identity and function of adult epithelial stem/progenitor cells remain Poster Board Number: 1011 poorly understood. To investigate these populations in mouse liver, pancreas and kidney, we developed a collection of new monoclonal antibodies recog- COMPARISON BETWEEN VARIOUS nizing cell surface antigens on mature and immature murine epithelial cell types and in vivo and ex vivo assays of epithelial progenitor function. Label- METHODS IN EVALUATION OF B-ISLET CELL ing with these cell surface markers permitted the isolation by FACS of mul- TRANSFORMATION tiple populations for study. A two-dimensional in vitro culture assay using Gabr, Hala, Abou El-Kheir, Wael defined, serum-free media was developed in order to test these populations for clonal growth capacity. Among liver, pancreas and kidney cells, progeni- Hematology, Faculty of Medicine, Cairo University, Cairo, Egypt, tor activity was found only in a subset of duct cells defined as CD45-/ Immunology, Military Medical Academy, Cairo, Egypt CD11b-/CD31-/MIC1-1C3+/CD133+/CD26-. Cells with this phenotype Background: Stem cell plasticity and transdifferentiation of stem cells from initiate colony growth at a frequency between 1/15 and 1/50 depending on various sources into betal islet cells has been documented by many research- the tissue type and external treatments. When pancreatic cells were assayed ers. Different methods, culture conditons and cytokine cocktails have been in a three-dimensional “organoid” assay, clonal growth was again initiated used. Evaluation of transformation can be done through morphological, only by cells with the progenitor phenotype. In these conditions extensive immunological or functional criteria of the tested cells. Researchers used self-renewal was observed; single cells form organoids comprising thousands insulin secretion as the core criterion for transformation. Insulin secretion can of cells and these structures can be dispersed and passaged to form several be evaluated by immunocytochemistry, insulin mRNA, response to glucose generations of daughter organoids. Investigation of the capacity of these challenge, flow cytometric detection of cytoplasmic insulin and correction cells to differentiate to endocrine and exocrine cell types is underway. of hyperglycemia in diabetic animals. Objectives: The aim of this study is Transcriptional profiling of the progenitor-enriched mouse liver cell fraction to compare between various criteria of islet transformation in sensitivity & has been performed by microarray; comparison with the equivalent popula- specificity. Materials & Methods: Bone marrow mesenchymal stem cells are tion of mouse pancreas has revealed substantial overlap in the expression separated by plastic adherence, subjected to transdifferentiation into beta of transcriptional regulators. The determination of a shared “epithelial stem islets through exendin-nicotinamide cocktail after priming by high glucose. cell” regulatory profile is in progress. Evaluation of insulin secretion was done by: 1. Insulin mRNA quantitation,( Poster Board Number: 1009 in relation to cell number); 2. Insulin level in culture supernatant, both basal and after glucose challenge (related to cell number); 3. Cytoplasmic insulin ISOLATING FETAL MOUSE ISLET PROGENITORS by immunocytochemistry; 4. Cytoplasmic insulin using flow cytometry. Comparison of all these parameters was done by correction of hyperglyce- WITH A NOVEL MONOCLONAL ANTIBODY mia in diabetic animal model after injection of standardized number of in- Dwyer, Benjamin, Jamieson, Emma, Jacoby-Alner, Tamara, Davern, sulin positive cells using flowcytometry. Tests were compared for sensitivity, Kathleen, Jiang, Fang-Xu, Yeoh, George, Morahan, Grant specificity& linearity. Results & Conclusions: Quantitative mRNA detection was the most sensitive method while cytoplasmic insulin using flowcytom- Centre for Diabetes Research, Western Australian Institute for Medical etry showed the best linearity. Research, Perth, Australia, Monoclonal Antibody Facility, Western Australian Institute for Medical Research, Perth, Australia, Biomedical, Poster Board Number: 1013 Biomolecular & Chemical Sciences School, University of Western Australia, Perth, Australia REPROGRAMMING PANCREATIC EXOCRINE Type 1 Diabetes is one of the major chronic diseases of childhood, and is CELLS TOWARDS BETA CELLS USING PTD- increasing in prevalence around the world. While insulin treatment can man- TRANSCRIPTION FACTORS age glucose levels, it is not a cure and is not sufficient to prevent diabetic complications in a third of subjects. Though islets can be transplanted for Lima, Maria J., Docherty, Hilary M., Docherty, Kevin therapy, the demand for donor material far outweighs availability. Currently, Institute of Medical Sciences, Aberdeen, United Kingdom stem cells are being investigated as a potential source of insulin produc- The in vitro generation of functional beta cells is currently accepted as ing beta cells for Type 1 Diabetes therapy. The efficient differentiation of one of the most promising sources of tissue for pancreatic transplantation stem cells to clinically viable beta cells is dependent on understanding the in diabetic patients. The use of protein transduction domains (PTDs) has mechanisms underlying beta cell development. We have established several proved successful in promoting the uptake of various transcription factors monoclonal antibodies that define populations of stem cells from the liver (TFs) in a range of cell lines, including stem cells and pancreatic explants. and pancreas. Here, we report the characterisation of one of these antibod- These proteins can be sequentially added to the cells, in a way that mimics ies, WAM-21, for the isolation and study of murine fetal islet progenitor the expression observed during pancreatic development. This work consisted cells. Flow cytometric analyses of e15.5 fetal mouse pancreas demonstrated on the generation and sequentially administration of key pancreatic PTD WAM-21 identifies subpopulations of cells positive for known islet progeni- containing TFs to endoderm enriched mouse embryonic stem (mES) cell tor cell surface markers, and using transgenic reporter mice, we established cultures for the differentiation of beta cells. In order to obtain a definitive that WAM-21 marks virtually all neurogenin-3 expressing islet precursor endoderm (DE) population, cells were initially cultured as embryoid bodies cells. Flow cytometric or magnetic cell separation of progenitors using and treated with Activin A and BMP-4 for 6 days. After DE formation, mES

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Thursday Poster Abstracts cells were plated as a monolayer and treated with different combinations of Poster Board Number: 1017 recombinant PDX1 and PTD-MAFA for 21 days. Both TFs were efficiently taken up into the cells nuclei. RT-qPCR studies revealed the up-regulation of THE ESTABLISHMENT OF A SCREENING beta cell genes such as Insulin 1 and 2, Pdx1, Pax4 and Glut2 when PDX1 SYSTEM FOR LOW MOLECULAR COMPOUNDS and TAT-MAFA were simultaneously added to the cultures. These differenti- ated cells were also positive for Pdx1 and Insulin, as assessed by immunocy- FOR β CELL INDUCING ACTIVITY tochemistry. It was additionally shown that the two PTD-TFs in conjunction Sakano, Daisuke, Shiraki, Nobuaki, Kataoka, Masateru, Kume, with PTD-Ngn3 and PTD-Pax4 were able to reprogramme the rat pancreatic Kazuhiko, Kume, Shoen exocrine cell line AR42J-B13 towards the beta cell phenotype. The four PTD-TFs were added simultaneously to the culture medium for 3 days. After Department of Stem Cell Biology, Institute of Molecular Embryology and the treatment, Insulin 1, Pdx1, Pax4 and NeuroD genes were up-regulated Genetics, Kumamoto University, Kumamoto, Japan and insulin was also detected at the protein level in these cells. These results The pancreatic transplantation and the pancreatic islet transplant are known demonstrate that PTD-transcription factors are able to generate insulin-pro- as effective treatments for type I diabetes. Donor shortage is an obstacle ducing cells from different cell types when supplied in a controlled manner, to the treatment of type I diabetes. The understanding of differentiation establishing them as a powerful tool for cell based therapies. mechanism from ES cell to β-cells might add to our knowledge of the Poster Board Number: 1015 mechanism of β-cell differentiation and also contribute to the development of the regenerative medicine. The purpose of this research is to establish an THE HISTONE DEMETHYLASE LSD1 IS efficientβ -cell induction system using low molecular compounds. First, we aimed to establish a feeder free culture condition, in which ES cells can be REQUIRED FOR ENDOCRINE CELL FORMATION induced into insulin producing cells in vitro. Feeder free system is useful to IN THE DEVELOPING PANCREAS study the effect of low molecular compounds. We previously reported that mouse ES cell is efficiently differentiated into pancreatic progenitor cells and Patel, Nisha A., D’Amour, Kevin, Robins, Allan, Rosenfeld, Michael yielded as high as 30% Pdx1-positive cells, when they are cultured on M15 G., Sander, Maike cells and added with growth factors (Shiraki et al., 2008). We also reported University of California, San Diego, La Jolla, CA, USA, ViaCyte, Inc., that M15 cell secretes extracellular matrices, which potentiate ES cells to San Diego, CA, USA, Howard Hughes Medical Institute, University of differentiate into Pdx1-expressing pancreatic progenitor cells. The extracellu- California, San Diego, La Jolla, CA, USA lar matrices can be reconstituted by a synthetic basement membrane (sBM) (Higuchi et al., 2010). Therefore, we speculate that culturing ES cells on a Much effort is being expended on attempts to derive transplantable insulin- suitable scaffold would potentiate the differentiation into insulin producing producing beta-cells from human embryonic stem cells (hESCs) for treating cells. In an attempt for establishing a high throughput screening assay, we diabetes mellitus. However, as current protocols still fail to support the tested the use of three-dimensional scaffolds. Here, we found that a novel generation of functional beta-cells, a better understanding of the molecular synthetic matrix as a scaffold can substitute sBM and ES cells cultured on mechanisms underlying beta-cell development will be necessary to achieve this novel synthetic matrix differentiated into insulin producing cells. To vali- this goal. The transcriptional network regulating neogenesis of beta-cells date the screening system, we tested several known drugs that potentiate and other endocrine cell types in the embryonic pancreas has been compre- insulin expression. We found that the present culture system is feasible for hensively dissected. However, chromatin remodeling events which regulate screening for drugs that potentiate the differentiation of ES cells into insulin the pancreatic transcriptional program to orchestrate proper endocrine cell expressing producing cells. development remain largely unexplored. The chromatin modifying enzyme, LSD1, has been shown to be required for lineage specification in several Poster Board Number: 1019 tissues. To examine the role of LSD1 in the developing pancreas, we utilized both mouse genetic approaches and a parallel hESC-based in vitro system of RETINOIC ACID AMELIORATES TYPE I pancreatic cell differentiation. Using the first model, we found that inactiva- DIABETES MELLITUS BY UPREGULATING tion of LSD1 in mice abrogates pancreatic endocrine cell differentiation. Ex- tending this finding, spatially-controlled inactivation of LSD1 in the develop- REGULATORY T CELLS AND BY PROMOTING ing pancreas revealed that endocrine cell development requires LSD1 activity BETA-CELL REGENERATION in multipotent, but not in endocrine-committed, pancreatic progenitor cells. Furthermore, chromatin-immunoprecipitation experiments on hESC-derived Shen, Chia-Ning, Yuan, Tze-An, Chien, Chiao-Yun, Wu, Ruei-Ren, multipotent pancreatic progenitors in our second model demonstrated that Lee, Hsuan-Shu LSD1 occupies regulatory sequences of the key transcription factors for en- Genomics Research Center, Academia Sinica, Taipei, Taiwan, Department of docrine fate commitment and differentiation. Taken together, these findings Biotechnology and Laboratory Sciences in Medicine, National Yang-Ming suggest that LSD1 governs pancreatic endocrine cell formation by regulating University, Taipei, Taiwan, Institute of Biotechnology, National Taiwan endocrine lineage-specific gene expression programs in multipotent pancre- University, Taipei, Taiwan atic progenitor cells. Type 1 diabetes mellitus (T1DM), which is a T cell-mediated autoimmune disease resulting from the selective destruction of the insulin-producing beta cells of the pancreas, affects 20 million people worldwide. Vitamin A defi- ciency was found to impair islet development in rats and hens and to be as- sociated with development of T1DM in patients. However, whether retinoic acid can be utilized to ameliorate type I diabetes mellitus remain unclear. We initially found that vitamin-A deficiency in embryogenesis affect pancreatic endocrine tissue development. Moreover, we also demonstrated a key regu- latory role of retinoic acid on pancreatic vascularisation and islet differentia- tion in a mouse model of pancreas development. To further address the question that whether retinoic acid can ameliorate type I diabetes mellitus, none-obese diabetes (NOD) mice was used in current study. The spontane- ous incidence of diabetes was 80-90% in females and 20-30% in males by

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25 weeks of age. And we found that treatment of mice with intraperitoneal and will offer opportunities for studies of hepatic differentiation and other injection of cyclophosphamide accelerated development of diabetes. In applications. contrast, treatment of diabetic mice, induced by cyclophosphamide injec- tions, with all-trans retinoic acid (ATRA) reduced the level of blood glucose. Poster Board Number: 1023 The amelioration was possibly mediated by increasing functional Treg cells DIFFERENTIAL METHYLATION PATTERNS THAT and by promoting beta-cell differentiation as the level of Foxp3 in Treg cells and the number of Ngn3-positive endocrine cell precursors were elevated. MODULATE STAGE-SPECIFIC DEVELOPMENT In summary, the current findings suggest retinoid treament exhibit beneficial OF LIVER LINEAGES effects on T1DM. And the effect of ATRA on ameliorating hyperglycemia is possibly mediated by increasing functional Treg cells and by promoting beta- Bandi, Sriram, Greally, John, Gupta, Sanjeev cell regeneration from Ngn3-positive endocrine cell precursors. Dept. of Medicine, Albert Einstein College of Medicine, Bronx, NY, USA, Dept. of Genetics, Albert Einstein College of Medicine, Bronx, NY, USA In freshly isolated human fetal epithelial cells from the liver and pancreas at LIVER CELLS 20 to 24 weeks of gestation, we observed capacity for multilineage gene expression, including epithelial, mesenchymal and stem cell genes. Under Poster Board Number: 1021 culture conditions, these fetal epithelial stem/progenitor cells underwent genetic reprogramming with regression along relatively more primitive EFFICIENT DIFFERENTIATION OF PLURIPOTENT meso-endodermal stages, which were characterized by greater expression HESC TO GENERATE HEPATOCTYES CAPABLE of mesenchymal genes, e.g., vimentin and alpha-smooth muscle actin, OF CELL THERAPY-BASED RESCUE IN ACUTE compared with the expression of epithelial genes, e.g., albumin, E-cadherin, G-6-P, glycogen, CK-19 or GGT, along with expression de novo of the regu- LIVER FAILURE latory transcription factor, FoxA2 (HNF-3). We considered that one way to Bandi, Sriram, Joseph, Brigid, Gupta, Sanjeev understand mechanisms in endodermal lineage specification and differentia- tion would be to study epigenetic gene regulatory changes in the context Dept. of Medicine, Albert Einstein College of Medicine, Bronx, NY, USA of liver development. For instance, genomic DNA methylation substantively For various applications in drug development and toxicity studies, devel- contributes to transcriptional regulation underlying mammalian develop- opment of disease models, as well as cell therapy, efficient processes to ment and/or cell differentiation. However, little is known about genome- differentiate pluripotential stem cells into hepatocytes expressing excellent wide variations in DNA methylation patterns during endodermal lineage hepatic functions is critical. To advance these goals, we studied direct- development. In this study, we used HpaII tiny fragment Enrichment by differentiation of hESC (WA-01) into hepatic endoderm without the steps of Ligation-mediated PCR (HELP) method for restriction digestion of genomic embryoid body formation, incorporation of animal feeder cells, or materials DNA followed by sequencing of fragments by the NimbleGen DNA methy- of animal origin. In preceding ontogenic studies, we established that devel- lation arrays to evaluate the extent and patterns of cytosine methylation opment of the endoderm (liver, pancreas) proceeds through unique conjoint with open-source bioinformatics tools in well-characterized freshly isolated meso-endodermal stages of committed stem/progenitor cells. This provided primary fetal, cultured primary fetal, and freshly isolated adult human liver an effective framework to demonstrate the onset and extent of endoderm cells. We found DNA methylation was regulated in these cells in a devel- differentiation. Therefore, we analyzed expression of genes characterizing opment stage-specific fashion. The pluripotency-associated genes, OCT4 stem/progenitor cells, along with genes expressed in mesenchymal and liver and Nanog, were hypermethylated in adult liver cells and hypomethylated epithelial cells. We developed novel protocols involving culture of hESC in cultured and primary fetal liver cells. By contrast, hepatic transcription under serum-free, feeder-free conditions, with medium containing complex factor and epithelial genes, HNF4, HNF1, Alb, AFP, or E-cadherin, were mixture of substances, including multiple proteins and growth factors. This hypomethylated in primary fetal and adult liver cells, consistent with hepatic rapidly induced epithelial morphology in 100% of hESC by 3d followed by functions, but became hypermethylated in cultured fetal liver cells, indicat- conversion to liver-like cells by 14d. Molecular analyses showed hESC- ing altered gene expression during lineage-specific changes. Remarkably, in derived cells acquired endoderm properties by 3-6 d, with onset of SOX17 cultured fetal liver cells, the mesenchymal gene, vimentin, was hypomethy- and FOXA2 expression and decline of pluripotency genes. By 14d, all lated, indicating regression of cells to the meso-endodermal stage, whereas hESC-derived cells showed hepatobiliary properties, i.e., E-cadherin, HNF4a, in adult and fetal liver cells, vimentin was hypermethylated. These results Alb, AFP, AAT, ASGPR, G6P, glycogen, GGT, and CK19, although mesenchy- demonstrated that the methylation pattern of various genes was dynami- mal markers, i.e., vimentin and alpha-SMA, were also coexpressed. Global cally regulated in a developmental stage-specific fashion in endodermal cells. miRNA profiling showed these hESC-derived hepatocytes diverged from un- Therefore, recognition of DNA methylation patterns and their specificity for differentiated hESC, and converged toward primary fetal human liver stem/ early and late stages of endoderm development and/or differentiation will progenitor cells, although the profiles were not identical to adult human be helpful in defining epigenetic markers for pathophysiological studies, as hepatocytes. Characterization of the extent of hepatic function in hESC- well as for obtaining potential interventions in hepatic disorders. derived cells included secretion of Alb in culture medium and urea synthesis, Poster Board Number: 1025 which compared favorably with fetal human hepatocytes. Also, hESC- derived cells expressed major drug-metablizing Cyp450 isoforms, including HUMAN ES CELLS ENGINEERED WITH A 3A4, 7A1, 1B1, 2E1, and 1A1. Next, we examined therapeutic capacity of hESC-derived cells in a NOD/SCID mouse model of drug-induced acute REPORTER IN THE CYP-3A4 GENE FOR liver failure (ALF) with 90-100% mortality over few days and regeneration HEPATOCYTE-BASED DRUG METABOLISM AND of liver after hepatic support from intraperitoneally transplanted mature hepatocytes. After inducing ALF, we transplanted hESC-derived cells i.p. TOXICITY SCREENING Compared with untreated mice, recipients of hESC-derived cells showed Bonham, Kristina, Xian, Hai-Qing, Ranade, Aarati, Blanco, Carmino, decreased encephalopathy, increased duration of survival, and improved Snodgrass, Ralph mortality, 50% v. 90% after 7d, p<0.05. Conclusions: We differentiated VistaGen Therapeutics, South San Francisco, CA, USA hESC with extremely high efficiency to generate hepatocytes recapitulating the meso-endodermal phenotype of natural fetal human liver stem/progeni- One of the leading causes of drug failures and drug recalls in the United tor cells. These hESC-derived hepatocytes possessed therapeutic potential States is drug-induced liver toxicity. These hepatotoxic effects can be caused

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Thursday Poster Abstracts either by the parent drug itself or its metabolite(s), produced during hepatic cells under our newly-developed culture conditions. One week after initiat- phase I and II metabolism. In addition, significant numbers of drug candi- ing the culture, a new type of stem cell colony appeared, with a morphol- dates are terminated during development due to the induction or repres- ogy different from the parental hESC or hiPSC. They did not form discrete sion of the hepatic phase I and II metabolizing enzymes, which can lead to borders at the edge of the colonies, and the cells were not packed at high serious drug-drug interactions and toxicity. Thus there is a significant need density. The new stem cells could be cryopreserved and passaged, and had for more predictive human systems for screening drug candidates, early in a doubling time of 5-7 days. To date, these cells have grown on feeder drug development, for liver toxicity and potential effects on liver-based drug layers for 27 generations without any phenotypic changes. In the presence metabolism. The traditional approach of drug screening in animal models is of retinoic acid and FGF7/FGF10, the morphology of the derived cell lines expensive, time consuming, and has limited predictive value due to species- evolved to round colonies with discrete edges; without any distinct density specific responses. The development of a reliable, clinically predictive, in change. The new stem cell lines did not express OCT4, NANOG or SOX2, vitro assay system that replicates human liver drug metabolism is critical for and were also negative for SOX17 and FOXA2, indicating that cell differ- developing safer drugs. Human cadaver hepatocyte-based systems offer entiation passed beyond DE with our culture conditions. Importantly, Prox1 some benefits, but have significant limitations due to availability, functional was expressed. Because Prox1 is one of the earliest markers of mammalian stability, quality, and pharmacogenomic variations. Embryonic stem cell- foregut endoderm cells, this indicated that our derived cell line was aged derived hepatocytes have the potential to address the limitations of primary at an early developmental stage of foregut endoderm with the potential human hepatocytes. to give rise to mature hepatic and pancreatic cell types. The cells express Various investigators have generated human ES-derived cells with some liver stem cell markers, EpCAM, NCAM, CK8, CK18, and CK19, but do not characteristics of hepatocytes, but these approaches have not been success- express AFP or ALB, indicating that they are hepatic cell precursors. Impor- ful in producing hepatocytes with the full range and levels of drug metabo- tantly, these lines co-express CK19 and PDX1, which are important markers lizing enzymes seen in fresh healthy adult liver cells. To address this issue, we for hepatic stem cells and pancreatic stem cells respectively. The differentia- have engineered K117, a human embryonic stem cell line, with a humanized tion potential of these stem cell lines into mature hepatic and pancreatic cell beta-lactamase (hBLA) reporter inserted in-frame in the CYP3A4 gene. We lineages is currently under investigation. These findings represent an initial believe that this CYP3A4-hBLA hES (“3A4Bla”) reporter cell line will be use- step in establishing human hepatopancreatic stem cell lines from hESC and ful for studying hepatocyte differentiation, and for hepatotoxic drug screen- hiPSC, free of totipotent progenitor cell contamination, for the purpose of ing. As a reporter, BLA has many advantages, such as a broad dynamic providing functional hepatocytes and pancreatic endocrine cells for cell- range, high level of sensitivity, suitability for multiple quantitative analytical based therapeutics, as well as for pharmacology and toxicology studies. assays, including solution-based quantitative enzymatic assays, cell-based fluorescence analysis and cell sorting. CYP3A4 is a major drug metabolizing Poster Board Number: 1029 enzyme isoform expressed preferentially in adult, versus fetal, hepatocytes, ISOLATION AND CLONOGENIC EXPANSION OF and is responsible for metabolizing a majority of drugs. In addition, CYP3A4 can be used as an important marker for mature hepatocyte function. These CD34+ STEM CELLS CAPABLE OF PRODUCING new 3A4Bla clones have normal karyotypes, are trypsin adapted, and dif- HUMAN HEPATOCELLULAR CARCINOMA ferentiate well into the three germ layers, endoderm, and hepatocytes. We will report on the use of these cell lines to optimize culture protocols that Duan, Yuyou, Saramipoor Bahbahan, Iman, Aujia, Tijess P., Zhang, enhance the maturation of the hES-derived hepatocytes leading to high lev- Guoli, Nguyen, Ngoc Tue, Lam, Alexander, Ramsamooj, Rajendra, els expression of albumin, CYP3A4, CYP1A7, and CYP1A2. We will discuss Ma, Xiaocui, Khoobyari, Shiva, Zern, Mark A. drug induction studies, and a novel 3D culture system designed to increase Internal Medicine, University of California Davis Medical Center, the maturation of hepatocytes compared with standard 2D cultures. Sacramento, CA, USA, Pathology & Laboratory Medicine, University of The CYP3A4 reporter cell line, in conjunction with improved efficient differ- California Davis Medical Center, Sacramento, CA, USA entiation protocols yielding mature human hepatocytes, will be an improve tool for optimizing hepatocyte differentiation protocols, and for in vitro CD34+ stem cells play an important role during liver development and screening of drug candidates for hepatotoxicity and potential drug-drug tumorigenesis. We hypothesized that hepatocellular carcinoma (HCC) interactions. cells might be derived from genetically mutated or epigenetically modified CD34+ stem cells. Employing flow cytometry, we found that the percent- Poster Board Number: 1027 age of CD34+ cells in PLC/PRF/5 hepatoma cell line (Alexander cells) was 5-10 fold higher when compared to six other hepatoma cell lines (Hep G2, DERIVATION OF HEPATOPANCREATIC STEM/ Hep 3B, C3A, Huh7, HLE and HLF). CD34+ cells sorted from Alexander PROGENITOR CELL LINES FROM HESC AND cells were placed on mouse fibroblast feeder cells under our derived culture HIPSC condition, after 7 to 10 days in culture, 2-3 different types of stem cell colo- nies were seen: the morphology of these colonies was round and packed Duan, Yuyou, Ahuja, Tijess P., Ma, Xiaocui, Saramipoor Behbahan, with high density. These CD34+ stem cell populations can be cryopreserved Iman, Nguyen, Ngoc Tue, Zern, Mark A. and passaged. Putative cancer stem cell markers such as CD90, EpCAM, University of California Davis Medical Center, Sacramento, CA, USA CD44, CD133, and liver stem cell markers like CK8, CK18, CK19, NCAM and OV6, were expressed by some of the CD34+ populations. By moving Tumorogenisity is still one of the main obstacles in the clinical application of the CD34+ cells to feeder free conditions, CD31+ cells could be enriched to human embryonic stem cells (hESC) and human induced pluripotent stem 60% after treatment with cisplatin, indicating drug resistance. After injecting cells (hiPSC). A major question is how to determine whether a differentiated 30,000 of cultured CD34+ stem cell populations, human liver carcinomas cell population derived from hESC or hiPSC is free from the presence of any were formed in both nude mice and NOD/SCID mice. These tumors showed early progenitor cells, and thus is safe to be used? Our aim is to establish HCC pathological and histologic features. By using 15 surface markers, we hepatopancreatic stem cell lines from hESC and hiPSC. If such a cell line can found that the tumor cells produced by CD34+ populations showed a high be established, it would be a relatively safe and readily employed cell source percentage of CD31+, and relatively homogenous populations for CD54+, for both pancreatic endocrine cell and hepatocytes. By mimicking liver and CD13+, and EpCAM+, and also elevated percentage for CD44+, CD133+ pancreatic development, we established culture conditions to induce defini- (designated type 1 tumor cells), while the tumor cells produced by the pa- tive endoderm (DE) from hESC and hiPSC. We then isolated specific cells at rental Alexander cells showed relatively homogenous populations of CD54+, the late stage of DE induction by sorting using two specific surface markers, CD13+, and a elevated percentage for CD31+, CD44+, CD133+, and EpCAM and NCAM. Sorted cells were placed on mouse fibroblast feeder EpCAM+ (designated type 2 tumor cells). The type 1 tumor cells grow as

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colonies, and the type 2 tumor cells proliferate and spread like their parental Poster Board Number: 1033 Alexander cells. The tumor cells from 2nd and 3rd generation tumors by series transplantation showed the same phenotype and colony-like prolifera- THE PLOIDY-CONVEYOR OF MATURE HUMAN tion as their parent tumor cells from 1st generation tumors produced by HEPATOCYTES AS A SOURCE OF EXTENSIVE CD34+ populations. The percentage of CD31+ and EpCAM+ cells decreased in culture over time. CD31 and EpCAM were not detected by immunochem- GENETIC DIVERSITY istry (IHC) in parental Alexander cells. Alpha 1-antitrypsin (alpha 1-AT) was Duncan, Andrew W., Strom, Stephen C., Grompe, Markus expressed in a high percentage of parental Alexander cells; however, only a Oregon Stem Cell Center, Papé Family Pediatric Research Institute, Oregon few cells were positive for albumin (ALB) and alpha -fetoprotein (AFP). The Health & Science University, Portland, OR, USA, Department of Pathology, type 2 tumor cells produced by parental Alexander cells are mixture, and the University of Pittsburgh, Pittsburgh, PA, USA hepatic gene expression pattern of these tumor cells was the same as the pattern in parental Alexander cells. Alpha 1-AT was expressed in almost all Hepatocytes are characterized by the dynamic ability to change ploidy colonies of type 1 tumor cells. Type 1 tumor cells with a relatively homog- during proliferation. The most highly described change is polyploidization, enous populations for alpha 1-AT+ and EpCAM+ were only generated by which has been shown in humans and rodents. The mechanism of poly- enriched CD34+ populations. We further found that all colonies of type 1 ploidization involves failed cytokinesis. Polyploid hepatocytes are either tumor cells strongly co-express alpha 1-AT, ALB, and AFP as relatively ho- mononucleated or binucleated, and they have 4n, 8n, 16n or higher nuclear mogenous population, indicating a hepatic phenotype. This is the first report content. Recently, we described additional ploidy changes by murine hepa- that a human HCC appears to be initiated and developed from CD34+ stem tocytes. Polyploid hepatocytes produce daughter cells with numerical aneu- cells, thus revealing a diversity of origins for human HCC. Moreover, the ploidy and/or reduced ploidy during proliferation. A common mechanism is in vitro culture of CSC provides an opportunity for analysis in the aspects responsible for both aneuploidy and ploidy reversal. Multipolar spindles are of genomics, epigenomics, proteomics, metabolomics among CSC, normal established by polyploid hepatocytes early in mitosis. In most divisions, tran- stem cells and primary cells to explore the mechanism of tumorigenesis and sient multipolar spindles are reoriented in a bipolar configuration, leading to cancer development. emergence of two daughter cells. Chromosome imbalances arise by failure of chromosomes to segregate completely to each pole. In approximately 5% Poster Board Number: 1031 of mitoses, multipolar spindles are maintained, leading to multipolar divi- HUMAN ADULT HEPATIC PROGENITOR CELLS, sions that generate three or four daughters. These reduced-ploidy daughter hepatocytes function normally. Collectively, the data support a dynamic BONE MORPHOGENIC PROTEIN AND GREMLIN model of hepatocyte polyploidization, ploidy reversal and aneuploidy, a IN THE PATHOGENESIS OF HEPATOCELLULAR phenomenon called the “ploidy-conveyor.” To determine whether the ploidy-conveyor is active in humans, we charac- CARCINOMA ON TOP OF CHRONIC HCV terized aneuploidy and mitotic structures in the human liver. First, normal INFECTION human hepatocytes isolated from surgical resections or cadaveric donors were karyotyped using standard G-banding techniques. Chromosomal gains Baddour, Nahed, ElKaffash, Dalal, Gumei, Maha, Taher, Yousry1, and losses were seen in 10-50% of metaphases analyzed. Secondly, because Abdou, Laila1 karyotyping selects for cells that proliferate the best, we also determined Pathology, University of Alexandria, Alexandria, Egypt, Clinical Pathology, chromosome copy number using a non-biased approach. Hepatocytes from University of Alexandria, Alexandria, Egypt male and female donors, ranging from 2-72 years old, were analyzed by Background: The role of hepatic progenitor cells (HPCs) HCC on top of fluorescence in situ hybridization (FISH). Samples were hybridized with chronic HCV is questionable. Our working hypothesis was that Grem- clinical-grade point probes corresponding to chromosomes 1/9/16 and lin which inhibits Bone Morphogenic protein-7 (BMP-7), is secreted by 13/18/21/X/Y. Consistent with the karyotypes, quantification of FISH sig- fibroblasts, will block BMP-7 differentiating effect on stem cells, arrest their nals in interphase nuclei revealed chromosomal gains and losses in hepato- maturation and promote proliferation in an immature state . Patients and cytes from donors of all ages. Finally, to survey the diversity of cell divisions Methods: Immunohistochemical expression of CK19, and FGF 2, and m RNA by human hepatocytes, mitotic structures were visualized in primary human expression of BMP-7 and Gremlin were studied in chronic HCV, cirrhosis and hepatocytes following in vitro expansion. Mitotic cells established multipolar HCC patients. Results : CK 19 positivity was higher in cirrhotics (0.009) and spindles in prometaphase/metaphase, and lagging chromosomes were seen correlated with grade r=0.64, p-0.009 in group I , r=0.75 , p=0.001 in group in anaphase/telophase. In summary, the ploidy-conveyor is active in humans II, and stage ( r=0.71, p=0.001). No CK19 positivity was observed in HCC -- human hepatocytes polyploidize, establish multipolar mitotic spindles and patients . Ductular reaction was higher in cirrhotics ( p=0.001). It correlated become aneuploid. We propose that the ploidy-conveyor is a fundamental with CK 19 positivity ( r=0.42, p=0.012), grade ( r=0.56,p=0.024) and stage characteristic of hepatocytes. Thus, ploidy changes and aneuploidy should (0.66, p=0.006) . FGF 2 expression increased in HCCs . FGF-2 correlated be considered in approaches to differentiate hepatocyte-like cells from pluri- with grade (r=0.6,p=0.013 ), stage ( 0.72, p=0.002), CK 19 (r=0.71, p= potent progenitor populations. .002) and ductular reaction (0.68,p=0.004). BMP7 was expressed in hepa- titic, cirrhotic and cancerous tissues. BMP-7 was higher in cirrhotics. It cor- related with ductular reaction (r=0.576, p=0.031) and negatively with CK19 (r=0.6, p=0.023). A (p= 0.009) higher number of cirrhotics and HCC cases showed positive Gremlin expression, which correlated with the stage (r=0.7, p=0.002). A positive correlation was noted between BMP-7 and gremlin mRNA expression (r=0.77, p=0.045). Thus, a negative feedback loop exists which controls BMP 7 expression. Gremlin expression correlated with CK19 , FGF2 positivity and steatosis in hepatitics (r=0.75 ,p=0.001). Conclusion: CK 19 is not a valid marker of stem cell activation in HCC on top of chronic HCV. Cirrhosis promotes carcinogenesis by fibroblast secreted gremlin block- ing BMP function and promoting stem cell activation and proliferation which may contribute to carcinogenesis in this setting.

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Poster Board Number: 1035 made available in different multi-well formats. We have studied the usability of hPSC-derived hepatocyte-like cells as a test system for use in various in DISEASE-CORRECTED HEPATOCYTES FROM vitro applications including toxicity testing of chemical compounds. We have HUMAN FAMILIAL HYPERCHOLESTEROLEMIA shown how global gene expression data from such studies could be used to identify toxic compounds, and moreover, how the mechanism of toxicity INDUCED PLURIPOTENT STEM CELLS can be unrevealed. Data also indicates that our hPSC-based models can Fattahi, Faranak, Asgari, Samira, Seifinejad, Ali, Baharvand, Hossein, give information about how liver-related toxicity pathways will be affected Hosseini Salekdeh, Ghasem by different (hepato) toxic agents. In conclusion, we have shown important aspects of routine mass production of hPSC-derived hepatocyte-like cells, Department of Molecular Systems Biology, Royan Institute for Stem and how these cells can be used in industrial applications and substance Cell Biology and Technology, ACECR,, Tehran, Iran, Islamic Republic of, screening. Department of Stem Cell and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, ACECR,, Tehran, Iran, Islamic Republic Poster Board Number: 1039 of, Department of Stem Cell and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, ACECR,, Tehran, Iran, Islamic MICROENVIRONMENT CHANGES BUT NOT Republic of DIRECT DIFFERENTIATION ASSOCIATE WITH The generation of induced pluripotent stem cells from an individual patient THE PREVENTION OF NON-ALCOHOLIC provides a unique tool for disease modeling, drug discovery and cell replace- STEATOHEPATITIS ONSET OBSERVED AFTER ment therapies. The patient specific pluripotent stem cells can be expanded in vitro and are thus suitable for genetic manipulations. To date several SYSTEMIC ADMINISTRATION OF MULTIPOTENT diseases have been modeled using patient-specific iPSCs and it has been MESENCHYMAL STROMAL CELLS INTO OBESE shown that genetic manipulation of somatic cells prior to their reprogram- ming can correct the disease phenotype in the iPSCs-derived differentiated MICE WITH METABOLIC SYNDROME. cells. Here, we present the generation of human familial hypercholester- Ezquer, Marcelo, Ezquer, Fernando, Simon, Valeska, Conget, olemia iPSCs and we show that genetically manipulated iPSCs can give rise Paulette to functional hepatocytes. The iPSCs were derived from the dermal fibro- Instituto de Ciencias, Facultad de Medicina, Clínica Alemana-Universidad blasts of a patient, homozygous for a mutation in the LDL receptor gene. del Desarrollo, Santiago, Chile The mutated gene encodes a truncated non-functional receptor and the hepatocytes differentiated from the defective iPS cells do not have the abil- The hepatic manifestation of metabolic syndrome is non-alcoholic fatty ity to uptake labeled LDL particles. In order to deliver the normal cDNA to liver disease, which progression-limiting step is non-alcoholic steatohepa- the defective cells, a lentiviral vector carrying the normal LDL receptor ORF titis (NASH). The clinical relevance of this condition is due to the high and was used to transduce the iPSCs. The transformed cells were expanded and increasing number of obese patients and the potential of NASH to evolve differentiated towards hepatocytes and LDL uptake assays showed their LDL towards end-stage liver diseases. Recently, we demonstrated that bone mar- uptake ability and the correction of disease phenotype. The functionality of row derived multipotent mesenchymal stromal cells (MSC) systemically ad- differentiated cells was also determined by ICG uptake assay, PAS staining, ministered into obese mice with metabolic syndrome, prevent NASH onset. inducible cyp450 activity and oil red staining. These data prove that iPS cell Here we describe the cellular and molecular mechanisms associated to this technology can be used for the generation of disease-corrected, patient- therapeutic effect. To assess whether systemically administered MSC home specific cells with potential value for cell therapy applications. into the liver, donor cells were isolated from isogenic mice that express GFP (MSCGFP) and intravenously administered into C57BL6/J mice. Seventeen Poster Board Number: 1037 weeks later, we found, by flow cytometry and immunohistofluorescence, ROUTINE PRODUCTION AND APPLICATIONS OF donor cells in the liver of obese mice with metabolic syndrome but not in normal mice. To determine whether donor cells differentiate into hepatic HUMAN PLURIPOTENT STEM CELL-DERIVED cells, liver sections obtained from obese mice with metabolic syndrome HEPATOCYTE-LIKE CELLS transplanted with MSCGFP were co-stained with anti-GFP, and anti-albumin or anti-MIC1/OC2, or anti-F4/80. We observed no donor cells express- Jensen, Janne, Brolén, Gabriella, Eriksson, Gustav, Synnergren, Jane, ing markers of hepatocyte, oval or Kupffer cells. To evaluate whether MSC Björquist, Petter administration modify the inflammatory process observed during NASH Cellartis AB, Gothenburg, Sweden onset, liver samples obtained from normal and obese mice with metabolic syndrome transplanted or not with MSC were analyzed by qRT-PCR. In the Human pluripotent stem cells (hPSC) hold an enormous promise for an liver of obese mice the expression of pro-inflammatory markers (IL1beta, unlimited source of specific cell types to be used as cell therapy in regen- IL18, TNFalpha, MCP1, LFA1 and ICAM1) was higher than in normal mice. erative medicine. However, currently the most intense interest for hPSC is At day 7 after MSC administration, most of these factors were expressed within direct industrial applications such as use as tools in drug discovery. at basal levels. Moreover, in MSC-transplanted mice, the expression of Cell based in vitro assays with high human relevance are urgently needed anti-inflammatory markers (IL4, IL5, IL10, and IL12p40) was higher than in for pre-clinical activities, spanning from target identification and validation, obese mice. To evaluate whether MSC administration modify the expres- screening of compound efficacy, to drug metabolism and safety assessment sion of trophic (IGF1, HGF, bFGF, SDF1) and vasculogenic (VEGF and ANG) studies. Hepatocytes are considered to be one of the most important cell factors, liver samples obtained from normal and obese mice with metabolic types for these processes. Additionally, sourcing of human hepatocytes will syndrome transplanted or not with MSC were analyzed by qRT-PCR. In the be of great interest for extracorporal liver devices and other regenerative liver of obese mice, the expression of trophic and vasculogenic factors was medicine applications. We have differentiated hESC and iPSC into hepato- lower than in normal mice. At day 7 after MSC-administration, the level of cyte-like cells by using a four-step differentiation protocol guiding the cells expression of these factors was restored. Although donor cells were found through discrete stages recapitulating liver development. The resulting cells in the liver of treated mice, they scarcity and failure to express hepatic cell morphologically closely resemble human hepatocytes and express hepatic markers suggest that MSC systemic administration prevents NASH onset not markers on mRNA and protein levels. Additionally, the cells show hepatic by functional complementation from direct differentiation to parenchyma. functionality like albumin and urea production and CYP activity. The hPSC- Nevertheless, it seems to be the consequence of microenvironment changes, derived hepatocyte-like cells are routinely produced in large quantities and

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which preclude the immune system-mediated destruction and promote the the therapeutic application of MSCs. survival and replacement of liver cells. Supported by FONDECYT 11085034 to M.E. Poster Board Number: 1043 Poster Board Number: 1041 MOLECULAR ANALYSIS OF THE ROLE OF CASPASE-8 IN ACTIVATION AND IN VIVO INTRAHEPATIC TRANSPLANT OF ALLOGENEIC SELECTION OF THE HEPATIC STEM CELL MOUSE LIVER DERIVED-MESENCHYMAL COMPARTMENT AND LIVER REGENERATION STEM CELLS PRODUCING HUMAN FACTOR IX CORRECTS THE HEMOPHILIA B PHENOTYPE Chaudhary, Kunal, Liedtke, Christian, Trautwien, Christian, Streetz, Konrad Fernandes, Andrielle C., Souza, Lucas E B, Fontes, Aparecida M., Dept. of Internal Medicine III, University Hospital Aachen, Aachen, Almeida, Danilo C., Neto, Mário S A, Melo, Fernanda U F, Covas, Germany Dimas T. Background: When the ability of hepatocytes to participate in the liver repair Department of Clinical Medicine, Faculty of Medicine of Ribeirão Preto, process is impaired due to chronic and acute live injury, caused by drugs, University of São Paulo, Institute of Science and Technology in Stem Cell toxins or viruses, a subpopulation of liver cells, termed hepatic stem cells Therapy - Brazil, Ribeirão Preto, Brazil (oval cells or liver progenitor cells (LPCs)), is induced to participate in the liv- Hemophilia B is an inherited X-linked disease and consists of the deficiency er regeneration process. Materials and Methods: We aim to investigate the of blood coagulation factor IX (FIX). The liver is the major site of protein FIX role of the Caspase8 pathway in oval cell response, by treating conditional synthesis and an alternative source for obtaining mesenchymal stem cells hepatocytes specific Caspase8 knockout (Casp8Δhepa) mice and wild type (MSCs). It has been postulated that MSCs are a promising target for delivery (WT) mice with DDC (3,5-diethocarbonyl-1,4-dihydrocollidine). This causes therapeutic proteins, being considered immunoprivileged and can engraft chronic sclerosing cholangitis and triggers the activation of the LPC compart- in allogeneic recipients with intact immune system. Thus, we tested the hy- ment. LPCs were isolated and characterized by flow cytometry (FACS) and pothesis that allogeneic MSCs derived from the liver (L-MSCs) and geneti- real time PCR. Liver tissue was further analysed by IHC. To further inves- cally modified to express recombinant human FIX (hFIX) correct the pheno- tigate the capacitiy to proliferate, differentiate and repopulate chronically type of hemophilia B mice. For this, L-MSCs were isolated from the liver of injured liver by LPCs, WT mice and conditional hepatocytes specific NEMO mice FVB/GFP and co-transduced to express hFIX and Luciferase as reporter knockout (NEMOΔhepa) mice were lethally irradiated and treated with ret- gene. Hence, L-MSCs/hFIX/Luc cells were characterized and intrahepati- rosine followed by intra-spleenic GFP positive LPCs transplantation for 2 and cally infused in hemophilia B mice (B6.129P2-F9tm1Dws/J) undergoing 4 weeks. Results: Higher transaminases and bilirubin levels were observed acute liver injury via carbon tetrachloride (0.265 g/Kg), and the correction of in WT mice compared to Casp8Δhepa mice 4 weeks after DDC-treatment. hemophilia phenotype was assessed. Finally, L-MSCs/hFIX/Luc in matrigel Additionally, histological analysis revealed reduced liver injury and immune- were implanted subcutaneously into two groups: syngeneic (FVB/GFP) and cell infiltration in Casp8Δhepa mice. TUNEL assay showed a significantly allogeneic (hemophilia B) models for cellular rejection analysis. The results increased apoptosis-rate during DDC-treatment in WT animals as compared showed that L-MSCs maintained fibroblastoid morphology, immunophe- to Casp8Δhepa animals. Correlating with enhanced liver injury in WT-mice notypic profile similar to murine MSCs and normal karyotype after genetic these animals also displayed stronger proliferation in periportal areas- where modification in vitro. In addition, L-MSCs/hFIX/Luc showed no tumorigenic LPCs emerge and reside (3-fold higher BrdU incorporation rate as compared potential in vivo. When cultured in defined medium, the L-MSCs/hFIX/ to Casp8Δhepa animals), while hepatocyte proliferation was reduced. We Luc cells were able to differentiate into adipocytes and osteocytes. After now aimed to analyse, whether the observed differences in liver apoptosis treatment with vitamin K, L-MSCs/hFIX/Luc secreted 80 mUI/106/24h of and proliferation was related to the degree of LPC induction to compensate hFIX biologically active. The allogeneic transplant demonstrated a strong the inability of hepatocytes to regenerate adequately. Quantitative analysis positive correlation (r = 0.84) between the bioluminescence emitted and the of LPCs by flow cytometry with sca-1, c-kit, OC-1, OC-2 and OC-3 oval cell biological activity of hFIX. Forty-eight hours after the transplantation the markers as well as different immune-histochemical (CK-19, OC-1) markers cell arrest in liver region (1.2 x 107 ± 1.1 x 107 photons/sec) and consider- unraveled a significantly stronger (3 to 4-fold increase) induction rate of able plasma levels of hFIX (0.45 ± 5.8 UI/mL) were observed. On the 4th progenitor cells in WT mice. CK-19, DMPT1, CD133 mRNA expression ki- day it was detected a significant increase of 79% of the bioluminescence netics were enhanced in WT mice as well. Transplantation and repopulation (1.7 x 107 ± 1.2 x 107 photons/sec) and 64% of the level of hFIX (0.65 ± studies of NEMOΔhepa and WT mice liver showed successful engraftment 16.8 UI/mL) compared to hemophilia B mice control (0.1 ± 0.45 IU/mL), of transplanted GFP positive hepatic stem cells in portal and periportal area indicating proliferation of transplanted cells. On the 10th and 15th day there as evidenced by GFP staining after 2 and 4 weeks of transplantation. Most was a sudden decrease of bioluminescence (10th day: 7.2 x 104 ± 3.8 x interestingly the number of GFP positive LPCs was higher in NEMOΔhepa 104; 15th day: 1.4 x 104 ± 8.6 x 103 photons/sec) and of hFIX (10th day: mice as compared to WT mice. Conclusion: Our results implicate that death 0.12 ± 1.1; 15th day: 0.5 ± 2 IU/mL), an indicative of cell death. Even with receptor-mediated hepatocyte apoptosis during chronic liver injury signifi- the cell loss, the biological activity of hFIX in transplanted mice was 5 times cantly contributes to the activation of the hepatic stem cell compartment. higher than in control mice. Regarding the experiment of cell implantation, Caspase8 in hepatocytes seems to prevent from liver injury, thus lowering the bioluminescence decreased 44% in the syngeneic model, whereas in the this otherwise important regenerative response. The exact contribution of allogeneic model the signal was not detected. Furthermore, the proportion caspase8 to the proliferation and gene regulation of LPC itself is currently of infiltrated T lymphocytes was higher in allogeneic [CD4 (2.4 ± 0.8%) investigated using isolated hepatic stem cells. We also conclude that chroni- and CD8 (5.3 ± 1.5%)] implants compared to their syngeneic counterparts cally liver damaged NEMOΔhepa mice may serve as a novel suitable model [CD4 (0.52 ± 0.1%) and CD8 (2 ± 0.7%)]. Thus, our results indicate that it to accept LPC donor cell grafts to study their in vivo repopulation potential. is possible to isolate MSCs from mouse liver, genetically modify them with viral vectors and detect significant levels of protein hFIX in vitro and in vivo. These data suggest a temporary phenotypic correction of murine hemophilia B by transplantation of allogeneic MSCs producing hFIX, indicating that MSCs are important carriers of recombinant proteins with therapeutic poten- tial. In addition, this work suggests the importance of histocompatibility for

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Thursday Poster Abstracts

Poster Board Number: 1045 Poster Board Number: 1047 NITRIC OXIDE ACTS VIA TWO INDEPENDENT KERATIN 7 AND PROMININ-1 EXPRESSION MECHANISMS TO REGULATE LIVER IS ASSOCIATED WITH LIVER DAMAGE AND DEVELOPMENT AND REGENERATION IN PREDICTS SHORT-TERM MORTALITY IN ZEBRAFISH PATIENTS WITH ALCOHOL HEPATITIS Cox, Andrew G., Kelsey, Peter, Harris, James M., North, Trista E., Sancho Bru, Pau, Altamirano, José, Rodrigo-Torres, Daniel, Bataller, Goessling, Wolfram Ramon Brigham & Women’s Hospital, Boston, MA, USA, Beth Israel Deaconess Liver Unit, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Medical Center, Boston, MA, USA Sunyer (IDIBAPS), Centre Esther Koplowitz, CIBERehd, Barcelona, Spain The liver plays a central role in lipid and protein homeostasis and produc- Background and aims. Alcoholic Hepatitis (AH) is a severe condition that tion of critical plasma proteins. Liver toxicity due to acetaminophen (Tylenol, may occur as a complication of alcohol liver disease. AH is characterized by a APAP) is the most common cause of acute liver failure in the U.S., resulting strong inflammatory reaction and reduced proliferative capacity of hepato- in liver transplantation and death. Identification of modulators of embryonic cytes. A ductular reaction has been associated to alcohol liver disease. How- liver development may help to elucidate conserved pathways that affect ever, little is known about the extent of liver progenitor cell proliferation and liver regeneration after toxic injury in the adult. In order to discover novel the clinical significance of the ductular reaction in AH. The aim of this study factors that regulate liver specification and differentiation, we screened a was to investigate the progenitor cell proliferation and ductular reaction in library of known bioactive compounds for effects on liver size in zebrafish. AH, and its correlation with severity of liver damage and short-term mortal- Chemicals affecting nitric oxide (NO) signaling modulated liver develop- ity. Methods. 59 patients with clinical and histological diagnosis of AH were ment, as assessed by in vivo fluorescence microscopy of reporter fish at 72 included in the study. The expression of EpCAM and Prominin-1, markers of hours post fertilization (hpf) and in situ hybridization for the hepatocyte- progenitor cells, and KRT7, a marker of ductular reaction, were assessed by specific marker liver fatty acid binding protein (lfabp). Embryos treated with qPCR and immunohistochemistry. Univariate and multivariate logistic regres- NO donors such as L-Arginine (24-72 hpf) exhibited a significant increase sion analysis was performed to investigate the association between progeni- in liver size, whereas embryos exposed to the NO synthase (NOS) inhibitor tor gene expression and 90-day mortality. Classification and regression L-NAME (24-72 hpf) had smaller livers; NO activity was confirmed in vivo trees (CART) analysis was used to identify the interaction of those genes using a novel fluorescent reporter. To identify the critical developmental time associated with mortality. Results. The expression of EpCAM, Prominin-1 period and target cell population of NO activity, time-course studies were and KRT7 were significantly increased in patients with AH compared to nor- performed which revealed that NO exerted its most prominent effects on mal liver (p≤0,01), virus C hepatitis (p≤0,01), and virus C-induced cirrhosis modulating lfabp+ liver size when embryos were treated during the phase (p≤0,01). There was a positive correlation between KRT7 expression and of liver specification (24-48 hpf) as compared to embryos exposed during liver function as assessed by MELD score (r=0.41, p =0.006). Moreover, the the outgrowth stage of liver development (48-72 hpf). The endodermal expression of KRT7 (HR1.14, p=0.004) and Prominin-1 (HR1.14, p=0.002), progenitor marker foxA3 and the hepatoblast markers hhex and prox1 were but not EpCAM (HR1.16, p=0.06), were identified as independent predictors upregulated by NO, confirming an effect of NO modulation at the level of 90-day mortality. The interaction analysis by CART generated a simple of the hepatoblast. FACS analysis revealed increased hepatocyte number algorithm based-on the combination of KRT7 and EpCAM gene expression after NO exposure, and histological evaluation demonstrated increased cell that stratified three groups of patients with different short-term mortality: a proliferation in the liver. Other endodermal organs, such as the intestine and high risk group with 89% mortality, an intermediate risk group with 33% of pancreas, were not significantly affected by NO modulation during this de- mortality, and a low risk group with 6% of mortality. The tree generated had velopmental time window. Knockdown of zebrafish nos1 (neuronal (nnos)/ a good predictive capability (AUROC 0.84). Discussion. This study suggest endothelial (enos)), but not nos2 (inducible (inos)), caused a significant that ductular reaction is an important feature of AH, characterized by the decrease in liver size, indicating that NO derived from adjacent and innervat- induction of progenitor gene expression. Liver progenitor cell markers posi- ing endothelium impacts liver maturation. NO can modulate cGMP activity tively correlate with severity of liver disease and are associated with short- as well as protein nitrosylation: embryos exposed to the soluble guanylate term mortality in AH patients. These data suggest that during an episode of cyclase (sGC) inhibitor ODQ (24-72 hpf) exhibited a significant decrease in AH, progenitor cell proliferation may be not sufficient to regenerate the liver liver size, suggesting that the effects of NO are mediated in part by activa- parenchyma. tion of sGC and resultant cGMP production to affect vascular tone. In addi- tion, increased global protein nitrosylation by knockdown of the metaboliz- ing enzyme GSNOR resulted in increased liver size. Both hepatic progenitor cells and the vascular endothelium contribute to organ repair after massive toxic injury, but the effect of NO on liver regeneration is controversial. NO donors (L-Arg) improved liver histology and survival in embryos and adult zebrafish after APAP exposure, even when administered with significant delay (18 hrs). The NO inhibitor L-NAME conversely increased hepatocyte necrosis and death. These results suggest that NO can modulate liver forma- tion and regeneration via two independent mechanisms: NO-induced cGMP likely increases vascular diameter and sinusoidal blood flow, while protein nitrosylation may directly affect hepatoblast function. Our work will lead to novel therapeutic approaches to improve regeneration after toxic liver injury.

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Thursday Poster Abstracts

confluence, the BEGM media was removed and cells were incubated for 16 LUNG CELLS hours with either PneumaCultTM-ALI or B-ALI Growth Medium (Lonza) as Poster Board Number: 1049 a control. Following this incubation, cells were harvested and seeded onto collagen-1-coated porous tissue culture inserts in either control differen- PEDIGREE TRACKING REVEALS REGIOSPECIFIC tiation medium or PneumaCult™-ALI in submerged cultures. Cells were maintained in submerged culture until completely confluent then differenti- STEM CELLS OF THE HUMAN AIRWAYS ated at the ALI for 21 days by replacing the basal compartment media every Hu, Yuanyu, Yamamoto, Yusuke, Neo, Boon Hoe, Sun, Yan, 48h. Trans-epithelial resistance (TER) was measured weekly post-ALI, and Kumar, Pooja Ashok, Tay, Seok Wei, Lim, Siew Joo, Daghers, Rania, at each time-point representative wells from each condition were separately Zielonka, Elisabeth, Wang, De Yun, Crum, Christopher P., McKeon, processed for sectioning or lysed for protein extraction. Cross-sections were stained using hematoxylin & eosin (H&E) to show general morphology and Frank, Xian, Wa periodic acid-schiff (PAS) to demonstrate mucous production. Western blot Stem cell and developmental biology, Genome Institute of Singapore, analysis revealed expression of Muc5AC, cytokeratin 18, E-cadherin, ZO-1, Singapore, Singapore, Dept. of Cell biology, Harvard Medical School, and β-casein. In separate experiments, ALI cultures grown in PneumaCult™- Boston, MA, USA, Institute of Medical Biology, Singapore, Singapore, ALI were treated with IL-13 (10 ng/ml) for 14 days to examine mucous Institut de Science et d’Ingenierie Supramoleculaires, University of hyperplasia by PAS and Western Blot. Results: ALI cultures grown in Strasbourg, Strasbourg, France, Department of Otolaryngology, Yong Loo PneumaCult™-ALI demonstrated TER values that were consistently higher Lin School of Medicine , National University of Singapore, Singapore, than inserts alone. TER values were stable for at least 28 days post-ALI Singapore, Department of Pathology, Brighan and Women’s Hospital, induction. In contrast to control conditions, H&E and PAS staining from Boston, MA, USA cultures grown in PneumaCult™-ALI show improved stratification and mu- Adult stem cells function in the repair and remodeling of airway epithelium cociliary differentiation at 14 and 21 days. Protein expression data confirm in chronic respiratory diseases, and yet major questions remain for their mo- that our formulation triggers increased expression of mucous cell (muc5AC) lecular diversity, commitment status, and lineage potential. To address these and ciliated cell (CK-18) markers compared to control. No difference was questions germane to adult stem cells in general, we cloned human airway observed in the expression of E-cadherin or ZO-1. Treatment of cultures stem cells from single cells and performed a pedigree-defined analysis of grown in PneumaCult™-ALI with IL-13 resulted in mucous hyperplasia as their lineage commitment, developmental potential, and gene expression observed by PAS staining and increased expression of Muc5AC. Conclusions: profiles. These studies reveal three regio-specific stem cell types united by a We have developed a novel defined media formulation, PneumaCult™-ALI, common, p63-expressing basal phenotype and yet distinguished by expres- that provides consistent, rapid, and improved mucociliary differentiation sion profiles. Stem cell pedigrees from the distal airways show a remarkable of primary HBECs in ALI cultures compared to a traditional formulation. ability in vitro to form either alveoli-like structures or bronchiolar epithelium, Cultures differentiated using our formulation show improved morphological while those of proximal airways beget upper airway cell types or squamous characteristics that are similar to that observed in the normal human airway. metaplasia. Pedigree tracking of single cell-derived clones permits the Furthermore, we demonstrate that PneumaCult™-ALI cultures can elicit decoding of adult stem cell repertoires to answer basic questions of lineage physiological responses to known stimuli, making our formulation a robust commitment, developmental plasticity, and their specific roles in repair and tool for in vitro lung epithelial cell research. disease. Poster Board Number: 1053 Poster Board Number: 1051 CANCER STEM CELLS IN A MOUSE MODEL OF PNEUMACULT™-ALI: AN IMPROVED MEDIA LUNG ADENOCARCINOMA FORMULATION FOR THE DIFFERENTIATION Belloni, Elena, Marino, Elena, Gerbino, Elvira, Fusar, Fulvia, OF PRIMARY HUMAN BRONCHIAL EPITHELIAL Stendardo, Massimo, Meani, Natalia, Mazzarol, Giovanni, Barbacid, CELLS AT THE AIR-LIQUID INTERFACE Mariano, Veronesi, Giulia, Di Fiore, Pier Paolo, Pelicci, Pier Giuseppe European Institute of Oncology and IFOM-IEO Campus, Milan, Italy, Riedel, Michael J., Wadsworth, Samuel, Eskandar Afshari, Andrea, CNIO, Madrid, Spain, Division of Thoracic Surgery, European Institute of Dorscheid, Delbert, Thomas, Terry E., Eaves, Allen C., Louis, Sharon Oncology, Milan, Italy A. The role of cancer stem cells is thought to be relevant for disease initia- STEMCELL Technologies, Inc., Vancouver, BC, Canada, UBC James Hogg tion, development, and propagation. Lung cancer is the leading cause of Research Centre, Providence Heart + Lung Institute, St. Paul’s Hospital, cancer deaths worldwide and there is great interest in identifying those Vancouver, BC, Canada, STEMCELL Technologies, Inc. and Terry Fox lung cells, which presumably are the target of genetic transformation (lung Laboratory, BC Cancer Agency, Vancouver, BC, Canada cancer initiating cells or lung cancer stem cells, LCSCs). In the normal lung Rationale: The human airway epithelium is a complex and highly differenti- tissue, the definition of lung stem cells is still a matter of debate. It has ated structure containing at least eight distinct specialized cell types. Differ- been established that at the junction between bronchioli and alveoli, there entiation of primary human bronchial epithelial cells (HBECs) to a physiologi- are cells showing stem properties, which have been indicated as Bronchio- cally relevant mucocilitated phenotype can only be achieved using air-liquid Alveolar Stem Cells (or BASCs). These have been defined as double-positive interface (ALI) culture methods with specialized media formulations. Current for both CCSP (Clara Cell Secretory Protein, specifically expressed by Clara ALI culture methods for HBEC differentiation often show poor stratification cells in the bronchiolar epithelium) and SP-C (Surfactant Protein C, specifi- and mucociliary differentiation and thus do not mimic the in vivo human cally expressed by type II pneumocytes, in the alveoli). Such cells have been airway. We have developed a defined, serum-free medium (referred to here also described to increase in number, when adenocarcinoma formation is as PneumaCult™-ALI) that provides consistent and improved growth of induced in a mouse model of lung cancerogenesis. We have chosen to study pseudo-stratified, mucociliated cultures of HBECs. Methods: HBECs from 3 the existence and role of LCSCs in the k-Ras(+/V12)/RERT(ert/ert) mouse normal donors were obtained from Lonza or the International Institute for model, where induction of oncogenic k-Ras results in a progressive lung the Advancement of Medicine (IIAM). These HBECs were initially expanded disease ultimately leading to adenocarcinoma formation. We started with in adherent cultures for 3 passages in BEGM (Lonza) in separate tissue the phenotypic characterization of the k-RasV12 induced lesions, defining culture-treated T-25 flasks. When the passage 3 cultures reached 70-80% the role of different proteins at different time points of the tumoral progres-

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Thursday Poster Abstracts sion (from hyperplasia, to adenoma, to adenocarcinoma). We are assaying Poster Board Number: 1057 markers already known to identify specific cells in the lung epithelium (such as CCSP and SP-C), and others with a defined relevance in different cancer TARGETED MURINE BONE MARROW CELL types. Preliminary interesting results have been obtained with the cell cycle THERAPY FOR CFTR RESTORATION IN THE inhibitor p21, which shows progressively growing levels of expression paral- lel to malignancy progression. At the same time, we used cells derived from LUNG primary k-RasV12 adenocarcinomas in limited-dilution serial transplantation Duchesneau, Pascal, Wong, Amy P., Waddell, Thomas K. experiments, to assess their stem cell content. By performing undercutane- Latner Thoracic Surgery Research Laboratories & McEwen Centre for ous lung cancer cells transplantations on immunodeficient NOD-SCID mice Regenerative Medicine, University of Toronto, Toronto, ON, Canada, (up to 5 subsequent serial passages have been realized), we estimated a Developmental & Stem Cell Biology, Hospital for Sick Children, Toronto, LCSCs content of about 1/2000. We next wanted to define an assay able ON, Canada, Latner Thoracic Surgery Research Laboratories & McEwen to isolate primary lung cancer stem cells in order to study their self-renewal Centre for Regenerative Medicine, University, University of Toronto, capacity and replicative potential. We applied the same in-vitro culture strat- Toronto, ON, Canada egy that we set up with normal lung tissue, which is able to select for cells with stem properties by isolating them in floating spherical structures (pneu- Cell therapy is a promising approach for treatment of lung disease such as mospheres). Preliminary data seem to indicate that normal pneumosphere- cystic fibrosis. However, previous studies reported low engraftment while constituting cells are able to repopulate the injured normal lung epithelium. the function of grafted cells remained unclear. Conditions to increase airway Unfortunately, our preliminary experiments show that primary k-Ras V12 engraftment and therefore cystic fibrosis trans-membrane receptor (CFTR) adenocarcinoma cells are unable to grow as floating spheroids and alterna- expression have not been described. We previously showed better cell tive strategies are needed in order to isolate lung adenocarcinoma stem cells retention in the lung for trans-tracheal delivery versus intravenous injection for their further characterization. using 7-day cultured plastic adherent bone marrow cells (BMC). We also optimized other delivery parameters such airway injury and timing of cell Poster Board Number: 1055 delivery which resulted in increased retention efficiency. Here we show that TRACKING MOUSE LUNG EPITHELIAL with our optimal BMC delivery protocol, donor cells were capable of long term engraftment. Using our optimal protocol we delivered wild-type BMC PROGENITORS DURING EMBRYOGENESIS AND to CFTR knockout mice and observed substantial retention efficiency as ADULTHOOD detected by Y chromosome PCR. We detected CFTR mRNA in the lung by real-time PCR and CFTR protein by western blot to levels approaching those Bilodeau, Melanie, Rossant, Janet of the wild-type animals. CFTR expressing cells were found in the airways Program in Developmental and Stem Cell Biology, Hospital for Sick by fluorescent microscopy. There was no CFTR detected in knockout mice Children, Toronto, ON, Canada which received BMC from CFTR knockout animals under the same optimal protocol conditions. We propose that improved BMC engraftment in the Asthma, cystic fibrosis, and other lung diseases, such as cancer, are patholo- lung will result in increased CFTR expression possibly leading to restoration gies for which the design of targeted treatments is limited by our current of chloride clearance and thus improving lung function in cystic fibrosis. knowledge of the molecular mechanisms regulating pulmonary cell behavior. Also, a better understanding of the mechanisms regulating the organogen- Poster Board Number: 1059 esis and adult homeostasis of the lung could be translated into optimized protocols to maintain and differentiate native pulmonary progenitors in vitro THE STUDY FOR THE ROLE OF PULMONARY and to derive their counterparts from pluripotent stem cells. Nkx-2.1 (Thy- STEM/PROGENITOR CELLS IN HYPEROXIA roid transcription factor 1) is one of the few molecular markers available for monitoring lung development. This essential transcription factor is broadly INDUCED LUNG INJURY expressed during the differentiation of the pulmonary epithelium. Therefore, Chen, Hung-Kuan, Chen, Huei-Wen, Chien, Chung-Liang, Yang, using gene targeting in mouse embryonic stem cells, we created two new Pan-Chyr, Ling, Thai-Yen knockin reporter alleles regulated by the endogenous Nkx-2.1 promoter. One targeting construct introduced a mCherry fluorescent marker gene in Graduate Institute of Anatomy and Cellular Biology, Taipei, Taiwan, the 3’UTR of the Nkx-2.1 locus (IRES-mCherry). The second construct intro- Graduate Institute of Toxicology, Taipei, Taiwan, Department of Internal duced, in addition to the mCherry reporter gene, a carboxy-terminal affinity Medicine, Taipei, Taiwan, Institute of Pharmacology, Taipei, Taiwan tag to Nkx-2.1 (tag-F2A-mCherry), to better characterize the molecular Survival among preterm infants has improved obviously in recent decades, pathways that Nkx-2.1 regulates under physiological conditions. Following but chronic lung disease, or bronchopulmonary dysplasia (BPD), is now germline transmission of both engineered alleles, expected Nkx-2.1 and the most important long-term pulmonary complication in these infants. mCherry expression patterns were confirmed in vivo and in primary cells Severe BPD is associated with inflammation, pulmonary hypertension, and at various developmental stages using complementary techniques: fluo- increased airway resistance, as well as abnormalities of lung developmet, rescent microscopy, in situ hybridization, cell sorting coupled to qRT-PCR, including disturbed alveolarization and angiogenesis. The disease might be and immunostaining. Importantly, Nkx-2.1/mCherry expressing cells could caused by supplementation of high concentrations of oxygen with mechani- efficiently be isolated during embryogenesis and adulthood and further used cal ventilation to support respiratory function for the patients. However, the for molecular studies (e.g. expression profiling) and clonogenic progeni- pathogenic mechanisms for the disease remain controversial. In previous tor assays (e.g. sphere formation assays). These newly engineered mouse studies, we have successfully isolated putative pulmonary stem/progenitor embryonic stem cell lines and mouse models are valuable tools to visualize cells (mPSCs) from mouse lung tissues, which expressed stem cell markers, and capture lung epithelial progenitors and to characterize the molecular such as Oct-4, Nanog and Sox-2. And, we also successfully developed an network associated with Nkx-2.1, a transcription factor aberrantly expressed in vitro-induction culture condition that mPSCs could differentiate to the in lung diseases and cancer. terminal stage for type-1 pneumocytes in alveolar linage, which expressed aquaporin-5 and T1- . In this study, in vitro-induction culture condition was uses as cell models to reveal the mechanism of pulmonary toxicity with extended exposure to high concentrations of oxygen. The results showed that hyperoxic condition ( > 50%) could induce cell differentiation arrest of mPSCs into alveolar lineage; and induce apoptosis of the differentiating

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Thursday Poster Abstracts

cells but neither the mPSCs themselves nor terminal differentiated type-1 Poster Board Number: 1063 pneumocytes. When treatment in 40% O2, a significant survival numbers for the cells were observed. In addition, apoptosis for the cells could also be LINEAGE SPECIFICATION OF MOUSE AND observed in the in vitro-induction culture by treating the cells with H2O2, HUMAN EMBRYONIC STEM CELL DERIVED which produced reactive oxygenspecies (ROS); and the apoptotic cells could be rescued by treatment with antioxidants, such as N-acetyl cysteine (NAC). CARDIAC MESODERM Taken together, we proposed that both mPSCs and type-1 pneumocytes Witty, Alec, Kattman, Steve, Keller, Gordon might possess protective response to oxidative stress; on the contrary, transi- McEwen Centre for Regenerative Medicine, Toronto, ON, Canada, Cellular tional differentiating cells were more susceptible to ROS hazards. Dynamics International, Madison, WI, USA During heart development the earliest cardiomyocyte, endothelial and vascular smooth muscle lineages arise from cardiac mesoderm that expresses CARDIAC CELLS Flk-1/KDR and PdgfR-α. Studies in both the chick and mouse embryo have Poster Board Number: 1061 shown BMP signaling is important in cardiomyocyte specification and prolif- eration, yet the mechanism for endothelial specification remains unknown. FUNCTIONAL COMPARISON OF LONG QT- Using both mouse and human embryonic stem cells to model cardiovascular ASSOCIATED CARDIAC ION CHANNELS (INA, development in vitro, we are able to recapitulate the critical developmen- tal stages including the generation of KRD+/PdgfR-α+ (K+/P+) cardiac IKR, IKS) IN HUMAN EMBRYONIC STEM CELLS mesoderm and its specification to the cardiac and vascular lineages. Using AND HUMAN INDUCED PLURIPOTENT STEM this in vitro model we have found that the BMP pathway is a key regulator of cardiac and vascular development from the K+/P+ cardiac mesoderm. CELLS Specification to the cardiac lineage is dependent on intermediate levels of Terrenoire, Cecile, Wang, George Kai, Keller, Gordon, Kotton, BMP signaling. Blocking the pathway or activating it with high levels of Darrell N., Kass, Robert S. agonist suppresses cardiomyocyte development and promotes the growth of adhesive cells that express genes indicative of smooth muscle, endothelial Pharmacology, Columbia University, New York, NY, USA, McEwen Centre and fibroblast lineage development. Given that these commitment steps for Regenerative Medicine, University Health Network, Toronto, ON, represent the earliest stages of heart organogenesis, these findings could ul- Canada, The Pulmonary Center, Boston University School of Medicine, timately generate important insights into diseases that affect cardiovascular Boston, MA, USA development as well as provide valuable approaches to generate enriched Human induced Pluripotent Stem cells (hiPS cells) have been developed as populations of cardiac derived tissues from ES cells. an alternative to human Embryonic Stem cells (hES cells) in order to carry Poster Board Number: 1065 out drug screenings and to study diseases such as the Long QT (LQT) syn- drome, a heritable cardiac arrhythmia linked to sudden death. To this date, CHARACTERIZATION OF NOVEL CIRCULATING the basic electrophysiological properties of cardiac cells derived from hiPS cells (hiPSC-CMs) have not been fully characterized and it is still not known MULTIPOTENT STEM CELLS DERIVED FROM how they compare with cardiac cells derived from hES cells (hESC-CMs). HUMAN PERIPHERAL BLOOD Here we report investigation of the electrophysiological properties of single hESC-CMs and hiPSC-CMs during the first 30-60 days of cytokine directed Yang, Han-Mo, Lee, Sahmin, Kim, Ju-Young, Lee, Joo-Eun, Kwon, differentiation with a focus on cardiac ion channels associated with Long QT Yoo-Wook, Cho, Hyun-Jai, Park, Myoung Hee, Oh, Byung-Hee, syndrome: INa (LQT3), IKr (LQT2) and IKs (LQT1). INa, defined as inward Kim, Hyo-Soo, Park, Young-Bae current blocked by TTX (30 μM), had a mid-point of steady-state inactiva- Department of Internal Medicine, Seoul National University Hospital, tion at -65.1 ± 2.4 mV (n=6) in hESC-CMs and at -68.9 ± 1.1 mV (n=9) in Seoul, Korea, Republic of, Dept of Internal Medicine, Seoul National hiPSC-CMs. The late sodium current, measured at the end of 100 ms-depo- University Hospital, Seoul, Korea, Republic of, Department of Laboratory larizing pulses to -10 mV (from -90 mV), represented 0.06 ± 0.03 % (n=6) Medicine, Seoul National University Hospital, Seoul National University and 0.03 ± 0.01 % (n=9) of INa peak current in hESC-CMs and hiPSC-CMs, College of Medicine, Seoul, Korea, Republic of respectively. The biophysical properties of IKr, defined as E4031-sensitive outward potassium current during prolonged depolarization, were very simi- Background: There has been only one study showing the presence of lar between the two cell types: the current-voltage curve measured during multipotent stem cells in bone marrow, called multipotent adult progenitor the test pulse was bell-shape with a maximum current observed at 10 mV cells (MAPCs). These cells can be differentiated not only into mesenchymal in both cell types. IKr tail current, measured at -40 mV after a depolarizing lineage cells, but also into cells with neuroectoderm or endoderm features. pulse to 50 mV, was 1.0 ± 0.1 pA/pF (n=11) in hESC-CMs and 0.6 ± 0.1 Until recently, however, there has been no report isolating and character- pA/pF (n=15) in hiPSC-CMs. IKs, defined as Chromanol 293B-sensitive out- izing these cells from human peripheral blood. Here, we hypothesized that ward potassium current during prolonged depolarization, had a mid-point of there might be circulating multipotent stem cells in human peripheral blood. activation at 9.1 ± 1.7 mV (n=11) in hESC-CMs and at 9.5 ± 3.1 mV (n=8) Methods and Results: From human peripheral blood mononuclear cells in hiPSC-CMs. This is the first comprehensive comparison of the biophysical adherent on non-coated dishes, we could identify new population of cells, properties of INa, IKr and IKs in hESC-CMs and hiPSC-CMs. Our study sug- which are quite distinct from previously reported other stem cells. RT-PCR gests that hiPSC-CMs constitute a relevant cellular model for studying ion showed that newly identified cells have the gene profiles such as Oct3/4, channels mutations that affect channels biophysical properties. KLF4, and c-Myc, suggesting multipotent stemness. Moreover, FACS analy- sis demonstrated that these stem cells have the property of mesenchymal stem cells. With diverse differentiation media, we could differentiate these cells into endothelial-like cells or vascular smooth muscle-like cells, which together could be called vascular progenitor cells. These stem cells have the properties of osteogenic, adipogenic, and myogenic differentiation. Moreover, these cells can be differentiated into ectodermal lineage cells such as neuron-like cells as well as endodermal lineage cells such as hepatocyte- like cells. When injected into the mouse heart in vivo, these newly identified

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Thursday Poster Abstracts stem cells could be differentiated into endothelial cells, smooth muscle cells, ible cardiac differentiation across a wide range of iPS and ES cell lines. This and cardiomyocytes. Conclusions: This study showed the presence of novel technology can enable research and future clinical applications using cardiac circulating multipotent stem cells in human peripheral blood, which shows lineages derived from human pluripotent stem cells. the quite similar features compared to bone marrow-derived MAPCs. These stem cells can be differentiated into mesodermal, ectodermal, and endo- Poster Board Number: 1069 dermal lineage cells. These findings suggest that these cells can be easily GENERATION AND CARDIOMYOCYTES obtained from human peripheral blood and used for regenerative medicine in real world. DIFFERENTIATION OF HUMAN INDUCED Poster Board Number: 1067 PLURIPOTENT STEM CELLS FROM POST- MYOCARDUAL INFARCTION HEAET FAILURE MATRIX-PROMOTED EPITHELIAL-TO- PATIENTS MESENCHYMAL TRANSITION LEADS TO Zwi-Dantsis, Limor, Huber, Irit, Winterstern, Aaron, Gepstein, EFFICIENT MESODERM FORMATION AND Amira, Arbel, Gil, Gepstein, Lior CARDIOGENESIS OF HUMAN PLURIPOTENT Technion – Israel Institute of Technology, Haifa, Israel STEM CELLS Background: Myocardial cell therapy strategies are hampered by the paucity Zhang, Jianhua, Lian, Xiaojun, Herman, Amanda M., Raval, Kunil of sources for human cardiomyocytes and by the expected immune rejection K., Wilson, Gisela F., Barron, Matthew R., Yu, Junying, Palecek, of allogeneic cell grafts. The ability to derive patient-specific human induced Sean P., Thomson, James A., Kamp, Timothy J. pluripotent stem cells (hiPSCs) by somatic cell reprogramming may provide a potential solution for these challenges. Aims: To derive hiPSCs lines from University of Wisconsin - Madison, Madison, WI, USA, Cellular Dynamics post-myocardial infarction heart failure (HF) patients, to induce their car- International, Madison, WI, USA diomyocyte differentiation, to define the phenotypic properties of the gener- Efficient and reproducible approaches for differentiating human iPS and ES ated hiPSCs-derived cardiomyocytes (hiPSCs-CMs) and to test their ability cells to cardiomyocytes (CMs) are needed for research and clinical ap- to integrate with preexisting cardiac tissue. Methods and Results: Dermal plications. Differentiation of human ES cells to cardiac lineages involves a fibroblasts from two HF patients were reprogrammed by retroviral delivery step-wise process which recapitulates the key stages found in embryonic of Oct4, Sox2, and Klf4. The generated HF-hiPSCs generated teratomas development. The epithelial-to-mesenchymal transition (EMT) during gas- in SCID mice and displayed fully reprogrammed properties as assessed by trulation is the first step required to form the mesodermal cells specified for immunocytochemistry and quantitative RT-PCR analysis. The HF-hiPSCs the cardiac lineages in development. The purpose of this study was to test were then coaxed to differentiate into the cardiac lineage using the em- the ability of extracellular matrix (ECM) in combination with growth factors bryoid body differentiating system. Gene expression for cardiac genes and to promote the EMT of the epiblast-like iPS and ES cells and thus promote immunostaining studies confirmed the cardiomyocyte phenotype of the HF- efficient mesoderm formation and cardiogenesis. Various iPS cell lines includ- hiPSC-CMs with comparable properties to hiPSC-CMs of healthy individual. ing vector and transgene free iPS lines (DF19-9-11T, DF6-9-9T) and virally The cardiac tissue developed a functional syncytium with stable pacemaker generated wild type iPS cells (IMR90 C4) as well as ES cell lines (H1 and H9) activity and action potential propagation. Adequate chronotropic responses were cultured on Matrigel in mTeSR1 media as a monolayer. The cultured were found after application of isoproterenol and carbamylcholine. Next, we iPS and ES cells exhibited a polarized epithelial cell phenotype with microvilli showed that electrically active donor CMs derived from HF-hiPSCs can func- on the apical surface and expression of E-cadherin. Application of a thin tionally integrate with primary cultures of neonatal rat ventricular myocytes overlay of Matrigel triggered the formation of multilayered foci in which and to induce synchronized electrical and contractile activities. Conclusions: the lower layer cells exhibited a mesenchymal cell phenotype as shown in Our study shows the ability to generate hiPSCs lines from HF patients and electron micrographs and expressed N-cadherin, while the upper layer cells to differentiate them into bone-fide cardiomyocytes that can integrate with still maintained expression of E-cadherin and a polarized epithelial organiza- pre-existing myocytes. This novel source for patient-specific heart cells may tion. Q-PCR showed a significant increase of gene expression for the EMT bring a unique value to the emerging field of cardiac regenerative medicine. markers including CDH2, SNAIL1, SNAIL2, GSC, VIM and FN1 in the matrix Poster Board Number: 1071 sandwich cells than the control. After the initial EMT step, we tested the combination of growth factors in TGF-β superfamily such as Activin A and VOLTAGE DEPENDENCE AND KINETICS BMP4, and the basic fibroblast growth factor (bFGF) to promote meso- derm formation and cardiac differentiation. We found an exposure of high OF ENDOGENOUS IKS CHANNELS IN dose Activin A (100 ng/ml) for 24 hours followed by low concentrations HUMAN EMBRYONIC STEM CELL DERIVED of BMP4 (< 10 ng/ml) and bFGF (5-10 ng/ml) for 4 days in the defined CARDIOMYOCYTES: IMPLICATIONS FOR media of RPMI plus B27 supplement without insulin generated high purity (60-90%) and high yield CMs (1 input iPSC or ESC to 5-10 CMs) measured SUBUNIT STOICHIOMETRY IN THE ASSEMBLED as cTnT positive cells by flow cytometry; however, higher concentration of CHANNEL BMP4 inhibited cardiogenesis. Q-PCR and RT-PCR analysis demonstrated the sequential up-regulation of mesendoderm and mesoderm markers (T, Wang, Kai, Terrenoire, Cecile, Sampson, Kevin J., Iyer, Vivek, MIXL1, MESP1 and GATA4,), followed by cardiac transcription factors (ISL1 Osteen, Jeremiah D., Lu, Jonathan, Keller, Gordon, Kass, Robert S. and NKX2-5) and cardiac myofilament proteins (TNNT2, TNNI3, MYL2, Department of Pharmacology, Columbia University Medical Center, New MYL7). By day 8 confluent contracting cells were observed and large CMs York, NY, USA, Department of Medicine, Columbia University Medical networks formed demonstrated by cTnT and MLC2a immunolabeling. At Center, New York, NY, USA, McEwen Centre for Regenerative Medicine, 15 days of differentiation, ~60% of these CMs still exhibited proliferation University Health Network, Toronto, ON, Canada potential measured by flow cytometry of cTnT and Ki-67. Sharp microelec- trode recordings demonstrated spontaneous action potentials typical of The IKs cardiac potassium channel, critical to human cardiac electrophysiol- nodal-, atrial- and ventricular-like iPS and ES cell-derived CMs as previously ogy, is a heteromultimeric protein consisting of four identical α-subunits observed with EB-mediated differentiation. Our results demonstrate that the (KCNQ1) assembled with auxiliary β-subunits (KCNE1). KCNE1 confers matrix sandwich method promotes EMT to enable efficient and reproduc- distinct biophysical properties on the channels including slowing of gating

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kinetics and shifting activation. Subunit stoichiometry recently has been re- Poster Board Number: 1075 ported to be variable and to impact channel function. Here we characterize endogenous IKs channels, expressed in single cardiomyocytes derived from GENE TARGETING IN HUMAN EMBRYONIC human embryonic stem cells (hESC-CMs), identified as Chromanol 293B- STEM CELLS UNDERPINNING THE STUDY OF sensitive current, and use channel biophysical properties to gain information about endogenous subunit stoichiometry. The half maximal activation volt- PRIMARY HEART FIELD IN VITRO age in hESC-CMs falls between that of HEK293 cell expression of KCNQ1 Ortmann, Daniel, Bernardo, Andreia S., Elliott, David A., Ortiz, channels with and without KCNE1. In order to rule out a hESC-CM specific Mariaestela, Rosen, Barry, Stanley, Edouard G., Elefanty, Andrew G., alteration in KCNQ1 gating, charybdotoxin (CTX)-sensitive KCNQ1 chan- Pedersen, Roger A. nels (CTX-KCNQ1) were expressed in hESC-CMs and HEK293 cells without any observed difference in biophysical properties. Expression of KCNE1 Surgery, University of Cambridge, Cambridge, United Kingdom, Monash alone in hESC-CMs markedly shifts and slows native IKs activation. These Immunology and Stem Cell Laboratories, Monash University, Clayton, results imply that additional KCNE1 subunits can assemble with endogenous Victoria, Australia, Wellcome Trust Genome Campus, Wellcome Trust channels suggesting submaximal baseline KCNQ1/KCNE1 stoichiometry. Sanger Institute, Hinxton, Cambridge, United Kingdom Simulations of channel activity are consistent with this hypothesis. These Human pluripotent stem cells offer unprecedented opportunities to generate results demonstrate the utility of hESC-CMs as platforms for assaying the a variety of cell types that can be used for toxicity testing, cell therapies or biophysical properties of wild type and mutant IKs channels, and the impor- developmental studies. Cardiomyocytes are amongst the most sought after, tance of KCNQ1/KCNE1 subunit stoichiometry as a determinant of channel especially those with a left ventricular identity, because of their therapeutic function in these cells. Our results further suggest that variation in the ratio relevance. We focused our study on the characterisation of cardiomyocyte of KCNE1 to KCNQ1 subunits in assembled channels may be a novel and progenitor cells, in particular those that constitute the primary heart field important mechanism of channel modulation in disease or tissue specific (PHF), which gives rise to the left ventricle. The lack of suitable surface manners. markers has so far prevented the isolation of a primary heart field (PHF) Poster Board Number: 1073 population. Using gene targeting in human embryonic stem cells, we have generated a double fluorescent reporter cell line for NKX2.5 (a pan cardiac UNIQUE IMMUNOLOGICAL PROPERTIES OF marker) and TBX5 (a transcription factor widely used for the identification of the PHF). We utilised this line to isolate and study double positive cells, HUMAN EMBRYONIC STEM CELL-DERIVED which we generated by employing our newly developed chemically defined, CARDIOMYOCYTES monolayer based cardiac differentiation protocol. This approach yields high percentages of NKX2.5- and TBX5- expressing cardiac progenitors in under Okamura, Ross M., Nishimoto, Kevin P., Dawes, Glenn N., 10 days. This population will be further characterised by deep sequencing Lebkowski, Jane S., Reddy, Anita and its developmental potency determined. This approach can potentially Geron Corporation, Menlo Park, CA, USA lead to identification of specific surface markers for PHF progenitors, which Human embryonic stem cells (hESC) are a novel source of differentiated will enable new insights into the specification of cardiac progenitors and cardiomyocytes with great promise for cardiovascular regenerative medi- their identity. This knowledge can also ultimately lead to improvments in ap- cine. We have developed an efficient and defined protocol for the scalable plications of stem cell derived cardiac cells. Furthermore, the study of TBX5 production of cardiomyocytes from hESC (GRNCM1) suitable for cardiac expressing cell populations can improve our understanding of Holt-Oram- cell replacement therapy. Since a critical factor in determining the clini- Syndrome, an autosomal dominant heart and limb disorder associated with cal utility of allogeneic human embryonic stem cell-based cell therapeutics TBX5 mutations. (Supported by British Heart Foundation, The Wellcome for heart disease will be the immunogenicity of the transplanted cells, we Trust, EU FP7 and Medical Research Council) evaluated the immunological properties of GRNCM1 in vitro. MHC class Poster Board Number: 1077 I antigen expression was low on GRNCM1, whereas surface MHC class II, CD80, CD86, and CD40 expression was undetectable. In allogeneic mixed HUMAN CARDIAC STEM CELLS ISOLATED lymphocyte reactions (MLR), GRNCM1 was unable to elicit either T cell FROM ATRIAL APPENDAGES STABLY EXPRESS proliferation or IFN-g production. In fact, GRNCM1 was found to inhibit the autologous MLR in one PBMC donor that was examined. This inhibitory C-KIT WITHOUT LINEAGE DEPLETION effect was further evaluated with the addition of GRNCM1 to PBMC re- He, Jia-Qiang, Vu, Duc M., Hunt, Greg, Chugh, Atul, Bhatnagar, sponding to either tetanus toxoid antigen or alloantigen in the PBMC MLR. Aruni, Bolli, Roberto GRNCM1 was found to suppress IFN-g responses to tetanus toxoid antigen and partially block allogeneic T cell responses when added as a third party University of Louisville, Louisville, KY, USA in the PBMC MLR. GRNCM1-mediated immune suppression was found to Background: In the past decade, compelling evidence has accumulated sug- be at least partially due to soluble factors and furthermore, expression of the gesting that the heart has regenerative potential through resident cardiac immunoregulatory molecule B7-H1/PDL-1 has been detected on GRNCM1. stem cells (CSCs). Update, at least five different types of CSCs (including Lastly, GRNCM1 was found to be resistant to human serum complement c-kit+/Lin-; Sca1+; Isl1+, cardiac side population/Abcg2+/MDR+, and mediated lysis in vitro and the complement inhibitory proteins CD46, CD55, cardiospheres/c-kit+/Sca1+/Flk1+) have been reported based on various and CD59 are each expressed by GRNCM1. These findings indicate that stem cell markers and isolation methods. These cells have shown some ben- cardiomyocytes derived from hESC and may possess immune privileged eficial results in in vivo study of myocardial infarct (MI), but the results are characteristics and may provoke only minimal immune-mediated rejection still controversial. One potential reason may be related with lacking of fully upon cell transplantation. understanding the properties of these cells before transplantation. In the present study, we aimed to systematically characterize the c-kit+/Lin- CSCs to examine how CSCs markers adapted to post-isolation, expansion, purifi- cation, differentiation, and whether there was any contamination of other cell types during procedure. Methods: Human CSCs were enzymatically isolated from atrial appendage (AA) specimens obtained during open heart surgery from patients. The samples were randomly divided into three groups with at least three samples from different patients in each group. In Group1,

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Thursday Poster Abstracts samples directly went to fixation and tissue section procedure; in Group 2, forces of 1.28±0.11mN at 26% stretch and exhibited a maximum capture the freshly isolated CSCs from AA were continuously cultured at 37oC up to rate of 5.5z. Conclusion: Whilst these values are lesser than those measured passage 10 (P10) without purification or Lin depletion procedures. In Group for neonatal rat myocardium this is the first demonstrated example of en- 3, isolated cells underwent c-kit magnetic activated cell sorting (MACS) and/ tirely stem cell-derived tissue capable of near-neonatal functional levels. or Lin depletion between P1 to P3. The isolated cells were passed up to P10 with cells being collected on every other passage for various biologi- Poster Board Number: 1081 cal analysis, including immunohistochemistry (IHC), immunocytochemistry A NOVEL CELL-LINE MODEL SYSTEM FOR (ICC), Flow cytometry with specific antibodies anti c-kit, Lin (CD2, 3, 14, 16, 19, 20, 24, 34, 45, 56, 68, 123, 133 & 235a), cardiac (Nkx2.5, GATA4, MOUSE CARDIAC PROGENITOR CELLS cTnI, & α-sarcomeric actin), endothelial (KDR & CD31), smooth muscle Freire, Ana, Nascimento, Diana, Forte, Giancarlo, Valente, Mariana, (MHC), fibroblasts, and mast cell markers. RT-PCR was also performed with Carvalho, Isabel, Di Nardo, Paolo, Pinto-do-Ó, Perpétua various primers to detect gene expression of cardiac lineages. All data was expressed as M±SE and p<0.05 was considered statistically significance. Re- NEWTherapies Group,Biomaterials Division, INEB-Instituto de Engenharia sults: By specific antibody staining, we demonstrated that tissue sections of Biomedica, Porto, Portugal, Laboratorio di Cardiologia Molecolare & AA specimens contained less than 1% c-kit+ cells among the total counted Cellulare,Dip. di Medicina Interna, Università di Roma Tor Vergata, Rome, nuclei and the freshly isolated cells from same or different tissues specimens Italy, IBMC- Instituto de Biologia Molecular e Celular, Porto, Portugal had a comparable percentage of c-kit+ cells. Interestedly, without MACS the The view of the adult heart as a terminally-differentiated organ has been percentages of c-kit+ cells gradually increased up to ~40% at P4-8 during challenged with reports of de novo cardiomyocyte generation and the culture, but not exceeding 80% unless c-kit MACS was carried out. The ad- identification of putative cardiac progenitor cells (CPCs). Despite the elusive dition of Lin depletion following c-kit MACS seemed not to further increase ontogenic origin of CPCs, their commitment to cardiac lineages has been c-kit+ % during culture up to P10. The resulting c-kit+ cells did not display tracked back to a myocardium scarce cell fraction(s) displaying the stem- KDR, CD31, 34, 45, 133 and Lin markers after c-kit MACS, and the Lin cell associated markers Sca-1 and/or c-Kit/MDR1. The aim of the herein depletion procedure seemed unnecessary for c-kit purification. Moreover, work was to address whether an immortalized line of Sca-1+CPCs replicate c-kit+ CSCs demonstrated cardiac lineage markers (such as Nxk2.5, GATA4, their native counterparts and could thus become an in vitro model-system MEF2c, KDR, vWF, & MHC) before differentiation, and strongly expressed for high-throughput studies. The Sca-1+CPC-line consistently expressed cardiac structure proteins (cTnI & α-sarcomeric actin) after differentiation. early-cardiac transcription factors and stem-cell associated molecules while Conclusion: We concluded that by using an enzymatic dissociation method, no transcripts characteristic of mature cardiomyocytes were found. Markers a large number, higher % of non-contaminated human CSC (c-kit+/Lin- of hematopoietic and endothelial affiliation were absent as shown by FACS. ) could be obtained from AA specimens within ~4 weeks following c-kit Expression of CD105 and CD106 did however suggest that these cells MACS without Lin depletion. This simple, but cost effective approach can might represent an uncommitted mesenchymal progenitor cell-line whose be used to obtain enough numbers of stably-expressed c-kit cells for clinical influence on cardiogenesis and response to injury needs to be assessed. trial in repairing MI. Thus, upon subcutaneous transplantation into Nude mice, the transplanted Poster Board Number: 1079 cells appeared contained within lumps that developed at the injection site up to 14 weeks post-injection. Immunohistochemical analysis of the lumps ASSEMBLY OF AN ELECTROMECHANICALLY indicated a recruitment of host cells likely induced by the grafted-cells. Sca-1+CPCs were not detected in immunocompetent mice on the same FUNCTIONAL 3D BIOSYNTHETIC TISSUE experimental setting. The CPC-line response to the native cardiac environ- USING MOUSE EMBRYONIC OR INDUCED ment was also evaluated following intramyocardial injection into sham- PLURIPOTENT STELL CELL-DERIVED CARDIAC operated and myocardial-infarcted (MI) syngeneic mice. Cardiac function was assessed by transthoracic echocardiography and MI size quantified on PROGENITOR CELLS Masson’s trichrome stains at 14 days posttransplantation. MI-transplanted Christoforou, Nicolas, Liau, Brian, Chakraborty, Syandan, hearts revealed higher shortening (FS) and ejection fractions (EF), and a Chellapan, Malathi, Bursac, Nenad, Leong, Kam W. reduced remodeling of the left ventricle, when compared to vehicle-injected MI animals. Moreover, transplanted cells could be identified in the infarcted Biomedical Engineering, Duke University, Durham, NC, USA hearts up to 21 days post-injection, despite the profound modifications on Background: We report the use of a single cell source: mouse embryonic the cardiac architecture, and were also detected in the hearts of trans- or induced pluripotent stem cell (ES or iPS cell) derived cardiac progenitor planted sham-operated animals. Examination on how the transplanted cells cells (CPCs) capable of differentiating into cardiomyocytes, smooth muscle, integrate/interact with the recipient cardiac cells is underway. Validation of and endothelial cells in combination with a novel cardiac tissue engineering the herein cell-line as adult cardiac progenitor cells would entail the first de- technique to generate highly functional 3D biosynthetic tissues. Methods scribed model for mesenchymal-derived CPC and thus a tool for dissecting and Results: We derived stably transfected ES or iPS cell clones allowing the further the role of such cells on cardiac homeostasis vs. pathology. Further- expression of puromycin N-acetyl-transferase under the control of a cardiac more, the CPC-line may be a source for high-throughput molecular analysis specific Nkx2-5 enhancer element. We optimized both the ES / iPS cell and a potential valuable platform for pharmacological screening. differentiation protocol and the antibiotic selection time period allowing the Poster Board Number: 1083 robust and efficient derivation of CPCs (>107 cells). Differentiating cardiac progenitors formed monolayers of electrically coupled cardiomyocytes. MITOCHONDRIAL DNA MUTATIONS AS USEFUL Using optical mapping of membrane potentials, we measured conduc- tion velocities of 19.2±0.2 cm/s, significantly higher than those previously MARKERS FOR FUNCTIONAL COMPETENCE OF reported for cardiomyocytes derived from mouse or human ES cells. The MOUSE ADULT STEM CELLS CPCs were suspended in a matrix hydrogel and placed in 3D microfabricated silicon molds allowing the formation of highly dense, aligned, and intercon- Lushaj, Entela, Anstadt, Emily, Kohmoto, Takushi nected tissue constructs. Using a custom-built force measurement setup, we University of Wisconsin, Madison, Madison, WI, USA measured isometric twitch amplitude and found that the tissue constructs Background: Despite the significant advances over the past two decades exhibited positive force-length and negative force-frequency relationships, in the management of human acute myocardial infarction, there remains characteristic of rodent tissue. Tissue constructs were capable of producing a large population of postinfarct survivors who will develop cardiac failure,

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leading to early death. Cardiac transplantation remains the only therapy CPCs as a therapeutic target for cardiac regeneration, it is critical to decipher available to these patients. The presence of immature cycling cardiomyo- the interplay between their proliferation and myogenic potentials. MicroR- cytes in human hearts after myocardial injury has raised the possibility that NAs are short, non-coding, single stranded RNA that are 20-23 nucleotides cardiac stem cells may, in fact, exist in the heart. Stem cells (SC) have the in length and may be involved in this delicate balance of cardiac prolifera- potential to regenerate tissue, and hold forth a promise of future benefit to tion and cardiogenesis. To test the hypothesis that microRNAs regulate the patients in many areas of diseases, and especially that of myocardial infarc- developmental growth of CPCs, we identified sets of differentially expressed tion, where tissue necrosis limits cardiac function. The successful use of SC microRNAs that predicted a differential growth phenotype of CPCs between lines for therapeutic cell replacement will require that cells exhibit physiologi- embryonic and adult human hearts. cal and molecular stability over extended periods of culture. At this time, Methods: Single-cell sorting was utilized to purify linneg c-kitpos CPCs from there are no established molecular and cellular markers to ascertain which neonatal and adult mouse hearts and microRNAs were extracted for dif- cell lines are defective or functionally competent. The key question is what ferential expression profiling. Flow cytometric analysis of CPCs size, Ki67 ex- functional characteristics, important for normal differentiation of these cells, pression, and DNA content from human and mouse hearts in vivo as well as should be used to select the healthiest lines for further in-depth studies, doubling times in mouse primary cultures were used to test the differential particularly prior to organ and tissue derivation. While most SC studies have phenotype predicted by the microRNA profiles. Results: Twenty microRNAs focused on the activity of the nuclear genome, characteristics of the mito- were differentially expressed between neonatal and adult mouse CPCs and chondrial genome have been largely ignored. It is of concern that mitochon- gene ontology analysis demonstrated that >40% of proteins targeted by drial DNA (mtDNA) mutations are found in a wide spectrum of cancers and these microRNAs were involved in cellular proliferation-related pathways. ageing tissues. Mitochondria are organelles of pivotal importance to the cells Five out twenty microRNAs were validated by RT-PCR with 100% fidelity to energy production, yet their genome is susceptible to damage. Objective: the profile. To further validate the prediction of the differential expression of Our work was based on the hypothesis that the efficiency of cardiac stem microRNAs, the phenotypic variance in proliferation of human and mouse cells in myocardial regeneration is causally associated with mtDNA mutations CPCs were tested as a function of development. The CPC’s mean forward and subsequent electron transport system dysfunction. MtDNA mutations scatters were not different and suggested identical sizes. In contrast, the hu- accumulate with each passage of cultured mouse adult cardiac stem cells man embryonic and mouse neonatal CPCs showed a 6-fold increase in Ki67 and play a role in their ability to differentiate and regenerate the myocar- expression when compared to adult CPCs of both species (n=6, p2N DNA dium. The goal of this study was to investigate the presence of mtDNA content consistent with transiting into S-G2-M phases of cell cycle. This deletion mutations in adult cardiac stem cells and their impact on growth represented a two-fold increase when compared to both adult cells (n=3, and differentiation capacity of these cells. Methods: Mouse adult cardiac p<0.05). To determine if the microRNA profile and molecular phenotyping stem cells were isolated from C57BL/6 mice of different ages. MtDNA was translate into differences in cell division, we examined the doubling time isolated from CSC of different passages and DNA primers specific for the of adult and neonatal mouse CPCs in primary culture. The neonatal CPCs mtDNA were utilized in standard PCR for deletion analysis and quantitative had a 7-fold increase (n=6, p<0.001) at 16 days when compared to adult PCR measurements of mutation frequency. Growth rate and differentiation cells. Conclusion: Taken together, our data demonstrate that the expression capacities of CSCs with mtDNA deletion mutations were measured on clon- pattern of 20 microRNAs predicts a differential proliferative phenotype in ally expanded CSCfrom single cells with known levels of mtDNA deletions. neonatal CPCs when compared to adult CPCs. Our findings suggest that a Results: MtDNA deletion mutations of various sizes were identified in all selected group of microRNA play a critical role in the regulation of organ- CSC of late passages. All mutations resulted in the partial or entire removal specific progenitor cells during development. Further studies into their regu- of the mitochondrial protein functional genes. The size of deletions ranged latory role in these processes may provide a needed breakthrough towards from ~ 6 - 9.5 kb. The highest rate of growth was observed in CSC without the goal of enhancing cardiac regeneration. deletion mutations and with low frequency of deletion mutations and the lowest rate of growth in CSC with high frequency of deletions. Conclusions: Poster Board Number: 1087 The successful analysis of mtDNA mutations is important because it will con- GCSF AND SITAGLIPTIN ENHANCE STEM CELL tribute greatly to understanding the requirements for stem cell maintenance, and could have other very useful applications in the translation of stem cell HOMING, CARDIAC FUNCTION AND SURVIVAL science from the laboratory towards human stem cell transplantation in the AFTER MI IN MICE clinic. Theiss, Hans D., Krieg, Lisa, Vallaster, Markus, David, Robert, Poster Board Number: 1085 Assmann, Gerald, Mueller-Hoecker, Josef, Franz, Wolfgang M. MICRORNA PROFILING PREDICTS A VARIANCE Department of Cardiology, University of Munich, Munich, Germany, University of Munich, Munich, Germany IN THE PROLIFERATIVE POTENTIAL OF Background: Cardiac homing of stem cells occurs via the SDF-1-CXCR4 CARDIAC PROGENITOR CELLS DERIVED FROM axis. Myocardial SDF-1 is cleaved by the extracellular protease CD26. We NEONATAL AND ADULT MOUSE HEARTS hypothesized that inhibition of CD26 by Sitagliptin (which is a clinically admitted antidiabetic drug) leads to an increase of myocardial SDF-1 thus Sirish, Padmini, López, Javier E., Wong, Andrew, Timofeyev, improving the homing of G-CSF-mobilized stem cells after myocardial infarc- Valeriy, Young, Nilas J., Chen, Huei-sheng Vincent, Chiamvimonvat, tion (MI) in a mouse model. Methods: We induced AMI in 10-11 weeks old Nipavan male C57BL/6 mice using surgical occlusion of the left descending artery. Division of Cardiovascular Medicine, University of California, Davis, CA, Mice were then treated either with G-CSF in combination with Sitagliptin USA, Division of Cardiothoracic Surgery, University of California, Davis, (“G-CSF+Sita”), G-CSF (“G-CSF”) or Sitagliptin (“Sita”) alone or saline CA, USA, Sanford Burnham Medical Research Institute, La Jolla, CA, USA (“control”). FACS analyses were used to determine cardiac stem cell popula- tions after 6 days. Cardiac function parameters were assessed using a Millar- Background: The existence of resident cardiac progenitor cells (CPCs) in Tip catheter system. Histology was performed to examine capillary density adult hearts has directly challenged the paradigm that myocardium is a (CD31+ cells) and infarct size 6 or 30 days after AMI. Apoptotic cardiomyo- terminally differentiated organ. Recent studies have provided exciting evi- cytes were assessed by TUNEL assays. Survival was analyzed by Kaplan- dence that stem cells may offer an enormous potential for replacing lost or Meier-method for 30 days (n=20 in each group). Findings: In the first step, diseased cardiac myocytes in cardiomyopathic hearts. Despite this promise, we established the optimal dosage of Sitagliptin administration using mass CPCs exist in small numbers with relatively limited myogenesis. To optimize spectrometry. As proof of principle, DPP-IV activity was decreased in G-

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CSF+Sita mice in a dose dependent manner. G-CSF + Sitagliptin administra- ever, this requires invasive cardiac biopsies. We reasoned that plakoglobin tion lead to a significantly improved cardiac homing of several CD34+ stem immunoflourescence studies of ARVC patient keratinocytes might lead to cell populations (also dose dependent) and a relevant induction of resident the development of a less invasive diagnostic test, and that derivation and cardiac stem cells in the G-CSF+Sita group. Besides, myocardial remodel- cardiac differentiation of patient-specific induced pluripotent stem (iPS) cells ing was reduced in the G-CSF+Sita group compared to the other treatment might allow more detailed study of ARVC disease mechanism. We have suc- regimes. Neovascularization (represented by CD31+ capillaries in the bor- cessfully established primary keratinocyte cultures from ARVC patient skin derzone) was enhanced and apoptosis reduced in G-CSF+Sita mice. Cardiac biopsies. These patient-specific keratinocytes have diminished desmosomal function was almost doubled in G-CSF+Sita mice after 30 days. Finally, G- plakoglobin when differentiated in calcium containing media. Importantly, CSF+Sita mice showed a significant increase of survival after 30 days (80% this desmosomal plakoglobin loss was also directly demonstrated in histo- versus 40% in G-CSF mice and 35% in the saline group). All the beneficial logical sections derived from ARVC patient skin biopsies. These findings mir- effects of G-CSF+Sita were reversed after application of AMD3100, which ror recent observations of plakoglobin loss in endomyocardial biopsies from is a CXCR-4-antagonist. This proves the specificity of our approach to ARVC patients and could be developed for minimally invasive diagnostic the SDF1-CXCR4-axis. Interpretation: This is the first study showing that purposes. We have also successfully established transgene-free iPS cells from combined application of G-CSF and Sitagliptin 1) enhances cardiac homing ARVC patients using a non-integrating RNA virus. These iPS cells can gener- of stem cells 2) reduces cardiac remodeling 3) induces neovascularization ate cells of all three embryonic lineages. We predict an in-vitro phenotype of 4) diminishes apoptosis 5) improves cardiac function 6) enhances survival ID plakoglobin loss in cardiomyocytes derived from these patient-specific iPS after acute myocardial infarction specifically to the SDF1-CXCR axis. Since cells on the basis of our finding of junctional plakoglobin loss in patient epi- Sitagliptin is a clinically used drug, our results have immediate impact on the dermal cells. Ongoing experiments will establish whether this in-vitro disease transfer of this therapeutic approach from bench to bedside. model recapitulates the pathobiology of the condition in patients. Poster Board Number: 1089 Poster Board Number: 1093 NOVEL SMALL MOLECULES FACILITATE HIGH A MODEL FOR LQT3 USING DISEASE-SPECIFIC EFFICIENT CARDIAC DIFFERENTIATION FROM IPS CELL-DERIVED CARDIOMYOCYTES ES AND IPS CELLS Baba, Shiro, Valdivia, Carmen R., Zhang, Jianhua, Spencer, C. Minami, Itsunari, Minami, Itsunari Ian, Nakamura, Kenta, Sears, Marie A., Aalto-Setälä, Katriina, iCeMS, Kyoto University, Kyoto, Japan Scheinman, Melvin, Yamanaka, Shinya, January, Craig T., Kamp, Timothy J., Makieski, Jonathan C., Conklin, Bruce R. The cardiomyocytes derived from human ES/iPS cells are important for drug Gladstone Institute of Cardiovascular Disease, San Francisco, CA, USA, discovery screening of a pharmacological QT prolongation test and a cardiac Departments of Medicine and Physiology, Cellular and Molecular cell transplantation therapy. However, cardiac model cell systems sufficient Arrhythmia Research Program, University of Wisconsin, Madison, WI, for using those tests are not developed so far, discovery of cardiac differen- USA, IBT, Institute of Biomedical Technology, Heart Center, University of tiation inducing/promoting factors has been expected. Therefore, we estab- Tampere, Tampere, Finland, Division of Cardiology, University of California, lished High Throughput Screening (HTS) system searching for compounds San Francisco, San Francisco, CA, USA, Center for iPS Cell Research and promoting cardiac differentiation of ES cells. We generated a transgenic Application, Kyoto, Japan monkey and human ES cell lines expressing a GFP driven by a cardiac specificα MHC gene promoter, and the ES cell lines were differentiated into Human skin fibroblasts can be retrovirally reprogrammed with Sox2, Oct3/4, cardiomyocytes in a cardiac differentiation medium. Then, we screened the Klf4 and c-Myc into induced pluripotent stem (iPS) cells that express pluri- compounds increasing a GFP fluorescent amount of the cardiomyocytes potency markers and differentiate into all the major cell lineages. Patient- from a chemical library containing about 10000 compounds. As a result, we specific iPS-derived cardiomyocytes (iPS-CMs) allow new opportunities to found two compounds, a compound X and a novel compound Y. Moreover, study congenital arrhythmias such as long QT (LQT) syndrome. We have it was confirmed that these compounds have remarkable effects to promote developed two separate iPS cell lines from a patient carrying a missense cardiomyocyte differentiation in human ES and iPS cell lines. Therefore, our mutation (N406K) in the cardiac sodium channel gene (SCN5A) resulting in HTS system is able to be applied with human ES/iPS cells, and expected to LQT type 3. Producing iPS-CMs using the spontaneous embryoid body dif- use for QT prolongation tests and transplantations of cardiomyocytes. ferentiation technique was inefficient, while directed differentiation using a matrigel sandwich method was >1000-fold more efficient, resulting in sheets Poster Board Number: 1091 of beating CMs. The N406K mutation was confirmed by DNA sequencing IN VITRO MODELING OF ARRHYTHMOGENIC of fibroblasts, iPS cells, and iPS-CMs. To study the biophysical properties of the N406K mutant iPS-CMs sodium current (INa), we used the patch- RIGHT VENTRICULAR CARDIOMYOPATHY clamp technique. We found that the peak current density and midpoints of Ayetey, Harold, Takashima, Yasuhiro, Grace, Andrew, Smith, Austin activation and inactivation were similar to control cells. Conversely, iPS-CMs expressing N406K had a mean late INa, that was significantly increased Centre for Stem Cell Research, University of Cambridge, Cambridge, United (p<0.05). In the mutant cells (n=9), late INa was 2.3±0.9% of peak INa Kingdom, Biochemistry, University of Cambridge, Cambridge, United compared to 0.5±0.2% of peak INa in control cells (n=7). Our results show Kingdom that increased late INa in a human iPS-CM model of LQT3 is consistent with Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a cardiac results from animal model and heterologous expression systems. Therefore, disease affecting 1-2 in 5000 people, up to 50% of who have heterozygous this human model could be useful for in vitro research, drug discovery, and mutations in desmosomal proteins. The condition is typified by adipocyte re- toxicology screening of LQT syndrome. placement of myocardium, cardiac rhythm abnormalities and sudden cardiac death typically in young adults. Diagnosis of ARVC is difficult due in part to its high clinical and genetic heterogeneity and requires satisfaction of com- plex clinical criteria. Recent studies have demonstrated diminished plako- globin at cardiac intercalated discs (ID) of patients with ARVC independent of their underlying genetic mutation. This promising finding has resulted in cardiac ID plakoglobin loss being proposed as a useful diagnostic tool. How-

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Poster Board Number: 1095 marine invertebrates, we identified a couple of natural chemicals (nCDC). Particularly, nCDC#1 showed potent cardiomyocyte induction at nanogram/ EPIGENETIC REGULATION OF CARDIAC milliliter (ng/ml) level. This active concentration (2 ng/ml) was 1000 times DIFFERENTIATION lower than cyclosporin-A. And nCDC#1 increased cardiomyocyte percentage and cell number that appeared from Flk-1 positive mesoderm cells approxi- Alexander, Jeffrey M., Wamstad, Joseph A., Delgado-Olguin, Paul, mately 20 times more than control. These findings would provide a clue for Holloway, Alisha K., Kattman, Steven J., Keller, Gordon, Pollard, cardiomyocyte differentiation mechanisms and offer novel cardiac regenera- Katherine S., Boyer, Laurie A., Bruneau, Benoit G. tive strategies. Gladstone Institute of Cardiovascular Disease, San Francisco, CA, USA, Poster Board Number: 1099 MIT, Cambridge, MA, USA, McEwen Center for Regenerative Medicine, University Health Network, Toronto, ON, Canada IN VIVO DIFFERENTIATION OF EPIGENETICALLY Complex networks of transcription factors regulate cardiac cell fate and REPROGRAMMED ENDOTHELIAL PROGENITOR morphogenesis, and dominant mutations in transcription factor genes lead CELLS INTO CARDIOMYOCYTES ENHANCES to most instances of inherited congenital heart defects (CHDs). The mecha- nisms underlying CHDs that result from these mutations is not known, but FUNCTIONAL AND ANATOMICAL POST- regulation of gene expression within a relatively narrow developmental INFARCT MYOCARDIAL REPAIR window is clearly essential for normal cardiac morphogenesis. In addition to transcription factors, epigenetic regulation via histone modifications, Thal, Melissa A., Krishnamurthy, Prasanna, Hoxha, Eneda, Lambers, chromatin remodeling, and non-coding RNAs have key roles in modulat- Erin, Verma, Suresh, Qin, Gangjian, Losordo, Douglas, Kishore, Raj ing gene expression programs. It is not known how these critical gene Feinberg School of Medicine, Northwestern University, Chicago, IL, USA regulation mechanisms specifically affect heart development, either directly Background: Currently, bone marrow derived endothelial progenitor cells or through interactions with transcription factor networks. We examined (BM-EPCs) are being used clinically to improve vascularization in patients the interplay between these interconnected mechanisms, primarily using with ischemic heart disease. While it is generally accepted that EPCs mouse models and ES cell-based in vitro models of cardiac differentiation. participate in vascular repair of the ischemic myocardium, there exists no To understand the function of chromatin-level regulation during differentia- convincing evidence that these cells are capable of trans-differentiating into tion, we utilized directed differentiation of ES cells towards cardiomyocytes, functional cardiomyocytes (CMC). Since ischemic heart disease leads to taking advantage of the stepwise differentiation of ES cells to precardiac substantial loss of CMC, cardiomyogenic plasticity of an existing autologous mesoderm, followed by mulitpotent cardiac precursors, and finally to highly cell therapy is of obvious import. EPCs and CMC both differentiate from purified beating cardiomyocytes. We combined with expression profiling and a common mesodermal progenitor cell however; during specific lineage genomic occupancy studies to generate an epigenetic map of the cardiac differentiation to EC phenotype CMC specific genes are epigenetically si- differentiation process. In parallel we examined the effect of deleting key lenced. We hypothesized that reprogramming of EPC using small molecules chromatin remodeling complex subunits in ES cells and in mice. Our results targeting key epigenetic repressive marks may recapitulate cardiomyogenic indicate a high degree of specificity for the function of chromatin remodel- potential in EPCs. Method and Results: Mouse Lin-Sca1+CD31+ BM cells ing complexes (exemplified by Brg1) and histone modifications (exemplified (EPCs) were sorted and treated with inhibitors of DNA methyltransferases by Ezh2) in regulating lineage decisions and differentiation programs during (5-Azacytidine), histone deacetylases (valproic acid) and G9a histone meth- the progressive stages of cardiac differentiation. yltransferase. Forty eight hour treatment led to the reactivation of pluripo- Poster Board Number: 1097 tency associated and CMC specific mRNA expression while EC specific genes were significantly up-regulated. When cultured under appropriate differen- SCREENING OF NOVEL CHEMICALS tiation conditions, reprogrammed EPCs showed efficient differentiation into EFFICIENTLY INDUCING CARDIOMYOCYTE CMC and vascular smooth muscle cells. Epigenetically, treatment of EPCs showed significant demethylation of Nkx2.5 promoter and hyper acetylation DIFFERENTIATION FROM PLURIPOTENT STEM of Histone 3 lysine 9 (H3-K9) and multiple H4 lysine residues. Intra-myo- CELLS cardial transplantation of reprogrammed eGFP-EPCs in an acute myocardial infarction mouse model showed significant improvement in LV functions Hiroyuki, Fukushima, Uosaki, Hideki, Jun, Yamashita K. compared to control EPCs and this was histologically supported by the de Department of Cell Growth and Differentiation, Center for iPS Cell novo CMC differentiation (GFP+sarcomeric actinin double positive) as well Research and Application, Kyoto University, Kyoto, Japan as increased capillary density and significantly reduced fibrosis. Conclusions: Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are Taken together, our results suggest that epigenetically reprogrammed EPCs promising cell sources for cardiac regenerative medicine. Previously, we es- display a more plastic phenotype and improve post-infarct cardiac repair by tablished a 2-dimentional culture-based sequential cardiovascular differenti- both neo-cardiomyogenesis and neovascularization. ation system from mouse ESC/iPSCs. This method is amenable to assess dif- Poster Board Number: 1101 ferentiation efficiencies at each differentiation stage (undifferentiated ESC/ iPSCs, mesoderms, cardiac progenitors, cardiomyocytes). Recently, chemical RGS4-LACZ EXPRESSION IS ASSOCIATED WITH biological approaches are starting to have an increasingly important role SPONTANEOUSLY BEATING CARDIOMYOCYTES in the field of stem cell biology. We reported that an immunosuppressant, cyclosporin-A, showed a novel potent cardiogenic effect specifically acting DURING SINOATRIAL NODE DEVELOPMENT on Flk-1 positive mesoderm cells to increase cardiomyocytes by 10 times. Yeung, Emily, Dubois, Nicole, Cifelli, Carlo, Keller, Gordon, Heximer, Here, we report an establishment of high-throughput screening (HTS) based Scott on our ESC cardiovascular differentiation system and chemical approach. Us- ing this HTS and mouse ES cells that carry α-myosin heavy chain promoter- Physiology, University of Toronto, Toronto, ON, Canada, McEwen Centre driven EGFP gene, we can accurately and efficiently identify chemicals for Regenerative Medicine, University Health Network, Toronto, ON, promoting cardiomyocyte differentiation from Flk-1 positive mesoderm. And Canada we successfully discovered several cardiomyocyte differentiation chemicals Parasympathetic-mediated hyperpolarization of sinoatrial-node (SAN) (CDCs) from chemical libraries. In a natural chemical library derived from myocytes results in a decreased heart rate. Regulator of G-protein signaling

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Thursday Poster Abstracts

4 (RGS4) is selectively expressed in SAN myocytes and has been shown to gel were seen in cells after stimulation. The contractions of the cells matched be an important regulator for parasympathetic signaling in those cells. Our the frequency of stimulation but settled back to original frequency when no study aims to determine the importance of RGS4 expression as a marker signal was present. Effects of stimulation on electrophysiological proper- of SAN cells during cardiac development. We here characterize changes in ties of cells could not be seen after stimulation. However, gene expression RGS4 expression in the heart through various stages of development in the of some cardiac proteins were stronger in stimulated cells than in controls mouse and mouse embryonic stem cells (mES cells). We hypothesize that which may indicate some cell maturation. In summary, surface topography RGS4 is highly expressed in early autorhythmic cardiomyocytes derived was a stronger determinant of cardiomyocyte orientation than the electrical from cardiac neural crest and mesenchyme progenitor and thus will be a field stimulation and therefore microarchitecture of the materials should be useful marker of both immature and mature SAN myocytes. Using the ES examined more in case of cell orientation. Instead, electrical stimulation and cell culture system and the RGS4-LacZ-gene-trap genetic mouse model, its duration seem to have some effect on cell maturation and this stimulation we have investigated the expression of RGS4 during mouse heart develop- system may be useful tool in the future when studying for example cardiac ment as well as during the differentiation of mES cells. RESULTS: 1.) Mouse differentiation of stem cells. hearts were collected starting at day E8.5 to P4.5, and were stained for LacZ expression. High expression of RGS4 was observed selectively in the SAN Poster Board Number: 1105 area starting at E11.5, whereas earlier time points showed ubiquitous RGS4 VESSEL-DERIVED STEM CELLS FOR THERAPY expression 2.) RGS4-LacZ mES cells were differentiated to cardiovascular lineage using a serum-free differentiation protocol. Cardiac monolayers OF SKELETAL AND CARDIAC MUSCLE IN consisting of cardiomyocytes, vascular smooth muscle cells, fibroblasts and DUCHENNE MUSCULAR DYSTROPHY endothelial cells were stained with LacZ. RGS4 expression was exclusively associated with cells located in contracting areas of the culture and strongly Berry, Suzanne E., Chun, Ju Lan, O’Brien, Robert, Wang, Lei, correlated with cardiac troponin-T staining. To ensure that RGS4 expres- Kamath, Anant sion corresponded to SAN type cells, immunohistochemistry for HCN Comparative Biosciences, University of Illinois, Urbana, IL, USA, Animal was performed in parallel with the lacZ stain. Together, this indicated that Sciences, University of Illinois, Urbana, IL, USA, Veterinary Clinical RGS4 expression is strongly associated with autorhythmic and/or beating Medicine, University of Illinois, Urbana, IL, USA, Cellular Engineering cardiomyocytes during development. Recently, RGS4-GFP was cloned into Technologies, Coralville, IA, USA E14 mES cells for additional studies with RGS4 expression and the SAN type cells. Further studies will be carried out to establish the nature of these cells and whether their isolation can be used to selectively induce specialized Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease SAN-like tissues in vitro and in vivo. affecting 1 in every 3500 boys born, and resulting in death of most patients before their third decade of life. DMD affects both skeletal and cardiac Poster Board Number: 1103 muscle. Although many studies have been done to repair and regenerate skeletal muscle in DMD, very few studies have focused on regenerative THE EFFECT OF ELECTRICAL FIELD therapy for the heart. DMD patients are living longer, as a result of home STIMULATION ON CARDIOMYOCYTE GROWTH ventilation and spinal fusion, and therefore greater numbers of patients are developing cardiomyopathy in the later stages of disease. We are using ON COLLAGEN GEL AND 2D-COATING myogenic, vessel-derived stem cells known as mesoangioblasts (ADM) for Kujala, Kirsi, Ahola, Antti, Kerkelä, Erja, Pekkanen-Mattila, Mari, therapy of both skeletal and cardiac muscle in animal models for DMD. We Aalto-Setälä, Katriina, Hyttinen, Jari have characterized the development of dilated cardiomyopathy in the mdx/ utrn-/- dystrophin and utrophin-deficient mouse model for DMD, finding it Institute of Biomedical Technology, University of Tampere, TAMPERE, is similar to patients, and we are currently examining whether ADM improve Finland, Department of Biomedical Engineering, Tampere University heart function or replace damaged cardiomyocytes in cardiac muscle in of Technology, TAMPERE, Finland, Institute of Biomedical Technology, this model. ADM survive up to 10 weeks following transplantation into the University of Tampere & Heart Center, Tampere University Hospital, heart, and some donor cells express cardiac proteins including troponin I TAMPERE, Finland and tropomyosin. Wild-type donor ADM also promote modest expression The ultimate goal in cardiac tissue engineering is to generate a heart muscle of dystrophin in the heart. Using echocardiography to study heart function with morphology of natural myocardium which means that its functional after ADM transplantation, we have observed an improvement in ejection properties are directly related to the cellular orientation and elongation. fraction and fractional shortening when cells are transplanted prior to onset However, the challenge in culturing cardiomyocytes is directing the cells to of cardiac dysfunction. Transplantation of ADM after the onset of cardiomy- establish the physiological structure and function of the tissue. Electrical field opathy in mouse models for DMD does not result in improvements in func- stimulation has been shown to improve cell differentiation, alignment and tional parameters, but prevents thinning of the ventricular wall characteristic functional properties. Here we studied the effect of electrical field stimula- of dilated cardiomyopathy. Our data indicate that vessel-derived stem cells tion on neonatal rat cardiomyocytes. A novel platform for electrical stimula- may therefore by good candidates for treatment of both skeletal and cardiac tion was designed for this study where cells were exposed both to long term muscle pathology in DMD patients. stimulation and short term pacing experiment. The goal of this study was to investigate whether it is possible to achieve cell orientation and matura- tion of cardiomyocytes and improve cells’ functional properties by electrical field stimulation. Cells were cultured on gelatin-coated microelectrode array chambers and on collagen gel. Microelectrode array technology (MEA) was used to determine electrophysiological properties of cells. Morphologi- cal properties were analyzed by microscoping and live/dead staining and protein and gene expressions were characterized by immunostaining and by polymerase chain reaction (PCR). Cells were viable after electrical field stim- ulation, but no orientation or other morphological changes were seen after stimulation. However, in some samples cell orientation was seen due to elec- trode lines in MEA dishes. Cells grew well on collagen gel and it adjusted to beating of cardiomyocytes but no differences between coating and collagen

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ENDOTHELIAL CELLS/HEMANGIOBLASTS Poster Board Number: 1109 Poster Board Number: 1107 SYNTHETIC PEPTIDE-ACRYLATE SURFACE FOR LONG TERM EXPANSION AND ENDOTHELIAL OPTIMIZATION OF A DERIVATION NICHE DIFFERENTIATION OF HUMAN INDUCED TO ELUCIDATE THE LINK BETWEEN HUMAN PLURIPOTENT STEM CELLS PLURIPOTENT STEM CELL ORIGIN AND Abdul Jalil, Rufaihah, Melkoumian, Zara, Hunter, Arwen L., Cooke, ENDOTHELIAL DIFFERENTIATION CAPACITY John P. Kusuma, Sravanti, Tan, Siah Hong, Mali, Prashant, Cheng, Linzhao, Cardiovascular Medicine, Stanford University Stanford, CA, USA, Corning Gerecht, Sharon Incorporated, Corning, NY, USA John Hopkins University Baltimore, MD, USA Human induced pluripotent stem cells (hiPSCs) hold great promise as a Recreating functional vasculature is a pivotal step in the development of source of differentiated cells for vast therapeutic implication. However, novel therapies in the field of regenerative medicine, providing innova- in order for hiPSCs and their derivatives to be clinically relevant, a robust tive treatment options for patients suffering from vascular disorders, and feeder-free culture conditions using chemically defined raw materials would generating functional and transplantable tissues that have been engineered be favorable. Here, we report the use of synthetic, xeno-free peptide- in vitro. Endothelial cells (ECs), which comprise the inner lining of the vas- acrylate surface, Corning® Synthemax™ Surface that supports expansion culature, are critical cells to these endeavors. Because vascular cells derived of hiPSCs in a chemically defined medium and directed differentiation into via biopsies may be diseased or have low proliferative capacity, research- endothelial cells (ECs). Synthemax surface was prepared by deposition of ers have turned to human pluripotent stem cells (hPSCs) as an unlimited carboxylic acid containing acrylate onto 6-well plates, followed by conjuga- source of progenitors from which vascular cells may be derived. Controlled tion of amine-containing cell adhesion-promoting peptide, derived from vit- and robust differentiation of hPSCs toward vascular lineages is critical for ronectin. hiPSCs were grown on Synthemax surface using mTeSR1 medium the advancement and future of patient-specific vascular therapeutics. Here and passaged routinely. Characterization of hiPSCs was performed using we use a two-dimensional, feeder-free platform to examine differentiation gene expression and immunostaining of pluripotency markers. Proliferation of hPSCs, i.e. human embryonic stem cells (hESCs) and human induced and viability of hiPSCs grown on Synthemax surface were assessed using pluripotent stem cells (hiPSCs), toward ECs and determine whether the cell trypan blue and the Live/Dead cell assay. Endothelial lineage differentiation origin type of hiPSCs affects endothelial differentiation. Previous derivation was initiated by adding serum-free differentiation medium supplemented techniques comprise co-culture with mouse cells, which is not clinically rel- with BMP-4, activin-A, bFGF and VEGF-A for the first 7 days of differentia- evant, or embryoid body formation, which can yield inconsistent results. Our tion and VEGF-A and TGF-β inhibitor, SB431542, from day 7 to day 14 of differentiation protocol overcomes these limitations and allows us to study differentiation. Real time gene expression analysis and immunostaining of multiple aspects of EC differentiation under controlled cues. We differenti- endothelial markers were performed. Functional EC characterization, such as ated hESCs and hiPSCs in monolayer in media supplemented with 10% fetal Matrigel tube formation, cell migration assay and in vivo Matrigel plug as- bovine serum to guide differentiation toward the mesoderm lineage. These say were also carried out. hiPSCs were successfully expanded on Synthemax cells were subsequently cultured in endothelial growth media supplemented surface for ten serial passages. Cells maintained characteristic morphol- with vascular endothelial growth factor (VEGF) to induce differentiation into ogy, expression of pluripotency markers, stable proliferation rate and high ECs. However, this differentiated population was hetereogenous in marker viability throughout the ten passages. Furthermore, Synthemax surface expression with only a small subset expressing endothelial markers such supported directed differentiation of hiPSC to ECs with typical cobbled- as vascular endothelial cadherin and VEGF receptor-2. Consequently, we stone morphology and expression of EC-specific markers, such as platelet examined various factors of the stem cell differentiation niche - specifically, endothelial cell adhesion molecule (CD31), vascular endothelial cadherin substrate compositions and biochemical components of the differentiation (CD144), endothelial nitric oxide (eNOS) and von Willebrand factor (vWF). media - to elucidate their effect on endothelial specification. The kinet- ECs functionality was confirmed by incorporation of acetylated low-density ics of endothelial marker expression and the differentiation products were lipoproteins and formation of tube-like structures in Matrigel in vitro and analyzed using RT-PCR, flow cytometry, and immunocytochemistry. These capillaries in Matrigel plugs in vivo. This study shows the feasibility of using results, as well as in vitro functionality assessments, confirmed the pheno- fully defined, xeno-free, synthetic surface for sustained culture of hiPSCs type of derived ECs. Finally, we used our differentiation niche to determine and directed differentiation into functional ECs, which can be useful for both the effect that cell origin (i.e. fibroblast, cord blood, or bone marrow) of research and clinical applications. hiPSCs has on endothelial differentiation. Our findings reveal that microen- Poster Board Number: 1111 vironmental cues influence EC differentiation efficiency and that these cues can be incorporated into a differentiation niche to understand the effect of DO HUMAN ENDOTHELIAL PROGENITOR hiPSC cell origin on endothelial specification. CELLS PARTICIPATE IN VASCULOGENESIS DURING FETAL DEVELOPMENT? Chong, Mark, Ng, Darice J., Choolani, Mahesh A., Chan, Jerry Dept of Obstetrics & Gynaecology, National University of Singapore, Singapore, Singapore Aim: Endothelial progenitor cells (EPC), first discovered in adult peripheral circulation, are found in cord blood at higher frequencies, and with greater proliferative capacity. We hypothesise that EPC in fetal circulation may play a crucial role in vasculogenesis during development. Here, we present data on the characterisation of fetal blood-derived EPC, as compared against their umbilical cord blood (UCB) derived counterparts, and evidence to sug- gest a role for circulating EPC in fetal vasculature development. Methods: EPC were isolated from umbilical cord blood and mid-gestation fetal blood

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Thursday Poster Abstracts by adhesion selection in endothelial growth media. Following expansion, differentiate them to the cardiovascular lineage in order to understand the the cells were assessed by immunocytochemistry (ICC) for expression of underlying molecular pathology of MFS cardiovascular defect in humans. endothelial markers. Colony forming capability was assessed. As a test of Methods and results: We generated an hESC line from an embryo deter- functionality, Matrigel differentiation assay was carried out to compare the mined by pre-implantation genetic diagnosis to have inherited a deletion vasculogenic potential of the cell sources. Subsequently, cells were injected resulting in a premature termination in the 5’ region of the FBN1 gene and into a murine model of hindlimb ischaemia to study the ability to effect conferring MFS. We also derived three hiPSC lines by reprogramming of rescue. Finally, genome-wide microarray analysis was carried out to compare skin fibroblasts of MFS patients with clinically important FBN1 mutations: (i) the expression profiles of both cell populations and identify significant gene one mutation causing a premature termination in the 5’ region of the FBN1 ontologies associated with vascular development. Results: Fetal EPC (fEPC) gene conferring a severe aortic defect, (ii) one mis-sense mutation resulting were found to be capable of generating endothelial-like progeny as assessed a cysteine substitution leading to a mild aortic defect, and (iii) one mutation by ICC. In comparison to UCB-EPC, fEPC were found to be more morpho- in the region of exons 24-32, which is associated with the severe neonatal logically heterogeneous, and FACS analysis found fEPC to express lower lev- form of MFS. All cell lines were fully characterized for the expression of els of CD31 (6.95 fold), CD34 (3.36 fold), CD133 (2.29 fold), HLA-1 (1.96 pluripotency markers, teratoma formation, normal karyotype, confirmation fold) and HLA-2 (79.2 fold). fEPC proliferate 1.6 times faster, and were of mutation in the FBN1 gene) and have been further differentiated in vitro found to have higher colony-forming potential, with 29.3% of fEPC capable and in vivo into vascular progenitors (VPCs), endothelial cells (ECs), smooth of generating colonies. In addition, 60% of these colonies were found to be muscle cells (SMCs) and fibroblasts. By comparison with non disease hESC greater than 5 mm in diameter, as compared to none in the UCB-EPC group. and iPSC progenies, we studied the effect of different classes of mutations In spite of 92.3% of fEPC colonies being capable of taking up acetylated on the dysregulation of cardiovascular development and function, and LDL (suggesting endothelial nature) the cells appear to transdifferentiate TGF-beta signaling. Conclusions:We described an in vitro ESC/IPSC model following extended culture, adopting a more spindle-shaped morphol- of MFS, hopefully paving the way for the discovery and evaluation of new ogy. Again, this was not observed in UCB EPC. When placed in Matrigel, drug candidates for the treatment of this disease. fEPC were found to be highly vasculogenic, and established more complex, macroscopic networks capable of secondary aniogenic sprouting. Increased Poster Board Number: 1115 functionality was also demonstrated in the ability to rescue ischaemia, with DIFFERENTIATION OF HUMAN INDUCED fEPC capable of restoring perfusion levels to 56% of normal within 16 days (Saline: 26.8%, UCB-EPC: 37.8%). Through microarray analysis, 2123 PLURIPOTENT STEM CELLS INTO FUNCTIONAL genes were found to differentially regulated by two-fold. Gene ontology CD34+ CELLS BY MODULATING MEK/ERK AND analysis revealed 56 of these genes to be known to participate in angiogen- esis and vascular development. Pathway analysis suggests the involvement BMP4 SIGNALING PATHWAYS of six pathways, including the NOTCH, TGFBR and Wnt pathways, which Park, Sang-Wook, Koh, Young Jun, Jeon, Jongwook, Choi, Chulhee, represent key pathways in stem cell fate determination. Conclusion: This Koh, Gou Young, Han, Yong-Mahn study demonstrates that EPC isolated from mid-gestation fetal circulation. KAIST, Daejeon, Korea, Republic of fEPC are phenotypically and functionally different from UCB-EPC. The more plastic nature and greater propensity for colony formation and proliferation Differentiation of human induced pluripotent stem cells (hiPSCs) into func- suggests that fEPC are more primitive. fEPC were found to be more vascu- tional cell types is a crucial step in stem cell therapy. Although hiPSCs are logenic in both in vitro and in vivo assays. Finally, microarray data identifies able to differentiate into many different cell types, differentiation of hiPSCs several upregulated genes associated with vascular development, further into a specialized cell type is still inefficient. Here, we demonstrate that indicating a role for fEPC in the developing fetus. hiPSCs could efficiently differentiate into functional CD34+ cells on a feeder- free system by regulating two signaling pathways. Inhibition MEK/ERK sig- Poster Board Number: 1113 naling and activation of BMP signaling synergistically enhanced expression HUMAN EMBRYONIC STEM CELLS AND of mesoderm-lineage markers in hiPSCs. The mesoderm-lineage cells could be further developed into CD34+ cells with high efficiency (10~20%) after HUMAN INDUCED PLURIPOTENT STEM CELLS culturing in the endothelial culture medium supplemented with VEGF-A FOR STUDYING THE CARDIOVASCULAR and bFGF for 9 days. After CD34+ magnetic sorting, hiPSC-derived CD34+ cells could differentiate into endothelial cells, but not hematopoietic cells. DEFECT OF MARFAN SYNDROME Moreover, they contributed directly to neovasculogenesis in ischemic mouse Marchand, Melanie, Anderson, Erica, Chiao, Eric T., Leonard, Brian, hindlimbs, thereby resulting in improved blood perfusion and limb salvage. Nguyen, Ha Nam, Francke, Uta, Reijo Pera, Renee A., Longaker, Our results indicate that combined modulation of signaling pathways is Michael T. effective on differentiation of hiPSCs into functional CD34+ cells. This work was supported by the Stem Cell Research Center of the 21st Century Fron- Stem Cell Biology and Regenerative Medicine, Stanford University, tier Research Program (SC2210). Stanford, CA, USA, Pluripotent Stem Cell Lab, Hoffmann-LaRoche, Nutley, NJ, USA, 3Johns Hopkins University, Baltimore, MD, USA, 4Genetics, Stanford University, Stanford, CA, USA, 5Surgery - Plastic and Reconstructive Surgery, Stanford University, Stanford, CA, USA Introduction: Marfan Syndrome (MFS) is a hereditary aortic aneurysm disorder caused by mutations in the Fibrillin 1 (FBN1) gene. Without treat- ment, individuals with MFS are at risk of death from dissection or tearing of the aorta. Both human Embryonic Stem Cells (hESCs) derived from human embryos and induced Pluripotent Stem Cells (iPSCs) generated by reprogramming of somatic cells offer promise for regenerative medicine and drug discovery. When generated from individuals with diseases, they represent a new cell model to study the mechanisms of the disease and to test drug treatments. Objectives: Our objectives were to generate MFS patient-specific ESC and iPSC lines with unique mutations in Fbn1 gene and

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Poster Board Number: 1117 Poster Board Number: 1119 MAJOR ROLE FOR P-SELECTIN DURING INDUCTION OF LYVE-1/STABILIN-2-POSITIVE HOMING OF TRANSPLANTED MOUSE LIVER SINUSOIDAL ENDOTHELIAL CELLS BONE MARROW MONONUCLEAR CELLS TO FROM MOUSE EMBRYONIC STEM CELL- ISCHEMIC HINDLIMB DERIVED EMBRYOID BODIES BY MODULATION Almeida, Brígida G., Castro, Tiago B. R., Menezes, Gustavo G., OF ADRENOMEDULLIN-RAMP2 SIGNALING Paula, Ana M., Marques, Fernanda F., Andrade, Silvia P., Pinho, Arai, Takuma, Sakurai, Takayuki, Kamiyoshi, Akiko, Ichikawa- Vanessa, Teixeira, Mauro M., Barcelos, Lucíola S. Shindo, Yuka, Koyama, Teruhide, Yoshizawa, Takahiro, Uetake, UFMG, Belo Horizonte, Brazil Ryuichi, Yamauchi, Akihiro, Okimura, Ayano, Kawate, Hisaka, Introduction: The stimulation of vascular growth by angiogenic stem cells Ogawa, Shinichiro, Miyagawa, Shinichi, Shindo, Takayuki transplantation is a promising alternative for the treatment of Peripheral Surgery, Shinshu University Graduate School of Medicine, Matsumoto, Artery Disease. Efficient migration to ischemic limbs is a prerequisite for Japan, Organ Regeneration, Shinshu University Graduate School of successful therapy. Adhesive mechanisms governing progenitor/stem cell Medicine, Matsumoto, Japan trafficking through the microcirculation of ischemic muscles are broadly un- Background and Aims: Embryonic stem cells (ESCs) are a useful source for known. Here, we evaluated the relevance of selectins for transplanted bone various cell lineages, including hepatocytes. So far, however, progress to- marrow-derived progenitor/stem cells-endothelium interactions in a model ward reconstitution of mature liver morphology and function has been lim- of limb ischemia. Hindlimb ischemia was induced by permanent occlusion ited. We have shown that knockout mice deficient in adrenomedullin (AM) of the femoral artery of C57Bl/6 mice. The method of sodic fluorescein or its receptor-activity modifying protein (RAMP2) die in utero due to poor diffusion into the bloodstream after plantar cushion injection was used for vascular development and hemorrhage within the liver. In this study, using evidence of ischemia. Rhodamine-labeled circulating cells-endothelium embryoid bodies (EBs)-culture system, we induced liver sinusoidal endothe- interactions were analyzed by intravital fluorescence microscopy (IVFM). lial cells (LSECs) by modulation of AM-RAMP2. Methods and Results: In an In another experimental setting, bone marrow-derived mononuclear cells EB-derived mesodermal/endodermal co-differentiation system, we found (BM-MNC) obtained from GFP transgenic mice were i.v. injected 24h post- that co-administration of AM and SB431542, an inhibitor of TGF-β receptor ischemia and just before IVFM recording. Results: The fluorescein peak in type 1, markedly enhanced differentiation of LYVE-1/stabilin-2-positive the systemic circulation before arterial occlusion occurred at 7.5±1.6 min endothelial cells. These cells possess fenestrae-like structure, a key morpho- and increased to 30±0.1 min just after occlusion (p<0.001, n=5/group), logical feature of LSECs, and also showed robust endocytosis of Ac-LDL and confirming the arterial Methods: occlusion. By using IVFM, we observed upregulated expression of LSEC-specific markers, including F8, Fcgr2b, and that the number of circulating cells rolling and adherent to the endothe- Mrc1. At later phase of EB culture, LYVE-1/stabilin-2-positive endothelial lium of the quadriceps muscle was higher at days 1 and 3 post-ischemia cells made contact with albumin-positive hepatocytes and expression of when compared with sham-operated animals (p<0.01 for rolling, n=5-7/ mature hepatocyte-specific genes was upregulated. In RAMP2-null liver, by group; p<0.01 for adherence at day 1 and p<0.05 at day 3, n=5-7/group). contrast, LYVE-1 was downregulated in LSECs, and the sinusoidal struc- No significant differences were observed in an earlier (12h) or later (7d) ture was disrupted. Conclusions: Our findings highlight the importance of time point. Similar results were obtained when analyzing gastrocnemius reconstituting the organ-specific vasculature for achievement of mature liver muscle. After i.v. transplantation of bone marrow-derived angiogenic stem/ regeneration, as well as the potential utility of AM-RAMP2 as a therapeutic progenitor cells, we observed a greater number of rolling and adherent target. We suggest that these results could serve as the basis for develop- GFP BM-MNC when compared with sham animals (p<0.01 and p<0.05, ment of techniques for the regeneration of liver, and could also be useful in respectively, n=6/group). This behavior was significantly reduced after i.v. a variety of other medical and research applications, including bioartificial injection of fucoidan (10 min before transplantation), a pan-selectin inhibi- liver systems. tor, when compared with untreated ischemic animals (p<0.05 and p<0.001 for rolling and adhesion, respectively, n=6/group). After i.v. injection of an Poster Board Number: 1121 anti-E-selectin blocking antibody (10 min before transplantation), we ob- served a significant reduction in rolling and adhesion when compared with DEVELOPMENT OF A HIGH-THROUGHPUT AND control group (p<0.05 and p<0.01, respectively, n=5/group). Interestingly, ROBUST VASCULAR DIFFERENTIATION ASSAY after P-selectin blockade, we observed a greater reduction in rolling (p<0.01 vs. both control and anti-E-selectin, n=5/group). Nevertheless, we did not USING MOUSE EMBRYONIC STEM CELLS observe any adhesion of transplanted cells after P-selectin blockade. Finally, Hammoud, Lamis, Rossant, Janet the blockade of host selectins prevented the reparative angiogenesis induced Developmental and Stem Cell Biology Program, Hospital for Sick Children, by BM-MNC transplantation as analyzed by histomorphometry of adduc- Toronto, ON, Canada, Developmental and Stem Cell Biology Program and tor sections. Conclusions: Our results suggest a possible behavior of rolling Department of Molecular Genetics, Hospital for Sick Children, Toronto, of transplanted BM-MNC cells to host-dependent selectins and uncover a ON, Canada major role for P-selectin. Support: FAPEMIG/CNPq. VEGF, acting through the Flk1/VEGFR2 receptor, is crucial for blood vessel formation and development. Many events that occur during embryonic vas- cular development are recapitulated during adult neo-angiogenesis, which is critical to tumour growth and metastasis. While the latest anti-angiogenic drugs, such as avastin (anti-VEGF), have been shown to prolong life expectancy in cancer patients, they have serious side effects. Furthermore, relapses often occur necessitating the need for novel therapeutic targets. The aim of this study is to optimize an in vitro vascular differentiation assay in a 96-well format that can be used in high-content genome-wide screens to identify and characterize novel genes modulating angiogenesis. Embryoid bodies (EBs) were formed by aggregating mouse embryonic stem

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Thursday Poster Abstracts cells (ES), containing a Flk1-eGFP reporter, in suspension using the hanging Poster Board Number: 1125 drop method. After 4 days, EBs were embedded in a 3 dimensional collagen type I gel in the presence of medium supplemented with 10% FBS and one HEMATOGENIC ENDOTHELIUM DERIVED or more of the 4 previously established angiogenic growth factors (VEGF, FROM EMBRYONIC STEM CELLS IN RHESUS bFGF, IL-6 and EPO), or in the absence of any factors (control). These were allowed to grow for 6-7 days in the collagen matrix, with Flk1 positive MONKEY sprouts becoming visible by day 4 and showing an increase in both number Lu, Bin and complexity by day 6-7. Our results showed that the mean number Kunming Institute of Zoology Chinese Academy of Sciences, Kunming, of Flk1 positive sprouts was significantly higher in the presence of growth China factors when compared with control. However there was no significant dif- ference in the mean number of Flk1 positive sprouts between VEGF treated The early stage of hematopoiesis during embryo development is impor- EBs and those treated with 2, 3 or the 4 aforementioned growth factors, tant in embryo development, but it is not so clearly in primates. Recently, suggesting that VEGF alone can account for the majority of the angiogenic a progenitor known as hematogenic endothelium has been identified in response induced by the growth factor combinations. This assay was further mice, which bears endothelial characters and hematogenic potential. In the validated using a gamma secretase inhibitor (L685458), which is known to present study, we established steady hematopoietic differentiation system induce excessive angiogenic sprouting and a Flk1 inhibitor (SU5416) known from rhesus monkey embryonic stem cells (rESC) with OP9 co-culture feeder to inhibit angiogenic sprouting. Our results showed that L685458 signifi- cells. Three different hematopoietic progenitors including hematogenic cantly increased the mean number of Flk1 positive sprouts in VEGF treated endothelium were obtained and identified in determinate time points with EBs, while SU5416 resulted in a significant decrease in angiogenic sprouts. identifiable markers. Briefly, on d4-5, the KDR+ hemangioblast cells were Furthermore, the cellomics array system was used to measure overall derived, which could be differentiated into early embryonic erythrocytes fluorescent intensity and extent of sprouting, both of which were higher in (γglobins+εgoblins+βglobins-) and endothelial lineage. On d8, KDR++(high) the L685458+VEGF treated EBs as compared to control, or SU5416+VEGF CD31+ hematogenic endothelium were purified, which were more matured treated EBs. In conclusion, we have developed and validated a robust vascu- progenitor and could be differentiated into most hematopoietic lineages and lar differentiation assay from mouse ES cells in a 96-well format. This assay endothelial lineage. Finally, on d14, we gained CD34+CD41+ hematopoietic can now be used to screen for novel modulators of angiogenesis. stem cells, which could be developed into all hematopoietic lineages. The results are firstly confirmed determinate markers and time point to identify Poster Board Number: 1123 the hematogenic endothelium derived from primate ESCs according to our IDENTIFICATION OF THE EMERGENCE OF knowledge. Furthermore, our study also demonstrated that reducing Notch signal pathway promote hematopoietic differentiation obviously. However, CLONALLY MULTIPOTENT HEMATOPOIETIC the more details of molecular regulation on hematogenic endothelium STEM AND PROGENITOR POPULATIONS IN THE development need further study. MOUSE EMBRYO Poster Board Number: 1127 Inlay, Matthew A., Serwold, Thomas, Mosley, Adriane, Weissman, SCL/TAL1 IS REQUIRED TO INHIBIT Irving L. CARDIOGENESIS FROM ENDOTHELIUM IN ISCBRM, Stanford University, Stanford, CA, USA, Joslin Diabetes Center, Boston, MA, USA HEMATOPOIETIC TISSUES In the adult hematopoietic system, all mature blood cells derive from the he- Montel-Hagen, Amelie, Van Handel, Ben, Ferrari, Roberto, matopoietic stem cell (HSC). However, HSCs are not the first blood-forming Sasidharan, Rajkumar, Nakano, Haruko, Org, Tonis, Zhou, Jamie, cells to appear during embryogenesis. Rather, erythrocyte-biased progenitors Li, Xinmin, Pellegrini, Matteo, Orkin, Stuart H., Nakano, Austin, can be identified in the yolk sac (YS) blood islands approximately a week af- Kurdistani, Siavash K., Mikkola, Hanna K.A ter fertilization. After the establishment of circulation, fully functional HSCs Department of Molecular, Cell and Developmental Biology, UCLA, Los can be identified within the aorta-gonad-mesonephros (AGM) and fetal liver Angeles, CA, USA, Department of Biological Chemistry, UCLA, Los Angeles, (FL) tissues. The origin and maturation of HSCs in embryonic development CA, USA, Department of Pathology and Laboratory Medicine, UCLA, Los remains an open and important question. To help address this issue, we Angeles, CA, USA, Dana-Farber Cancer Institute, Boston, MA, USA sought to identify and isolate the blood-forming populations within these embryonic tissues at the stages when hematopoietic potential first appears. Hematopoietic stem and progenitor cells originate from hemogenic Using a modified OP9 stromal line that can yield 8 hematopoietic lineages endothelial precursors from the major blood vessels in the embryo and ex- (B, T, NK, DC, granulocyte, macrophage, platelet, and erythrocyte), we iden- traembryonic tissues. However, it is not known how this unique endothelium tified populations with multipotent potential at the clonal level in all tissues. is specified. Here we show that the establishment of the hematopoietic tran- While these embryonic multipotent cells share many of the surface markers scriptional program in the endothelium is driven by the bHLH factor Scl/tal1. that define adult HSCs, there are also distinct tissue-specific differences. Fur- Furthermore, the failure to express Scl leads to ectopic cardiomyocyte differ- thermore, these populations lack the ability to engraft into adult irradiated entiation from endothelium in hematopoietic tissues. Ectopic cardiogenesis in recipient mice, indicating their development into mature HSCs is incomplete. Scl deficient embryos was associated with a dramatic upregulation of cardiac Our data support a model by which the ability to generate all hematopoietic transcription factors and structural proteins in the yolk sac endothelium. lineages emerges prior to the ability to engraft. Consistent with the gene expression, we found cells co-expressing CD31, an endothelial marker, and cardiac specific troponins in the vasculature of mutant yolk sacs. Strikingly, lack of Scl resulted in generation of spontane- ously beating cardiomyocytes in the yolk sac. CD31+PDGFRα+ cardiac precursor cells accumulated in Scl-deficient embryos in all hematopoietic tissues and the heart, but not in the head region, demonstrating widespread, yet anatomically restricted, misspecification of embryonic endothelium. These results reveal a surprising developmental plasticity in the endothelium of embryonic hematopoietic tissues such that lack of a single transcription factor, Scl/tal1, can lead to the conversion of hemogenic endothelium to the

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cardiogenic fate. extracted and cultured in differentiation medium. In vivo, single GFP+ blast colonies were transplanted into a lethally irradiated wild-type recipients. Poster Board Number: 1129 At 12 weeks and 12 months after reconstitution, the blood, bone marrow, OVER EXPRESSION OF DOMINANT spleen and other organs were harvested and the stemness and long-term self-renewal potential of the transplanted blasts were evaluated by flow NEGATIVE RETINOIC ACID RECEPTOR-ALPHA cytometery, immunofluorescent staining, spleen colony assay and colony- (DNRARΑ) IMMORTALIZES EMBRYONIC forming unit assay. The source of the hemangioblasts was determined by bone marrow-uterine tracking. Results: The CD34+/c-kit- uterine cells in HEMANGIOBLAST-LIKE PRECURSORS WITH blast cell differentiation media differentiated into two phenotypically distinct HEMATOPOIETIC AND ENDOTHELIAL CELL cell types: adherent and non-adherent. The adherent cells assumed endothe- DEVELOPMENTAL POTENTIAL lial cell characteristics with a high expression of FLK-1, CD31 and factor VIII as well as uptake of acetylated-LDL. Non adherent cells expressed he- Ortiz, Mariaestela, Salcedo, Rosalba, Tsai, Schickwann, Keller, matopoietic cell markers, such as GATA 1, β-H1, and β-Major globin as well Jonathan as leukocyte markers: CD45, CD13/CD16/Mac3, and CD4/CD8. In the in Basic Research Program, SAIC-Inc., SAIC Frederick, and the Pediatric vivo study, single GFP+ blast colonies were transplanted into lethally irradi- Oncology Branch, Center for Cancer Research, National Cancer Institute, ated wild-type recipients and GFP+ cells were identified in the hematopoietic and Division of Hematology, University of Utah, Frederick, MD, USA, compartments in 29 of 32 the recipients after 12 weeks and 12 months. At Division of Hematology, University of Utah, Salt Lake City, UT, USA, 12 weeks, 1-2% of the cells in the recipient BM, blood and spleen expressed The Anne McLaren Laboratory for Regenerative Medicine, University of GFP detected by FACS analysis. At 12 months, approximately 1% cells in Cambridge, Cambridge, United Kingdom, Basic Research Program, SAIC- the hematopoietic compartments were GFP+. These GFP cells also expressed Inc., SAIC Frederick, and the Pediatric Oncology Branch, Center for Cancer hematopoietic lineage markers at both 12 weeks and 12months. GFP+ Research, National Cancer Institute, Frederick, MD, USA colony-forming units were observed 7 days after the recipient BMCs were cultured in the MethoCult media. Vascular differentiation was demonstrated Hematopoietic and endothelial cells arise from a common precursor, the by the colocalization of GFP and factor VIII one year after reconstitution in hemangioblast, in the developing embryo. Our current understanding of the the vascular structures of several organs. A second reconstitution was per- molecular and cellular mechanism(s) that regulate hemangioblast develop- formed with GFP+ CD34+/c-kit-blast colony cells from mice transplanted 12 ment is limited due to the scarcity of this cell population. Over expression weeks or 12 months earlier and GFP+ cells were identified in spleen nodes of a dominant negative form of the retinoic acid receptor alpha (dnRARα) in the secondary recipients. Conclusions: We identified adult hemangioblasts in bone marrow cells imposes a developmental block and immortalizes (CD34+/c-kit-) residing outside the BM, with the capacity for bilineage dif- primitive hematopoietic progenitor cells. Therefore, we evaluated if over ferentiation into hematopoietic and vascular cells. This clonogenic cell was expression of dnRARα could induce a developmental block and immortalize capable of long-term self-renewal and hematopoietic reconstitution. These cells from the yolk sac (YS) and aorta gonad mesonephros (AGM) region at results provide the first proof of an extramedulary reservoir of hemangio- an earlier stage of development. Bi-potential stem cell factor (SCF) -depen- blast stem cells in the adult. dent YS- and AGM- progenitor cell lines (PCLs) were immortalized, which express hematopoietic cell surface markers (c-Kit, Sca-1, CD34, PECAM-1, Poster Board Number: 1133 and CD41), and endothelial markers (MECA-32, VE-Cadherin, Flk-1, Tie-2, CD-105, endothelium VonWillebrand factor, and incorporated Dil-Ac-LDL). ANGIOGENIN-1 IS NECESSARY FOR THE YS- and AGMPCLs could be induced to differentiate into neutrophils, mac- GENERATION OF ENDOTHELIAL PROGENITOR rophages, erythrocytes, megakaryocytes, and endothelial cells in vitro and CELL COLONIES vascular networks in vivo. Thus, dnRARα promotes a developmental block in bi-lineage hematopoietic/endothelial progenitors, which provides ad- U-pratya, Yaowalak, Sudchada, Sakchai, Kheolamai, Pakpoom, ditional evidence that these distinct cell lineages share a common ancestor, Supokawej, Aungura, Manochantr, Sirikul, Tantrawatpan, Chairat, and represents a novel model to examine the mechanism(s) that regulate Issaragrisil, Surapol hematopoietic/endothelial cell fate decisions. Division of Hematology, Department of Medicine, Faculty of Medicine Poster Board Number: 1131 Siriraj Hospital, Mahidol University, Bangkok, Thailand, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, ADULT UTERINE HEMANGIOBLASTS: Bangkok, Thailand, Division of Cell and Molecular Biology, Department EXTRAMEDULLARY SELF-RENEWING AND of Pre-clinical Sciences, Faculty of Medicine, Thammasat University, Pathumthani, Thailand, Department of Clinical Microscopy, Faculty of BILINEAGE Medical Technology, Mahidol University, Bangkok, Thailand Sun, Zhuo The origin of endothelial progenitor cells (EPCs) in umbilical cord blood is Cardiovascular surgery department, Toronto General Hospital, Toronto, ON, unknown. Previous studies showed that culturing CD14+ or CD14- sub- Canada populations produced no EPC colony whereas co-culturing these two sub- populations yielded EPC colony formation. We explored the origin of EPCs Introduction: In adult mammals, organ repair is frequently facilitated by derived from umbilical cord blood by culturing CD14+ and CD14- separately resident multipotent stem cell populations. While such a population has or co-culturing both subpopulations direct contact or with the transwells. not been definitively characterized in the adult uterus, we hypothesize that We found no colony formation when culturing CD14+ or CD14- alone, but resident multipotent stem cells contribute to the new tissue formation that EPC colonies were observed when co-culturing both subpopulations either characterizes the cyclic regeneration of uterine endometrium. Here, we with or without transwells. We observed the presence of EPC colonies from show that blast colony-forming cells originating from the adult mouse uterus CD14- population but not from CD14+ population in transwell experiments. exhibit bilineage (hematopoietic and vascular) potential and long-term We interpret that CD14- population is the origin of EPC colonies, however, self-renewal which is the first in vitro and in vivo evidence of an adult he- cytokines releasing from CD14+ population may be necessary for support- mangioblast retained in the adult uterus. Methods: CD34+/c-kit- cells were ing the growth of EPCs. We screened 42 cytokines involving in angiogenesis isolated from mouse uterus and cultured with blast formation conditioned from the conditioned medium collected from CD14+ and CD14- subpopu- medium. In vitro blast colonies formed in two weeks and single blasts were lations using an ELISA array. We found the presence of angiogenin-1 in

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Thursday Poster Abstracts all conditioned media collected from CD14+ cells whereas there was no examine cytoprotective effects of autophagy on survival of EPCs under angiogenin-1 in the conditioned media of CD14- cells as observed in five hypoxia condition. CD34+CD133+VEGFR-2+ cells were isolated from independent experiments. We reconfirmed the result by supplementing mononuclear cells of human umbilical cord blood. The cells were divided angiogenin-1 in the culture medium of CD14- subpopulation and found that into control, hypoxia 1, hypoxia 2, hypoxia 3 and 3-MA groups. In hypoxia angiogenin-1 can induce the formation of EPC colonies in the CD14- sub- groups, the culture dishes containing the cells were treated in an incubating population. We concluded that EPCs in umbilical cord blood is confined to chamber filled with 95% N2 and 5% CO2 for 30 min, 1 h and 2 h respec- CD14- subpopulation and angiogenin-1 secreted from CD14+ subpopula- tively. In 3-MA (3-methyladenine) group, the cells were pretreated with 5 tion may be an important cytokine promoting EPC colony formation. mmol/l 3-MA for 1 h and then treated with hypoxia for 1 h. Autophago- some precursors, autophgosomes and autophagolysosomes of the cells Poster Board Number: 1135 were viewed with transmission electron microscope. Autophagic structures VEGFR-3 SIRNA NANOPARTICLES INHIBIT were detected with MDC staining and LC3 immunostaining. Expression of Autophagy-related gene Beclin-1 was analyzed with RT-PCR. Survival of the LYMPHANGIOGENESIS OF LYMPHATIC cells was evaluated with AO/EB staining. Relation of autophagy with apop- ENDOTHELIAL PROGENITOR CELLS tosis of the cells was examined. After hypoxia treatment, abilities of growth and differentiation of the cells were decreased. Numbers of autophagosome Wang, Hai-Jie, Li, Ting, Tan, Yu-Zhen precursors, autophgosomes and autophagolysosomes in hopoxia groups Shanghai Medical School of Fudan University, Shanghai, China were greater than that in control group. MDC and LC3 positive-structures increased significantly in hopoxia groups. Lysosomes labeled with LysoSen- Lymphatic endothelial progenitor cells (LEPCs) is involved tumor lymp- sorTM green also increased. After treatment with hypoxia, expression of hangiogenesis. VEGF-C/VEGFR-3 signaling pathway plays an important Beclin-1 was enhanced. With blocking in 3-MA, numbers of autophagic role in proliferation, migration and differentiation of LEPCs. VEGFR-3 structures and the level of autophagy decreased remarkably. After au- expressed on LEPCs may be a novel target for inhibiting tumor lymp- tophagy was inhibited with 3-MA, apoptotic cells increased obviously in hangiogenesis. This study is to investigate targeting effects of VEGFR-3 hypoxia groups. These results demonstrate that the Beclin-1 expression and siRNA-loaded PEI-alginate nanoparticles on inhibiting LEPCs differentia- autophagic activities are upregulated under hypoxia condition. Autophagic tion. CD34+CD133+VEGFR-3+ cells were sorted from mononuclear cells of structures sequester the damaged organells into lysosomes for degradation. cord blood with a fluorescece-activated cell sorter. With VEGF-C induction, The enhanced autophagic activities are beneficial for EPC survival through the cells differentiated into lymphatic endothelial cells expressing LYVE- hypoxia tolerance. 1. VEGFR-3 siRNA-loaded PEI-alginate nanoparticles were characterized by assessing the surface charge, size and shape. After transfection with Poster Board Number: 1139 siRNA-nanoplexes, expression of VEGFR-3 mRNA and protein of LEPCs was inhibited significantly. Under induction with VEGF-C, LEPCs transfected with TISSUE PLASMINOGEN ACTIVATOR IMPAIRS siRNA-nanoplexes could not differentiate towards lymphatic endothelial STEM CELL FUNCTION, THUS LIMITING cells. Apoptotic cells in siRNA-nanoplex group were more than that in the control group. Numbers of the proliferated cells decreased obviously after RECOVERY FOLLOWING STROKE transfection with the nanoplexes. Migration and tube formation of the cells Afzal, Aqeela, Ansari, Saeed, Kellner, C P., Sosunov, S A., Hoh, were examined using transwells and three-dimentional collagen gel. Number Brain, Scott, Edward, Connolly, E Sander, Mocco, J of the migrated cells and area of tubes formed by the cells in nanoplex group were greater than that in the control group. On another experiment, Neurosurgery, University of Florida, Gainesville, FL, USA, Neurosurgery, effects of siRNA-nanoplex on proliferation, migration and tube formation of Columbia University, New York, NY, USA, Neurosurgery, University of lymphatic endothelial cells (LECs) isolated from circumcision specimen were Florida, Gainesville, FL, USA examined. Lymphangiogenic abilities of the cells reduced in nanoplex group. Background: Intravenous Tissue Plasminogen Activator (tPA) is the only The models of breast tumor were established with inoculating Hs578T cells FDA approved pharmacological recanalization therapy for stroke, however subcutaneouly in athymic mouse. At two week after injection of siRNA- tPA has been associated with deleterious effects on the blood brain barrier nanoplexes into the tumor, tumor lymphangiogenesis was examined with in experimental models. A potential contributor to vascular integrity and/or LYVE-1 immunostaing. Density of lymphatic vessels in tumor tissue and repair following stroke are Hematopoietic Stem Cells (HSCs)/ Hematopoietic surrounding tissue of the tumor in nanoplex group was lower than that in Progenitor Cells (HPCs), circulating bone marrow derived mononuclear cells the control group. These results show that PEI-alginate nanoparticles are that promote repair in areas of injury. HSC/HPC’s have recently been shown useful carriers for delivering siRNA. LEPCs and LECs can be transfected with to mobilize to the peripheral circulation from bone marrow in response to VEGFR-3 siRNA-loaded nanoparticles effectively. Application of this tech- stroke. Furthermore, increasing levels of circulating HSC/HPCs have been enique is potential for inhibiting tumor lymphangiogensis. demonstrated to correlate with improved neurological function following Poster Board Number: 1137 stroke, suggesting a potentially critical role for HSC/HPC’s in limiting stroke injury and/or facilitating stroke recovery. Stromal Derived Growth Factor CYTOPROTECTIVE EFFECTS OF AUTOPHAGY 1-Alpha (SDF1-A) along with its receptor CXCR4 is a potent chemo attrac- tant released by areas of injury. SDF1-A has been shown to mobilize HSC/ ON SURVIVAL OF HYPOXIA-INDUCED HPC from the bone marrow to the blood and lead to ‘homing’ of the cells ENDOTHELIAL PROGENITOR CELLS to an area of injury. We hypothesized that tPA inhibits HSC/HPC’s function, potentially limiting tPA’s beneficial effects in acute stroke therapy. Meth- Zhang, Dan, Tan, Yu-zhen, Wang, Hai-jie ods: Animals (n=10) were euthanized 24 hours post ischemia/reperfusion Department of Anatomy, Histology and Embryology, Shanghai Medical following a murine intraluminal filament model. Infarction was confirmed School of Fudan University, Shanghai, China using TTC staining of 2 mm sections of the brain. HSC/HPC were harvested In ischemic diseases, endothelial progenitor cells (EPCs) are mobilized from from bone marrow and blood using LIN negative and SCA1 Positive labeled marrow into peripheral blood and then incorporate into local vessels for nanoparticles. The harvested cells were counted using a hemacytometer; the angiogenesis. On other hand, EPCs are transplanted into ischemic tissue to HSC/HPC were then either left untreated or treated with 10nM TPA and improve local microcirculation. However, the fate of EPCs under ischemic migrated towards SDF1-A in a Boyden Chamber. The level of the SDF1-A condition is unknown. Recently, many studies suggest that autophagy is receptor (CXCR4) was also assessed by real time PCR of the untreated and involved in survival of ischemic tissues. This investigation was designed to tPA treated HSC/HPC. Experiments, and their analysis, were performed in a

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blinded manner. Results: Mean infarct volume was 43±10%. Pre-treatment the purpose of this study was to determine if BMDC can be recruited to sites with 10nM tPA reduced HSC/HPC migratory capability towards SDF1-A of epithelial injury in an animal model, and subsequently to determine the (100±1.3% versus 173±1.0%, p<0.05). Pre-treatment with 10nM tPA also ability of exogenous stem cells, introduced systemically or within the injury reduced expression of CXCR4 from 100±7.9% to 35.8±7.1%, p<0.05). site, to enhance the injury repair response. Age was observed to be a factor Conclusion: These data indicate that exposure of HSC/HPC’s to tPA abro- in both donor cells and recipient. In younger animals healing was faster, hair gates their migratory response to SDF1-A. mRNA analysis of HSC/HPC cells growth area was larger and the scar generating healing center was smaller. following treatment with tPA demonstrates a down regulation of the CXCR4 Additionally, BMDC from younger donor animals were associated with receptor. These results suggest that tPA may reduce the ability of the HSC/ more efficient healing when cells were applied to the wound to supplement HPC to home to ischemic brain following stroke and possibly interferes with healing. This suggests that autologous bone marrow is a good candidate for repair mechanisms associated with HSC/HPC. additional study of clinical wound healing applications. Poster Board Number: 1145 EPIDERMAL CELLS FINE-TUNING OF MOUSE EPIDERMAL STEM Poster Board Number: 1141 CELL BEHAVIOR BY THE MOLECULAR CLOCK PROTEIN BMAL1 EMBRYONIC STEM CELL LIKE CELLS FROM Janich, Peggy, Pascual, Gloria, Aznar-Benitah, Salvador HUMAN FORESKIN FIBROBLASTS Differentiation and Cancer, Center for Genomic Regulation, Barcelona, Aflatoonian, Behrouz,Sadeghian, Fatemeh, Fesahat, Farzaneh, Spain Soleimani-Salehabadi, Mohammad, Aflatoonian, Nastaran The natural daily cycles of light and dark have played a fundamental role Centre for Stem Cell Biology, Yazd Institute for Reproductive Biomedical in shaping the development of an adaptive intrinsic clock mechanism Sciences, Yazd, Iran, Islamic Republic of required for regulating homeostasis in organisms. An intrinsic molecular Previously, we have reported the co-culture of skin-derived precursors oscillatory system allows mammals to coordinate the function of multiple (SKPs) with mouse embryonic fibroblasts (MEFs). SKPs in co-culture with organs by setting the correct circadian timing of cellular functions includ- MEFs have formed colonies similar to human embryonic stem cells (hESCs). ing proliferation, DNA damage response, metabolism, and chromatin In this study, we’ve modified the culture condition and cultured ES-like cells remodeling, among others, according to external entraining cues such as without MEF feeder layer. The morphology (high ratio of nuclei to cyto- light, temperature and feeding. Circadian periodicity is established through plasm with multi-nucleolus) and colony formation of these cells are similar multiple interconnected transcriptional and translational networks that to hESCs. After 10-15 days the whole flask (T25) become confluent of cells generate rhythmic negative feedback loops. In this sense, the transcrip- which shows their capacity of self-renewal and proliferation. For passaging tion factors CLOCK-BMAL1 drive the expression of Periods (Per1-3) and of the cells both Trypsin/EDTA (T/E) and Collagenase IV were applied. Cells Cryptochromes (Cry1-2) among others that translocate back to the nucleus become fibroblast like using T/E and with Collagenase IV between fibroblast to inhibit CLOCK-BMAL1 transcriptional activity thereby repressing their like cells and ES like cells. Therefore, for the characterization of these cells, own expression. In mammalian skin, homeostasis is ensured by epidermal immuno-florescence (IF) localization with SSEA4 was applied in the original stem cells (epSCs). EpSCs localize to specialized niches where they un- flask (T25) of the new sample. While, the ES-like colonies were negative to dergo cycles of quiescence and proliferation, followed by egression from express SSEA4, the surrounding cells (fibroblast- like cells) were positive for the niche to subsequently contribute to the differentiated lineages of the SSEA4. This result maid us to test other ES cell markers (SSEA1, SSEA3, TRA- tissue. Several pathways are known to play essential roles in epSC function; 1-60, TRA-1-81, TRA-2-49, TRA-2-54) for fibroblast like cells in 24 well however, how are these pathways spatiotemporaly coordinated, and why plate. The result was negative for all of these markers. I summary, we’ve not all stem cells within the niche behave in the same manner, is still poorly found a population of cells in human foreskin which are similar to hESCs in understood. We have analyzed the role of the molecular clock in fine-tuning aspects of morphology and proliferation, but, there is difficulty to passage the behavior of epidermal stem cells. Using a fluorescent reporter mouse them in undifferentiated state. There are some cells which express SSEA4, model, we demonstrate that the dormant epidermal stem cell compartment but, characterization with other hESC markers was failed. We need to find a contains two co-existing populations of stem cells at opposite phases of the solution for passaging of these cells and also try to characterize these puta- clock. Global comparative transcriptome analysis indicated that each clock tive ES like cells with other markers and methods such as PCR. We hope to population corresponds to a distinct predisposition state of response towards be able to find an easy access source of human pluripotent stem cells for the stem cell activating and dormancy cues. We provide evidence that BMAL1/ future application in cell therapy. CLOCK bind to regulatory elements in the promoters of several of these stem cell homeostatic genes, thus being directly responsible for creating Poster Board Number: 1143 these two stem cell clock states. Unbalancing this clock driven equilibrium of epSCs, through conditional deletion of Bmal1 in the epidermis, resulted in a ROLE OF AGE IN MOUSE STEM CELL long-term progressive accumulation of non-responsive stem cells, premature MEDIATED WOUND HEALING tissue aging, and a significant reduction in the development of cutaneous squamous cell carcinomas. Our results indicate that the molecular clock Badowski, Michael, Hilgaertner, Jianhua, Harris, David machinery fine-tunes the spatiotemporal behavior of epidermal stem cells Immunobiology, University of Arizona, Tucson, AZ, USA within their niche, and that perturbation of this mechanism affects tissue homeostasis and the predisposition to neoplastic transformation. In previous wound healing studies bone marrow stems cells (BMSCs) have been found in regenerating tissues at the site of injury. Some C57BL/6 mice in these experiments had chronic cutaneous wounds, which we found hap- pened more often in aged mice. It seemed that older mice had a higher ratio of severe wound-healing impairments. Studies seeking to clarify the cellular populations involved in the epithelial injury repair response have shown that bone marrow derived cells (BMDC) are an unexpected, yet significant portion of the regenerating epithelial tissue. Based on these observations,

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Poster Board Number: 1147 changes in crucial intrinsic factors between bulge stem cells and its progeny that leads to cell fate change which is reflected by long-term self-renewal IGF1 SIGNALING CONTRIBUTES TO BULGE ability and responsiveness to active signals. One of the bulge stem cells EPIDERMAL STEM CELL HOMEOSTASIS AND specific transcription factors is involved in regulating HF SCs activation both during development and wound healing. IS REQUIRED FOR THEIR ACTIVATION IN AGED MICE IN RESPONSE TO WOUNDING. Poster Board Number: 1151 Roy, Edwige, Linay, Fabien, Holzenberger, Martin, Aractingi, Selim, IMPACT OF CRYOPRESERVATION ON Khosrotehrani, Kiarash EPITHELIAL STEM CELLS GROWN IN Experimental Dermatology Group, University of Queensland Centre for MONOLAYER CULTURE AND IN TISSUE Clinical Research (UQCCR), Brisbane, Australia, INSERM UMR 938-UPMC University Paris VI, Paris, France, INSERM UMR_S938, Brisbane, France ENGINEERED SKIN Epidermal stem cells are a well-defined population of keratinocytes with Goyer, Benjamin, Lavoie, Amélie, Fugère, Claudia, Larouche, multipotent capabilities. They are required constantly to maintain tissue Danielle, Germain, Lucie homeostasis and to repair damage after wounding. The influence of the Université LAVAL, Québec QC, QC, Canada Centre LOEX de l’Université underlying dermis and dermal fibroblasts on the phenotype and homeostasis Laval, Génie tissulaire et régénération : LOEX - Centre de recherche FRSQ of the epidermis is established but the molecular mechanisms involved in this du Centre hospitalier affilié universitaire de Québec, and Département de process are complex and they are still poorly understood. IGF-1 signalling Chirurgie, Faculté de Médecine, Université Laval, Québec, QC, Canada. is essential in epidermal development and interacts with the rho-GTPase The presence of stem cells within tissue-engineered skin substitutes is Rac to maintain the interfollicular epidermis (IFE). However its role in adult required for their long-term survival after grafting. It is therefore essential homeostasis of bulge stem cells and their contribution to skin wound is that skin stem cells be preserved during the in vitro steps leading to the unclear. To address this question, we used Keratin 14 and Keratin 15 driven production of tissue-engineered skin substitutes such as the elaboration of a conditional deletion of the IGF1 receptor (IGF1R) to abrogate IGF-1 signal- skin cell bank by cryopreservation. To evaluate the effect of cryopreservation ling in the epidermis and more specifically in the bulge stem cells in an in liquid nitrogen by a slow-cooling method involving dimethyl sulfoxide inducible manner in 3 week old mice. In steady state, we did not observe (DMSO) on the preservation and functionality of epithelial skin stem cells, any major histological changes in young mice (5 weeks old) and aged mice human epithelial cells were extracted from skin samples, amplified in culture (6 months old). Detailed quantitative analyses of epidermal subpopula- and used to produce tissue-engineered skin substitutes, before cryopreser- tions based on CD34 and alpha-6 integrin staining using flow cytometry, vation as well as after thawing. Keratin (K) 19 was used as a marker to revealed a significant reduction of IFE epidermal cells in K14 driven deletion evaluate the preservation of stem cells in vitro by flow cytometry and as opposed to an outer rout sheath (ORS) reduction in K15 driven deletion immunofluorescence analyses, while the growth capacity was assessed by suggesting the importance of IGF1 signalling in the maintenance of each colony forming efficiency (CFE). To investigate the presence and function- compartment. In both situations there was a reduction in the proportion ality of stem cells within tissue-engineered skin substitutes, slow-cycling of CD34 high bulge stem cells. To further analyse the contribution of these cells were identified using 5-bromo-2’-deoxyuridine (BrdU). We found that different compartments to skin wound re-epidermisation, we performed the proportion and the growth potential of stem cells in monolayer culture 6mm excisional wounds in young and aged mice. K14 driven IGF1R deletion remained constant before and after cryopreservation. Epithelial stem cells resulted in significant delay in wound closure at any age. In mice with IGF1R (BrdU and K19 double positive cells) were established and maintained in the deficiency in bulge cells, wound healing was normal in young animals but tissue-engineered skin substitutes and their proportion was not significantly was significantly delayed at older age. In conclusion, our results show the affected after cryopreservation. We conclude that upon our cryopreservation importance of IGF-1 signalling in adult skin homeostasis after the comple- process, human skin stem cells are adequately preserved and are suitable tion of developmental processes of stratification and hair follicle formation. for the production of tissue-engineered skin substitutes dedicated to clinical This pathway significantly affected epidermal stem cell maintenance and applications. activity both in the hair follicle bulge and presumably in the IFE. In particular, our results show the reduced capacity of bulge stem cells deficient in IGF1R Poster Board Number: 1153 to contribute to skin wound healing over time. ADULT SALIVARY GLAND DERIVED Poster Board Number: 1149 PROGENITOR CELLS AND THEIR POTENTIALITY REGULATING HAIR FOLLICLE STEM CELL TO DIFFERENTIATE INTO ENDODERM MAINTENANCE, ACTIVATION AND SELF- ORIGINATED CELL TYPES RENEWAL Baek, Hyunjung, Yeon, SooIn, Lee, Heekyung, Choi, Young Wook, Chen, Ting, Fuchs, Elaine Kwon, Heechung Rockefeller University, New York, NY, USA Division of Radiation Oncology, Korea Institute of radiological & medical sciences, Seoul, Korea, Republic of In the adult skin, hair follicles (HFs) undergo cycles of new hair growth followed by destruction and rest. The process is fueled by follicle stem cells Radiotherapy as a treatment for head and neck cancers causes irreversible (SCs) that reside in a permanent niche (bulge) and undergo cycles of qui- damage to salivary glands, such as oral dryness, impairment of normal oral escence and activation. Understanding the signaling pathways and intrinsic functions, and decreased lubrication of the mucosal surfaces and of ingested factors that control the maintenance, activation and differentiation of SCs is food. Progenitor/stem cells have been isolated in several adult tissues for not only relevant to basic SC biology, but also has clinical significance, par- treatment in injuries of endoderm originated tissues involving liver and pan- ticularly in human cancers, where these mechanisms go awry. Our previous creas. However, there is a little known about salivary gland progenitor/stem study shows that HF regeneration happens as a two-step process involving cells that are able to differentiate into endodermal lineages. In this study, molecularly distinct epithelial compartments: bulge and hair germ (HG), we isolated the progenitor cells from submandibular gland in normal adult where HG cells fuel initial steps in hair regeneration, while the bulge is the rat and examined if the cells include the major abilities of stem cell, what is engine maintaining the process. Our current results indicated that there are the self-renewal and multipotent. The salivary gland progenitor cells express

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both pluripotent stem cell markers such as Oct-4 and nestin, and mesenchy- effect on epidermal formation: it is leading to hyperproliferation and finally mal stem cell markers such as c-kit, CD44, and CD49f. Proliferative potency to spontaneous tumour formation, clonal expansion of basal cells which exit of cultured salivary gland progenitor cell is higher than human mesenchymal the basal layer and move to suprabasal areas. Downstream effectors of Rac1 stem cell. In addition, Cells were proliferated more than 50 passages main- might be affected by ROS levels as Rac1 is part of the NADPH-oxidase com- taining epithelial-like morphology. Furthermore, we analyzed the capacity plex NOX2/pg91phox, the most relevant in skin and keratinocytes. Further of their culture to differentiate into endoderm lineage cell types such as epxeriments will show how ROS levels may interfere with adherent junction acinar, ductal, and pancreatic cells. In culture on Matrigel, morphology of formation or TGF-β responsiveness. progenitor cells were changed to acinar-ductal like structure and these cells expressed acinar cell marker, α-amylase, both in cytoplasm and the cultured media. RT-PCR data showed increased expression level of pancreatic cell markers involving insulin, Pdx1, Ppy, and Ngn3, When cells formed clusters INTESTINAL/GUT CELLS in the presence of retinoic acid, activin A, and exendin-4. These data dem- Poster Board Number: 1157 onstrated that adult salivary progenitor/stem cells are used for a potential source for cell therapy of salivary gland hypofuction and diabetes. THE COLORECTAL CANCER STEM CELL Poster Board Number: 1155 MARKER, CD166/ALCAM PLAYS AN ESSENTIAL ROLE IN MAINTAINING THE NORMAL MOUSE ROLE OF RAC1 IN INTERCELLULAR AND HUMAN INTESTINAL STEM CELL NICHE COMMUNICATION WITHIN THE EPIDERMIS Wong, Melissa H., Levin, Trevor, Davies, Paige S. Quist, Sven R., Miremadi, A., Schreiner, A., Sirokmany, G., Watt, F. M. Oregon Health & Science University, Portland, OR, USA, Cell and Developmental Biology, Oregon Health & Science University, Portland, OR, Cambridge Research Institute, Cancer Research UK, Cambridge, United USA, Dermatology, Oregon Health & Science University, Portland, OR, USA Kingdom, Department of Histopatholog, Addenbrookes Hospital, and Cancer Research UK, Cambridge, United Kingdom, University of The tight regulation of cell-cell communication and adhesion within the Cambridge, Welcome Trust Centre for Stem Cell Research, Cambridge, intestinal stem cell niche is paramount for coordination of the proliferation, United Kingdom, University of Cambridge, Welcome Trust Centre for Stem asymmetric cell division and cell migration capacities integral to maintaining Cell Research, and Cancer Research UK, Cambridge, United Kingdom functional intestinal homeostasis. While the dynamic regulation of cell adhe- sion in the base of the crypt undoubtedly plays a critical role to maintain the Objectives: Stratified human epidermis consists of multiple layers of ke- stem cell niche, little is known about which adhesion molecules are specifi- ratinocytes that are maintained by stem cells, which have the capacity of cally expressed among stem and progenitor cells; and more specifically, if self-renewal, and their progeny, the transit amplifying cells. In squamous cell they function beyond their adhesive role. Intriguingly, CD166 is an adhesion carcinoma (SCC) or hyperproliferative skin diseases (Psoriasis), the organi- molecule of the immunoglobulin-like family, notably described as a he- sation of these stratified layers of keratinocytes is disrupted and integrin matopoietic stem cell niche factor important in maintaining a stem-like state expression, normally confined to the basal layer is abnormally expressed and recently identified on colorectal cancer stem cells. These observations by suprabasal cells. Terminally differentiating cells can communicate with provide the basis for testing the hypothesis that CD166 plays an important the basal cell compartment including the stem cells to stimulate or inhibit role in maintaining the intestinal epithelial stem cell niche. We have previ- expansion of mutant stem-cell clones involved in the earliest steps of skin ously shown a novel expression pattern for CD166 within the crypt-base of carcinogenesis as shown by two-stage chemical carcinogenesis experiments. the small and large intestine. Further, while CD166 expression is restricted to Rac1, a small GTPases of the Rho-superfamily, which has been found up- the lower 1/5 of cells in the crypt, it is broadly expressed on both putative regulated in SCCs, relays signals downstream of integrins to the cytoplasm stem cells and the intervening differentiated Paneth cell population, suggest- and is involved in the regulation of cell movement, polarity, adhesion, gene ing that this adhesion molecule may be a key component of the stem cell transcription, cell cycle progression and enzyme activity. Methods: One way niche. Here we reveal that surprisingly, mice with ablated CD166 are viable. to study basal-suprabasal communication is by using mouse models which Overall there is decreased proliferation within the epithelia and the intestinal express genes of interest under the control of the involucrin promoter tar- stem cell niche is disorganized. Primarily, there are fewer and misshapen in- geting specifically terminally differentiated cells. We created a mouse model tervening differentiated Paneth cells, suggesting that CD166 acts to regulate expressing eGFP together with Rac1 QL, an active form of Rac1 under the cell shape and anchorage within the stem cell niche. Additionally, loss of control of the involucrin promoter. We analyzed this mouse model using CD166 expression correlates with downregulation of crypt-based EpCam ex- immunofluorescence, electron microscopy, gene expression and stratification pression and is linked to a decrease in the Wnt signaling pathway. Together, experiments of differentiated cells overlying basal cells transfected with a these findings support the important role of adhesion molecules like CD166 Smad 2/3-luciferase reporter. Results: Rac1 overexpression by differentiated for regulating stem cell microenvironments. cells lead to epidermal acanthosis, hyperkeratosis, parakeratosis, hyper- granulosis, spongiosis and mild dermal lymphatic infiltration. At the age of >8 months spontaneous tumour formation was observed in about 0.2 % of transgenic mice including squamous cell carcinoma (from moderate differentiated SCCs to spindle cell carcinoma) and papilloma. Increase of cell colonies in number and size were demonstrated by colony forming assay using feeder cells. Cell growth was enhanced and expansion of Keratin 14 positive basal cells was noticed. Transmission electron microscopy showed strong increase in number of desmosomes, especially in the basal-suprabasal zone, clumped keratin filaments as well as strong interaction with the base- ment membrane. Serveral members of desmosomes were uregulated includ- ing desmoplakin, periplakin and desmocollin-2. Stratification with TG but not WT differentiated cells pertubated Smad2/3-responsiveness to TGFβ. Conclusion: Suprabasal expression of the small-GTPase Rac1 using a mouse model expressing active Rac1 driven by the involucrin promoter has a clear

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Poster Board Number: 1159 Poster Board Number: 1161 CRITICAL ROLE OF THE MACROPHAGE TELOMERASE-EXPRESSING MOUSE COLONY-STIMULATING FACTOR RECEPTOR INTESTINAL STEM CELLS ARE ACTIVATED IN (C-FMS) IN MOUSE INTESTINAL STEM CELL RESPONSE TO FASTING MAINTENANCE THROUGH PANETH CELLS Richmond, Camilla, Montgomery, Robert, Ambruzs, Dana, Carlone, Akcora, Dilara -., Malaterre, Jordane, Lightowler, Sally, Ramsay, Diana, Breault, David Robert G. Pediatrics, Children’s Hospital Boston, Boston, MA, USA Pathology, Melbourne University, Melbourne, Australia, Differentiation Telomerase (mTert) expression is associated with resistance to cellular senes- and Transcription Laboratory, Peter MacCallum Cancer Centre, Melbourne, cence, stem cell activation and is a marker for self-renewal divisions. Using Australia two lines of reporter mice (mTert-GFP and mTert-CreER::R26R) we have re- Traditionally, macrophage colony-stimulating factor (M-CSF, CSF-1) has cently reported on a population of slowly cycling intestinal stem cells (ISCs). been considered to be the most pleiotropic macrophage growth factor act- This population is long-lived, multipotent and distinct from rapidly cycling ing through its unique proto-oncogene tyrosine kinase receptor, c-Fms (Csf- Lgr5+ ISCs. Under normal homeostasis mTert-expressing cells can give rise 1R). CSF-1 is produced by a variety of cell types such as fibroblasts, stromal to Lgr5+ cells indicating a lineage relationship between these populations. cells, endothelial cells, smooth muscle cells as well as activated macrophag- Interestingly, while Lgr5+ cells and putative ISCs present at the traditional es. All these cells are in close proximity to intestinal crypts. The intestinal “crypt position +4” are sensitive to the effects of ionizing radiation, mTert- stem cell niche, which supports stem cell maintenance and regulates the expressing cells are resistant to high-dose radiation and contribute to the function of progenitor cells via secreted proteins or by direct connection via regenerative response following injury. These findings lead us to propose the basement membrane, is composed of an overlapping group of cell types: that the intestinal crypt contains a dormant population of self-renewing ISCs epithelial cells, myofibroblasts, macrophages, dendritic cells, endothelial which transiently express mTert and give rise to terminally differentiated cells cells, lymphocytes, smooth muscle cells, and neural cells. Additionally, in the via both Lgr5-dependent and independent pathways. To investigate the role small intestine (SI) stem cells are intermingled between Paneth cells at the of mTert-expressing cells in response to physiologic stimuli, mice were sub- bottom of crypts. Paneth cells, which are important for innate immunity in jected to a protocol of fasting and re-feeding. As it is well established that the SI, appear to affect intestinal cellular homeostasis by secreting a number fasting induces villus atrophy through decreased proliferation and increased of antimicrobial molecules and cytokines. Recently, it has been shown that apoptosis, we hypothesized that mTert expression would be down-regulated Paneth cells are required for the growth of Lgr-5+ve stem cells both in vitro during fasting and activated upon re-feeding. Adult mTert-GFP and mTert- and in vivo. In addition, we have shown that c-Fms and M-CSF are required CreER::R26R mice were divided into 3 groups: control (ad libitum fed), fast- for Paneth cell development and intestinal homeostasis by using CSF-1 ed (48h) and fasted/refed (48h/24h). Fasted animals lost, on average, 22% deficient osteopetrotic mice and knock-out csf1r mice. Importantly, we of their total body weight and demonstrated a 15% decrease in villus height also showed such mice had markedly reduced Lgr5 expression in SI crypts. (ANOVA p<0.0001), consistent with previous observations. Surprisingly, We took this study further to determine the precise role of c-Fms in mouse flow cytometric analysis revealed a dramatic 10-fold induction in mTert- intestine in vivo by using c-fms conditional knock-out mice and in vitro by GFP+ intestinal crypt cells in the fasted group (1.93±0.59%) compared SI organoid culture. Immunofluorescence staining for c-Fms showed that with control mice (0.13±0.06%), with levels of GFP+ cells returning to near it is expressed within the stem cell niche of SI and lower parts of crypts. To baseline levels in the refed group (0.27±0.15%). These results were further examine whether c-fms expression is restricted to Paneth cells in SI, epithe- supported by lineage-tracing data, which demonstrated a 7-fold increase lial cell adhesion molecule (EPCAM) and carbohydrate adhesion molecule in the percentage of LacZ-marked crypts (fasted 0.073±0.025% vs. control (CD15) antibodies were used to specifically sort Paneth cells using FACS. 0.012±0.006). In conclusion, telomerase-expressing ISCs rapidly respond to qRT-PCR data revealed that EPCAM+ve, CD15+ve cells had Cryptidin-1 nutrient deprivation, undergoing stem cell activation despite concomitant and Lysozyme expression compared to EPCAM+ve, CD15-ve cells. Perhaps villus atrophy. This paradoxical induction raises important questions about EPCAM+ve, CD15+ve cells also expressed Lgr-5 expression due to the the mechanisms regulating intestinal homeostasis and suggests that mTert- possibility that Lgr-5+ve cells were co-sorted with Paneth cells. These pos- expressing ISCs are poised to contribute to intestinal regeneration following sible Lgr-5+ve stem cell and Paneth cell doublets had both c-fms and m-csf physiologic stress. mRNA expression. To address this more directly we performed organoid cul- Poster Board Number: 1163 ture from 4 weeks old c-fms conditional knock-out and wild type (WT) mice and used 4-OHT to remove c-fms expression. The number of organoids was MYB CONTROLS INTESTINAL STEM CELL decreased in c-fms knock-out cultures compared to WT and cell viability was GENES AND SELF RENEWAL found to be slightly decreased. Furthermore, we treated 6 weeks-old c-fms conditional knock-out/villinCREERT2 mice with Tamoxifen for four weeks Ramsay, Robert G. to delete c-fms in the intestines (n=3). In the SI, although the loss of c-fms Peter MacCallum Cancer Centre, Melbourne, Australia resulted in a reduction of the number of Lysozyme+ve Paneth cells, prolif- eration in general did not seem to be affected. As it is known that the life Rapid advances have been made in the understanding of how the highly span of Paneth cells can be as long as 8 weeks, we speculated that 4 weeks proliferative gastrointestinal tract (GI) epithelium is regulated under homeo- of Tamoxifen treatment may not have been enough to deplete Paneth cells. stasis and disease. The identification of putative intestinal stem cell (ISC) This was born out by waiting longer before mice were investigated such that genes and the ability to culture ISC capable of generating all four lineages at 8 weeks Paneth cells were demonstrably lost. Interestingly, in colon the plus the architecture of small intestinal (SI) crypts has been transformative. loss of c-fms also led to a decrease in Lgr-5 expression. Consequently, c-Fms Here it will be shown that transcription factor Myb governs ISC gene expres- seems to be important to the stem cell niche and our study further highlights sion, particularly Lgr5. Lgr5 is associated with cells that have the capacity to the key role of Paneth cells in this compartment. generate all cell lineages in “organoid” cultures and colorectal cancer (CRC) cells with over-expression of MYB. Wnt signaling and Myb co-operate in maximal Lgr5 promoter activation; conversely hypomorphic Myb mutant (plt4/plt4) mice have decreased Lgr5 expression. After ionizing radiation (IR) ISC gene expression is elevated however, in plt4/plt4 mice this response is

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repressed. ISC genes bmi-1 and olfm4 were expressed at sub-normal levels These properties make them very attractive therapies for MS in that they in plt4/plt4 mice and bmi-1 is induced with IR to half the level in mutant may dampen CNS inflammation, reduce damage and may possibly have mice compared to wt mice. dcamkl-1 and olfm4 fail to recover after IR in neuro-regenerative potential. Accordingly, we have investigated the poten- both wt and mutant mice. cyclinE1 is employed to assist cells in the emer- tial immuno-regulatory effects of AECs as a possible therapy for an MS-like gence from a quiescent state (an expectation of quiescent ISC following IR) disease, EAE (Experimental Autoimmune Encephalomyelitis) in mice. and is over-expressed after IR in wt mice but does not change from a very We found that AECs can suppress both specific and non-specific T-cell prolif- low base in plt4/plt4 mice. Self-renewal assays using “organoid” cultures eration. Moreover we found that AECs decrease the amount of pro-inflam- further highlighted the dependence of ISC on Myb. Our results provide matory cytokines produced by T cells. In vivo studies have also revealed new insights into the influence of Myb on the GI and suggest that Myb is a that AECs can not only suppress the development of EAE but also prevent master regulator of GI ISC. relapse. EAE-protected mice had reduced MOG-specific T cell responses and produced less pro-inflammatory cytokines, upon antigen specific and non- Poster Board Number: 1165 specific stimulation. Further investigations also revealed that AEC injected IN VIVO GENERATION OF FUNCTIONAL mice had increased percentages of Th2 cells in the CNS. While the mecha- nism of action by which AECs produce their immuno-regulatory effect is as INSULIN-PRODUCING CELLS IN THE GUT BY yet to be fully determined, current results suggest that this effect is in part FOXO1 KNOCKOUT mediated by an anti-inflammatory response within the CNS. These results suggest that AECs hold some promises for the treatment of autoimmune Talchai, Chutima, Accili, Domenico disease like MS. Medicine, Columbia University, New York, NY, USA Poster Board Number: 1169 Restoration of endogenous insulin production is the ultimate goal of type 1 diabetes therapy. Neurogenin3-positive (Neurog3+) endocrine progenitor HUMAN PLACENTA DERIVED MESENCHYMAL cells give rise to insulin-producing (β) cells in the pancreas, but not in the STEM CELLS MODULATE ALLOGENEIC gut. It is unclear what determines this difference. Here we show that somatic ablation of Foxo1 in Neurog3+ enteroendocrine progenitors results in the IMMUNE CELL RESPONSE appearance of intestinal insulin-positive (Ins+) cells that express markers Kim, Hye Sun, Jang, Moon Ju, Lee, Hey Jin, Kim, Kyung Sul, of mature β-cells, including those required for glucose sensing, glucose Chung, Hyung-Min metabolism, and proinsulin biosynthesis and processing. Enteric Ins+ cells can be labeled using an Insulin2-Gfp knock-in allele, and secrete insulin and CHA Stem Cell Institute, Seoul, Korea, Republic of, Internal Medicine, C-peptide in response to glucose and sulfonlyureas. Moreover, acid-ethanol School of Medicine CHA University, Seoul, Korea, Republic of extracts from gut of Neurog3-Foxo1lox/lox mice lower blood glucose when Mesenchyma stem cells (MSCs) are one of the most attractive therapeutic injected into newborn mice. And unlike pancreatic β-cells, gut Ins+ cells resources in clinical application due to their multipotent capacity. Previous regenerate in vivo following ablation by the β-cell toxin, streptozotocin, and studies have been shown that MSCs have an important function of sup- reverse hyperglycemia in mice. The data indicate that Neurog3+ enteroen- pressing graft-versus-host disease (GVHD) after allogeneic transplantation; docrine progenitors have the potential to differentiate into insulin-producing however, the mechanisms studies are still unclear. Here, we derived human cells, but this fate is physiologically restricted by Foxo1 during development. placenta originated mesenchymal stem cells (PDMSCs) from the chorionic Our results provide impetus to develop Foxo1 inhibition in enteroendocrine plate (CP) of placenta and investigated the immune-regulatory proper- cells as a means to restore insulin production in type 1 diabetes. ties of PDMSCs in controlling GVHD mice model. PDMSCs inhibited the proliferation and activation of mononuclear cells (MNCs) in response to PHA (mytogen) and allogenenic T cells, and arrested cell cycle in G0/G1 phase. They especially reduced T cell population which was activated by PHA. EPITHELIAL CELLS (NOT SKIN) Moreover PDMSCs were altered the cytokine secretion by co-culturing with Poster Board Number: 1167 activated MNCs, directly and indirectly. Nextly, we verified the accessibility of PDMSCs in preventing acute GVHD on mice model, which reflected by THERAPEUTIC POTENTIAL OF HUMAN AMNION enhanced survival rate and reduced damage degree of each organ such as EPITHELIAL CELLS FOR AUTOIMMUNE- small and large intestine, skin and liver. In this result, PDMSCs restored the survival rate and organ damage, dose-dependently. As the result, we could MEDIATED DEMYELINATION conclude that PDMSCs modulate the immune response in vitro and vivo McDonald, Courtney, Payne, Natalie, Sun, Guizhi, Herszfeld, owing to arresting the cell cycle in G0/G1 phase and altering the cytokine Daniella, Moussa, Leon, Siatskas, Christopher, Jenkin, Graham, secretion. These data offer understanding of mechanisms in MSC medi- ated induction of tolerance, which modulated by inflammation though the Bernard, Claude interactions between allogenic MSCs and immune cells, for reducing GVHD. Monash Immunology & Stem Cell Laboratories, Monash University, With respect to the field of cell therapy, these results may give us a crucial Clayton, Melbourne, Australia, Ritchie Centre, Monash Institute of Medical clue to cure of immune related diseases. Research, Clayton, Melbourne, Australia Poster Board Number: 1171 Multiple Sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system (CNS). The cause of MS is as yet unknown, but THE ROLE OF ΔNP63Α IN HUMAN is believed to involve both cell-mediated and humoral immune responses directed against components of the CNS. Current treatments available for CYTOTROPHOBLAST STEM CELL MS patients are predominantly non-specific immuno-modulators only effec- PROLIFERATION AND LINEAGE SPECIFICATION tive in 30% of RR-MS patients, have no long-term beneficial effects and are Li, Yingchun, Sullivan, Stephen, Moretto Zita, Matteo, Parast, Mana often associated with significant side effects. In recent years there has been a strong push towards using stem cells to treat disease like MS. It has been Pathology, University of California San Diego, La Jolla, CA, USA found that cells such as mesenchymal stem cells (MSC), and more recently The placenta is a transient organ, necessary for proper fetal development. amnion epithelial cells (AEC), have the ability to suppress immune responses Its main functional component is the trophoblast, which is derived from in vitro and in vivo and can differentiate into many cell lineages in vitro.

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Thursday Poster Abstracts extraembronic ectoderm. Little is known about the trophectoderm in the and HLA-DR expression. Gene expression profiling based on microarray human embryo, as human trophoblast stem cells have yet to be isolated analysis revealed a difference in gene expression in particular with the down and characterized. p63, a member of the p53 family of nuclear proteins, has regulation of the epithelial markers. Conclusion: We conclude that under the been shown to be involved in stem cell maintenance in skin and other strati- influence of TGF-β, mature AEC undergo epithelial-to-mesenchymal transi- fied epithelia. We have previously identified p63 as a marker of proliferative tion and acquire a mesenchymal cell-like phenotype. EMT greatly enhances trophoblast in human placental tissues. Specifically, based on immunohis- cell mobility and may thus help optimize AEC for use in cardiovascular cell tochemical staining, p63 is expressed in all Ki67-positive trophoblast, includ- therapy. ing cytotrophoblast in the floating chorionic villi and cytotrophoblast cell columns in anchoring villi; however, it is absent in differentiated trophoblast, Poster Board Number: 1175 both syncytiotrophoblast of the chorionic villi and the invasive extravillous TENDON HEALING INDUCED BY trophoblast in the placental bed. Here, we have further studied its role in trophoblast proliferation, using the human trophoblast cell lines, BeWo and ALLOTRANSPLANTED OVINE EPITHELIAL HTR8, and primary cytotrophoblast (CTB) isolated from first trimester or AMNIOTIC CELLS term placental tissue. BeWo cells can be induced to differentiate into syncy- tiotrophoblast by treatment with forskolin, while primary CTB differentiate Barboni, Barbara, Muttini, Aurelio, Curini, Valentina, Berardineli, spontaneously to syncytiotrophoblast over a 7-day period in culture. We Paolo, Mattioli, Mauro, Russo, Valentina, Bernabò, Nicola show that p63 expression in all these cells is limited to the ΔNp63α isoform Dept. Biomedical Science, University of Teramo, Teramo, Italy (from here on referred to as p63) and is lost as the cells (both BeWo and primary CTB) differentiate into syncytiotrophoblast. We generated lentivirus, The role of tendons is to transfer muscular forces to bone allowing body movements. Tendons are constantly exposed to mechanical loads proning expressing the ΔNp63α isoform, and showed that overexpression of this protein leads to increased trophoblast proliferation, as measured by BrdU them to acute injuries (e.g. sports activities) or chronic overuse. Although uptake. In addition, when cells are cultured under low oxygen tension, con- spontaneous healing can occur, more often inflammatory or degenerative ditions which promote CTB proliferation and inhibit syncytiotrophoblast dif- diseases result in scar thus compromising tendon biomechanical properties. ferentiation, p63 expression is enhanced. Finally, p63 expression is induced Since poor clinical outcome is related to the limited regenerative capac- in human embryonic stem cells, following differentiation into trophoblast ity of tendons, growing interest is addressed to stem cell-based therapy. lineage by short-term treatment with BMP4. We hypothesize that p63 plays An emerging source of stem cells is represented by amniotic epithelial cells an important role in both trophoblast proliferation and lineage specification. (AEC) that for their high plasticity and unique hypoimmunogenic proper- ties could be advantageously used also in allotransplantation settings. This Poster Board Number: 1173 research has been designed to assess whether AEC can be used to improve tissue healing. To this aim, AEC isolated from the epithelial layer of amniotic EPITHELIAL TO MESENCHYMAL TRANSITION membranes of 2- 3 month old fetuses, expressing specific stemness markers OF HUMAN AMNION EPITHELIAL CELLS ( CD14-, CD 31-, CD45-, CD58-, CD49f, CD29, CD166, OCT 4, Sox 2, Nanog, TERT), were allostranplanted into defects experimentally induced in Roy, Rajika, Brodarac A, Nitschke M, Stachelscheid H, Tschoepe C, Achille’s tendon by using a large animal model, the sheep, that has a great Kang SK, Stamm C potential for translational research for its weight, size and joint - ligament Regenerative Therapies, Berlin-Brandenburg School for Regenerative structures. AEC (1x 106 cells/animal) marked with the fluorescent vital cell Therapies , Deutsches Herzzentrum Berlin, Berlin, Germany linker dye, PKH26, were suspended in fibrin glue (Tissucol) and then im- planted into surgically induced tendon defects (3mm in diameter and depth) Objective: Amnion epithelial cells (AEC) are a readily available cell source of 20 sheep under general anesthesia. Controlateral tendon lesions used as for potential use in regenerative medicine. Subpopulations of AEC may control (Ctr) received only fibrin glue. Tissue regeneration was monitored express embryonic stem cell markers such as SSEA-3/4, TRA1-60, TRA1- after 15 and 30 days (d) by evaluating the process of matrix remodeling and 81, OCT-3/4, NANOG, SOX-2 and display stem cell behaviour, but usually the biomechanical properties of explanted tendons isolated after euthanasia. AEC behave like mature epithelial cells. We propose that AEC which are Tissue microarchitecture (HE and Herovici staining) revealed a more rapid transformed towards a mesenchymal phenotype have a higher capacity for tissue organization in AEC treated tendons, where abundant and oriented tissue regeneration due to their enhanced migratory capacity. In this study fibers were visible already after 15 d differently from Ctr. Immunohistochem- we sought to induce epithelial-to-mesenchymal transition (EMT) in AEC to istry confirmed that extracellular matrix remodeling was more rapid in AEC improve their capacity for cardiovascular regeneration. Methods: Human treated tendons, where, after 30 d, immature fibers (COLIII) had been AEC’s were derived from full-term placenta and expanded in customized completely replaced by mature ones (COLI). Higher COLI expression was media. To induce EMT, transforming growth factor-β (TGF- β) was added then confirmed by RT-PCR carried out on mRNA extracted from defect sites to the cell culture medium. Cultured cells were then analysed at regular by laser-capture microdissection. Moreover, statistical analysis showed that intervals for changes in cell morphology by light microscopy. Induced and AEC treated tissues exhibited a prompt recovery with a cellularity, vascular non-induced cells were stained with mouse anti-human N-cadherin conju- area and cd45 positive cells infiltration similar to those of healthy tendons gated to Alexa 488. A transwell migration assay was performed in a 24-well and lower than Ctr. Despite these findings, the proliferation index in the format using 8μm pore size transwell inserts. FACS analysis was performed healing area was significantly higher in AEC than in Ctr. AEC (PHK26 posi- using a BD FACS Calibur and microarray analysis was done using Affymetrix tive) could be found either at 15 or 30 d, mainly localized at the periphery HG-U133A chip. Furthermore the in vivo effects of the EMT were assessed of healing area: some of these cells were in mitosis (Ki67 positive), a low in a mouse model of myocardial infarction by ligating the LAD, for which the percentage (20 %) expressed CD45 (phagocytated PHK26 cells) and, most study is presently in progress. Results: Following expansion in customized interestingly, the cells closer to site of transition from the healing to the media, AEC expressed stem cell markers but lost Oct-4 expression at higher healthy tendon were fusiform shaped and entrapped within newly deposited passages and behaved like mature epithelial cells. 25ng/ml TGF- β added to fibers. Moreover, these cells expressed COLI thus showing a direct involve- the medium for 5-6 days induced striking changes in AEC morphology. Cells ment of transplanted AEC in the regenerative process. Finally, biomechanical acquired an elongated, fibroblastoid shape, equivalent to that of mesen- test demonstrated that AEC treatment improved the recovery of mechanical chymal stem cells. TGF-β induced AEC showed up-regulation of N-cadherin properties with a maximum failure load and stiffness 2 and 1.5 times higher, and irregular migration pattern that lead to accelerated wound closure and respectively, than Ctr. These results demonstrate, for the first time, that al- only stimulated AEC migrated through an 8μm transwell membrane. FACS lotransplanted AEC can be used to improve tendon healing. analysis showed an increase in CD 90 expression and a decrease in CD14

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Poster Board Number: 1177 been maintained in xeno-free conditions were differentiated in serum-free media containing xeno-free media supplements. At various timepoints, LACRIMAL GLAND REGENERATION PTENTIAL cells were tested for the expression of markers of retinal development using BY THEIR PROGENITOR CELLS immunocytochemistry and RT-PCR analyses. Results: hiPSCs maintained in either traditional or xeno-free culture conditions were capable of repeated Kawakita, Tetsuya, Okada, Naoko, Kawashima, Motoko, passaging and maintenance of a pluripotent state as determined by the Shimmura, Shigeto, Mishima, Kenji, Ito, Masataka, Saito, Ichiro, expression of pluripotency genes. Upon differentiation in xeno-free condi- Tsubota, Kazuo tions, cells acquired advancing features of retinal development, including cell Ophthalmology, Keio University School of Medicine, Tokyo, Japan, populations analogous to the eye field and optic vesicle/cup stages of devel- Ophthalmology, TsurumiUniversity, Tsurumi, Japan, National Defense opment. Furthermore, this differentiation in xeno-free conditions occurred Medical University, Tochigi, Japan, Tsurumi University, Tsurumi, Japan in a manner similar to those cells differentiated using previously established protocols. Conclusions: Results from this study demonstrate that hiPSCs Objective: Previous report showed that mouse lacrimal gland epithelial can be maintained and directed to differentiate under xeno-free conditions cells were cultured successfully only in case of newborn mouse lacrimal similarly to previously established protocols using feeder cells and animal gland. This study was performed to cultivate and characterize adult mouse products. This capability will facilitate future efforts to develop hiPSC-based lacrimal gland epithelial cells, and investigate regeneration potential of these therapies for retinal disorders. cells by sphere culture method. Materials and Methods: Lacrimal glands were removed from newborn mice (C57B/6) and isolated lacrimal gland Poster Board Number: 1181 epithelial cells were seeded on a tissue culture-treated or non-coating dish in Cnt-07 culture medium with or without cholera toxin. Cultivated cells were EX VIVO EXPANSION OF AUTOLOGOUS characterized by pan-cytokeratin, α-smooth muscle actin, and lactoferrin HUMAN CONJUNCTIVAL EPITHELIAL CELL immunostaining. Lacrimal gland cells from 7-weeks old GFP and non-GFP GRAFT FOR TREATMENT OF RECURRENT (C57B/6) mice were seeded on non-coating dish or on 3T3 feeder layer for colony forming potential. Sphere which was generated from newborn lacri- PTERYGIUM: A MULTI-CENTRIC CLINICAL mal gland epithelial cells were embedded in collagen and injected into lac- STUDY REPORT rimal gland recession mouse model to evaluate their regeneration potential. Results: The lacrimal gland epithelial cells of newborn mice could be selected Vasania, Viraf, Hari, Aarya, Tandon, Radhika, Shah, Sanjay, by cholera toxin, which was confirmed by pan-cytokeratin (+) andα -smooth Haldipurkar, Suhas, Shah, Smitesh, Sachan, Shailendra, muscle actin (-). With this medium and low adherent culture dish, adult mice Viswanathan, Chandra generated sphere formation from single cells. Such cells could also form Reliance Life Sciences Pvt. Ltd., Navi Mumbai, India, Dr. R. P. Centre for colony formation on 3T3 feeder cells. Spheres generated from new born lac- Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, rimal gland could be transplanted into injured lacrimal gland, and tended to India, King Edward Memorial Hospital, Pune, India, Laxmi Eye Institute, recover corneal injured score, but not secretory function. Conclusion: Adult Panvel, India, Dr. Shah’s Laser Eye Institute, Kalyan, India mouse lacrimal gland epithelial cells were successfully cultivated and these cells could generate the sphere formation. Such spheres could be transplant- Pterygium is a fibrovascular overgrowth of altered conjunctiva over the able to injured lacrimal gland. cornea and can normally be treated by surgical excision. However, recurrent pterygium is one of the major constraints and repeated excision may cause severe conjunctival scarring resulting in insufficient conjunctival epithelium. Transplantation of limbal and conjunctival autografts for treating pterygium EYE OR RETINAL CELLS has been practiced worldwide. However, availability of healthy tissue from the donor eye still remains a limiting factor. We examined the feasibility of Poster Board Number: 1179 using autologous human conjunctival epithelial cell (hCjEC) graft at multiple DIFFERENTIATION OF RETINAL CELLS FROM centers for treating moderate to severe recurrent pterygium. The present study is a prospective, open-label, single-arm, multi-centric trial conducted HUMAN INDUCED PLURIPOTENT STEM CELLS to evaluate mainly the safety followed by the efficacy of ex vivo expanded UNDER XENO-FREE CULTURE CONDITIONS autologous hCjEC graft in patients with recurrent pterygium. Twenty-five subjects meeting all eligibility criteria were enrolled in the study after receiv- Meyer, Jason S., Melkoumian, Zara, Sridhar, Akshayalakshmi, ing approval from their respective Ethics committee. Conjunctival biopsy Steward, Melissa M. was excised from the donor eye and transported to Reliance Life Sciences Department of Biology, Indiana University Center for Regenerative Biology (RLS) for further processing under cGMP conditions. The biopsy was cut and Medicine, Indianapolis, IN, USA, Corning Life Sciences, Corning, Inc, into pieces and seeded equally as explants on two denuded human amniotic Corning, NY, USA membranes and allowed to expand in culture for 2-3 weeks. The grafts were characterized for the expression of conjunctival epithelial markers p63, AE-1, Purpose: Human induced pluripotent stem cells (hiPSCs) have received AE-3, CK4, CK7, CK19, MUC4 etc. In-process testing was done to check for attention for their potential in the field of regenerative medicine. However, sterility, endotoxin and mycoplasma contamination. The hCjEC grafts were established methods for deriving retinal phenotypes from hiPSCs typically placed in indigenously designed stainless steel receptacles and transported involve the use of feeder cells or other animal products. In order for hiPSCs to the site for transplantation. Validation of transport conditions for delivery to realize their therapeutic potential, it will be necessary to demonstrate of conjunctival biopsy and the graft at respective centers were established. the ability to derive mature phenotypes in the absence of animal products. The patients were followed up at days 1, 7, 14, 30, 90 and 180 as per the Therefore, we sought to develop a simple method of deriving retinal cells study schedule post-transplant. The clinical outcome was assessed by local from undifferentiated hiPSCs under xeno-free conditions. Methods: hiPSCs examination, visual acuity, slit lamp examination, imprint cytology, fluores- were initially maintained through traditional methods on a layer of irradiated cein/ rose bengal staining, schirmer’s test and photographic evaluation to MEF cells, or through their growth in xeno-free medium on synthetic xeno- rule out the recurrence of pterygium. Out of the 25 evaluable patients, two free Corning® Synthemax™ Surface culture plates. After a minimum of 5 were lost to follow up during the study. Conjunctival inflammation subsided passages in a particular environment, cells were differentiated with minor to the normal level in 16 patients (69.6%) within 7 days and 18 patients modifications to our previous retinal differentiation protocol. Cells that had (78.3%) within 14 days. At day 90, 22 patients (95.7%) showed no con-

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Thursday Poster Abstracts junctival inflammation in the operated eye except for one patient till the end Poster Board Number: 1185 of the study. Goblet cells were detected in 19 patients (82.6%) at day 180 indicating that implanted hCjEC graft was anatomically similar to healthy na- SUBRETINAL TRANSPLANTATION OF NEURAL tive conjunctiva. Adequate hydration with presence of tears along with wet PROGENITORS DERIVED FROM HUMAN cornea was observed in all the patients confirming the normal physiological functioning of the conjunctiva. There were no recurrences of pterygium EMBRYONIC STEM CELLS INTO RETINAL seen in 18 patients. However, mild to moderate recurrence in the area of DEGENERATION RAT MODEL pterygium excision were observed in the remaining patients. There were no secondary infections and other complications reported in the study. Based on Park, Jung Hyun, Park, Un Chul, Cho, Myung Soo, Ku, Seung-Yup, our observation, we conclude that transplantation of hCjEC graft at multiple Choi, Young Min, Moon, Shin Yong, Yu, Hyeong Gon centers appears to be safe and effective for the treatment of recurrent ptery- Ophthalmology, Inje University, College of Medicine, Seoul, Korea, gium. Further, this study provides the initial basis of exploring the potential Republic of, Ophthalmology, Seoul National University, College of clinical application of hCjEC grafts for various conjunctival disorders. Medicine, Seoul, Korea, Republic of, Ophthalmology, Research and Development Center, Jeil Pharmaceutical Co., Ltd., Yong-In, Korea, Poster Board Number: 1183 Republic of, OB/GYN, Seoul National University, College of Medicine, MICRORNA-145 REGULATES THE Seoul, Korea, Republic of DIFFERENTIATION OF HUMAN CORNEAL STEM Purpose: To investigate the survival, integration and differentiation of CELLS hESCs-derived neural progenitors transplanted into the subretinal space Yam, Gary Hin-Fai of retinal degeneration rat model. Methods: The human embryonic stem cells (hESC) were induced to form embryoid body (EB) and underwent Dept. Ophthalmology & Visual Sciences, The Chinese University of Hong suspension culture for 4 to 7 days. Cultured EB were attached on the Kong, Hong Kong, Hong Kong matrigel-coated culture dish and expanded to form neural rosette and neural MicroRNAs are important epigenetic regulator in the renewal and differ- tube-like structure. The spherical neural masses (SNMs) were formed from entiation of adult epithelial stem cells and their progenies. Human corneal these structures by mechanical dissection. The expression of markers for epithelial progenitor cells (CEPCs), located in the basal limbus, maintain cor- photoreceptor precursor in neural progenitor was confirmed by immuno- neal homeostasis throughout one’s life. They respond to injury and normal staining and RT-PCR. The cell clumps with neural structures were isolated wearing by generating the corneal epithelium, though factors controlling and expanded in suspension. Retinal degeneration was induced by sodium this homeostatic switch are still largely unknown. This work was to identify iodate intravenous injection (70mg/kg) via tail vein of Spraque Dawley specific microRNAs participating in CEPC proliferation and differentiation. rats. A week after intravenous injection, destruction of retinal pigment We profiled the mature microRNA population in CEPC-enriched limbal/ epithelium and photoreceptor layers was identified. Approximately 2 x 105 peripheral corneal epithelium (LPC) containing CEPCs and early transit of neural progenitors derived from SNMs were injected into the subretinal amplifying cells by Human MicroRNA Microarray analysis (Agilent), and space of rats. Immunohistochemistry study was performed up to 8 weeks validated by quantitative polymerase chain reaction and in situ hybridiza- after transplantation with antibodies against human nuclei and beta-tubulin tion. We discovered differential expression of 18 microRNAs against central III. Results: After subretinal transplantation, the neural progenitors were corneal epithelia (CC), which contain late transit amplifying cells and termi- found to survive in the subretinal space up to 8 weeks and integrated in nally differentiated cells. Among them, the cluster miR-143/145 expressed the host retina. Teratoma or tumors were not observed. The expression of strongly in LPC but at low levels in CC (P=0.0004, Mann-Whitney U-test). neural retinal cell markers was confirmed with confocal microscopy in the Human CEPCs transfected with Lenti-miR-145 generated much thinner and transplanted cells. Conclusions: Transplanted neural progenitors migrate and substandard epithelium in vitro than those with Lenti-miR-143 or scrambled integrate into the retina of retinal degeneration rat model, expressing neural sequences. The cells had higher expression of corneal differentiation markers retinal cell markers. These results support the feasibility of the potential cytokeratin-3/12 and connexin-43. By oligo microarray analysis, miR-145 clinical application of hESCs-derived neural progenitors for treating retinal transfection in human corneal epithelial cells markedly reduced integrin degenerative diseases. beta-8 and up-regulated interferon beta-1 expression. Our study indicates the importance of miR-145 in promoting CEPCs to corneal epithelial differ- Poster Board Number: 1187 entiation. It could also be involved in the maintenance of epithelial integrity A SIMPLE AND EFFICIENT PROTOCOL FOR and immune protection of the cornea. DIFFERENTIATION OF RETINAL PIGMENTED EPITHELIAL CELLS FROM HUMAN INDUCED PLURIPOTENT STEM CELLS Zahabi, Azadeh Stem Cell, Royan, Tehran, Iran, Islamic Republic of Purpose: Human induced pluripotent stem cells (hiPSC) have the potential to differentiate into any cell type such as retinal pigmented epithelial (RPE) cells, rendering them a potential source for the treatment of a wide range of diseases such as age-related macular degeneration, retinitis pigmentosa, and Stargardt disease. Here, we describe a new efficient protocol for dif- ferentiation of hiPSCs to RPE cells efficient for transplantation. Materials and methods: The Royan hiPSC1 line was efficiently induced to RPE cells in an adherent and defined condition. Pigmented area then was isolated and expanded to form a differentiated RPE monolayer. The hiPSC-RPE cells were analyzed by electron microscopy, RT-PCR, Real-Time PCR, immunofluores- cent staining, flow cytometry, and patch clamp analysis. Results: We found

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that differentiated hiPSC-RPE cells were hexagonal and morphologically sim- developed and optimized. Efficacy of the reporter iPS clones are being tested ilar to mature human RPE cells. Data analysis indicates that retinal progeni- by growing them according to established methods that favor photorecep- tor specific genes significantly increased over two weeks and retinal mature tor generation and differentiation. Towards the goal of screening a small genes increased significantly over four weeks. The differentiated cells highly molecule library to improve efficiency of photoreceptor cell generation and expressed ZO1, a RPE marker, MITF, (57%), CRALBP (30), pigmentation differentiation we have first focused on developing the conditions necessary markers and RPE65 (39%), BEST (39%), specific RPE markers. Additionally, to grow stem cells as monolayer cultures, which is critical for quantifying the the forebrain markers, SIX3 and PAX6 and retinal precursor cells, RX, MITF, high content screen. For the mouse ES/iPS cells we used established proto- and CHX10 were expressed two weeks after induction. The pigmentation- cols in the presence of factors known to promote PhR generation in vitro. related genes, DCT, TYR and RPE-related genes, CRALBP, RPE65, BEST, and Such protocols, however, rely on differentiation as embryoid bodies which PEDF were detected highly over 4 weeks. The expression of RPE65 increased grow as 3D masses of cells that vary in their exposure to morphogens, significantly by continuing culture. The patch clamp analysis of differenti- cell-contact factors, show significant cell heterogeneity and are difficult to ated RPE cells at weeks 9-11 showed functionality of Ca2+ L-type channels. image in a high content fashion. To tailor this protocol to suite our needs Conclusion: These findings indicate a new short time, simple, and efficient we utilized existing knowledge of mouse development to successfully derive protocol for differentiation of hiPSCs to RPE cells. Moreover, hiPSCs provide neural enriched monolayer cultures. Similar work is underway with human an unlimited source for RPE cells not only for drug development and screen- ES and iPS cells based on adaptations to previously published work. ing and in-vitro disease modeling but also for cell-replacement therapy in eyes with retinal degenerative diseases due to primary RPE dysfunction. Poster Board Number: 1191 Poster Board Number: 1189 EXOGENOUS FACTORS ENRICH THE PROPORTION OF NEURAL RETINAL INDUCTION OF HUMAN AND MOUSE STEM PROGENITORS DERIVED FROM ADULT MOUSE CELLS TOWARDS A PHOTORECEPTOR-LIKE RETINAL STEM CELLS FATE Ballios, Brian G., Shoichet, Molly S., van der Kooy, Derek Wahlin, Karl J., Maruotti, Julien, Tucker, Budd A., Wallace, Kyle, Institute of Medical Science, University of Toronto, Toronto, ON, Canada, Gamm, David M., Michael, Young J., Zack, Donald J. Department of Chemical Engineering and Applied Chemistry, University Ophthalmology, Johns Hopkins School of Medicine, Baltimore, MD, USA, of Toronto, Toronto, ON, Canada, Department of Molecular Genetics, Carver College of Medicine, Iowa City, IA, USA, Waisman Center Stem Cell University of Toronto, Toronto, ON, Canada Research Program, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA, Department of Ophthalmology and Visual Recent reports have debated the existence of the adult RSC and the ability Sciences, University of Wisconsin School of Medicine and Public Health, of its progeny to differentiate into rod photoreceptors. We found that com- Madison, WI, USA, Ophthalmology, Harvard Medical School, Boston, MA, binations of taurine, retinoic acid and FGF2/heparin (T+RA+FH) added to USA differentiating RSC colonies can greatly increase the number of rod photore- ceptors to greater than 80% of all progeny. In contrast, only 10% of differ- Human embryonic stem cells (hESCs) and induced pluripotent (iPS) cells can entiated RSC progeny produce photoreceptors when cultured in serum. Our be encouraged to differentiate into rod and cone photoreceptor-like cells results for Nrl and rhodopsin expression profiles in vitro using Nrl.gfp report- (PhRs) by several published methods that rely on exposure to extracellular er mice suggest that the ontogenic stages of rod photoreceptor gene expres- factors that influence cell differentiation. A combination of IGF-1, noggin sion in vivo can be recapitulated in differentiating RSCs. The conservation (a BMP inhibitor), and Dkk1 (a Wnt/ß-catenin antagonist) followed by of the identical temporal sequence of Nrl/rhodopsin expression regardless exposure to bFGF is the basis for one protocol that takes approximately of the presence of a two-week expansion of retinal precursors from RSCs one month to obtain PhRs. Another uses aFGF, bFGF, taurine, Shh and RA in 1%FBS+F/H ([1%FBS+F/H] / [T+RA+F/H] sequence) supports a fate for induction and takes significantly longer to generate PhRs (six months as change hypothesis, but does not in itself rule out a selective photorecep- opposed to one). While promising both methods are slow, inefficient and tor survival effect. The differentiation time course shows steadily increasing generally yield low numbers of PhRs thus limiting their utility in stem cell re- rhodopsin-positive cell counts over 44 days of culture. Combined with the placement therapy. With the combined goals of improving our understand- similar proliferative rates between [1%FBS+F/H] / [T+RA+F/H] and 1%FBS ing the mechanisms underlying the differentiation of stem cells into PhR s alone, this strongly argues for a fate change effect of taurine/retinoic acid. and increasing the efficiency of the process for possible future therapeutic To further investigate the mechanism through which taurine/retinoic acid in- application, we are developing a high content screen for small molecules and fluences the differentiation of RSC progeny along a rod lineage, we treated pathways that promote PhR differentiation. As a step toward these goals, primary cultures of dissociated ciliary epithelium from pigmented/YFP adult we are generating fluorescent reporter cell lines for detection of photore- mice (ACTN.YFP) with T+RA+FH or FH-only (normal RSC sphere growth ceptors generated de novo. We have generated PhR specific reporter cell media) for 7 days. There was no statistical difference in the number of RSC lines from mice carrying fluorescent reporter genes and are in the process of spheres derived from the ciliary epithelium in either condition (p<0.05, doing so with human iPS cell lines also. For the mouse lines tail fibroblasts N=3). Numbers of RSC spheres were similar to those reported previously from transgenic reporter mice expressing green fluorescent protein under from wild-type pigmented animals. Qualitative differences were noted in the the control of the PhR specific promoters human CRX (early stage PhRs), degree of pigmentation of spheres: spheres derived in T+RA+FH exhibited bovine rhodopsin, or mouse red/green cone opsin (mature rods and cones) less pigmentation (and higher YFP expression) than spheres derived in were reprogrammed with the four ‘Yamanaka factors’ delivered by lentiviral FH-only. This may be due to the selection of (non-pigmented) neural retinal transduction. Preliminary analysis revealed the hallmarks of traditional iPS progenitors over (pigmented) retinal pigment epithelium progenitors during cells including teratoma formation, some of which even express GFP in iso- proliferation of RSC progeny in the forming sphere. When these T+RA+FH- lated clusters suggesting production of ‘photoreceptor-like’ cells. For human derived spheres were differentiated in 1%FBS+FH for 40 days, the percent- reporter iPS cell lines we are generating fluorescent reporters by coupling age of cell expressing rhodopsin and RPE65 were increased and decreased, photoreceptor cell specific promoters (NRL and IRBP) to cherry or GFP tags respectively, compared to FH-derived spheres differentiated in 1%FBS+FH. inserted into self-inactivating lentiviral (SIN LV) constructs and/or piggyBac There were no differences in the minor numbers of cells expressing Pax6 transposase reporter constructs. The ability to monitor differentiation with- (a retinal progenitor marker), calbindin (interneurons), or CRALBP (Müller out the need for immunocytochemical analysis should increase the through- glia), and no difference in the total number of cells after 40 days. This sup- put of the high content screen for which culture conditions are still being ports an effect of taurine/retinoic acid on the selective proliferation and/or

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Thursday Poster Abstracts survival of a neural retinal progenitor from adult RSCs. One cannot rule out Poster Board Number: 1195 the possibility of taurine/retinoic acid encouraging the survival of an early rod photoreceptor-specific progenitor (i.e. one which does not express Nrl LOSS OF P-CADHERIN NEGATIVELY AFFECTS or rhodospin). The existence of such a proliferative photoreceptor-specific MOUSE RETINAL STEM CELLS IN VITRO, BUT progenitor is controversial, and no specific marker exists for such a cell. On a broad scale, our study confirms the importance of exogenous (non-cell NOT IN VIVO. autonomous) signals directing the fate of retinal progenitors. Coles-Takabe, Brenda, Donaldson, Laura, van der Kooy, Derek Poster Board Number: 1193 Molecular Genetics, University of Toronto, Toronto, ON, Canada, IMS, University of Toronto, Toronto, ON, Canada A BIOENGINEERED DELIVERY SYSTEM FOR The adult mouse retinal stem cell (RSC) is a rare quiescent cell in the ciliary THE TRANSPLANTATION OF MOUSE RETINAL epithelium (CE) of the retina. The CE is made up of a non-pigmented inner STEM CELL-DERIVED ROD PHOTORECEPTORS and a pigmented outer cell layer, however the stem cells have been shown to reside in the pigmented layer using both FACS and clonal cell plating DIRECTLY ENCOURAGES CELL SURVIVAL strategies. The pigmented CE expresses the cell surface marker P-Cadherin Ballios, Brian G., van der Kooy, Derek, Shoichet, Molly S. (P-Cad) and the non-pigmented layer expresses N-Cadherin (N-Cad). The Institute of Medical Science, University of Toronto, Toronto, ON, Canada, clonal RSC colonies that arise from the CE proliferate from single pigmented Department of Molecular Genetics, University of Toronto, Toronto, ON, cells to form spheres that are made up of both pigmented and non-pig- Canada, Department of Chemical Engineering and Applied Chemistry, mented cells, and subsequently the spheres express both N-Cad and P-Cad University of Toronto, Toronto, ON, Canada mRNA. The RSC can be enriched in the CE population through sorting for medium size pigmented CE cells that express medium levels of P-Cad. In Adult retinal stem cells (RSCs) derived from the ciliary epithelium of mice order to test whether these cell surface antigens were important to the can give rise to all retinal cell types, as well as integrate into early postnatal formation of RSC sphere colonies, we used function blocking cadherin an- retinae. RSC progeny, directed to differentiate toward a photoreceptor fate, tibodies. We dissociated CE from mice, plated them at 10 cells/µl, and then have demonstrated functional recovery in mouse models of disease. The treated (at the time of dissociation into single cells) with function blocking potential of RSC progeny in adult mouse transplantation models has been antibodies at various concentrations to generate dose response curves. limited by poor cell survival, distribution and integration into host tissue. An Both the P- and N-Cad treated cells showed a significant decrease in clonal injectable and biodegradeable hydrogel-based material, a blend of hyaluor- sphere formation compared to controls, however the combination of P- and nan and methylcellulose (HAMC), provides an alternative cell delivery strat- N-Cad antibodies was not additive. Interestingly, the few spheres that do egy for RSCs and progeny and has shown promise in overcoming the barrier arise in both conditions are similar in size and morphology to the control of cell distribution following injection. Here we report a mechanism through spheres. When P- and N-Cad antibodies were added to CE cultures on day which HAMC directly supports the survival of newborn post-mitotic RSC- 3 after dissociation (when small multi cell colonies were present), there was derived rods. RSC progeny were pre-differentiated in vitro for 28 days on no decrease in the number of clonal RSC spheres as compared to controls. laminin substrate in the presence of either taurine/retinoic acid (rod photore- These results suggest that P- and N-Cad may facilitate the adherence of the ceptor-induction media) or 1% FBS (pan-retinal cell type induction media). cells in the formation of the clonally derived spheres; however, the primary At 28 days, induction factors were removed and the cells were exposed to effect of the P-Cad and N-Cad appears to be on the initial formation of the either HAMC reconstituted in serum free media (SFM), or SFM alone for 5 sphere, possibly due to adherence of the cells after the first divisions. To test days. Survival was assessed with ethidium homodimer and phenotype by whether P-Cad is required for RSC maintenance in vivo, we used P-Cad-/- immunocytochemical staining for Pax6, RPE65, and rhodopsin. It was found mice. P-Cad-/- mice have 3 times more clonal RSC spheres per mouse than that the post-mitotic photoreceptors showed significantly improved survival the control mice; however the frequencies for a CE derived cell to form a over 5 in HAMC (90%) relative to media alone (60%), with no change in clonal sphere are similar to +/+ mice. The ciliary epithelium was not enlarged mature photoreceptor phenotype as determined by rhodopsin expression. in the PCad-/- mice; therefore this suggests that many more P-Cad-/- cells Cells pre-differentiated in 1%FBS showed no change in phenotype, but are able to survive the dissociation procedure (presumably because they showed similar overall survival in both HAMC and SFM alone. When cul- don’t adhere to each other so much during the harsh dissociation into tured in 1%FBS for 28d, a significant number of RSC-derived cells continue single cells). These experiments demonstrate that the RSCs that reside in to express the mitotic, multipotent marker Pax6 (20%), and cell prolifera- the pigmented CE layer and express P-Cad, which may be important to the tion could account for the trend toward increased cell numbers over 5 days formation of adherent sphere colonies in vitro but is not essential for RSC in vitro. In addition, it has been previously reported that multipotent RSC behavior in vivo. progeny secrete their own growth factors (such as FGF2), which support cell survival and proliferation in a paracrine fashion. Investigating survival Poster Board Number: 1197 in the presence of HA and MC components individually demonstrated that the survival effect could be accounted for by the presence of HA alone. GENERATION OF RETINAL PIGMENT Immunocytochemical analysis revealed the expression of the hyaluronan re- EPITHELIUM FROM ADIPOSE-DERIVED STEM ceptor CD44 on RSC-derived rods. The binding of HA to CD44 is known to CELLS influence a number of downstream signaling pathways with diverse effects on cellular migration and survival. On a broad scale, the study points to the Ashley, Rebekah K., Hinman, Cassidy R., Carbone, Catherine, therapeutic potential of adult stem cell therapy enhanced by the application Clegg, Dennis O.1 of a bioengineered delivery vehicle. Molecular Cellular & Developmental Biology, University of California Santa Barbara, Santa Barbara, CA, USA, Biology, Brown University, Providence, RI, USA Adipose-derived stem cells (ASC) have become a valued stem cell popula- tion for use in regenerative medicine. A plentiful, autologous cell popula- tion isolated from adult adipose tissue, ASC use for cell-based therapies presents minimal ethical considerations in comparison to human embryonic stem cells (hESC). ASC populations demonstrate multipotency characteris-

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tics and are currently being implemented in numerous tissue replacement Poster Board Number: 1201 treatments as a source of differentiated bone, cardiac muscle, and cartilage cells. An appealing, novel use for ASC-based therapies is the treatment of RESVERATROL PROTECTS RETINAL STEM Age-Related Macular Degeneration (AMD). In the US alone, AMD is the CELLS FROM OXIDATIVE STRESS VIA leading cause of blindness in the elderly population, and is characterized by the degeneration of retinal pigment epithelium (RPE). This RPE dysfunc- EXPRESSION OF SIRT1 tion results in the death of rod and cone photoreceptors found in the eye’s Peng, Chi-Hsien, Chiou, Shih-Hwa macula inducing progressive vision loss. During development, RPE arise from National Yang-Ming University, Taipei, Taiwan the eye field subset of cells within the anterior neural plate following neural induction. Previous studies have shown that neural stem cell (NSC) derived Purpose: Silencing information regulator (SirT1) is an essential mediator for neurospheres have been successfully differentiated into functional RPE cells. longevity, and the level of SirT1 was evaluated for self-renewal and aging Independently, it has been demonstrated that neurosphere-like neuronal process abilities of retinal stem cells (RSCs). Methods: The level of SirT1 precursors can be derived from ASC populations. Thus ASCs are speculated mRNA expression in RSCs from rats of different ages was collected. Results: to be candidates for further differentiation from neuronal lineages. We com- SirT1 expression was significantly decreased in in vivo aged eyes, associated pared the RPE differentiation capability of ASCs and hESCs using a two-step with poor self-renewal abilities. Additionally, SirT1 mRNA levels were dose- approach of neurosphere differentiation followed by RPE differentiation. The dependently increased in resveratrol- treated RSCs. The expression of SirT1 first step entailed culturing both cell lines as neurospheres for 10 days. Then on oxidative stress-induced damage was significantly decreased, negatively the neurosphere-differentiated cells were passaged onto Matrigel coated correlated with the level of intracellular reactive oxygen species production. plates and cultured for 21 - 35 days in media supplemented with specific Treatment with resveratrol could effectively further reduce oxidative stress growth factors related to ocular differentiation; IGF-1, Dkk-1 and noggin. induced by H2O2 treatment in RSCs. Importantly, the anti-oxidant effects of Initial results indicate that ASCs can differentiate into neural precursors dem- resveratrol in H2O2-treated RSCs were significantly abolished by knock- onstrating neurosphere morphology and upregulation of specific neural stem down of SirT1 expression (sh-SirT1). Conclusion: SirT1 expression provides cell markers, an important preliminary step in differentiation to RPE. a feasible sensor in assessing self-renewal and aging process in RSCs. Res- veratrol can prevent reactive oxygen species-induced damages via increased Poster Board Number: 1199 retinal SirT1 expression LIMBAL STEM CELL CULTURE’S JOURNEY TO Poster Board Number: 1203 WIDESPREAD ADOPTION TRANSFER OF MRNA AND MICRO-RNA Foley, Lucy A., Ahmad, Sajjad BY EMBRYONIC STEM CELL-DERIVED Newcastle University, Newcastle Upon Tyne, United Kingdom MICROVESICLES INDUCES GENE EXPRESSION The cornea is the clear front of the eye and its epithelial surface is renewed CHANGES IN RETINAL PROGENITOR MüLLER by stem cells which can become damaged by chemical burns to the eye. Such damage results in loss of corneal clarity, persistent eye pain and blind- CELLS ness. Over the past few years we have developed a therapy using animal Katsman, Diana, Farber, Debora cell and product free cultured corneal epithelial stem cells to treat patients with considerable success, often restoring their vision. The challenge now is Ophthalmology, University of California Los Angeles, Los Angeles, CA, USA to be able to treat patients who are not able to travel to us for this stem cell Retinal degenerative diseases are among the leading causes of irreversible therapy. We have evaluated the different routes to widespread availability of blindness in the world. Therapies to slow retinal degeneration and augment this limbal stem cell treatment by considering cost, ease of implementation repair could reduce disability and improve the quality of life of millions of and regulatory requirements. Each route challenges the whole bioprocess affected individuals. Müller cells are emerging as good candidates for retinal (from biopsy to implantation) in different ways. These challenges include progenitor cells: they are multipotent and differentiate along multiple retinal process throughput, systems for cell culture, cell preservation and cell ship- neural lineages. Currently, a new concept of employing stem cell-derived ment. Limbal stem cell treatment is an autologous cell therapy which is more microvesicles (MVs) to induce the endogenous regenerative capacity of aligned with a service model structure than the mass production model of tissues has shown promising results. MVs are plasma membrane-derived pharmaceuticals and biopharmaceuticals. For autologous cell therapies wide- vesicles released into the extracellular environment by most cell types. MVs spread availability is achieved through scaling-out the production process. contain proteins, lipids, and mRNA molecules present in the parent cells. Our We have evaluated the economics and challenges of this scale-out approach laboratory has shown that embryonic stem cell-derived MVs (ESMVs) are with those identified in the scale-up approach used in pharmaceutical enriched in mRNA for several early transcription factors and in micro-RNAs and biopharmaceutical production and suited to allogeneic cell therapies. (miRNAs) that are essential for regulation of mRNA expression. Thus, ESMVs Widespread availability does not necessarily mean a therapy will be adopted may stimulate regeneration by inducing endogenous progenitor cells to by healthcare providers. We have gone some way to evaluating limbal stem repopulate and repair damaged tissue. Here, we describe the transfer of ESC cell therapy in terms of health economics in order to facilitate the adoption miRNA and mRNA to cultured Müller cells by ESMVs and the morphologi- process. This presentation will describe the results of our research, namely cal and gene expression changes induced in Müller cells by ESMV exposure. the most appropriate models for scaling out an autologous treatment, how ESMVs isolated from the supernatant of cultured ESCs were added to Müller this compares with scale up of an allogeneic equivalent and the associated cell culture medium. ESMV-exposed Müller cells were examined for morpho- bioprocessing and adoption challenges that we are addressing. logical changes. Their RNA was isolated at 8, 24, and 48 hours post expo- sure and expression of transcripts important for maintenance of stem cell pluripotency as well as stem cell-specific miRNAs was analyzed by qRT-PCR. Global gene expression changes induced by ESMV exposure were analyzed by microarrays. Müller cell cultures exposed to ESMVs showed morphologic heterogeneity, multiple cellular processes, and stellar-shaped cells growing as individual cells, when compared to the bipolar, homogeneous, spindle- like Müller cells growing in adherent “sheets” in control cultures. qRT-PCR analysis indicated the presence of miRNA 292 and 295-3p transcripts in

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ESMVs and Müller cells at 24 and 48 hours post ESMV exposure, with trace Poster Board Number: 1207 expression levels detectable 120 hours post exposure, indicating that miRNA is stable and not degraded for an extended period of time after transfer. TAKING STEM CELLS INTO THE CLASSROOM - Moreover, the levels of miRNA let 7a, which promotes and maintains cel- TAILORED EDUCATIONAL MATERIAL TO ASSIST lular differentiation, were found to be reduced in Müller cells post ESMV exposure. Oct-4 mRNA was found in ESMVs and in Müller cells 8, 24, and AUSTRALIAN TEACHERS 48 hours post exposure. Conclusions: ESMVs transfer genetic information Munsie, Megan, Sanderson, Aimee, Skinner, Rebecca (mRNA and miRNA) to Müller cells of the retina; the transcripts are retained Australian Stem Cell Centre, Clayton, Melbourne, Australia in Müller cells for days after transfer, and Müller cells in culture demonstrate morphological and gene expression changes after ESMV exposure. Studies Stem cell science attracts a large amount of public attention and enjoys a of global gene expression changes in Müller cells after ESMV exposure may high level of support in Australia, whether this equates to an understand- uncover the de-differentiation transcriptome and identify novel ways of ing of the ‘science behind the headlines’ is more difficult to gauge. A recent inducing endogenous retinal regenerative capacity. survey conducted by Australian Scientific and Technological Societies high- lighted what has been described as a “disturbing ignorance about science”. Further evidence of this lack of understanding can be seen in recent Austra- lian Government surveys putting Australian support for stem cell research ETHICS AND POLICY (including human embryonic stem cell research) consistently over 70% but Poster Board Number: 1205 putting understanding of the research at about 38%. If public support is not based on a sound understanding then it could be subject to exploitation or HOPEFUL JOURNEYS: EXPERIENCES OF adverse change. These findings, coupled with the fact that the number of AUSTRALIANS TRAVELLING OVERSEAS FOR science teachers in Australian schools is declining, raises issues about how we are going to teach science to the next generation of Australians. Due STEM CELL TREATMENT AND IMPLICATIONS to its high profile and therapeutic potential, introducing stem cells into the FOR STEM CELL SCIENCE AND REGENERATIVE classroom is one way to trigger students’ interest in science. Over the last five years, the Australian Stem Cell Centre has been developing educa- MEDICINE tional material and programs for students. This has included fact sheets, a Munsie, Megan, Skinner, Rebecca, Seear, Kate, Petersen, Alan dedicated interactive website the Stem Cell Channel, direct responses to questions asked by students and teachers and establishing a ‘Back to Schools Australian Stem Cell Centre, Clayton, Melbourne, Australia, School of Program’, where stem cell scientists have visited more than 2850 students Political and Social Inquiry, Monash University, Clayton, Melbourne, in Victorian high schools. While these efforts have been welcomed by Australia teachers and students alike, feedback from teachers to the ASCC has stated Stem cell science is a field that promises to revolutionise healthcare. At pres- that what they really need is easy to use, curriculum relevant lesson plans. ent the only clinically recognised stem cell treatments available in Australia Teachers are then able to use these lesson plans to build educational units are for patients with haematological malignancies and immune deficiencies. around topical events, or a visit to the school by a scientist. In response to Although clinical trials are underway here and overseas, stem cell based these requests, in 2010 the ASCC developed a Stem Cell Teachers Kit which therapies (SCT) for a wider range of conditions and disabilities are yet to be features over 30 activities matched to state and national curricula. Endorsed proven safe and effective. In stark contrast, many clinics and companies, by Australian science education networks, the Kit has been popular with in usually based outside of Australia, are already marketing a range of stem excess of 600 downloads from a protected area of the ASCC website since cell based treatments at high cost and with little, if any, medically recognised its launch. The challenge now is to ensure that this resource remains widely evidence supporting their claims. This rise of direct-to-consumer marketing available and continues to contain content that reflects the developments in of SCT, often through internet based social networking, presents a signifi- the field in a manner that will engage young Australians in science. cant challenge as patients are left largely on their own to decide whether to pursue advertised treatments. To better understand how Australians Poster Board Number: 1209 navigate this challenge, we conducted qualitative interviews (n=16) to document the experiences, motivations and expectations of patients and INFORMING THE PUBLIC - STEM CELL their careers who have travelled abroad to receive SCT. One of the most sig- THERAPIES, NOW AND IN THE FUTURE nificant findings relates to the process of weighing benefits against the risks. Skinner, Rebecca, Herbert, Kirsten, Munsie, Megan Most participants did not report feeling “duped” by overseas providers and/ or promised miracles that did not eventuate. Instead, they were prepared to Australian Stem Cell Centre, Clayton, Melbourne, Australia, Peter “experiment” with treatment on the basis that it might work, or because of MacCallum Cancer Centre, Melbourne, Australia other “risks” associated with a deteriorating condition or reduced life span. The Australian Stem Cell Centre (ASCC) receives hundreds of enquiries each It was also reported that participants perceived Australia had “fallen behind” year from members of the public seeking information on stem cell therapies in this area due to political, cultural or ethical reasons, and that they had “no and clinical trials. Many of these enquiries are triggered by newspaper or choice” but to leave the country for treatment. The findings of this study television stories of remarkable recovery following stem cell treatments. provide the first insight into the experience of Australians travelling overseas More recently there has also been an increase in the number of enquiries as for unproven SCT. Gaining a greater understanding of what shapes patients a result of information obtained on the internet. With claims of instant cures expectations is essential to effectively counter the aggressive marketing for a wide range of conditions, yet with little detail presented on the actual strategies being employed and the risk unregulated SCT use poses to regen- treatment including evidence of clinical benefit and safety, patients or their erative medicine. loved ones are searching for more information on what they see as their only hope. To assist the Australian patient community in their quest to un- derstand more about their options, the ASCC created a Patient Handbook, based on the International Society for Stem Cell Research (ISSCR) Patient Handbook but tailored for the Australian audience. Published in December 2009, and endorsed by Australian patient groups, the ASCC Patient Hand- book has become the ASCC’s key document for interacting with patients

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and the public. It is the most downloaded item on the ASCC’s website, has autonomy and privacy of donors. Stem cell research is a global enterprise. received international recognition in Nature Medicine and has been widely The international exchange of samples, particularly in the context of clinical reported on within patient group newsletters and other Australian media. translation, is predicated on obtaining donor information. Hence, trace- While the ASCC Patient Information Handbook has proved popular, the ability is essential for the above to occur. Moreover, traceability of samples challenge is to evaluate its effectiveness as a means of informing the public constitutes one of the cornerstones of stem cell banking. Traceability has about stem cell science and gather more data to better understand individu- been defined as ‘tracking an individual through their medical history’. It not als’ motivations and information needs. As such, in 2010 the ASCC was only promotes safety and quality, but also provides a system for the track- involved in a qualitative study where 16 patients and/or their careers who ing of handling and storage conditions and of ethical provenance. Indeed, had travelled outside of Australia for an unproven stem cell treatment were traceability is an essential component of the quality management system of interviewed about their experiences, motivations, understanding and expec- stem cell banks. Across jurisdictions, policies governing the traceability of tations. A number of the interview questions focused on the background samples and stem cell lines vary. Moreover, the regulations adopted in some research the patients and careers undertook, including their knowledge and countries make traceability unfeasible by mandating the anonymization of experiences of the ASCC Patient Handbook, prior to making the decision (allogeneic) stem cell lines. The latter has negative consequences in terms to travel. The information gathered provides insight into how patients and of the utility of the lines. From an international comparative perspective, careers evaluate risk, where they gather information from and what sources this presentation analyzes convergence and divergence in policy and ethical they trust. These insights will be used to ensure the maximum effectiveness approaches to the traceability of samples and data. Issues surrounding the of the ASCC Patient Handbook for Australian patients and their careers. transnational sharing of stem cell lines, informed consent and privacy re- quirements will be studied in the context of stem cell banking. Furthermore, Poster Board Number: 1211 we will propose policy recommendations, building on lessons learned in the EXPERIMENTAL TREATMENT, STEM CELLS general context of biobanking. AND SPINAL CORD INJURIES: IS THERE Poster Board Number: 1215 A BALANCED RIGHT BETWEEN RISK AND REGULATION OF STEM CELL-BASED LUXURY? THERAPIES IN CANADA: A SURVEY OF Eijkholt, Marleen, Illes, Judy CURRENT ISSUES AND CONCERNS Institute of Science Ethics and Innovation, University of Manchester, Nguyen, Thu Minh, von Tigerstrom, Barbara, Knoppers, Bartha Manchester, United Kingdom, Neurology, University of British Columbia, Maria Faculty of Medicine, BC, Canada Dept. of Human Genetics, Faculty of Medicine, Centre of Genomics and Practical changes on the landscape of stem cell therapy for spinal cord injury Policy, Montreal, QC, Canada, University of Saskatchewan, College of Law, (SCI) have created an urgent need to revisit the ethical and legal question of Saskatoon, SK, Canada the right to experimental treatment. On the one hand, clinical trials involving stem cells for spinal cord injuries have been launched. They involve age-old Stem cell therapies offer enormous potential for the treatment of a wide challenges associated with recruitment and enrollment eligibility, capacity, range of diseases and conditions. Despite the excitement over such ad- consent, and disclosure, but equally entail new challenges associated with vances, regulators are faced with the challenge of determining criteria to unparalleled medical risks and the nature of stem cell research itself. On the ensure stem cells and their products are safe and effective for human use. other hand, the relentless medical tourism trade highlights the impatience of However, stem cell-based products and therapies present unique regula- prospective beneficiaries of therapies, the demand for empowerment, and tory challenges because standard drug development models do not wholly the ability to acquire access through financial elitism. Tensions are mount- apply given the complexity and diversity of these products and therapies. As ing between interest in autonomy and the interest in public health as result, a result, regulatory requirements are often unclear and ambiguous creating and have created an imperative to explore whether individuals with spinal unnecessary barriers for research. As part of a Canadian Stem Cell Network- cord injuries have a right to experimental treatment with stem cells. For funded project entitled: “Regulation of Stem Cell-Based Therapies: Mapping terminally ill patients and for life saving purposes, some jurisdictions have and Exploring the Canadian Landscape,” our team sought feedback from developed regimes of ‘compassionate use’ (CU) for experimental drugs. stakeholders regarding areas of uncertainty or concern about existing Given the dynamic environment for stem cells and SCI and the potential for regulatory oversight of cell therapies. A selection of Canadian researchers life-improving benefit, should the CU regime be extended to the SCI and and clinicians working in the area of stem cell research were interviewed to stem cell context? We will discuss the dilemmas of the CU, examine factors assess certain key questions: 1) whether current regulatory requirements are that must be considered in access to experimental treatment for spinal cord easily accessible and well understood; 2) whether regulatory requirements injury, and propose some pragmatic solutions seen through the legal and create important challenges or barriers; and 3) whether there is a need for neuroethics lens. further guidance on the issue. The results of this survey will be analyzed and compared to issues and concerns experienced in other countries, as reported Poster Board Number: 1213 in the literature, to identify challenges which may be on the horizon for Canada and possible solutions for regulatory reform. TRACKING DONORS IN STEM CELL RESEARCH AND BANKING: POLICY CONSIDERATIONS Knoppers, Bartha Maria, Isasi, Rosario Human Genetics, McGill Univeristy, Montreal, QC, Canada Stem cell banks are repositories supporting transnational access to qual- ity- controlled and ethically sourced stem cell lines of different origin (e.g. embryonic, iPS cells) and of varying grades (e.g. research vs. clinical). Like biobiobanks generally, stem cell banks have as a core objective to avoid redundancy in research projects and to eliminate the need for the collection and derivation of additional human materials. Moreover, they both face sim- ilar challenges in ensuring safety through traceability, while protecting the

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Poster Board Number: 1217 Poster Board Number: 1219 GERMANY’S SEARCH FOR NEW STEM CELL A HISTORY OF THE GUIDELINES FOR CLINICAL RESEARCH RULES BETWEEN THE POLES OF TRANSLATION OF REGENERATIVE MEDICINE THE RECENT PREIMPLANTATION GENETIC RELEASED BY MINISTRY OF HEALTH, LABOR DIAGNOSIS RULING OF THE FEDERAL AND WELFARE, JAPAN SUPREME COURT AND THE IMPACT OF Matsuyama, Akifumi REPROGRAMMED STEM CELLS Foundation for Biomedical Research and Innovation, Kobe, Japan Faltus, Timo A medical system is anticipated, where high-quality medical services are University of Leipzig, Translational Centre for Regenerative Medicine, accessible without anxieties whenever we are ill. The innovative regenerative Leipzig, Germany medical products, or tissue-engineered medical products, have enabled us to overcome some life-threatening diseases, however, challenges still remain Research with all kinds of stem cells, including human embryonic and for others. The Ministry of Health, Labor and Welfare, Japan has implement- induced pluripotent stem cells, is permissible in Germany. However, the ed “The guideline for clinical research using human stem cells” (Notification destructive use of early human embryos to yield embryonic stem cells is not No.425. 2006. MHLW, Japan.) and has launched on Sep1, 2006. The major allowed in Germany, but the import of those cells from abroad is permis- goal of the guideline is to reinforce and ensure safe on investigational new sible. On the one hand, a recent decision of Germany’s Federal Supreme regenerative medical products. After the guideline had launched, Minister Court, which now allows discarding certain pre-implantation genetic of MHLW, Japan has approved over 30 protocols in which human somatic diagnosis (PGD) embryos, sheds new light on Germany’s stem cell law and stem/progenitor or precursor cells have applied for patients. In major parts may facilitate to ease still existing restrictions in stem cell research. On the of the approved protocols, bone marrow-derived mesenchymal stem cells other hand, the discovery of induced pluripotent stem cells (iPSC) coincided have been used for clinical investigation. On 3 Dec 2008, ISSCR has released with the latest amendment of Germany’s stem cell law and could have been Guidelines for the Clinical Translation of Stem Cells. The guideline has urged almost a reason to legally cancel further research with embryonic stem cells the Japanese government to revise the guideline (Notification No.425. 2006. in Germany, because iPSC were seen as a scientific alternative for the use of MHLW, Japan.). The government had invited the academic experts in the human embryonic stem cells. On July 6, 2010, the German Federal Supreme field of regenerative medicine, and the committee proposed that the clinical Court decided that PGD is permissible under the German Embryo Protec- studies using iPS cells shall be allowed, because recent advance of stem tion Act. That decision is surprising because one consequence of PGD is the cell research hinted us to the epoch in which iPS cells could be used for the necessity of discarding those embryos in which genetic disorders have been clinical studies. On Nov 1, 2010, “The guideline for clinical research using detected. This finding seems contrary to the central provision in the German human stem cells” has been revised as Notification No.380. 2010. MHLW, Embryo Protection Act, which states that every usage of the embryo that Japan. On the revised guideline, clinical investigators can apply autologous does not guarantee the maintenance of the embryo in vitro is prohibited. and/or allogenic iPS cells as investigational cellular products, but not hES Therefore, this ruling may pave the way for a new legal understanding of cells. From Sep, 2006 to Nov, 2010, FDA has released a line of guidelines in vitro embryos and, therefore, may influence future stem cell legislation in for cellular therapy, and EMA has proposed some directives for somatic Germany. If it is now permissible to discard certain PGD embryos, it must be stem cellular therapy. FDA approved hES cells as IND for the patients with clarified whether those embryos must be discarded or if they could be used subacute severe spine injury. Till 2010, biomedical researchers in Japan could for research purposes. If it were permissible to use certain PGD embryos use ES cells only in basic research but not in clinical studies and/or trials. for research purposes, why should it not also be permissible to use surplus This news made Japanese government felt the need for revision of “The embryos created by in vitro fertilisation for research purposes. The German guideline for clinical research using human stem cells” (Notification No.380. Stem Cell Act states the provisions for the import and work with imported 2010.) and the government will do so in 2011. In this study, I give an human embryonic stem cells in Germany. After political debates and pres- overview of the history for clinical and/or translational research using human sure by the scientific community, German lawmakers amended the German stem cells in Japan. Stem Cell Act in 2007. The amendment changed the cut-off date for the import to May 1, 2007. That amendment coincided with the creation of less ethically charged iPSC. The discovery of iPSC during the amendment process of the German Stem Cell Act could have put a total stop to German human MUSCLE CELLS embryonic stem cell research. For example, some politicians and church Poster Board Number: 1221 representatives were of the opinion that the discovery of the iPSC as an al- ternative for embryonic stem cells would be the right time to exit completely PAX7 EXPRESSING CELLS CONTRIBUTE TO from the research with human embryonic stem cells in Germany. Instead of accepting this opinion the lawmakers only changed the cut-off date. How- DERMAL WOUND REPAIR; REGULATING ever, since its first announcement, the German Stem Cell Act has contained SCAR SIZE THROUGH A β-CATENIN MEDIATED a subsidiary provision, which states that the import of human embryonic PROCESS stem cells to Germany is not permitted if there is a scientific alternative for the use of human embryonic stem cells. The scientific and medical success Amini-Nik, Saeid, Glancy, Dylan, Boimer, Corey, Whetstone, of reprogramming research could therefore legally inhibit the further import Heather, Keller, Charles, Alman, Benjamin of and research with human embryonic stem cells in Germany, if iPSC were Dev Biol and Stem Cells, Hospital for Sick Children, Toronto, ON, Canada, an alternative for the use of human embryonic stem cells. The subsidiary Oregon Health & Science University ., Portland, OR, USA provision could therefore also be a possibility to stop or to hinder the further embryonic stem cell research in Germany without any changes in the law. During skin wound healing, fibroblast like cells reconstitute the dermal com- ponent of the repaired skin filling the wound gap. A subset of these cells are transcriptionally acitive for β-catenin /TCF signaling during the proliferative phase of the repair process, and β-catenin levels control the size of the scar that ultimately forms by regulating the number of dermal fibroblasts. Here

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we performed cell lineage studies to reveal a source of the dermal cells in Poster Board Number: 1225 which β-catenin signaling is activated during wound repair. Using a reporter mouse, we found that cells in the early wound in which TCF dependent CLONED MURINE SATELLITE CELLS transcription is activated express marker of myocytes. Using mice in which DISPLAY HETEROGENEITY AND DISTINCT cells express Pax7 (muscle progenitors) or Mck (differentiated myocytes) are permanently labelled, we showed that one quarter of dermal cells in the DIFFERENTIATION POTENTIAL BOTH IN VITRO healing wound are derived from Pax7 expressing progeny, but none from AND IN VIVO Mck expressing progeny. Removing one allele of β-catenin in the Pax7 ex- pressing progeny resulted in a significantly smaller scar size with fewer Pax7 Urbani, Luca, Repele, Andrea, Franzin, Chiara, Piccoli, Martina, expressing progeny contributing to wound repair. During wound healing, Solagna, Francesca, Pozzobon, Michela, De Coppi, Paolo β-catenin activation causes muscle satellite cells to adopt a fibrotic pheno- Department of Pediatrics, University of Padova, Padua, Italy, Department type and this is a major source of dermal cells in the repair process. of Pediatrics, University of Padova and Città della Speranza Foundation, Padua, Italy, Città della Speranza Foundation, Padua, Italy, Department of Poster Board Number: 1223 Pediatrics, University of Padua and University College London and Città ORIGIN OF MUSCLE SATELLITE CELLS IN THE della Speranza Foundation, London, United Kingdom XENOPUS EMBRYO Background: Several studies have raised the possibility that the satellite cell (SCs) compartment may be heterogeneous. Recently, we identified and Daughters, Randy, Chen, Ying, Slack, Jonathan clonally characterize two main subpopulations of SCs in rat skeletal muscles Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA which were distinct by their growth rate in low proliferative clones (LPC) and high proliferative clones (HPC). In this study we investigated whether Muscle satellite cells are the stem cells of skeletal muscle. They proliferate LPC and HPC can be found also in mouse skeletal muscles and investigated and contribute to new muscle formation during the growth of the animal, whether they could have a different role for muscle regeneration. Methods: and then become quiescent in the adult. The biology of satellite cells in the Clones of SCs were obtained from mouse Extensor Digitorum Brevis and So- adult has been well studied, but their origin in the embryo remains unclear. leus myofibers. After enzymatic digestion, muscle fibers were collected and Previous studies have indicated that satellite cells originate from the somites triturated using a 18 G needle to disengage SCs. The resulting suspension and in particular from a Pax3/7-positive population of cells located in the was diluted in proliferative medium and dispensed into 96-well petri dishes central region of the dermamyotome of the somite. But these studies did not with limiting dilution (0.5 cell/well). MTS proliferative assay was performed indicate the origin of the satellite cells prior to somitogenesis, nor whether at 5, 10 and 15 days of culture. Results: The two different proliferation rates they come from the same or different region as the myoblasts that form the were tested with MTS assay that highlighted a 6-fold increase in the dye primary and secondary myofibers. The Xenopus embryo offers an opportu- processing by HPC compared to LPC after 10 days in culture, that became nity to investigate this problem because the neurula stage embryo is easily 9 times higher at day 15 of culture. Interestingly, when transplanted in accessible and is well suited to microsurgical experiments. Fate mapping at cardiotoxin-injured Tibialis Anterior (TA) muscles, mouse GFP-positive LPC the open neural plate stage was carried out using orthotopic grafts from pools were able to reach a better engraftment than HPC, as evidenced by transgenic embryos expressing GFP. These studies showed that most satel- GFP detection through PCR analysis. Moreover, transplantation with single lite cells originate from the dorsolateral plate rather than from the paraxial clones cultured for 10 days has been performed. Only TA injected with LPC mesoderm. Specification studies were made by isolation of explants from the and not HPC were positive for GFP. Conclusions: These results support our paraxial and dorsolateral regions of neurulae and these also indicated that suggestion that different proliferation and differentiation potential of HPC the satellite cell progenitors arise from the dorsolateral plate. Muscle satel- and LPC mirror two distinct satellite cells states in the muscle fibre. Their in lite cells express Pax7, but overexpression of Pax7 in blastomeres of whole vivo regenerative ability may have implication for cell therapy and tissue embryos that populate the myogenic areas does not induce the formation of engineering applications. additional satellite cells. Moreover, a dominant-negative construct, Pax7EnR, does not reduce satellite cell formation. Neither Pax7 nor other myogenic Poster Board Number: 1227 transcription factor genes will induce satellite cell formation in animal caps treated with FGF. However, BMP RNA or protein will do so, both for FGF- A MICRORNA CONTROLS THE SKELETAL treated animal caps and for paraxial neurula explants. Conversely, the induc- MUSCLE / BROWN FAT SWITCH tion of Noggin in dorsolateral explants from HGEM-Noggin transgenic neu- rulae, will block formation of satellite cells, showing that BMP signaling is Yin, Hang, Pasut, Alessandra, Soleimani, Vahab, Bentzinger, Florian, required in vivo for satellite cell formation. We conclude that, in contrast to Rudnicki, Michael the currently held belief of a paraxial mesoderm origin, satellite cell progeni- Sprott Centre for Stem Cell Research, Ottawa Hospital Research Institute, tors are initially specified in the dorsal part of the lateral plate mesoderm and Ottawa, ON, Canada later become incorporated into the myotomes. In addition, the initial speci- fication occurs at the neurula stage and depends on the ventral to-dorsal Two functionally distinct adipose tissues exist in mammals: white adipose BMP gradient in the early embryo. We are currently investigating whether tissue (white fat) stores energy by synthesizing triglyceride, while brown the same mechanisms are also found in mouse satellite cell formation. adipose tissue (brown fat) expends energy in form of body heat by fatty acid oxidation. In human adults, the presence of brown fat is reversely correlated with obesity. Interestingly, skeletal muscles and brown fat, but not white fat, are derived from the same group of progenitor cells in the embryo. How- ever, it remains poorly understood whether such lineage plasticity of brown fat and skeletal muscles exists in adult and, if so, the molecular mechanism underlying their lineage determination. Here, we report that adult muscle stem cells (satellite cells) can spontaneously differentiate into functional brown adipocytes. We further demonstrate a microRNA controls the lineage plasticity between brown fat and skeletal muscles both in vitro and in vivo. This study provides a potential alternative approach to preventing / treating obesity by increasing energy expenditure rather than limiting calorie intake.

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Thursday Poster Abstracts

Poster Board Number: 1229 separate role from motor neuron dysfunction in the etiology of SMA. SMALL MOLECULE SCREENING FOR MOUSE Poster Board Number: 1235 SATELLITE CELL PROLIFERATION A LONG-TERM SELF-RENEWABLE POPULATION Wagner, Amanda K., Hayhurst, Monica, Arvanites, Anthony, IN ACTIVATED SATELLITE CELLS IN MOUSE Davidow, Lance S., Wagers, Amy J., Rubin, Lee L. SKELETAL MUSCLE Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, Ono, Yusuke, Miyagoe-Suzuki, Yuko, Takeda, Shin’ichi USA Department of Molecular Therapy, National Center of Neurology and Satellite cells are a population of skeletal muscle stem cells that are located Psychiatry, Tokyo, Japan beneath the basal lamina surrounding the muscle fiber and are required for muscle growth and muscle repair after injury or exercise. Satellite cell Satellite cells are skeletal muscle stem cells, playing important roles for post- number and function are affected by normal aging and in several diseases, natal muscle growth and repair/regeneration of adult muscle. Satellite cells resulting in progressive muscle wasting or inefficient recovery after injury. are mitotically quiescent in healthy muscles but are activated immediately Examples include Duchenne muscular dystrophy, in which satellite cells are following muscle damage, in order to proliferate for supplying myonuclei depleted through constant use, and sarcopenia, where satellite cells may and self-renew for maintaining satellite cell pool. Although fast-dividing and both be depleted and adversely affected in their proliferative capacity by slow-dividing cell populations in activated satellite cells have been observed, changes in their environment. Finding treatments that would expand the the functional differences between them remain unclear. To understand the endogenous population of satellite cells could aid greatly in treating these relation between the proliferation behaviours and stem cell function, we debilitating diseases. To this end, we conducted an image-based screen of isolated satellite cells from mice and labelled with a fluorescent lipophilic dye selected small molecules for their ability to increase proliferation in satellite PKH26, to chase the frequency of cell division. The time-lapse videomicro- cells isolated from adult mouse muscle tissue. Satellite cells were isolated us- scopic analysis revealed that a slow-dividing small population retaining high ing cell surface markers, cultured in the presence of small molecules for four level of PKH26 dye exists even in the growth factor-rich culture condition. days, and then analyzed using automated confocal microscopy to determine Both of fast-dividing PKH26low and slow-dividing PKH26high popula- cell number. Using this procedure, we have confirmed several compounds, tion sorted by FACS underwent myogenic differentiation and self-renewal, operating through defined signaling pathways, which are capable of whereas only PKH26high population could give rise to highly proliferative enhancing proliferation in cultures of primary mouse satellite cells. The progeny having long-term self-renewal ability after passage. Id1, a negative myogenic capacity of treated cells is currently being tested through marker regulator for bHLH transcriptional factors, is known to be important for analysis and differentiation assays, as well as in vivo muscle grafting. We an- maintaining undifferentiated state of satellite cells by preventing myogenic differentiation.Poster The level of Id1 expression Withdrawn was varied within PKH26high ticipate that compounds capable of proliferating cells without adversely af- fecting their differentiation potential could become useful drugs for treating population, and long-term self-renewable population was restricted to the diseases that include a skeletal muscle defect as one of their components. high level of Id1 expression (Id1high) compartment. Finally, genome-wide gene expression analysis identified that molecular characteristics of PKH- Poster Board Number: 1233 26high/Id1high population are more quiescent and undifferentiated state compared to others. Our observations therefore suggest that the slow- A MUSCLE PRECURSOR DEFECT ASSOCIATED dividing population expressing high level of Id1 protein retains long-term WITH LOW LEVELS OF SURVIVAL OF MOTOR self-renewal potential and may be indispensable for efficient repeated NEURON PROTEIN. regeneration of adult muscle. Hayhurst, Monica, Cerletti, Massimiliano, Wagner, Amanda K., Poster Board Number: 1239 Wagers, Amy J., Rubin, Lee L. TRANSPLANTATION OF MESENCHYMAL STEM Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, CELLS FROM MOUSE ES CELLS USA AFTER MUSCLE INJURIES Spinal muscular atrophy (SMA) is the leading genetic cause of mortality in infants and toddlers. The disease is caused by low levels of Survival of Motor Ninagawa, Nana, Isobe, Eri, Murakami, Rumi, Kobayashi, Mami, Neuron (SMN) gene expression and is characterized by the degeneration of Torihashi, Shigeko spinal cord motor neurons and muscular atrophy. Several lines of evidence Nagoya University Graduate School of Medicine, Nagoya, Japan suggest that muscle cells may be affected directly by reduced SMN and that they may contribute indirectly to poor motor neuron survival. Here Background and Aims: It is well known that mesenchymal stem cells (MSCs) we examine the behavior of satellite cells, muscle stem cells that play an have a high potential for differentiation into mesenchymal cells. Adipose essential role in muscle growth and repair, which we isolated from a severe tissues have proved to be a useful and rich source of adipose tissue-derived mouse model of SMA (Smn-/-; SMN2+/+). The mice die within the first mesenchymal stem cells (ADSCs). Although ADSCs have shown therapeutic week after birth and, already by postnatal day 2, have muscles that are small efficiency in repairing damaged mesenchymal tissues, their isolation and in size compared to wildtype (Smn+/+; SMN2+/+) muscle. Surprisingly, purification from adult adipose tissue still involves complex and troublesome we found similar numbers of satellite cells in the two types of muscles at procedures. Moreover, they readily differentiate into adipocytes, osteocytes that stage of development. To study these cells in more detail, we isolated and chondrocytes but not into muscular cells. Mouse ES cells, on the other them from skeletal muscle by FACS using a previously established set of cell hand, are pluripotent, and their induction of adipogenesis has been well surface markers. When cultured under high serum conditions, isolated satel- described. We recently established a novel method for the induction and lite cells from wildtype and SMA muscle showed comparable survival and collection of MSCs using a typical cell surface marker, CD105, through proliferation rates. However, the SMA muscle satellite cells differentiated adipogenesis from mouse ES cells without genetic manipulation. Thus, our abnormally, as revealed by the premature expression of committed myoblast aim in the present study is to evaluate the potential of MSCs derived from markers, and reduced efficiency in forming myotubes when switched to low ES cells (E-MSCs) for differentiation into skeletal muscles and promotion serum culture medium. These differences may explain the small muscle size of functional recovery in injured muscle of mice in which our E-MSCs were associated with SMA and suggest that muscle defects play a significant and transplanted. Materials and Methods: ES cells (G4-2; kind gifts of Dr. Niwa)

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Thursday Poster Abstracts

carrying the enhanced green fluorescent protein _EGFP_ gene under the were harvested, trypsinized and cultured on iMEFs in ECM media. Pluripo- control of cytomegalovirus/chicken β-actin promoter, expanded their popu- tency was tested via teratoma formation, immunocytochemical staining, lation, and embryoid bodies (EBs) were formed in hanging drops. EBs were Western Blot and RT-PCR. Results: iPSC colonies were obtained using each cultured in a retinoic acid-containing medium. After washing, they settled on of the above 5 infection conditions. In the 4-factor STEMCAA (Oct4, Sox2, culture dishes and were maintained with adipogenesis medium (insulin/tri- cMyc, Klf4) group initial colonies appear by 6 days post-infection, in the iodo-thyronine). After the increase of CD105+ cells, we isolated and sorted remaining 4 conditions colonies began to appear at 13 days post-infection. them by a magnetic cell sorter (MACS; Miltenyi). CD105-positive E-MSCs While iPSC colonies were generated via induction of Sox2 or Oct4 alone the were transplanted into the injured tibialis anterior muscles of SCID mice 24 number of colonies generated was approximately 20 fold lower as compared hours after clamping. At 1, 2 and 3 weeks, respectively after transplanta- to the 4-factor condition. Although several small “background” colonies tion, myogenic differentiation was examined by M-cadherin and/or skeletal appeared in the negative control condition, they differed in morphology, did muscle myosin heavy-chain (MHC) immuno-staining, respectively. We then not continue to grow, and were not passageable. Conclusion: Exogenous counted EGFP expressing E-MSCs among regenerating muscle cells, fol- Sox2 or Oct4 are sufficient for induction of pluripotency in hRPCs. lowed by comparing the functional performance of transplanted and sham transplanted animals using a functional analyzer Cat Walk XT (Noldus). Re- Poster Board Number: 1245 sults and Conclusion: MSCs derived from mouse ES cells (E-MSCs) expanded USING HUMAN IPS AND INDUCED NEURONAL by themselves without any changes in their characteristic features and karyotypes. High telomerese activation was demonstrated. E-MSCs showed CELLS (IN CELLS) TO INVESTIGATE high potential for differentiation into mesenchymal cell types, especially skel- PSYCHIATRIC DISEASE RISK GENES etal muscles in vitro and in vivo. When E-MSCs were transplanted into the injured tibialis anterior muscles of SCID mice, most of them differentiated Singh, Karun, Yoshimizu, Takao, Madison, Jon, Perlis, Roy, Tsai, into skeletal muscles in vivo. In addition, transplanted E-MSCs promoted Li-Huei and accelerated functional recovery of injured muscles. Thus, our E-MSCs Massachusetts Institute of Technology, Cambridge, MA, USA, Stanley proved to be suitable cell sources for skeletal muscle tissue regeneration. Center for Psychiatric Research, Broad Institute, Cambridge, MA, USA, Massachusetts General Hospital, Boston, MA, USA Psychiatric disorders such as Schizophrenia (SZ) and Bipolar Disorder (BPD) NEURAL CELLS are debilitating diseases that affect a large proportion of the general popula- tion. It is known that genetics play a major role in the risk for these diseases, Poster Board Number: 1243 however until recently the risk factors were unknown. However, large REPROGRAMMING OF HUMAN RETINAL human genetic studies have revealed a number of genes and loci that are substantial risk for factors for SZ and BPD. To study the underlying biology PROGENITOR CELLS TO PLURIPOTENCY VIA of the genetic association signals, we have taken advantage of induced LENTIVIRAL INDUCTION OF SOX2 pluripotent stem cells (iPSCs) and induced neuronal cells (iNCs) technologies to create disease models in vitro using patient-derived fibroblasts. We have Baranov, Petr Y., Regatieri, Caio, Yao, Jing, Ma, Jian, Tucker, Budd, specifically focused on DISC1 and CACNA1C, which are risk for schizo- Mostoslavsky, Gustavo, Young, Michael phrenia and bipolar disorder, respectively. Here we will present evidence The Schepens Eye Research Institute, Boston, MA, USA, University of Iowa, to demonstrate that iPSCs and iNCs are useful tools to uncover in vitro Iowa City, IA, USA, Boston University, Boston, MA, USA phenotypes that are specific disease-associated haplotypes. Specifically, we are examining how disease-associated haplotypes affects parameters such as Introduction: Since Takahashi and Yamanaka originally reported in 2006 that morphology, gene expression and electrophsyiological properties in neurons. forced expression of the four transcription factors OCT4, SOX2, KLF4 and We believe these cellular models will serve as important tools for translation- c-MYC were able to induce pluripotency in terminally differentiated fibro- al genetics and will allow the development of novel drugs therapeutics. blasts, an explosion of research focusing on a variety of different cell types and reprogramming cocktails have been explored. The main goals of these Poster Board Number: 1247 subsequent studies were to avoid using the potentially oncogenic genes Klf4 and c-Myc and increasing iPSC generation efficiency. One study in particular DEVELOPMENT OF AN ASSAY TO IDENTIFY demonstrated that a more potent cell source, the neural stem cell, could be ACTIVATORS OF TRKB SIGNALING USING reprogrammed via forced expression of the gene OCT4 alone. A multipotent cell type that we have used quite extensively in our lab that could potentially HUMAN INDUCED PLURIPOTENT STEM CELL be utilized for a similar purpose is the human retinal progenitor cell (hRPC). DERIVED NEURONS Like neural stem cells, we show here that hRPCs express endogenously Engle, Sandra J., Kimmel, Lida H., Lanz, Thomas A., Friedrich, Amy many of the originally reported reprogramming factors (KLF4, c-MYC and SOX2, OCT4) as such these cells may potentially be more amenable to re- M., Weber, Mark, Prior, Faith, Lazzaro Jr, John T., Efremov, Ivan, programming, requiring fewer of the original transcription factors for induc- Kleiman, Robin J. tion of pluripotency. Materials and Methods: hRPCs were isolated using es- GeMM RCoE, Pfizer, Groton, CT, USA, Neuroscience Research Unit, Pfizer, tablished methods from human fetal eyes at 10-18 weeks of gestation. Cells Groton, CT, USA, Primary Pharmacology RCoE, Pfizer, Groton, CT, USA, were expanded in low oxygen conditions (3%) in serum-free media. hRPCs Neuroscience Chemistry -Worldwide Medicinal Chemistry, Pfizer, Groton, were infected with either the lentiviral vectors STEMCAA (OCT4, SOX2 CT, USA KLF4, c-MYC), STEMCAA cherry (OCT4, SOX2, KLF4), SOX2 and OCT4, Brain derived neurotrophic factor (BDNF) - TrkB signaling is a key regula- SOX2 alone or OCT4 alone for 24 hours. As a negative control hRPCs were tor of brain development and function. Binding of BDNF to TrkB induces cultured on iMEFs in embryonic stem cell media (ECM) (DMEM/F12, bFGF, dimerization of the receptor leading to autophophorlyation and activation EGF, -mercaptoethanol, HI FBS). After infection cells were washed and β of three signaling cascades: Ras-mitogen-activated protein kinase (MAPK) passaged at density 1000 cells/cm2 on inactivated MEFs, fed for three days pathway, the phosphatidylinositol 3-kinase (PI3K)-Akt pathway and the with hRPC media (Ultraculture media, bFGF, EGF) then switched to ECM for phospholipase C gamma - calcium (PLCγ-Ca2+) pathway. Genetic and the duration of the study. At 30 days post-infection colonies were counted post-mortem evidence suggests that BDNF-TrkB signaling is dysfunctional (via alkaline phosphatase staining), cut and passaged. Secondary colonies in brains from patients with a variety of CNS diseases, including schizophre-

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Thursday Poster Abstracts nia, autism, depression, bipolar disorder, anorexia, and neurodegenerative Furthermore, human disease-related mtDNA mutations have not been diseases. Facilitating BDNF signaling has been suggested as a potential successfully generated in mouse models, and therefore study materials therapeutic approach to these diseases. To identify compounds that activate have been limited to skin and muscle cells. We have generated iPS cells or potentiate BDNF mediated TrkB signaling, we developed a high content from patients with “mitochondrial recessive ataxia syndrome” (MIRAS) and assay to evaluate phosphorylation of ERK, a member of the MAPK pathway, with “mitochondrial myopathy, encephalomyopathy, lactic acidosis, and using human induced pluripotent stem cell (iPSC) derived neurons. An stroke-like episodes” (MELAS) syndrome, as well as from control individu- iPSC line was generated from male neonatal fibroblasts and characterized als. MIRAS patients have a nuclear defect in the polymerase gamma gene, for markers of pluripotency. Differentiation protocols yielding GABAergic leading to a defect in mitochondrial DNA maintenance and depletion of neurons were developed and resulting preparations were characterized mtDNA in the brain. MELAS patients have a heteroplasmic tRNA mutation electrophysiologically and immunocytochemically for expression of neuronal in mtDNA, leading to disruption of mitochondrial protein synthesis. Both of properties. iPSC derived neurons exhibited phosphorylated ERK in response these disorders manifest with neurologic and metabolic problems, MIRAS to BDNF and other neurotrophin treatments in a dose dependent manner. being one of the most common hereditary ataxias and MELAS the most Similar responses were observed following treatment with TrKB activat- common disease caused by pathological mtDNA point mutations world- ing antibodies. Interestingly, the neurons did not show a response to many wide. We have further differentiated these patient-derived iPS cells towards compounds that had been reported previously in the literature to activate neuronal fate and shown that we can generate functional patient-derived TrkB signaling. The validated assay was used to screen a library of natural neurons. We report here the first generation of iPS-derived human neurons products and identified several molecules. from mitochondrial encephalopathies, as well as the initial characterization of the disease phenotype in these neurons. Poster Board Number: 1249 Poster Board Number: 1253 OLIGODENDROCYTE PROGENITORS DERIVED FROM HUMAN INDUCED PLURIPOTENT STEM DIFFERENTIATION AND CHARACTERIZATION CELLS IMPROVE RECOVERY OF RAT MODEL OF RETT SYNDROME PATIENT-SPECIFIC OF OPTIC CHIASM DEMYELINATION NEURAL PROGENITOR CELLS Pouya, Alireza, Satarian, Leila, Kiani, Sahar, Javan, Mohammad, Kwok, Showming, Sheridan, Steven D., Petravicz, Jeremy C., Reis, Baharvand, Hossein Surya, Anguera, Monserrat C., Lee, Jeannie T., Haggarty, Stephen J., Sur, Mriganka Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology & Technology, Tehran, Iran, Islamic Republic of, Department of Brain and Cognitive Sciences, Picower Institute for Learning Department of Physiology, Faculty of Medical Sciences, Tarbiat Modares and Memory, Massachusetts Institute of Technology, Cambridge, MA, USA, University, Tehran, Iran, Islamic Republic of Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA, Department of Molecular This study aimed to differentiate hiPSCs into oligodendrocyte precursors Biology, Massachusetts General Hospital, Harvard Medical School, Boston, (OPs) and assess their recovery potential in a rat demyelinated optic chiasm MA, USA model. We have generated a homogenous cell population of OPs from hiPSCs as well as human embryonic stem cells (hESCs) by using an embryoid Rett syndrome is a neurodevelopmental disorder caused by mutations in a body (EB) formation in a defined medium supplemented with a combination transcriptional modulator gene, methyl CpG-binding protein 2 (MECP2). of factors, positive selection and mechanical enrichment. Immune-fluores- Mouse models with MeCP2 deletion recapitulate Rett-like symptoms. cence staining for (Olig2, NG2, PDGFRα, Sox10, O4, GalC, MBP, MAP2 and Reintroduction of MeCP2 in adult mice was shown to rescue the mutant GFAP) and Real-time PCR studies for (Oct4, Sox2, Pax6, Tuj1, Olig2 and phenotype, pointing to the possibility that Rett syndrome is a treatable PDGFRα genes) showed that the stage-specific markers were well expressed disorder even if MeCP2 or its downstream signaling pathway is restored following the differentiation procedure and enrichment of the oligodendro- in adulthood. Administration of a tripeptide form of IGF1, a growth factor cyte lineage relative to neurons or astrocytes was noted. These results were that is important for neuron survival and synaptic maturation, was found to comparable with the hESC-derived oligodendrocyte lineage cells in their ex- partially reverse Rett symptoms in a mouse model. However, the detailed pressions of stage-specific marker genes. Transplantation of hiPSCs-derived disease mechanisms and affected signaling pathways remain elusive, making OPs (hiPSC-OPs) into the lysolecithin (LPC)-induced demyelinated optic identification of therapeutic targets for treating Rett patients difficult. To chiasm of the rat model resulted in recovery from symptoms, integration overcome these limitations, we are using pluripotent stem cell approach to and differentiation into oligodendrocytes. These results showed that OPs derive mature neurons from reprogrammed adult somatic cells from Rett pa- generated efficiently from hiPSCs can be used for future biomedical studies tients for studying disease mechanisms and discovering therapeutics. Using once the safety issues have been overcome. viral-mediated delivery of reprogramming factors, we have generated sev- eral lines of patient and control fibroblast-derived induced pluripotent stem Poster Board Number: 1251 cells (iPSCs) that carry various mutations in MECP2. Neural progenitor (NP) cells were successfully derived from these iPSC clones and were character- IPS CELL-DERIVED MODELS FOR HUMAN ized using immunocytochemical methods that demonstrated that these cells MITOCHONDRIAL DISORDERS are homogenous and express stage-appropriate markers. Upon long-term culturing on laminin coated surface up to 10 weeks, these NP cells differ- Hämäläinen, Riikka H., Manninen, Tuula, Trokovic, Ras, Otonkoski, entiate into neurons as demonstrated by the detection of lineage specific Timo, Suomalainen, Anu proteins using immunocytochemistry. We are able to further examine these Research Program Unit, Molecular Neurology, University of Helsinki, neurons with patch clamp recording and calcium imaging techniques to as- Helsinki, Finland sess function and the extent of their maturation. Finally, we are looking into specific signaling pathways that underlie disease mechanisms. Based upon Mitochondrial dysfunction is the most common cause of inherited neurode- these findings, we are developing high-throughput cellular screening assays generation, and a common cause of children’s metabolic disorders, including using automated microscopy and pathways-selective reporter genes to encephalomyopathies and cardiomyopathies. Patient-derived fibroblasts are decipher differences in signaling mechanisms between Rett patient specific often available to study the disease phenotypes and pathogenesis, but the neurons and healthy controls. highly cell-type specific disorders manifest only sometimes in fibroblasts.

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Poster Board Number: 1255 Poster Board Number: 1259 DISEASE RELEVANT PHENOTYPE IN MOTOR DERIVATION OF HUMAN NEURAL NEURONS DERIVED FROM AMYOTROPHIC PROGENITORS FROM FRAGILE X SYNDROME LATERAL SCLEROSIS (ALS) PATIENT IPS CELLS INDUCED PLURIPOTENT STEM CELL LINES Martinez, Fernando J., Burkhardt, Matthew, Volfson, Dmitri, WITH VARIATION IN CGG REPEAT, DNA Shoukat-Mumtaz, Uzma, Martinez, Rita, Gai, Hui, Ramos, Carla, METHYLATION, AND EXPRESSION OF THE Grskovic, Marica, Strulovici, Berta, Griswold-Prenner, Irene, FMR1 LOCUS Javaherian, Ashkan Sheridan, Steven D., Theriault, Kraig, Haggarty, Stephen J. Ipierian, Inc., South San Francisco, CA, USA Center for Human Genetic Research, Massachusetts General Hospital, Amyotrophic Lateral Sclerosis is a neurodegenerative disease character- Harvard Medical School, Boston, MA, USA ized by the progressive loss of the motor neurons necessary for voluntary motion. Many hereditary and environmental factors have been implicated The creation of patient-specific stem cell models of human neuropsychiatric as contributors to the disease process. In addition, there are significant disorders holds the potential to transform the discovery and characterization variations in disease onset, progression, and clinical pathology in patients, of the underlying molecular mechanisms as well as facilitate the discovery making ALS a challenging target for disease modeling and drug discovery. of novel therapeutic interventions aimed at preventing or reversing disease In order to better understand the etiology and progression of ALS, we have pathophysiology. Moreover, genetically accurate lines derived from multiple generated a large cohort of iPS cells from patients with many forms of ALS. patients will facilitate the elucidation of potential inter-patient variability These iPS lines are shown to differentiate into several disease relevant cell in relevant pathways tractable to therapeutic intervention as well as their types. Using these patient-derived cells as tools, we established a battery variable responses. To create cell models to support the investigation of the of scalable assays designed to reveal ALS phenotypes in vitro. These assays pathophysiology and treatment of autism spectrum disorders (ASDs), we include measuring protein and RNA expression levels of disease related report here the development and characterization of induced pluripotent genes, assessing mitochondrial membrane potential, and perturbing multiple stem cells (iPSCs) from multiple patients with Fragile X syndrome (FXS), disease pathways through the application of small molecule compounds. We the most common inherited cause of intellectual disability and the most describe disease relevant differences in neurons derived from ALS patients common known genetic cause of autism. FXS is known to be predominantly compared to healthy subjects. Our data demonstrates how reprogramming caused by an expanded CGG repeat in the 5’ untranslated region of the technology combined with a parallelized discovery approach can be used Fragile X Mental Retardation (FMR1) gene. Upon reprogramming of FXS to establish an in vitro disease model for a non-monogenic, multifactorial patient fibroblasts into iPSC clones, the resulting clones exhibited variation disease, with complex etiology. with respect to the length of the CGG repeat in the FMR1 gene, its DNA methylation status and the expression of the FMR1 gene. In one instance, Poster Board Number: 1257 iPSC clones of a mosaic patient having both normal and pre-mutation length CGG repeats resulted in genetically matched iPSC lines differing in their GENERATION AND MAINTENANCE OF NEURAL CGG repeat lengths. In two cases, we report that some of the iPSC clones PROGENITOR CELLS FROM HUMAN INDUCED contain predominant repeat lengths that are shorter than what could be ob- PLURIPOTENT STEM CELLS served in corresponding input population of fibroblasts. We also report, for the first time, the derivation of homogenous, self-renewing neural progeni- Hatami, Maryam, Nemati, Shiva, Kiani, Sahar, Hemmesi, Katayoon, tor (NP) cells from FXS iPSCs that are positive for neural stem cell markers Gourabi, Hamid, Masoudi, Najmeh, Alaie, Sara, Baharvand, Hossein (e.g. nestin, SOX1, PAX6, PSA-NCAM). These iPSC-NP cells can be readily Department of Stem Cells and Developmental Biology, Royan Institute expanded as monolayer cultures and subsequently differentiated into neu- for Stem Cell Biology and Technology, Tehran, Iran, Islamic Republic of, rons that express multiple markers of post-mitotic neurons. We demonstrate Department of Genetics, Royan Institute for Reproductive Biomedicine, that these iPSC-NP cells retain the FMR1 CGG repeat, its DNA methylation Tehran, Iran, Islamic Republic of status and FMR1 mRNA expression of the iPSC clone from which they were derived. Unlike the starting fibroblast and iPSC stages, these iPSC-NP cells Human induced pluripotent stem cells (hiPSCs) have led to an impor- express metabotropic glutamate receptor 5 (mGluR5), a key target protein tant revolution in stem cell research and regenerative medicine. To create to FXS pathogenesis. By providing a highly pure population of progeni- patient-specific neural progenitors (NPs), we have established a homog- tor cells that can be used for comparisons of the phenotypic properties of enous, expandable, and self-renewable population of multipotent NPs defined neuronal and glial cell types, these iPSC-NP cell models of FXS will from hiPSCs, using an adherent system and defined medium supplemented facilitate the in-vitro investigation of the mechanisms of FXS pathogenesis with a combination of factors. The established hiPSC-NPs highly expressed over developmental time periods. Nestin and Sox1. These NPs were continuously propagated for ∼1 year without losing their potential to generate astrocytes, oligodendrocytes, and functional neurons and maintained a stable chromosome number. Volt- age clamp analysis revealed outward potassium currents in hiPSC-NPs. The self-renewal characteristic of the NPs was demonstrated by a symmetrical mode of Nestin-positive cell division. Additionally, these hiPSC-NPs can be easily frozen and thawed in the presence of Rho-associated kinase (ROCK) inhibitor without losing their proliferation, karyotype stability, and develop- mental potential. The characteristics of our generated hiPSC-NPs provide the opportunity to use patient-specific or ready-to-use hiPSC-NPs in future biomedical applications.

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Thursday Poster Abstracts

Poster Board Number: 1261 Poster Board Number: 1263 CHARACTERIZATION OF WNT/GSK-3 DEVELOPMENT OF A NOVEL HTS AMENABLE SIGNALING IN HUMAN NEURAL PROGENITOR ASSAY PLATFORM FOR VISUAL ASSESSMENT CELL MODELS OF FRAGILE X SYNDROME OF HUMAN EMBRYONIC STEM CELL DERIVED DERIVED FROM INDUCED PLURIPOTENT STEM NEURAL PROGENITOR MIGRATION CELLS Powe, Allan C., Hodges, Kathryn L., Chilton, Jamie M., Herber, Theriault, Kraig, Sheridan, Steven D., Cheng, Chialin, Karmacharya, Renee L., Hulkower, Keren I., Stice, Steven L. Rakesh, Haggarty, Stephen J. ArunA Biomedical, Inc., Athens, GA, USA, Platypus Technologies, LLC, Center for Human Genetic Research, Massachusetts General Hospital, Madison, WI, USA Harvard Medical School, Boston, MA, USA Neural progenitor migration is an important process for the proper develop- The creation of patient-specific stem cell models of human mental disor- ment and maintenance of the nervous system. Derived from proliferative ders holds the potential to transform the discovery and characterization of zones within the brain, neural progenitors migrate to specific destinations the underlying molecular mechanisms as well as facilitate the discovery of guided by various extracellular cues. Dysfunctional neural progenitor migra- novel therapeutic interventions aimed at preventing or reversing disease tion is evident in a number of clinical syndromes, such as lissencephalies. pathophysiology. Moreover, genetically accurate cell models created from Exposure to neurotoxins during development can interfere with neural multiple patients will facilitate the elucidation of potential inter-patient vari- progenitor migration and lead to nervous system defects. In fact, certain ability in relevant pathways tractable to therapeutic intervention as well as toxicants are known to interfere with neural stem cell migration. Recent their variable responses. We report here the development and characteriza- publications advocate the development of in vitro cell culture systems to tion of neural progenitor (NP) cells derived from induced pluripotent stem identify and prioritize potential human developmental neurotoxins among cells (iPSCs) generated from multiple patients with Fragile X syndrome (FXS), >80,000 untested commercial chemicals. To that end, we are developing a the most common known genetic cause of ASD and the most common high throughput screening (HTS) amenable assay to measure the migration inherited cause of intellectual disability. FXS is known to be caused predomi- of human embryonic stem cell (hESC) derived neural progenitors (hNP1; nantly by an expanded CGG repeat (>200 repeats) in the 5’ untranslated ArunA Biomedical) by using a novel 96-well based cell migration assay region of the Fragile X Mental Retardation (FMR1) gene leading to a loss platform (Oris Cell Migration Assay; Platypus Technologies). Stoppers create of expression of the fragile X mental retardation protein (FMRP).,Recent central exclusion zones within the wells; cells are plated outside the zones pre-clinical and subsequent clinical findings strongly support that lithium, a and migrate inward once the stoppers are removed. At the end of the as- known GSK-3 inhibitor, may have therapeutic efficacy in the treatment of say period, cells that have migrated into the central zones are stained and FXS, supporting a role in GSK-3 mediated signaling in response to the loss of detected using fluorescence plate readers and/or visualized by microscopy. FMRP. However, GSK-3 signaling plays many important roles in neurons and Using the Oris assay, we demonstrated that cytochalasin D, a disruptor of glia, and the basis and consequence of altered GSK-3 activity in FXS remains actin microfilaments, inhibits hNP1 migration with an IC50 of ~15 nM. We poorly understood. Furthermore, lithium is known to have multiple targets also show that neurotrophic factors, such as basic fibroblast growth factor in addition to GSK-3 and provides a narrow therapeutic index in part due to (bFGF), can accelerate neural progenitor migration as high as two-fold. the high doses required. To address these issues, our efforts have focused on Thus, this assay can be used to identify factors that either inhibit or promote using iPSC-NP cell models of FXS to characterize small-molecule probes of neural progenitor migration. Together, these data demonstrate an assay sys- the canonical Wnt/GSK-3/ß-catenin signaling pathway. To develop a robust tem that can be used to readily identify novel factors that promote or inhibit cell-based assay capable of supporting high-throughput screening, we have neural progenitor cell migration. The combination of this novel cell migration utilized a TCF/LEF-dependent luciferase reporter gene system. This reporter assay and neural stem cells provides a powerful tool for understanding gene was stably transfected in both unaffected control iPSC-NP cell lines as proper nervous system development, identifying neurotoxins, and develop- well as several iPSC-NPcell lines derived from FXS patient fibroblasts. Using ing therapies for migration defects in neurological syndromes. this TCF/LEF luciferase reporter-based assay, we demonstrate that Wnt- Poster Board Number: 1265 conditioned media and the potent GSK-3 inhibitor, CHIR-99021, strongly activate TCF/LEF-mediated transcription in undifferentiated iPSC-NP cells MMP-9 ASSOCIATED WITH THE TETRASPANIN and in the case of CHIR-99021 cause a significant accumulation and nuclear CD82 AND REGULATED THE MIGRATION TO translocation of β-catenin background, demonstrating the utility of this patient derived iPSC-NP cell model to investigate the GSK-3 pathway in HUMAN NEURONAL STEM CELL patient-derived human neurons. Interestingly, differences in reporter re- Lee, Jin-Sung, Lee, Soo Youn, Yang, Jae Won, Choi, Jeong Woo, sponse to known GSK-3 activity modulators were observed in control versus An, Jeung Hee FXS affected iPSC-NP cell reporter lines. This suggests disease-specific path- way differences that could be used in combination with chemical libraries Chemical & Biomolecular Engineering, Sogang University, Seoul, Korea, with known targets to dissect the components of the Wnt/GSK-3 pathway Republic of affected by the disease, as well as possible methods of correcting the differ- Recently neural stem cells (NSCs) were reported to migrate to damaged ences discovered in the disease state. brain area, such as brain tumor or ischemic region. So, we hypothesized that human glioma or ischemic tissues secreted chemo-attract molecules that induce NSC migration toward that area. In present study, we reported one candidate protein, MMP-9, commonly expressed from microarray and proteomic data. Imunostaining showed that expression of MMP-9 is up- regulated in all glioma tissues compared to normal brain tissues. In addition, MMP-9 dramatically induced migration of NSCs, but not HEK 293 cells in a dose-dependent manner, and showed the maximum activity at a concentra- tion of 100 ng/ml. This induction of NSC-specific migration may be derived from the difference of MMP-9 receptor expression because F3 highly

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Thursday Poster Abstracts

expressed all MMP-9 receptors, but HEK 293 cells do not. After then, to Poster Board Number: 1269 investigate if CD82, one of MMP-9 membrane receptors, mediate NSC mi- gration by MMP-9 on the cell surface, we established CD82-knockdown F3 USING HUMAN IPSC DERIVED NEURAL CELLS cell lines using a vector-based small hairpin RNA (shRNA) strategy. Interest- FROM GENETICALLY RELATED INDIVIDUALS ingly, CD82 knockdown blocked almost all NSC migration by MMP9 in vitro migration assays. In addition, MMP-9 mediated migration mediated through TO UNDERSTAND BIPOLAR DISORDER. ERK and Cdk5 signaling pathway, as PD98059 and roscovitin reduced the Madison, Jon, Zhou, Fen, Wolf, Pavlina, van der Ven, Karlijn, Hsu, migration by 45% and 40%, respectively. All these results demonstrated Jenny, Theriault, Kraig, Sheridan, Steven, Reis, Surya, Reuckert, that MMP-9 is a powerful chemo-attract molecule for NSC, and may be Erroll, Daheron, Laurence, Sklar, Pamela, Haggarty, Stephen promising drug for brain tumor therapy. Stanley Center for Psychiatric Research, Broad Institute, Cambridge, Poster Board Number: 1267 MA, USA, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA, USA, HSCI iPS Cell Core Facility, Harvard University, DOUBLECORTIN EXPRESSION PROMOTES THE Cambridge, MA, USA, Center for Human Genetic Research/Stanley Center MIGRATION OF HUMAN EMBRYONIC STEM for Psychiatric Research, Massachusetts General Hospital/Broad Institute, CELL-DERIVED NEURONS AND STIMULATES Cambridge, MA, USA THE CONSTRUCTION OF NEURAL PROGENITOR Bipolar disorder and schizophrenia pose a significant burden to patients, families and society. Despite evidence for high heritability, the etiology of SCAFFOLDS THAT GUIDE MIGRATION these psychiatric disorders remains poorly understood. Recently, several Filipovic, Radmila, Santhosh Kumar, Saranya, Fiondella, Chris, groups, including ours, observed that copy number variants and several sta- LoTurco, Joseph tistically compelling risk loci contribute to disease risk. With what is proving to be a complex genetic landscape, existing approaches that model changes Physiology and Neurobiology, University of Connecticut, Storrs, CT, USA in small numbers of genes across development are not well suited to capture Human embryonic stem cell derived neuronal progenitor cells (hNPCs) the multiple genetic factors interacting in an individual to cause disease risk. potentially provide a valuable cell source for cellular replacement following Recent advances in stem cell technology may offer a solution to investigate neurodegenerative diseases. Upon appropriate placement in damaged areas, this genetic complexity in disease relevant cells by allowing the derivation hNPCs should be able to migrate, differentiate, and integrate in the host of pluripotent stem cells (IPSCs) from bipolar and schizophrenic patient cells environment. One of the greatest challenges in cell replacement therapies and subsequent derivation of neural cells. This approach will enable both the is the ability of neuronal progenitor cell populations to migrate into and investigation of the functional variation that contributes to risk for psychi- integrate within existing neuronal circuitry. We hypothesized that migration atric disorders, and will also allow us to develop phenotype-based assays of hNPCs could be promoted by genetically manipulating them to express for chemical screening. To develop a framework to investigate psychiatric genes that promote migration during normal development. The DCX gene disorders, we have reprogrammed fibroblasts from a family segregating sev- encodes doublecortin, a microtubule-associated protein expressed in migrat- eral forms of psychiatric illness including Type I bipolar disorder. To attempt ing neuronal precursors which stabilizes microtubules and promotes cell to control for phenotypic variability between individuals due to background migration. Our present study focused on the effect of human Dcx (hDcx) heterogeneity, we have characterized multiple independent iPSC lines expression on hNPCs using vitro migration models. For stable transgenesis from two healthy parents and their two bipolar sons. From each of these of hNPCs we used a piggyBac transposon system, which allows for viral-free iPSC lines we have used adherent monolayer cell culture and fluorescence insertion of genes. We constructed DNA plasmid vectors 5’TR-CAGGS-hD- activated cell sorting to generate proliferative neural progenitor cell lines cx-IRES-GFP’TR (hDcx-PB) and 5’TR-CAGGS-GFP’TR (GFP-PB) by cloning (NPCs). NPCs from each of these individuals were capable of differentiat- the full-length human Dcx cDNA or GFP cDNA into the 5’TR-CAGGS-3’TR ing into neurons as defined by immunocytochemistry using lineage-specific vector. hNPC were derived from the H9 cell line (UCHC Stem Cell Core, markers. We are currently using both these NPCs and NPC derived neurons Farmington, CT). Neural rosettes were trypsinized and nucleofected either to identify and study disease associated phenotypes and gene expression with (hDCx-PB) or GFP-PB. Similarly sized neurospheres generated from ge- differences. We hope these approaches will provide novel insights into the netically modified rosettes were plated either on Matrigel or rat brain slices etiology of these complex psychiatric disorders and paths toward novel prepared from rat neocortex. Migration of cells out of the neurospheres targeted therapeutics for their treatment. was assessed after 12h, 24h, 48h and 120h. In both assays, hESC-dNP Poster Board Number: 1271 transfected with hDCX-PB migrated significantly further than control cells (GFP-PB transfected). When overlayed on the top of cortical rat slices, hNPC AZACYTIDINE INDUCED DNA DEMETHYLATION transfected with hDCx-PB migrated 340.97±17.24 μm vs. 153.45±14.6 μm for GFP-PB transfected neurospheres. Additionally, a network of nestin fibers PROMOTES ASTROCYTIC DIFFERENTIATION was found to develop and integrate into host tissue from neurospheres that OF NEURAL PROGENITORS DERIVED FROM were transfected with hDCx-PB. hDCX expressing neural progenitors that HUMAN EMBRYONIC STEM CELLS migrated into tissue were invariably found associated with the infiltrating nestin expressing cellular fibers suggesting that enhanced migration into Majumder, Anirban, Dhara, Sujoy K., Stice, Steven L. neural tissue following DCX expression is mediated by induced growth of Regenerative Bioscience Center, University of Georgia, Athens, GA, USA, migration scaffolds from neural progenitors. In summary, our results show Division of Animal Biotechnology, Indian Veterinary Research Institute, that hDcx expression significantly improves migration of hNPCs. Future ex- Bareilly, India periments are in progress to test whether in vivo transplanted cells migrate more into tissue when transected with hDcx. Astrocytes play significant roles in many aspects of nerve function and human embryonic stem (hES) cells could potentially provide an unlimited supply of non-transformed astrocytes for a variety of applications. Human neural progenitor (NP) cells derived from hES cells as adherent monolay- ers, however produce largely neuronal phenotypes upon withdrawal of the mitogenic signal FGF2. GFAP or S100B expressing astrocytes are rarely formed. Moreover, these NP cells do not produce astrocytes in response

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Thursday Poster Abstracts to the proastrocytic signals Bone morphogenetic protein 2 (BMP2) and Poster Board Number: 1275 Leukemia inhibitory factor (LIF) inspite of expressing their receptors. Only after prolonged culture without FGF2 these cells give rise to a small number ACCELERATED DIFFERENTIATION OF HUMAN of astrocytes. Interestingly, with prolonged culture without FGF2 they also NEURAL STEM CELLS BY A CELL-CONTACT acquire the ability to respond to BMP2 and LIF to generate astrocytes. Here we demonstrate that the early neurogenic tendency is possibly due to a MEDIATED MECHANISM lack of adequate activation of Notch and the consequent lack of demethy- Krishnan-Kutty, Viknish, Rizk, Pamela, Udolph, Gerald lation of pro-astrocytic gene promoters. We show that the proastrocytic Division of Bioengineering, National University of Singapore, Singapore, genes are hypermethylated at their promoter regions in the NP cells even Singapore, Institute of Medical Biology, Singapore, Singapore though notch signaling components are present at progenitor stage, and they remain methylated even after prolonged culture and passaging. The Neural Stem Cells (NSCs) have the potential of differentiating into the three requirement of Notch signaling can be bypassed by chemical treatment with neural lineages found in the brain: astrocytes, oligodendrocytes and neu- the methyl tranferase inhibitor Azacytidine. Azacytidine, along with the rons. This makes them attractive candidates in the treatment of neurologi- histone deacetylase inhibitor Trichostatin A (TSA), and coincident signaling cal disorders. However, the mechanisms by which these cells differentiate from LIF and BMP2 results in early expression of the astrocyte progenitor into specific neural cell types are not well understood. Some of the known marker CD44 and several other astrocytic markers including S100B. Using methods necessitate genetic interventions to either over-express or down this treatment, expression of CD44 goes up 3 fold and that of the astrocyte regulate critical factors so as to induce the cells to differentiate into certain marker S100B goes up 20 fold within 5 days of treatment, reaching 4 and lineages. While this may be useful in studying the innate property of par- 25 fold respectively at 15 days. On the other hand differentiation of NP ticular factors, they cannot be utilized for cellular therapy. During develop- cells by withdrawal of FGF2 alone leads to downregulation of both markers. ment, NSCs from the proliferating neural tube differentiate to make-up the Here we have described a method for enhanced derivation of astrocytes and entire nervous system via extrinsic factors: diffusible factors released by determined mechanisms involved in astrocytic differentiation of human NP cells, ECM molecules deposited by cells and direct cell-cell contact. As such, cells, facilitating future studies on astrocytes and their interaction with NP there is a possibility that these factors could be used to induce specific neural generated neuronal cells. differentiation. Our data show that NSCs accelerate neuronal differentia- tion when co-cultured with pre-differentiated NSCs. We hypothesize that Poster Board Number: 1273 molecules expressed by pre-differentiated neuronal cells promote neuronal SCALABLE HUMAN NEURAL STEM CELLS differentiation by means of a cell contact related mechanism. GFP-tagged NSCs were induced to differentiate either on a laminin substrate (control) PRODUCE ROBUST NEURAL MODEL or on a layer of pre-differentiated NSCs. Our results show that within 24 Malavarca, Richard, Roach, Marsha, Cacace, Angela, Fennell, Myles hours of co-culture, GFP-tagged NSCs down-regulate NSC makers (Nestin, SOX2) by about 80% and up-regulate neuronal markers (β-Tubulin III, GigaCyte, LLC, Branford, CT, USA, Applied Genomics, Bristol-Myers PSA-NCAM, MAP2,) by up to 20% compared to cells in control conditions. Squibb, Wallingford, CT, USA, Applied Genomics, Bristol-Myers Squibb, Morphologically, an increase (~10 fold) in neurite length and an increase Pennington, NJ, USA (~8 fold) in neurite branching are observed when compared to the control. In neuroscience drug discovery today there is a major unmet need for differ- Experiments have ruled out the sole role of diffusible factors released in the ent types of mature human neurons that can be utilized to investigate drugs medium, the sole role of ECM deposition or a combination of both in elicit- for the treatment of neurological disorders such as Alzheimer ’s disease. ing this response. Moreover, this rapid differentiation is not observed when Consequently, pharmaceutical companies are in search of more physiologi- NSCs are co-cultured with a non-differentiated feeder layer or with a feeder cally relevant pre-clinical models that can reliably predict the safety and layer of fixed (inactivated) cells. In conclusion, direct cell contact between efficacy of new pharmacological agents. With increasing attrition rates NSCs and a living differentiated feeder layer of cells results in accelerated and associated development costs of new compounds in clinical trials, a neuronal differentiation of NSCs. At present, various cell contact molecules great need exists to improve the early-stage selection process of candidate and mechanisms are being studied for their role in inducing this effect. compounds in order to limit the number of costly failures in late phase clini- These experiments will shed some light on how cell contact might accelerate cal trials. Thus we have developed human stem cell-derived neural models neuronal differentiation of NSCs and perhaps such knowledge could be used to meet that need. Recent advances in our stem cell technology and media toward directing cells to a particular neural fate faster than conventional formulations have led to the development of a scalable human neural stem/ protocols, without the need of any genetic intervention. progenitor cell (NSPCs) culture system that enables neuroscience research- Poster Board Number: 1277 ers to produce mature human neurons in a -well format that is compatible with automation and high throughput screening. Here we will demonstrate ELECTROPHYSIOLOGICAL PROPERTIES that human NSPCs can be expanded to a scale greater than cells that can be stored in a cryo-bank so that a screen of full file chemical libraries can be AND EXPRESSION OF VOLTAGE-GATED ION performed from one bank of cells. Upon thawing, the human NSPCs can be CHANNELS IN HUMAN EMBRYONIC STEM reproducibly differentiated into mature neural populations that are at least CELLS DURING NEURAL DIFFERENTIATION -% neurons with mature synapses. Kiani, Sahar, Shahbazi, Ebrahim, Hemmesi, Katayoun, Janahmadi, Mahyar, Mirnajafi-Zadeh, Seyad-Javad, Baharvand, Hossein Stem Cell and Developmental Biology, Royan, Tehran, Iran, Islamic Republic of, Physiology, Shahid Beheshti University, Tehran, Iran, Islamic Republic of, Physiology, Tarbiat Modares University, Tehran, Iran, Islamic Republic of We have investigated the profile of membrane currents in human embryonic stem cells (hESCs) during neuronal differentiation using whole-cell patch clamp technique in a voltage clamp mode. hESCs were induced to the neural lineage in a feeder-free in defined media supplemented with growth factors. Our protocol seems to recapitulate the early steps of nervous system devel-

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opment in vivo in that undifferentiated hESCs organized into rosettes and erative (>90% of total cells were Ki67 positive), maintained differentiation then neural tube-like structures are formed. Differentiating cells expressed properties and stable karyotypes after multiple passages, and were able to neuroectodermal and mature neuron markers during neural plate and tube cryopreserved. Furthermore, at high density, these cells were spontaneously formation and maturation. The differentiated cells were expressed neuron- reassembled into the rosette structure, indicating their distinct early neural specific antigens such as TUj1 and, MAP-2. During neural differentiation the stem cell characteristics. Therefore, they were feasible to induce into specific relative expression of alfa subunit of ion channels (calcium, sodium and po- regional identities such as ventral midbrain by inductive cues (e.g, signaling tassium channels) showed many changes. In matured cells KV 1.2, KV 2.1, molecules or small molecules). Through this procedure, we induced func- KV 3.1, KV 10.1, KV 4.2, and Nav 1.2, Nav 1.1, Cav 2.2 were significantly tional neurons exhibiting midbrain dopaminergic phenotypes from hESCs as up-regulated (p<0.01, ANOVA, Tukey HSD test) and Cav 1.4 was signifi- well as Parkinson’s disease patient specific iPSCs. These results indicate that cantly down-regulated compared to hESCs (p<0.05, ANOVA, Tukey HSD magnetic based-cell purification enable to enrich and expand homogenous test), as indicated by real-time RT-PCR. The resting membrane potential of population of early stage neural precursors and could provide a promising hESCs during differentiation in to neuronal cells was more negative progres- strategy for cell transplantation therapy of neurodegenerative disorders. This sively. in matured cells RMP was significantly more negative (-49.13±1.72 research was supported by grants (SC1110) from the Stem Cell Research mV) than rosette structures (-25±1.24 mV, independent sample t test, Center of the 21th Century Frontier Research Program funded by the Minis- t<0.001) and hESCs (-8.66±0.87 mV, independent sample t test, t<0.001). try of Education, Science and Technology, Republic of Korea. During differentiating into neurons, input resistance progressively decreased. The value of input resistance in hESCs was 11.94±0.23 MΩ that significantly Poster Board Number: 1281 higher than rosette (9.12±0.134 MΩ, n=20, p<0.001, independent sample IDENTIFYING BIOMARKERS OF FETAL t test) and neural tube like structures (9.2 ± 0.34 MΩ, n=20, p<0.001, in- dependent sample t test). On every stage membrane capacitance estimated ALCOHOL EXPOSURE USING METABOLOMICS in voltage clamp mode, in hESCs membrane capacitance was higher (0.146 AND DERIVATIVES OF HUMAN EMBRYONIC ± 0.551 nF) than Rosette (0.620 ± 0.166 nF, independent sample t test, t<0.001) and neural tube stage (0.092 ± 0.017 nF, independent sample t STEM CELLS test, t<0.001). Therefore input resistance and membrane capacitance in Palmer, Jessica A., Poentitzsch, Ashley, Smith, Susan, Conard, matured cells were not significantly difference with hESCs (NS, independent Kevin, West, Paul, Cezar, Gabriela G. sample t test). In voltage clamp experiments some outward currents were Animal Science, University of Wisconsin-Madison, Madison, WI, USA, recorded in hESCs and rosette structures that increased progressively with Nutritional Sciences, University of Wisconsin-Madison, Madison, WI, USA, positive voltages. These outward currents were inhibited by TEA but not Stemina Biomarker Discovery, Inc, Madison, WI, USA 4-AP in hESCs and 4-AP but no TEA could inhibit outward currents in ro- sette structures. These channels did not show inactivation there were no in- Fetal alcohol exposure (FAE) causes life-long cognitive, behavioral, and sen- ward currents in hESCs and rosette stage. In neuronal cells, inward currents sorimotor deficits in children with fetal alcohol spectrum disorders (FASD). were recorded. These currents were inhibited by Nifedipine and QX-314. Of great importance is to understand why developing neurons are vulner- These data show that hESCs have special electrophysiological properties that able to ethanol’s damage. A significant challenge to FASD prevention and changed during neuronal differentiation. Voltage dependent delayed rectifier treatment is the absence of clear biomarkers to identify those with in utero K currents that are sensitive to TEA but not 4-AP which existing in hESCs. alcohol-induced damage. To identify such biomarkers in an unbiased man- In rosette stage, outward potassium currents are sensitive to 4-AP but not ner, we developed a novel paradigm using derivatives of human embryonic TEA. It seems that a type potassium channel currents were exist in rosette stem (hES) cells and metabolomic analysis. The hES cell lines WA01 and structures. Inward currents were recorded in neuronal cells that sensitive to WA09 were differentiated into neural progenitors or neurons. Each stage Nifedipine and QX-314. It seems that there were sodium and calcium volt- underwent a four day challenge with 0, 0.1, or 0.3% ethanol, and the spent age dependent channel currents in matured neuronal cells. culture media was analyzed thereafter using LC-ESI-QTOF mass spectrom- etry. Differential small molecule metabolites were identified using ANOVA. Poster Board Number: 1279 Significant features, defined as an entity detected by the mass spectrometer EXPANSION AND DIFFERENTIATION OF PSA- that is a combination of exact mass, retention time and abundance, were then putatively annotated by comparison to compounds found in publically NCAM POSITIVE NEURAL PRECURSORS searchable databases and the identities of four compounds were confirmed DERIVED FROM HUMAN PLURIPOTENT STEM using comparative MS-MS against their chemical reference standard. We found that ethanol treatment induced statistically significant abundance CELLS changes to various features in human embryoid bodies (180 features), Kim, Dae-Sung, Lim, Bo Young, Yoo, Jeong-Eun, Kang, Hoon-Chul, neural progenitors (76 features) and neurons (42 features). There were no Kim, Dong-Wook shared significant features between cell types. A total of 16 features showed a dose-response to ethanol. We confirmed the identity of four significant Dept. Physiology. #401, Yonsei University College of Medicine, Seoul, features, 5’-methylthioadenosine (MTA), L-thyroxine, L-kynurenine, and Korea, Republic of, Dept. Pediatrics, Yonsei University College of Medicine, indoleacetaldehdye (IAA). Kynurenine and IAA are tryptophan metabolites Seoul, Korea, Republic of and kynurenine was previously identified in a biomarker screen of valproate- The generation and expansion of homogenous neural precursors derived treated hESCs. MTA is an intermediate in cysteine/methionine metabolism from human pluripotent stem cells (hPSCs; hESCs and hiPSCs) are crucial for and is involved in polyamine synthesis. One significantly increased feature the successful implementation of therapeutic hPSC transplantation and serve with putative annotation was succinyladenosine, which is a biomarker for as the platform for human developmental studies and new drug discovery. adenylosuccinate lyase deficiency. Several additional features were highly In this report, we used magnetic-activated cell sorting (MACS) to purify selective to ethanol treatment but have not been annotated. We conclude hESC-derived neural precursors expressing poly-sialated neural cell adhesion that alcohol neurotoxicity induces statistically significant changes to the molecule (PSA-NCAM), which initially presented at neural rosette stage abundance of metabolites in human embryoid bodies, neural progenitors and homogeneous PSA-NCAM positive (~90%) neural precursors then and neurons. Because several of these metabolites are endogenous in hu- expanded as monolayer culture in N2B27 medium supplemented with bFGF man serum, studies are underway to establish which of these are altered in by enzymatic passages. Purified neuronal precursors extensively expressed whole animal models of FAE. These findings also elucidate the biochemical neural markers (>80%) such as nestin, sox1, sox2, and musashi-1, but not pathways affected by ethanol in the developing nervous system. Supported oct3/4 or nanog. During expansion, neural precursors were highly prolif- by R21 AA16958 to GGC.

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Thursday Poster Abstracts

Poster Board Number: 1283 Poster Board Number: 1285 METABOLOMICS OF HUMAN EMBRYONIC METFORMIN ACTIVATES ATYPICAL AND INDUCED PLURIPOTENT STEM CELLS PKCS TO PROMOTE RODENT AND TO PREDICT DEVELOPMENTAL TOXICITY: A HUMAN NEUROGENESIS AND ENHANCE COMPARISON HIPPOCAMPUS-DEPENDENT LEARNING Palmer, Jessica A., Weir-Hauptman, April M., Smith, Alan M., West, Wang, Jing, Gallagher, Denis, DeVito, Loren, Cancino, Gonzalo, Paul R., Conard, Kevin R., Fontaine, Burr, Donley, Elizabeth L.R., Tsui, David, Keller, Gordon, Frankland, Paul, Kaplan, David, Miller, Cezar, Gabriela G. Freda Cell Biology, Stemina Biomarker Discovery, Inc, Madison, WI, USA, Developmental and Stem Cell Biology, Hospital for Sick Children Research NanoOncology, Madison, WI, USA, Animal Science, University of Institute, Toronto, ON, Canada, Neurosciences and Mental Health, Hospital Wisconsin-Madison, Madison, WI, USA for Sick Children Research Institute, Toronto, ON, Canada, Stem cell and Developmental Biology, Ontario Cancer Institute, Toronto, ON, Canada, Birth defects are the largest cause of infant morbidity and mortality in the Cell Biology, Hospital for Sick Children Research Institute, Toronto, ON, United States. Exposure to developmental disruptors has a significant role Canada in the pathogenesis of defects in human development. Teratogens, defined as substances that cause one or more fetal abnormalities during devel- While endogenous recruitment of adult neural stem cells has been proposed opment, are responsible for 5-10% of all birth defects. More predictive as a therapeutic strategy, clinical approaches for achieving this recruitment developmental toxicity screens would reduce the prevalence of birth defects are lacking. Here, we show that metformin, a widely-used drug, promotes and increase pharmaceutical and chemical safety. The majority of preclini- neurogenesis from rodent and human neural stem cells and enhances learn- cal efficacy and toxicity testing of pharmaceuticals are currently performed ing. Specifically, we show that atypical PKCs are essential for neural precur- using animal models, which are costly, time-consuming, and the subject of sors to generate neurons, and that metformin-mediated activation of aPKCs ongoing ethical debate. Most importantly, rodent models for developmental promotes rodent and human neurogenesis in culture. Metformin also en- toxicity testing do not adequately correlate to human response, resulting hances hippocampal neurogenesis in mice, and in so doing, facilitates spatial in only 62% concordance to humans. Human embryonic stem (hES) cell reversal learning in a water maze task. Thus, metformin, by activating aPKC, technology is an innovative and robust alternative to predict developmental recruits neural stem cells and enhances neural function, thereby providing a toxicity of chemicals during human pregnancy. We have developed the first candidate pharmacological approach for nervous system therapy. all-human in vitro developmental toxicity screen that utilizes hES cells and metabolomics to discover metabolite biomarkers of developmental toxicity: Poster Board Number: 1287 devTOX. Induced pluripotent stem (iPS) cells are derived from the genetic NEUROTROPHIN SIGNALING IN NEURONAL manipulation of human somatic cells. These cells are being investigated for use in place of hES cells due to the moral, ethical and political controversies DIFFERENTIATION OF surrounding their use. Human iPS cells are phenotypically and genetically MOUSE AND HUMAN EMBRYONIC STEM similar to hES cells in many respects (i.e. morphology, proliferation, gene expression). However, the metabolic similarity between hES cells and iPS CELLS IN 2D AND 3D MODELS cells is not known. It is vital to understand this information when considering Zhang, Dawei, Herland, Anna, Cebers, Gvido, Cotgreave, Ian, using iPS cells in the same manner as hES cells, such as for human toxicity Teixeira, Ana I. testing. We measured the secreted metabolites (secretome), across three hES Karolinska Institutet, Stockholm, Sweden, Safety Assessment, AstraZeneca cell lines and two iPS cell lines using liquid chromatography mass spectrom- R&D, Södertälje, Sweden etry (LCMS), to determine what differences exist in the secretome between cell types. Additionally, we exposed all five cell lines to 23 compounds with Neuronal differentiation of embryonic stem cells has the potential to be known teratogenicity to test the hypothesis that iPS cells exhibit a similar used in models of neural tissue in health and disease. In this work we focus response to that of hES cells when exposed to non-teratogen and terato- on using such models to study interactions between neurons and their gens. The spent culture media was analyzed following treatment using microenvironment, but also see to the prospective use of these models for LC-ESI-QTOF mass spectrometry with electrospray ionization. Differential drug evaluation. We report on long-term neuronal differentiation of human small molecule metabolites were identified using univariate and multivariate and mouse embryonic stem cells to achieve mature post mitotic cultures. statistical analysis. Significant mass features (an entity detected by MS that is Specifically we concentrate on evaluating the expression of neurotrophin a combination of exact mass, retention time and abundance, prior to being and neurotrophin receptors in different differentiation protocols. In variants identified as an annotated compound) were annotated by comparison with of the widely used Bain protocol, relying on formation of cellular aggregates Stemina’s internal metabolite database. Our initial comparisons between and neuronal induction with retinoic acid, the majority of the neuronal the three genetically distinct hES cell lines have shown that no metabolites population is glutamatergic or GABAergic. TrkB expression is as anticipated were unique to a single cell line and that few statistical differences in the verified in these cultures. In more recent protocols from Hovatta and co- abundance of secreted features were observed. However, we did uncover worders addition of neurotrophins during differentiation results in forebrain a difference in individual cell line responses to treatment. In addition to cholinergic neurons protocols, with expression of TrkC. Although neurotro- metabolomics analysis, we evaluated the cytotoxicity of the compound test phins are secreted soluble proteins, it is well established that BDNF (brain set using a cell viability assay. The results of these assays have shown a dif- derived neurotrophic factor) binds to α-2,8-linked polysialic acid (polySia), ference between the viability of iPS cells and hES cells following treatment. the major post-translational modification of NCAM (neural cell adhesion Many compounds are more cytotoxic to iPS cells than hES cells. molecule). PolySia is known to exert an important influence of the develop- ment and functions of the nervous system and is temporally regulated to promote neurogenesis, migration, axonal outgrowth and synaptic plasticity. The fundamental function of polySia is further emphasized by the finding that mice lacking the NCAM gene show a mild phenotype, whereas mouse models lacking polysialyltransferase demonstrate a lethal phenotype. We report on the development of tissue culture surfaces and 3D fibrous tissue

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culture models with a polySia coating for neuronal differentiation of mouse impaired in the aged microenvironment. Additionally, unilateral injuries, in and human embryonic stem cells. These culture models are used to evaluate particular, result in predominant loss of function on the ipsilateral side but the effect of polySia in BDNF signalling in terms of downstream signalling may also alter the contralateral niche. Thus, this type of injury could provide events and intracellular neurotrophin trafficking. insight into demyelination/pathogenesis in SCI. In the present study, we: 1) characterize pathology in young vs aged animals in a newly developed Poster Board Number: 1289 unilateral cervical injury model that combines contusion and compression of MAMMALIAN GLIAL CELLS MISSING GENES the spinal cord; and 2) test the capacity of human neural stem cells (hCNS- SCns) to promote recovery/repair in this model. Immunodeficient Rag2- INDUCE HES5 EXPRESSION BY ACTIVE DNA gamma(c)-/- mice (young: 3-4 mos old; aged: 16-17 mos old) received DEMETHYLATION IN EARLY MOUSE EMBRYOS 30kD unilateral contusion injuries with an additional 5 second compression on the same side as the contusion at the C4 vertebral level. Nine days post- Hitoshi, Seiji, Ishino, Yugo, Ikenaka, Kazuhiro injury, aged mice received either hCNS-SCns or vehicle while young animals Division of Neurobiology & Bioinformatics, National Institute for only received vehicle. Forelimb function and kinematic gait parameters were Physiological Sciences, Okazaki, Japan analyzed terminally using the horizontal ladderbeam and CatWalk tasks. Allodynia/altered hindlimb senstivity were assessed using von Frey testing. Notch signaling plays pivotal roles in early development of mammalian Analysis of CatWalk testing revealed improved stepping pattern (P=0.05) embryos and in the generation and maintenance of various stem cell and higher maximum contact area in aged animals receiving hCNS-SCns populations. Based largely on studies of Drosophila and C. elegans, a Notch in comparison to vehicle (P=0.05). Analysis of Von Frey testing revealed receptor-mediated lateral inhibition model has been proposed, in which cells no evidence of allodynia/altered hindlimb sensitivity in cell-treated versus that are initially equivalent but eventually express ligands more than others vehicle, or young versus old, animals. In parallel, horizontal ladderbeam data send signal to neighboring cells. The cells that receive ligand stimuli activate revealed a significant reduction in the number of forelimb errors made by canonical Notch pathway to upregulate effector genes, Hes1 and 5, which aged animals receiving hCNS-SCns compared to vehicle (P<0.05). Interest- in turn suppress the expression of ligand genes. The lateral inhibition model ingly, no significant differences in either ladderbeam or CatWalk of vehicle- may be valid to explain how neural stem cells in developing mammalian treated young vs aged mice were detected, suggesting that age did not brains are kept undifferentiated; neural stem cells receive signal from their affect spontaneous recovery. Histological data for cell engraftment, lesion progeny that differentiate and express ligands, Dll1 and Jagged. However, size, and the demyelinated niche will be presented. These data extend our recent observations that Hes1 levels oscillate in neural precursors, and that previous findings demonstrating cell-mediated recovery of motor function in Hes1 oscillation in turn induces the inverse oscillation of Dll1 and a proneu- young, thoracic contusion SCI animals; together the data suggest the capac- ral gene Neurogenin2, have challenged the validity of the lateral inhibition ity of hCNS-SCns to improve motor function in animals at multiple ages and model. Therefore, it is critical to clarify molecular mechanisms how the SCI types. canonical Notch pathway is initially activated and fixed in stem-producing cells in the early embryo. Here we show that the promoter region of Hes5 Poster Board Number: 1293 gene is under the epigenetic regulation and that mammalian Glial cells miss- ing (Gcm) genes are involved in this process. Analysis of mutant embryos ADULT NEUROGENESIS AFTER ACUTE SGN for Gcm 1 and 2 revealed that the methylation status of the Hes5 promoter DEGENERATION IN THE MOUSE INNER EAR remains high in Gcm1/2-/- mutants at 8-12 somite stages. Accordingly, Hes5 expression is greatly reduced in those mutants. Our results suggest Lang, Hainan, Samuvel, Devadoss J., Kilpatrick, Lauren A., Zhu, that mammalian Gcms could be a switch to activate the canonical Notch Juhong, Krug, Edward L. signaling in stem-forming cells in early embryos. Pathology and Laboratory Medicine, Medical University of South Carolina, Poster Board Number: 1291 Charleston, SC, USA, Otolaryngology-Head and Neck Surgery, Medical University of South Carolina, Charleston, SC, USA, Regenerative Medicine, HUMAN NEURAL STEM CELLS PROMOTE Medical University of South Carolina, Charleston, SC, USA RECOVERY OF FUNCTION IN A MOUSE Spiral ganglion neurons (SGNs) are primary auditory afferent neurons that UNILATERAL CERVICAL SPINAL CORD INJURY deliver signals from the inner ear to the brain. Loss of SGNs occurs with ex- posure to ototoxic drugs and noise, genetic mutations and age, resulting in MODEL permanent sensorineural hearing loss. Recent studies have shown that stem Hooshmand, Mitra J., Nishi, Rebecca, Lucero, Jovanny, Huang, cells are able to be isolated from vestibular and auditory sensory epithelia and spiral ganglia of the developing inner ear. However, it is unclear whether Kevin, Jadhaw, Neera, Perez, Harvey, Uchida, Nobuko, Anderson, neural stem/progenitor cells are present in the adult auditory nerve. The Aileen J. goal of the present study is to evaluate the potential of adult neurogenesis Institute for Memory Impairments and Neurological Disorders, University in the mouse inner ear. We have established an animal model of acute SGN of California, Irvine, Irvine, CA, USA, Christopher and Dana Reeve degeneration by applying ouabain to mouse inner ear. In addition to selec- Foundation, Irvine, CA, USA, CIRM Stem Cell Research Biotechnology tively removing type I SGNs, hyperplasia and hypertrophy of glia-like cells Training Program, California State University, Long Beach, Long Beach, CA, occur in the ouabain-injured auditory nerves. The transcription factor Sox2 is USA, StemCells, Inc., Palo Alto, CA, USA predominantly expressed in undifferentiated neural precursors during adult The average age of the clinical population of spinal cord injury (SCI) is 34 neurogenesis in the brain. A subset of glial cells up-regulated Sox2 and de- years old. Yet biological mechanisms of recovery/repair in most animal mod- differentiated shortly after ouabain treatment, suggesting that glial cells may els are generally addressed in young rodents between 8-12 weeks. Further, be a source for neurogenesis after acute SGN injury in the adult mouse inner while the thoracic contusion paradigm is the experimental model of choice ear. In this study, we examined the capability of neurogenesis in adult mouse for SCI, the clinical incidence of cervical SCI is greater than thoracic. Injuries inner ear after acute SGN degeneration using an in vitro approach. The in the human population are frequently accompanied by vertebral compres- results showed that more neurosphere-like clusters were generated when sion and, consequently, ischemia. Notably, oligodendrocytes are particularly cultured from ouabain-injured mice than from normal mice. The majority of vulnerable to ischemia/compression injuries, and chemical demyelination cells from these clusters were stained positively for BrdU and nestin, a neural models demonstrate an age-related failure of remyelination by endogenous stem/progenitor cell marker. Our results suggest that neurogenesis occurs in oligoprogenitors. This suggests that endogenous remyelination may be the adult inner ear and the acute SGN degeneration enhances this capability.

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Thursday Poster Abstracts

Poster Board Number: 1295 month-2M) versus middle-aged (12 month-12M) mice. Previously, we iden- tified a critical period between young adult and middle-age during which IS ALTERED LIPID REGULATION NSCs become quiescent. Furthermore, biochemical analyses revealed that INVOLVED IN AGE RELATED DEFICITS IN 2M and 12M NSCs have differences in their levels of a number of key intra- cellular signalling pathways. To study the molecular mechanisms regulating ADULT NEUROGENESIS IN THE MURINE NSC quiescence in the aging brain, we performed a phospho-proteomic SUBVENTRICULAR ZONE? analysis of microdissected SVZ niches of young adult versus middle-aged mice. Protein phosphorylation status provides us with precise changes in Hamilton, Laura K., Avrith, Nita, Aumont, Anne, Calon, Frédéric, protein properties that affect many critical cellular processes. Aging-associ- Fernandes, Karl J.L. ated changes in multiple proteins were identified, including regulators of cell Pathologie et Biologie Cellulaire, Universite de Montreal, Montreal, cycle, cell-cell interactions, proliferation and cellular migration. We are cur- QC, Canada, Centre Hospitalier de l’Université Laval Research Center, rently exploring the importance of candidate regulators of NSC aging using Universite Laval, Quebec, QC, Canada multiple in vitro and in vivo manipulations of signalling pathways. Primary NSC cultures are being treated with pharmacological inhibitors to study their It is well established that a stem cell’s microenvironment critically regulates impact on NSC expansion and differentiation. We are also using adult brain its behaviour. In the adult brain, there are two niches that maintain acon- in vivo electroporation to establish the role of these pathways as intrinsic tinuous stem cell pool, the subventricular zone (SVZ) surrounding the lateral modulators of NSC quiescence. These experiments will provide novel in- ventricles and the dentate gyrus of the hippocampus. Within these niches, sights into the global changes in intracellular signalling pathways regulating neurogenesis occurs throughout life and is necessary for cognitive plasticity NSC aging and quiescence, and provide a basis for future work aimed at and cell replacement. With aging and metabolic disorders, lipid accumula- appropriately modulating stem cell activity to ameliorate the cognitive symp- tions are observed in a variety of tissues. However, the impact of altered toms of aging. Supported by the Canadian Institutes of Health Research. lipid regulation on stem cell behaviour is not well understood. In the present study, we aim to understand if changes in lipid regulation within the CNS are Poster Board Number: 1299 involved in age-associated decreases in neural stem cell activity, neurogen- esis and cell replacement in the SVZ. Using Oil Red O, a lysochrome stain JMJD3-MEDIATED EPIGENETIC REGULATION predominantly used for demonstrating triglycerides, we have observed that OF NEUROGENESIS FROM ADULT MOUSE during aging there are dramatic increases in lipid droplet number and size within the ependymal cells of the SVZ. Ependymal cells line the lateral ven- NEURAL STEM CELLS tricles and make up 25% of the cells in the SVZ niche. Moreover, ependymal Park, Dae Hwi, Sun, Shawn, Incorvaia, Elisabetta, Burgold, Thomas, cells direct CSF flow and secrete factors that are critical for the maintenance Testa, Giuseppe, Lim, Daniel A. of the SVZ stem cell niche. Thus, perturbation of the ependymal cells could have a significant impact on the stem cell niche at its activity. To determine if Neurological Surgery, University of California, San Francisco, San Francisco, these lipid droplets interfere with ependymal cell function, we are develop- CA, USA, European Institute of Oncology, Milan, Italy ing novel in vitro assays to evaluate the influence of increased local lipid One of the fundamental questions in stem cell biology is how multipo- accumulations on ependymal cell function. Since it is possible that changes tent stem cells selectively activate and repress sets of genes to orches- in local lipid regulation could directly impact SVZ neural precursors, we are trate tissue development and homeostasis. In embryonic stem (ES) cells, also using colony-forming “neurosphere” cultures of SVZ stem cells to assay activation of genes required for lineage commitment correlates with rapid the impact of increased circulating lipids on neural stem cell proliferation, loss of the repressive chromatin modification, trimethyl histone H3 lysine self-renewal and differentiation. Finally, to understand the impact of altered 27 (H3K27me3), at their loci. However, it remains to be elucidated how lipid regulation on overall signalling changes within the SVZ niche in vivo, H3K27me3-mediated epigenetic mechanisms regulate lineage specification we are establishing a complementary animal model in which lipids accu- to achieve homeostasis in adult stem cells. Previously we have shown that mulate within the SVZ of young adult mice. Together, these in vitro and in the chromatin remodeling factor Mll1 is required for proper expression of vivo models will enable us to identify the role of lipids in regulating stem cell a neurogenic gene, Dlx2, during neuronal differentiation in the postnatal behaviour. Understanding whether all or only isolated stages of neurogen- mouse subventricular zone (SVZ). Chromatin immunoprecipitation (ChIP) esis are affected and the mechanisms involved will provide important clues data are consistent with a mechanism in which MLL1 recruits a H3K27me3 as to how lipids may be involved in regulating neurogenesis under normal specific demethylase to neurogenic loci such as Dlx2. There are two known and pathological conditions. H3K27me3 specific demethylases, UTX and JMJD3. While UTX expression Supported by: the Canadian Institutes of Health Research was found to be ubiquitous in adult mouse brain, we found JMJD3 expres- Poster Board Number: 1297 sion to be enriched in SVZ neural stem cells as well as the neuronal daughter cells. In this study, we report a critical role of JMJD3 in lineage specification MOLECULAR REGULATION OF NEURAL STEM using postnatal subventricular zone (SVZ) neural stem cells as a model sys- tem. Quantitative gene expression analysis shows that Jmjd3 is expressed in CELL QUIESCENCE IN THE AGING MURINE neurogenic subventricular zone and transiently increased in the early stage BRAIN of neurogenesis in adult brain. Consistent with its gene expression profile, JMJD3 is found to be localized mainly in DLX2 positive transit amplifying Paliouras, Grigorios N., Bouab, Meriem, Aumont, Anne, Fernandes, cells (type C cells) and neuroblasts (type A cells) in vivo as well as in vitro. Karl JL Knockdown of Jmjd3 using shRNAi decreases Tuj1 positive neuroblasts and Pathology and Cellular Biology, University of Montreal, Montreal, QC, reduces expression of Dlx2 in adult SVZ neural stem cells (NSCs). Further- Canada more, JMJD3 downregulation also reduces the number of DLX2 positive cells during neurogenesis without a concomitant increase in cell death. The discovery of adult neurogenesis and neural stem cells (NSCs) has Taken together, these results indicate the functional requirement of JMJD3 in opened new realms of possibilities for promoting brain health and repair. inducing the expression of Dlx2 for postnatal neurogenesis. It also supports The well-documented loss of NSCs in the aged brain may be a contributing the idea that JMJD3 plays a pivotal role in the activation of developmental factor to the development of age-related cognitive impairments and neuro- regulators during lineage specification. degeneration. To gain insights into the initial stages of age-related NSC loss, we studied the subventricular zone (SVZ) stem cell niche of young adult (2

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Poster Board Number: 2001 and size; however, limiting dilution and differentiation analyses revealed that IGF-II was superior to IGF-1 in promoting neurosphere number. Knockdown LIF-DEPENDENT PRIMITIVE NEURAL STEM of either the IR or IGF-1R using shRNAs supported the conclusion that the CELLS IN THE ADULT MOUSE SUBEPENDYMA IGF-1R promotes progenitor proliferation whereas the IR is important for self-renewal. Q-PCR revealed that IGF-II increased Oct4, Sox1 and FABP7 COMPRISE A DISTINCT POPULATION THAT CAN mRNA levels in neurosphere cells. Altogether our data support the conclu- BE MANIPULATED INDEPENDENT OF ADULT sion that IGF-II promotes the self-renewal of neural stem/progenitors via the DEFINITIVE NEURAL STEM CELLS IR. By contrast, IGF-1R functions as a mitogenic receptor that increases cell cycle progression of progenitors. Supported by a Dean’s Grant from NJMS Leeder, Rachel, Sachewsky, Nadia, Rose, Keeley L., DeVeale, Brian, awarded to SWL and TLW and F31NS065607 awarded to ANZ. Morshead, Cindi M., van der Kooy, Derek Poster Board Number: 2005 University of Toronto, Toronto, ON, Canada We recently identified a novel population of LIF-dependant neural stem cells VENTRAL HIPPOCAMPUS AS THE ORIGIN FOR (NSCs) in the adult mouse subependyma, termed adult-derived primitive THE MOUSE SUBGRANULAR NEURAL STEM NSCs (AdpNSCs). AdpNSCs comprise a rare population of GFAP-negative CELLS AT ALL LEVELS cells that are similar to embryonic primitive NSCs as they give rise to LIF-de- pendent neurospheres in vitro. AdpNSCs can be passaged in vitro to self-re- Li, Guangnan, Fang, Li, Pleasure, Samuel new or give rise to the more common GFAP-positive EGF- and FGF-depen- Neurology, University of California San Francisco, San Francisco, CA, USA dent adult definitive (d)NSCs. AdpNSCs were shown to express low levels of Oct4 and contribute to chimeric blastocysts, both markers of pluripotency Hedgehog (Hh) signaling is required for the establishment and maintenance not exhibited by other adult dNSCs. Currently, we are investigating strate- of the subgranular zone dentate progenitors and genetic fate-mapping anal- gies to isolate and functionally manipulate this rare AdpNSC population to ysis shows that quiescent neural stem cells (NSCs) in the adult hippocam- better understand its role in the adult mouse brain. Adult dNSCs expressed pus can be distinguished by their responsiveness to Hh signal. However, high levels of nestin, an intermediate filament protein, whereas AdpNSCs the anatomic origin of the dentate NSCs hasn’t been determined yet. The arose from a distinct lower nestin expressing population by FACS sorting. existing model in general assumes that the ventricular zone of the dentate Second, mice with a floxed Oct4 allele excised by Cre expressed under primordium is the common origin for both adult hippocampal NSCs and the Sox1 promoter gave rise to fewer LIF-dependent clonal neurospheres. the embryonically produced granule neurons that form the nascent dentate This indicates a requirement for Oct4 in AdpNSC function or survival that granule cell layer, but it has been called into question by the aggregate chi- is not required by adult dNSCs to form EGF- and FGF-dependent clonal mera analysis. Here we show that embryonic NSCs from the ventricular zone neurospheres in vitro. Finally, a previous mass spectrometry based screen of of the ventral hippocampus initially respond to Shh from the amygdala and in vitro ESC-derived primitive neurospheres identified cell surface markers then spread in a temporal to septal direction. The descendants of these cells selectively upregulated in primitive LIF-dependent cells compared to ESCs are the primary source for hilar NSCs covering all dentate anatomic levels. and definitive neurospheres. We are testing these cell surface markers to Subsequently, local Shh sources further play an essential role in maintenance identify factors that selectively affect the ability of AdpNSCs to form clonal of the subgranular NSCs. In contrast to the prevailing view in the literature, LIF neurospheres, without affecting other adult dNSCs. Specifically, Gleevec, our findings demonstrate that adult dentate NSCs have a more defined em- a c-kit pathway inhibitor, significantly increased LIF-dependent AdpNSC bryonic origin and follow a newly recognized temporal to septal migratory derived neurosphere formation while attenuating EGF- and FGF-dependent route to establish the subgranular zone. adult dNSC derived neurosphere formation. Together, these experiments Poster Board Number: 2007 indicate that AdpNSCs, aside from being GFAP-negative and LIF-responsive, can be isolated from other adult NSCs based on nestin expression, Oct4 MASH1 IS A NOVEL TARGET OF GLI2 DURING dependence and c-kit function. These characteristics will enable selective GLI2-INDUCED NEUROGENESIS IN MURINE manipulation of AdpNSCs to better understand their function in the adult mouse brain. P19 EMBRYONAL CARCINOMA STEM CELLS Poster Board Number: 2003 Voronova, Anastassia, Fischer, Anna, Ryan, Tammy, Al Madhoun, Ashraf, Skerjanc, Ilona S. INSULIN RECEPTOR ACTIVATION PROMOTES Biochemistry, Microbiology and Immunology, University of Ottawa, MURINE NEURAL PRECURSOR STEMNESS Ottawa, ON, Canada Ziegler, Amber N., Wood, Teresa L., Levison, Steven W. The Sonic Hedgehog (Shh) signaling pathway is important for neurogenesis Neurology and Neuroscience, UMDNJ-New Jersey Medical School, Newark, in vivo. Gli transcription factors, effector proteins of Shh signaling path- NJ, USA way, have neurogenic properties in vivo, which are still poorly understood. To study the molecular basis of neurogenic properties of Gli2, we used a The insulin-like growth factor (IGF) system plays a critical role in brain well-established embryonic stem cell model, the murine P19 embryonic development and growth. IGF-I and IGF-II both activate the IGF-1R. In carcinoma (EC) cell line, which can be induced to differentiate into neurons contrast, IGF-II, but not IGF-I can activate a splice variant of the insulin in the presence of retinoic acid (RA). We found that, in the absence of RA, receptor (IR) known as IR-A. We hypothesized that IGF-II will exert distinct expression of Gli2 induced P19 EC cells to differentiate into neurons, but not effects on neural stem/progenitor cells (NSPs) than IGF-I. IHC revealed astrocytes. To our knowledge, this is the first indication that the expression that IGF-II is expressed as a gradient in the neural stem cell niche. Q-PCR of Gli factors can convert EC cells into neurons. Furthermore, Gli2 upregulat- analysis revealed that IGF-II mRNA is highly expressed by the choroid ed expression of the neurogenic basic helix-loop-helix (bHLH) factors, such plexus. Additionally, Q-PCR showed that the IGF-1R and the IR isoforms as NeuroD, Neurog1 and Ascl1/Mash1 in P19 EC cells. Using chromatin are differentially expressed between NSPs and more lineage restricted cells, immunoprecipitation assay, we showed that Gli2 bound to multiple regula- with IR-A being predominant in NSPs. IGF-II promoted NSP cell expansion tory regions in the Ascl1 gene, including promoter and enhancer regions better than either IGF-1 or standard culture medium (containing super- during Gli2-induced neurogenesis. In addition, Gli2 activated the Ascl1/ physiological levels of insulin). A combination of IGF-I and IGF-II mimicked Mash1 promoter in vitro. Using the expression of a dominant-negative form standard neurosphere growth conditions in terms of neurosphere number

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Thursday Poster Abstracts of Gli2, fused to the Engrailed repression domain, we observed a reduction Poster Board Number: 2011 in gliogenesis and a significant downregulation of the bHLH factors Ascl1/ Mash1, Neurog1 and NeuroD, leading to delayed neurogenesis in P19 EC CHEMOTAXIS OF MOUSE NEURAL STEM CELL- cells, further supporting the hypothesis that Ascl1/Mash1 is a direct target of DERIVED NEUROBLASTS IS STIMULATED BY Gli2. Our results provide mechanistic insight into the neurogenic properties of Gli2 in vitro, and offer novel plausible explanations for its in vivo neuro- CXCL12 N-TERMINAL PEPTIDES genic properties. Porcionatto, Marimelia A., Filippo, Thais R. M., Barnabe, Gabriela Poster Board Number: 2009 F., Ariza, Carolina B., Galindo, Layla T., Moreira, Caroline M., Mello, Luiz E., Juliano, Maria A., Juliano, Luiz TRANSGENIC ENRICHMENT OF MOUSE Bioquimica, Universidade Federal de São Paulo, São Paulo, Brazil, EMBRYONIC STEM CELL-DERIVED Fisiologia, Universidade Federal de São Paulo, São Paulo, Brazil, Biofisica, PROGENITOR MOTOR NEURON CELLS Universidade Federal de São Paulo, São Paulo, Brazil Traumatic brain injury (TBI) is a leading cause of mortality and morbidity McCreedy, Dylan A., Silverman, Cara R., Gottlieb, David I., worldwide. The major causes for TBI are violence, automotive accidents, Sakiyama-Elbert, Shelly E. sports, and war. Up to date there are few therapeutic strategies to reduce Biomedical Engineering, Washington University in St. Louis, St. Louis, TBI sequelae, but no complete recovery is possible yet. The central nervous MO, USA, Anatomy and Neurobiology, Washington University School of system (CNS), contrary to peripheral nervous system, shows little spontane- Medicine, St. Louis, MO, USA ous recovery which depends on the extension and location of the injury. Embryonic stem (ES) cells provide a unique opportunity to produce an abun- The cellular response to brain injuries includes secretion of inflammatory dant supply of committed or terminally differentiated cells for biomedical mediators at the injury site, as well as secretion of axonal growth inhibi- and therapeutic applications. Directed differentiation of mouse and human tors by reactive glial cells, mainly astrocytes and oligodendrocytes. A few ES cells into spinal motoneurons (MNs) can be accomplished by exposure days after a TBI, chemokines secreted at the injury site attract neuroblasts to retinoic acid (RA) and sonic hedgehog (Shh). In this process, ES cells are originated from neural stem cells present in the subventricular zone. Among first induced to become progenitor motor neurons (pMNs) expressing the the cytokines secreted at the injury site is CXCL12, a chemokine that attracts basic helix-loop-helix transcription factor Olig2. The resulting pMNs can CXCR4+ cells, such as the neuroblasts. In the present work, we show data then commit to the MN fate, expressing Ngn2 and initiating expression of a new approach to increase chemotaxis of neuroblasts originated at the of transcription factors Isl1 and Hb9. Using this pMN induction protocol, subventricular zone. Using an animal model for TBI we report on synthetic approximately 60% of cells express Olig2, of which 30-50% become com- peptides that are analogous to the N-terminal of CXCL12. Our results show mitted MNs (Hb9+). This commonly reported heterogeneity is problematic that a 21aa long peptide, with the exact sequence of CXCL12 N-terminal is because transplantation of additional neuronal subtypes resulting from low capable of promoting chemotaxis of neuroblasts. We also show that when purity cell populations can mask the specific effects of Hb9+ MNs. Higher the two cysteins needed for CXCL12 activity were replaced by two alanines, purity cultures can be obtained by fluorescence-activated cell sorting (FACS) the peptide lost the chemotactic activity but showed downregulation of of transgenic ES cell lines expressing green fluorescent protein (GFP) under GFAP expression, a marker for reactive astrocytes. This last result could the Olig2 or Hb9 promoters. However, these methods are limited due to the indicate that the peptide with the alanine substitution might be decreasing requirement for expensive equipment and the possible risk of contamina- the local inflammatory response exerted by reactive glia. We propose a new tion. To provide a readily available, inexpensive method to enrich for pMNs, strategy to be tested for TBI therapy that includes a combination of the two we generated a transgenic mouse ES cell line (P-Olig2) that expresses the synthetic peptides to increase neuroblast chemotaxis towards the injury site puromycin resistance gene, puromycin N-acetyl-transferase (PAC), under and to reduce neuroinflammation at the injury site. the Olig2 promoter. The PAC gene was targeted to the protein-coding Poster Board Number: 2013 region of Olig2 to provide specific expression in pMNs. Induction of P-Olig2 ES cells was performed using a 2-/4+ RA/Shh induction protocol where a FOXP2 REGULATES NEUROGENESIS IN THE Shh signaling pathway agonist (purmorphamine) was substituted for Shh. EMBRYONIC MURINE CORTEX ES cells were aggregated into embryoid bodies (EBs) for two days on non- adhesive dishes (2-). EBs were then transferred to gelatin-coated 6-well Tsui, David, Burns, Sarah, Kaplan, David R., Miller, Freda D. plates and induced with 2 µM RA and 1.5 µM purmorphamine for four Developmental & Stem Cell Biology, The Hospital for Sick Children additional days (4+). Puromycin (2 ng/ml) was added to P-Olig2 cultures Research Institute, Toronto, ON, Canada from day 4 (2-/2+) to day 6 (2-/4+) to select for Olig2+ pMNs. After induc- tion and selection, cultures were analyzed using flow cytometry or plated on Rare mutations involving FOXP2 are associated with impairments in the laminin-coated wells and allowed to differentiate for two weeks. At the end learning and production of sequences of oral movements that lead to of the induction period, puromycin selection resulted in a significant increase speech. Thus far, it is the only gene implicated in a Mendelian form of in Olig2+ pMNs cells from 21.9 ± 5.9% to 48.7 ± 7.3% (n = 3, p < 0.007). human speech and language dysfunction. However, it is still unclear how Hb9+ committed MNs increased from 20.6 ± 7.9% to 58.5 ± 1.5% (n = 3, FOXP2 mutations disturb speech and language development. Because p < 0.002). Plated cells differentiated into cholinergic MNs and oligodendro- FoxP2 is highly conserved in many vertebrates and shows comparable neural cytes, two common progeny of pMNs. These results demonstrate that trans- expression patterns, its function can be assessed in animals and could be genic selection can provide efficient and inexpensive enrichment of pMNs similar in human. Several mouse models carrying different FoxP2 muta- and committed MNs for use in drug screening or cell replacement strategies. tions have been developed and mutant pups demonstrated impairments in innate behaviour called ultrasound vocalization, leading us to hypothesize that FoxP2 plays an essential role in embryonic cortical development. Here, we show that FoxP2 is expressed in the embryonic cortex at embryonic day 11 when almost all of the cells comprising the cortex are neural precur- sors. Knockdown of FoxP2 in cortical precursors in vivo with three shRNAs with different target sequences in the gene all showed a robust reduction in the percentage of transfected cells in the cortical plate 3 days after in utero electroporation. This inappropriate localization was due to enhanced

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maintenance of radial precursors at the expense of differentiated progeny; whether ependymal cells give rise to functional neurons in the damaged the proportion of Pax6+ radial precursors was increased, with a concomi- striatum and generate neuronal progeny after stroke in rats. Development tant reduction in both Tbr2+ basal progenitors and βIII-tubulin+ neurons. and good survival of functional neurons from ependymal cells are necessary Consistent with these findings, initial results indicate that overexpression of if these cells should be of any importance for the symptomatic recovery after a human version of FoxP2 resulted in an increase in the percentage of trans- stroke. We have developed a method for tracing the progeny of ependymal fected cells in the cortical plate, with a decrease in Pax6+ radial precursors, cells in adult rats. The objective of the present study has been to follow the and an increase in both Tbr2+ basal progenitors and βIII-tubulin+ neurons. fate of ependymal cells lining the lateral ventricular wall after stroke in rats. Thus, FoxP2 is apparently necessary for the appropriate differentiation of The ependymal cells are labeled with fluorescent reporter gene constructs radial precursors into neurons in the embryonic cortex, thereby providing for fate mapping by in vivo electroporation. Experiments are in progress one potential explanation for the cognitive deficits noted when it is mutated with middle cerebral artery occlusion in electroporated adult rats. The in humans. molecular and morphological changes in the labeled ependymal cells after inducing stroke will be analyzed. Poster Board Number: 2015 Poster Board Number: 2019 STEM CELL TRANSPLANTATION IMPROVES AXONAL CONDUCTION IN THE SPINAL CORD AMYGDALA REGULATION OF ADULT BY ALTERING BK CHANNEL EXPRESSIONS IN NEUROGENESIS AND FEAR-RELATED MEMORY THE RAT IN RATS. Buttigieg, Josef, Ye, Hui, Wan, Yudi, Fehlings, Michael Kirby, Elizabeth D., Friedman, Aaron R., Covarrubias, David, Ying, Carl, Sun, Wayne G., Goosens, Ki A., Sapolsky, Robert M., Kaufer, Toronto Western Hospital, University Health Network, Toronto, ON, Canada Daniela Helen Wills Neuroscience, UC Berkeley, Berkeley, CA, USA, Integrative Although spinal cord injury (SCI) typically results in a significant loss of Biology, UC Berkeley, Berkeley, CA, USA, Molecular and Cell Biology, neuronal tissue, often there are surviving axonal tracts at the subpial rim of UC Berkeley, Berkeley, CA, USA, McGovern Institute for Brain Research, the spinal cord. These spared axonal tracts are characteristically dysmyeli- MIT, Cambridge, MA, USA, Department of Biology, Stanford University, nated and express a significant decrease in action potential (AP) conduction Stanford, CA, USA velocity, AP amplitude, an increase in the refractory period, and impaired responses to high-frequency stimulation. While it has been suggested that Impaired regulation of emotional memory is a feature of several affective stem cell transplantation may reverse these deficits, it is poorly understood disorders, including depression, anxiety and post-traumatic stress disorder. as to how stem cell transplantation alters axonal ion channel physiology post Such regulation occurs, in part, by interactions between the hippocam- transplantation. Here we describe the effects of stem cell transplantation on pus and the basolateral amygdala (BLA). Hippocampal neurogenesis may the expression and function of a newly identified potassium channel, the support emotional memory, but the responsiveness of adult neurogenesis large-conductance, voltage- and Ca2+-activated K+ channel (BK channel). to emotional input from the BLA is unknown. We show that BLA lesions Using the combined tools of immunocytochemistry and electrophysiology, suppress adult neurogenesis, while lesions of the central nucleus of the we report that in the healthy cord the BK channel is typically located in amygdala do not. Similarly, we show that reducing BLA activity through the juxtaparanodal region of the axon, covered by the myelin sheath, and viral vector-mediated overexpression of an outwardly rectifying potassium play no apparent role in axonal conduction. In contrast to the non-injured channel also suppresses neurogenesis. Last, using immediate early gene cord, the BK channel is exposed post-SCI due to the demyelination of expression following contextual fear conditioning, we show that BLA lesions axonal tracts. The activation of these exposed ion channels contributes to a prevent selective activation of immature newborn neurons. These results decrease in AP amplitude, and AP duration. After transplantation of neural demonstrate that BLA activity regulates adult hippocampal neurogenesis precursor cells, obtained from the sub ventricular zone of the mouse brain, and the fear context-specific activation of newborn neurons. Together, these there is an increase in myelinated axonal tracts. These remyelinated tracts findings denote functional implications for recruitment of new neurons into demonstrate an improvement in axonal physiology. There is an increase emotional memory circuits. in AP amplitude, and a decrease in the refractoriness. In addition we also Poster Board Number: 2021 observed that, in the remyelinated axonl tracts, the BK channel is located primarily in the juxtaparanodal region, as seen in healthy tissue. These data VASCULAR NICHE FACTORS REGULATE suggest that stem-cell induced remyelination may enhance function conduc- tance of the spared axons by resume BK channel morphology. RODENT ADULT CAROTID BODY STEM CELL ACTIVITY. Poster Board Number: 2017 Platero-Luengo, Aida, Durán, Rocío, López-Barneo, José, Pardal, TRACING NEURONAL PROGENY OF ADULT RAT Ricardo EPENDYMAL CELLS USING ELECTROPORATION Instituto de Biomedicina de Sevilla-IBiS, Sevilla, Spain Devaraju, Karthikeyan, Heider, Fanie Barnabé, Clementson, Preliminary observations in our lab identified the mammalian carotid Christine Ekdahl, Frisén, Jonas, Kokaia, Zaal, Lindvall, Olle body (CB) as a neurogenic niche in the peripheral nervous system with a Clinical Sciences, Division of Neurology; Lund Stem Cell Center, Lund recognizable physiological function in adult life. This chemoreceptor organ University, Lund, Sweden, University of Montreal, Montreal, QC, Canada, is responsible for the detection of hypoxemia and is able to adapt to a per- Cell and Molecular Biology, Karolinska Insititute, Stockholm, Sweden sistent stimulus by increasing the number of neuronal, type I cells. We have previously shown that this neurogenesis process depends on the prolifera- Ependymal cells, lining the lateral ventricles of the brain, give rise to tion and differentiation of a quiescent subpopulation of glia-like stem cells or neuroblasts after stroke in adult mice. Thus, similar to subventricular zone type II cells which are activated in chronic hypoxia. The CB neurogenic niche neural stem/progenitor cells, ependymal cells participate in the neurogenic shares important characteristics with central nervous system neurogenic response to stroke and may be part of a self-repair mechanism. Virtually all niches. The presence of quiescent and activated progenitors and the close of the neuroblasts derived from ependymal cells die in mice. It is unknown interaction of these cells with a profuse vessel network are among them.

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We are interested in the molecular and cellular mechanisms underlying the provided by S. Kriks (SKI, NYC), that expresses YFP from the Pitx3 locus thus activation and deactivation processes in the CB neurogenic niche; how stem facilitating the quantification of dopamine neurons in the selected popula- cell proliferation is triggered; which are the stimuli and how important is the tions by flow cytometry. We conclude that lentiviral mediated transduction niche context for this process. We first wanted to know whether the stem of reporter genes under specific promoters is a valid and useful strategy cells are sensitive to the hypoxic stimulus by themselves or need commu- for selection of dopamine neurons derived from pluripotent stem cells for nication from the surrounding vascular cells. Our recent data suggest that transplantation and other applications. hypoxia is not a direct stimulus activating CB stem cell (CBSC) proliferation, but rather vascular niche factors are released during the hypoxic stimulus to Poster Board Number: 2025 activate neural progenitors. Furthermore, hypoxia seems to be important on HIGH-THROUGHPUT SCREENING OF SMALL inducing dopaminergic differentiation in CBSCs, as it has been described in different types of neural progenitors. Regarding the role of vascular factors, MOLECULES THAT DIRECT DOPAMINERGIC endothelin-1 (ET-1) has a potent effect on increasing proliferation in CBSCs. NEUROGENESIS IN NEURAL PROGENITOR We have analyzed ET-1 production in different CB cell types and have deter- mined endothelial cells as the main source of this factor. Moreover, we have CELLS quantified the angiogenesis occurring in the organ in response to hypoxia to Chan, Shing Fai, Sances, Sam A., Mercola, Mark, McKercher, Scott highlight the increase in production of ET-1 during the stimulus. This work R., Lee, Hyojin, Lipton, Stuart A. helps to understand the importance of the niche in stem cell biology, and Del E. Webb Center for Neuroscience, Aging, and Stem Cell Research, specifically the implications of vascular cells on directing proliferation and Sanfod Burnham Medical Research Institute, San Diego, CA, USA, Muscle cell fate in the neural progenitors. Understanding the physiology of CBSCs Development and Regeneration Program, Sanfod Burnham Medical is crucial not only to learn more about adult neurogenic niches but also to Research Institute, San Diego, CA, USA improve the use of these cells for therapeutics and to better understand the pathophysiology of the occasional tumorigenic growth in the organ. Parkinson’s disease (PD) is a devastating neurodegenerative disease characterized by muscle rigidity, tremor, slowness of physical movement Poster Board Number: 2023 (bradykinesia) and even loss of physical movement (akinesia). PD results OPTIMIZATION AND VALIDATION OF A primarily from the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta in the mesencephalon, causing a loss of the basal ganglia SELECTION STRATEGY FOR ES-DERIVED circuit that supplies neurons in the striatum with dopamine. Recently, there MIDBRAIN DOPAMINE NEURONS FOR have been increasing efforts to develop therapies to decrease Parkinsonian symptoms and delay or even prevent their onset. One promising therapeutic TRANSPLANTATION AND DISEASE MODELING strategy for PD is to replace damaged or deficient DA neurons using em- IN PARKINSON DISEASE bryonic stem cell (ESC)-derived neural progenitor cells (NPC) by implanta- tion therapy. Lentiviral transduction of neurogenic genes in NPCs has been Aguila, Julio Cesar, Sousa, Amaia, van Arenbergen, Joris, Blak, shown to yield DA neuron-enriched progeny that survive and are resistant Alexandra, Sonntag, Kai C., Sanchez-Pernaute, Rosario to teratoma formation. To avoid known caveats to random viral integration, Stem Cells and Neural Repair, Inbiomed, San Sebastian, Spain, Universidad including tumorigenesis, the current study focuses on screening for small de Barcelona, Barcelona, Spain, McLean Hospital, Belmont, MA, USA molecules that activate expression of these neurogenic transcription factors Cell selection is critical for the application of pluripotent stem cells to cell endogenously. Utilizing a luciferase reporter gene assay, high-throughput therapy and disease modeling. For dopamine neurons derived from pluripo- screening (HTS) was conducted on several small molecule libraries to probe tent stem cells, selection procedures are not well established. We have used for increased expression of neurogenic transcription factors. HTS was mouse ES cells to optimize and validate the use of lentiviral-mediated ex- conducted on several model NPC types, including rat adult neural progeni- pression of reporter genes under the control of a transcription factor Foxa2 tor cells and P19 mouse embryonic carcinoma cells. We have found initial (hepatocyte nuclear factor 3beta) that is expressed in midbrain dopamine candidate small molecules that activate endogenous neurogenic programs in neurons. To drive the expression of GFP or PAC reporter genes we designed these models in a dose-dependent manner. Secondary screening will be per- several lentiviral constructs using either a 400bp sequence of the Foxa2 formed using a high content assay to assay for the DA phenotype. Positive promoter (small promoter) or a long promoter that included 1200bp of the hits will be tested for DA enriched neurogenesis in human ESC and induced first exon. First we verified the functionality of these vectors in a human pluripotent stem cells (iPSCs) in vitro, and then assessed in animal models as hepatocarcinoma cell line (HepG2) that constitutively expresses Foxa2. Then potential drug therapies for PD. we examined the specificity of the two promoters comparing GFP expres- Poster Board Number: 2027 sion in HEK 293, a cell line that does not express Foxa2. We confirmed that the vector with the short promoter sequence does not express GFP in 293, NEURAL PRECURSOR CELLS IN while the vector carrying the long promoter showed some expression of NEUROSPHERES SHOW GLIAL COMMITMENT GFP. Based on these results we chose the small promoter region for driving the expression of reporter genes in human and mouse ES cells in the rest of IN EMBRYONIC NEUROGENIC ENVIRONMENTS the experiments. To optimize transduction and selection procedures using Covarrubias, Luis, Baizabal, José-Manuel, Cano, Agustina, Valencia, these vectors we used mouse ES cell lines differentiated following the five Concepción, Santa-Olalla, Jesús, Bartlett, Perry F. stage protocol (Lee et al., 2000). We have tested different times and condi- tions for transduction. We have found that lentiviral infection at late stage 4 Department of Developmental Genetics and Molecular Physiology, Instituto (neural progenitors) gives the best results with up to 40% of GFP expression de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, when transduction is done in suspension. FACS sorting 4-5 days later (early Morelos , Mexico, Queensland Brain Institute, University of Queensland, stage 5) is feasible with good viability and an average final yield around 7%. Brisbane, Qld. Australia, Australia Earlier transduction times resulted in a much lower proportion of GFP ex- Neural Precursor Cells (NPCs) with high proliferative potential are com- pressing cells, probably related to faster expansion of non-transduced cells. monly expanded in vitro as neurospheres. As a population, neurosphere Selection based on puromycin resistance was validated in rodent primary cells show long-term self-renewal capacity and multipotentiality in vitro. fetal ventral midbrain neurons. We also examined the efficiency and efficacy However, it remains to be determined whether mesencephalic NPCs change of this approach using a BAC transgenic mouse ES cell line (K20), kindly their differentiation potential after neurosphere formation or rather they

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require specific culture conditions or patterning cues to achieve efficient Poster Board Number: 2031 dopaminergic differentiation. In order to contribute to solve this issue, we used a previously reported explant culture to expose ventral mesenceph- DIRECT CONVERSION OF FIBROBLASTS alic neurospheres to the niche of dopaminergic neurogenesis in the early TO A MULTIPOTENT NEURAL PRECURSOR embryonic midbrain. We found that mesencephalic NPCs expanded as neu- rosphere showed poor neurogenesis and dopaminergic specification in their POPULATION WITH DEFINED FACTORS original niche of differentiation. Remarkably, after just one passage in vitro, Lujan, Ernesto, Wernig, Marius neurosphere cells completely lost their capacity to generate new neurons in Program in Genetics, Stanford University, Stanford, CA, USA, Institute for the embryonic tissue. Instead, neurospheres cells showed niche-independent Stem Cell Biology and Regenerative Medicine, Department of Pathology, differentiation to the astrocytic lineage, indicating an intrinsic developmental Stanford University, Stanford, CA, USA restriction to glial fates. Furthermore, neurosphere cells were largely unable to acquire region-specific positional markers at different dorso-ventral levels Recently a set of three transcription factors has been shown to directly and of mesencephalic explants, suggesting that neurosphere formation signifi- abundantly convert mouse embryonic and adult fibroblasts to functional cantly abolish responsiveness to patterning cues. Using the same explant neurons. Although this conversion has been demonstrated to be direct with culture approach, we present data supporting a fate-switch from neurogenic few to no cell divisions in transit, we hypothesized that an intermediate neu- potential to glial commitment of adult NSCs expanded as neurospheres. Our ral precursor population (NPC) could also be produced directly from mouse data suggest that neurosphere formation does not expand the endogenous embryonic fibroblasts (MEFs) under appropriate conditions. In an attempt to neurogenic NPCs but rather promotes amplification of gliogenic precursors achieve such a state directly from fibroblasts, MEFs derived from Rosa-RTTA, that do not respond to niche-derived signals of cellular specification and Sox2-GFP (RS2G) knock-in mice were infected with a set of 11 transcription differentiation. factors (11F) with functions important for neural development and shown to be highly expressed in NPCs. As Sox2 is highly expressed in NPCs, we rea- Poster Board Number: 2029 soned that cells induced to a NPC state could be identified via the Sox2-GFP STAUFEN 2 REGULATES THE MAINTENANCE reporter. Twenty-four days after transgene induction, Sox2-GFP+ colonies were observed from MEFs infected with the 11F pool but not from MEFs AND DIFFERENTIATION OF NEURAL infected with RTTA alone. qPCR analysis of genes selectively expressed in PRECURSORS DURING MAMMALIAN NPCs but not present in MEFs showed NPC specific gene induction in 11F Sox2-GFP+ MEFs at levels similar to those seen in NPCs. Further refine- CORTICAL DEVELOPMENT ment of this 11F pool resulted in a minimal pool of 2 factors that was able Vessey, John P., Burns, Sarah E., Kaplan, David R., Miller, Freda D. to directly induce a NPC state as demonstrated by their ability to self-renew Development and Stem Cell Biology, The Hospital for Sick Children, and differentiate into TUJ1+, MAP2+ neurons and GFAP+ astrocytes as Toronto, ON, Canada, Cell Biology, The Hospital for Sick Children, Toronto, clonogenic cultures after more than 10 passages. The direct conversion of a ON, Canada differentiated cell type to a multipotent neural precursor state with only two factors demonstrates the potential of forced transcription factor expression RNA-binding proteins involved in mRNA localization in Drosophila are in determining cell fate and its implications for regenerative medicine. critical regulators of asymmetric cell division in the developing fly nervous system. One such protein is the double-stranded RNA-binding protein Poster Board Number: 2033 Staufen, which localizes prospero mRNA to the ganglionic mother cell from the asymmetrically dividing neuroblast. Although the mammalian Staufen EVALUATING STEM CELL-BASED PLASTICITY IN genes have been studied in the mature nervous system, their contributions A MODEL OF CERVICAL SPINAL CORD INJURY during development have not been investigated. We hypothesized that the Satkunendrarajah, Kajana, Wilcox, Jared T., Ruff, Crystal, Fehlings, brain enriched, mammalian Staufen 2 (Stau2) plays a similar role in regulat- ing neurogenesis from the cortical precursor cell pool of the developing brain Michael as it does in Drosophila. Development of the cortex relies on asymmetric cell Genetics and Development, Toronto Western Research Institute, Toronto, divisions to generate both neurons and glia from the cortical precursors lin- ON, Canada, Toronto Western Research Institute, Toronto Western ing the ventricles of the embryonic brain. We found that Stau2 is expressed Research Institute, ON, Canada during the neurogenic phase in both dividing precursor cells and in newly Background: Spinal cord injury (SCI) represents significant burden of disease born, post-mitotic neurons. Its expression in precursor cells is enriched at the with substantial individual and societal impacts. The majority of SCI occurs apical surface of the ventricular zone, the location of the apical end feet of at the cervical level; however, appropriate cervical SCI models have not radial precursor cells. The expression of proteins such as DDX3 and RNAs been established. Objective: To develop a cervical SCI model represent- such as β-actin, that are known to associate with Stau2 in mRNA gran- ing clinically relevant forelimb deficits for use as a platform for evaluating ules also are enriched in this region. Knockdown of Stau2 in E13 cultured therapeutic transplantation of adult neural precursor cells (aNPCs). Methods: progenitors led to significant increases in both neurons and basal progenitors To establish a cervical SCI model, spinal cords of Wistar rats were injured and a concomitant decrease of dividing precursors. Knockdown of Stau2 in using severe (35g) or moderate (23g) clip compression for 60 seconds vivo via in utero electroporation also led to a greater number of neurons at at C6 following C7/C6 laminectomy. To evaluate the effects of stem cell the expense of dividing precursors. Current experiments aim to elucidate the transplantation, aNPCs (4 x 10e5) were administered 2 weeks following molecular mechanisms whereby Stau2 regulates maintenance of the radial moderate (23g) or severe (35g) C6 SCI. Cyclosporine and minocycline was precursor state versus differentiation into neurons. administered to all groups. Animals were followed for 8 weeks for neurobe- havioural assessment of sensorimotor function using: grip strength meter, BBB locomotor scale, BBB Subscore, inclined plane, paw contracture scale (WARF); and electrophysiological assessments: motor-evoked potentials (MEP), somatosensory-evoked potentials (SEP) and spinal cord-evoked potentials (SCEP). Results: All motor assessments demonstrated significant increases following severe C6 SCI from 1 to 6 weeks post injury, including grip strength (37g to 292g), BBB (3.3 to 9.5), Subscore (0 to 1.6), inclined plane (18.7° to 35.7°) (all p<0.0001). Motor function was substantially

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Thursday Poster Abstracts higher in sham controls than injured animals for each test and at each Poster Board Number: 2037 timepoint (p<0.0001). Paw contracture (WARF: 0, complete contracture; 6, full function) improved from 3.4 to 4.6 (p<0.001) in injured animals, where PPARS ROLES IN MOUSE ADULT NEURAL sham animals scored 6 throughout. Behavioural measures exhibited a pla- PRECURSORS CELLS teau effect in all groups, with no significant increases observed across weeks 6 to 8. All three electrophysiological assessments (MEP, SEP and SCEP) re- Bernal, Carolina A., Araya, Claudia, Bronfman, Miguel vealed a significant decline in axonal conduction as measured by both peak Cellular and Molecular Biology, Pontificia Universidad Católica de Chile, latency and amplitude in the injured groups compared to sham. Conclusions: Santiago, Chile This cervical (C6) clip compression model is a viable model to examine the efficacy of stem cell transplantation. Preliminary data of aNPC transplanta- PPARs are a group of ligand-activated transcription factors for which three tion indicates behavioural and electrophysiological improvements in severe isotypes (alpha, beta/delta and gamma) have been described. In rodent (35g) C6 SCI with greater improvements in moderate (23g) cervical injury. embryonic development, PPARgamma presents two peaks of expression; The C6 SCI model creates significant deficit in the forelimb and paw with at E13.5-E15.5 in the nervous system and at E18.5 in the adipose tissue, significant and reliable incremental increases before reaching plateau at ~6 where this transcription factor is necessary and sufficient for adipogenesis. weeks, providing a platform for relevant evaluation of potential therapeu- In adult brain, PPARgamma is mainly localized in neurogenic regions, but tic interventions. Transplantation of aNPCs into cervical SCI could provide little is known about its function. PPARbeta/delta is expressed ubiquitously neurological benefits representing hindlimb function, forepaw strength and in all rodent and human tissue tested. PPARs are key control elements in axonal conduction. cell proliferation, differentiation and apoptosis, and most genes known to be regulated by these receptors are involved in fatty acid metabolism and Poster Board Number: 2035 homeostasis. In this work we evaluated the possible roles of PPARbeta/ delta and PPARgamma in adult neural precursor cells (aNPC) in vitro and SKIN-DERIVED PRECURSORS DIFFERENTIATED also in a model of diet-induced diabetes in mice, condition known to INTO SCHWANN CELLS (SKP-SCS) decrease neurogenesis. For in vitro assays, neurospheres were prepared from the sub-ventricular zone (SVZ) of C57bl/6 adult mice. Proliferation TRANSPLANTED EIGHT WEEKS POST SPINAL was evaluated by BrdU incorporation assays. For in vivo assay, rosiglitazone CORD CONTUSION IMPROVE RECOVERY OF (RGZ) was administered in the diet, 5 mg per Kg of animal body weight. MOTOR AND BLADDER FUNCTION AFTER Mice were injected with 100 mg BrdU per Kg of animal body weight. BrdU incorporation in SVZ cells was evaluated by confocal immunofluroscence. INJURY. Ethics Commission of PUC approved animal handling procedures. RGZ Assinck, Peggy, Dworski, Shaalee, Sparling, Joseph, Duncan, Greg and F-moc, two PPARgamma agonists that have different mechanisms of J., Wu, Di L., Jiang, Yuan, Liu, Jie, Lam, Clarrie K., Kwon, Brian, action, induces aNPC proliferation. RGZ slows down differentiation process of aNPC in vitro and increased BrdU positive cells in the SVZ region in vivo. Miller, Freda, Tetzlaff, Wolfram PPARgamma agonist or its over-expression, increased EGFR levels and this ICORD, UBC, Vancouver, BC, Canada, Hospital for Sick Children/University effect was reversed by PPARgamma antagonist. On the other hand, PPAR- of Toronto, Toronto, ON, Canada, ICORD, UBC/ICORD, Vancouver BC, beta/delta agonist increased proliferation too, but do not increased EGFR BC, Canada levels. PPARbeta/delta agonists increased Sox-2 level, whereas, PPARbeta/ Cell transplantation has emerged as a promising candidate therapy for spinal delta antagonists decreased Sox-2 basal levels in aNPC and revert agonist cord injury (SCI). However, the best candidate cell for a transplantation- effect. Our results suggested a potential role of PPARs in aNPC self-renewal. based treatment of SCI remains a matter of intense debate. Our laboratories Activation of PPARs is not sufficient for maintenance of neural stem cell have previously shown that Schwann cells differentiated from skin-derived phenotype in differentiation conditions, but induces proliferation and slows precursors (SKP-SCs), when transplanted 7 days after contusion injury, down the differentiation process, two important issues in the maintenance promote histological and functional recovery in rats. SKPs are potentially of the neural stem/precursors cells pools. We propose as a working model suitable for autologous transplantation but the therapeutic potential of that PPARgamma might be a target of the classical pathways of self-renewal SKP-SCs in the chronic injury environment has not yet been investigated. of adult neural stem cells increasing EGFR level and inducing proliferation; Here, we transplanted one million cells into the lesion site of rats at 8 weeks whereas, PPARbeta/delta could be a target of EGFR in these contributing post T9/T10 contusion injury and allowed survival until week 29. Behavioral to the regulation of Sox-2 levels, an important factor in the maintenance de data indicate that SKP-SC transplantation prevented the decline of forelimb phenotype and proliferation of the adult neural precursor cells. and hindlimb stride length (Catwalk) and elicited a trend towards higher BBB Poster Board Number: 2039 scores, which reached significance in week 19, 21, and 23. Furthermore, the media control treated animals show a significant increase in bladder FGF-2 CAN RECRUIT VEGF SIGNALING wall thickness as compared to the SKP-SC treated animals. Up to 18.3% of the transplanted SKP-SC survived for 21 weeks post transplantation. THROUGH ACTIVATION OF FLK-1 IN VIVO TO Cellular bands of SKP-SCs bridged the lesion in predominantly rostro-caudal PROMOTE NEUROGENESIS orientation and were massively filled with axons ensheathed by P0-positive Bernal, Giovanna M., Peterson, Daniel A. (Schwann cell) myelin of endogenous as well as transplant origins as confirmed via immunohistochemistry. These results highlight the potential Neuroscience, Rosalind Franklin University, North Chicago, IL, USA of SKP-SC transplantation as a possible treatment strategy for chronically Endogenous neural stem cells are a constant source for new neurons in the injured SCI patients. adult dentate gyrus. Under normal conditions, these new cells contribute to cognitive functions like memory and learning. Under abnormal conditions, neural stem cells may become candidates for recruitment to regenerate damaged tissue. Neural stem cell maintenance and neuronal lineage com- mitment are regulated by both autonomous and exogenous cues, the latter provided by other cells forming this neurogenic niche. Clearly, an orchestra- tion of these signals is required to guide this multi-step process of neuro- genesis. It is therefore important to determine the identity of regulatory

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factors, their sources, and the extent of interaction between these factors, Poster Board Number: 2043 as this will aid in developing therapies recruiting endogenous stem cells for repair. GFAP-positive cells, including mature astrocytes and neural stem cells, PROPERTIES OF ADULT NEURAL STEM CELLS represent a significant population that supplies a wide array of growth fac- ARE REVEALED BY ALTERATIONS IN DISC1 tors to the neurogenic dentate gyrus. Two astrocytic growth factors, vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2), Chandran, Jayanth S., Kazanis, Ilias, Clapcote, Steven J., Porteous, are of special interest due to their mitotic and neuroprotective effects in the David, ffrench-Constant, Charles central nervous system. We asked if the co-expression of VEGF and FGF-2 Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, by GFAP-positive cells results in a synergistic mitotic effect between these United Kingdom, Department of Veterinary Sciences, University of growth factors. Synergistic stimulation by VEGF and FGF-2 is observed Cambridge, Cambridge, United Kingdom, Institute of Membrane and outside the CNS, and we addressed here whether FGF-2 requires VEGF to Systems Biology, University of Leeds, Leeds, United Kingdom, Medical mediate its mitotic effects. First, we show that in vivo delivery of FGF-2 into Genetics Section, Molecular Medicine Centre, Institute of Genetics and the adult rat brain enhances the expression of VEGF, which parallels GFAP Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom, expression, indicating astrocyte reactivity and identifying the likely source of Centre for Regenerative Medicine, Centre for Multiple Sclerosis Research, VEGF. Second, we show that FGF-2 delivery enhances the expression of the University of Edinburgh, Edinburgh, United Kingdom main VEGF receptor Flk-1, which may potentiate the effects of VEGF signal- ing. Finally, we show that the well-documented increase in cell prolifera- Neural stem cell (NSC) populations generate neurons in two discrete tion within the dentate gyrus following FGF-2 infusions can be diminished, microenvironments in the adult mammalian brain, termed niches, through though not abolished, with an antibody blocking Flk-1 activation, indicating a tightly regulated process that controls the differentiation of multipotent that VEGF mediates the mitogenic effects of FGF-2. We conclude there is an progenitors into their more fate restricted daughter cells. The signaling interaction between endogenous growth factors in vivo, with FGF-2 recruit- pathways governing differentiation of NSCs in the adult subependymal ing VEGF signaling through activation of Flk-1 to promote neurogenesis in zone (SEZ), which resides along the lateral ventricle, and the subgranular the adult hippocampus in vivo. zone (SGZ), that resides below the granule cell layer (GCL) of the dentate gyrus, remain poorly understood. RNAi mediated knockdown of Disrupted- Poster Board Number: 2041 in-schizophrenia-1 (DISC1), a risk factor for a spectrum of mental illnesses, has been reported to decrease cell proliferation in the adult SGZ and ac- THE P53 FAMILY MEMBER P63 IS REQUIRED celerate neuronal migration through the GCL indicating that DISC1 likely FOR THE MAINTENANCE OF ADULT NEURAL plays a critical role in adult neurogenesis. Here we used missense Disc1 PRECURSORS AND COGNITIVE FUNCTION mouse mutants previously described with unique behavioral phenotypes and compromised protein binding at the amino terminus to enable us to Cancino, Gonzalo I., Yiu, Adelaide P., Josselyn, Sheena A., Miller, evaluate the role of Disc1 in NSC maintenance and differentiation in both Freda D., Kaplan, David R. adult niches. Within the adult SEZ, homozygous mutants from both mis- Developmental and Stem Cell Biology, Cell Biology, The Hospital for Sick sense strains (Disc1100P/100P and Disc131L/31L) have similar deficits in Children, Toronto, ON, Canada, Institute of Medical Sciences, University generating neuroblasts (NBs) compared to controls despite similar numbers of Toronto, Toronto, ON, Canada, Neurosciences and Menthal Health of NSCs and transit amplifying progenitors (TAPs) suggesting a role for Disc1 Program/Department of Physiology, Institute of Medical Science, The in regulating division of TAPs into NBs. In the adult SGZ, Disc131L/31L Hospital for Sick Children/University of Toronto, Toronto, ON, Canada, mice, described previously with depressive-like phenotypes, have a three- Developmental and Stem Cell Biology/Molecular Genetics, The Hospital for fold increase in NSCs and a two-fold deficit in TAPs compared to control Sick Children/University of Toronto, Toronto, ON, Canada, Cell Biology/ mice. Both the Disc131L/31L and the Disc1100P/100P mice, previously Molecular Genetics, Institute of Medical Science, The Hospital for Sick described with schizophrenic-like phenotypes, have fewer NBs in the SGZ. Children/University of Toronto, Toronto, ON, Canada The Disc1100P/100P mice, however, show an increase in neurons that pre- maturely integrate out of the GCL, indicating that different signals govern We recently reported that the p53 family member deltaNp63 promotes the differentiation of progenitors and migration of postmitotic neurons in the survival of embryonic neural precursor cells, by antagonizing the apoptotic SGZ. These results identify unique roles for DISC1 in generating neuronal role of p53. Here, we asked whether p63 plays a similar role in adult neuro- precursors in the SEZ, and in controlling multiple aspects of NSC differentia- genesis. To assess this, we evaluated the number of adult precursor cells in tion in the SGZ, and imply that NSC differentiation is conserved between the the hippocampus and olfactory bulb in 3 month old wild type and p63+/- two niches after specification of TAPs. mice. p63+/- brains had fewer BrdU positive cells in the dentate gyrus of the hippocampus and olfactory bulb than wild type mice, and a significant Poster Board Number: 2045 decrease of newly-born neurons as assessed by doublecortin staining. To ask whether this was due to the depletion of precursors, we generated clonal THE EFFECTS OF PHYSICAL VS. COGNITIVE neurospheres from the lateral SVZ, where the relevant precursors reside, and ACTIVITY ON ADULT NEUROGENESIS: found fewer numbers of primary and secondary neurospheres from p63+/- UNRAVELLING THEIR MECHANISMS OF than wild type mice. To determine whether the depletion of neurospheres was due to enhanced apoptosis, we grew secondary neurospheres in the ACTION presence of a caspase-3 inhibitor, Z-VAD, and found that Z-VAD rescued the Grégoire, Catherine-Alexandra, Aumont, Anne, Fernandes, Karl J.L. depletion of p63+/- neurospheres. Finally, to evaluate whether the reduced Pathology and Cellular Biology, CENUM, GRSNC, Université de Montréal, neurogenesis in p63+/- mice has a consequence on learning and memory, Montréal, QC, Canada we performed a Morris water maze assay. We found that p63+/- mice showed higher latency escape values and more travel distance than wild In the adult brain, neural stem cells continue to produce new neurons within type mice, and performed worse in the probe test, suggesting that p63 is the dentate gyrus of the hippocampus, where these neurons are implicated important in cognition processing. These results suggest that p63 is required in the processes of learning and memory. Remarkably, this process of adult for the maintenance of the adult neural precursor pool, likely by preventing neurogenesis is highly stimulated by environmental enrichment (EE), such as ongoing cell death. by providing rodents access to a voluntary running wheel. This neurogene- sis-promoting effect of running wheels has generally been attributed to in- creased physical activity. Intriguingly, however, our recent work has led us to

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Thursday Poster Abstracts hypothesize that physical activity primarily regulates the post-mitotic survival aged proteins, such as the difficult to degrade and age-associated 2,4-hy- and maturation of newly generated neurons, while EE-induced cognitive droxynonenal (HNE), and propose that the stem cell niche is involved in this stimulation may be responsible for the activation of quiescent hippocampal segregation. HNE co-localizes with DE-cadherin in all three stem cell systems stem cells. In the present study, we are testing this hypothesis by attempting and a transporter of DE-cadherin, Myosin 10a, is also enriched in areas with to dissect i) the differential contributions and ii) the mechanisms of action of intense HNE staining in neuroblasts. Furthermore, mechanical disruption of physical activity versus cognitive stimulation during adult neurogenesis. First, the stem cell niche and mutation of the extracellular domain of DE-cadherin using adult mice, we are establishing new housing paradigms to evaluate abolishes HNE asymmetry in neuroblasts. Surprisingly, only the intestinal the effects of moderate physical activity, high physical activity, social hous- stem cell protects itself by segregating HNE to differentiating progeny while ing, isolation, environmental complexity, and combinations of these, on indi- the neuroblast and germline stem cell retain HNE during division. We pro- vidual stages of the adult neurogenesis pathway. Second, we are attempting pose that lifespan determines the cell that receives the majority of damaged to correlate the observed changes in adult neurogenesis with alterations in proteins during division and term this the lifespan asymmetry hypothesis. stress levels and functional differences in spatial learning and memory. Third, we are using genetic and biochemical approaches to identify intracellular Poster Board Number: 2051 regulatory pathways responsible for stage-specific effects of different forms THE AVIAN TRUNK NEURAL CREST COMPRISES of EE on the hippocampal stem cell niche. Together, these studies should provide novel insights regarding the mechanisms and molecules through MULTIPOTENT PROGENITORS WITH which environment enrichment can act on adult hippocampal neurogenesis. NEUROGENIC, OSTEOGENIC AND ADIPOGENIC Poster Board Number: 2047 CAPACITIES IN VIVO VISUALISATION OF MIR-124 IN Dupin, Elisabeth, Coelho-Aguiar, Juliana M., Le Douarin, Nicole M. NEURAL STEM CELLS USING LENTIVIRAL Center of Psychiatry and Neurosciences U Team Glial Plasticity, INSERM, Paris, France, UPR3294 Laboratoire NED, Institut de Neurobiologie Alfred TRANSGENESIS Fessard, CNRS, Gif-sur-Yvette, France, Académie des Sciences, Institut de Åkerblom, Malin, Sachdeva, Rohit, Jakobsson, Johan France, Paris, France Experimental science, Wallenberg neuroscience center, Lund, Sweden A wide range of cell types originate from the neural crest (NC), a transient structure formed in the dorsal neurectoderm in Vertebrates. All along the Micro RNA (miRNA) possess central and complex roles in gene regulation rostro-caudal axis, the NC cells become migratory and give rise to neurons by post transcriptional suppression of mRNA. The role of miRNA in the and glial cells of the peripheral nervous system and to melanocytes. In ad- brain has primarily been investigated in vivo by conditional Dicer knockouts, dition, the cephalic NC yields mesenchymal cell types, which form the facial an enzyme crucial for the production of all miRNA. Although the studies skeleton and most of the head dermis, fat tissue and vascular smooth muscle conclude an indispensable function of miRNA, alternative methods must cells. In the trunk of amniote vertebrates, mesenchymal tissues derive be used to study individual miRNA, an issue that due to technical reasons from the mesoderm, not from the NC. In contrast, the trunk NC of lower is complicated. To start to address this issue we have generated a novel vertebrates produces some mesenchyme, such as the caudal fin skeleton in transgenic mouse model. This model exploits a lentiviral vector designed to zebrafish. The question thus arises as to whether the ability of NC cells to visualise the expression of a specific miRNA. The vector expresses a green yield mesenchyme has been lost in the trunk during evolution or if it can still fluorescent protein (GFP) reporter gene regulated by the endogenous be elicited in amniotes under specific conditions. To address this question, miRNA. A strong promoter drives the transcription of the GFP element that we have investigated the mesenchymal differentiation capacity of trunk NC is linked to four exact complementary sites of a miRNA. When the miRNA cells (TNCCs) isolated from the quail embryo and cultured in vitro. In mass is present in the cell, it binds the transgene that is suppressed and no GFP is cultures of TNCCs, immature osteoblasts expressing the bone-specific early expressed. However, if the miRNA of interest not is present in the cell, the marker gene Runx2 were observed from culture day-7 whereas adipocytes transgene remains viable expressing GFP that easily can be detected. We with cytoplasmic lipidic droplets started to differentiate from day-10. Later have generated a transgenic mouse displaying miR-124a activity as well as on, subsets of osteoblasts underwent maturation, showing synthesis of a positive control vector with no miRNA target sites. In this work we show osteoid matrix proteins (collagen-1, bone sialoprotein and osteonectin) and that the vector efficiently reports the activity of the individual miRNA in vivo expression of alkaline phosphatase in calcified areas. We characterized the and in neural stem cell in vitro, demonstrating the value of this technique. mesenchymal progenitors using in vitro clonal cultures of TNCCs. After The expression pattern of miR-124a was previously well characterized; it is 10 days of culture on a feeder-layer of 3T3 fibroblasts, more than 60% of brain specific, expressed in all neurons, but not expressed in neuronal stem clonogenic TNCCs generated multiphenotypic clones containing glial cells, cells, astrocytes as well as in microglia. The expression is initiated as stem adrenergic neurons and osteoblasts. In addition, adipocytes differentiated cells differentiate towards neurons. In this work we confirm and extend this together with glial cells, myofibroblasts and melanoblasts in colonies derived expression pattern. from TNCCs grown in adipogenesis-promoting conditions. These results thus Poster Board Number: 2049 show that the quail TNCCs comprise multipotent progenitors endowed with both neural and osteogenic/adipogenic potentials. Therefore, although not DAMAGED PROTEIN SEGREGATION DIFFERS expressed during development in vivo, mesenchymal differentiation poten- BETWEEN ASYMMETRICALLY DIVIDING STEM tials are present in TNCCs of amniote vertebrates and they can be elicited after in vitro culture. Similar to the cephalic NC cells, the TNCCs are able CELL POPULATIONS to differentiate in vitro into mesenchymal cells, specifically adipocytes and Bufalino, Mary Rose, DeVeale, Brian, van der Kooy, Derek osteocytes. Importantly, mesenchymal phenotypes arise from multipotent progenitors both in cranial and trunk regions of the avian NC, suggesting University of Toronto, Toronto, ON, Canada that the original NC stem cell of the early vertebrates was endowed with Asymmetric division of damaged proteins during mitosis has been linked to both neural and mesenchymal fates. the protection of one cell from aging in yeast and bacteria. Recent evidence suggests that stem cells may employ a similar mechanism; however, there is no in vivo evidence suggesting this occurs in healthy adult stem cells. We report that stem cells in the Drosophila larval neuroblast, adult female germline and adult intestinal lineages each asymmetrically segregate dam-

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Poster Board Number: 2053 Poster Board Number: 2055 THE EFFECT OF POST-MI TIMING ON THE INTEGRATED PLATFORM FOR CREATION OF ENGRAFTMENT AND COLONIZATION OF ASSAY-READY NEURAL CELLS MYOCARDIAL SCAR TISSUE BY A PRIMITIVE Fontes, Andrew D., Quintanilla, Rene, Hermanson, Spencer, Bi, NEURAL CREST-LIKE STEM CELL POPULATION Kun, Rao, Mahendra, Lakshmipathy, Uma DERIVED FROM ADULT ORAL MUCOSA Primary and Stem Cell Systems, Life Technologies, Carlsbad, CA, USA Pitaru, Sandu, Gafni, Yosef, Rachima, Heled, Marinka-Kalmani, The ability to genetically modify neural cell precursors and progenitor cells Keren, Blatt, Alexander, Vered, Zvi offers an enabling tool for dissecting basic cellular and molecular biology of the nervous system, with potential extended application in drug discov- Dept. Oral Biology, Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel, ery. Creation of such cells relies on efficient and nontoxic gene delivery Department of Oral and Maxillofacial Surgery, Sheba Medical Center, Tel methods. We have developed a platform that utilizes a non-integrating viral Aviv, Israel, Department of Cardiology, Asaff Haroffe Medical Center, Tel method for rapid and efficient creation of assay-ready neural cells. BacMam Aviv, Israel are modified baculoviruses used in mammalian expression and known to Ischemic heart failure is a leading cause for mortality and morbidity world- enable efficient gene delivery into hard-to-transfect cells. We have as- wide. Stem cells (SC) hold promising therapeutic capacity in the treatment sembled diverse targets and gene elements to create expression vectors and of ischemic heart diseases and their sequel. Neural crest SC (NCSC) are non-integrating transient BacMam virus that can express single or multiple migratory and have been recently identified in the mammalian heart as a targets of interest. Using these viruses we show consistent transduction dormant SC population capable to respond to myocardial infarction (MI) of neural stem cells and neural precursor cells at very high efficiency with stress. We have recently identified a novel primitive NCSC-like population minimal toxicity. In addition, neural cell types differentiating from ESC cells in the oral mucosa lamina propria (hOMSC). hOMSC are highly expandable along different stages of neurosphere formation can be labeled to high (trillions of cells are generated from a tiny biopsy); are positive for the NCSC degree. BacMam therefore provides an integrated platform that enables the markers p75 (95%) and nestin (80%) and for the pluripotency associ- creation of assay-ready cells at different neural development stages. Using ated markers Oct4, Sox2 (70%) and Nanog (40%); differentiate into cell this method, we have expressed fluorescent proteins, pathway reporters and lineages of the 3 germ layers in vitro; and upon dexamethasone stimulation mutant LRRK2 proteins in neural cell types. The ability to rapidly generate and implantation in scid mice form teratoma-like tumors that consists of uniformly labeled neural cells with diverse gene content allows its extended ectodermal and mesenchymal lineages. Major prerequisites for SC therapy application in creation of disease models for dissecting basic biology or as a are substantial SC engraftment and retention into the myocardial scar and drug discovery tool. homogenous colonization of the scar tissue by the engrafted cells, implying Poster Board Number: 2057 migration and proliferation. These processes might be affected by the nature of the myocardial scar tissue as determined by the post-MI remodeling EFFICIENT INDUCTION AND PURIFICATION process. The aim of this study was to test the effect of the post-MI timing on the colonization of cardiac scar tissue by hOMSC. An in vivo/in vitro OF NEURAL PROGENITORS DERIVED FROM experimental model was developed. MI was induced in rats. Myocardial scar LINEAGE SPECIFIC KNOCKIN HPSC REPORTERS tissue was collected at T0,T1, T3, T7, T14 and T28 days post-MI. DiI labled hOMSCs (5*105/100µl) were injected into the center of cylinders (3mm Liu, Ying, Wang, Yu-Chieh, McCann, Anna, Lynch, Candace, Xue, diameter) prepared from the scar tissue obtained at each time point and Haipeng, Laurent, Louise, Loring, Jeanne maintained in organ culture. Specimens harvested at 0, 3, 7, 14, and 28 days Reproductive Medicine, University of California, San Diego, San Diego, CA, of culture were frozen and cryosectioned perpendicularly to the cylinder USA, The Scripps Research Institute, La Jolla, CA, USA axis. The circular sections were stained with either H&E or DAPI. The total Human pluripotent stem cells (hPSCs), including human embryonic stem cell number (labeled &unlabeled)/mm2 and the labeled cell number (LCN)/ cells (hESCs) and induced pluripotent stem cells (hiPSCs), have the potential mm2 was determined by fluorescent image analysis in 5 fields in each of 4 to be unlimited sources of material for cell replacement therapy for spinal sections from each sample - a central field and 4 peripheral fields located at cord trauma and neurodegenerative diseases. For our studies, we used equidistant distances on the circle arc. The results indicate open inflamma- transgenic reporter hPSC lines made by a homologous recombination-based tion between T3-T7 followed by scar formation. At culture day 0, the total knock-in strategy and carrying GFP driven by neuronal- and oligodendro- cell number decreased by 3.5 fold between T0-T28 post MI. The highest cytic-specific promoters. We were able to directly differentiate these cells hOMSC engraftment occurred in T0 post-MI specimens and the lowest toward motoneuron progenitors and oligodendrocyte progenitors in defined in T7 ones (0.3% of T0 value) with a recovery to 27% of the T0 value in culture conditions. These differentiated cells were purified by fluores- T28 specimens. The LCN decreased in the central fields and increased in cence activated cell sorting (FACS) at different stages of differentiation, as the peripheral ones pointing to cell migration from the injection site to the indicated by GFP reporter expression, and harvested as pure populations. periphery. The highest and lowest peripheral increases in LCN were found We conducted gene expression and proteomic profiling of these cells, and in T0 and T28 specimens, 49 and 3.6 folds, respectively. A 5 fold peripheral compared these data to those obtained from human fetal tissue-derived increase in LCN was detected in T3, T7 and T14 specimens. Except at T3, motoneuron precursors and oligodendrocyte precursors. Our results indi- the total LCN at the other post-MI time points doubled during the culture cated that the properties of hPSC-derived neural populations corresponded period suggesting hOMSC proliferation in the scar tissue. hOMSC consti- well with primary human fetal cells at different developmental stages. This tuted 50% of the cell population in T28 specimens after 28 days of culture. strategy is useful for identification of novel biomarkers that can be used for Within the limitation of this model system, the results indicate that hOMSC purification and quality control of hPSC-derived neural cell populations for proliferate and migrate to colonize the myocardial scar. The findings also use in drug development and cell replacement therapy. This work is sup- point that for engraftment and colonization the pre-inflammatory phase is ported by California Institute for Regenerative Medicine (CIRM). the best timing for hOMSC transplantation, the second best timing being the early stage of scar formation and the worst the inflammatory stage. These findings have important implications for optimizing the timing for cardiac SC therapy.

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Thursday Poster Abstracts

Poster Board Number: 2059 embryonic development. However, in stem cell therapy all temporal control of post-transplantation events is lost. We report on the development of an DIFFERENTIATION OF INDUCED PLURIPOTENT electrochemically controlled polymer for spatial and temporal control of the STEM CELLS TO NEURAL - LIKE CELLS stem cell microenvironment. We demonstrate the use of this polymer for controlling rat neural stem cell state in vitro and its integration in scaffolds WITHOUT GROWTH FACTORS suitable as support for stem cell transplantation. In vivo instructive cues such Mossahebi Mohammadi, Majid, khamisipour, Gholamreza, as growth factors are commonly presented to cells in an anchored form by Pourfatolah, Ali Akbar, Soleimani, Masoud, Kaviani, Saeed, Abroun, various biopolymers. In fact, when anchored the effect of the growth factor is often amplified compared to when free in solution. We have synthesized Saeed, Forozandeh Moghadam, Mehdi a conjugated polymer that can electronically alter the redox state and thus Hematology and blood banking, Tarbiat Modares University, Tehran, Iran, the microenvironment of stem cells cultured on this material. By controlling Islamic Republic of, Immunology Department, Tarbiat Modares University, the redox state we were able to control the stem cell differentiation, i.e. Tehran, Iran, Islamic Republic of, Biotechnology Department, Tarbiat keep the cells in their proliferating stem cell state or initiate differentiation. Modares University, Tehran, Iran, Islamic Republic of The precise temporal control of the electrochemical switching gives an exact Introduction: Generation of stem cells through differentiation of human onset of differentiation. We anticipate that this method can provide a foun- embryonic stem cells started from past two decades. But because of some dation for temporal control in stem cell transplantation. ethical and biological limitations , using them in clinical trials isn’t possible . Poster Board Number: 2063 Meanwhile, most of stem cells transplantations weren’t successful because of using of unrelated and histoincompatible sources and occurrence of GVH- CELLULAR ORGANIZATION OF THE FOREBRAIN Ds . In recent years , by production of embryonic like stem cells ( induced pluripotent stem cells) from somatic differentiated cells by using ectopic ex- NEUROGENIC NICHE IN ADULT ZEBRAFISH pression of embryonic specific genes which resembles embryonic stem cells Lindsey, Benjamin W., Darabie, Audrey, Tropepe, Vincent can differentiate into three germ layers , achieving to patient specific stem Cell and Systems Biology, Centre for the Analysis of Genome Evolution and cells and autologous transplantation was permitted. In the present study , Function, University of Toronto, Toronto, ON, Canada We produced safer induced pluripotent stem cells( iPSs ) with recombinant adenoviruses carrying embryonic genes ( Sox2 , Oct4 , Klf4 and c-myc) and Adult neural stem/progenitor cells reside in discrete proliferative niches in evaluate effect of neural derived mesenchymal stem cells from rat without vertebrates and harbour the potential to give rise to functional neurons. growth factor in culture , on differentiation of human iPS cells to neural like Vertebrate models of adult neurogenesis propose that the proliferative cells. Method and materials: In current study, we used iPS cells, which is capacity of stem/progenitor cells is intimately related to both the cellular produced by recombinant adenoviruses carrying embryonically active genes and non-cellular components comprising the niche. Here, we examine the ( Sox2 , Oct4 , Klf4 and C-myc) . After passaging these cells on mitotically adult zebrafish (Danio rerio) forebrain to describe the cellular architecture inactivated mouse embryonic fibroblast cells as feeder layer, we formed of six highly proliferative niches, and to investigate whether heterogeneity embryoid bodies (EBs) by culture of harvested cells in low attachment plates exists between these regions. Analyses of dividing cells using bromodeoxyu- with embryonic medium without human bFGF . Then, we co-culture the ridine labelling demonstrated that the highest percentage of proliferating formed EBs with PC12 cell line, and neural derived mesenchymal stem cells cells throughout the forebrain are within the dorsal telencephalic area (D), (MSCs) . We study morphological changes and also we extracted RNA from its medial (Dm) and lateral (Dl) zones, the dorsal (Vd) and ventral (Vv) differentiated iPS cells and after cDNA synthesis we analyse the expression nuclei of the ventral telencephalon, and the anterior parvocellular preoptic of NSE , β-tubuline and MAP2 by specific primers for qRT-PCR. Results: We nucleus (PPa), and that the density of cycling cells varied by region along showed that the mesenchymal stem cells could cause differentiation of iPS the rostrocaudal axis. Each niche was distinguished by a dense collection cells through Neural-like cells . In morphological observation , typical view of of cell bodies located within the periventricular zone adjacent the forebrain neural cells was seen . Also, by quantitative expression analysis and imuno- ventricles. Immunohistochemistry showed that many niches were highly histochemical staining of those three genes, we confirm the differentiation vascularized and composed of several types of glia. At least six different cell of iPS cells to Neural-like cells. Conclusion: As previously described , MSCs types were identified by examining the ultrastructural organization using produced NGF which can convert PC12 cell line to neural cells .we found transmission electron microscopy (TEM). Classic ependymal cells (type 1) that co-culture of neural MSCs result in differentiation of iPS cells to neural- were restricted to a single row spanning the dorsal roof of the telencephalic like cells , as well culture with filter insert showed that the condition media ventricle. In all regions a single layer of discontinuous, ependymal-like cells can also cause differentiation to neural-like cells. These findings demonstrate (type 2) lined the proliferative niche. Type 2 cells were considerably larger the direct and indirect (by secreted factors) differentiating effects of neural than type 1, and contained nuclei with dispersed chromatin, few cilia, and derived mesenchymal stem cell , on embryonic like stem cells(iPS). were most abundant across niches. Type 3 cells also shared ependymal-like features, but unlike type 2 cells, were infrequent and contained numerous Poster Board Number: 2061 cilia. Glial-like cells (type 4) had dark nuclei, irregular contours, and sparse cytoplasm, and were most abundant within the Ppa and Vv. Some type PRECISE TEMPORAL CONTROL OF THE 4 cells displayed elongated nuclei characteristic of stem/progenitor cells. MICROENVIRONMENT TO STEER NEURAL Preliminary immuno-TEM data further showed that cell types 2-4 label with STEM CELL DIFFERENTIATION glial markers (S100, glutamine synthetase), and that a subset of type 4 cells are immuno-positive for the stem cell marker Sox2. Putative microglia (type Meneghello, Giulia, Herland, Anna, Persson, Kristin, Lundin, 5) were rarely observed, while neurons (type 6) often bordered the niche Vanessa, Berggren, Magnus, Jager, Edwin W.H., Teixeira, Ana laterally. Finally, lineage tracing showed that stem/progenitor populations Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden, within niches gave rise to adult-born neurons as early as two weeks. Our Department of Science and Technology, Linköping University, Linköping, data provides the first comparative study of the cellular architecture of Sweden proliferative niches in the adult zebrafish forebrain where neural stem cells reside. We reveal five main cell types making up each niche (types 2-6), that Stem cell maintenance and differentiation during embryonic development there exists heterogeneity in the organization and frequency of cells types is regulated by instructive cues from the microenvironment with precise between niches, and that a subpopulation of glial-like cells appears to retain temporal control. Strategies for stem cell differentiation in vitro typically stem cell properties. Moreover, our findings suggest that the architecture of involve exposing cells to temporal sequences of growth factors, mirroring

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the forebrain ventricular zone across vertebrates is not entirely conserved, as genes from the core LSC transcriptional program we previously established shown by the absence of classic ependymal cells throughout this region in as novel candidate regulators of stem cell function. The LSC transcriptional the adult zebrafish brain. program was identified by fractionation of 16 primary human acute myeloid leukemia (AML) samples into four populations and these were subjected to sensitive in vivo LSC analysis. mRNA expression analysis was performed on each fraction and a global LSC-specific signature was determined from CANCER CELLS functionally defined LSCs. Similarly, using human cord blood, a hematopoi- Poster Board Number: 2067 etic stem cell (HSC) enriched gene signature was established. Bioinformatic analysis identified a core transcriptional program that LSCs and HSCs share, CHARACTERIZATION OF HUMAN ACUTE revealing the molecular machinery that underlies stemness properties. The MYELOID LEUKEMIA STEM CELLS LSC and HSC signatures have prognostic significance independent of other factors when assessed against a large group of cytogenetically normal AML Chen, Weihsu Claire, Eppert, Kolja, Mitchell, Amanda, Popescu, samples. We have initiated experiments to determine the role of the 17 Andreea, McLeod, Jessica, Minden, Mark, Dick, John, Wang, Jean selected candidate stem cell regulators using functional in vivo and in vitro assays. Preliminary results show that over-expression of two of 17 genes Division of Stem Cell and Developmental Biology, Ontario Cancer Institute, increase engraftment capability of HSCs in immune-deficient recipients com- Princess Margaret Hospital, University Health Network, Toronto, ON, pared to control, suggesting a role in stem cell regulation for these genes. Canada, Dept. of Medicine, University of Toronto, Toronto, ON, Canada In conclusion, our data established that determinants of stemness influence Acute myeloid leukemia (AML) is a heterogeneous disease sustained by a clinical outcome of AML patients. Moreover, we have identified multiple subset of cells, termed leukemia stem cells (LSCs) that are distinct from the novel candidate stem cell-related genes and generated support for a role in bulk of the tumor. Increasing evidence suggests that LSCs have biological stem cell regulation for two genes. properties that may permit them to survive anti-leukemia therapies, lead- ing to eventual relapse. Better understanding of the biology of LSCs could Poster Board Number: 2071 therefore lead to improved prognostic tools and therapy that could ulti- mately result in improved outcomes. The goal of this study is to characterize miR-126 BIOACTIVITY MARKS THE PRIMITIVE the molecular regulators that govern the self-renewal and developmental COMPARTMENT IN AML AND REGULATES program of LSCs, and correlate LSC signatures with clinical parameters in HUMAN LEUKEMIA STEM CELL NUMBERS order to determine the prognostic and therapeutic value of LSCs. Global gene expression profiles of LSCs will be generated by microarray analysis of Lechman, Eric R., Van Galen, Peter, Gentner, Bernhard, Eppert, flow cytometry-sorted subpopulations of AML patient samples. Additionally, Kolja, Hiramatsu, Hidefumi, Hope, Kristin, Takenaka, Katsuto, the tumor-initiating ability of the sorted fractions will be confirmed through Minden, Mark, Naldini, Luigi, Dick, John E. xenotransplantation in immunodeficient mice. We have screened a total of Cell and Molecular Biology, University Health Network, Toronto, ON, 196 primary AML samples in NOD.SCID mice, and identified 76 engraft- Canada, San Raffaele Telethon Institute for Gene Therapy, Milan, Italy, ing samples. To identify an LSC signature, we have sorted 45 engrafting Stem Cell and Regenerative Medicine, McMaster University, Hamilton, samples into 4 fractions based on CD34 and CD38 expression and injected ON, Canada, Department of Medicine and Biosystemic Sciences, Kyushu each fraction into NOD.SCID IL2Rγnull (NSG) mice at limiting dilution to University, Fukuoka, Japan, Medical Biophysics, University of Toronto, determine LSC frequency. Our preliminary results show that LSC are found Toronto, ON, Canada at higher frequency in the CD34+ cell fractions (engraft NSG mice at lower cell doses) compared to the CD34- fractions. For these functionally validated In order to elucidate the role of miRNA in leukemia stem cells, we performed patient samples, RNA was extracted from each sorted fraction as well as miRNA profiling on fractionated subpopulations of primary AML patient from bulk AML cells for microarray analysis. We have completed testing of samples. Supervised analysis guided by the in vivo SCID leukemia initiat- the first batch of RNA samples (collected from 16 patients) by Illumina gene ing capacity (SL-IC) of each sub-population generated a unique miRNA expression analysis. The data reveals that our LSC gene expression profile signature associated with LSC enriched fractions. An in vitro antagomir- correlates positively to the pre-existing LSC and HSC gene signatures. This based functional miRNA knockdown screen identified miR-126, our top study will utilize a large cohort of AML patient samples to identify a func- array candidate, for further study. After RT-PCR validation, the biological tionally validated LSC-specific gene signature that is expected to be clinically activity of miR-126 was confirmed at single cell resolution by using a novel relevant. bidirectional lentivirus miRNA reporter system in the 8227 cell line in vitro and within primary AML patient samples xenografted into immune-deficient Poster Board Number: 2069 NSG mice. These data suggest that primitive AML cells may express high levels of bioactive miR-126 relative to more “differentiated” blast popula- IDENTIFICATION AND CHARACTERIZATION OF tions. To test the hypothesis that AML stem cells are marked by high miR- HUMAN LEUKEMIA STEM CELL FUNCTIONAL 126 bioactivity, we FACS sorted miR-126 genetic reporter vector transduced REGULATORS primary AML patient samples and transplanted these populations into immune-compromised secondary mouse recipients. The results of these Hermans, Karin G., Eppert, Kolja, Dick, John E. proof-of-concept experiments demonstrates our ability to prospectively Cellular and Molecular Biology, University Health Network, Toronto, ON, isolate LSC enriched fractions in all 4 AML patient samples tested using only Canada a single biomarker, miR-126. Finally, to understand the functional relevance of miR-126 expression within primitive AML cells, stable enforced expression Emerging evidence shows that many cancers are organized as cellular and knockdown of miR-126 was achieved using lentiviral vectors. Enforced hierarchies and are sustained by a rare subpopulation of cancer stem cells expression of miR-126 in CD34+CD38- 8227 cells resulted in reduced (CSC). It is thought that CSC properties influence therapy response, overall AML blast colony formation, an increase/maintenance of CD34+ cells and survival, and relapse of the disease. However, little is currently known about a decrease in differentiation marker positive (CD14, CD15) AML blasts. the molecular pathways that control stem cell behaviour. Our goal is to Similarly, enforced miR-126 expression in 4 primary AML xenografts resulted identify and characterize the molecular regulatory networks that govern in a several fold increase of CD34+CD117+ lentivirus marked leukemia cells the self-renewal and developmental program of human leukemia stem after 12 weeks. In addition, the miR-126 cells showed reduced differentia- cells (LSC). To expand our understanding of LSC function, we selected 17 tion marker expression (CD14, CD15) with no significant differences in AML

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Thursday Poster Abstracts graft size. To determine if the expanded population had SL-IC activity or was Poster Board Number: 2075 a downstream leukemic progenitor, limiting dilution assays were performed by transplantation of FACS sorted lentivirus marked cells into secondary TRANSCRIPTION PROGRAM OF LEUKEMIA recipient mice for 12 weeks. A 3-20 fold increase in LSC activity was ob- STEM CELL DEVELOPMENT IN MLL-ENL- served in 3 AML patient samples with miR-126 forced expression compared to control vector expressing cells. These data suggest that high levels of INDUCED LEUKEMIA IN MICE miR-126 bioactivity may support self-renewal/maintenance of primitive Takacova, Sylvia, Luzna, Pavla, Stranecky, Viktor, Slany, Robert, AML cells at the cost of aberrant differentiation. Conversely, in vitro knock- Bartek, Jiri, Divoky, Vladimir down of miR-126 in CD34+CD38- 8227 cells increased AML blast colony formation and reallocated colony forming burden from the CD34+CD38+ Department of Biology, Faculty of Medicine, Palacky University, Olomouc, progenitor subpopulation to the primitive CD34+CD38- population. In vivo Czech Republic, Institute of Inherited Metabolic Disorders, First Faculty targeting of miR-126 resulted in a reduction of CD117+ primitive cells along of Medicine, Charles University in Prague, Prague, Czech Republic, with concomitant increase in differentiation marker expression in 2 out of Department of Genetics, University Erlangen, Erlangen, Germany, Institute 3 xenotransplanted AML patient samples, while secondary LDA experi- of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark ments showed a modest 2 fold reduction in LSC activity. In summary, these During the multistep pathogenesis of leukemia, a pool of leukemia stem cells experiments demonstrate that miR-126 is more abundantly expressed and (LSCs) emerges that is capable of limitless self-renewal and ensuring disease biologically active within the leukemia stem/progenitor cell compartment of maintenance. There is an interest to study the molecular mechanism that the AML functional hierarchy and serves to regulate AML stem cell numbers. controls the development of LSCs and kinetics of cellular transformation. Poster Board Number: 2073 Using an MLL-ENL-ERtm mouse model, we identify that the activated onco- gene initiates aberrant proliferation in committed myeloid progenitors. We IDENTIFICATION OF HUMAN LEUKEMIA identify a fail-safe mechanism mediated by cooperation of the DNA damage response (DDR) machinery and inflammatory tissue microenvironment lead- STEM CELL GENES BY IN VIVO RETROVIRAL ing to activation of the ATR/ATM-Chk1/Chk2-p53/p21 checkpoint and cel- INSERTIONAL MUTAGENESIS lular senescence at early stages of cellular transformation. We demonstrate that experimental DDR inhibition accelerates the transition to immature cell Wienholds, Erno, van Galen, Peter, Dobson, Stephanie M., Barabé, states, acquisition of LSC properties and acute leukemia development. We Frédéric, Kennedy, James, Dick, John E. have profiled gene expression across disease stages in vivo and identified a Division of Stem Cell and Developmental Biology, Campbell Family hierarchical activation of a transcription signature that underlines transi- Institute for Cancer Research / Ontario Cancer Institute, Princess Margaret tion of pre-LSCs to a LSC state. Our study provides insight into the role of Hospital, University Health Network, Toronto, ON, Canada cellular barriers in pre-leukemia cell fate decisions between senescence and self-renewal in MLL-ENL-induced leukemia in mice. Insertional mutagenesis screens in murine models have been proven to be extremely valuable for the discovery of new genes involved in vari- Poster Board Number: 2077 ous types of cancers. However, it has never been used in primary human hematopoietic cells. We have recently developed a xenograft assay to A2B5-DEFINED TUMOR PROGENITORS model the initiation and progression of genetically induced human B cell DERIVED FROM HUMAN GLIOMAS EXHIBIT acute lymphoblastic leukemia (B-ALL) in vivo. In this assay primary human umbilical cord blood stem/progenitor cells are infected with retroviruses A CORE SET OF DYSREGULATED GENES AT that express mixed lineage leukemia (MLL) fusion genes and transplanted ALL STAGES OF TUMOR PROGRESSION, into immuno-deficient mice. To test if this model can be used for a in vivo IN ADDITION TO GRADE-ASSOCIATED insertional mutagenesis screen for co-operating cancer genes we determined the integration sites for 74 unique retroviral insertions that were present in TRANSCRIPTIONAL CHANGES CONSISTENT 58 MLL-ENL and MLL-AF9 leukemias. Approximately half of the insertions WITH EPITHELIAL-TO-MESENCHYMAL were found in or close to genes that are known to be affected in leukemia or other types cancer and/or have a function in stem and progenitor cells, TRANSITION suggesting a co-operating function. In addition we identified several com- Auvergne, Romane M., Sim, Fraser, Wang, Su, Chandler-Militello, mon insertion sites, which suggests that there is a limited set of co-operating Devin, Burch, Jaclyn, Li, Xiaojie, Pilcher, Webster, Walter, Kevin, cancer genes in MLL leukemia. Currently we are verifying a subset of the Johnson, Mahlon, Achanta, Pragathi, Quinones-Hinojosa, Alfredo, target genes by expression analysis and combinatorial infection experiments. Natesan, Sridaran3, Goldman, Steven A.1 In addition we are setting up a large-scale screen in which we aim to gener- ate a total of 500-1,000 human MLL leukemias. Neurology, University of Rochester Medical Center. Center of Translational Neuromedicine, Rochester, NY, USA, Johns Hopkins University, Baltimore, MD, USA, Sanofi-Aventis, Cambridge Genomics Center, Cambridge, MA, USA Glial progenitor cells (GPCs) of the adult human white matter, which express gangliosides recognized by monoclonal antibody A2B5, are a potential source of glial tumors of the brain. We used A2B5-based sorting to extract progenitor-like cells from a range of human glial tumors spanning WHO stages II-IV, that included low-grade glioma, oligodendroglioma, oligo-astro- cytomas, anaplastic astrocytoma, and glioblastoma multiforme. The A2B5+ tumor cells proved tumorigenic upon orthotopic xenograft, and the tumors generated reflected the phenotypes of those from which they derived. Ex- pression profiling revealed that A2B5+ tumor progenitors expressed a cohort of genes by which they could be distinguished from A2B5+ GPCs isolated from normal adult white matter. Most of the genes differentially expressed by glioma-derived A2B5+ cells varied as a function of tumor stage, such that

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with tumor progression, A2B5+ tumor progenitors exhibited the differential ior, we performed gene expression analyses of tumor-derived stem cells and up-regulation of genes associated with both the TGF-β pathway, and with primary brain tissue. Our results show that reactivation of embryonic signal- the epithelial-to-mesenchymal transition (EMT), as higher grade A2B5+ iso- ing is a prominent component of high grade glioma formation. In particular, lates differentially exhibited a mesenchymal phenotype. Nonetheless, a small we show that the hox family of transcription factors are overexpressed number of genes were invariably differentially expressed at all stages of during tumor development and are important for glioma stem cell growth gliomagenesis; these transcripts appeared with initial gliomagenesis in WHO and survival. These studies further validate the critical role of developmental stage II-derived A2B5+ cells, and persisted as differentially dysregulated in signaling in the pathogenesis of gliomas and will likely pave the way for grade III and IV tumors as well. This conserved set of glioma progenitor- novel, more effective therapeutic approaches for these devastating cancers. associated genes included CD24, SIX1 and EYA1, which were up-regulated at all stages of gliomagenesis, and MTUS1 and SPOCK3, which were Poster Board Number: 2083 down-regulated at all stages of tumor progression. qPCR and immunolabel- OVEREXPRESSION OF CD133 PROTMOTES ing confirmed the differential expression of these genes in primary gliomas, while pathway analysis permitted their segregation into differentially active DRUGS RESISTANCE IN U87 GLIOMA CELLS signaling pathways. By comparing the expression patterns of glial tumor Chiang, Yung-Hsiao, Chen, Kai-Yun, Lin, Jia-Wei, Chiu, Wen-Ta, progenitors across anaplastic progression to their identically-isolated normal Wu, Chung-Che homologues, we have thus identified a discrete set of genes associated with both initial glial tumorigenesis and later progression, whose selective target- Department of Neurosurgery, Taipei Medical University Hospital, Taipei, ing might influence glioma growth at all stages of tumor progression. Taiwan, Department of Neurosurgery, Taipei Medical University-Shuang Ho Hospital, Taipei, Taiwan Poster Board Number: 2079 Malignant gliomas are common primary brain tumors which have poor ADAPTATION OF HUMAN GLIOMA STEM-LIKE prognosis. Even though recent advances in chemotherapy prove to be ef- fective in controlling the disease progression, the duration of disease control CELLS TO SEVERE HYPOXIA is still limited. Limitation of chemotherapy effect is frequently observed in Golebiewska, Anna, Sanzey, Morgane, Vallar, Laurent, Bjerkvig, cancer stem cells, which in many aspects are similar to normal stem cells. Rolf, Niclou, Simone P. Despite the rarity of cancer stem cells, they have very different characteris- tics such as their resistance to chemotherapy and radiotherapy. CD133 was Norlux Neuro-Oncology laboratory, Centre de Recherche Public Santé, identified as an important cancer stem cell marker, with increased expres- Luxembourg, Luxembourg, Microarray Center, Centre de Recherche Public sion in various human malignancies, including gliomas. Here, we show that Santé, Luxembourg, Luxembourg ectopic overexpression of CD133 in U87 glioma cells leads to significant Progression of glioblastoma multiforme (GBM), a malignant brain tumor, is reluctant to undergo apoptosis from carboplatin and temozolamide (TMZ). believed to be triggered by a cancer stem-like cell population, which is char- ATG5, LC3 and Beclin levels were significantly lower in CD133+ cells after acterized by increased self-renewal property and tumor initiation capacities. drugs treatment. Furthermore, tissue proliferation related factors such as GBM are characterized by intratumoral hypoxic regions, which may overlap Akt, Erk1/2, c-Raf were also evaluated. The up-regulation of p-Erk1/2 and with putative cancer stem-like cell niches. It has been postulated that glioma Ser338 c-Raf suggests that G-protein coupled receptor pathway is involved stem-like cells (GSCs) are able to survive and adapt to a hypoxic microenvi- in the drug resistance of CD133+ cells. In conclusion, the CD133+ cancer ronment, contributing to their resistance to radio- and chemotherapy. How- stem cells display strong capability of tumor’s resistance to carboplatin and ever, the mechanisms permitting cancer stem-like cell survival under severe TMZ. This resistance is involved in down-regulation of autophagy-related hypoxic conditions are poorly understood. Using gene expression and flow proteins and up-regulation of tissue proliferation related factors. Future cytometric profiling we provide a detailed functional and molecular analysis treatment should target CD133+ cancer stem cells in gliomas to improve of GSCs grown under severe hypoxia. We show that GSCs can survive even survival of patients. at extremely low oxygen levels (0,1% O2), where they undergo both cell selection and molecular adaptation to anaerobic conditions. Cells survive by Poster Board Number: 2085 adjusting the proliferation rate and adapting their metabolism at the tran- HOXA10 IS A POSTERIORIZING SIGNAL scriptomic and proteomic level. Comparison of global transcriptomic profiles between GSCs and non stem-like glioma cells and normal human neural THAT CONTRIBUTES TO THE TUMORIGENIC stem cells grown under varying oxygen levels, reveals cellular pathways re- PROPERTIES OF GLIOMA-INITIATING CELLS sponsible for cell survival and point to potential novel therapeutic strategies. Gallo, Marco, Ho, Jenny, Clarke, Ian D., Ward, Ryan J., Lee, Lilian, Poster Board Number: 2081 Meyer, Mona, Head, Renee, Dirks, Peter B. HOX GENES ARE ESSENTIAL FOR GLIOMA Developmental & Stem Cell Biology, Arthur and Sonia Labatt Brain Tumor Research Centre, The Hospital for Sick Children, Toronto, ON, Canada, STEM CELL SURVIVAL AND TUMORIGENICITY Developmental & Stem Cell Biology, Arthur and Sonia Labatt Brain Tumor Alcantara Llaguno, Sheila R., Yang, Jingsheng, Xie, Yang, Burns, Research Centre, The Hospital for Sick Children, University of Toronto, Dennis, Bachoo, Robert, Parada, Luis F. Toronto, ON, Canada Developmental Biology, University of Texas Southwestern Medical Center, Homeobox (HOX) domain genes are known to play an important role in Dallas, TX, USA, Clinical Sciences, University of Texas Southwestern the etiology of some cancers, most prominently leukemias. We investigated Medical Center, Dallas, TX, USA, Pathology, University of Texas the possibility that HOX genes have a role also in human gliomas. In a Southwestern Medical Center, Dallas, TX, USA, Neurology, University of genome-wide expression study, we found that some HOX genes, particu- Texas Southwestern Medical Center, Dallas, TX, USA larly 5’ HOX, were consistently up-regulated in cultured patient-derived glioma-initiating cells compared to human fetal neural stem cells. One of the Malignant gliomas are highly infiltrative and aggressive brain tumors that are most up-regulated genes was HOXA10, which is normally expressed during resistant to conventional therapies. Our laboratory has previously developed development of vertebrate and invertebrate animals to specify the fate of mouse models based on conditional inactivation of human glioma-relevant posterior regions. We validated the micro-array data by studying HOXA10 genes Nf1, p53, and Pten, wherein mutant mice develop tumors with 100% expression by qRT-PCR and HOXA10 protein levels in a panel of patient- penetrance. To determine the mechanisms involved in its malignant behav- derived glioma-initiating cells. In all cases, we observed a clear increase in

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HOXA10 transcription and protein levels in the malignant stem cells. Similar in each group. The multiplex PCR reaction with single cells is performed to its role in leukemia-initiating cell proliferation, we found that HOXA10 with the AmpliSpeed slide cycler (Advalytix) and analyzed by automated down-regulation by shRNA treatment decreased cell proliferation in several fluorescent capillary electrophoresis to determine the presence or absence of glioma-initiating cell lines. In order to gain a better understanding of the a gene. Differential correlation analysis is performed for all comparisons. The biological function of HOXA10 in brain tumors, we knocked-down HOXA10 benign and cancer stem cell profile is developed by comparing expression expression in human glioma-initiating cells and injected them orthotopically of each gene in the multiplex from single side population and ALDHbr cells in the forebrains of NOD-SCID mice. Our survival experiments showed that from benign and cancer specimens and cell lines. For each gene, normalized down-regulation of HOXA10 reduced the ability of glioma-initiating cells to log signal will be modeled as a function of benign/cancer effects, account- form tumors and significantly improved mouse survival. We also assessed ing for patient level variation by mixed modeling. Thus, these studies seek HOXA10 expression in glioma samples freshly resected from patients. We to identify a benign and cancer stem cell gene expression profile in order to found that the freshly sorted CD15(+) population, which enriches for the distinguish cancer stem cells from benign stem cells to enhance prognostic stem cell population, strongly over-expressed HOXA10 compared to the value of quantitating cancer stem cells in prostate specimens containing CD15(-) population and human fetal neural stem cell controls. We were areas of pathologic prostate cancer. also able to detect HOXA10(+) cells in sectioned paraffin-embedded human glioma tumor samples. By immunohistochemistry, we showed that HOXA10 Poster Board Number: 2089 co-localized with SOX2-expressing cells, suggesting that HOXA10(+) cells CONSTITUTIVE NF-KB SIGNALING MARKS A represent a fraction of the cancer stem cell population with tumorigenic potential in vivo. The cyclin-dependent kinase inhibitor p21, an established SUBSET OF STEM-LIKE HUMAN PROSTATE direct transcriptional target of HOXA10, co-localized with HOXA10 in TUMOR-INITIATING CELLS human glioblastoma samples. Furthermore, we showed with knock-down experiments that MLL is at least partially responsible for the up-regulation Rajasekhar, Vinagolu K., Studer, Lorenz, Gerald, William, Socci, of HOXA10 in glioma-initiating cells. We propose a mechanism of tumor Nicholas D., Scher, Howard I. initiation and maintenance caused by aberrant, MLL-dependent epigenetic Stem Cell Center, Dev. Biology and Dept. Medicine, Memorial Sloan changes that up-regulate HOXA10 expression in glioma-initiating cells. Kettering Cancer Center, New York, NY, USA, Stem Cell Center, HOXA10 is therefore an important molecular player in the etiology of brain Developmental Biology, Memorial Sloan Kettering Cancer Ctr, New York, tumors and may represent a target for future therapies. NY, USA, Medicine, Memorial Sloan Kettering Cancer Center, New York, Poster Board Number: 2087 NY, USA, Computational Biology, Memorial Sloan Kettering Cancer Center, New York, NY, USA, Medicine (Genitourinary Oncology Service), Memorial DEVELOPMENT OF HUMAN PROSTATE BENIGN Sloan Kettering Cancer Center, New York, NY, USA AND CANCER STEM CELL SPECIFIC GENE Anti-androgen therapy is a key therapeutic strategy for human prostate cancer that has become the second leading cause of cancer deaths in men of EXPRESSION PROFILES the Western World. However, a small number of prostate cancer cells often Gangavarapu, Kalyan J., Bshara, Wiam, Kazim, A L., Foster, Barbara escapes this treatment and eventually regenerates the parent-like tumor as A., Huss, Wendy J. has been the case with many other cancers. Here, we present studies on the cell of origin in human prostate cancers and possible therapeutic resistance Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY, mechanisms associated with these cells. We demonstrate that human pros- USA, Pathology, Roswell Park Cancer Institute, Buffalo, NY, USA, Cell Stress tate tumors harbor a minor, but distinct subpopulation of cells which do not Biology, Roswell Park Cancer Institute, Buffalo, NY, USA express differentiated cell markers of the prostate gland such as AR, PSA, Benign and cancer stem cells potentially share many properties, thus unique and NKX 3.1. These cells possess sphere-forming capacity and multipotency identifiers remain elusive. Quantifying prostate cancer stem cells in prostate as demonstrated by surrogate self-renewal assays in vitro and the initiation cancer may provide prognostic insight, but only if cancer stem cells can be of serially transplantable parent-like prostate tumors in animals, respectively. distinguished from benign stem cells. In the present study, two stem cell These tumor-initiating sphere cells exhibit a pattern of cytokeratin expression enrichment assays were tested to determine the level of enrichment for cells intermediate between those of basal and luminal cells of the prostate gland. with stem cell properties in each population; side population assay based on However, detailed biochemical characterizations revealed that these cells are ABCG2 transporter activity and ALDEFLUOR® assay based upon aldehyde of a largely undifferentiated basal cell subtype with an AR-/PSA-/NKX3.1-/ dehydrogenase enzyme activity. The aim of the study is to test stem cell SOX9+/p63-/CK5+/CK8+/CK18- phenotype. These cells can also be more properties using tissue recombination technique and determine unique stem specifically enriched by prospective purification from human prostate tumors cell gene signatures within single cells. Several cell lines were analyzed for based on their novel expression of human pluripotent stem cell surface side and ALDEFLUOR® (ALDHbr) population. While CWR-R1 prostate markers such as TRA-1-60 along with two other known epithelial cancer cancer cells have an average side population of 3.3% and 3% ALDHbr stem cell markers, namely CD151 and CD166. When xeno-transplanted, cells; DU145 prostate cancer cells have an average side population of 1.6% these marker-positive cells recapitulate the parent tumor heterogeneity and 1.5% ALDHbr cells; and RWPE-1 non-tumorigenic prostate cells have comprised of both the marker-positive and marker-negative cells indicating an average side population of 0.3% and 3% ALDHbr cells. The average a tumor cell hierarchy in human prostate cancer development. These stem- side population is 0.4% (non-tumor) and 0.2% (tumor) in freshly digested like tumor-initiating cells are enriched with a functional MET/PKC/NFKBIA/ human prostate specimens and the specimens have an average of 2% NF-kB-signaling pathway as revealed by genome-wide messenger RNA and (non-tumor) and 2% (tumor) of ALDHbr cells. Tissue recombination with micro RNA expression profiling studies, and additional biochemical analyses. urogenital mesenchyme showed that side population cells from human pros- By exploiting the above sequential sphere-formation ability of these stem- tate clinical specimen are more enriched with cells demonstrating stem cell like cells, we designed a prototype screen for small molecule pathway- properties, human prostatic ductal growth up to 3 generations, compared specific inhibitors and identified the anti-apoptotic pathway as a targetable to the ALDHbr cells that showed minimal growth after first generation. To functional mechanism in this study. Additional data on these stem-like identify the gene expression profile of the cells isolated based upon the side tumor-initiating cells and the associated NF-kB pathway will be discussed population and ALDEFLUOR® assays, 14 different genes were selected that comparing clinical patient data obtained from biochemical recurrence of the are associated with the stem cell biology and prostate differentiation. Based human prostate cancers and their metastases. All of these approaches may upon the initial results from multiplex PCR, the genes selected for identifying be translatable to other organ tumor types including the identification of the stem cell profile are grouped into 4 different groups with 1 control gene additional biomarkers as well as for future high throughput drug discovery in

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overcoming therapeutic resistance. cells, different assays are performed: label retaining with PKH membrane staining, EdU DNA labeling, and alpha tubulin staining for microtubules in Poster Board Number: 2091 sphere formation, colony formation, and tissue recombination assays. Sphere AMENDMENT OF THE HUMAN PROSTATE and colony formation assays are used to enrich human and murine adult prostate stem cells in vitro and stem cell properties are fully tested in vivo STEM CELL MODEL: ACTIVE ANDROGEN by prostate tissue generation in tissue recombination experiments. Once the RECEPTOR IS EXPRESSED AT ALL STAGES OF pattern of prostate stem cell divisions in spheres and colony formation assays are determined, cells expressing putative stem cell markers will be evaluated PROSTATE DIFFERENTIATION for stem cell properties using stem cell division patterns as an initial criterion. Williamson, Stuart C., Armstrong, Kelly, Rigas, Anastasia, Robson, Poster Board Number: 2095 Craig, Heer, Rakesh Solid Tumour Target Discovery, Northern Institute for Cancer Research, ANDROGEN ABLATION MITIGATES DEFECT Newcastle Upon Tyne, United Kingdom OF B CELLS TO A PROSTRATE CANCER AND Introduction: Tumour initiating cancer stem cells in human prostate cancer INCREASE SURVIVAL RATE OF TRAMP MICE. have been described according to the expression of CD133. However, in these descriptions of stem and cancer stem cells, the lack of androgen Altuwaijri, Saleh A. receptor expression has been controversial, as androgen receptor activity is Clinical Research, Saad Specialist Hospital, Al Khobar, Saudi Arabia an established central mechanism in prostate cancer initiation and cancer Androgen ablation is the most commonly used therapy for prostate cancer. progression. In this study, we revisited and investigated expression of the However, the effects of androgen ablation on immune system, especially B androgen receptor in CD133+VE and CD113-VE primary human prostate cell distribution are not well understood. We have used transgenic adenocar- epithelia. Methods: We developed a highly sensitive detection method cinoma mouse prostate (TRAMP) mice to characterize the B cell distribu- for the androgen receptor using flow cytometry to screen EpCAM+VE tion in periphery blood and spleen upon androgen ablation. We found a CD133+VE and CD133-VE α2β1HI progenitor cells from primary benign decrease of the B cell population in wild type TRAMP mice compared to prostate samples. Androgen receptor activity was determined by the expres- normal wild type mice. Furthermore, the B cells increased when in castrated sion of measuring androgen regulated gene transcripts with real time PCR. TRAMP mice compared to the wild type TRAMP mice. Interestingly, we Results: Androgen receptor transcript was confirmed in EpCAM+VEα 2β1HI found an increase in immature B cells in the spleen of castrated TRAMP CD133+VE and CD133-VE cells. With flow cytometry we confirmed that mice, which might be due to the resistance to apoptosis during B cell matu- 54.95% EpCAM+VE α2β1HI CD133-VE cells and 80.58% of EpCAM+VE ration. Our data suggest that androgen ablation might play an important α2β1HI CD133+VE cells express the androgen receptor. Activity of the an- role in the regulation of B cell tolerance. drogen receptor was confirmed in prostate progenitor cells by the expression of androgen receptor regulated genes with up-regulation of these transcripts Poster Board Number: 2097 with increasing differentiation (PSA p value =0.03, KLK2 p-value=0.005, TMPRSS2 p value =0.009). Conclusion: The expression of the androgen PARATHYROID HORMONE-RELATED PROTEIN receptor in prostate progenitor cells allows integration of the prostate cancer (PTHRP) PROMOTES THE ACQUISITION OF stem cell theory, with the established importance of the androgen receptor in prostate cancer. Further study of specific androgen receptor function in CANCER STEM CELL-LIKE PROPERTIES IN prostate stem and cancer stem cells may highlight novel therapeutic options. PROSTATE CANCER Poster Board Number: 2093 Rahimy, Elham, Kiang, Alan, Abhold, Eric, Yi, David, Burton, Doug, Deftos, Leonard J., Kuo, Selena, Ongkeko, Weg M. CELL DIVISION IN MOUSE AND HUMAN Department of Surgery, University of California, San Diego, San Diego, CA, PROSTATE STEM CELLS USA, VA Medical Center, San Diego, San Diego, CA, USA Samant, Mugdha D., Huss, Wendy J. The mechanism by which tumor cells become invasive and eventually meta- Pharmacology and Cancer Therapeutics, Roswell Park Cancer Institute, static is a crucial question in cancer biology, especially in prostate cancer. Buffalo, NY, USA The metastatic process requires that a cell acquire a motile phenotype to penetrate tissue and reach the vasculature and lymphatics. Parathyroid hor- Human prostate is the site of two of the most frequent medical problems in mone-related protein (PTHrP), an oncoprotein which is commonly expressed elderly men: benign prostatic hyperplasia (BPH) and prostate cancer. Adult at high levels in prostate cancer tissue and cell lines, has been implicated prostate epithelial stem cells are proposed to be involved in the etiology in the pathogenesis and progression of several cancers. In this study, we of both BPH and prostate cancer. The exact location of stem cells in the compared the parental prostate cell lines with their corresponding derivatives prostate and the pathway of lineage differentiation of different types of cells that stably overexpress PTHrP. We observed in in vitro experiments that namely; luminal, basal and neuroendocrine cells are still unknown. There are PTHrP-overexpressing derivatives were more invasive using a Matrigel inva- different cell surface markers which are currently being used to enrich for sion assay. In a mouse model of prostate cancer, we demonstrate that PTHrP stem cells, however; no marker is unanimously agreed upon as a prostate resulted in extensive metastasis, but not in mice that received the parental stem cell marker. Hence, compared to other adult organs, prostate stem controls. Even more important, the PTHrP derivatives promoted epithelial- cells are poorly defined and a more systematic determination of stem cell to-mesenchymal transition (EMT). A central developmental event during markers is needed to be done. In the stem cell niche, stem cells can divide embryogenesis, EMT describes the changes in an epithelial cell as it gains either symmetrically (both cells remain stem cells) or asymmetrically (one cell mobility and invades the extracellular matrix. Cells that overexpressed cell moves away from stem cell niche and differentiates while the other cell PTHrP had decreased levels of the epithelial marker E-cadherin, increased maintains stem cell properties). It was proposed that in cancer, a balance levels of the mesenchymal marker vimentin, and increased levels of the EMT between these two types of division is lost in the stem cell compartment. In regulator Snail, as measured by qPCR. These results were supported by im- hematopoietic system, intestine and in mammary tissue, it has been shown munofluorescence data. Recent evidence further suggests that the induction that carcinogenesis accompanies with more symmetrical divisions in the stem of EMT in cancer cells leads to the development of cancer stem cell traits. cell compartment. In order to monitor stem cell division in prostate stem Interestingly, cells that had high levels of PTHrP not only exhibited induc-

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Thursday Poster Abstracts tion of the stem cell genes CD133, Oct4, and Nanog, but also had increased Poster Board Number: 2101 ability to form tumorspheres compared to the parental control. DU145 prostate cancer cells that normally are incapable of forming tumors when ATTACKING NEUROBLASTOMA CANCER STEM implanted subcutaneously into immunocompromised mice are transformed CELLS WHERE THEY RESIDE: NICHE-SPECIFIC to highly tumorigenic cells when engineered to express high levels of PTHrP. These very same properties characterize cancer stem cells, that are thought APPROACHES FOR RELAPSE METASTATIC to not only be capable of initiating tumors, but also are believed to be criti- DISEASE cal for cancer invasion and dissemination. Hansford, Loen M., Angelini, Paola, Grinshtein, Natalie, Morozova, Poster Board Number: 2099 Olena, Lipman, Tatiana, Marrano, Paula, Smith, Kristen M., NOVEL MURINE CELL LINES ISOLATED Marrano, Marco A., Goldenberg, David M., Thorner, Paul S., Baruchel, Sylvain, Irwin, Meredith S., Kaplan, David R. FROM SELF-RENEWING PTEN-/-TP53-/- Hospital for Sick Children, Toronto, ON, Canada, Genome Science Centre, PROTOSPHERES HAVE MULTIPOTENT Vancouver, BC, Canada, Center for Molecular Medicine and Immunology, PROGENITOR ACTIVITY AND FORM Garden State Cancer Center, Belleville, NJ, USA ADENOCARCINOMA AND METASTASES. Fifty per cent of neuroblastoma (NB) patients have metastases at diagnosis, of which 90% will die after metastatic relapse. In contrast to NB tumors, Abou-Kheir, Wassim, Hynes, Paul, Yin, Ivy, Liu, Yen-Nien, Martin, we find that tumor-initiating cells (TICs) from bone marrow metastases of Philip, Seng, Victoria, Kelly, Kathy high-risk patients display a mixed neural and hematopoietic phenotype, NIH/NCI, Bethesda, MD, USA including expression of the neural crest stem cell marker nestin and the B- cell markers CD20, CD22, CD74, and IgH. Suppression of CD74 by shRNA Prostate cancer is the most commonly diagnosed cancer in men. PTEN or anti-CD74 (Milatuzumab) inhibited TIC survival, and CD74 shRNA and/or TP53 deletions in prostate cancer are associated with aggressive- increased tumor latency and survival in vivo. Anti-CD20 (Rituximab) also ness, castration-resistance, and poor prognosis. One origin of progressive, inhibited NB TIC survival in vitro and suppressed tumor growth in vivo. NB treatment-resistant prostate cancer has been hypothesized to be androgen- TICs from bone marrow metastases exhibited constitutive B-cell, mTor, and independent, prostate cancer stem cells. In order to investigate molecular PLK1 signaling, and this signaling was required for TIC survival. The mixed mechanisms linking specific gene mutations to tumorigenic properties, we neural-hematopoietic phenotype appears to be “plastic,” in that incubation have established and characterized two prostate luminal epithelial cell lines of metastatic cells from the bone marrow with brain cells suppresses the with Pten/TP53 deletions, derived from a prostate epithelial stem/progeni- expression of hematopoietic-specific genes. We suggest that metastatic TICs tor-enriched cell population and maintained in a serum-free media. from NB adopt gene expression signatures of the niche where they reside, Using a genetically engineered mouse model containing a tamoxifen-in- which may aid diagnosis and the development of novel treatments. We ducible Cre and floxed Pten and TP53 alleles, we isolated prostate epithelial hypothesize that drugs used for lymphoma may be efficacious therapeutics cells and subsequently generated Pten-/-TP53-/- protospheres in vitro. Cells for metastatic NB. Towards this end, we have initiated a North American were propagated as spheres for three subsequent generations in order to Phase I clinical trial for the mTor inhibitor rapamycin with vinblastine (Sylvain enrich for stem/progenitor cells. A polyclonal cell line was then grown from Baruchel, principal investigator), and our preclinical data indicates that cells dissociated from the protospheres and subsequently a clonal daughter clinically used anti-CD20, anti-CD74, and PLK1 inhibitors may also be ideal line was established. The cell lines, termed Plum-P and Plum-C (Prostate candidates for NB clinical trials. Luminal- Parental or Clone), demonstrate a luminal phenotype in vitro (prevalent presence of CK8, AR, and NKX3.1) while retaining a capacity Poster Board Number: 2103 for multi-lineage differentiation in vitro and in vivo. Both cell lines formed adenocarcinoma tumors, composed mainly of luminal (CK8+) cells, but cells IN SEARCH OF THE ELUSIVE NEUROBLASTOMA expressing markers characteristic of basal (CK5+) and neuroendocrine (syn- (NBL) TUMOR INITIATING CELLS (TIC) IN aptophysin) cell lineages also were observed. Moreover, the same lineages were identified in vitro following growth in 3-dimensional culture condi- PERIPHERAL BLOOD HEMATOPOIETIC STEM tions. In addition, a subpopulation of cells demonstrated self-renewal ability CELLS (PBHSC) COLLECTIONS AFTER G-CSF as shown by protosphere propagation for at least six generations. Intra- MOBILIZATION IN PATIENTS WITH NBL IN cardiac injection of both lines resulted in brain and lung metastasis, while intra-tibial injection induced osteoblastic bone formation, recapitulating the REMISSION. bone metastatic phenotype in human prostate cancer. Interestingly, although Kletzel, Morris, Huang, Wei, Elizabeth, Sato, Olszewski, Marie androgen was not required for growth in vitro, the cells express androgen Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, receptor and were sensitive to bicalutamide treatment. Withdrawal of an- IL, USA, Pediatrics, Children’s Memorial Hospital, Chicago, IL, USA drogens from established orthotopic tumors resulted in tumor regression and eventually castration-resistant growth. We have generated tumorigenic and Neuroblastoma TIC may be present in PBHSC of patients with NBL in metastatic prostate epithelial cell lines containing deletion of Pten and TP53, remission. NBL patients with advance disease have a tendency for relapse genetic aberrations associated with progressive human prostate cancer. after high dose chemotherapy followed by autologous hematopoietic stem These cell lines will be a useful tool in investigating the molecular mecha- cell rescue. Hematopoietic stem cells harvested for this purpose might be nisms contributing to prostate tumor progression and metastasis. contaminated with TIC. Several test have been use to determine the pres- ence of NBL cells Tyrosine Hydroxylase (TH) gene expression and, Immuno- cytochemistry (ICC). In this study, we investigated if NBL TICs are present in PBHSC collections. Materials & Method: Eighteen samples were obtained from harvested HPBSC by aphaeresis after mobilization with G-CSF were recovered from residual blood cells left over from the collections. Samples were characterized at D-0 using CD133+, CD34+, CD56+ GD2+, CD9+ by flow cytometry (FAC Sort) and by the gene expression of Tyrosine Hydroxy- lase (TH) and MYCN by quantitative real time PCR (Applied Biosystem):

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2x10^6 cells were cultured in Liquid Media RPMI 1640 for 8 weeks, and tion through ligand/receptor interactions. Physical variables include the 3D compare to 4 cell lines (NGP, IMR5, LAN-5, SKNSH) and non NBL mobilized architecture and mechanical properties of the microenvironment. In this PBHSC at which time the same characterization performed at day 0. Results: work, we are investigating the mechanisms of regulation of cancer stem cells On day 0 there was no gene expression of TH or MYCN in-patient samples by extrinsic signals through the use of 3D scaffolds that provide systematic compare to 1.8 x 100 and 1.4 x10-1 in negative controls vs. 2.9x106 and control of microenvironmental cues presented to cancer stem cells. Neuro- 9.x102 for TH and MYCN respectively in the cell lines. At week 8 there was blastoma is the most common extracranial tumor in children. Neuroblastoma a 6.5x 103 fold increase in TH and 1.6x103 fold increase in MYCN gene cancer stem cell-like cells, tumor initiating cells (TICs), have been previously expression in patient samples while the negative controls show no increase isolated from neuroblastoma bone marrow metastasis by growing tumor expression and the cell lines show similar gene expression. In Table 1 we cells as a sphere culture in conditions normally used to culture neural crest show the comparison results of the flow cytometry. Conclusion: These stem cells. Additionally, neuroblastoma cell lines have been shown to contain data suggests the presence of NBL TICs in collected HPBSC because of the TICs. The neuroblastoma cell line IMR-32 cultured in the same culture appearance of NBL specific markers (increase GD2, CD56, CD9 cell surface conditions as the patient derived TICs aquires a phenotype resembling these expression and increase in gene expression of TH and MYCN) cells. We are investigating the effects of modulation of growth factors and extracellular matrix components in the 3D scaffold to mimic communication Poster Board Number: 2105 between TICs and thePoster microenvironment. Withdrawn Furthermore we aim at varying KRUPPEL-LIKE FACTOR 4 SUPPRESSES the elasticity of the scaffold material, resembling the elasticity of the sites of neuroblastoma metastases. NEUROBLASTOMA GROWTH BY PROMOTING SMOOTH-MUSCLE DIFFERENTIATION Poster Board Number: 2109 Tsoi, Liz, Lau, SingTing, Tam, Paul, Ngan, Elly THE PROGNOSTIC ROLE OF HUMAN ADULT Dept of Surgery, University of Hong Kong, Hong Kong, Hong Kong CANCER STEM CELL MARKER PROFILES Neuroblastoma (NB) is an embryonic tumor and possesses a unique propen- IN AGGRESSIVE METASTATIC COLORECTAL sity to exhibit either a spontaneous regression or an unrestrained growth. CANCER Growing evidence suggests that NB comprises heterogeneous populations of improperly differentiated neural crest cells and a small subset of NB cells Hessman, Crystal J., Anderson, Eric C., Wong, Melissa H. behaves as stem cells. Commitment of NB stem cells to the fibromuscular Oregon Health & Science University, Portland, OR, USA lineage may give a favorable outcome, while to the neuronal lineage results Background: Despite advances in screening and treatment, metastatic col- in a malignant tumor progression. Krüppel like factor 4 (KLF4) is one of orectal cancer (CRC) remains the third-leading cause of cancer-related death the key reprogramming factors. Intriguingly, it also possesses paradoxical in the United States. CRC most commonly metastasizes to the liver, and functions in cancers, either as an oncogene or tumor suppressor dependent once this occurs, survival rates are tremendously impacted. A small subset of cell context. In this study, we elucidated the roles of KLF4 in the lineage of patients may achieve long-term survival with metastasectomy. However, determination of NB stem cells and tumor progression. Quantitative RT-PCR the majority of patients develop a disease recurrence within 2 years, and showed that loss of KLF4 expression was frequently found in the high-stage only one-third achieve 5-year survival. Several studies have proposed that NB (Stages III and IV). In particular with the high-risk factors such as age of cancer stem cells (CSC) may be the functional effectors of tumor metastasis, patient >1 year, N-myc amplification and low TrkA expression, the decreased treatment resistance and recurrence. However, no one to date has examined expression of KLF4 was significantly associated with an unfavorable NB whether the differential expression of CSC markers can be used as prognos- outcome. Subsequent targeted down-regulation studies using a NB cell line tic indicators of recurrence and overall survival in metastatic CRC. Methods: (SK-N-SH and Be(2)-C) directly demonstrated that reduced KLF4 expression Formalin-fixed, paraffin-embedded metastatic CRC tissue samples were ob- favors the growth of NB cells and tumorigenesis. In concordance with this, tained from 96 patients treated with metastasectomy for liver disease. Using overexpression of KLF4 profoundly suppressed proliferation and induced standard immunohistochemical techniques, tissue samples were stained with apoptosis of NB cells (SH-SY-5Y). At the molecular level, KLF4 directly up- antibodies to previously characterized CSC markers CD166, CD44, CD26, regulated the cell-cycle inhibitor protein p21CIP and induced cell cycle arrest and Aldehyde Dehydrogenase 1. Cellular expression patterns for solitary and and cell death. In addition, KLF4 overexpressing cells have lost their neuro- multiple CSC markers were captured with confocal microscopy and analyzed blastic phenotypes, they were epithelial-like, strongly substrate-adherent, using an automated open source image quantification program (CellProfiler expressing smooth muscle marker and became non-tumorigenic. Moreover, 2.0). Expression profiles were then correlated to patient outcomes (disease KLF4 knockdown clones were not able to committed to fibromuscular lin- recurrence and overall survival). Results: Variable expression of CSC markers eage, suggesting that KLF4 expression is crucial for lineage determination of was seen between long- and short-term survival as well as rate of recur- NB stem cells. Collectively our work showed that decreased KLF4 expression rence. Patients with an overall survival of greater than 2 years showed a is associated with poor disease outcome and KLF4 can directly mediate the higher level of CD166 expression. A loss of CD44 expression correlated to growth and lineage determination of NB cells. an increased disease-free survival of greater than 2 years. Conclusion: In Poster Board Number: 2107 this study, we identify a subset of CSC markers that correlates to disease behavior and tumor biology in metastatic CRC. Further delineation of CSC MODELING THE NEUROBLASTOMA TUMOR profiles may give valuable clues to therapeutic resistance as well as offer INITIATING CELL MICROENVIRONMENT IN 3D new therapeutic targets in the treatment of this disease. CULTURE Pinheiro, Irina, Lundin, Vanessa, Herland, Anna, Teixeira, Ana Cell Molecular Biology, Karolinska Institute, Stockholm, Sweden Cancer stem cells are tumor subpopulations capable of self-renewal and differentiation into all cell types present in the tumor. Accumulating evidence suggests that cancer stem cells are influenced by the surrounding microen- vironment. ChemicalPoster cues present Withdrawn in the microenvironment include small molecules, growth factors, extracellular matrix, and cell-cell communica-

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Poster Board Number: 2111 intestinal cell populations including ISCs. Comparison of the expression profiles of different crypt cell types versus CRC patient samples led us to EXPRESSION OF SSEA-1/CD-15 IN DIFFERENT identify a gene program shared between normal ISCs and the most aggres- COLON CANCER CELL LINES sive CRCs. 30 to 40% of CRC patients undergoing curative therapy will ex- perience disease relapse usually in the form of metastasis. We demonstrate Ling, Qing-Dong, Su, I-Chang, Chen, Yi-Shiou, Yang, Ting, that high expression levels of ISC genes in primary tumors are associated Higuchi, Akon, Lee, Hoong-Chien with high risk of recurrence. Furthermore, expression of ISC genes identi- Cathay Medical Research Institute, Cathay General Hospital, New fies an ISC-like tumor cell niche capable of tumor initiation upon injection in Taipei, Taiwan, Division of Neurosurgery, Department of Surgery, Sijhih immuno-deficient mice. Our findings have profound implications not only Cathay General Hospital, New Taipei, Taiwan, Department of Chemical for the understanding of CRC biology but also for the clinical management & Materials Engineering, National Central University, Chungli, Taiwan, of CRC patients Graduate Institute of Systems Biology and Bioinformatics, National Central Poster Board Number: 2115 University, Chungli, Taiwan Many studies have revealed that Site-specific embryogenic antigen-1 (SSEA- HUMAN CANCER STEM CELLS FROM 1) (or known as CD-15) not only is a molecular marker to be expressed on CHEMORESISTANT OVARIAN EPITHELIAL the surface of mouse embryonic stem cells, but it be used to identify for CANCER: A PERSONALIZED MODEL FOR several cancer stem cells (CSC) such as glioblastoma, lung cancer and colon cancer cells. Recent clinical researches reported that the ectopic expression OVARIAN CANCER THERAPY SCREENING of SSEA-1/CD-15 in colon cancer patients markedly related to the metastatic Aguilar-Gallardo, Cristobal, Martinez-Arroyo, Ana, Riboldi, Marcia, ability of cancer cells. However, whether this antigen is expressed on differ- Poo, Maria, Sanchez, Eva, Hidalgo, Juan, Domingo, Santiago, ent origins of colon cancer cells has been unknown. In this study, we dem- onstrated expression of SSEA-1/CD-15 on cancer stem-like cells that were Simon, Carlos derived respectively from different colon cancer cell lines including Caco-2, Prince Felipe Res Centre, Valencia, Spain, University Hospital La Fe, DLD-1, HT-29, T-84, HCT-116 and Colo-205. We used an in vitro method Valencia, Spain “spheroid colony formation” that is one of several different approaches to Introduction: Ovarian cancer is the leading cause of death from gynecologi- identify CSCs. The spherical colonies were produced after several weeks in cal malignancies. Epithelial ovarian carcinoma (EOC) is the most frequent culture with serum-free media containing epidermal growth factor (EGF) and variety diagnosed at an advanced stage with disseminated disease. Despite basic fibroblast growth factor (FGF). The immunofluorescent experiments efforts to promote optimal therapy, patients relapse in 70% of cases with a for SSEA-1/CD-15 and other CSC markers were processed for comparison survival rate of barely 33%. Accumulated evidence demonstrates that Can- of each spherical colony which was derived from different cell lines. In our cer Stem Cells (CSCs) possess several properties that allow them to develop results, SSEA-1/CD-15 was markedly expressed on the surface of cells within resistance to conventional therapies. The objective of this study is to isolate, the spheres for HT-29 and T-84, but weakly expressed for Colo-205, while and obtain an in vitro model of CSCs to be used with a drug screening plat- the other cell lines did not. This suggests that although SSEA-1/CD-15 has form, as targets for new therapy approaches. Drugs effective against CSCs been used as a surface marker to identify CSCs, but it might be expressed would be tested using an in vivo model. Methods: CSCs Isolation and Char- non-specifically in colon cancer tissues from different origins. acterization: Tumor samples diagnosed as EOC were disaggregated to single Poster Board Number: 2113 tumor cells and were sorted with FACS using antibodies against CD133/ CD326 fluorochrome-conjugated. Subcutaneous xenograft was obtained INTESTINAL STEM CELL-LIKE CELLS IN by injecting CD133+/CD326+ cells into NOD-SCID mice. Transcriptional COLORECTAL CANCER RELAPSE expression was evaluated for undifferentiated markers, such as membrane transporters involved in chemoresistance. Image-based analysis of nuclear Merlos-Suárez, Anna, Barriga, Francisco M., Jung, Peter, Iglesias, translocation of transcription factors was performed by in-flow image Mar, Céspedes, María Virtudes, Rossell, David, Sevillano, Marta, analysis using a flow Imaging Cytometer (IC) (Amnis, Seattle, US). Ovarian Hernando-Momblona, Xavier, da Silva-Diz, Victoria, Muñoz, Cances Spheroid (OCS) in vitro model. 0.5.104 cells/cmts2 were seeded in Purificación, Clevers, Hans, Sancho, Elena, Mangues, Ramón, Batlle, 96 well plates and placed under undifferentiating conditions by culturing in low-serum in hypoxia to facilitate sphere formation. OCS were seeded in Eduard 96-well plates and cultured 72h at 37°C and then MTS colorimetric assay Oncology, Institut de Recerca Biomèdica de Barcelona (IRBB), Barcelona, was performed according to the supplier’s instructions. Prestwick Chemical Spain, Pathology, Hospital Universitari del Mar, Universitat Autònoma de Library permits probes compounds with known pharmacological activity at Barcelona, Barcelona, Spain, Institute of Biomedical Research (IIB Sant 10µM concentration. In Vivo Imaging Design: OCS were marked with Red Pau) and Networking Research Center on Bioengineering, Biomaterials Flourescent Protein (RFP) by lentiviral transformation for in vivo monitoriza- and Nanomedicine (CIBER-BBN, MICINN), Barcelona, Spain, Biostatistics, tion by a subcutaneous xenograft with RFP-OCS. Disease progression and Institut de Recerca Biomèdica de Barcelona (IRBB), Barcelona, Spain, cell tracking were carried out by the Optical Imaging IVIS® Spectrum (MA, Catalan Cancer Epigenetics and Biology Program (PEBC), Bellvitge USA). Results: CD133+/CD326+ cells from EOC recapitulated the parental Biomedical Research Institute (IDIBELL), Barcelona, Spain, Hubrecht tumor phenotype and are composed by a hybrid population of cells with Institute and University Medical Center Utrecht, Utrecht, The Netherlands, tumorogenic potential showing both mesenchymal (Vimentin, CD44) and Institute of Biomedical Research (IIB Sant Pau) and Networking Research epithelial markers (EpCAM). Ovarian CSCs cultured as OCS overexpressed Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN, stem cell markers and several membrane transporters related to chemore- MICINN), Barcelona, Spain sistance. Using IC analysis system (AMNIS, USA), we quantified nuclear Mature differentiated cells of the intestinal tract are constantly renewed by translocation of Oct-4, Sox2, and Nanog. As OCSs are structurally similar to the progeny of Intestinal Stem Cells (ISCs). We report that the organiza- the avascular microtumor nodules, we recreated the tumor features that per- tion of most human colorectal cancers (CRCs) is reminiscent of that of the mit the chemoresistance. We performed an in vitro drug screening in order normal intestinal epithelium. CRC cells display phenotypes similar to either to demonstrate resistance to classical chemotherapeutic drugs. Transfection ISCs or intestinal differentiated cells and recreate the formation of crypt-like of OCS with RFP and the injection into NOD-SCID mice made it possible structures within tumors. We have developed a method to purify normal to use an in vivo imaging approach to monitor for tumor appearance and

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invasive/metastatic events and response to therapy. Conclusions: Under- vivo xenotransplantation assays. Current work in our lab focuses on explor- stand epithelial ovarian cancer as a stem cell disease opens the possibility ing these findings in further cell lines and primary tumors, cell cycle analyzes to revolutionize its therapeutic approaches. Our results establish a model of ALDH+ and ALDH- cells, as well as analysis of the relevance of coexpres- that permits isolation, characterization and development of new therapeutic sion of ALDH with previously reported ovarian CSC markers (CD133, CD24, strategies directed specifically against ovarian CSCs. This model maintaining CD44/CD117). CSCs as Ovarian Cancer Spheroids (OCS) has proved to be a useful model to recreate the features of the parental tumor. Poster Board Number: 2119 Poster Board Number: 2117 THE ROLE OF PERICYTES AS CANCER ASSOCIATED FIBROBLASTS ALDEHYDE DEHYDROGENASE, A WIDELY- USED MARKER TO ISOLATE CANCER STEM Chong, Lynn, Schluter, Holger, Haviv, Izahk, George, Joshy, Bowtell, David, Kaur, Pritinder CELLS IN VARIOUS TUMORS, SEEMS NOT Peter MacCallum Cancer Centre, Melbourne, Australia, Baker IDI Heart and TO ENRICH THE CSC-FRACTION IN OVARIAN Diabetes Institute, Melbourne, Australia CANCER Cancer associated fibroblasts (CAFs) are a heterogeneous population of cells Bareiss, Petra M., Fehm, Tanja, Grauer, Matthias, Kokorsch, Philipp, with a broadly similar phenotype, but with the common ability to promote Grimm, Sabrina, Bühring, Hans-Jörg, Staebler, Annette, Fend, Falko, cancer growth. In this report, we have specifically studied the role of a particular stromal cell type: the pericyte, in cancer progression. Pericytes are Wallwiener, Diethelm, Kanz, Lothar, Lengerke, Claudia well-known to stabilise blood-vessel endothelial cell function both during Med Ctr II, Hematology & Oncology, University of Tuebingen, Tuebingen, homeostasis and tumour-associated angiogenesis. Here, we demonstrate a Germany, Women’s Hospital, University of Tuebingen, Tuebingen, novel role for these cells as cancer-associated fibroblasts. Specifically, ovar- Germany, Institute of Pathology, University of Tuebingen, Tuebingen, ian cancer patients carrying the pericyte gene signature are predisposed to Germany having a higher risk of relapse and lower overall survival suggesting that Background: Ovarian cancer is a highly aggressive tumor with particularly pericyte involvement is a strong predictor of recurrent cancer and mortality. high mortality rate due to the fact that diagnosis is often made in a meta- Importantly this high risk patient group was distinct from the groups identi- static stage of the disease. During the last years, there have been reports on fied by an angiogenic signature suggesting a more direct role for pericytes cancer stem cells (CSC) as subpopulations of tumor cells with self-renewal in increasing tumour growth. Consistent with this, co-injection of an ovarian capacity that mediate disease initiation, metastasis as well as therapy resis- cancer cell line (OVCAR-5), with pericytes resulted in accelerated tumour- tance and relapse in different types of tumors, including ovarian carcinomas. forming ability in vivo. Pericytes were also able to enhance the intrinsic The phenotype and molecular properties of ovarian CSC are however still migratory and invasive capacity of OVCAR-5 cells in vitro. Taken together, unclear and require further investigation. Aldehyde dehydrogenase (ALDH) these results suggest that CAFs can originate from pericytes and the genes activity characterizes (cancer) stem cells in different tissues. In some cancers, they express may be useful prognostic tools in predicting patient survival. such as breast carcinoma, elevated ALDH1 is a marker of poor prognosis. In Current work is aimed at identifying the cellular and molecular events by ovarian carcinomas, ALDH expression has been reported by different groups, which pericytes contribute to tumour development. The data obtained will yet contradictory data are presented on ALDH levels in putative ovarian assist in developing potential targets for anti-cancer therapy. CSC. Methods: We used Aldefluor-staining to assess ALDH activity in dif- Poster Board Number: 2121 ferent ovarian carcinoma cell-lines and patient samples. ALDH positive and negative cells were isolated by FACS and analyzed functionally by in vitro ONCOLYTIC VIRUSES FOR ERADICATION OF ovarian spheres cultures and in vivo tumorigenicity assays by xenotransplan- tation of human cells into NOD SCID gc-/- mice. Furthermore, sorted cells CANCER STEM CELLS were compared on a molecular basis regarding expression of stemness mark- Borrego-Diaz, Emma, Gerst, Jennifer, Liu, Zhengian, Esfandyari, ers such as OCT4, SOX2 and NANOG, and analyzed for cell cycle properties Tuba, Farassati, Faris as well as ATP-activity and apoptosis in chemo- and radio- sensitivity assays. Department of Medicine, Divisions of Gastroentrology and Hematology/ Results: ALDH+ cells were more able to form clonal spheres in suspension Oncology- Molecular Medicine Lab, The University of Kansas Medical in comparison to ALDH- cells (OVCAR-3 cell line: ALDH+ 272±30, ALDH- Centre, Kansas City, KS, USA 71±29, p=1.5E-10; patient sample #1: ALDH+ 44±10, ALDH- 4±0.3, p=0.0027; #2: ALDH+ 192±27, ALDH- 27±2.3, p=0.0004; #3: ALDH+ Oncolytic viruses are a novel class of anti-cancer therapeutics with many of 39±12, ALDH- 15±9.2, p=0.15), while ALDH low cells showed intermedi- their members entering phase I-II clinical trials. An oncolytic virus is a virus ate numbers of spheres. Notably, larger spheres (> 5.000 µm²) were formed that preferentially infects and lyses cancer cells. Oncolytic viruses have obvi- only by ALDH+ cells and not seen in the ALDH- population. In in vivo ous potentials for cancer therapy, both by direct destruction of the tumor tumorigenicity assays performed with ALDH+ and ALDH- isolated cells from cells, and, if modified, as vectors for expression of anticancer proteins to the cell line OVCAR-3, tumors were observed from both transplanted cell be delivered specifically to the tumor site. Ideally, oncolytic viruses should fractions. However, ALDH+ cells generated tumors more rapidly and overall replicate only in cancer cells and lead to their lysis and destruction. Older induced larger tumors (ALDH+ 4.515 mg in total, ALDH- 2.238 mg in total, versions of oncolytic herpes have cleared phase I clinical trials for treatment 10 weeks after transplantation when starting with a total 1.000.000 cells in of different cancers. Our laboratory has gained a significant level of exper- n=15). Interestingly, dissection of tumors initiated by both fractions showed tise in developing novel oncolytic viruses with capability of targeting cancer a mixture of ALDH+ and ALDH- cells. On a molecular level, no difference in cells in a specific manner. We have developed oncolytic herpes viruses for expression of stemness markers was noted between the two fractions and targeting two stem cell markers, CD133 and Cripto-1. This was achieved similar induction of apoptosis was documented following treatments with by controlling the expression of ICP4, a gene necessary for viral replication, chemo- (Paclitaxel, Cisplatin, Carboplatin) and radiotherapy. Discussion and using promoters for CD133 or Cripto-1. The rationale for developing these Outlook: Taken together, our data suggest that, unlike in other tumor enti- viruses was based on the idea of eradicating cancer stem cells and reducing ties such as breast carcinoma, ALDH activity cannot be used as a marker to and/or eliminating the self-renewal capacity of the tumor. We have now enrich CSC in ovarian cancers. However, ALDH+ may indicate a cell fraction collected evidence that elimination of CD133+ cells in different models such with higher proliferative capacity, especially in ovarian sphere culture and in as colorectal and hepatic cancer as well as elimination of Cripto-1+ cells in

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Thursday Poster Abstracts glioma, medulloblastoma and ovarian cancer can lead to a significant de- the ability to create a whole tumor and hence it’s the only targeted cell. The crease in the viability of the tumor cell population, loss of invasiveness and cancer stem cells are able to resist toxic substances, such as cancer drugs. in-vivo tumor growth. Upon treatment with these viruses, the percentage Hence, through drugs it may become impossible to eradicate the cancer of CD133+ or Cripto-1+ cells was significantly reduced and the capability of stem cells. Cancer immunotherapy is considered to be the possible approach these cells to invade was also diminished. Further studies are underway to of using our own immune system to attack cancer cells directly. The current evaluate the effects of treatment with CSC-targeting oncolytic viruses on cell cancer therapies are targeting the wrong cells. Unfortunately, in the majority cycle progression and EMT markers in order to understand the mechanism of cancers, cancer immunotherapy has proven ineffective. There are two of action of these agents in more details. possibilities, cancer stem cells either suppress the ability of the immune system to respond or fail to fully activate the immune system. DNA micro Poster Board Number: 2123 arrays or “gene chips” will be used to identify genes that are active in the CHARACTERIZATION AND TARGETING OF cancer-causing cells compared to benign tumor cells. Using the molecular mechanisms of p53 gene and the “antigen-Adjuvant System” combination CANCER STEM CELLS IN HUMAN MPNST will potentially produce a more effective immune response to target mutated Borrego-Diaz, Emma, Wise, Amanda, Taylor, Sarah, Esfandyari, progenitors of the cancer stem cells. This whole approach will reverse the mechanism of the cancer stem cell into normal stem cell & hence the cancer Tuba, Farassati, Faris will be eradicated automatically. The advantages of this kind of cancer im- Department of Medicine, Divisions of Gastroentrology and Hematology/ munotherapy will increases the durability of the anti-cancer response with Oncology- Molecular Medicine Lab, The University of Kansas Medical no severe side effects, as the immune system can select cancer stem cells & Centre, Kansas City, KS, USA change it to become the normal cells. Although monoclonal in origin, most tumors appear to contain heteroge- Poster Board Number: 2127 neous populations of cancer cells. One possible explanation of this tumor heterogeneity is that human tumors are not merely monoclonal expansions NATURAL KILLER CELL LINES PREFERENTIALLY of a single transformed cell, but rather caricatures of normal tissues, and their growth is sustained by cancer stem cells (CSCs). Cancer Stem Cells TARGET CLONOGENIC MULTIPLE MYELOMA (CSCs) are believed to be the main regenerative pool of cells in charge of CELLS repopulating tumors after exposure to therapeutic modalities. Therefore, Swift, Brenna, Martinez, Joaquin, Keating, Armand gaining knowledge about the characteristics of CSCs is important not only in terms of understanding the biology of tumors but also in develop- Princess Margaret Hospital, University Health Network, Toronto, ON, ing novel anti-cancer therapies. We have identified a subpopulation of Canada cells positive for CD133 (a recognized CSCs marker) from human primary Multiple myeloma (MM) is an incurable plasma cell malignancy. Current malignant peripheral nerve sheath tumors (MPNST). Non-malignant human therapies including dexamethasone, lenalidomide and bortezomib, target Schwann cells were mainly void of this antigen. CD133 was also found to MM plasma cells to reduce tumor burden but are ineffective in eradicating be expressed in mouse MPNST cells. The CD133+ enriched population was the disease, suggesting that clonogenic MM cells are resistant to treatment studied based on CSC-related characteristics and Ras pathway signaling (Matsui et al., 2008). Novel approaches are therefore needed to eliminate profile. CD133+ cells were capable of forming spheres upon being cultured the MM stem cells responsible for the maintenance and progression of MM. in non-adherent/serum free conditions. The spheres were also positive for We have investigated the cytotoxicity of the natural killer (NK) cell lines, CD24 (another CSC marker). The activation levels of Ras (Ras-GTP) and NK-92 and KHYG-1, against a variety of tumor targets and have undertaken its down-stream effector pathways such as ERK, JNK, PI3K, p38K and RalA a phase I trial with the former in patients with refractory blood cancers. We were significantly increased in this population. Moreover, the CD133+ cells were particularly interested in assessing the efficacy of these cell lines against showed enhanced capabilities for invasiveness through matrigel which was MM. Chromium release and flow cytometry cytotoxicity assays were used to mechanistically linked to the increased expression of β-Catenin and Snail, quantify MM cell death over a 4 hour co-incubation. In the flow cytom- two molecules involved in the epithelial to mesenchymal transition (EMT) etry cytotoxicity assay, NK cells were differentiated from MM cells with a and to Paxilin, a focal adhesion protein. Among other important character- monoclonal antibody for CD7 and MM cell death was identifed by 7-AAD istics of the CD133+ population, Endoplasmic Reticulum Stress (ER-Stress) and Annexin V positivity. To evaluate the in vivo cytotoxicity of NK-92, we marker IRE1α was decreased, implying the potential sensitivity of CD133+ transduced the U266 MM cell line with GFP and luciferase (U266eGFPluc) CSCs to the accumulation of unfolded proteins. Interestingly, apoptotic to establish a xenograft MM model in NOD/SCID γ null mice. Two million indicators seemed to be majorly unchanged in CD133+ cells as compared U266eGFPluc cells were injected intravenously, followed by 10x106 NK-92 to the wild population MPNST cells. Finally, in order to test the possibility cells at day 7 for a total of 5 doses, each 5 days apart. The tumor burden of targeting CD133+ CSCs with Ras pathway pharmacological inhibitors, was evaluated using IVIS to quantify bioluminescence and flow cytom- we exposed these cells to an ERK inhibitor. The wild population was more etry to enumerate GFP positive cells in the bone marrow. Clonogenic cell sensitive to inhibition of proliferation by this inhibitor as compared with the death was evaluated using the same 4 hour co-incubation procedure, then CSCs population, thus supporting the theory that CSCs are more resistant to injecting the cells into methylcellulose and counting colony formation after conventional therapies. 7-14 days. Each treatment group was normalized to the colony growth in Poster Board Number: 2125 a control sample where the targets and effectors were incubated separately for 4 hours then injected together into methylcellulose. The NK cell lines AMAZING CANCER STEM CELLS AND THE NK-92 and KHYG-1 exhibit cytotoxicity in vitro against the MM cell lines RPMI 8226, NCI-H929 and U266 by chromium release and flow cytometry MOST DESIRABLE CANCER IMMUNOTHERAPY cytotoxicity assays. At an effector to target ratio of 10:1, the cytotoxicity of Edward, Christina the three tested MM cell lines is between 50-90% after treatment with NK- Madurai Kamaraj University, Chennai, India 92 or KHYG-1. Preliminary results in a xenograft mouse model also suggest efficacy of NK-92 cell therapy in vivo. Notably, NK-92 and KHYG-1 have The desire of my theoretical research is to instigate methods of targeting shown preferential cytotoxicity against the clonogenic population in the MM the cancer stem cells by our own immune system. The one and only cells cell line RPMI 8226 in a methylcellulose cytotoxicity assay. At an effector to within an organism that live long enough to accumulate the mutations target ratio of 10:1, about 50% of the bulk tumor cells were killed while NK- which leads to cancer are the cancer stem cells. The cancer stem cells have 92 and KHYG-1 killed 70% and 90% of the clonogenic cells, respectively

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(NK-92 P value=0.0024; KHYG-1 P value=0.0001). The residual colonies samples. Objective: We sought to examine tumor initiation in a series of after treatment formed significantly fewer colonies compared to the control primary human HCCs. Methods: Primary subcutaneous tumor xenografts in a secondary replating (NK-92 P value=0.0186; KHYG-1 P value=0.0007). were generated by implanting fragments of freshly resected human HCCs This suggests that the residual colonies have a reduced clonogenic capacity. into immunodeficient NOD/SCID (non-obese diabetic/severe combined We are investigating the mechanism responsible for the preferential killing immunodeficiency) or NSG (NOD/SCID/interleukin-2 receptor gamma null) of clonogenic MM cells by considering the functional relevance of different mice. Xenografts were subsequently passaged in vivo by subcutaneous im- ligands and receptors on MM and NK cells. This study demonstrates preclini- plantation of tumor fragments or cell suspensions. Primary human HCC cells cal efficacy of the NK line therapy against MM. The preferential cytotoxicity were propagated in tissue culture for in vitro assays of sphere and colony of clonogenic MM cells by NK-92 and KHYG-1 infers a more effective thera- formation as a surrogate for in vivo tumor initiation. Histopathology, RT- py for refractory MM and suggests that clinical trials with these cell lines are PCR, flow cytometry and immunocytochemistry were utilized for analysis of warranted in patients with refractory disease. tumor tissues and cultured cells. Results: Stable xenografts from 15 different HCC specimens were generated. The majority of serially passaged HCC xe- Poster Board Number: 2129 nografts resembled parent tumors on histopathological analysis and retained A NOVEL FORKHEAD-ASSOCIATED SIGNALING expression of hepatocyte markers. Limiting dilution assays demonstrated that in vivo tumor initiation consistently required subcutaneous implantation DOMAIN CONTAINING PROTEIN ENHANCES of between 103 and 104 bulk HCC cells. Similarly, cultured primary HCC SURVIVAL OF HUMAN EMBRYONIC STEM cells retained epithelial morphology and expression of hepatocyte markers. Only a small fraction of cultured cells were capable of sphere or colony for- CELLS VIA CD30-DEPENDANT ACTIVATION OF mation in vitro. The proportion of cells expressing CD133 and EpCAM was CANONICAL NFκB SIGNALING highly variable between different primary HCC xenografts and tumor initiat- ing capability was not restricted to cells expressing these markers in vivo. Thakar, Nilay Y., Ovchinnikov, Dmitry, Chung, Tung-Liang, Similarly, cultured primary HCC cells demonstrated variable expression of Wolvetang, Ernst J. CD133 and EpCAM, and sphere/colony formation in vitro was not restricted Stem Cell Engineering Group, University of Queensland, Brisbane, Australia to cells expressing these markers. Conclusions: Our observations of primary CD30 is a biomarker for embryonal carcinoma cells, Hodgkin’s lymphoma human HCCs support the concept that tumor initiation is driven by a small and anaplastic large cell lymphoma and its activation of the NFkB signaling subset of tumor cells in this disease. Our data suggest that the phenotype pathway is believed to be causative for the Hodgkin’s lymphoma pathol- of tumor-initiating cells is heterogeneous in primary HCC, and not restricted ogy. All hESC and iPS cells that are cultured in the presence of ascorbate will to CD133+ or EpCAM+ cells. We propose that tumor-initiating cells bearing express CD30 and we have shown previously that this activates NFkB signal- additional novel markers need to be identified in human HCC in order to ing, inhibits apoptosis and enhances single cell survival of hESC. We now gain a comprehensive understanding of the biology of this disease. report that both hESCs that endogenously express CD30 and those that are Poster Board Number: 2133 engineered to over-express CD30 variant (CD30v), the ligand-independent intracellular signaling domain of CD30, show increased ERK phosphoryla- INHIBITION OF AKT/PI3K PATHWAY INCREASES tion as well as nuclear translocation of both AKT and , unexpectedly, CD30v LOW RHODAMINE 123 RETENTION CELLS itself. By introducing mutations and small deletions in the CD30 signaling domain we demonstrate that in both hESCs and 293FT cells two specific WITH CHARACTERISTICS OF CANCER STEM threonine residues within CD30v control NFKB activation through an FHA CELLS IN HUMAN MELANOMA domain-containing protein. We have also shown, via western blot analy- sis, that over-expression of CD30v within hESCs results in preferentially Touil, Yasmine, Zuliani, Thomas, Wolowczuk, Isabelle, Mortier, increased activation of the canonical NFkB pathway which explains the Laurent, Kuranda, Klaudia, Segard, Pascaline, Masselot, Bernadette, reduced apoptosis and enhanced single cell survival phenotype of these Quesnel, Bruno, Formstecher, Pierre, Polakowska, Renata hESCs. In contrast, CD30-negative hESCs show strong activation of the non- Centre de Recherche Jean-Pierre Aubert -Inserm U837, Inserm, Lille, canonical NFkB pathway and higher OCT-4 protein levels. We will report France, UMR 8199 Institut Pasteur de Lille, CNRS, Lille, France, Service de on the identification of this novel FHA domain-containing NFkB activating Dermatologie, Centre Hospitalier et Universitaire de Lille, Lille, France protein that binds to CD30 and reveal its binding partners in the cytosol and nucleus of hESC. Our data identify a novel NFkB signaling pathway in hESC Melanoma is one of the most aggressive tumors. Once it has progressed to downstream of CD30 that is also operational in CD30 expressing lympho- the metastatic stage it becomes extremely resistant to conventional thera- mas where it can be further investigated for therapeutic purposes. pies and the prognosis is poor. In the last few years, many lines of evidence suggest that tumor initiation, development and recurrence after treatment Poster Board Number: 2131 is driven by a subset of surviving cancer stem cells that can regenerate the tumor. Cancer stem cells appear to overexpress ABC transporters efficiently PHENOTYPIC HETEROGENEITY OF TUMOR- excluding xenobiotics from the cell and conferring their multidrug-resistance INITIATING CELLS IN PRIMARY HUMAN phenotype. This property has been used to develop the dye efflux assay HEPATOCELLULAR CARCINOMA identifying stem cells within the side population (SP) consisting of low dye- retention cells. In the absence of unambiguous melanoma stem cell surface Chen, Kui, Ahmed, Sharif, Naik, Pooja, Ghanekar, Anand markers, we exploit this assay to functionally detect stem cells in metastatic Toronto General Hospital, University Health Network, Toronto, ON, Canada melanomas and melanoma cell lines. By employing Rhodamine123 (Rh123), the specific substrate of the ABCB5 and ABCB1 transporters, we discovered Background: Accumulating evidence supports the model that many cancers, that all of 6 tested melanoma tumors and 4 cell lines contained a small sub- including solid epithelial tumors, are hierarchically organized and are propa- set (0.6-6.4%) of cells with the Rh123 low (Rh123low)-fluorescent profile. gated and maintained by a small subset of tumor-initiating cells. Recent Subsequently, we investigated in detail characteristics of the Rh123low studies suggest that tumor-initiating cells in human hepatocellular carcinoma cells in Mel4M cell line generated in our laboratory from one of the tested (HCC), the most common form of primary liver cancer, can be identified by tumors. We determined that the Rh123low fraction, but not the Rh123high surface expression of CD133 or EpCAM (epithelial cell adhesion molecule, fraction, was greatly enriched for cells with stem cell-like activities. This CD326). Many of these studies have been based on observations of im- included their quiescence, the ability to self-renew and produce non-stem mortalized HCC cell lines or very small numbers of primary human HCC

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Thursday Poster Abstracts cell progeny, to form melanospheres that recapitulated the phenotypic explain some of the heterogeneity observed in mesenchymal neoplasms. profile of the original tumor, and to form tumors in immunodeficient SCID mice. In addition, we found that Rh123low cells grown as monolayer and/ Poster Board Number: 2137 or melanosphere cultures overexpressed Hif 1α, OCT4 and the ABCB5 trans- RESVERATROL INHIBITS PANCREATIC CANCER porter which is a marker of melanoma stem cells, but expressed lower levels of Cyclin D1 than Rh123high cells. We also showed that Rh123low cells STEM CELL CHARACTERISTICS IN HUMAN AND were more resistant than Rh123high cells to anticancer drugs currently used KRASG12D TRANSGENIC MICE BY INHIBITING in melanoma therapy, including cisplatine and fotemustine. Interestingly, the pool of Rh123low cells was significantly increased (3 fold) by LY294002, PLURIPOTENCY MAINTAINING FACTORS AND an inhibitor of the AKT/PI3K pathway, but not by rapamycin or PD98059 EPITHELIAL-MESENCHYMAL TRANSITION. inhibitors of mTOR and MAPK, respectively. The LY294002-specific increase Srivastava, Rakesh K., Tang, Su-Ni, Rodova, Mariana, Nall, Dara, of the Rh123low cell compartment was accompanied by the increase in quiescent cells (2 fold), the reduced ability to form spheres, and a decrease Shankar, Sharmila in phosphorylated FOXO3A, all suggesting that inhibition of the AKT/PI3K Pharmacology, Toxicology and Therapeutics, and Medicine, University of pathway reverses active melanoma stem cells to their quiescent state and, Kansas Medical Center, Kansas City, KS, USA therefore, may render them more resistant to anticancer treatment. In con- Background: Cancer stem cells (CSCs) can proliferate and self-renew clusion, our data strongly imply that the Rh123low subset of cells encom- extensively due to their ability to express anti-apoptotic and drug resistant pass melanoma stem cells and that their activation is AKT/PI3K-dependent. proteins, thus sustaining tumor growth. Therefore, the strategy to eradicate Poster Board Number: 2135 CSCs might have significant clinical implications. The objectives of this study were to examine the molecular mechanisms by which resveratrol inhibits DISCOVERY OF NEW CELL SURFACE MARKERS stem cell characteristics of pancreatic CSCs derived from human primary TO IDENTIFY TUMOR INITIATING CELLS IN tumors and KrasG12D transgenic mice. Methodology/Principal Findings: Human pancreatic CSCs (CD133+CD44+CD24+ESA+) are highly tumori- HUMAN MESENCHYMAL TUMORS genic and form subcutaneous tumors in NOD/SCID mice. Human pancreatic Wei, Qingxia, Hyatt, Elzbieta, Wang, Chang Ye Yale, Zheng, Feifei, CSCs expressing high levels of CD133, CD24, CD44, ESA, and aldehyde dehydrogenase also express significantly more Nanog, Oct-4, Notch1, Azarnier, Ronak Ghanbari, Sato, Shingo, Han, Ilkyu, Ailles, Laurie, MDR1 and ABCG2 than normal pancreatic tissues and primary pancreatic Wunder, Jay S, Alman, Benjamin cancer cells. Similarly, CSCs from KrasG12D mice express significantly high The Hospital for Sick Children, Toronto, ON, Canada, University Health levels of Nanog and Oct-4 than pancreatic tissues from Pdx-Cre mice. Network, Toronto, ON, Canada, Mount Sinai Hospital, Toronto, ON, Resveratrol inhibits the growth (size and weight) and development (PanIN Canada lesions) of pancreatic cancer in KrasG12D mice. Resveratrol inhibits the self- Tumor initiating cells (TICs) are found in multiple tumor types. While specific renewal capacity of pancreatic CSCs derived from human primary tumors cell surface markers selectively expressed on TICs are often used to isolate and KrasG12D mice. Resveratrol induces apoptosis by activating capase-3/7 these cells, no such markers are known to prospectively identify TICs in and inhibiting the expression of Bcl-2 and XIAP in human CSCs. Resveratrol mensenchymal tumors. Our previous study demonstrated that a subpopu- inhibits pluripotency maintaining factors (Nanog, Sox-2, c-Myc and Oct-4) lation of cells in mesenchymal tumors, which exclude Hoechst dye and and drug resistance gene ABCG2 in CSCs. Inhibition of Nanog by shRNA referred to as “side population” (SP) cells when subjected to cell sorting, are enhances the inhibitory effects of resveratrol on self-renewal capacity of enriched for tumor initiating potential. Specific cell surface markers that are CSCs. Finally, resveratrol inhibits CSC’s migration and invasion and markers enriched within this cell population might be used to prospectively identify of epithelial-mesenchymal transition (Zeb-1, Slug and Snail). Conclusions/ TICs in mesenchymal tumors. To identify specific cell surface markers on SP Significance: These data suggest that resveratrol inhibits pancreatic cancer cells, a high throughput screen with monoclonal antibodies was performed. stem cell characteristics in human and KrasG12D transgenic mice by inhibit- Cells from three individual primary mesenchymal tumors were stained with ing pluripotency maintaining factors and epithelial-mesenchymal transition. Hoechst dye and labeled with one of the 238 fluorescence-conjugated In conclusion, resveratrol can be used for the management of pancreatic monoclonal antibodies. These cells were then analyzed through BD LSRII cancer. flow cytometry. Five cell surface markers were found to be enriched within Poster Board Number: 2139 the SP in all three mesenchymal tumors. Thirteen other surface markers were enriched within the SP in two out of three tumors. CD146, a cell REGULATION OF MOUSE ADULT STEM surface marker, which is also expressed by pericytes, was one of the mark- TO PROGENITOR CELL TRANSITION BY ers identified. Pericytes from multiple human organs have MSC properties. CD146 expression was found in all primary mesenchymal tumors analyzed, MICRORNAS ranging from a subpopulation of 0.04% to 44.72% of cells in the forty tu- Arnold, Christopher P., Zhou, Baiyu, Yue, Si-Biao, Chen, Caifu, mors analyzed. Double staining of tumor cells with Hoechst dye and CD146 Chen, Chang-Zheng demonstrated that CD146+ cells were enriched for SP up to seven fold. To investigate whether CD146 can be used to isolate TICs from mesenchymal Department of Microbiology and Immunology, Stanford University, tumors, both CD146+ and CD146- cells were sorted from ten primary hu- Stanford, CA, USA, Department of Statistics, Stanford University, Stanford, man mesenchymal tumors, including five osteosarcomas and five malignant CA, USA, Molecular Biology Systems Division, Life Technologies, Foster fibrous histiocytomas and injected into immunodeficient NSG mice at the di- City, CA, USA lution of 10, 102, 103,104 and 105 cells per injection site. Tumor formation Adult stem cells support tissue development and regeneration through- was monitored and mice were sacrificed three month post-injection. The out animal life but are often quiescent or slow cycling and are limited in data demonstrated that as few as 10 CD146+ cells were able to initiate tu- numbers. A conserved transit amplification process employed by adult mor formation in NSG mice while 1000 or more cells were needed from the stem cells of different tissues and organisms enables the rare stem cells to CD146- population. The serial transplantation experiments showed that in produce large number of progenitors with the ability to differentiate albeit the secondary transplantation there was a similarly enrichment with CD146+ with reduced self-renewal potential. It was postulated that transit amplifica- cells. CD146 is a potential marker for TIC from a variety of mesenchymal tion might also protect stem cells from extensive cycling and thus prevent tumor types. Unique biologic properties of this subpopulation may help accumulation of mutations. Importantly, this transition marks the change

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of genetic programs from self-renewing and quiescent in stem cells to Matrigel and treated with 3 biweekly injections of Adox or vehicle. Tumors rapid proliferating and differentiating in progenitor cells. Understanding the were resected after 9 weeks. Results: Both freshly isolated and transformed mechanisms underlying this transition may shed insight into the genetic pro- ICC-SC expressed mRNA and protein for PcG members Suz12, Ezh2 and grams that control the critical properties of stem cells, such as self-renewal Eed. In transformed ICC-SC, Suz12, Ezh2 and H3K27me3 were found to co- and quiescence. Emerging evidence suggests that microRNAs (miRNAs), an occupy the Kit promoter, implying direct epigenetic control of Kit expression abundant class of ~22-nt small regulatory RNAs, play key roles in control- by PcG repressive complexes. RNAi-mediated knock-down of Suz12 caused ling the post-transcriptional genetic programs in stem and progenitor cells. a significant upregulation of Kit mRNA. Pharmacological inhibition of Ezh2 Here we examined the regulation of miRNA expression during stem and methyltransferase activity with Adox (2.5 µM; 3 days) led to significantly progenitor cell transitions in the muscle and blood and identified a set of reduced global H3K27me3 levels, reduced specific H3K27me3 occupancy of miRNAs that are coordinately regulated during this transition (designated as the Kit promoter, and upregulated Kit protein expression. Adox inhibited the stem-to-progenitor transition miRNAs -- SPT-miRNAs). These miRNAs are proliferation of transformed ICC-SC in vitro and made them susceptible to likely to control the genetic programs initiated during the transition, such as further inhibition by imatinib. Adox also inhibited tumor growth from xeno- the regulation of self-renewal, quiescence and proliferation. Supporting this grafts of transformed ICC-SC, revealing that its antiproliferative effects are notion, we showed that the SPT-miRNA signature predicts the effects of ge- anti-tumorigenic in vivo. Conclusions: In transformed ICC-SC, Kit expression netic perturbations and leukemic transformations, such as loss of PTEN and is maintained in an epigenetically repressed state by PcG-mediated silencing. the Rb family genes, AML1-ETO9a expression and MLL-AF10 transforma- Reversal of this repression upregulates Kit mRNA and protein, and restores tion, on self-renewal and proliferation potentials of mutant stem/progenitor the sensitivity of transformed ICC-SC to imatinib. Our results demonstrate a cells. Furthermore, we demonstrated that some of the SPT-miRNAs control novel paradigm for the regulation of Kit expression and the utility of “epige- the self-renewal rate of embryonic stem cells and the reconstitution poten- netic drugs” for targeting therapy-resistant cancer stem cells in GIST. tial of hematopoietic stem cells (HSCs), indicating that these miRNAs might control conserved programs in distinct stem cell types. Finally, we demon- Poster Board Number: 2143 strated that the SPT-miRNAs coordinately regulate targets known to play TUMORIGENIC CELLS ARE ABUNDANT IN roles in controlling HSC self-renewal, such as Hoxb6 and Hoxa4. Together, these analyses reveal the miRNA programs that control key processes in dur- MOUSE MALIGNANT PERIPHERAL NERVE ing the stem to progenitor transition and set the foundations for dissecting SHEATH TUMORS BUT THEIR FREQUENCY post-transcriptional regulatory networks in stem cells. DEPENDS UPON TUMOR GENOTYPE AND Poster Board Number: 2141 EXPOSURE TO EXTRACELLULAR POLYCOMB-MEDIATED EPIGENETIC MATRIX REPRESSION OF KIT EXPRESSION UNDERLIES Buchstaller, Johanna, McKeever, Paul, Morrison, Sean THERAPY RESISTANCE OF MOUSE STEM CELLS Life Science Institute, University of Michigan, Ann Arbor, MI, USA, FOR GASTROINTESTINAL STROMAL TUMORS Pathology, University of Michigan, Ann Arbor, MI, USA Tumor-initiating cells have been suggested to be rare in many solid cancers Asuzu, David T., McGehee, Cordelia, Hayashi, Yujiro, Bardsley, but common in melanoma, raising the question of whether melanoma is Michael R., Lomberk, Gwen A., Urrutia, Raul A., Ordog, Tamas unique among solid cancers and what parameters influence tumorigenic Physiology & Biomedical Engineering, Mayo Clinic College of Medicine, cell frequency. We tested this in mouse malignant peripheral nerve sheath Rochester, MN, USA, Gastroenterology Research Unit, Mayo Clinic College tumors (MPNSTs). Mouse MPNSTs contained a similarly high frequency of Medicine, Rochester, MN, USA of tumorigenic cells irrespective of whether they were transplanted into Background & Aims: Gastrointestinal stromal tumors (GIST) are the most immuno-competent or immuno-compromised mice, demonstrating that as common sarcomas of the gastrointestinal tract. Most GIST arise from the long as transplants were performed among histocompatible mice, immune lineage of interstitial cells of Cajal (ICC), pacemaker and neuromodulator status was not a major determinant of tumorigenicity. In MPNSTs from cells of the gut, due to activating mutations in Kit receptor tyrosine kinase. Nf1+/-;Ink4a/Arf-/- mice, 18% of primary cells and 49% of serially trans- Pharmacological inhibition of Kit signaling with e.g. imatinib mesylate is planted cells formed tumors after transplantation. However, in MPNSTs from currently the only treatment option for metastatic GIST. However, this treat- Nf1/p53+/- mice only 1.8% to 2.6% of primary or serially transplanted ment is not curative. In murine GIST models we found that this is due in part cells formed tumors, demonstrating that the tumor genotype influenced to transformed Kitlow ICC stem cells (ICC-SC), which are insensitive to Kit the frequency of tumorigenic cells that could be detected in some assays. blockade and can serve as imatinib-resistant cancer stem cells. Polycomb Culture on laminin increased the frequency of tumorigenic MPNST cells from group (PcG) proteins are epigenetic master regulators of gene expression Nf1/p53+/- mice, but not from Nf1+/-;Ink4a/Arf-/- mice, to 19% by bind- and differentiation in stem cells. Our expression profiling studies in ICC-SC ing ß1-integrin-containing receptors. Cells with tumor-forming potential are indicated upregulation of 12 key PcG members and downregulation of 272 therefore common in mouse MPNSTs, but tumors of different genotypes, potential PcG target genes including Kit. Therefore, we hypothesized that even with the same malignant phenotype or histopathologic classification, Kit may be a PcG target in both normal and transformed ICC-SC, and that require different assay conditions to detect the full range of cells capable of the Kitlow phenotype of transformed ICC-SC can be reversed by disrupting contributing to cancer progression. PcG-mediated repression, thus sensitizing these cells to imatinib therapy. Methods: Freshly isolated and spontaneously transformed murine ICC-SC were studied. Expression of Kit and the PcG members Ezh2, Suz12 and Eed was analyzed by microarrays, RT-PCR, Western immunoblotting and immu- nocytochemistry. PcG activity was inhibited by RNAi-mediated knock-down of Suz12 and by pharmacological inhibition of Ezh2-mediated trimethylation of lysine 27 of histone 3 (H3K27me3) with adenosine dialdehyde (Adox). PcG protein and H3K27me3 occupancy of the Kit promoter (-1 to -750 bp) was detected by chromatin immunoprecipitation. Cell proliferation was measured by colorimetric MTS assay. Athymic NCr-nu/nu (nude) mice were injected s.c. with 5x106 transformed ICC-SC in growth factor-reduced

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Thursday Poster Abstracts

Poster Board Number: 2145 indicated by the low expression of several osteogenic differentiation marker genes. Bone morphogenic protein (BMP) family, especially BMP-2, plays MURINE HEMATOPOIETIC STEM CELLS ARE an important role in bone formation by promoting osteogenic differentia- ACTIVELY CYCLING. tion from progenitor cells. There are also evidences suggesting that several osteosarcoma cell lines produce large amount of Wnt inhibitor, Dickkopf- Goldberg, Laura R., Dooner, Mark S., Papa, Elaine, Pereira, Mandy, related protein 1 (dkk1), which inhibits osteogenic differentiation. The Del Tatto, Michael, Aliotta, Jason, Quesenberry, Peter J. present study aims at investigating the role of BMP-2 and dkk1 inhibitor Medicine, Warren Alpert Brown Medical School, Providence, RI, USA (anti-dkk1) in osteosarcoma cell line, Saos-2, undergoing normal osteogenic differentiation. Saos-2 cells were treated with 50 ng/ml of BMP2 and/or Background: It is widely believed that hematopoietic stem cells (HSCs) exist 1 μg/ml of anti-dkk1. The degree of osteogenic differentiation of Saos-2 predominantly in a dormant state. Indeed, it has been shown that highly cells after BMP-2 and anti-dkk1 treatment was assessed by measuring the purified long-term hematopoietic stem cells (LTHSC) repopulate the bone alkaline phosphatase activity, gene expressions and matrix mineralization, marrow of irradiated hosts when they are primarily in a quiescent, G0 which represent early and terminal differentiation markers of osteoblasts. state. However, current HSC purification protocols are often biased toward The combination of BMP2 and anti-dkk1 could significantly increase the isolation of dormant cells, rendering studies on highly purified HSC popula- alkaline phosphatase activity, gene expressions and matrix mineralization tions potentially misleading with regard to cell cycle status. In the studies in Saos-2 cells compared with controls. In conclusion, BMP2 and anti-dkk1 presented here, we explored the cell cycle status of the total population of could induce the differentiation of poorly differentiated osteosarcoma cells HSCs in unseparated whole bone marrow (WBM) and found a large popula- Saos-2 toward osteocytes. These findings might have an implication for the tion of actively cycling HSCs capable of long-term multilineage engraftment. development of effective strategy for osteosarcoma treatment in the future. Methods: WBM was either separated into cell cycle-specific fractions using Hoechst 33342/Pyronin Y or exposed to tritiated thymidine suicide and Poster Board Number: 2149 then competitively engrafted into lethally irradiated mice. Percent donor chimerism was measured using flow cytometry to evaluate donor engraft- TOXICITY OF HEAVY METALS IN STEM CELLS ment. Results: These studies revealed a large percentage of actively cycling Ciavattone, Nicholas, Maurino, Christopher, Mortens, Daniel, cells capable of long-term multi-lineage engraftment. In some studies, McKee, Christina, Bloda, Martin, Dinda, Sumi, Chaudhry, G. Rasul over 50% of the long-term engrafting cells were in S/G2/M. In addition, we are studying the kinetics of LTHSC flux through cell cycle in vivo using Dept of Biological Sciences, Oakland University, Rochester, MI, USA, bromodeoxyuridine (BrdU). Our preliminary data show that the percentage School of Health Sciences, Oakland University, Rochester, MI, USA of LTHSC that have progressed through S-phase and returned to G0/G1 Every year millions of people are exposed to toxic and carcinogenic heavy within 24 and 48 hours was 43% and 86%, respectively. This suggests that, metals through contaminated food and water or workplace hazards. These although highly purified LTHSC predominantly engraft while in a quiescent contaminants can cause a wide range of health problems including meta- state, this same population of cells are actually actively cycling in vivo with bolic defects, diseases and even cancers. For example, chronic exposure of relatively rapid kinetics. Conclusions: These data show that hematopoietic arsenic is known to cause transformation of cells. However, little is known stem cells found in unseparated marrow are an actively cycling population. about the effect of heavy metals on the early development and differentia- These cycling stem cells are continually changing phenotype as they transit tion processes. We hypothesized that stem cells that mimic embryonic devel- cell cycle and would therefore be inaccurately represented by current HSC opment can be used to study the hazardous effects of heavy metals such as purification strategies that rely on isolating cell populations based on stable arsenic, nickel, mercury, cadmium and chromium. In our preliminary studies epitope expression. In addition such phenotypic variability during cell cycle arsenic was found be cytotoxic at concentration 1x10-6 M and no cell sur- progression, in combination with stimuli from the microenvironment includ- vived at 20x10-6 M and higher, while cadmium and mercury inhibited cell ing cytokines and microvesicles, likely plays an important role in stem cell growth started at 1x10-8 M and higher. Exposure of nickel displayed toxicity plasticity and HSC fate determination. only at concentration 2x10-6 M. The cells exposed to these metals also Poster Board Number: 2147 displayed differentiation, particularly when exposed to arsenic for a period longer than 2 weeks. More chronic exposure to arsenic and mercury lead to BMP2 AND dkk1 INHIBITOR INDUCE detachment and rounding up of cells. Studies are underway to characterize the potential differentiation and molecular changes that resulted due to their OSTEOGENIC DIFFERENTIATION OF chronic exposure to the metals, results will be presented. OSTEOSARCOMA CELLS Chayosumrit, Methichit, Phetfong, Jitrada, Supokawej, Aungkura, Pakpoom, Kheolamai, U-Pratya, Yaowalak, Manochantr, Sirikul, Tantrawatpan, Chairat, Issaragrisil, Surapol Siriraj Excellence Center for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand, Department of Clinical Microscopy, Faculty of Medical Technology, Mahidol University, Bangkok, Thailand, Division of Cell Biology, Department of Pre-clinical Sciences, Faculty of Medicine, Thammasat University, Pathumthani, Thailand, Division of Hematology, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand Osteosarcoma is the most common malignant bone tumor consisting of poorly differentiated cells, which are highly invasive. During the last decade, osteosarcoma cell lines have been used as a cellular model to study the biology of osteoblasts and osteocytes. However, the accumulated evidence suggests that the phenotypic characteristics and the molecular mechanisms, which regulate the biology of osteosarcoma cells, are radically different from those of normal osteoblasts. The osteosarcoma cell is poorly differentiated as

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ysis reveal that the putative germ cell isolated from differentiating hESCs ex- GERMLINE CELLS press germ cell markers, including VASA, STELLA, DAZL and meiotic markers Poster Board Number: 2153 SCP1 and SCP3. Further, fluorescence in situ hybridization (FISH) analysis confirms the haploid status of the hESC-derived germ cells. Together, our re- THE TRANSDIFFERENTIATION OF THE HUMAN sults demonstrate that the combination of marker selection and growth fac- tor regime can successfully lead to the generation of highly-enriched human FORESKIN FIBROBLASTS TO FORM GERM meiotic germ cells that can be further used to generate mature germ cells as CELLS USING RETINOIC ACID well as providing opportunity to dissect the signaling pathway important for germ cell formation, development and niche-germ cell interaction. Aflatoonian, Behrouz,Sadeghian, Fatemeh, Fesahat, Farzaneh, Khorad-Mehr, Arezoo, Janan, Arghavan, Aflatoonian, Reza, Poster Board Number: 2157 Aflatoonian, Abbass GERMLINE DIFFERENTIATION OF HUMAN Centre for Stem Cell Biology, Yazd Institute for Reproductive Biomedical Sciences, Yazd, Iran, Islamic Republic of, Department of Endocrinology and EMBRYONIC STEM CELLS Female Infertility, Royan Institute for Reproductive Biomedicine, Tehran, Panula, Sarita P., Takahashi, Kazutoshi, Nakamura, Michiko, Iran, Islamic Republic of Hovatta, Outi, Reijo Pera, Renee A., Yamanaka, Shinya Fibroblasts are the main cells of stromal tissue and play a crucial role in Center for iPS Cell Research and Application, Kyoto University, Kyoto, regenerating of damaged organs. Here we report preliminary data that hu- Japan, Department of Clinical Science, Intervention and Technology, man foreskin fibroblasts (HFFs) after inducing with Retinoic Acid (RA) reveal Karolinska Institutet, Stockholm, Sweden, Institute for Stem Cell Biology & gene expression pattern characteristic of the germ cells. HFF cell lines were Regenerative Medicine, Stanford University, Stanford, CA, USA derived by mechanically and enzymatically (Collagenase I and IV) treating of human neonatal foreskin samples and culturing the cells in DMEM/FBS in Germ cells are crucially important for any species that multiplies through T25 flasks. After several passages, HFFs were cultured with germ-line stem sexual reproduction as they are in charge of transmitting the genetic infor- cell media 1 (GSC-M1) which is consistent of; DEME/F12, knock-out serum mation and therefore functioning as a link between generations. In humans, replacement (KO-SR), basic fibroblast growth factor (bFGF) and RA. HFFs germ cell specification, proliferation and early differentiation begins shortly were cultured in GSC-M1 for a week before RNA extraction for RT-PCR. In after the implantation of the embryo. Thus, the analysis of early germ cell parallel, cells from testicular sperm extraction (TESE) samples were cultured differentiation in vivo is difficult and impracticable due to ethical consid- in similar condition as a control group. RT-PCR results of germ cell specific erations. The ability to drive germline differentiation of human embryonic gene expression for TESE-derived cells and HFFs cultured in GSC-M1 were stem cells (hESCs) offers a valuable tool to study development and disease. fairly comparable. In this study we looked at the expression of NANOG, Efficient differentiation of hESCs would make the otherwise rare germ cell SOX2, AFP, STAT3, DAZL, VASA, STELLA, BETA-ACTIN and GAPDH genes. populations accessible for studies of germ cell development. Current dif- Our preliminary data shows that RA may have a role to induce transdiffer- ferentiation methods rely heavily on the usage of fetal bovine serum (FBS), entiation of HFFs to germ cells which need to be proved by other techniques which creates undefined conditions vulnerable for batch-to-batch variation such as immuno-fluorescent (IF) localization and flow-cytometry, to isolate and exposes the human cells to animal components. However, serum-free the cells and study the morphology of the HFF-derived germ cells. differentiation has been shown to decrease the efficiency considerably. Here, we sought to find an efficient, chemically defined method to differentiate Poster Board Number: 2155 hESCs towards the germ cell lineage. We cultured hESCs on Matrigel-coated plates with serum-free mTeSR1 medium and tested the effect of bone MEIOTIC GERM CELLS ENRICHED FROM morphogenetic protein 4 (BMP4), forskolin, retinoic acid and basic fibroblast DIFFERENTIATING HUMAN EMBRYONIC STEM growth factor (bFGF) on germ cell differentiation. We found that only after 7 days of exposure, retinoic acid and bFGF together increased the expression CELLS VIA TRANSGENIC MANIPULATION, of germ cell specific VASA gene 5-fold. To isolate and further analyze these SURFACE MARKER SELECTION AND GROWTH putative germ cells, we generated a hESC reporter line expressing GFP under FACTOR STIMULATION the control of the human VASA promoter, using bacterial artificial chromo- some (BAC) transgenesis. Our preliminary results show that about 5 % of Lin, I-Ying, Chuang, Ching-Yu, Lin, Kuo-I, Kuo, Hung-Chih the hESCs cultured in our serum-free conditions are GFP positive, analyzed Genomic Research Center, Academia Sinica, Taipei, Taiwan by flow cytometry. Thus, reaching similar efficiency as reported for FBS in- duced differentiation. The reporter cell line provides a powerful tool to study Human embryonic stem cells (hESCs) are currently the only system available human germ cell development in vitro and allows the isolation of putative for studying early human development experimentally. While the transgenic germ cells from the heterogeneous differentiation population for further mouse ES cells have been broadly used for understanding gene function and analysis. Thus, offering new insights into the poorly understood human fer- the mechanism underpinning mouse germline development, the demon- tility. Furthermore, our novel serum-free differentiation method offers a step stration of utilization of transgenic hESCs in the aforementioned purposes towards clinically relevant system that may ultimately be used as a treatment is still limited due to the difficulty of transferring transgene into hESCs and for various infertility causes. the subsequent clonal selection. Apart from the transgenic approach, the only way to identify human germ cell from differentiated hESCs is through surface marker selection. Here, we describe the generation of OCT4-eGFP reporter hESCs and demonstrated its possibility to isolate early germ cells by this system. Surface marker profiling on the differentiated OCT4-eGFP+ cells has led us to identify novel gemline surface markers that highly expressed in human fetal gonads and allow in vitro tracking of germline lineages during in vitro differentiation of hESCs. Furthermore, the Oct4-eGFP transgenic hESC system has allowed us to compare the effect of different growth fac- tors/cytokines on the germ line differentiation of hESCs. Combination of marker selection and growth factor stimulation has significantly improved the yield of germline lineage from differentiating hESCs. IF and RT-PCR anal-

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Poster Board Number: 2159 (pre-puberty) and 8-weeks (adult) old BALB-C type of mice were used. For the investigation of the presence of possible stem cells, the expression CHARACTERIZATION OF A NOVEL LIF- profiles of three well known stem cell markers, Oct-4, Nanog and Sox2 were RESPONSIVE, EMBRYONIC-LIKE, PRIMITIVE determined in the ovaries of two different age groups by real time RT-PCR (qRT-PCR). Protein expression levels and their localization in the ovary cells NEURAL STEM CELL IN THE ADULT MOUSE were immunohistochemically evaluated on fresh-frozen ovary tissue sections BRAIN REVEALED FOLLOWING THE TARGETED by using monoclonal antibodies specific to Sox2, Nanog and Oct-4. The ABLATION OF GFAP EXPRESSING ADULT gene expression levels of Oct-4 and Nanog were found to be significantly differentiated between 2-weeks old and 8-weeks old mice. On the other NEURAL STEM CELLS. hand no significant difference was observed in the expression level of Sox2 Xu, Wenjun, Sachewsky, Nadia, Rose, Keeley, Leeder, Rachel, van between two age groups. Immunohistochemical results showed the pres- ence of both Sox2 and Oct-4 protein in the cytoplasm of ovarian epithelial der Kooy, Derek, Morshead, Cindi cells, granulosa cells, oocytes and theca cells. Nanog protein was observed University of Toronto, Toronto, ON, Canada only in the nucleus of the oocytes and additionally the expression of Nanog Neural stem cells (NSCs) in the adult forebrain are thought to comprise a was higher in eight weeks samples compared to two weeks old ones which subpopulation of GFAP expressing subependymal (SE) cells. In vivo, GFAP+ corresponds to the qRT-PCR results. These results suggest for the first time NSCs proliferate and generate progeny that proliferate and migrate along that Nanog protein is expressed both in adult and pre-puberty mouse ova- the rostral migratory stream to the olfactory bulb (OB) where they differ- ries and locate at the nucleus of oocyte and to the best of our knowledge entiate into interneurons. In vitro, GFAP+ NSCs give rise to clonally derived this is the first study that shows the differential expression of Oct-4, Nanog colonies of stem and progenitor cells termed neurospheres. During develop- and Sox2 in pre-puberty and adult mouse ovaries by qRT-PCR. Collectively ment, the neural stem cell lineage includes a LIF-responsive NSC (primitive our results may suggest that both pre-puberty and adult mice ovaries ac- NSC, pNSC) as early as E5.5 in the developing embryo that gives rise to a commodate cells carrying stem cell features. FGF dependent definitive NSC by E8.5. Herein we describe the isolation Poster Board Number: 2163 and characterization of a novel LIF-responsive cell in the periventricular region of the adult forebrain that can generate self-renewing, multipotent CROSSTALK OF HYPOXIA AND INSULIN-LIKE free-floating colonies in vitro in the presence of LIF alone. The adult derived LIF-responsive cell is extremely rare, with a frequency of ~5 colonies per GROWTH FACTOR-1 RECEPTOR SIGNALING mouse forebrain; however, the population can be increased up to 4 fold IN SELF-RENEWAL PROLIFERATION AND following a stroke lesion or intraventricular infusion of LIF. The adult derived MIGRATION OF PLURIPOTENT MOUSE LIF-responsive cell does not express GFAP but gives rise to GFAP express- ing NSCs as LIF colonies can be passaged into EGF/FGF. We have shown GERMLINE STEM CELLS that these adult LIF-responsive cells express the pluripotency marker Oct4 Huang, Yen-Hua, Ling, Thai-Yen both in vitro and in vivo (by PCR, immunohistochemistry and transgenic Dept Biochemistry, Department of Biochemistry, School of Medicine, Taipei mouse models). Further, LIF-responsive cells are capable of integration into Medical University, Taipei, Taiwan, Institute of Pharmacology, College of the inner cell mass of blastocyst chimeras. We hypothesize that these LIFR+ Medicine, National Taiwan University, Taipei, Taiwan cells are lineally related to the neurogenic, GFAP+ neurosphere forming cells. Using a transgenic GFAP-tk mouse model that permits the selective ablation Recent studies using a transgenic mice model have demonstrated that of proliferating GFAP+ cells in the presence of ganciclovir (GCV), we have knockout of hypoxia inducible factor 2α (HIF-2α-/-) decreases primordial demonstrated that GFAP+ NSCs can be completely lost in numerous ablation germ cell (PGC) survival. However, little is known about hypoxia-interacting paradigms but invariably, neurosphere formation in vitro, and proliferating endocrinal regulation of PGC development. Herein, we demonstrate a neuroblasts in vivo, return with longer survival times. These findings support crosstalk of hypoxia and endocrinal insulin-like growth factor-1 receptor the hypothesis that the LIF-responsive cell is repopulating the GFAP+ adult (IGF-1R)/Akt signaling in pluripotent germ cell self-renewal proliferation and NSC population. To definitively address the lineal relationship, LIF colonies migration. We previously generated PGC-like mouse alkaline-posphatase- grown from YFPxGFAP-tk mice in the presence of GCV (thereby eliminating positive germline stem cells (AP+GSCs) in vitro using a serum-free cul- GFAP+ adult NSCs) have been transplanted into the anterior SE. We predict ture system. Hypoxia greatly increased the colony size, Cyclin D1/c-Myc that YFP+ cells will be observed in the in the OB over time. These findings expression, and nuclear BrdU+ cell population of AP+GSCs. As well, hypoxia will confirm the lineage relationship between the LIF-responsive cells and enhanced the stemness (Oct-4, Sox2, Nanog, Klf-4) and nuclear Oct-4/ the well characterized adult NSCs and support the hypothesis that a LIF HIF-2α expression. Importantly, hypoxia significantly increased secreted- responsive cell is upstream of the neurosphere forming, GFAP+ adult NSC. IGF-1 production, activated IGF-1R/Akt signaling, and enhanced migration of AP+GSCs. PPP (specific IGF-1R phosphorylation inhibitor) and LY294002 Poster Board Number: 2161 (specific PI3K inhibitor) significantly suppressed the hypoxia-induced HIF-2α INVESTIGATION OF STEM CELLS IN ADULT and Oct-4 expressions and cell migration. A mouse renal capsule engraft model revealed the hypoxic AP+GSCs successfully differentiating into c-Kit+, AND PRE-ADULT MOUSE OVARIES Vasa+, and Scp3+ spermatozoa-like cells in vivo. In conclusion, the present Esmaeilian, Yashar, Gur Dedeoglu, Bala, Atalay, Arzu, Erdemli, Esra study demonstrates a crosstalk of hypoxia and endocrinal IGF-1R/Akt sig- naling in activation of self-renewal proliferation and migration of PGC-like Biotechnology Institute-Ankara University, Ankara, Turkey, Histology- mouse AP+GSCs. This finding provides insights into the niche endocrinology Embryology, Medical Faculty-Ankara University, Ankara, Turkey underlying early pluripotent germ cell development. The progress in stem cell research has changed the knowledge of formation and development of human oocyte which was acceptable until recently. The possible presence of oocyte and granulosa cells originated from stem cells in the adult mammalian ovaries was claimed by some studies which will lead to major changes in reproductive biology and infertility treatments. Purpose of this research is to investigate the possible existence and the location of the potential stem cells in mouse ovaries. In this study the ovaries from 2-weeks

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Poster Board Number: 2165 spindle orientation relative to the surrounding tissue is a strategy utilized by many adult stem cells to divide asymmetrically. Drosophila melanogaster IMPACT OF A SINGLE DOSE OF 12 GY male germline stem cell (GSC) system is a premier model to study stem cell- IRRADIATION ON BOVINE SPERMATOGONIAL niche interactions in vivo because of its simple anatomy, defined signaling and cellular markers. Somatic hub cells constitute the GSC niche and provide STEM CELL SURVIVAL AND RECOVERY the fundamental signals required for maintenance of stem cell identity. Herrid, Muren, Davey, Rhonda, Stockwell, Sally, Schmoelzl, Sabine, In the asymmetric division of GSCs, one daughter cell that maintains the Uphill, Grant, Poirier, Valerie, Hope, Shelly, Hill, Jonathan, Holland, contact with the hub retains the stem cell identity, while the other that is displaced away from the hub initiates differentiation. This is achieved by Michael, Lehnert, Sigrid proper centrosome and spindle orientation perpendicular to the hub. We re- CSIRO Food Futures National Research Flagship, Armidale, NSW, Australia, cently demonstrated that GSCs with mis-oriented centrosomes do not enter CSIRO Food Futures National Research Flagship, Brisbane, QLD, Australia, mitosis until their centrosomes are re-oriented, pointing to the presence of Brisbane Veterinary Specialist Clinic, Brisbane, QLD, Australia, School of a checkpoint mechanism that monitors interphase centrosome orientation Veterinary Science, University of Queensland, Australia, Brisbane, QLD, prior to mitosis (the centrosome orientation checkpoint). Furthermore, we Australia identified Par-1 kinase as a key component of this checkpoint mechanism. In Transplantation of testis stem cell populations has broad applications from this study, we demonstrate a novel function of a polarity protein Bazooka/ treatment of infertility in males with testicular cancer to commercial applica- Par-3 in the centrosome capturing (“centrosome engagement”), which ap- tions in breeding of livestock. In livestock, testis germ cells can be trans- pears to be monitored by the centrosome orientation checkpoint. Bazooka planted between individual animals and even across breeds. The success rate is localized at the hub-GSC interface forming a small “patch“ structure, in livestock is low, with only a small percentage of donor spermatozoa in the which closely associates (“engages”) with the apical centrosome in the late recipient’s semen. This may be improved by the availability of vacant stem G2 cells right before mitotic entry. We also present evidence that Khc-73, a cell niches induced by irradiation of the testis of the recipient bull before plus-end-directed kinesin heavy chain, plays a critical role in the centrosome- stem cell transplantation. In this study, the effect of a single dose of radia- Baz engagement. Together, we propose that the correct orientation of the tion on depletion of testis cells and recovery of sperm production in recipient centrosome is monitored by the engagement between the Bazooka patch, testis was investigated. The testes of recipient bulls at the pre-pubertal stage which is localized to the hub-GSC interface, and the apical centrosome. were treated with a single dose of 12 Gray (Gy) irradiation followed by germ Poster Board Number: 2169 cell transplantation either 4 or 8 weeks later in two individual experiments. In both experiments, biopsy samples of testes tissue were collected and pro- THE TRIM-NHL PROTEIN MEI-P26 REGULATES cessed for immunohistology at various time points following irradiation and germ cell transfer. The body weight and scrotal circumferences (SC) were BOTH GERMLINE STEM CELL SELF-RENEWAL recorded at biopsy. The testis sections were stained with different antibodies AND DIFFERENTIATION THROUGH DISTINCT to assess the proportion and absolute numbers of stem and Sertoli cells and MECHANISMS tubule diameter (µm) was measured. Histological results from both experi- ments showed an irreversible degeneration of seminiferous tubules. From Li, Yun, Maines, Jean, McKearin, Dennis, Buszczak, Michael 2 weeks post irradiation, a dramatic reduction in tubule diameter (50%), Molecular Biology, UT Southwestern Medical Center, Dallas, TX, USA, numbers of spermatogonia (30%-70%) and Sertoli cell numbers (70%) was Howard Hughes Medical Institute, Chevy Chase, MD, USA observed. This reduction was sustained for up to 20 weeks post irradiation. This observation has serious implications for the availability of functional In the Drosophila female germline, the selective translational repression of stem cell niches. Transplantation at a longer interval between irradiation and specific mRNAs governs both stem cell maintenance and the differentiation transplantation (8 weeks) had lower negative impact on the growth of the of their progeny. However, the exact mechanisms that control and coordi- scrotal circumference than transplantation at a shorter interval (4 weeks). nate different modes of translational repression in this lineage remain poorly After the bull calves reached puberty (15-20 months after transplantation), understood. TRIM-NHL domain proteins have emerged as critical regula- semen from ten recipients in two experiments was collected to test for tors of translation. Here we reinvestigate the function of the Drosophila transplantation success (detection of DNA from transplanted stem cells using TRIM-NHL protein Mei-P26 in ovarian germline stem cells (GSCs) and their microsatellite markers). None of the semen samples were found to contain differentiating daughters. We find loss of mei-P26 results in GSC deple- donor DNA in the ejaculates, indicating the microenvironments of recipi- tion despite a downstream block in differentiation. Within GSCs, Mei-P26 ent testis treated with a single dose of 12 Gy irradiation are not optimal for associates with the miRNA pathway components Ago1 and GW182. Both supporting donor stem cell colonization and/or proliferation. It is possible Mei-P26 and Ago1 associate with orb mRNA and repress its translation in that this radiation dose, delivered to the prepubertal bull testis, may have GSCs, suggesting Mei-P26 promotes miRNA mediated silencing in specific damaged the stem cell niches irreversibly. Due to practical considerations, contexts. Within early differentiating stem cell daughters, miRNA pathway irradiation of bulls at a later developmental stage is not feasible, therefore activity ceases and Mei-P26 becomes repurposed in a miRNA independent lower doses of radiation are currently under evaluation. translational repression complex. We find Mei-P26 associates with Bam, Bgcn and Sxl and represses the translation of nanos and orb mRNA dur- Poster Board Number: 2167 ing early cyst development. Our data support a model in which Mei-P26 mediates both GSC self-renewal and GSC daughter differentiation through FUNCTION OF BAZOOKA/PAR3 IN distinct modes of translational repression that depend on its differential as- ASYMMETRIC STEM CELL DIVISION AND THE sociation with Bam. CENTROSOME ORIENTATION CHECKPOINT IN DROSOPHILA MALE GERMLINE STEM CELLS Inaba, Mayu, Yamashita, Yukiko Life Science Institute, University of Michigan, Ann Arbor, MI, USA Asymmetric cell division is widely utilized by many adult stem cells to bal- ance self-renewal and generation of differentiated, short-lived cells. Mitotic

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Thursday Poster Abstracts

TECHNOLOGIES FOR STEM CELL RESEARCH Poster Board Number: 2173 Poster Board Number: 2171 DIAGNOSTIC MICROBIOREACTOR ARRAYS FOR MULTIPLEXED MICROENVIRONMENTAL A MULTI-STEP APPROACH FOR TARGETED SCREENING OF PLURIPOTENT STEM CORRECTION OF THE ENDOGENOUS MUTATED CELL EXPANSION, MAINTENANCE AND GENE IN SCID-X1 FIBROBLASTS AND DIFFERENTIATION GENERATION OF REPROGRAMMING FACTOR- Titmarsh, Drew, Hudson, James, Hidalgo-Gonzalez, Alejandro, FREE IPS CELLS. Turner, Jennifer, Wolvetang, Ernst, Cooper-White, Justin J. Lombardo, Angelo, Lo Riso, Pietro, Di Stefano, Bruno, Amendola, Australian Institute for Bioengineering and Nanotechnology, The University Mario, Holmes, Michael C., Gregory, Philip D., Gennery, Andrew R., of Queensland, Brisbane, Queensland, Australia, Australian Institute for Broccoli, Vania, Naldini, Luigi Bioengineering and Nanotechnology and, The University of Queensland, Brisbane, Queensland, Australia San Raffaele Telethon Institute for Gene Therapy and Vita-Salute San Raffaele University, Milan, Italy, Stem Cell and Neurogenesis Unit, San Self-renewal and differentiation of stem cells are processes which depend on Raffaele Institute, Milan, Italy, San Raffaele Telethon Institute for Gene a delicate balance of key signalling pathways. Accurate control of these fates Therapy, Milan, Italy, Sangamo BioSciences Inc., Richmond, CA, USA, is paramount for cellular therapies based on stem cells, however, this is lim- Newcastle Upon Tyne Hospital, Newcastle Upon Tyne, United Kingdom ited by several factors, including the use of undefined media and substrata, autocrine/paracrine action of cultured cells, temporal variation in microen- Patient-derived, gene-corrected induced Pluripotent Stem Cells (iPSCs) have vironmental composition, and downstream cross-talk between signalling the potential to substitute for autologous cells in the treatment of several pathways. To address these issues while progressing towards highly-con- human genetic diseases, particularly those in which the isolation, ex vivo trolled, multiplexed screening platforms for stem cells, we have fabricated a expansion and/or genetic correction of target cells are technically challeng- scalable, full-factorial microbioreactor array generating every combination ing. To date, the most efficient approach to derive and gene-correct iPSCs of 3 concentrations each of 3 soluble factors (27 discrete culture conditions exploits randomly integrating vectors, which, however, have the following in total). This was combined with serial replication of 250 μm-high culture limitations: i) uncontrolled expression of the therapeutic transgene; ii) stable chambers to present 270 culture wells in a 23 × 63 mm area. This platform transfer of the oncogenic transcription factors used for reprogramming has been combined with in situ immunofluorescence and validated EOS and and their potential re-activation in the iPSC progeny; iii) the potential for MIXL1 reporter hESC lines to create a rapid screening platform under com- insertional mutagenesis. We have addressed these issues by providing proof- pletely defined surface and soluble conditions, with accurate spatiotemporal of-principle of a combined strategy that allows for targeted correction of the control of the cellular microenvironment. MEL-2 human embryonic stem endogenous malfunctioning gene and safe reprogramming of patient cells. cells (hESC) were screened for maintenance of pluripotency markers TG30 SCID-X1 (caused by mutations in the IL2RG gene) was chosen as a model and Oct-4 against b-FGF and TGF- 1 in a chemically-defined medium back- to assess the feasibility of this approach, as HSC-based gene therapy trials β ground, with retinoic acid included as an internal pro-differentiation control. showed major clinical benefits but also unacceptable rate of leukemia due to Factorial analysis revealed the main and interaction effects of the supplied insertional mutagenesis and uncontrolled transgene expression. Using Zinc factors on pluripotency marker expression, which was also strongly depen- Finger Nucleases (ZFNs) targeting the human IL2RG gene, we knocked- dent on sequential position within a column of serial culture chambers. The in a functional IL2RG cDNA downstream of its endogenous promoter in markers were more highly-expressed in the initial chambers, and expres- B-lymphoblastoid cells and primary fibroblasts from normal male donors sion was attenuated in downstream chambers, an effect which is thought and SCID-X1 patients. To select gene corrected fibroblasts, which do not to be best explained by accumulation of paracrine factors which negatively express IL2RG, we included downstream of the IL2RG cDNA a LoxP-flanked modulate pluripotency. The microbioreactor array was then utilised to inves- GFP expression cassette driven by the retrovirus-derived SFFV promoter. tigate differentiation of hESCs to a MIXL1+, primitive streak-like population. Molecular analyses performed on the GFP+ sorted cell pools and 30 single A MIXL1 gene reporter was activated in specific combinations of BMP-4, cell-derived clones confirmed targeted integration into IL2RG. Importantly, Activin A, and BIO (a canonical Wnt activator) treatment, and was addition- FACS and RT-PCR analyses performed on the B-lymphoblastoid cells showed ally dependent on the position within a column of serial culture chambers. physiologic expression of the edited IL2RG gene. For fibroblast reprogram- Significant expression was not observed in the initial chambers, regardless ming to iPSC, we have developed a novel lentiviral vector (LV) containing of the factors supplied to cells, but was activated effectively in downstream LoxP sites in the LTR and expressing the human OCT4, KLF4 and SOX2 chambers within the device for certain conditions of supplied factors. genes from a mono-cistron that was recoded for robust expression, allowing Strikingly, this suggests accumulation of paracrine factors was required to for single-copy LV reprogramming. Unedited and gene-corrected SCID-X1 efficiently activate MIXL1 expression. Direct action by BMP, Activin and/ fibroblasts transduced at single-copy with this LV were reprogrammed at or canonical Wnt signals was not sufficient to activate robust expression. efficiencies (up to 0.03%) comparable to those obtained with standard Inhibitor experiments in static culture also identified non-canonical Wnts as multiple-copies retroviral vectors. Derived iPSC clones homogeneously candidates for paracrine effectors of MIXL1 activation. This work showed expressed pluripotency markers and contained on average 1.2 LV copies per the utility of the microbioreactor array for dissecting both pluripotency and cell. Upon transient Cre delivery, the reprogramming LV and the GFP cas- differentiation processes starting with a “blank slate” soluble microenviron- sette were excised from ~95% of the iPSC clones. In conclusion, these data ment free of paracrine signals, then allowing accumulation of paracrine show the feasibility of generating gene-corrected, vector- and reprogram- signals along spatially-separated culture chambers - a feature not available ming factor-free iPS cells from SCID-X1 patients, thus alleviating the risks in static culture protocols. This approach thus enables the discernment of associated with integrating vectors and paving the way to the development direct-acting soluble factors, which is essential to comprehensively decipher of safer iPSCs for regenerative medicine. microenvironmental control of stem cell fate and improve the efficiency of directed differentiation protocols, ultimately unlocking the future potential of pluripotent stem cells.

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Poster Board Number: 2175 of 32-47 hrs. Cells cultured on Synthemax Surface yielded similar cell num- ber, viability, and morphology to cells cultured on the freshly coated laminin A NEW EXTRACELLULAR MATRIX FOR FEEDER- control. Importantly, cells retained normal karyotype, high levels of pheno- FREE GROWTH OF PLURIPOTENT STEM CELLS typic markers (nestin and musashi) and ability to differentiate into neurons and astrocytes after long-term multi-passage culture on Synthemax Surface. Dvash, Tamar, Dodla, Mahesh, Zhong, Bonan, Lyons, Ian Interestingly, cells grown on laminin but not Synthemax Surface began to R&D, Sigma-Aldrich, St. Louis, MO, USA show a steady decline in nestin/musashi positive cell population around passage 7, indicating more permissive conditions for long-term expansion of Pluripotent stem cells hold great promise for application in regenerative multipotent NSC population on Synthemax Surface. Our results suggest that medicine. Derivatives of patient- or disease-specific induced pluripotent Synthemax Surface, in combination with serum-free medium, provides a stem cells (iPSCs) may also provide valuable tools for toxicity assays and complete solution for expansion and differentiation of NSC under xeno-free, for pharmaceuticals development. Homogeneity of undifferentiated iPSC defined culture conditions. We believe that Synthemax Surface will be useful culture is required for efficient differentiation, and requires culture systems for both research purposes and therapeutic applications of NSC. able to maintain stable expansion of undifferentiated cells over time. Mitoti- cally inactive mouse embryonic fibroblasts (MEFs) were initially and are Poster Board Number: 2179 still used as attachment surfaces for iPSCs, however incomplete removal of MEFs may adversely influence differentiation. Here, we report an alterna- SYNTHETIC SURFACES FOR THE CULTURE OF tive and inexpensive extracellular matrix, which primarily contains laminin, HUMAN EMBRYONIC AND ADULT STEM CELLS collagen type IV, heparan sulfate proteoglycan and entactin generated from Engelbreth-Holm-Swarm murine sarcoma (ECM gel, Cat.1270 Sigma). We Sams, Alexandria, Li, Zhensheng, Qunitanilla, Rene H., Powers, show that this new substrate stably supports the growth of undifferentiated Mark, Lakshmipathy, Uma human embryonic stem cells and multiple lines of iPSCs in conjunction with Life Technologies, Frederick, MD, USA, Life Technologies, Carlsbad, CA, mTeSR1 medium. Pluripotent stem cells grown on this matrix have typical USA morphology and expression of stem cell markers (TRA1-81, TRA1-60, SSEA3, SSEA4, Oct4). Additionally human ES cells and iPSCs cultured on this Utility of stem cells for downstream clinical applications primarily rely on the ECM gel robustly proliferate over 15 passages with no evidence of genetic ability to expand cells in large scale under defined media and matrix condi- abnormality, and exhibit good differentiation capacity into multiple lineages tions. To address that need, several chemically defined media systems have in vitro. The consistency and low cost of this commercially available ECM been reported for the maintenance and proliferation of pluripotent stem 1270 provides a useful platform for large scale expansion of iPSCs towards cells. An equally critical component is the use of an extracellular matrix that various applications. is animal-origin free and chemically defined. An ideal synthetic matrix would not only create a microenvironment ideal for stem cell expansion but would Poster Board Number: 2177 also be modular such that it can be adapted to support robust differentia- tion into lineage of choice. Using a combination of various biomaterials NOVEL SYNTHETIC, XENO-FREE SURFACE with unique mechanical and thermal properties attached via novel linkers FOR EXPANSION AND DIFFERENTIATION OF to functional biomolecules, novel coated surfaces can be created. We have evaluated several defined synthetic surfaces in comparison to traditional HUMAN NEURAL STEM CELLS IN SERUM-FREE ECM molecules. Cell based assays were adapted to monitor efficiency of cell MEDIUM. attachment to specific surfaces in a high-throughput format. Matrices that supported efficient attachment of ESC were further evaluated for prolifera- Melkoumian, Zara, Baker, Wendy A., Pai, Sadashiva K., Dolley- tion. ESC expanded on experimental matrices for multiple passages were Sonneville, Paula J., Petzold, Petzold N. assessed for pluripotency based on surface marker expression and ability to Life Sciences, Corning Incorporated, Corning, NY, USA differentiate into the three representative germ layers. Using the estab- Human neural stem cells (NSC) are multipotent progenitor cells with the lished streamlined process of evaluation, evaluation was extended to novel ability to differentiate into several cell types, such as neurons, oligodendo- biomaterials with unique mechanical and thermal properties for 2 and 3 cytes, and astrocytes. There is great interest in NSCs as a source for special- dimensional cell culture. ized cells for basic neuroscience, drug discovery applications, and cell based Poster Board Number: 2181 therapies for a variety of neurodegenerative diseases including Alzheimer’s, Huntington’s, and Parkinson’s. Neural stem cells can be expanded in vitro APPLICATION OF SYNTHETIC MEDIUM AND either as adherent monolayers or neurospheres. Adherent NSC cultures GMP COMPLIANT HUMAN FEEDER CELLS IN require pre-coating of culture surfaces with biological materials, such as laminin or CELLstart™ to enable cell adhesion in serum-free conditions. THE MAINTENANCE, EXPANSION AND CLONAL These biological materials have limited shelf life, lot-to-lot variability and the RECOVERY OF HUMAN EMBRYONIC STEM potential for contamination with adventitious agents. To address the limita- tions of biological growth substrates, Corning Life Sciences, in collaboration CELLS. with Geron Corporation, developed a novel, xeno-free, synthetic surface, McRae, Scott, Ferguson, Linda, Prathalingam, Nilendran, Herbert, the Corning® Synthemax™ Surface, for stem cell culture applications. This Mary, Jones, Michael surface is comprised of an acrylate polymer, functionalized with a short Cell Guidance Systems, Cambridge, United Kingdom, Newcastle University, peptide sequence derived from the vitronectin protein, to mimic biological Newcastle, United Kingdom, Newcastle University, Newcastle, United ligands for cell adhesion. Our earlier data demonstrated successful long-term Kingdom self-renewal of multiple human embryonic stem cell lines on Synthemax Surface in xeno-free media. In this study, we describe the application of the In driving hES cell technology towards therapeutic application considerable Synthemax Surface for the long-term culture and differentiation of ReNcell effort has been focused on the generation of cGMP compliant conditions. VM neural progenitor stem cells in serum-free medium. Cell performance on The use of feeder based protocols for the establishment, expansion and Synthemax Surface was compared to cells on freshly coated laminin surface. banking of hES cells, and for clonal recovery during genetic modification, Our results demonstrate efficient expansion of NSC on Synthemax Surface are still relevant despite advances in feeder free culture technology. One key over the course of nine serial passages (40 days), with stable doubling time issue needing to be addressed is the presence of serum derived components

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Thursday Poster Abstracts present in many commercially available chemically defined media. Here we Poster Board Number: 2185 demonstrate the partnership of a fully synthetic medium alongside a human fibroblast feeder line generated under cGMP compliance for the mainte- SINGLE-CELL BASED XENO-FREE CULTURE OF nance and expansion of human embryonic stem cells. Following extended HUMAN EMBRYONIC STEM CELLS passage the cells retain the undifferentiated phenotype as evidenced by expression of markers of the undifferentiated state and capacity for in-vitro Chen, Kevin G., Mallon, Barbara S., Hamilton, Rebecca S., Kozhich, differentiation into the three germ lineages. In addition we can demonstrate Olga, Park, Kye-Yoon, McKay, Ronald D.G. high efficiency clonal recovery following single cell dissociation as compared NIH Stem Cell Unit, National Institute of Neurological Disorders and to knockout serum based media. These components and protocols are Stroke, National Institutes of Health, Bethesda, MD, USA, Laboratory of intended to aid researchers in protocol development for processes targeted Molecular Biology, National Institute of Neurological Disorders and Stroke, towards cell therapy which will ultimately require to be cGMP compliant. National Institutes of Health, Bethesda, MD, USA, NIH Stem Cell Unit Poster Board Number: 2183 and Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA THE CORNING® SYNTHEMAX™ SURFACE: Regenerative medicine, relying on human embryonic stem cell (hESC) tech- A COMPLETE SOLUTION FOR RECOVERY, nology, opens promising new avenues for therapy of many severe diseases. EXPANSION, AND DIFFERENTIATION OF However, this approach is restricted by limited production of the desired cells due to the refractory properties of hESC growth in vitro. It is further HUMAN EMBRYONIC STEM CELLS hindered by insufficient control of growth rates and also by heterogeneous Kelley, Jessica J., Dolley-Sonneville, Paula, Weber, Jennifer, Yang, cellular states under current culture conditions. In this study, we report a single-cell-based culture (SCC) of hESCs in the presence or absence of either Jiwei, Brandenberger, Ralph, Melkoumian, Zara a ROCK inhibitor Y-27632 or JAK inhibitor I on different matrices. This Life Sciences, Corning, Inc., Corning, NY, USA, Geron Corporation, Menlo SCC was initially developed using MEF conditioned medium on 7.5% BD Park, CA, USA Matrigel-coated plasticware, it was readily adapted to a defined medium, Scalable, reproducible, low cost and regulatory-friendly technologies must mTeSR™1, on 2.5% Matrigel-coated plastic ware. Ten hESC lines were be developed to enable clinical use of human embryonic stem cell (hESC)- adapted to SCC under this growth condition. In general, cells with higher based therapeutics. hESC culture methods use complex, animal-derived single cell plating efficiency exhibit an exponential growth pattern between products, such as mouse feeder layers, Matrigel™, murine laminin or 24 to 72 hr, followed by a delayed growth period and then a stage with de- human-derived biological substances as surfaces to which the hESCs attach. creased cell numbers. Microarray data suggest that hESCs under SCC condi- Most of these materials are costly, of limited scalability, have batch to batch tions retained global mRNA expression patterns characteristic of hESCs. Flow variability and are a potential source of adventitious agents. Corning Life cytometric analysis and immunostaining showed that these cells expressed Sciences in collaboration with Geron Corporation developed and com- all examined hESC markers. Furthermore, most hESC lines under SCC have mercialized a fully synthetic, xeno-free surface, Synthemax Surface, for the normal karyotypes and gene copy numbers. This approach has been shown culture of hESC. Our previous studies demonstrated successful long-term to homogenize cellular states, precisely control growth rates, significantly self-renewal of multiple hESC lines (H7, H1, H9, BGO1v/hOG) in several increase cell production, and enhance hESC recovery rate from cryopreserva- defined commercial media with stable proliferation rate, expression of phe- tion without compromising chromosome integrity. It also facilitates xeno- notypic markers (Oct4, Tra-1-60, SSEA4), normal karyotype and retention free culture of hESCs that are needed for stem-cell-based therapy. Moreover, of pluripotency. Synthemax Surface also supported directed differentiation the SCC system is simple, robust, scalable, and suitable for high throughput of H7 cells to functional cardiomyocytes. The successful development of screening and drug discovery. hESC-based therapeutics requires the production of cell banks and develop- Poster Board Number: 2187 ment of cryopreservation and thaw methods. The objective of this study was to examine the recovery of cryopreserved hESC on Synthemax Surface GENERATION OF NEW MONOCLONAL in defined medium. Cryopreserved H9 cells were thawed and propagated for five serial passages on Synthemax Surface and Matrigel-coated plates in ANTIBODIES ELICITED AGAINST HUMAN mTeSR1 medium. Our results demonstrate efficient attachment and growth PLURIPOTENT STEM CELL DERIVATIVES AS of cryopreserved H9 cells on Synthemax Surface. Cell morphology, doubling MARKERS FOR EARLY STAGES OF HUMAN time, viability and expression of pluripotency markers were comparable to cells recovered on Matrigel. Normal karyotype at the end of five passages DEVELOPMENT was confirmed by g-banding analysis. To our knowledge, Synthemax Sur- Aguila, Héctor L., Gibson, Jason, Nelson, Craig E., Root, Sierra H. face is the only commercially available, synthetic, non-biological surface that Immunology, University of Connecticut Health Center, Farmington, CT, provides a complete solution for recovery, expansion, and differentiation USA, Molecular and Cell Biology, University of Connecticut, Storrs, CT, of hESC in chemically-defined media. We believe that Synthemax Surface USA will be useful for both research purposes and production of cells for cellular therapies. The ability of Human Embryonic Stem Cells (hESCs) to differentiate into all adult human tissues makes them an ideal source of cells for regenerative therapies. However, the direct use of these cells is deleterious due to tumor formation and uncontrolled differentiation. Therefore, hESCs must be differ- entiated to restricted intermediate progenitors. This should be accomplished in a controlled and reproducible fashion combined with techniques allowing the identification and isolation of such intermediates. Using coculture sys- tems with mouse stromal cell lines, we have developed differentiation proto- cols for hESCs that we hypothesize recapitulates very early stages of human development. Analysis of differential expression of various cell surface markers, using multi color flow cytometry, indicated that these derivatives are heterogeneous, they have lost the expression of markers associated to pluripotency, and can be dissected into discrete subpopulations. Using anti-

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CD34 and anti-CD44 antibodies these cultures generated at least 6 discrete bead and fluorescence-based cell sorting. We are investigating peptide subpopulations. Purification of these fractions by fluorescence activated cell receptor targets using database homology searching and affinity chromatog- sorting and analyses of their gene expression through low-density arrays, raphy. The data indicate that it is feasible to use phage display to identify cell together with reactivities against other cell surface markers, have identified selective peptides that can discriminate between progenitor cell types. The populations expressing signatures features of progenitors for mesenchy- combination of surface receptor identification and functional lineage fate mal, hematopoietic and vascular lineages. Moreover, the potential of these mapping of the progenitor cell lines will facilitate identification of peptide populations to progress towards hematopoietic and vascular pathways combinations (bar codes) for lineage-defined enrichment of progenitor have been validated in in vitro differentiation assays. We have used these cell lines from any differentiating pluripotent cell population. These tools heterogeneous populations of hES derivatives as antigens to generate new will facilitate the selection, expansion, and differentiation of well-defined monoclonal antibodies using standard hybridoma technology. We had done therapeutic cells from patient-derived iPS cells for individualized regenerative that with the idea that these populations should contain reactivities against therapies. novel epitopes not recognized by commercially available antibodies, which have been mostly raised against adult or transformed cells. A battery of over Poster Board Number: 2193 150 monoclonal antibodies has been generated and initially characterized DEVELOPMENT OF REPORTER GENE PET by relative reactivity against hESCs coculture derivatives, undifferentiated hESCs and their isotypes have been defined. Within these antibodies there IMAGING TECHNIQUES FOR LONG-TERM are many of them that are able to recognize discrete populations within the ASSESSMENT OF TRANSPLANTED HUMAN 6 populations initially defined by CD44 and CD34 markers. These novel monoclonal antibodies are being purified, conjugated to multiple fluoro- CIRCULATING PROGENITOR CELLS chromes and we expect they should allow us to further characterize and Zhang, Yan (Mary), Kuraitis, Drew, Burgon, Patrick G., Crowe, isolate more defined populations of cells for better dissection of early human Suzanne, Vulesevic, Branka, Thorn, Stephanie, DaSilva, Jean N., developmental pathways leading to the identification of tissue specific stem deKemp, Rob A., Beanlands, Robert S., Suuronen, Erik J., Ruel, cells for regenerative therapies. Supported by State of Connecticut Stem Cell Marc Program (Grants 06SCC04 and 08-SCD-UCHC-003). Cardiac Surgery, University of Ottawa Heart Institute, Ottawa, ON, Canada, Poster Board Number: 2189 Cardiovascular Endocrinology, University of Ottawa Heart Institute, Ottawa, ON, Canada, National Cardiac PET Centre, University of Ottawa BAR-CODING HUMAN EMBRYONIC Heart Institute, Ottawa, ON, Canada PROGENITOR CELL LINES USING PHAGE Introduction: Direct cell radiolabeling methods with positron emission DISPLAY tomography (PET) have been used to track transplanted stem cells non- invasively. However, this technique can only monitor the cells for several Bignone, Paola A., Krupa, Rachel A., Chin, Alan, Parsons, hours due to the short half-life of PET radioisotopes. To further investi- Stephanie, Das, Shreyasi, Funk, Walter D., West, Michael D., gate cell fate and function in vivo by PET imaging, we transduced human Snyder, Evan Y., Larocca, David circulating progenitor cells (CPCs) with reporter genes for monitoring cell Mandala Biosciences LLC, San Diego, CA, USA, Sanford-Burnham Medical homing and engraftment over the long-term. Methods: A self-inactivating Research Institute, La Jolla, CA, USA, BioTime Inc., Alameda, CA, USA versatile lentiviral particle, carrying a triple-fusion reporter (TFR) construct consisting of PET (herpes simplex virus type 1 truncated thymidine kinase; The self-renewal and differentiation potential of human pluripotent stem HSV1- sr39tk), fluorescence (monomeric red fluorescence protein; mRFP), cells offers a virtually unlimited source of therapeutic replacement cells and bioluminescence (firefly luciferase) reporter genes, was produced by to treat a variety of degenerative diseases including heart disease, Par- transient co-transfection into the 293FT cells with pFUUFRTW, psPAX2, and kinson’s, Alzheimer’s, arthritis, osteoporosis, and macular degeneration pMD2.G plasmids by using the calcium-phosphate precipitation method. to name a few. The sine qua non for regulatory approval of pluripotent Human CPCs were transduced with the concentrated viral particle at stem cell-derived therapeutics is safety which is in turn dependent on cell increasing multiplicities of infection (MOI) in RetroNectin-coated plates. purity and identity. Therefore, there is a critical unmet need for methods After 2 days, lentiviral transduction was repeated by adding viral superna- of isolation and scale-up of well-defined cell populations from the highly tant to the plated cells. The transduction efficiency was determined by flow diverse mixed cell populations that are derived from hES and hiPS cultures. cytometry analysis of RFP expression, and the expression of HSV1-tk protein Ideally, therapeutic cells for patient specific therapy would be isolated and in the transduced CPCs was assessed by Western blot. The morphology and expanded from patient derived induced pluripotent stem (iPS) cells. Toward viability of transduced CPCs were measured. Fluorescence-activated cell these goals, we are developing methods and reagents to identify and isolate sorting analysis isolated the stably transduced populations. The effect of TFR clonally pure and scalable lineage-defined progenitor cells from differentiat- gene transduction on cellular functions was evaluated by in vitro migra- ing pluripotent stem cell cultures. Our aim is to produce a peptide-based tion and angiogenesis assays. Results: The mean transduction efficiency bar-code system for labeling and identifying progenitor cell lines with was 2.6%, 2.2%, 3.3%, 6.7%, 12.4% and 17.3% at MOIs of 0.1, 1, 5, defined lineage potential. The potential of hES cells to generate scalable 25, 50 and 100, respectively. Western blot analysis confirmed HSV1-tk lineage-restricted progenitor lines has been demonstrated using a combina- protein expression in transduced CPCs. Both transduced and control CPCs torial cell cloning approach, termed the ACTCellerate initiative. This effort exhibited similar morphology. No significant difference was observed in cell led to the production of over 140 unique clonally pure embryonic progenitor viability between the transduced CPCs (80.6±11.8% at MOI 50) and the (EP) cell lines. We are using phage display technology to identify peptides untreated controls (82.8±10.3%) at the low level of MOI; however, there that bind these hES derived EP cell lines and have selected a 12-mer phage was a reduction in transduced CPC viability (66.2±9.5%, p<0.05) at MOI displayed peptide library on 14 distinct EP cell lines. Analysis of a database of 100. The migration potential (66.2±21.3 cells/field of view (FOV)) and of all selected peptides revealed a pattern of novel peptides or motifs that capillary tube length (5.6±0.6mm) of cells were not adversely affected by were unique to a single target cell line and additional peptides or motifs the transduction process compared to the control (61.1±19.6 cells/FOV and that were shared among 2 or more target cell lines. We assayed peptides for 5.7±0.5mm; p=0.7 and p=0.8, respectively). RFP expression was maintained cell binding by ELISA, qPCR, and immunocytochemistry to identify peptide for at least 4 weeks post-transduction. After 4 weeks, more than 80% of combinations suitable for cell-type specific staining and FACS sorting. These the sorted transduced CPCs continued to express RFP, and could still be cell-binding peptides are being used to demonstrate feasibility of separating visualized under the fluorescence microscope. Conclusion: Quiescent human targeted progenitor cell lines from a complex mixture of cells using magnetic

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CPCs transduced with a lentiviral vector show stable expression of the triple system, which allows us to identify and isolate the subpopulation of hPSCs reporter genes for at least 4 weeks. The TFR gene approach can now be de- that expresses high levels of the pluripotency-associated cell surface antigens veloped for tracking transplanted CPCs, while preserving the cell phenotype, CD9 and GCTM-2. These data, combined with our membrane polysome viability, migration and angiogenesis capabilities. These techniques may be translation state analysis that identifies all proteins likely to be expressed on used for the development of reporter gene PET imaging, to monitor the fate the surface of pluripotent cells, led us to a final selection of candidate cell of transplanted cells noninvasively, longitudinally, and quantitatively, and to surface proteins, which had not been previously associated with pluripotent thereby elucidate the mechanisms and effectiveness of cell-based therapies. cells, but which we observed were switched off very rapidly with differentia- tion. For candidate proteins where commercial antibodies were available, Poster Board Number: 2195 these antibodies were tested for their ability to recognize epitopes on live ANTIBODY-DIRECTED LENTIVIRAL GENE cells using FACS. The antibodies that passed this filter are being coupled to beads for use in purification of viable populations of purified hPSCs, and TRANSDUCTION FOR LIVE-CELL MONITORING are being conjugated to toxins for use in purging pluripotent cells from AND SELECTION OF HUMAN IPS AND HES mixed cell populations. We have generated antigens for forty candidate cell surface proteins for which commercially available antibodies were either not CELLS available or did not detect epitopes on live hPSCs by FACS analysis. These Roth, Monica, Wu, Dai-tze, Seita, Yasunari, Zhang, Xia, Lu, Chi-Wei antigens were generated via peptide synthesis or protein expression in both Biochemistry, UMDNJ-RWJMS, Piscataway, NJ, USA, Ob/Gyn, UMDNJ- prokaryote and eukaryote cells. Following immunisation and high through- RWJMS, Piscataway, NJ, USA put FACS screening of hybridoma cultures, we have identified a number of new monoclonal antibodies (MAbs) capable of detecting novel pluripotency- The identification of stem cells within a mixed population of cells is a major associated epitopes on live human pluripotent cells. These new MAbs are hurdle for stem cell biology, in particular in the identification of induced currently being characterised, and will be coupled to beads and to toxins for pluripotent stem (iPS) cells during the reprogramming process. Based on the testing in our hPSC purification and purging assays. selective expression of stem cell surface markers, a method to specifically infect stem cell through antibody-conjugated lentiviral particles has been Poster Board Number: 2199 developed which can deliver both visual markers for live-cell imaging as well as selectable markers to enrich for iPS cells. Antibodies recognizing SSEA4 TRANSIENTLY TRANSFECTED HUMAN and CD24 mediated the selective infection of the iPS cells over the parental MESENCHYMAL STEM CELLS PROVIDE A human fibroblasts, allowing for rapid expansion of these cells by puromycin READY-TO-USE PLATFORM FOR CELL-BASED selection. Adaptation of the vector also allows for the selective marking of hES cells and their removal from a population of differentiated cells. The ASSAYS ability to specifically target gene delivery to human stem cells has broad ap- Atze, Kristin, Valentin, Silke, Ortmann, Bodo, Breuer, Nadine, plications in tissue engineering and stem cell therapies. Kuehn, Mathias, Franke, Steffi, Nasello, Cara Marie, Batchelor, Alex, Poster Board Number: 2197 Faust, Nicole Research & Development, Lonza Cologne GmbH, Koeln, Germany, ADDRESSING THE SAFETY BOTTLENECK FOR Department of Genetics, Rutgers University, Piscataway, NJ, USA USE OF HUMAN PLURIPOTENT STEM CELLS In drug discovery research cell-based assays are a widely accepted tool for IN CLINICAL APPLICATIONS: DEVELOPMENT high throughput screening of potential drug candidates. Typically, immor- OF ANTIBODIES TO NOVEL EPITOPES ON LIVE talized cell lines are used in cell based assays. They can be easily cultivated and modified in vitro, but they bear some limitations as they are (1) often HUMAN PLURIPOTENT STEM CELLS not originated from the actual tissue or native cell of interest, (2) sometimes O’Brien, Carmel, Zhou, Qi, Chy, Hun, Lynch, Candace, Lambshead, non-human (e.g. hamster derived CHO cell line), and (3) often express the Jack, Abdelrahman, Sara, Adams, Tim, Wang, Yu-Chieh, Laurent, transfected targets at non-physiological levels. For these reasons, there is a growing demand for primary cells such as human Mesenchymal Stem Cells Louise C., Loring, Jeanne F., Laslett, Andrew L. in secondary and even primary drug screens. A major drawback has been Department of Anatomy and Developmental Biology, Monash University, the unreliable availability of high quality primary cells and the lack of appro- Materials Science & Engineering, CSIRO, Clayton, Australia, Materials priate transfection methods. With the AmaxaTM NucleofectorTM Technol- Science & Engineering, CSIRO, Clayton, Australia, Department of Chemical ogy it is now possible to transfect these stem cells with high efficiency with- Physiology, Center for Regenerative Medicine, The Scripps Research out affecting their functionality. This enables expression of biosensors for Institute, La Jolla, CA, USA, Materials Science & Engineering, CSIRO, second messengers such as Ca2+ or cAMP. Biosensors can therefore be used Parkville, Australia, Department of Reproductive Medicine, University of to monitor specific signal transduction events such as protein kinase A (PKA) California, San Diego., Department of Chemical Physiology, Center for activation during osteogenic differentiation. Here we show that PoieticsTM Regenerative Medicine,The Scripps Research Institute, La Jolla, CA, USA primary hMSCs can be used in high throughput formats (i.e. 96-well or 384- Human pluripotent stem cells (hPSCs), including human embryonic stem well plate) to monitor intracellular Ca2+ and cAMP fluxes. The cells were cells (hESC) and human induced pluripotent stem cells (hiPSC), have the transiently transfected with the luminescent calcium biosensor iPhotina® abilities to indefinitely self-renew and to differentiate into essentially all cell or cAMP biosensor GloSensorTM. After subsequent loading either with types. While these characteristics make them potential sources of material iPhotina® substrate coelenterazine or GloSensorTM cAMP reagent the cells for a wide range of clinical applications, they also pose potential risks from can be used directly or cryopreserved and reactivated as needed. Stimulation uncontrolled self-renewal and off-target cellular differentiation. We are through various agonists and antagonists of endogenously expressed surface generating antibodies to novel epitopes on live hPSCs and using them to receptors lead to dose-dependent Ca2+ and cAMP responses. This ready-to- develop efficient and scalable methods to produce highly enriched popula- use cell based assay system represents an excellent tool to study agonist and tions of live human pluripotent cells that can be used as inputs for differ- antagonist effects on calcium and cAMP signalling in hMSCs and will open entiation protocols and to remove residual pluripotent cells from target cell new roads for more predictable drug screenings. populations after differentiation. We have characterised multiple hPSC lines using our previously published FACS-based immuno-transcriptional profiling

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Poster Board Number: 2201 thermore, cells are able to differentiate into osteoblasts, chondrocytes and adipocytes after the expansion. Therefore StemXVivo Mesenchymal Stem HIGHLY EFFICIENT EXPANSION OF HUMAN Cell Serum Free Expansion Media provide excellent serum free environment MESENCHYMAL STEM CELLS IN A NOVEL for human MSCs expansion, and serve as a good substitute for serum- containing medium. SERUM FREE, ANIMAL COMPONENTS FREE AND CHEMICALLY DEFINE MEDIUM Poster Board Number: 2205 Wang, Ing-Kae, Hsieh, Sing-Ying, Wu, Cheng-Yi, Lin, Meng-Hsueh, SYNTHETIC SURFACE FOR LONG-TERM Huang, Jun-Jae, Liao, Chih-Ching, Lin, Su-Yo, Hsu, Hsiang-Chun, CULTURE OF HUMAN MESENCHYMAL Lai, Hsing-Hua, Chen, Wannhsin, She, Bin-Ru STEM CELLS IN SERUM-FREE, XENO-FREE Biomedical Technology and Device Research Laboratories, Industrial CONDITIONS Technology Research Institute, Hsinchu, Taiwan, e-Stork Reproductive Center, Hsinchu, Taiwan Dolley-Sonneville, Paula J., Myrglot, Katelin S., Melkoumian, Zara Mesenchymal stem cells (MSCs) are adult multipotent cells which have been K. isolated from almost every type of connective tissue. They can self-renew Corning Inc, Corning, NY, USA and have the capacity to differentiate into adipogenic, chondrogenic and Human mesenchymal stem cells (hMSCs) are multipotent progenitor cells osteogenic lineages under appropriate in vitro conditions. Human mesen- with the ability to differentiate into several cell lineages, such as adipocytes, chymal stem cells (hMSCs) are promising candidates for stem cell therapy osteoblasts and chondrocytes. There is a great interest in application of and regenerative medicine. However, despite the remarkable clinical ad- hMSC in cell therapy and tissue engineering. Human MSCs are traditionally vancements in this field, most applications still use traditional culture media expanded in vitro using serum-supplemented culture medium. However, the containing fetal bovine serum. The highly variable nature of traditional presence of serum introduces variability in cell culture systems and is highly culture media remains a challenge. Although there are several commercially undesirable for clinical applications. Several serum-free media conditions for available serum-free media, the cell expansion efficiency is low or required expansion of hMSCs were recently described in the literature. In these stud- ECM coating. Here, we developed a serum-free, animal components-free ies cell culture ware was coated with biological materials to enable hMSC and chemically define medium for efficient expansion of hMSCs without adhesion in serum-free medium. The biological materials have limited shelf ECM coating. The medium is comprised of a chemical defined basal medium life, lot-to-lot variability and the potential for contamination with adventi- supplemented with human recombinant growth factors, growth hormones, tious agents. To address the limitations of biological growth substrates, lipids, amino acids, vitamins and antioxidants. Using this medium, hMSCs Corning Life Sciences, in collaboration with Geron Corporation, developed derived from Wharton’s jelly, adipose or bone marrow were expanded 5 to a novel, xeno-free, synthetic surface, the Corning® Synthemax™ Surface, 10 passages with low cell seeding density (0.5~1x103/cm2) on non-coated for stem cell culture applications. This surface is comprised of an acrylate culture plates. These cells increased about 35~50, 40~50 and 15~25 folds polymer functionalized with a short peptide sequence derived from the in this serum-free medium for 5~7 days at each passage, respectively. The vitronectin protein, to mimic biological ligands for cell adhesion. Earlier data medium has higher cell expansion capacity compared to serum containing demonstrated successful long-term self-renewal of multiple hESC lines on medium or commercially available medium. After several passages, the ex- Synthemax Surface in xeno-free media. This study illustrates the applica- panded cells maintained CD73+ CD90+ CD105+ CD34- CD45- phenotype tion of Synthemax Surface for long-term culture of bone marrow-derived and exhibited the ability to differentiate into adipogenic, chondrogenic or hMSCs in serum-free MesenCult®-XF medium. Cell performance on the osteogenic lineages. This serum-free, animal components-free and chemi- Synthemax Surface was compared to cells cultured a) on a biological coating cally define medium provides a substitute for serum-containing medium for recommended by the manufacturer of MesenCult®-XF medium; and b) in hMSC expansion that has broad applicability for basic research and future classical hMSC culture conditions on tissue culture treated (TCT) plastic in clinical applications. serum-containing medium. The results demonstrate efficient expansion of Poster Board Number: 2203 hMSCs on Synthemax Surface over the course of 9 serial passages (47 days) with average doubling time of 38 hrs and average 12-fold cell expansion in DEVELOPMENT OF SERUM FREE DEFINED one passage. Throughout the multi-passage study on Synthemax Surface, MEDIUM FOR HUMAN MESENCHYMAL STEM cells maintained high viability, typical spindle-shaped morphology and phenotypic marker profile characteristic for multipotent hMSCs (high levels CELL EXPANSION of CD166, CD44, negative for CD14, CD19). Similar cell performance was Tang, Yixin, Aho, Joy, Cross, Rosie, Herr, Greg, Tousey, Susan, observed for cells cultured on the biological surface in serum-free medium. In contrast, cells cultured on TCT in 10% fetal bovine serum (FBS) exhib- Andersen, Marnelle, Reyna, Kristen, Asfaw, Dawit, Ni, Jessie H.T. ited much slower growth rate, with average doubling time of 189 hrs and R&D Systems, Minneapolis, MN, USA average 3-fold expansion in one passage. After 6 passages, cell growth rate Human mesenchymal stem cells (MSCs) are multipotent and able to dif- significantly decreased with concomitant decline in expression of phenotypic ferentiate into a variety of cell types, including: osteoblasts (bone cells), markers (CD166 and CD44), confirming replicative senescence of cells upon chondrocytes (cartilage cells) and adipocytes (fat cells). Given the limited expansion in TCT/10% FBS condition. In summary, the results of this study number of cells one can get from adult tissue, in vitro propagation is abso- suggest that Synthemax Surface, in combination with serum-free medium, lutely essential for stem cell therapy research and applications. For conven- provides a complete solution for long-term expansion of hMSCs under tional MSC culture, fetal bovine serum (FBS) is required for cell stimulation xeno-free, defined culture conditions. We anticipate that the Synthemax and proliferation. The main disadvantage of FBS is its ill-defined elements, Surface will be valuable for both research purposes as well as therapeutic which leads to inconsistent lot to lot performance. Here we developed a applications of hMSCs. fully defined, serum free MSC expansion medium: StemXVivo Mesenchymal Stem Cell Serum Free Expansion Media. This media can efficiently propagate human MSCs for 5 continuous passages. After expansion, cells maintain positive expression of MSC markers (CD105, CD90, CD73 and CD44) and negative expression of hematopoietic markers (CD45 and CD11b). Fur-

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Poster Board Number: 2207 Mobius™ CellReady 3L bioreactor for the scale up of human MSCs grown on microcarriers. ENHANCEMENT OF MESENCHYMAL STEM Poster Board Number: 2211 CELL YIELDS FROM HUMAN UMBILICAL CORD TISSUE EFFICIENT PRODUCTION OF HUMAN Kandoi, Sangeetha, Thoppe, Karthiklal, Raghunathan, Srividhya, OLIGODENDROCYTE-LINEAGE CELLS FROM A Visweswaran, Malini, Rajasekar, Reena, Kumar, Ajit NEW TYPE OF HUMAN NEURAL STEM CELLS R&D, LifeCell International Pvt. Ltd., Chennai, Tamil Nadu, India Kido, Tsuneo Enhancing mesenchymal stem cell yields from any given source is of vital Stem Cell Medicine, LLC, Germantown, MD, USA importance. The umbilical cord is a very good source of mesenchymal stem Oligodendrocytes are glial cells that form myelin in the central nervous cells. Enzymatic methods for tissue dissociation need to be optimized as they system. Myelin is an electrically insulating material that forms a layer, the can affect cell viability and batch-to-batch variations in enzymatic activity myelin sheath, usually around only the axon of a neuron. It enables saluta- also necessitate optimization with each new batch. Further, while enzymatic tory conduction of neuronal action potentials and is essential for the proper methods could result in the release of all cells from the tissue matrix, explant function of neuron. Oligodendrocytes are known to play a critical role in cultures are biased towards cells that have the capacity to migrate from the the pathogenesis of many neurological disorders including spinal cord injury, tissue explant. Here we demonstrate that coating the growth surface with multiple sclerosis, schizophrenia, and congenital demyelinating diseases 0.1% gelatin significantly enhances mesenchymal stem cell yields from ex- like Krabbe disease. Traumatic injury to the spinal cord causes demyelina- plant cultures of human umbilical cord tissue. Cells grown on gelatin-coated tion of intact axons near the trauma site, which robs them of their capacity surfaces were indistinguishable from those grown on uncoated tissue culture for neural transmission. Many scientists believe that impairments will be surfaces. Immuno-phenotyping revealed typical mesenchymal stem cell improved if oligodendrocytes are supplied and remyelination occurred in markers such as CD90 and CD105. Real-time PCR analysis revealed similar injured region and proved it in animal model. Geron Corporation started a expression of smooth muscle α-actin and calponin from cells grown on un- clinical trial to treat patients with spinal cord injury using human embryonic coated and gelatin-coated surfaces. Cells grown on gelatin-coated surfaces stem cell-derived oligodendrocyte precursor cells. Oligodendrocyte precur- maintained the immuno-phenotype over several passages and displayed sor cells (OPCs) are capable to make oligodendrocyte efficiently but their typical differentiation characteristics. In sum, explant cultures of human proliferation potential and migration potential may be inferior to neural stem umbilical cord tissue on gelatin-coated surfaces could be used for enhancing cells (NSCs). However, NSCs may not be differentiated into oligodendrocyte mesenchymal stem cells yields. so efficiently as OPCs. I has looked for the cells that have high prolifera- Poster Board Number: 2209 tion and migration potential as NSCs and that can be differentiated into oligodendrocyte as efficiently as OPCs and developed a chemically defined SCALING UP HUMAN MESENCHYMAL STEM media for human NSCs that are prone to differentiate into oligodendrocyte- CELLS ON MICROCARRIERS IN SUSPENSION lineage cells. Human NSCs derived from human fetal central nervous tissue were expanded using 10 ng/ml of bFGF and 10 ng/ml of EGF in DMEM/ FOR CULTURE IN SINGLE-USE BIOREACTORS F12 with N2/B27 supplements on a petri dish. After neurospheres were Kehoe, Daniel E., Schnitzler, Aletta C., Simler, Jancie L., DiLeo, formed, they were collected by gravity sedimentation. Then, they were cultured on poly-L-ornithin-coated dishes in the proprietary media (Pat- Anthony ent pending). Cells were passaged every 1 or 2 weeks in the same media. EMD Millipore, Woburn, MA, USA Cells showed homogeneous morphology after 2 or 3 passages. Cells could As the FDA continues to approve clinical trails using human stem cells in be passaged more than 15 times and expanded more than 100,000 times. patients, there is an increasing necessity for the scale-up of stem cell produc- Most cells were CD133- and nestin-positive. When they were differenti- tion. Conventional stirred tank bioreactors utilizing microcarriers have been ated in serum-containing media, MAP-2-positive cells (neurons), GFAP- shown to be capable of stem cell expansion. The research presented here positive cells (astrocytes), and O4-positive cells (oligodendrocytes) could be examines the utility of a single use stirred tank bioreactor for mesenchymal observed. These results (neural stem cell marker expression and multipotent) stem cell expansion and comprehensively compares the characteristics of the indicate that they retain neural stem/progenitor cell character in this condi- product cells with those grown in 2-D culture. Human mesenchymal stem tion. When they were differentiated by reducing growth factor concentra- cells (MSCs) derived from bone marrow that express H-CAM (CD44), THY-1 tion, many cells became process-bearing pro-oligodendrocytes (O4-positive, (CD90) and STRO-1 were used as a model in this study. Optimal condi- GalC-negative) or oligodendrocytes (GalC-positive) within a week but only tions for seeding density (5,000 cells/cm2) and hypoxic oxygen levels were few astrocytes or neurons were seen. I developed a new method to expand found. Nutrient consumption and waste product profiles were monitored human NSCs that were prone to differentiate into oligodendrocyte-lineage dynamically with NOVA analyzer to assess the metabolic state of the cells. cells, including OPC, pro-oligodendrocytes, and oligodendrocytes. This new Growth profiles over multiple passages were observed until the onset of type of NSCs could be expanded >100,000 times in homogeneous manner. senescence. The expression of undifferentiated markers of the MSCs was These features are thought to be important for a cell therapy product. They verified, as well as differentiation of these cells to multiple phenotypes (adi- will be tested their potency and efficacy in vivo in the near future. In addi- pocytes and osteoblasts). Collagen coated microcarriers from Solohill were tion, this method enables to make enough number of OPCs, pro-oligoden- selected for the bioreactor studies since they yielded the highest attachment drocytes, and oligodendrocytes for screening drugs to treat multiple sclerosis and cell recovery for all the beads evaluated. Bioreactor operating conditions and/or various demyelination diseases. were selected to match those in 2-D culture. Mixing rates were established by measuring the shear (energy dissipation rates, EDR) at which apoptosis of individual cells was observed in a single-cell shear microfluidics chamber. Dynamic nutrient consumption and waste product profiles were moni- tored for comparison to 2-D culture. MSCs recovered from the beads were analyzed for the mesenchymal markers H-CAM, THY-1, and STRO-1, and the ability of the recovered MSCs to differentiate into several phenotypes was assessed. This research will demonstrate the utility of the single-use

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Poster Board Number: 2213 for triggered release to enhance transfection efficiency. Following a single transfection, gene expression was found to last up to 8 days. The leading A 3D CULTURE SYSTEM FOR MEDIUM-SCALE polymers identified for each fibroblast cell-type were used for reprogram- PRODUCTION OF HUMAN EMBRYONIC STEM ming with the oriP/EBNA vectors with varying stoichiometric combinations of the reprogramming factors. Multiple transfection doses are administered CELLS to sustain the gene expression for the initial phase of reprogramming. Bradley, Cara, Scott, Heather, Schmidt, Uli In preliminary trials, colonies with embryonic stem cell-like cobblestone morphology that stain positive for Oct-3/4 are observed 2 months following Sydney IVF Stem Cells, Sydney, Australia first transfection, demonstrating the potential of degradable polymeric vec- Human embryonic stem cells (hESCs) are a promising source of cells for tors for non-viral generation of iPSCs. Modification of transfection condi- regenerative medicine and cell replacement therapy, as well as a tool for tions and the addition of small molecule epigenetic modulators like valproic toxicity and drug development studies. In order to use stem cells for research acid can further enhance nanoparticle-based reprogramming. and development as well as clinical applications, it is critical to culture hESCs and their relevant differentiated progenitors in an efficient, reproducible Poster Board Number: 2217 and scalable manner. Here, we demonstrate medium-scale production of COMPARATIVE PROTEOMIC AND hESCs and neural precursor cells (NPCs) utilizing the BioLevitator™, a com- mercially available, fully automatable three-dimensional culturing platform. PHOSPHOPROTEOMIC PROFILING OF HUMAN hESCs grown using the BioLevitator in combination with proprietary Sydney EMBRYONIC STEM CELLS AND THEIR PURE IVF feeder-free hESC medium maintained the characteristics of hESCs over multiple passages, including expression of pluripotency marker proteins such PAX6+ NEUROECTODERMAL DERIVATIVES as Nanog and Oct4. Additionally, hESCs could be efficiently differentiated in Brill, Laurence, Singec, Ilyas, Crain, Andrew, Tobe, Brian, Hou, the BioLevitator to NPC by culture in Sydney IVF NPC Medium; cells were Junjie, Snyder, Evan >80% Sox2+, >70% Nestin+ and MAP2+, showing greater efficiency and Sanford-Burnham Medical Res Institute, La Jolla, CA, USA reproducibility than comparable cultures in flasks. This culturing platform combined with Sydney IVF’s proprietary media can provide a reliable cell One obstacle to use of human embryonic (hESCs) and induced pluripo- supply of hESCs and NPCs for cell-based high-throughput drug screening, as tent stem cells for translational studies, including comprehensive cell type well as for future clinical applications. characterization, has been the need for rapid, controlled, and cost-effective differentiation strategies, and an understanding of their control. Combined Poster Board Number: 2215 (phospho) proteomic analyses of stem cells can shed light on the molecular NON-VIRAL STRATEGIES FOR ENHANCEMENT basis of pluripotency and multipotency. Total proteins were obtained from pure populations of pluripotent hESCs and multipotent neural stem cells OF HUMAN INDUCED PLURIPOTENT STEM (NSCs), reduced, alkylated, digested and peptides separated by strong cation CELL GENERATION exchange chromatography (SCX). Phosphopeptides from SCX fractions were enriched, and fractions from the enrichments run in duplicate by LC-MS/MS Bhise, Nupura S., Green, Jordan J. on an LTQ Orbitrap Velos equipped with ETD. Searches were against a ipi. Biomedical Engineering, Johns Hopkins School of Medicine, Baltimore, HUMAN database using Sorcerer-SEQUEST, filtered to a false discovery rate MD, USA of 0.005-0.01 using ProteinProphet, and protein families and pathways were analyzed. Neural lineage entry (NSCs; ~97% PAX6+/Nestin+) in only 6 days Reprogramming differentiated human somatic cells to induced pluripotent was induced by simultaneously blocking TGFβ superfamily and canonical stem cells (hiPSCs) offers an embryo-free source of pluripotent cells with WNT signaling with small molecules. We delineated comparative (phospho) potential applications in basic biology, drug development and regenera- proteomic profiles from undifferentiated hESCs (OCT4+/PAX6-) vs. the tive medicine. From a clinical perspective, patient specific iPSCs offer an earliest definitive human NSCs (OCT4-/PAX6+) identifying 12,904 proteins, unlimited source of genetically matched cells that bypass the problem of 76.4% of which were phosphorylated on a total of 59,680 sites. This is the immune rejection. The use of viruses for reprogramming presents risks of largest comparative (phospho) proteomic resource to date in any biological genomic integration, immunogenicity, and mutagenesis that hinder the system. Although many additional quantitative differences were obtained, development of clinical therapies. To overcome this, recent efforts have there was a 75.9% overlap of proteins identified between hESCs and NSCs, focused on developing non-integrating non-viral vectors with high efficiency but only 25.7% of the localized phosphorylation sites overlapped between at reprogramming. Thompson’s group demonstrated the generation of the cell populations, suggesting that more regulation of protein activities transgene-integration free iPSCs from human fibroblasts using oriP/EBNA1 was at the level of phosphorylation than expression. A significantly lower episomal plasmids with a modest reprogramming efficiency of about three percentage of the phosphoproteins vs. those lacking detectable phosphory- to six colonies per million input cells. In this study, we investigate the use of lation were enzymes in both hESCs and NSCs, whereas in both cell popula- cationic degradable poly(beta-amino ester)s (PBAE)s as delivery vectors for tions a significantly higher percentage of the phosphoproteins vs. proteins transfecting the EBNA plasmids to fibroblasts. These polymers electrostati- lacking detectable phosphorylation were kinases, receptors and channels. cally self-assemble with plasmid DNA to form gene delivery nanoparticles In addition, hESCs contained a significantly higher percentage of phospho- of about 100 nm. A nanoparticle-based reprogramming protocol has been rylated transcription factors than transcription factors lacking detectable optimized for enhanced hiPSC generation. The polymers were screened phosphorylation. Most of the known TGFβ and WNT pathway members using a high-throughput 96-well plate transfection assay to select polymers were identified, most with phoshorylation, and some were identified in only with high transfection efficacy to IMR90 human fibroblasts and to four one cell population or the other. We detected surprisingly early expression patient specific cell lines from patients with neurodegenerative diseases. and phosphorylation of proteins associated with later-stage neuronal func- Leading polymers were more effective for gene delivery than commercially- tion (e.g. synaptic vesicle proteins) and age-dependent neurological diseases available reagents including Lipofectamine 2000 and FuGENE HD. The best (e.g. alpha-synuclein, amyloid precursor protein). These findings shed light performing polymer transfected 37.5±2.5 % of IMR-90s in a single dose on pluripotency and human neural fate choice, their molecular basis, and and showed increased transfection following multiple doses. Interestingly, should facilitate the rapid and systematic production of large numbers of polymer chemical structure was found to tune gene delivery efficacy in a defined neural phenotypes for developmental studies, disease modeling, cell-type and patient-specific manner. Hybrid bioreducible/hydrolytic de- drug discovery and systems biology. grading polymers show promise as vectors with ‘smart’ chemical structures

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Poster Board Number: 2219 Poster Board Number: 2221 ANALYSIS OF THE HUMAN EMBRYONIC STEM DEFINING THE ADHESION CHARACTERISTICS CELL DEPOSITOME OF HUMAN EMBRYONIC STEM CELLS TO Hughes, Christopher, Postovit, Lynne, Lajoie, Gilles RECOMBINANT ECM FRAGMENTS USING Biochemistry, University of Western Ontario, London, ON, Canada, SURFACE PLASMON RESONANCE University of Western Ontario, London, ON, Canada Chong, Fenny, Prowse, Andrew B., Tellier, Helena, Munro, Trent P., Human embryonic stem cells (hESCs) have exciting potential in regenerative Gray, Peter P. medicine due to their ability to self-renew indefinitely and differentiate into The Australian Institute for Bioengineering & Nanotechnology, St Lucia, any of the cells of the three primary germ layers. Self- renewal and pluripo- Australia tency of hESCs is highly dependent on their microenvironment. Research has so far focused on the in-solution portion of the microenvironment. Analysis The creation of defined surfaces that support human embryonic stem cell of hESC interactions with the extracellular matrix portion of the microenvi- (hESC) growth and maintenance is essential for future therapeutic ap- ronment is understudied. This is partially due to the complexity of commonly plications. The extracellular matrix (ECM) proteins Vitronectin (VN) and used hESC matrices that can be difficult to analyze using current proteomic Fibronectin (FN) are promising candidates. Both molecules promote cell methodologies. Our research aims to detect proteins that are deposited in spreading and adhesion through interactions with cell surface integrins. We the extracellular matrix by hESCs during their growth in vitro. H9 human have recently shown that hESC lines MEL1, MEL2 and HES3 attach to sur- embryonic stem cells were grown on plates coated with Matrigel™ in de- faces functionalised with recombinantly produced fragments of FN and VN. fined SILAC medium containing [13C6, 15N4] - arginine and [13C6, 15N2] The VN fragment consists of the somatomedin B (SMB) domain and integrin - lysine. After the hESCs had reached confluency, they were removed from associating RGD motif while the FN fragment consists of type III domains the Matrigel™ layer by incubating the culture in a buffer designed to detach 7-10. We had previously shown that the VN SMB fragment is capable of but not lyse cells for 1 hour followed by gentle scraping. Cells were spun hESC support for 10 or more passages with equivalent pluripotency marker out of the resulting solution and the supernatant concentrated using C18 expression, proliferation and differentiation potential compared to controls desalting columns. Protein samples were fractionated using a combination grown on the current ECM standard derived from an Engel-Breth-Holm of strong anion and cation exchange chromatography and 1D-SDS-PAGE. Swarm mouse tumour (GeltrexTM). VN and FN interact with cells through Peptides were analyzed by LC MS/MS and identified using a combination of specific cell surface integrins α( Vβ1, αVβ3, αVβ5, αVβ6, αVβ8, αIIbβ3, Mascot, XTandem, PEAKS, and OMSSA with Scaffold. Our group previously α3β1, α4β1, α5β1, α8β1, α9β1, α4β7 ) that are known to influence dif- published characterization of matrices used for the growth of hESCs. Analy- ferentiation, proliferation and cell survival in a variety of different cell types. sis of these matrices represented a significant challenge due to their large However, the binding characteristics of FN and VN have not been extensive- dynamic range of protein abundance and gel-like nature. After evaluating ly studied in hESC. In this study, we used a Biacore T200 to directly measure several methods for protein and peptide based fractionation, an optimized the binding interactions of 3 hESC lines with the recombinant VN and FN protocol that uses a combination of strong anion and cation exchange fragments. The analysis revealed different binding affinities not only for chromatography, along with 1D-SDS-PAGE was chosen for matrix analysis. individual cell lines between the recombinant fragments but also between Using these methods we carried out analysis of conditioned matrix from H9 different cell lines on the same fragment. Integrin blocking studies identified hESCs. A total of 256 labeled proteins were identified. Using gene ontology the key integrins involved in binding to the FN and VN fragments namely to filter the data for proteins known to be secreted or associated with the αV, α5, β1 and β5. We also directly observed the cells growing on FN and extracellular matrix reduced this list to 50 protein candidates. To validate the VN surfaces. Examination of actin filaments revealed significant structural most promising candidate, secreted frizzled related protein 2 (sFRP2), further differences between cells attaching to each ECM with extended filopodia biological assays were performed. Investigation into the expression pattern and organised actin cytoskeleton appearing within 1 hour of binding to of sFRP2 revealed that it is highly expressed in hESCs but cannot be found FN in comparison to greater than 2 hours for VN. Our results indicate that in any other cell types assayed, including Hs578t, NIH-3T3, HuVEC, JAR, significant changes are induced by the underlying attachment matrix and JEG, BeWo, HTR8-SVNeo, MDA-MB231, MDA-MB465, C8161, T47D, and that this differs even between different hESC lines. This research will have hDfs. These results are in contrast to previous reports suggesting expression significant impact on the optimization of defined culture processes resulting of sFRP2 in Hs578t and NIH-3T3 lines. Using recombinant versions of sFRP2 in effective scale-up of hESC for therapeutic applications. and Wnt3a the effect of supplementation into culture media or growth Poster Board Number: 2223 matrix during hESC growth was tested. Using flow cytometry to monitor ex- pression of the pluripotency marker SSEA-4 and differentiation marker SSEA- LATE PASSAGE HUMAN FORESKIN 1, we found that sFRP2 has a significant effect on hESC phenotype that can be rescued through the addition of Wnt3a. Inducible sFRP2 knockdown H9 FIBROBLASTS SUPPORT HUMAN EMBRYONIC and CA1 hESC lines were generated to test the effect of reducing expression STEM CELLS IN CULTURE on stem cell phenotype and the composition of the hESC conditioned ma- trix. This research represents the first work into analyzing matrix deposited Aflatoonian, Behrouz,Fesahat, Farzaneh, Sadeghian, Fatemeh, proteins using MS-based proteomics. This work also presents one of the first Aflatoonian, Ali, Khorad-Mehr, Arezoo, Aflatoonian, Abbass works to employ a fully defined media system in a SILAC experiment with Centre for Stem Cell Biology, Yazd Institute for Reproductive Biomedical hESCs. Sciences, Yazd, Iran, Islamic Republic of, Medical School, Tehran University, Tehran, Iran, Islamic Republic of Human embryonic stem cells (hESCs) were derived from the inner cell mass (ICM) of the preimplantation embryos in blastocyst stage, in co-culture with mouse embryonic fibroblasts (MEFs) in present of basic growth factors (bFGF) in vitro. These cells are important as they have the capacity to form all cell types of the body in a proper culture condition. This potential of hESCs is promising of their application in cell therapy and regenerative medi- cine. One of the challenges in their application in cell therapy is to culture

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them in an animal-free condition. One of the solutions is to replace human cancer cells. Together with other transcription factors, Nanog can also re- fibroblasts instead of MEFs. human foreskin fibroblasts (HFFs)are the most program somatic cells, producing induced pluripotent stem cells. Therefore, common cells which have been applied to achieve this aim. In this study identifying cells with Nanog expression is crucial in cancer investigation and we have derived a new cell line (YAZD2)on MEFs, and passaged it from the clinical diagnostic. Immunohistochemistry (IHC) and in situ Hybridization early passages to form a sub-line in a late passage (P-44) HFF cell line. Late (ISH) are the techniques used to examine the expression and localization passage (P_44+) HFFs support YAZD2 early passages colonies as the safety of Nanog in tissue. Actually, there are several monoclonal and polyclonal of this method was investigated with characterization of the colonies cul- antibodies against Nanog offered by different companies. Only some are tured on HFF late passage with hESC markers such as OCT4, SSEA4, SSEA3, suitable for being used on mouse paraffin embedded tissue. In Uniprot TRA-1-60, TRA-181, TRA-2-54 and TRA-2-49. YAZD2 has been frozen us- database, it is suggested the existence of two Nanog isoforms in mouse. ing vitrification method and thawed. That would be interesting to compare They both are codificated from the same Nanog gene. The isoform 1 is the epigenetics pattern of YAZD2 sub-lines (on MEFS and on HFFs) in later the longest sequence (305 AA) and it is considered as canonical sequence. passages to investigate the effect of different feeders on the characteristics Isoform 2 (280 AA) is produced by alternative splicing and the residues 1 to of a hESC line. 25 are missed. Therefore, both isoforms show different N-terminal domains but the same C-terminal sequence. Nanog antibodies, which are optimized, Poster Board Number: 2225 recognize N-terminal epitope, only of isoform 1 and C-terminal epitope of S-SHIP PROMOTER EXPRESSION IDENTIFIES A both isoforms. Validation of the specificity of Nanog expression will be com- pared with ISH label. Three different probes are used and they complement SUBPOPULATION OF MOUSE PROSTATE BASAL with the uncommon N-terminal sequence, N-terminal common sequence CELLS WITH STEM CELL CHARACTERISTICS and C-terminal common sequence of Nanog mRNAs, respectively. On the other hand, applied protocols show as important steps as the permeabiliza- Bourette, Roland P., Chmelar, Renee, Greenberg, Norman M., tion of tissue because the Nanog mRNA are in cytoplasm but Nanog protein Rohrschneider, Larry R. is a molecule with nuclear localization, and the specific antibody has to go Institut de Biologie de Lille - CNRS UMR 8161, LILLE Cedex, France, Fred through the cytoplasmatic and nuclear membrane to reach the nucleus. This Hutchinson Cancer Research Center, Seattle, WA, USA and other protocol parameters have to be modified to search a reliable and high quality results. The ability to isolate epithelial stem cells of the adult prostate (PrSC) is crucial in terms of understanding their biology and possible involvement in Poster Board Number: 2229 normal and cancer development of the prostate. Previous isolations of PrSCs were based on subdividing total prostate epithelial cells by flow cytometry ESTABLISHMENT OF MOUSE EMBRYONIC using antibodies to cell surface markers. In this study we have used a novel STEM CELLS CULTURE IN 96 WELL PLATE FOR approach shown previously to successfully identify mammary stem cells (MaSCs). This method employs the s-SHIP promoter to tag presumptive HIGH-THROUGHPUT SCREENING stem cells with GFP in a transgenic mouse model, called Tg11.5kb-GFP. Cho, JaeJin, Cho, Mee-Young, Lee, Gene, Lee, Dong-Sup Here we show that in Tg11.5kb-GFP mice, epithelial GFP+ cells are present School of Dentistry, Seoul National University, Seoul, Korea, Republic of, transiently during early mouse prostate development and localize to the Department of Medicine, Seoul National University, Seoul, Korea, Republic basal cell layer of the epithelium. Cell surface marker analyses demonstrate of an antigenic profile characteristic of basal epithelial prostate cells: Lineage- CD24+ Sca-1+ CD49f+. Cells identified by GFP expression are capable of Embryonic stem (ES) cells are a powerful source for the development of self-renewal and enhanced growth potential in sphere-forming assay in novel cell therapies for regenerative medicine and animal biotechnology vitro, and transplantation assays demonstrate reconstitution of prostate using transgenic technology. ES cells are revolutionizing the laboratory cell ducts containing both basal and luminal cells in renal grafts. We also show biology and will provide much improved cell culture models not only for that s-SHIP expression occurs in many prostate cell lines and in xenograft discovery and development of drugs but also for fundamental studies of the prostate tumors. These results demonstrate that s-SHIP promoter expression genetic basis of diseases and toxicity tests. Especially, ES cells can be valuable is a new marker for basal epithelial prostate cells exhibiting stem/progenitor to monitoring of differentiation processes and to improved applications in cell properties. Thus, the Tg11.5kb-GFP mouse model enables PrSC in situ basic developmental biology. Human ES cells may be safer and more ef- identification and isolation via a consistent single parameter with applica- fective drug for human diseases and for basic developmental biology. The tions for further analyses of normal and potential cancer stem cells. application of hES cells in basic developmental biology and drug discovery is expected to have a direct impact on medical research, but to date, such Poster Board Number: 2227 an approach has primarily been used with mouse ES cells. This study, the optimal culture system of undifferentiated mES cell on 96well plates provides NANOG EXPRESSION ON MOUSE PARAFFIN a tool that enables monitoring of the interaction between cells and their mi- TISSUE croenvironment by cells based HTS assay. This tool may be useful in devel- oping models for drug discovery and development of drug and in the studies de Arriba, María del Carmen, Piazzolla, Daniela2, Serrano, of cell biology and toxicity test. Especially, this culture condition may be Manuel2, Cañamero, Marta1 expected to provide a much improved monitoring system of differentiation Comparative Pathology, CNIO, Madrid, Spain, Tumour Suppression, CNIO, pathway of mES cells by specific factors, such as growth factors, cytokines Madrid, Spain and transcription factors. Nanog, a homeodomain-bearing transcriptional factor, is a key molecule in- volved in the signaling pathway for maintaining the capacity for self-renewal and pluripotency. Its expression is specific to early embryos and pluripo- tential stem cells. Nanog-deficient embrionary stem cells lose pluripotency and differentiate into extraembryonic endoderm lineage. Thus it is one of the molecular markers more used for recognizing the undifferentiated state of stem cells in the mouse and human. Nanog is also expressed in tumor cells which show stem cell-like characteristics and addition, a critical process of cancer invasion and metastasis, is associated with stemness property of

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Poster Board Number: 2231 cells that can be analyzed and sorted by FACS. In this study, we designed molecular beacons to target mouse Sox2 mRNA. To deliver the molecular MULTIPLEX SINGLE CELL REAL TIME PCR FOR beacons to live cells, we used a PEI-based delivery vehicle. To demonstrate STEMNESS MARKERS IN MOUSE EMBRYONIC that cells could be sorted specifically for mouse Sox2-mRNA expressing cells, we assayed several Sox2-positive cell populations including mouse embry- STEM CELLS onic stem cells and mouse neural stem cells and compared them with mouse Franzin, Chiara, Piccoli, Martina, Bertin, Enrica, Solagna, Francesca, embryonic fibroblasts that are Sox2-negative. Next, we treated mouse em- Shaw, Sheng-Wen Steven, De Coppi, Paolo, Pozzobon, Michela bryonic stem cells for 4-days with retinoic acid which causes the embryonic stem cells to differentiate. Using the molecular beacons, cells were sorted Department of Pediatrics, University of Padova, Padova, Italy, Department that were specific for the Sox2-molecular beacon. These sorted cells had of Pediatrics, University of Padova and Fondazione Città della Speranza, the capacity to form undifferentiated mouse embryonic stem cell colonies, Padova, Italy, Ormond Street Hospital and Institute of Child Health, while negative sorted cells had a lower capacity to form undifferentiated University College of London, London, United Kingdom mouse embryonic stem cell colonies. These results show the vast potential Background: Quantitative evaluation of gene expression by Real-Time PCR of the molecular beacon technology, in it its capacity to sort specific live cell is a valuable tool for tissues or cells characterization. A limit of this tech- populations based on mRNA expression of cells. nique is related to the analysis of cell population, that could lead to faulty Poster Board Number: 2235 conclusions because it does not take in account the expression profile of each cell, e.g. the co-expression of some genes, which may imply a different A TECHNOLOGY PLATFORM FOR THE cell behavior or fate. Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst, that need to maintain the expression ISOLATION AND EXPRESSION PROFILING OF of specific stemness genes to preserve their pluripotency. Undifferentiated RARE CELL POPULATIONS FROM DEVELOPING mouse ES (mES) cells have been extensively characterized in terms of gene MOUSE EMBRYOS expression profile and since it seems they are not a homogeneous popula- tion, expression analysis at single-cell level may help on defining more accu- Lee, Wenqing Jean, Yap, Sook Peng, Kraus, Petra, Lufkin, Thomas rately different mES cell sub-populations. Method: Multiplex single-cell Real- Genome Institute of Singapore, Singapore, Singapore Time PCR assay was set up for the following murine markers of stemness: klf4, nanog, oct4, sca1, sox2, ckit, cmyc, and 28S as housekeeping gene. The analysis of gene expression changes in developing tissues is valuable to Primers have been designed and tested following Peixoto et al. (2004). the understanding of the various stages of differentiation. Global expres- mES cells were sorted using a FACS Aria I equipped with an automatic cell sion profiling of the developing mouse embryo has been carried out, mostly deposition unit (Becton Dickinson) and collected in individual PCR tubes generating data coming from major fluxes in gene expression. Specific cell containing 5 μL of PBS-DEPC 0.1%. Results and Conclusions: Efficiency populations especially those in low abundance tend to be masked in such and competition tests confirmed the effectiveness of our set of primers. studies. Thus, we aimed to establish a technology platform to study the We analyzed single mES cells, which displayed a high heterogeneity in the transcriptome of small cell populations in vivo. A transgenic mouse line with expression of the considered markers, although they were positive for all the an enhanced green fluorescent protein (EGFP) endogenously tagged to a genes when analyzed at population level. These results could help identify- developmental gene with a known expression was made. The expected ing different sub-populations with possibly distinct differentiative potential EGFP expression pattern were observed in the developing embryos and among mES cells. Moreover, this multiplex single-cell assay could become a these embryos were used for our subsequent studies. By dissociating the useful tool to deepen the characterization of stem cells other than mES. EGFP-expressing embryos to a single-cell suspension and using fluorescent activated cell sorting (FACS), we managed to efficiently isolate and enrich Poster Board Number: 2233 our cell population of interest by at least 30-fold. RNA was extracted from these sorted cells and microarray profiling studies were performed. From our SORTING LIVE MOUSE EMBRYONIC AND results, we have found the minimum RNA required for reproducible gene MOUSE ADULT STEM CELLS BASED ON MRNA expression studies. By comparing the expression profiles of the EGFP-posi- EXPRESSION tive population and the EGFP-negative population, we identified novel and known genes that are highly expressed in the specific cell lineage of interest. Larsson, Hans Mattias, Lee, Seung Tae, Roccio, Marta, Velluto, A robust strategy to isolate rare cell population to high purity for the analysis Diana, Frey, Peter, Hubbell, Jeffrey A. of spatio-temporal changes in the transcriptome of any specific develop- Laboratory for Regenerative Medicine & Pharmacobiology, Institute ing tissue or organ in vivo has been established and should provide greater of Bioengineering, Lausanne, Switzerland, Laboratory for Stem Cell insight to the understanding of differentiation and development. Biomodulation, KangWon National University, Seoul, Korea, Republic of, Laboratory of Stem Cell Bioengineering, Institute of Bioengineering, Lausanne, Switzerland, Laboratory of Experimental Pediatric Urology, Centre hospitalier universitaire vaudois, Lausanne, Switzerland Live cell sorting based on surface markers is limited in its selectivity of cell populations since many cells share similar markers. Transgenic reporter sys- tems in animal models can increase the selectivity of the sorted cells but are time-consuming and expensive to produce. Molecular beacons offer the po- tential to target any mRNA produced inside a cell, thereby increasing the se- lectivity of the live-sorted cell populations. Molecular beacons are modified DNA strands that form hairpin structures with a fluorophore on the 5’-end and a quencher molecule on the 3’-end. The loop sequence of the hairpin is designed to be specific to the mRNA of interest. After entry into the cell, the molecular beacon opens its stem and hybridizes to the corresponding mRNA of interest if it is present in the cell. This unfolding event releases the Cy3- fluorophore from the quencher and allows the emission of light from the

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Poster Board Number: 2237 tion of Gsk3 progressively depletes HSCs through activation of mTOR. This long-term HSC depletion is prevented by mTOR inhibition and exacerbated ISOLATION OF MOUSE HEMATOPOIETIC by ß-catenin knockout. Thus GSK3 regulates both Wnt and mTOR signal- STEM CELL POPULATIONS USING SPKLS IN ing in HSCs, with opposing effects on HSC self-renewal such that inhibi- tion of Gsk3 in the presence of rapamycin expands the HSC pool in vivo. COMBINATION WITH HIGH-THROUGHPUT These findings identify unexpected functions for GSK3 in HSC homeostasis, SINGLE CELL GENE ARRAYS SHOW DISTINCT suggest a therapeutic approach to expand HSCs in vivo using currently DIFFERENCES IN STEM CELL MARKER available medications that target GSK3 and mTOR, and provide a compel- ling explanation for the clinically prevalent hematopoietic effects of lithium, EXPRESSION INVOLVED IN HEMATOPOIETIC a widely prescribed GSK3 inhibitor. In the following study, we found that the DEVELOPMENTAL PATHWAYS combination of Gsk3 inhibitor and mTOR inhibitor can expand phenotypic HSCs in vivo and maintain functional HSC in ex vivo culture. This study will Reynolds, Susan, Renin, Gil, Mason, Clark, Mir, Alain, Isailovic, Lisa provide the basis for a new clinical approach to improve the efficiency of BD Biosciences, San Jose, CA, USA, Fluidigm, South San Francisco, CA, bone marrow transplantation. USA Poster Board Number: 2241 Cells of the hematopoietic system develop in the bone marrow from a population of self-renewing progenitor cells known as hematopoietic stem EXPANSION AND CHARACTERIZATION OF cells (HSCs). Recent evidence suggests that the developmental pathways CORD BLOOD-DERIVED CD34+ CELLS ON 3-D in the hematopoietic system may be maintained by a combination of HSC subtypes with distinct phenotypic, functional and molecular characteristics. NANEX NANOFIBER SCAFFOLD Goodell et al, described a technique to identify the most primitive HSCs Fischer, Stephen E., Chung, Meng-Hong, Sodha, Anirudhasingh, present in bone marrow based on their specific ability to efflux the dye Zhao, Yukang, Sakthivel, Ramasamy Hoechst 33342.1 These cells make up only 0.01-0.1% of the bone marrow. This population of HSCs is defined as “side population (SP) cells” and have Arteriocyte, Inc., Cleveland, OH, USA been shown to be able to replenish normal bone marrow function in vivo. In recent years, umbilical cord blood (UCB) has become recognized as an Osawa at al. developed an alternative method to identify and purify HSCs important source of hematopoietic stem cells (HSCs). Compared to mobi- based on cell surface marker expression.2.3 This combination of markers is lized peripheral blood (MPB) and bone marrow (BM), UCB contains a higher comprised of CD34-/lowc-kit+Sca-1+Lin- and the cells are referred to as frequency of HSCs possessing a less mature phenotype. Such cells demon- KLS. Using these markers it is possible to achieve a high degree of HSC en- strate both increased proliferative capacity and decreased immune reactivity, richment of an extremely small and homogeneous population of long-term which are critical advantages for numerous research and clinical applications. (LT)-HSCs. Recently, Goodell at el. have shown that HSCs, in particular, my- Unfortunately, the absolute number of these primitive HSCs is significantly eloid-based HSCs (My-HSCs) and lymphoid-based HSCs (Ly-HSCs) can be lower in UCB than it is in MPB or BM, which has limited the broader use purified based on their capacity for Hoechst dye efflux in combination with of cord blood as a cell source. In order to overcome the limited supply of HSC markers (SPKLS)CD150+ 4. This combination can show populations of HSCs available in UCB, a number of ex vivo culture strategies have been HSCs with distinct phenotypical and molecular characteristics with regards to undertaken with the goal of expanding the primitive stem cell population. In hematopoietic development. Variation in phenotype and gene expression at many ex vivo expansion systems, HSCs are regarded as suspension cells and the single cell level may be pivotal in the differentiation of stem cells. . Here, cultured in the presence of various media additives that act to enhance cell we present a protocol to discriminate different HSC subtypes on the basis of proliferation while reducing differentiation. An often-overlooked factor in- Hoechst dye efflux and SPKLS surface staining. Single cells from the Ly-HSC fluencing HSC growth and fate decisions is the interaction of these cells with and My-HSC sub-populations were isolated using a BD FACSAriaTM III flow a substrate. In the natural bone marrow microenvironment, HSCs maintain cytometer and differences in gene expression were found between the two close contact with a complex network stromal cells and extracellular matrix, populations using the Biomark TM System. Some of these differences can be likely indicating that cell-cell and cell-matrix interactions play an important correlated to surface phenotype differences. These results demonstrate the role in maintaining their stem cell phenotype. With the goal of mimicking powerful combination of flow cytometry and single cell gene expression as a the bone marrow stem cell niche, Arteriocyte, Inc. has developed its 3-D research tool and the need for robust protocols for both single cell isolation NANEX nanofiber cell culture substrate. The NANEX substrate is designed and analysis when studying the hematopoietic developmental pathway. to provide topographical and substrate-immobilized biochemical cues that Poster Board Number: 2239 act in synergy with media additives to enhance HSC proliferation. Here, we present our recent work with the NANEX platform towards achieving an PIVOTAL ROLE FOR GSK3 IN HEMATOPOIETIC efficient ex vivo expansion of UCB-derived CD34+ cells suitable for research and clinical applications. We show that both freshly isolated and cryopre- STEM CELL HOMEOSTASIS served CD34+ cells can be expanded to a considerable extent on the NANEX Huang, Jian, Klein, Peter substrate (250-fold in 7-10 day serum-free culture). We compare NANEX to a variety of commercially available products and demonstrate that NANEX Medicine, University of Pennsylvania, Philadelphia, PA, USA significantly reduces the phenotype loss commonly observed during ex vivo Hematopoietic stem cells (HSCs) maintain the ability to self-renew and to culture. Additionally, through the use of flow cytometry and CFU assays, differentiate into all lineages of the blood. The signaling pathways regulating we characterize NANEX-expanded cells and show that they maintain their hematopoietic stem cell (HSCs) self-renewal and differentiation are not well stem cell phenotype. Our data indicates that NANEX technology provides a understood. We are very interested in understanding the roles of glycogen robust ex vivo expansion of HSCs and offers great promise for increasing the synthase kinase-3 (Gsk3) and the signaling pathways regulated by Gsk3 in use of cord blood as an HSC source. HSCs. In our recent study (Journal of Clinical Investigation, December 2009) using loss of function approaches (inhibitors, RNAi, and knockout) in mice, we found that Gsk3 plays a pivotal role in controlling the decision between self-renewal and differentiation of HSCs. Disruption of Gsk3 in bone marrow transiently expands HSCs in a ß-catenin dependent manner, consistent with a role for Wnt signaling. However, in long-term repopulation assays, disrup-

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Thursday Poster Abstracts

Poster Board Number: 2243 36 of the 87 ECM-related proteins identified in EHT by proteomic profiling were detected only in FB-supplemented EHTs. Most of these differentially SUR8 INVOLVES BOTH INHIBITION OF expressed proteins (including Pcoc1, Plod2, Plod3, P4HA1, P4HA2, decorin DIFFERENTIATION AND MAINTENANCE OF and biglycan) are pivotal in collagen modification, fibrillogenesis, and ECM organization. Histological analysis of FB-supplemented EHTs showed SELF-RENEWAL OF NEURAL STEM CELLS VIA longitudinally orientated alignment of collagen fibers decorated with thick MODULATION OF ERK SIGNALING cardiac troponin-I-, f-actin-, and a-actinin-positive muscle bundles. In contrast, tissue lacking FBs did not exhibit a structurally organized ECM Moon, Byoung San, Choi, Kang Yell and contained mainly rounded ESC-CMs. We next cultured immature CMs Yonsei University, Seoul, Korea, Republic of in purely FB-derived 3D-ECM to directly investigate its cardio-instructive Sur8/Shoc2 is a scaffold protein that regulates the Ras-ERK pathway. properties. Under these conditions ESC-CMs displayed remarkably advanced However, the roles of Sur8 in cellular physiologies are poorly understood. morphological features (i.e. regular cross-striation, in strictly rod-shaped cell In the present study, Sur8 was severely repressed in the course of neural bodies with centrally localized nuclei), contrasting the low degree of matura- stem cells (NSCs) differentiation in the cerebral cortex of developing rat tion observed in classical monolayer ESC-CM or embryoid body cultures. embryos. Similarly, Sur8 was also critically reduced in cultured NSCs which Conclusion: We provide evidence for the capacity of ESC-CM to undergo were induced differentiation by removal of basic fibroblast growth factor organotypic maturation in vitro if co-cultured with cardio-instructive FBs. (bFGF). Sur8 regulation occurs at the protein level rather than at the mRNA Further dissection of the FB-derived ECM proteome provided a snap-shot of level as revealed by both in situ hybridization and RT-PCR analyses. The role potential cardiogenic stimuli. Ongoing studied in EHT address the question of Sur8 in NSC differentiation was confirmed by lentivirus-mediated Sur8 whether single or cooperative ECM-stimuli support advanced cardiomyocyte knock-down, which resulted in increased differentiation, while exogenous maturation. expression of Sur8 inhibited differentiation. Contrastingly, NSC proliferation Poster Board Number: 2247 was promoted by overexpression, but was suppressed by Sur8 knock- down. The role of Sur8 as an anti-differentiation factor in the developing LARGE PARTICLE FLOW CYTOMETRY FOR CELL rat brain was confirmed by an ex vivo embryo culture system combined CLUSTERS, MICROCARRIERS AND OTHER 3-D with the lentivirus-mediated Sur8 knock-down. The numbers and sizes of neurospheres were reduced, but neuronal outgrowth was enhanced by CELL CULTURE ENTITIES the Sur8 knock-down. The Ras-ERK pathway is involved in Sur8-mediated Pulak, Rock, Thompson, Julia, Bongaarts, Rico, Smet, Francis, regulations of differentiation, as the treatment of MEK inhibitors blocks the Portier, Nate, Bae, Weon effects of Sur8. The regulations of NSCs’ differentiation and proliferation by the Ras-ERK pathway were also shown by the rescues of the effects of Union Biometrica Inc, Holliston, MA, USA, Union Biometrica Inc, Geel, bFGF depletion, neuronal differentiation and anti-proliferation by epidermal Belgium growth factor (EGF). In summary, Sur8 is an anti-differentiation factor that A growing trend in biomedical research is to study cells in a three-dimen- stimulates proliferation for maintenance of self-renewal in NSCs via modula- sional collection, where cells are organized as a functional cluster or as cells tion of the Ras-ERK pathway. interacting with a substrate that mimics the extracellular matrix. There are Poster Board Number: 2245 several important reasons for this change in emphasis. The conventional approach of studying pure populations of individual cells, usually of one FIBROBLAST-DERIVED EXTRACELLULAR cell-type, does not address a number of important biological questions. By contrast, these newer approaches explore the interactions of cells in cell MATRIX CONTROLS MATURATION OF ESC- clusters, tissues, and organs addressing elements of cell-cell communication, DERIVED CARDIOMYOCYTES the response to cues from neighboring cells, as well as responses trig- gered by signals from their surrounding environment. Understanding these Christalla, Peter, Didié, Michael, Didangelos, Athanasios, Mayr, interactions is critical to the development of new successful therapies. These Manuel, Zimmermann, Wolfram-Hubertus considerations are especially relevant to the research areas of regenerative Pharmacology, University Medical Center Goettingen, Goettingen, medicine, stem cell biology and solid tumor cancers. Flow cytometry that Germany, Cardiovascular Division, The James Black Centre King’s College, accommodates these larger structures brings the power of multiparametric London, United Kingdom analysis and sorting to these studies. Additional benefits include analysis of Extracellular stroma-associated factors have been suggested to function as large sample sizes with interrogation of each individual particle in the sample key regulators of organ development. In the heart, however, little is known in an unbiased fashion and a level of automation that provides a high about the dialog between heart cells and stroma. Here we used a novel in throughput feature to these studies. Union Biometrica’s COPAS and BioSort- vitro model of heart muscle development, namely embryonic stem cell- er flow cytometers fulfill these needs and allows for the analysis and sorting based engineered heart tissue (EHT), to test the hypothesis that fibroblasts of many different multicellular particles for their characterization and use in (FBs) establish an extracellular environment capable of inducing advanced high throughput large-scale multi-cellular studies. The COPAS and BioSorter maturation in immature embryonic stem cell-derived cardiomyocytes (ESC- flow cytometers are designed to analyze and dispense biological materials CMs). Methods: Murine ESC-CMs were generated from αMHC-NeoR-ESCs from 10 micron to 1500 micron. These instruments work at low pressures in bioreactor cultures under G418-selection. Subsequently, EHTs were resulting in low shear forces. The sorting mechanism involves a pneumatic generated from ESC-CMs and 0-50% FBs (1.5x10E6 total cell number/EHT) method that provides a gentle sorting event. We tested a variety of biologi- as well as type 1 collagen (0.8 mg; volume: 450 µl). EHTs were character- cal samples including mouse embryoid bodies, human neurospheres, tumor ized by isometric force measurements, confocal laser scanning microscopy, spheroids, pancreatic islets and several types of hydrogel-encapsulated cells. gene expression analyses (qPCR), and proteomic profiling. Results: EHTs Biological samples were identified and dispensed into wells of multi-well generated from purified ESC-CMs alone failed to develop functional cardiac plates on the basis of size and optical properties such as optical density and tissue (n=35). In contrast, supplementation with 25-50% neonatal cardiac fluorescence. Dispensed samples were analyzed by microscopy to verify FBs facilitated the formation of a functional synzytium (n=63). Optimal accuracy and viability. Some samples were incubated further and analyzed force development (242 ± 0.1 µN; n=4) was achieved in the presence of for continued growth. For example, dispensed neurospheres were tested for 25% FBs. qPCR analysis during EHT maturation revealed strong extracel- DNA replication and metabolic activity. Other samples were analyzed for lular matrix (ECM) synthesis (eg. col1-a1 and -a2) in FB-supplemented EHTs. retention of function, such as the glucose response to insulin for dispensed

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porcine islets. COPAS and BioSorter flow cytometers are able to analyze and Poster Board Number: 2251 dispense large biological particles such as cell clusters (mouse embryoid bod- ies), organotypic culture samples (pancreatic islets), spheroids and hydrogel HIGH CONTENT ANALYSIS OF NEURAL STEM microcarriers. The low pressures and gentle sorting mechanism result in high CELL EXPANSION AND DIFFERENTIATION viability and normal physiological response for the study of these three- dimensional biological samples. Sirenko, Oksana, Powe, Allan C., Stice, Steven L., Cook, Karen, Callamaras, Nick, Hesley, Jayne, Jiang, Xin, Tang, Roger, Cromwell, Poster Board Number: 2249 Evan F. IRON IN NEURAL STEM CELLS CAN BE Molecular Devices, Inc., Sunnyvale, CA, USA, ArunA Biomedical, Inc., TRACKED WITH SYNCHROTRON X-RAY Athens, GA, USA FLUORESCENCE MICROPROBE. Neurogenesis is an important process for nervous system development and maintenance. During neurogenesis, neural stem cells generate neural Auriat, Angela M., Zheng, Weili, Nichol, Helen, Kelly, Michael, progenitors that then mature into functional neurons. Pharmacological Guzman, Raphael enhancement of neurogenesis represents a potential therapeutic approach Neurosurgery, Stanford University, Stanford, CA, USA, Radiology, Wayne for neuronal loss in neurodegenerative diseases, such as stroke, brain dam- State University, Detroit, MI, USA, Anatomy and Cell Biology, University age, Parkinson’s, and Alzheimer’s. Accordingly, there is great interest in using of Saskatchewan, Saskatoon, SK, Canada, Neurosurgery, University of neural stem cells as tools for screening neurogenic compounds during early Saskatchewan, Saskatoon, SK, Canada drug development. A key concern for neural stem cell use in drug discovery is determining the best type of stem cell for such development efforts. Adult Introduction: Intra-arterial (IA) cell transplantation is quickly becoming neuronal progenitor cells (NPC), embryonic (ESC), or induced pluripotent a popular treatment for stroke. Magnetic resonance imaging (MRI) of (iPSC) stem cells can all be directed to form different types of neurons and superparamagnetic iron oxide (SPIO) labeled stem cells is widely used to represent potential sources of neural progenitors for use in neurogenic detect cells in vivo but the MRI signal must meet a minimum detection compound screens. Evaluation of these potential sources involves assess- threshold. However, when cells are transplanted IA they are more widely ment of purity, quantity, maturity, and functionality of the stem cell derived distributed and thus may not reach the threshold for detection with MRI. neurons produced, especially for high throughput applications, such as high Here we explored synchrotron X-ray fluorescence (XRF) imaging using a content imaging. Currently available assays are complex and not appropriate hard X-ray microprobe in continuous rapid scanning mode to detect iron for the high-throughput compound screening. Here we describe automated in SPIO labeled stem cells after IA and IP transplantation in an experimen- assay methods for monitoring neural stem cell expansion and differentiation tal stroke model. XRF allows element-specific quantitative mapping at the that greatly increase reproducibility and throughput and that are suitable for resolution of individual cells. Methods: Wistar rats were subjected to a 1hour screening of pharmacological agents. The first method is a neurosphere as- middle cerebral artery occlusion (MCAO) stroke with reperfusion. On day 3 say which can monitor number and size of undifferentiated NPC clusters, or following stroke 500,000 SPIO labeled mouse neural progenitor cells (NPCs, neurospheres in multi-well plates without fluorescence labeling. The second C17.2) expressing a reporter gene containing a synthetic Renilla luciferase is a neuronal differentiation assay which automatically counts and character- and eGFP construct were transplanted either IA or IP. Control stroked izes different types of developing neuronal cells within a population. This animals received either an IP or IA injection of saline. Bioluminescence (BLI) method also evaluates the maturity of developing neurons and integrity of imaging was performed 24 hours after cell injection and once on the day of neuronal networks by a combination of cell scoring and neurite outgrowth euthanasia. The animals were euthanized 1, 10 or 30 days following trans- algorithms. We demonstrate applications of these assays by measuring plantation. Ex vivo MRI images of the brain were acquired using a FIESTA impact of erythropoietin (EPO), basic fibroblast growth factor (bFGF), brain- sequence with 100 µm isovoxels. Brains were then sectioned at 30 µm. XRF derived neurotrophic factor (BDNF) and Sonic Hedgehog (Shh) on neuronal mapping of iron in the injured hemisphere was carried out at the Stanford proliferation and differentiation using either adult human neuronal progeni- Synchrotron Radiation Lightsource on beamline 2-3. Specific regions of tors (Lonza) or embryonic stem cell derived hNP1 TM neural progenitors interest were imaged at a 3 µm resolution with a dwell time of 100 ms using and hN2 TM neuronal cells (ArunA Biomedical, Inc.). Multiple parameters an incident energy of 13 keV. Adjacent sections were stained with GFP, Iba-1 were evaluated by Principal Component Analysis. Also, the impact of and DAPI. Iron concentration in stem cells was also assessed in vitro. C17.2 neuroactive compounds, such as nitric oxide, glutamate or Antimycin A, on mouse NPCs, identical to those used in the in vivo study, were cultured on proliferation, differentiation and neurite outgrowth were quantitated and metal free coverslips. Cells were labeled with SPIO and PFA fixed. Iron was appropriate IC50s evaluated. mapped with XRF as described above. Iron was quantified by comparing the signal strength of the iron fluorescence with an iron calibration standard Poster Board Number: 2253 designed for XRF. Results: MRI demonstrated significant homing of NPC to the ischemic hemisphere but not to the contralateral hemisphere. Typical CHARACTERIZATION OF TRANSGENIC areas of susceptibility artifact were seen in the cortex and striatum. Iron XRF HUNTINGTON MONKEY NEURAL PROGENITOR maps showed distinct areas of iron signal distributed in the ischemic hemi- CELLS DERIVED FROM INDUCED sphere correlating with MRI findings. High-resolution scans depicted single cells in clusters with an average iron content of 2.4 pg ± 2.0 SD, which was PLURIPOTENT DENTAL PULP STROMAL CELLS similar to the concentration identified with the in vitro analysis. Intraparen- Carter, Richard L., Huang, Anderson H.C., Chan, Anthony W.S. chymal cell grafts appeared as focal signal on MRI and XRF. IP saline controls produced a needle shaped artifact on MRI, which correlated to an iron signal Human Genetics/Neuroscience, Emory University/Yerkes National Primate on XRF. Immunohistochemisty and Prussian Blue staining confirmed the Research Center, Atlanta, GA, USA, Oral Pathology, Kaohsiung Medical presence of SPIO labeled stem cells in the brain areas of interest. Conclu- University, Kaohsiung, Taiwan sions: The synchrotron X-ray microprobe allows high-resolution quantitative Recent literature illuminates the promise of induced pluripotent stem (iPS) iron specific imaging of single cells. Here we demonstrate its application in a cells as tools for basic research and biomedical applications. The potential multimodality-imaging paradigm to track SPIO labeled stem cells in a stroke of iPS cells to differentiate to neuronal lineages is an attractive feature for model. We found excellent correlation between MRI, XRF and histology. the study of neurodegenerative diseases such as Huntington’s disease (HD) The Synchrotron Medical Imaging team is funded by the Canadian Institutes and Alzheimer’s disease, and for the development of personal cell therapy. of Health Research and the Heart and Stroke Foundation of Canada. Issues that limit the advancement of these studies include poor cell culture

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Thursday Poster Abstracts homogeneity of differentiated neurons and the lack of animal models for at varying initiating cell density to determine the best condition for optimal therapeutic development. We propose the use of neural progenitor cells proliferation. Results: About 9-12 days after blastema incubation, some (NPCs) as a stable, rapid, and expandable means to generate neuronal cell cells migrated from explants and became the origin of cell population in types as platforms for pathological and therapeutic study. Non-human pri- culture. At primary culture the cells possessed fibroblastic as well as epithelial mates with similar genetic constitution and neurological function as humans morphology. After three rounds of subculture pure fibroblastic cells has are considered as one of the best model systems for studying neurodegen- emerged. These cells appeared to be highly colonogenic so that average of erative diseases such as Huntington’s disease. We have derived NPCs from 771 colonies was produced per 1000 cells being plated in 10 mm Petri dish. iPS cells of an HD monkey (HD-iNPCs). HD-iPS cells were established from The most interesting feature of blastema cells was their ability to generate dental pulp stromal cells of a transgenic HD monkey by expressing Oct4, osteoblastic, chondroblastic and adipogenic cell lineages. According to our Sox2 and Klf4. HD-iPS cells develop distinct cellular features of HD. These findings blastem-derived cells tended to expand in a relatively rapid rate features include the accumulation of mutant huntingtin (htt) aggregates and when being plated at the density of 100 cells/ml in presence of 10% FBS. the formation of intranuclear inclusion as they differentiate toward neuronal Conclusions: Taken together it could be concluded that blastema from rabbit lineages. The homogeneity of HD-iNPCs was determined by detecting the ear contains a population of fibroblastic cells much similar in characteristic to expression of neural progenitor markers using flow cytometry, quantitative mesenchymal stem cells isolated and described from various tissue. real time PCR (qRT-PCR), and immunocytochemistry. HD-iNPCs are positive for Nestin (81%) by FACS analysis. By qRT-PCR analysis we detect the ex- Poster Board Number: 2257 pression of Nestin along with additional neural progenitor markers including ULTRASENSITIVE IMAGE-BASED PROFILING Sox2 and Musashi in HD-iNPCs. Immunostaining further confirmed the ex- pression of neural progenitor markers Nestin and Sox2. HD-iNPCs were suc- TO FORECAST STEM CELL FATE AND IDENTIFY cessfully differentiated into neurons and glial cells by using a combination of CELLULAR HETEROGENEITY neurotrophins and growth factors, which was confirmed by the expression of neuronal markers [[Unsupported Character - Symbol Font ]]-III Bushman, Jared, Liu, Er, Kulea, Tony, Kohn, Joachim, Moghe, Tubulin and Map-2. One of the key cellular features in HD pathogenesis is Prabhas the formation of mutant htt aggregates and intranuclear inclusions. We can Department of Chemistry and Chemical Biology, Rutgers University, detect increased expression of both a soluble form of mutant htt aggregates Piscataway, NJ, USA, Department of Biomedical Engineering, Rutgers in the cytoplasm and the accumulation of intranuclear inclusion in differenti- University, Piscataway, NJ, USA ated neurons from HD-iNPCs. Our HD-iNPC model holds great promise as an in vitro system for the rapid expansion and differentiation of neuronal Even the most purified and selected proliferating cell populations drift cell types with HD cellular phenotype. This system will serve as a powerful in toward heterogeneity over time. Cues such as differentiation signals, attach- vitro platform for investigating the pathogenesis of HD. This system will also ment to substrates, or genomic stress cause cells to alter behavior and gene serve as a means to study and develop new drug/cell therapies with a key expression, resulting in cell sub-populations with diverging functions and advantage that new findings can be directly investigated in the transgenic responses to environmental stimuli. Traditional methods, such as immu- HD monkey from which the cell line is derived. nostaining or Q-PCR, are low content in nature and are often incapable of detecting the early and often subtle changes in cellular behavior indicative of Poster Board Number: 2255 the establishment of sub-populations. We have developed a method based on high-content imaging that is capable of parsing out early changes in ISOLATION OF MESENCHYMAL STEM CELL- cellular behavior and segmenting out cell sub-populations that are otherwise LIKE CELL POPULATION FROM BLASTAMA OF indistinguishable. The image based profiling method relies on fluorescent images where multiple cellular descriptors are extracted and computationally RABBIT PINNA integrated to determine cellular identity and heterogeneity. By staining cells Baghaban Eslaminejad, Mohamadreza, Bordbar, Sima only for DAPI and the nuclear mitotic apparatus (NuMA) as examples, we Stem Cell and Developmental Biology, Royan Inst. for Stem Cell Biology & have applied this method to integrate over 50 visual descriptors and identify Technology, Tehran, Iran, Islamic Republic of subtle changes in cellular heterogeneity. Two disparate cellular systems were tested; Mesenchymal stem cells (MSCs) in the early stages of differentia- Background and purpose: Regeneration could be considered as a new tion or oncogenic transformation could be identified, and oligodendrocyte growth of cells similar to lost cells following an injury. This process could be precursor cells (OPCs) from differing brain regions could be distinguished studied in rabbit pinna in which an experimentally-created hole regenerates despite displaying identical antigenic characteristics. These two examples by forming of a blastema. This tissue consists of a group of undifferentiated highlight the wide utility of this method and suggest that high-content im- cells capable of dividing and differentiating into varying cell types in vivo. aging paired with computational integration is an innovative means whereby The exact nature of blastema cells is not well-known hence the purpose subtle cellular changes can be detected and cellular fate forecasted. of the present study is to isolate and characterize these cells in terms of some well-known criteria for mesenchymal stem cells including colonogenic Poster Board Number: 2259 activity and differentiation potential into skeletal cell lineages. In this study culture requirement of the isolated cells also explored. Methods: Five News MICRO FLUIDIC BASED CELL MANIPULATION land white male rabbit with 3-6 month ages were used in this study. The CHIP FOR STEM CELL SCREENING pinna of the animals was locally anesthetized using lidocaine and a circular Hidari, Kentaro, Saito, Masato, Yamaguchi, Yoshinori, Tamiya, Eiichi hole with 2 mm dimensions was created in the middle of pinna and allowed forming blastema. After two days whole blastema ring was removed and Osaka University, Suita, Japan incubated in DMEM at an atmosphere of 37 C and 5% CO2. Migrating [Introduction]It is well known that similar cells could show different activities adherent cells from blastema explants were then lifted and propagated by separating into individual cells as revealed through further analyzing through several successive subcultures. assaged-3 cells were evaluated with steps. Therefore isolate, analyze and recover individual cells is particularly respect to their colonogenic activity using colony forming unit-fibroblast important in the medical field such as providing safe cellular materials (CFU-F) assay. Furthermore isolated cells from passage 3 were cultivated at for regenerative medicine, or drug screening and development of human osteogenic, chondrogenic and adipogenic conditions to find out whether or antibodies. Established isolating methods often use laser or DEP, but they not the cells belong to mesenchymal stem cell population. Finally blastema both caused the bad effects due to applying heat and electricity to the cells. cells were plated under different concentrations of fetal bovine serum (FBS) In this study, in order to develop microfluidic device that can recover only

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the isolated target cells, we designed a microchannel to trap and release Poster Board Number: 2265 polystyrene (PS) beads as a cell-mimicking model, only by the simple fluid controlling at room temperature. [Experiments and results]10 µm diameter NANOTECHNOLOGY APPROACHES FOR semicircular trap site is fabricated on the side of the 100 µm wide main INVESTIGATING MICROENVIRONMENTAL CUES channel connected to 3 µm wide passage and then 10 µm wide passage to the pumps. SU-8 was spin-coated on a silicon wafer to form the 10 µm REGULATING STEM CELL FATE thick layer, then, UV light was irradiated through a metal mask cured and Lee, KiBum, Solanki, Aniruddh, Shah, Shreyas, Kim, Cheoljin developed in SU-8 layer. Polydimethylsiloxane (PDMS) was dropped into Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, USA the mold, then cured by heating for 10 minutes at 120°C. After oxygen plasma treatment, glass substrate was bonded to PDMS. Three channels of This presentation will focus on the interface of nanotechnology and stem the pump are connected to the inlet of the main channel and two trap chan- cell biology. Although stem cell fate is controlled by dynamic interactions nels, respectively. Flow rate of each channel can be changed. The outlet of with microenvironmental cues, understanding the functions of microen- the main channel is released. The sample solution contains 10 µm PS beads vironments and the genetic manipulation of stem cells are hampered by in 0.5% polyvinylpyrrolidone solution. After optimizing various conditions limitations of current methods and technology. For example, the ability of such as flow rate, we could pumped PS beads containing solution from inlet, embryonic stem cells (ESCs) and/or neural stem cells (NSCs) to differenti- and we confirmed that it was possible to trap PS beads. On the other hand, ate into neurons and glial cells can provide essential sources of engraftable pumping in the opposite direction from trap sites, trapped PS bead was re- neural cells for the neuro-degenerative diseases such as Parkinson’s and leased immediately. The actual single cell trap and release is under progress Alzheimer’s disease and spinal cord injury. Nevertheless, two primary chal- in our laboratory. lenges in the context of stem cell-based neuro-regenerative medicine: i) to identify the optimal cues which can guide higher number of stem cells to Poster Board Number: 2261 differentiate into neurons and ii) to improve the survival rate of transplanted AN IMPROVED WORKFLOW FOR qRT-PCR stem cell-derived neurons when implanted in vivo, must be addressed. For the full investigation of stem cell neuro-differentiation in vitro or in vivo, GENE EXPRESSION STUDIES OF SINGLE both approaches from nanotechnology _ the “top-down” patterning of CULTURED CELLS extracellular matrix (ECM) and signal molecules in combinatorial ways (e.g. ECM compositions, pattern geometry, pattern density and gradient patterns) Pease, Jim, Meis, Judy E., Khanna, Anu on biodegradable/biocompatible substrate, and the “bottom-up” synthesis EPICENTRE Biotechnologies, Madison, WI, USA of multifunctional nanoparticles and their surface modification with specific signal molecules_should be combined synergistically. To address the afore- The variation in the gene expression profile of single cells can provide mentioned challenge, our research mainly focuses on three approaches: i) a powerful insight into our understanding of how stem cells differenti- the development of combinatorial arrays of microenvironmental signal mol- ate. Quantitative reverse-transcription-PCR (qRT-PCR) offers a rapid, ecules on biodegradable substrates for investigating stem cell neuro-differ- high-throughput process for quantifying the expression of selected genes entiation; ii) the synthesis and utilization of multifunctional nanoparticles as (transcripts). However, qRT-PCR studies from single cells is challenging for non-viral gene delivery tools; and iii) the development of a microfluidic assay a variety of reasons. These include: difficulty in purifying extremely small platform to identify the optimal conditions for stem cell neuro-differentiation amounts of RNA; the limited number of qRT-PCRs that can be performed; and axonal growth. More specifically, to generate transplantable neurons lack of sensitivity, especially when detecting low-expression transcripts; and for neurodegenerative diseases, we initially utilized the combinatorial signal the need to collect samples often. We describe an improved process that arrays to study the spatio-temporal effect of insoluble/physical cues on eliminates the difficulties associated with single-cell qRT-PCR studies. Our neuro-differentiation of stem cells in the absence of soluble cues promoting process first amplifies the mRNA (poly(A) RNA) directly from the lysate of as stem cell neurogenesis. From this study, we were able to identify a combi- little as one cell without having to purify RNA. The amplified mRNA is then nation of insoluble/physical cues (pattern geometry/dimensions, and ECM converted to cDNA for qPCR. This process greatly increases the number of compositions) that induced neurogenesis. We also observed that these cues qRT-PCRs, and hence the number of transcripts that can be analyzed from ultimately led to pattern-geometry dependent and dimension-dependent a single cell, as well as improving the sensitivity and reproducibility of gene neuronal differentiation. This research effort is currently being extended to expression analysis. generate a three-dimensional ECM culture system, which can better mimic Poster Board Number: 2263 the natural stem cell microenvironment. Furthermore, we have synthesized novel non-viral gene/drug delivery systems such as inorganic and poly- UNCOVERING THE DIVERSITY OF INDIVIDUAL meric nanoparticles to efficiently co-deliver siRNA molecules and chemical CELLS: GENE EXPRESSION PROFILING WITH inducers of neurogenesis (e.g. retinoic acid and suberoylanilide hydroxamic acid) to significantly enhance neuronal differentiation of the hESCs/hNSCs THE BIOMARK SYSTEM with minimum cytotoxicity. In parallel research efforts, we have developed Livak, Kenneth J., Datta, Krishnalekha, Batchu, Chandana, a high throughput screening method based on microfluidics to study human Hamilton, Amy, Mir, Alain pluripotent stem cell (hPSCs) responses toward multiple microenvironmental cues at the single cell level. In this presentation, a summary of the results Fluidigm Corporation, South San Francisco, CA, USA from these efforts and future directions will be discussed. Fluctuations in gene expression at the single cell level could be key for generating developmental signals and for understanding the progression of tumors. Data needs to be collected from a statistically significant number of single cells in order to determine the range of gene expression present in a population of cells. Furthermore, transcripts need to be quantified for a number of genes in order to obtain meaningful cell signatures. BioMark™ arrays provide a convenient and cost-effective system for performing multiple RNA expression assays on multiple single-cell samples. This system has been used to study single cell gene expression in embryonic stem cells, hematopoietic stem cells, cancer stem cells, and early stage embryos.

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Thursday Poster Abstracts

Poster Board Number: 2267 Poster Board Number: 2269 BIODISTRIBUTION OF TRANSPLANTED MODULAR MICROFLUIDIC BIOREACTOR BONE MARROW MONONUCLEAR CELLS IN PLATFORM FOR THE AUTOMATED SUBACUTE STROKE PATIENTS CULTIVATION AND TIME-LAPSE IMAGING OF Rosado de Castro, Paulo Henrique, Schmidt, Felipe R., de Freitas, EMBRYONIC STEM CELLS Gabriel R., Gutfilen, Bianca, Souza, Sergio A. L., Battistella, Valeria, Reichen, Marcel, Ruban, Ludmila, Veraitch, Farlan S., Szita, Nicolas Goldenberg, Regina C. S., Silva, Rafaella M., Maiolino, Angelo, Biochemical Engineering, University College London, London, United Gasparetto, Emerson L., Andre, Charles, Mendez-Otero, Rosalia, Kingdom Barbosa da Fonseca, Lea Mirian A modular microfluidic bioreactor platform for the automated cultivation of Department of Radiology, Federal University of Rio de Janeiro, Rio de embryonic stem cells is presented. Automation encompasses the tempera- Janeiro, Brazil, Department of Neurology, Federal University of Rio de ture control for the cell culture individual, the delivery of culture medium Janeiro, Rio de Janeiro, Brazil, Instituto de Biofísica Carlos Chagas Filho, at desired flow rates (from temperature-controlled storage containers) and Federal University of Rio de Janeiro, Rio de Janeiro, Brazil, Department of with different gaseous tensions, a switch between two media types during Hematology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil cultivation, and the on-line time-lapse imaging of cell morphology and Background: The main goal of this study is to compare two routes of admin- pluripotency markers on an inverted microscope. The microfluidic bioreac- istration of bone marrow mononuclear cells (BMMC) labeled with 99mTech- tors themselves have a culture chamber with two compartments which netium (99mTc) in patients with ischemic stroke in the territory of the middle allows to monitor simultaneously and under identical culture conditions cerebral artery (MCA) within 90 days after the beginning of symptoms. two GFP-reporter mES cell line. Additionally, they can be configured with Methods: Twelve patients between 24 e 68 years have been included in the different culture substrates, for example tissue culture polystyrene (TC-PS). study, from 19 to 89 days after the stroke. Seven patients received cells by Furthermore, pre- and post- cell cultivation, the microbioreactors seamlessly intra-arterial (IA) and 5 by intravenous (IV) route. The National Institutes of integrate with typical cell biology laboratory procedures: cells are seeded Health Stroke Scale (NIHSS) varied from 4 to 19 and they were treated ac- in static culture, and for end-point assays, cells can be fixed on a standard cording to a defined protocol (NCT00473057). Bone marrow aspiration with microscope slide. We have characterized the automated platform and show BMMC separation was performed and approximately 10% of the cells were preliminary culture results. labeled and injected in the MCA or intravenously. Labeling efficiency with Poster Board Number: 2271 99mTc was high (>85%) and cell viability superior to 95% in all cases The total number of cells varied from 1.0 to 5.0x108. Whole body and planar A NEW PHOTOPOLYMERIZABLE HYALURONAN- scintigraphies and single photon emission computed tomography (SPECT) were acquired after 2h and 24h. Patients were evaluated with blood tests, BASED HYDROGEL FOR STEM CELL CULTURE NIHSS, modified Rankin Scale and Barthel index, 1, 7, 30, 60, 90, 120 and AND TRANSPLANTATION 180 days after transplantation. Magnetic Resonance Imaging and Electro- encephalograms were performed before cell therapy and after 90 and 180 Doty, Nathaniel J., Amin, Caitlin, Atzet, Sarah K., Hynes, Richard days. Results: None of the patients showed any complication during the pro- F., Cady, Nathaniel, Toepke, Michael W., Schwartz, Michael P., cedure and 9 of them have completed the protocol without adverse events Murphy, William, Zarembinski, Thomas related to the cells. Cells migrated to the lesioned hemisphere in all patients Glycosan BioSystems, Inc., Salt Lake City, UT, USA, SUNY Albany, Albany, at 2h, however there was great variability among patients., The uptake in NY, USA, University of Wisconsin, Madison, WI, USA the brain region compared with the uptake in whole body varied from 0.4% to 5.0% in the IA group and from 0.7% to 1.0% in the IV group. Quan- Hyaluronic acid (HA) is one of the most abundant biopolymers in the mam- tification of SPECT images showed that uptake was higher in the lesioned malian extracellular matrix (ECM) and plays a key role in guiding stem cell hemisphere when compared with the contralateral side (55.5% to 97.8% behavior during development. HA is particularly rich in embryonic tissues of the total uptake in the brain in the IA group and from 50.5% to 60.5% as well as adult tissues such those found in the brain, eye, and cartilage. in the IV group). In comparison to IV administration, IA route led to greater Recently, biocompatible hydrogel matrices based on crosslinking thiol- uptake in the liver (34,0±9,1% vs 14,7±2,6%) and spleen (6,1±3,6% vs modified hyaluronate (HA-SH) and thiol-modified gelatin (Gel-SH) with 2,8±1,1%) and lower uptake in the lungs (7,8±2,0% vs 16,2±4,2%) 2h polyethylene glycol diacrylate (PEGDA) have been developed for fundamen- after cell therapy. The uptake in the brain region was not statistically differ- tal and applied research and have been successfully used as a customizable ent between both groups when correcting for cranial uptake in whole body substrate for in vitro studies as well as a cellular and protein delivery vehicle images (0,74±1,57 vs 0,12±0,08 for IA and IV groups, respectively). Conclu- in vivo. HA-SH/Gel-SH/PEGDA mixtures form hydrogels on a time scale sion: Our preliminary results indicate that cell therapy with 99mTc labeled comparable with natural ECM matrices such as collagen or Matrigel (tens cells is safe and allows monitoring of cell biodistribuiton and homing for of minutes) while offering more precisely defined starting materials and up to 24h in patients with subacute ischemic stroke. There were significant improved versatility due to the ability to strictly control the relative content differences in organ distribution and in relative uptake between hemispheres of matrix components important for guiding cell behavior. However, a when comparing IA and IV groups. However, total uptake in the brain was limitation of spontaneously gelling materials such as the naturally derived low and without statistical difference between both groups, what may have materials commonly used and the HA-SH/Gel-SH/PEGDA system is the lack important implications for choosing the route of administration in future of spatial and temporal control over gel formation such as that provided larger clinical studies. by biocompatible photoinitiated polymer systems such as PEGDA and HA-based materials. Herein we describe the evaluation of a photoinitiation strategy for polymerization of the HA-SH-Gel-SH materials described above using a crosslinker composed of 4-arm polyethylene glycol functionalized with norbornene moieties (PEG norbornene) as a replacement for PEGDA. A photoinitiated reaction between thiol groups on HA-SH/Gel-SH and C=C bonds on PEG-norbornene (1 wt %) (“thiol-ene” reaction) leads to gelation in as little as 15 seconds, with a Young’s modulus that reaches at least 1500

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Pa. Biocompatibility and control over materials properties using this platform Myogenin and Myosin Heavy Chain (MHC). We then assessed the in vivo for applications such as the formation of bioprinted arrays for high-content regenerative potential of these human-derived myogenic progenitors by stem cell screening and stiffness gradient materials for studying how stiffness transplanting them into cardiotoxin pre-injured Tibialis Anterior (TA) muscles guides stem cell differentiation in a single material will be presented. The of immunodeficient recipient mice. Our results show that these cells are able thiol-ene photopolymerization strategy offers substantially more control to engraft efficiently, as evidenced by the abundant number of human- over the formation of 2D and 3D cellular microenvironments by providing derived myofibers expressing human-specific dystrophin. This is the first spatial and temporal control over the formation of physiologically relevant, report on the efficient generation of engraftable human-derived myogenic biocompatible, and versatile HA/Gelatin-based ECM materials. precursors from ES and iPS cells, and provide basis for further in vivo studies to enable their future therapeutic application in muscular dystrophies. Poster Board Number: 2273 Poster Board Number: 2277 IN VIVO CLONAL ANALYSIS OF ADULT RADIAL GLIA-LIKE NEURAL PRECURSORS TISSUE ENGINEERING OF THREE- REVEALS SELF-RENEW AND MULTIPOTENT DIMENSIONAL CONTRACTILE SKELETAL CHARACTERISTICS MUSCLES FROM HUMAN PLURIPOTENT STEM CELLS Bonaguidi, Michael, Song, Hongjun, Ming, Guo-li Neurology, Johns Hopkins School of Medicine, Baltimore, MD, USA Huang, Yen-Chih, Rolong, Andrea, Rath, Sasmita Biomedical Engineering, Florida International University, Miami, FL, USA Neurogenesis and gliogenesis continue in discrete regions of the adult mam- malian brain. A fundamental question remains whether new cells are gener- Since the development of human embryonic stem cells, the potential of ated in vivo from distinct lineage-restricted progenitors or adult neural stem using those stem cells for therapeutic purpose had attracted a lot of studies. cells capable of both self-renewal and multipotent differentiation. Here we Later, the excited advancement of human induced pluripotent stem cells developed a genetic marking strategy for lineage-tracing of individual, qui- further broadened the possibility of stem cell therapies. The combination escent, nestin-expressing radial glia-like (RGL) precursors in the adult mouse of stem cell research and tissue engineering provided us a versatile tool to dentate gyrus. Clonal analysis identified multiple modes of RGL activation, make functional tissues for exploring the potential of clinical application as including both asymmetric and un-appreciated symmetric self-renewal. well as investigating the mechanism of diseases. We are interested in devel- Long-term lineage-tracing in vivo further revealed a significant percentage opment of functional skeletal muscles from human pluripotent stem cells. of clones that contain RGL(s), neurons and astrocytes, indicating capacity Based on our previous developed protocol, we were able to induce both hu- of individual RGLs for both self-renewal and multi-lineage differentiation. man embryonic stem cells (BG01V) and induced pluripotent stem cells (iPS Furthermore, conditional deletion of PTEN in RGLs promotes their activa- - DF19-9-7T) into skeletal muscles in serum-free culture condition. From the tion, paradoxically resulting in increases of symmetric self-renewal and astro- data of qRT-PCR and immunofluorescent staining with specific skeletal mus- cytic terminal differentiation in the adult hippocampus. Our study identifies cle markers confirmed the formation of skeletal muscles. The progenitor cells RGLs as self-renewing and multipotent neural stem cells and provides novel of skeletal muscle in culture appeared as small spindle shape cells and were insights into into neural stem cell properties in the adult mammalian brain. able to fuse together into myotubes with spontaneous contraction. In order to prepare engineered skeletal muscle constructs, we detached the cells with collagenase and seeded them on the surface of fibrin hydrogels in silicone elastomer coated 35mm culture dishes. After the attachment of seeded cells, TISSUE ENGINEERING fibrin hydrogels were slowly degraded in 7~10 days. Finally the cells self- Poster Board Number: 2275 organized and compacted into three-dimensional constructs. For evaluation of the contractile properties of engineered skeletal muscles, force transducer EFFICIENT SKELETAL MUSCLE REGENERATION was connected to one end of engineered skeletal muscle construct and the FROM HUMAN ES AND IPS CELLS active forces were measured under different frequency of electrical stimulus. The engineered human skeletal muscle constructs exhibited similar force- Darabi, Radbod, Irion, Stephan, Dimos, John, Grskovic, Marica, frequency relationship like the rat model we developed. They also could Kyba, Michael, Perlingeiro, Rita C.R. produce the contractile force in the range of hundreds of micronewton. In summary, we have developed tissue engineered skeletal muscles from hu- University of Minnesota, Minneapolis, MN, USA, iPierian, Inc., South San man pluripotent stem cells. The model could offer a versatile tool for basic Francisco, CA, USA research of understanding the molecular and cellular mechanisms of skeletal A major obstacle in the application of cell-based therapies for the treatment muscle physiology and disease studies as well as for translational research of neuromuscular disorders is obtaining the appropriate number of stem/ like drug discovery, testing and transplantation studies. progenitor cells to produce effective engraftment in vivo. The use of myo- genic progenitors derived from pluripotent ES or iPS cells could overcome this hurdle. However to date, an efficient protocol for the derivation of en- graftable skeletal muscle precursors from human ES or iPS cells has not been documented. We have previously shown in the mouse ES/iPS system that controlled expression of Pax3 or Pax7, key transcription factors in skeletal muscle development, results in a population of myogenic precursors en- dowed with robust regenerative potential. Here we describe a novel method for the application of this approach in human ES and iPS cells. By using a lentiviral based Tet-On system and controlled expression of Pax7 in human ES and iPS cells, we are able to derive a homogeneous population of myo- genic precursors, characterized by the expression of the satellite cell marker CD56 (NCAM), which can be easily expanded in vitro. Upon myogenic differentiation, both human ES- and iPS-derived myogenic progenitors un- dergo final maturation, giving rise to multinucleated myotubes that express

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Thursday Poster Abstracts

Poster Board Number: 2279 with the vasculotropic bacterium B. henselae. Our data demonstrate that EPCs are highly susceptible to B. henselae and that the infection does not DETECTION OF PRO-ARRHYTHMIC EFFECTS interfere with their differentiation towards endothelial cells. Upon infection WITH HUMAN ENGINEERED HEART TISSUE EPCs show a strong activation of hypoxia inducible factor-1 (HIF-1), the key transcription factor in angiogenesis. This is followed by the signature HIF-1- Hansen, Arne, Schaaf, Sebastian, Shibamiya, Aya, Mewe, Marco, dependent pro-angiogenic cell response including production of cytokines Eder, Alexandra, Stoehr, Andrea, Hirt, Marc, Zimmermann, Wolfram such as vascular endothelial growth factor (VEGF) and adrenomedullin Hubertus, Conradi, Lenard, Eschenhagen, Thomas (ADM). Furthermore, B. henselae prevents apoptosis of EPCs and induces Experimental Pharmacology, University Hospital Hamburg Eppendorf, cell migration along a stromal cell-derived factor (SDF)-1 gradient, both Hamburg, Germany, Pharmacology for Pharmacists, University Hospital essential functional components of the angiogenic response. Finally, when Hamburg Eppendorf, Hamburg, Germany, Department of Cardiovascular culture plates are coated with a basement membrane which simulates the Surgery, University Hospital Hamburg Eppendorf, Hamburg, Germany extra-cellular matrix (Matrigel), infected EPCs proliferate and assemble into complex, lumen containing vessel-like structures in vitro. Preliminary experi- Human embryonic stem cells (hESC) are pluripotent cells, they have the ments examining these structures reveal strong upregulation of endothelial ability to differentiate into all cell types of the body. HESC-derived cardio- marker genes vascular endothelial growth factor receptor-2 (VEGF-2), myocytes have the potential to improve drug development and predictive endothelial nitric oxide synthase-3 (eNOS) and vascular endothelial cadherin toxicology applications. This study analyzed the use of hESC-derived cardio- (VE-Cadherin) and positive staining for the intermediate filament vimentin myocytes in engineered heart tissues (EHT) to detect pro-arrhythmic drug indicating that the vessel-like structures are formed by endothelial cells. effects. Methods and results: To differentiate cardiomyocytes, embryoid We have recently shown that heat-killed B. henselae can also induce the bodies (EB) were generated from undifferentiated hESC colonies. Cardio- building of vessel-like structures in vitro suggesting the involvement of some myocyte differentiation was achieved by a growth factor-based three stage yet-unknown outer membrane element. Cumulatively, our data demonstrate protocol. Differentiated cardiomyocytes displayed immaturity with poorly that infection with B. henselae can improve the angiogenic capacity of EPCs organized sarcomeric structure and random orientation. To promote final and even induce vessel-like growth in vivo. It has often been suggested that maturation EBs were dissociated into single cells and fibrin-based EHTs were pro-angiogenic pre-treatment of EPCs may assist in overcoming the limita- generated. In this format hESC-derived cardiomyocytes developed a high tions of current progenitor cell therapy and improve its effectiveness. Further degree of differentiation, sarcomeric organisation and alignment. Further- elucidation of the phenomenon described here may reveal new concepts for more, human EHTs displayed a regular beating pattern for several weeks. such treatment. Electrophysiological characterisation indicated that EHT-derived cardiomyo- cytes have action potential durations similar to adult cardiomyocytes but Poster Board Number: 2283 have upstroke velocities and maximal diastolic potentials of immature car- diomyocytes. Pharmacological characteristics and responses to pro-arrhyth- THE ROLE OF FOXO3A IN THE REGULATION mic compounds were evaluated by automated video-optical recording and OF HUMAN MESENCHYMAL STEM CELL analysis of spontaneously beating human EHTs. These experiments revealed SENESCENCE UNDER HYPOXIA that pro-arrhythmic compounds led to a concentration dependent decrease in relaxation velocity and onset of irregular beating pattern. Threshold Palumbo, SunMi, Li, Wan-Ju values for reduced relaxation velocities matched well with findings in hERG Orthopedics and Rehabilitation, University of Wisconsin, Madison, channel assays. Conclusion: hESC-derived EHTs are a promising platform for WI, USA, Orthopedics and Rehabilitation and Biomedical Engineering, automated toxicology screens. University of Wisconsin, Madison, WI, USA Poster Board Number: 2281 Human mesenchymal stem cells (hMSCs) are a promising cell source for tissue engineering due to their extensive proliferation and multi-lineage INFECTION OF HUMAN ENDOTHELIAL differentiation potential. However, hMSCs lose such potential and be- PROGENITOR CELLS WITH BARTONELLA come senescent over a period of in vitro culture, which is one of the major obstacles in hMSC-based tissue engineering. Several studies have reported HENSELAE INDUCES VESSEL GROWTH IN that cells, including hMSCs, grown in the atmospheric oxygen condition VITRO (normoxia) become senescent after several passages and that low oxygen (hypoxia) conditions extend the cellular lifespan compared to the normoxia O’Rourke, Fiona, Mändle, Tanja, Urbich, Carmen, Dimmeler, condition. However, it is not fully understood how hypoxic conditions Stefanie, Lauber, Kirsten, Schaller, Martin, Kempf, Volkhard A.J. delay the onset of cellular senescence. FOXOs are the O subclass members Institute for Medical Microbiology and Infection Control, University of of the forkhead family of transcription factors involved in the regulation Frankfurt, Frankfurt am Main, Germany, Institute for Medical Microbiology of metabolism, apoptosis, stress resistance, and longevity. The activity of and Infection Control, Eberhard-Karls-University, Tuebingen, Germany, FOXOs is mainly affected by transcription/stability, sublocalization, and Molecular Cardiology, Department of Internal Medicine III, University posttranslational modifications, and AKT and JNK are critical effectors for of Frankfurt, Frankfurt am Main, Germany, Department of Molecular regulating their activity. Specifically, AKT phosphorylates FOXOs, resulting in Onkologie, University of Munich, Grosshadern, Germany, Department of the inactivation of FOXOs, whereas JNK phosphorylates FOXOs, including Dermatology, Eberhard-Karls-University, Tuebingen, Germany those pre-phosphorylated by AKT, to keep FOXOs active. However, FOXOs Endothelial progenitor cells (EPCs) are a heterogeneous mixture of adult phosphorylated solely by JNK or by both JNK and AKT activate different pat- stem cells that play an essential role in revascularization after vascular dam- terns of gene expression, which ultimately determines the cellular responses age with a high potential in regenerative therapy for ischemic injury. Despite to the environmental stimuli, such as hypoxia. A recent study reported that the overall success in increasing functional recovery for such patients the the levels of FOXO3a are inversely correlated with the cellular age, and the need for improvement in progenitor cell survival, homing and engraftment upregulated expression of FOXO3a rescues the cells from senescence. Taken has been expressed. In our work we investigated an unconventional method together, we hypothesize that hypoxia delays the onset of senescence of of improving the angiogenic potential of EPCs through bacterial infection. hMSCs via FOXO3a regulated by AKT and/or JNK. To investigate the role Bartonella spp. are facultative intracellular pathogens and the only known of FOXO3a in regulating hMSC senescence, we first compared the growth bacteria to induce angiogenesis in humans (bacillary angiomatosis). Here rate and expression of pluripotency and senescence marker genes of hMSCs we describe for the first time the course of a bacterial infection of EPCs maintained in 21% and 1% O2. Our results showed that hMSCs in the 1%

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O2 condition increased the lifespan and pluripotency gene expression, and bioprocesses incorporating the stirred-suspension cultivation of hPSCs are decreased the senescence gene expression compared to those in the 21% promising for generating suitable quantities of therapeutically useful heart O2 condition. Western blot analysis on hMSCs at passages 3 and 7 (p3 and cells. p7) showed that the levels of total FOXO3a were similar in p3 hMSCs and decreased in p7 hMSCs in both O2 conditions. However, the cell passage- Poster Board Number: 2287 dependent decrease of FOXO3a was more significant in hMSCs in 21% O2 EXTRACELLULAR MATRIX DEPOSITED BY than that in 1% O2. This observation suggests that FOXO3a plays a role in regulating senescence of hMSCs and in extending lifespan of the cells under HUMAN BONE MARROW STROMAL CELLS hypoxic culture conditions. Importantly, while the levels of total FOXO3a FACILITATES STEM CELL EXPANSION AND of p3 hMSCs in both O2 conditions were similar, the active FOXO3a levels were substantially higher in 1% O2, indicating that the activity of FOXO3a TISSUE-SPECIFIC LINEAGE DIFFERENTIATION at an early cell passage may be an important factor for delaying the onset of POTENTIAL senescence in hypoxic culture. Furthermore, both JNK and AKT were active Pei, Ming, He, Fan, Kish, Vincent L. in p3 hMSCs under hypoxia while only JNK was active in 21% O2. This suggests that the JNK and AKT-mediated posttranslational modifications of West Virginia University, Morgantown, WV, USA, Orthopaedics, West FOXO3a are different between the O2 conditions at an early cell passage, Virginia University, Morgantown, WV, USA and FOXO3a may modulate senescence in hypoxic culture by activating a Objective: To assess the rejuvenation effect of extracellular matrix (ECM) hypoxia-specific gene expression pattern. Our observations show that a sub- deposited by human bone marrow stromal cells (hBMSCs) on hBMSC stantial difference in the expression pattern and activity of FOXO3a under expansion and chondrogenic capacity. Methods: Passage 5 hBMSCs were hypoxic and normoxic conditions and FOXO3a may play a role in delaying expanded on ECM or conventional flasks (Plastic) for one passage. Cell the onset of hMSC senescence under hypoxia through the activation of both number was counted and immunophenotype profiles were assessed using JNK and AKT. flow cytometry. The selected integrins and proliferation-related pathway Poster Board Number: 2285 signals were assessed using western blot. The expanded cells were further evaluated for their chondrogenic potential in a pellet culture system with BIOPROCESSING HUMAN PLURIPOTENT STEM TGF-β3-containing chondrogenic medium using gross morphology, histol- ogy, immunostaining, biochemical analysis, real-time PCR, Western blot, CELLS FOR CARDIAC CELL THERAPY and biomechanical testing. ECM expanded hBMSCs were further evalu- Parikh, Abhirath, Jing, Donghui, Tzanakakis, Emmanuel S. ated for their osteogenic potential using Alizarin Red S staining and alkaline Chemical and Biological Engineering, State University of New York at phosphatase activity assay, and adipogenic potential using Oil Red O stain- Buffalo, Buffalo, NY, USA ing. Results: ECM expanded hBMSCs exhibited an enhanced proliferation capacity and expanded hBMSCs acquired a robust chondrogenic potential Efficient and reproducible bioprocessing of human pluripotent stem cells compared to those grown on Plastic. During cell expansion, ECM decreased (hPSCs) to implantable cardiomyocytes entails the development of culture intracellular reactive oxygen species and increased SSEA-4 expression in systems capable of satisfying clinical demand. hBMSCs. Our data also suggested that ECM expansion upregulated integ- We focused on utilizing stirred-suspension vessels for the cardiogenic dif- rins α2 and β5 and induced a sustained activation of Src kinase, ERK1/2, and ferentiation of hPSCs. Different modes of stem cell culture afforded in these cyclin D1. Interestingly, upregulation of TGF-β receptor II during cell expan- culture systems were investigated. Human ESCs were seeded on microcar- sion and chondrogenic differentiation might be responsible for an enhanced riers at 1×105 cells/ml and expanded without differentiating for one week. chondrogenic potential in ECM expanded hBMSCs. ECM expanded hBMSCs Subsequently, the cells were exposed to differentiation medium containing also displayed a promoted osteogenic potential and decreased adipogenic factors including activin and bone morphogenetic protein-4 (BMP4). A novel capacity. Conclusion: ECM deposited by hBMSCs may be a promising ap- protocol was developed for guiding human ESCs (hESCs) in static cultures proach to expand BMSCs from elder patients for the treatment of large-scale through mesendoderm, mesoderm, early cardiac cell phenotypes to heart bone defects through endochondral bone formation. muscle cells. This protocol was also applied to bioreactor cultures. Within 2 weeks of differentiation in stirred suspension, the differentiating cells were Poster Board Number: 2289 probed for the expression of cardiac muscle cell markers such as NKX2.5, GATA4, atrial natriuretic factor (ANF) and myosin heavy chain (MHC) by TISSUE ENGINEERING OF A HUMAN VESICAL quantitative PCR, flow cytometry and immunocytochemistry. Over 45% EQUIVALENT USING ADIPOSE-DERIVED STEM of differentiated hESCs co-expressed cardiac troponin I (cTnI) and car- CELLS diac MHC. When plated, these cells formed beating bodies responding to chronotropic agents such diltiazem and phosphodiesterase inhibitors in Rousseau, Alexandre, Bernard, Geneviève, Marceau Fortier, an organotypic fashion. The directed differentiation of stem cells encap- Guillaume, Gauvin, Robert, Bouhout, Sara, Fradette, Julie, Bolduc, sulated in alginate capsules and cultivated in a stirred-suspension vessel Stéphane was also investigated. A method was developed for liquefying the bead Chirurgie, LOEX, Québec, QC, Canada core thereby allowing stem cells to form aggregates of specific size while reducing resistance for the exchange of nutrients and factors between the Introduction and Objective: For several years, fibroblast cells have been cells and the bioreactor medium. Moreover, the beads were engineered so primarily used for tissue engineering but adipose-derived stem/stromal cells as to be permeable to differentiation factors. Such factors were then added (ASCs) show promising potential due to their facility to obtain, their capacity to the culture medium for coaxing cells toward cardiac cell progeny. Cells to differentiate and their ability to secrete mediators. Our group previously expressed cardiac cell proteins including MHC, TBX-20, NKX2.5 and GATA4. reported on the production of a bioengineered vesical equivalent using The percentage of NKX2.5-positive cells resulting from encapsulated cells dermal fibroblasts without exogenous matrix. The aim of this study was in the bioreactor was higher than in dishes demonstrating the advantages therefore to evaluate the possibility of engineering an autologous vesical of this scalable culture modality. Current efforts are focused on translating equivalent with human ASCs in order to validate if our model can benefit our findings to the directed differentiation of induced pluripotent stem cells from the attributes of the ASCs. Material and Methods: ASCs were obtained (iPSCs). These efforts entail the investigation of bioreactor culture variables from lipoaspirated adipose tissue and fibroblasts were extracted from a der- and conditions to improve differentiation efficiency. Our results suggest that mal biopsy. These human cells were cultured with serum and ascorbic acid to stimulate the formation of extracellular matrix and obtain cell sheets. Cells

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Thursday Poster Abstracts were cultured with constant media motion (GyrotwisterTM, Woodbridge, physical contact with NIH 3T3 cells overexpressing Fos, Sox4, Klf10 or Ski. NJ) during three weeks and then three cell sheets of ASCs or fibroblasts These results confirm that some factors are secreted by engineered NIH 3T3 were superimposed. After 4 days of maturation allowing cell sheet fusion, in the culture media to promote HSC expansion. In order to identify the human urothelial cells were seeded on top of the construction and matured mediators of HSC expansion, we performed gene expression profiling of at the air/liquid interface. The vesical equivalents were characterized by NIH 3T3 cells transduced with each of the NCA factors previously identi- histology, immunofluorescence as well as mechanical and suture resistance fied. Firstly, our results reveal a circular transcriptional regulatory network tests. Results: Complete vesical equivalents were obtained with ASCs or between Sfpi1, Fos, Klf10. Moreover, upregulated mRNAs corresponding to fibroblasts. The histology clearly showed that cell sheets forming the ASC factors that are secreted were considered as potential candidate agonists of vesical equivalents featured a strong cohesion between cell sheets and were HSC self-renewal. Also, a high degree of overlap was observed between the 1.8 fold thicker than the fibroblast vesical equivalents. Immunolabelings of sets of factors secreted by NIH 3T3 engineered to overexpress Fos, Sox4, the mature constructions showed the presence of cytokeratin 8\18, a dif- Klf10 or Ski. Studies are on going to identify these new factors able to ex- ferentiation marker for urothelial cell; and collagen 1 and 3, which are the pand human HSCs. New innovative strategies are needed to expand human major components of the extracellular matrix. The ASC vesical equivalents HSCs for clinical applications. We have identified new potential pathways were easy to manipulate resistant enough to suture, therefore allowing the to expand HSCs using a novel murine screen which has been validated on 3D reconstruction of a bladder shaped tissue engineered substitute. Conclu- human HSCs. Identification of these NCA factors has a clear potential for sions: Human vesical equivalents were successfully produced using either translational medicine. dermal fibroblasts or ASCs, without the use of exogenous scaffolding com- ponents. The ASC vesical equivalents could sustain suturing without tearing. Poster Board Number: 2293 Considering their accessibility, abundance and increased matrix production CAN BETA-BOSWELLIC ACID SYNERGIZE ACSs therefore represent a great cell source to further optimize our innova- tive model for vesical reconstruction. DERIVATION OF DOPAMINERGIC NEURON Poster Board Number: 2291 FROM MOUSE EMBRYONIC ES CELLS CULTURED ON MATRIGEL COATED EX VIVO EXPANSION OF MURINE AND HUMAN ELECTROSPUN PLGA/PCL NANOFIBROUS HEMATOPOIETIC STEM CELLS WITH NON-CELL SCAFFOLD AUTONOMOUS FACTORS Babaloo, Hamideh1, Abasi, Mozhgan1, Riazi, Gholamhossein2, Deneault, Eric, Wilhelm, Brian T., Berthiaume, Sara, Bergeron, Anne, Barzin, Jalal3, Massumi, Mohammad4 Barabé, Frédéric, Sauvageau, Guy 1National institude of genetic engineering and biotechnology, Tehran, Iran, IRIC, University of Montreal, Montreal, QC, Canada, Centre de recherche Islamic Republic of, 2Department of Biochemistry, Institute of Biochemistry du CHUL, Université Laval, Québec, QC, Canada and Biophysics, Tehran University, Tehran, Iran, Tehran, Iran, Islamic 3 The transplantation of hematopoietic stem cells (HSCs) as a life-saving pro- Republic of, Biomaterials Department, Iran Polymer and Petrochemical 4 cedure is one of the major medical discoveries of the 20th century. However Institute, Tehran, Iran., Tehran, Iran, Islamic Republic of, Department up to one third of patients in need of this procedure lack a suitable donor. of Animal and Marine Biotechnology, National Institute of Genetic Human cord blood (CB) represents an alternative source of readily available Engineering and Biotechnology, Tehran, Iran., Tehran, Iran, Islamic Republic HSCs for the vast majority of patients in need. However most CB harvests of are deemed inadequate for clinical use due to their paucity in the relevant Parkinson’s disease (PD) is a neurodegenerative disorder associated with the (HSCs) cell type causing delayed engraftment with life-threatening infec- relatively selective loss of nigro-striatal dopaminergic (DAergic) neurons and tions. Therefore, even a modest expansion of CB or adult HSCs would have a reduction in striatal dopamine. Cell replacement therapy can surmount a profound impact on the number of patients that would become eligible the obstacles related to current PD chemotherapy, and tissue engineering for a transplant therapy. Toward this goal, we recently reported a novel in will be indispensable compartment thereof. In this study, we evaluated the vitro→in vivo functional screen, which identified a series of nuclear fac- effect of Beta-Boswellic Acid (BBA) soluble factor, which could enhance the tors inducing high levels of murine HSC activity similar to those previously hippocampal neurites outgrowth and branching, on DAergic-differentiating- achieved with our positive control Hoxb4 (Deneault et al., Cell 2009). In ES cells cultured on different nanofibrous scaffolds including PCL, PLGA , total, 18 new determinants have emerged, several of which showed a non- composite of PCL/PLGA or PCL/PLGA as coax. To end that, we generated cell autonomous (NCA) influence on HSC activity (i.e., Fos, Tcfec, Hmgb1 a mouse GFP-expressing ES cell line to monitor the behaviour of the cells on and Sfpi1) using a transduction-free system in which engineered NIH 3T3 scaffolds. Then the cells were plated on different scaffolds as embryoid body cells served as feeder cells. We subsequently provided evidences that seven (EB) and they were coated by Matrigel as a natural extra cellular matrix. The additional factors, i.e., Smarcc1, Vps72, Sox4, Klf10, Ski, Prdm16 and Erdr1 BBA was added in different concentrations from 10-50 nM in neural basal significantly enhance unmanipulated HSC activity through a NCA pathway. medium containing 3% serum replacement and media were changed every Important intrinsic differences have been previously shown between murine other day. The cells were cultured for two weeks and were analyzed by and human HSCs, calling for the investigation of the non-cell autonomous real time RT-PCR, Immunocytochemistry for neuroectermal, mesencephalic effect of these factors on human cord blood HSCs. Using the same experi- or DAergic neuron-related markers. Our preliminary results, showed best mental design (transduced NIH 3T3 cells as feeder cells producing NCA attachment and infiltration in composite PCL/PLGA nanofibrous scaffolds factors), human CD34+ CB cells were expanded for 10 days ex vivo and (80%) in comparison to other scaffolds, and coating of Matrigel improved then tested for their repopulation potential in vivo in immunodeficient mice. attachment regardless to the type of scaffold. Monitoring of cells on scaf- We found that three of these factors, i.e., Vps72, Fos and Klf10, induce the folds showed more cell survival in presence of Matrigel. The adding of expansion of human HSCs. These results suggest that one or more media- BBA could increase the transcripts of mesencephalic neurons markers. The tor molecules are produced by our engineered NIH 3T3 feeder cells and analysis of synthesis and secretion of dopamine is in progress by reverse amplify human HSC activity in vitro. In order to identify these mediators, we HPLC in supernatant of differentiated cells. In conclusion, the composite of tested whether physical contact with engineered NIH 3T3 cells is required PCL/PLGA nanofibrous scaffolds in combination with Matrigel can provide to expand HSCs. To this end, we used a nanopore membrane between a suitable in vitro niche for DAergic differentiation of mES cells and BBA can engineered NIH 3T3 cells and murine HSCs to prevent any cell-cell contact. increase the efficiency of differentiation. Interestingly, expansion of HSCs was clearly shown to not depend on the

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Thursday Poster Abstracts

Poster Board Number: 2295 cells impedes the effectiveness of this technique, with cell survival typically limited to less than 4%. To improve cell survival, we are using a polymeric DO MATRIGEL-EMBEDDED TRANSGENIC cell delivery system composed of hyaluronan and methylcellulose (HAMC). MOUSE EMBRYONIC ES CELLS CULTURED HAMC is both injectable and inverse thermogelling, thus allowing con- trolled cell delivery to the brain. Mouse adult NSPCs were isolated from the ON ELECTROSPUN PLGA-PCL SCAFFOLDS subependymal region of adult yellow fluorescent protein (YFP) transgenic EFFICIENTLY DIFFERENTIATE TO mice forebrains and expanded in culture in the presence of growth factors DOPAMINERGIC NEURONS? (EGF and FGF2). The resulting NSPC colonies were dissociated into single cells, suspended in either HAMC or artificial cerebrospinal fluid (aCSF) and Terraf, Panieh, Safi, Mojtaba, Barzin, Jalal, Massumi, Mohammad injected 1mm below the surface of the brains of C57BL/6 mice. The total Department of Animal and marine Biotechnology, national institute of numbers and location of transplanted cells were analysed at 0, 3 and 7 days genetic engineering and biotechnology, Tehran, Iran, Islamic Republic of, post-transplantation. For both cells suspended in aCSF and HAMC, we Biomaterials Department, Iran Polymer and Petrochemical Institute, Tehran, observed YFP+ cells in the transplanted cortex immediately following injec- Iran, Islamic Republic of tion (at t=0). In contrast, YFP+ cells were observed in 50% of aCSF+NSPC transplanted animals versus 100% of animals that received HAMC+NSPC Parkinson’s disease (PD) is a degenerative disorder of the central nervous transplants, at both 3 and 7 days. This demonstrates that HAMC can system that impairs motor skills, cognitive processes, and other functions enhance viability of transplanted cells. Moreover, the distribution of the which is caused by the relatively loss of dopaminergic (DAergic) neurons cells in the cortex was different between the HAMC and aCSF groups. Cells and consequently a reduction in the amount of dopamine. Cell therapy can transplanted in HAMC were observed throughout the injection site, whereas be a good approach for treating animal models of PD. Stem cell treatment those injected in aCSF were primarily observed the surface of the brain. At 7 has especially been in the center attention of scientist on the other hand days post-transplantation, both the aCSF+NSPC and HAMC+NSPC groups, tissue engineering has introduced very promising methods for increasing the surviving YFP+ cells had extensive processes, suggesting differentiation the rate of differentiation taken place on stem cells. Here R1 and CGR8, into neural phenotypes. These data demonstrate that HAMC is advanta- feeder dependent and independent embryonic stem cell lines respectively geous for the delivery of stem cells to the brain after injury, enhancing cell which overexpressing Nurr1 transcription factor, GPX1 and Pitx3 will be viability and cell-tissue integration over time. Ongoing work to delineate plated on different electrospun PCL/PLGA nanofibrous scaffolds with 300 the differentiation profile of the transplanted cells and their efficacy in injury nm thickness as embryoid bodies (EB) . Then they will coax to DAergic models is currently underway. neuron differentiation in a standard protocol. In addition to PCL/PLGA we will use Matrigel as “on top” which by mimicking the extracellular matrix Poster Board Number: 2299 will help our ES cells to differentiate. We generated a mouse GFP-expressing ES cell line to monitor the behaviour of the cells on scaffolds. The result of ACCELERATED RECONSTRUCTION OF MOUSE differentiation will be analyzed in the level of mRNA using Real time RT-PCR CALVARIAL DEFECT BY THE ADIPOSE-DERIVED and in the protein level using Immunofluorescent for DAergic neuron mark- STEM CELL SEEDED ONTO POLYLACTIC ACID ers such as TUJ1 and TH. The amount of the dopamine secretion will be measured by Reverse phase HPLC. In our preliminary research we examined SCAFFOLD the effect of Nurr1¸GPX1 and Pitx3 alone on the differentiation of the R1 Dinarvand, Peyman, Farhadian, Shirin, Soleimani, Masoud and CGR8 stem cells, in our present study we will demonstrate the synergic affect of over expressing the genes and recruiting the scaffolds accompanied Stem Cell Biology Department, Stem Cell Technology Research Center, by Matrigel together on the differentiation rate of ES cells. In our previous Tehran, Iran, Islamic Republic of, Semmelweis University, Budapest, research the PCL/PLGA we used was as composite in one case and as coax Hungary, Hematology Department, Faculty of Medical Science, Tarbiat in another, and as a result we had the best attachment and infiltration in Modares University, Tehran, Iran, Islamic Republic of the composite PCL/PLGA with solvent 1, so in the present study this is the Bone defects and deformities are very common among clinical diseases scaffold which will be used. Matrigel improved attachment regardless to the and are still remained as major problems despite the improvements in the type of scaffold, monitoring of cells on scaffolds showed more cell survival knowledge of bone tissue engineering. Recent studies have shown the in the presence of Matrigel. We are expecting that nanofibrous scaffolds promising potential of stem cells and different types of biomaterials for bone accompanied by Matrigel will induce efficient differentiation of ES cells into regeneration applications. Adipose tissue-derived mesenchymal stem cells DAergic neurons. (AT-MSC) without osteogenic differentiation have been already shown as a Poster Board Number: 2297 supportive cell resource for bone regeneration. Also, some studies demon- strated that bone marrow-derived mesenchymal stem cells (BM-MSC) and IMPROVEMENT OF ADULT MOUSE NEURAL calvarial-derived osteoblasts (CDO) can be useful in the enhanced regen- eration of bone. We aimed to compare the regenerative effect of AT-MSC, STEM CELL DELIVERY TO THE BRAIN USING A BM-MSC, CDO and tricalcium phosphate (TCP) engrafted in bone calvarial BIOMATERIAL COMPOSED OF HYALURONAN defects. These types of cells and tricalcium phosphate as a bone void filler AND METHYLCELLULOSE (HAMC) standard, were seeded on polylactic acid (PLA) scaffold and implanted into calvarial critical-sized defect in mice. Full-thickness calvarial defects (4 mm Cooke, Michael, Morshead, Cindi, Shoichet, Molly in diameter) were created in an inbred Balb/c mice model. We evaluated Department of Chemical Engineering and Applied Chemistry, University healing of calvarial defects in mice 8 weeks after the initial implantation by of Toronto, Toronto, ON, Canada, Department of Surgery, University of three dimensional computed tomography (3D CT) scan images, digital x ray Toronto, Toronto, ON, Canada and histopathological evaluation. Near complete reossification was detected in the groups which received AT-MSC-seeded PLA scaffolds. 3D CT scan Following injury to the central nervous system (CNS) there is a cascade images showed that increase in volume of healed bone in the animals that of events that leads to cell death, tissue loss and consequently functional received ASCs on PLA had more bone formation compared to TCP, BMSs deficit. In response to injury, the CNS host repair mechanism is insufficient and CDO on PLA (P < 0.05). Also, histological examination demonstrated to restore damaged tissue. Our strategy involves the transplantation of that the defects were repaired by trabecular bone in all of the animals and exogenously expanded adult mouse neural stem/progenitor cells (NSPCs) calvarial defects implanted with AT-MSC on PLA were filled with more new into the injured CNS to promote repair. Significant death of transplanted trabecular bones compared to other groups (P < 0.05). These data reveal

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Thursday Poster Abstracts that the AT-MSC on PLA without osteogenic differentiation treat calvarial- Poster Board Number: 2305 sized defects to a maximum. This combination of stem cell-biomaterial is an efficient complex for bone tissue engineering application. DECELLULARIZATION OF KIDNEYS TO BE Poster Board Number: 2301 USED AS A TISSUE SCAFFOLD FOR IN VITRO CULTURE OF WHOLE ORGANS INDUCTION OF CORNEAL ENDOTHELIAL CELLS FROM MOUSE ADULT NEURAL CREST STEM Chua, Shawn, Whiteley, Jennifer, Li, Mira, Rogers, Ian Samuel Lunenfeld Research Institute, Toronto, ON, Canada CELLS As of January 2011, approximately 88,000 patients were registered on the Shimmura, Shigeto, Hatou, Shin, Yoshida, Satoru, Higa, Kazunari, kidney transplant waiting list at the United Network for Organ Sharing Miyashita, Hideyuki, Tsubota, Kazuo (UNOS) in the United States. The average wait time for a kidney transplant Ophthalmology, Keio University School of Medicine, Tokyo, Japan is 5-7 years. Due to the lack of available donor organs, engineering whole functional kidneys could benefit a large number of patients. For organ Purpose: Corneal endothelial cells form a monolayer on the inner surface manufacturing to become a reality, it is essential to bridge the gap between of the cornea and play a vital role in maintaining corneal transparency. We the current success to be able to grow stem cell derived tissue monolayers previously isolated neural crest stem cells from adult mice corneal stroma and the creation of complex three-dimensional organs. In this work, we (Cornea-derived Precursor (COPs)), which has the ability to differentiate hypothesize that the formation of a biological niche within a mechanical into several neural crest lineages including keratocytes, glial cells and neural niche will provide support and guidance for the development of a three- cells. In this presentation, we report the induction of corneal endothelial cells dimensional organ in vitro. Interspecies cell-cell and cell-niche interactions from COPs. Methods: COPs cultured as spheres were passaged and cultured are being studied to determine what conditions are required for growing as adherent cells in specific endothelium-deriving medium with 1% fetal human cells on decellularized animal scaffolds in vitro. Therefore, whole bovine serum for one week. RT-PCR was performed to detect markers char- adult mouse kidneys as well as aborted human fetal kidneys were perfusion- acterizing corneal endothelial function (Na,K-ATPase α1-subunit, carbonic decellularized and recellularized with either i) human multipotential stem anhydrase, Na,HCO3 co-transporter, collagen IV, collagen VIII). The pump cells (MPSC) derived from umbilical cord blood, ii) human fetal kidney cells function attributable to Na,K-ATPase activity of confluent monolayers of or iii) mouse fetal kidney cells. The results show that the decellularizing these cells was measured with an Ussing chamber. Pump function was calcu- method effectively removes all host cells. MPSCs can survive and integrate lated as the difference in short-circuit current measured before and after the into the extracellular matrix of a decellularized mouse kidney after 7 days addition of ouabain. Primary cultured mouse corneal endothelial cells and in culture. These findings demonstrate the potential use of decellularized 3T3 cells were used as control. Results: After one week culture, hexagonal animal kidneys as a scaffold to generate functional human kidneys. mosaic pattern monolayer cells were obtained from COPs. RT-PCR revealed the expression of all of the above markers. Na,K-ATPase pump activity of Poster Board Number: 2307 these cells was 32.9±6.3 µA/cm2, whereas mCE was 29.3±9.5 µA/cm2 and 3T3 was 9.7±2.8 µA/cm2. Conclusions: We successfully derived corneal INFANTILE HEMANGIOMA A TUMOR DERIVED endothelial cells from mouse corneal stromal cells (COPs), which have equal FROM TROPHOBLASTS pump function with primary cultured endothelial cells. Itinteang, Tinte, Day, Darren John, Guthrie, Siobhan, Tan, Cherise Poster Board Number: 2303 E., Brasch, Helen D., Tan, Swee T. TRANSPLANTATION OF AUTOLOGOUS BONE Victoria University of Wellington, NZ, Wellington, New Zealand, School of Biological Sciences, Victoria University of Wellington, NZ, Wellington, New MARROW DERIVED MONONUCLEAR CELLS Zealand, Gillies McIndoe Research Institute, Wellington, New Zealand COMBINED WITH XENOGRAFTS IN ADVANCED Background: Infantile hemangioma (IH), regarded as a tumour of the micro- MAXILLARY AND MANDIBULAR ATROPHY vasculature, is predominantly composed of proliferating endothelial cells, but also contains progenitor cells of mesenchymal and haematopoietic lineages. Bulgin, Dmitry, Hodzic, Enes, Komljenovic-Blitva, Danijela, Senzel, The similarities in expression profile between IH and placenta including the Stanislava expression of GLUT-1, FcγRII, Lewis Y antigen and type 3 iodothyronine ME-DENT The Center for Regenerative Medicine, Rovinj, Croatia deiodenase, suggest a trophoblastic origin of IH. Aims & Objectives: To Replacing missing bone or adding mass to existing bone is often essential investigate the expression of trophoblast-associated proteins in IH. to the success of a dental implant. An implant needs a critical mass of bone Methods & Observations: IH biopsies from 8 patients with proliferating and surrounding it in order to bind (osseointegrate) to it and deliver sufficient 3 patients with involuted IHs were (i) processed for IHC staining for the tro- strength and stability. If in the location where the implants are intended phoblast-associated proteins human chorionic gonadotrophin (hCG), human there is low mass of bone (width or height) a bone graft must be applied placental lactogen (hPL), and cytokeratin 7 (CK7)); (ii) used to determine the in order to maintain this critical bone mass. A large variety of graft materi- relative expression of transcripts for hCG, hPL and CK7 by qRT-PCR. Results: als have been used for maxillary and mandibular atrophy. To date there has IHC showed that the cells that line the capillary endothelium of proliferating been no graft material which can be regarded as completely satisfactory. IH expressed the trophoblast associated markers, hCG and hPL but not CK7. Our experience with autologous bone marrow derived mononuclear cells qRT-PCR confirmed the IHC data as abundant expression mRNA transcripts combined with Xenografts for augmentation of the extremely atrophied for the same proteins that were detected in IH tissue. Conclusion & Discus- maxilla and mandible to induce bone to augment the height and width, and sion: The mRNA and protein expression of hCG and hPL in proliferating IH to enable dental implant placement is presented. supports previous observations demonstrating the expression of proteins common to both IH and placenta. The clinical observations of increased incidence of IH in infants associated with pre eclampsia following chorionic villous sampling and amniocentesis, coupled with our recent data highlight- ing a cellular origin for IH downstream of ESC suggest a trophoblast origin of IH.

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Poster Board Number: 2311 Poster Board Number: 2313 FUNCTIONAL VALIDATION OF INTERVERTEBRAL DISC-DERIVED STEM CELLS: DECELLULARIZED EXTRACELLULAR MATRIX IMPLICATIONS FOR REGENERATIVE MEDICINE PREPARATIONS FOR EX-VIVO CULTURE OF AND NEURAL REPAIR HEMATOPOIETIC STEM/PROGENITOR CELLS Erwin, William M., Islam, Diana, Eftekarpour, Eftekhar, Inman, Duryagina, Regina, Prewitz, Marina, Thieme, Sebastian, Wobus, Robert D., Fehlings, Michael G. Manja, Werner, Carsten, Brenner, Sebastian, Bornhäuser, Martin Surgery, University Health Network, Toronto, ON, Canada, Genetics Medical Clinic and Policlinic I, University Hospital Dresden, Dresden, and Development, University Health Network, Toronto, ON, Canada, Germany, Leibniz Institute for Polymer Research, Max Bergmann Centre Physiology, University of Manitoba, Winnipeg, MB, Canada, Immunology, for Biomaterials, Dresden, Germany, Department of Pediatrics, University University Health Network, Toronto, ON, Canada Hospital Dresden, Dresden, Germany Introduction: Degenerative disc disease (DDD) is the most common form of Hematopoietic Stem/Progenitor cells (HSC) are of great interest for clinical spinal stenosis, a leading cause of spinal neurological disability in those over application. In case of limited availability effective strategies for their ex-vivo 60 yrs of age. Cervical spondylotic myelopathy is most commonly caused by expansion are needed. The HSC pool in-vivo is regulated by components of advanced DDD and may to tetraparesis or even death. It is known that the the bone marrow hematopoietic niche. One major component is extra- nucleus pulposus (NP) of human intervertebral discs (IVDs) contain multipo- cellular matrix (ECM). Besides its structural function, ECM may bind and tent progenitor cells that have been shown to have mesenchymal-like dif- present active proteins produced by stromal cells, in particular osteoblast and ferentiation capacity in vitro. We elected to test the hypothesis that nucleus mesenchymal stromal cell (MSC). We have previously established a novel pulposus progenitor cells (NPPCs) would within the neural niche, differenti- method for generation of cell-free ECM from human bone marrow-derived ate into neural precursor cells for use in neural repair. Methods: We obtained MSC using polymer-based scaffolds which help to optimize the recovery of NP cells from non-chondrodystrophic (mongrel) canine IVDs, following ECM after Triton-X treatment. To evaluate if cell-free ECMs can be used as enzymatic digestion and generated populations of self-renewing cell colo- an approach for direct culture and expansion of HSC this study is focused nies and evaluated them for the expression of stemness genes, their ability on: a) if functionally active signalling molecules are maintained incorporated to differentiate along chondrogenic, adipogenic, osteogenic and neurogenic within the ECM after the decellularization process and b) if ECM could be lineages in vitro. We then GFP-transfected them and determined their enriched with signalling proteins involved in HSC self-renewal. Therefore, ability to differentiate into neural precursor cells within the compact myelin we generated a human cell line overexpressing Jagged-1 protein. For this deficient shiverer mouse brain. Results: NPPCs express genes associated with purpose an immortalized MSC (single-cell-derived hTERT expressing human ‘stemness’ including Sox2, Oct4, Nanog, CD133, Nestin and NCAM. NPPCs MSC line [Scp-1]) were transduced with a lentiviral construct containing the are capable of differentiation into chondrogenic, adipogenic and neural human open read frame of Jagged-1 protein and GFP. After transduction lineages in vitro however canine NPPCs do not demonstrate osteogenic cells have been sorted by FACS and overproduction of Jagged-1 protein has differentiation. Following injection into the shiverer mouse brain NPPCs been proved by immunofluoresence staining. For decECM generation, Jag- differentiate into the major classes of neural precursor cells such as neuron ged-1 over expressing cells and non-transduced Scp-1 cells have been cul- and astro-glial cells. Most significantly we detected GFP expressing cells that tured for 10 days in DMEM 10% FCS without additional supplements. ECM demonstrated immunoreactivity to CNPase a marker of oligodendrocyte from Jagged-1 over expressing cells (JagECM) and Scp-1 cells (ScpECM) precursor cells, as well as GFP positive cells immunopositive for myelin basic have been immunostained with antibody against Jagged-1protein. By im- protein. Discussion: Progenitor cells obtained from the intervertebral disc munofluorescence microscopy Jagged-1 protein could be clearly visualised have the capacity to be used not only for cartilage and IVD repair, but also on JagECM. In contrast, the amount of Jagged-1 on ScpECM was too low for neural repair strategies and most importantly and of profound potential to be detectable with the same settings. To evaluate functional potential significance, offer the possibility of use in the case of the injured spinal cord. of Jagged-1 that is incorporated with JagECM we monitored activation of NPPCs obtained from the canine IVD do not demonstrate osteogenic dif- Notch pathway in CD34+ HSC cultured on both ECMs and TCP. Expres- ferentiation capacity, an observation that may provide valuable insight into sion levels of Notch target genes Hes-1 and Hey-1 have been determined the potential for these cells for tissue repair. with quantitative PCR in whole population of cells in 2 weeks in culture. Poster Board Number: 2315 Both JagECM and ScpECM induced the expression of Hes-1 and Hey-1 in comparison with TCP. Nevertheless, gene expression of Hes-1 was higher TISSUE ENGINEERED BIPHASIC CERAMICS FOR in HSC cultured on JagECM. In summary, these data suggest that our novel OSTEOCHONDRAL DEFECT SHORT STUDY technology of producing cell-free ECM preparations allows binding and presentation of MSC-derived proteins in a functional manner. Hence, ECM Fernandez, Francis, Shenoy, Sachin, Varma, Hari Krishna, John, from MSC overexpressing various cytokines and morphogens could become Annie a novel approach for HSC ex-vivo expansion. Sree Chitra Tirunal Institute for Medical Sciences & Technology, Trivandrum, India Introduction: Damage to articular cartilage is difficult to heal due to the avascular nature of the tissue and low cell viability in the area. Surgical techniques to remedy the same result in the creation of an osteochondral (crossing the cartilage - bone border) defect to stabilize the injury and also provide a stable anchoring point to the scaffold; this will prevent healing inhibiting micro movements. The purpose of this study was to evaluate scaffold type suitability along with the use of adipose derived stromal cells in the development of a biphasic osteochondral graft. The selection of a suit- able scaffold is critical as it plays a major role in controlling differentiation, acts as a cell carrier & optimizes the healing process. Materials & Methods: Hydroxyapatite (HA) is well known as a biocompatible bioactive ceramic in

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Thursday Poster Abstracts bone replacement, altered solubility rates in HA can be achieved by inducing garded as potential bioactive materials for bone regeneration. Strontium (Sr) varying degrees of crystalline components. The study focuses on three de- as a trace element in the human body has been found to have beneficial ef- rivatives: hydroxyapatite coated with silicon, beta tri calcium phosphate and fects on bone formation as Sr has been reported to induce osteoblast activity hydroxyapatite. All scaffolds were fabricated in house. While earlier studies by stimulating bone formation and reducing bone resorption. The in-house have focused on the use of bone marrow stromal cells in combination with developed SrCaPO4 is a similar bioactive ceramic intended to mend cases scaffolds we have chosen adipose derived stromal cells for their easier avail- of trauma and tumor resections in Orthopedic Surgery, due to its relevance ability and less invasive recovery. Evaluation of scaffolds with adipose de- in radiopacity in view of easy clinical radiographical evaluation. Neo-osteo- rived stem cells will validate their use in the bony region of an osteochondral genesis of the tissue-engineered construct was validated in vivo against bare scaffold. Desirable scaffold architecture includes porosity and pore intercon- SrCaPO4 (without cells) in rabbit ulna mid diaphyseal segmental defect (1.5 nectivity. This was evaluated by using Scanning Electron Microscopy. Also cm) - 3 months post-implantation through radiography, computed tomogra- FT-IR and XRD techniques were used to generate material characterization phy and histology of plastic sections. A press fit method was adopted for the data. Adipose stromal cells isolated from rabbits were confirmed to be of surgical procedure rather than the use of internal/external fixators of bone mesenchymal lineage via differentiation. Cytocompatibility analysis was car- plates/screws which often leads to revised surgeries. Good osteointegration ried out by seeding the scaffolds with stromal cells and evaluation adhesion and osteoconduction was observed in the tissue-engineered SrCaPO4 with and cyto-toxicity at various time points. Scaffolds were also evaluated for lamellar bone organization of newly formed bone throughout the defect in suitability with short term implantation studies. Surface studies carried out par with material degradation. Regeneration of segmental defects is a real with enhanced Micro-CT technique. Results and Discussion: Cells were dif- clinical challenge in skeletal reconstructive surgery. Moreover, subcutaneous ferentiated into osteoblastic lineage and confirmed by staining. SEM images adipose tissue with a high number of MSC-like elements forming colonies of depicted an elaborate porous interconnected architecture (50-500 microns fibroblastoid cells may be successfully expanded in culture and osteogenical- pore sizes) ideal for the infiltration of cells into the internal voids of the ce- ly-induced on scaffolds for experimental and clinical needs. The successful ramic. X-ray diffraction (XRD) pattern exhibited crystallinity with hydroxyap- repair of critical-sized bone defects via the current approach substantiates atite as the major phase in all the materials. The outer coating layer in HASi the potentiality of using ADMSCs with strontium bioceramic scaffolds for showed peaks for calcium silicate, tricalcium phosphate and hydroxyapatite. bone regeneration. Surface differences were observable between the scaffolds implanted. Conclusions/Summary: Hydroxyapatite, silica-coated hydroxyapatite and Poster Board Number: 2319 tricalcium phosphate proved to be non-cytotoxic and cytocompatible. Now REGENERATION OF ELASTIC CARTILAGE FROM a combination of these bioactive ceramics as a single or dual unit for the fabrication of biphasic scaffolds may be possible as promising candidates for ELASTIC CARTILAGE DERIVED CHONDROCYTES osteochondral grafts. Rabbit adipose derived stromal cells have proved their Lee, EunAh, Nam, Seungwoo, Cho, Wheemoon, Lim, SunKi, Son, osteogenic potential. So attempts will now focus on osteogenesis and chon- Youngsook drogenesis of RADMSC on these cytocompatible biphasic ceramic scaffolds separately/co-culture for the fabrication of osteochondral grafts ensuing in Kyung Hee University, Yongin, Geonggi-do, Republic of Korea their validation in vivo for pre-clinical trials in goats. Recent approach of cartilage regeneration employs autologous chondro- Poster Board Number: 2317 cyte transplantation (ACT) using patient’s own chondrocyte. In the case of articular cartilage regeneration, autologous chondrocytes can be obtained IN VITRO AND IN VIVO INVESTIGATIONS from non-load bearing site. However, in the case of elastic cartilage regen- eration, there is limited supply of chondrocytes of elastic cartilage due to the ON RADIOOPAQUE STRONTIUM CALCIUM fact that elastic cartilage is not obtainable without harming the function or PHOSPHATE CELL SEEDED SCAFFOLDS FOR external shape. Because of this, previous approaches to reconstruct auricular EASY CLINICAL EVALUATION cartilage have been tried with chondrocytes from hyaline cartilage. In search of cell source for cartilage repair, we found a vestigial cartilage tissue located Mohan, Beena, Shenoy, Sachin, Varma, Hari krishna, John, Annie at the apical end of xiphoid process. By histological staining and immuno- Sree Chitra Tirunal Institute for Medical Sciences & Technology, histochemical staining of elastin expression, we confirmed that the proximal Trivandrum, India half is composed of hyaline cartilage and the distal half is composed of elastic cartilage. The chondrocytes obtained from the elastic cartilage Segmental bone defects from trauma, pathology or excision of bone tumor showed better cell yield, expansion, and GAGs production in vitro. When represents a formidable clinical problem. For the repair of such defects, the clump-cultured scaffold-free xiphoid process chondrocytes (XCs) were autologous bone grafts are preferred as they possess inherent osteoconduc- subcutaneously transplanted to immunocompromised mice, they showed tivity, osteogenicity and osteoinductivity. An attempt to solve the dilemma in vivo ectopic reconstruction of elastic cartilage, which was confirmed by of limited supply of such grafts and in turn the donor site morbidity, tissue the presence of elastic fiber and elastin expression which was visualized by engineering in Regenerative Medicine appears to be a promising approach. Elastica van Gieson staining and immunohistochemical staining, respectively. Mesenchymal stem cells (MSCs) have the capability for renewal and dif- This results show that XCs can be a nice cell source for autologous cell-based ferentiation into various lineages of mesenchymal tissues and bone marrow elastic cartilage reconstruction. This study was supported by a grant from represents one of the main available sources of MSCs. Contemporarily, adi- Korean Ministry of Health & Welfare (A040003). pose derived stem cells (ADMSCs) have shown to be effective by their mul- tipotency and proliferative efficiency. Herein, the objective was (1) to fabri- cate a tissue-engineered construct in vitro using a bioactive ceramic such as strontium calcium phosphate (SrCaPO4) in combination with ADMSCs and (2) to investigate its potential in the healing of the ulna segmental defect in New Zealand White Rabbits. Mesenchymal stem cells isolated from rabbit adipose tissue were seeded onto SrCaPO4 scaffolds and induced to dif- ferentiate into the osteogenic lineage in vitro. Scanning electron microscopy revealed adhesion, proliferation and spread-out cells with time which even- tually formed a cell-sheet like canopy over the scaffold. Viability of cells on the scaffold was assessed using Acridine Orange and Ethidium Bromide by Confocal Microscopy. Generally, strontium incorporated bioceramics are re-

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trophectoderm specification have been extensively studied, little is known TOTIPOTENCY/EARLY EMBRYO CELLS about PrE formation. Current model involves specification of PrE vs. EPI of Poster Board Number: 2321 the inner cell mass (ICM) cells and then segregation of these two lineages that lead to two morphologically distinct compartments by the time of IDENTIFICATION OF MOUSE STEM CELL- implantation. By combining live imaging of embryos and embryo-derived stem cells expressing a histone H2B-GFP fusion reporter under the control of SURFACE PROTEINS ENABLES ISOLATION Platelet-derived growth factor receptor alpha (Pdgfra) regulatory elements OF LINEAGE PROGENITOR CELLS DIRECTLY with the analysis of lineage-specific markers, we have determined that FROM THE MOUSE BLASTOCYST USING FLOW Pdgfra expression coincides with GATA6, the earliest expressed transcrip- tional regulator of the PrE lineage. In addition, our data show that GATA6 CYTOMETRY is required for the activation of Pdgfra expression. Based on these observa- Cox, Brian J., Rugg-Gunn, Peter, Lanner, Fredrik, Sharma, Parveen, tions, we propose a model whereby initiation of Pdgfra expression requires Ignatchenko, Vladimir, Garner, Jodi, Gramolini, Anthony, Rossant, GATA6, and its maintenance requires GATA4 and GATA6. Using pharmaco- logical inhibition and genetic inactivation, we have addressed the role of the Janet, Kislinger, Thomas PDGF pathway in the PrE lineage as well as its derivative tissue, the visceral Program in Stem Cell and Developmental Biology, Hospital for Sick endoderm. Our results reveal a role for PDGF signaling in the establishment Children Research Institute, Toronto, ON, Canada, Physiology, University and proliferation of XEN cells, isolated from mouse blastocyst stage embryos of Toronto, Toronto, ON, Canada, Ontario Cancer Institute, Toronto, ON, and representing the PrE lineage. While implanting Pdgfra mutant blasto- Canada cysts exhibit a reduced number of PrE cells, an effect that is exacerbated by Prospective isolation of epiblast, primitive endoderm and trophectoderm delaying implantation. We noticed in mutant embryos an abnormal increase cells from mouse blastocysts would enable new ways to study how these of apoptosis in PrE cells prior cell sorting. This is tempting to speculate that lineages are regulated. However, this is currently a major technical challenge PDGF signaling may be required for cell survival and/or cell sorting during because few cell-surface proteins are known that can be used to distinguish the segregation of EPI vs. EPI lineages. Furthermore, tissue-specific inactiva- and isolate each cell type. The small size and quantity of mouse embryo tion of Pdgfra in the visceral endoderm reveals an unanticipated role for does not make them readily amenable to current proteomic techniques. We PDGF signaling in early postimplantation development. Taken together, our reasoned that embryonic, trophoblast and extraembryonic endoderm stem data provide new insights into the roles of PDGF signaling in the establish- cells which are derived from the early blastocyst could be used as a model ment and propagation of the extraembryonic lineage lineage. for their originating cell types. We applied an integrated proteomic and Poster Board Number: 2325 bioinformatic strategy to identify proteins expressed at the cell surface of these three stem cell lines. Cell-surface proteins were selectively labeled with CRITICAL REQUIREMENT OF FGF SIGNALING a cleavable biotin tag, affinity purified and identified using mass spectrom- TO PRIMITIVE ENDODERM LINEAGE etry. Over 3000 proteins were detected in the three cell types and machine- learning algorithms extracted a set of 400 high-confidence cell-surface COMMITMENT IN PREIMPLANTATION proteins. This list contains both common and cell type specific proteins, EMBRYOS and provides an important stem cell resource comprising many signalling receptors, cell adhesion and migration proteins, many not previously shown Piliszek, Anna, Artus, Jerome, Kang, Minjung, Hadjantonakis, Kat to be expressed in stem cell lines. Confocal microscopy revealed that many Developmental Biology, Sloan-Kettering Institute, New York, NY, USA newly identified stem cell specific cell-surface proteins were also expressed At the time of implantation, the mammalian embryo comprises three in a lineage-appropriate manner in blastocysts. Again using the three stem distinct lineages: the pluripotent epiblast (EPI) and extraembryonic primitive cell lines mixtures of Ab were optimized that separated all three lines by endoderm (PrE) and trophectoderm. The mechanisms responsible for PrE flow cytometry. We then prospectively sorted cells of the blastocyst into vs. EPI specification are poorly understood, but FGF/Erk signaling has been separate lineages by flow cytometry. Sorted populations and single cells suggested as a key signaling pathway facilitating this decision. Through the were examined for gene expression levels of 48 key genes with a Biomark analysis of mutants, we show critical requirement for FGF4, a member of (Fluidigm). This demonstrated that epiblast and primitive endoderm initially the fibroblast growth factor family, in PrE specification and in the establish- show substantial overlap in protein and gene expression patterns, before ment of embryo-derived stem cells. Implanting Fgf4 mutant embryos lack a acquiring distinct, lineage-specific expression profiles. In contrast, trophec- primitive endoderm lineage entirely, and whole inner cell mass is comprised toderm segregates from epiblast and primitive endoderm lineages prior to of Nanog-expressing epiblast cells, a phenotype that can be rescued by blastocyst formation. Functionally, we show that flow sorted E3.5 epiblast addition of FGF2 or FGF4 ligands. Surprisingly, by combining analysis of and primitive endoderm cells of the blastocyst inner cell mass can generate key-transcriptional regulators and live imaging with a PrE-specific reporter embryonic and extraembryonic endoderm stem cells respectively, but are we observed that, in absence of Fgf4, the initial period of stochastic marker lineage restricted by E4.5. This study will enable new avenues for character- expression is established, but the subsequent restriction phase, where fate izing early progenitor cells and our proteomic approach should be applicable commitment is acquired, is impaired in the PrE lineage, suggesting that FGF to other developmental and stem cell systems. signaling is not required in vivo for the initiation of expression of lineage- Poster Board Number: 2323 specific transcription factor but is critical for lineage commitment. Address- ing the role of FGF signaling in embryo-derived stem cells we note that Fgf4 SUCCESSIVE ROLES OF PDGF SIGNALING IN deficient embryonic stem (ES) cells can be derived in much higher efficiency THE EXTRAEMBRYONIC ENDODERM OF THE than wild-type, and while refractory to epiblast lineage differentiation these ES cells can be directed towards a PrE fate by key transcription factor misex- MOUSE EMBRYO pression. By contrast, mutant XEN cells cannot be established and inhibition Artus, Jerome, Hadjantonakis, Kat of FGF signaling impairs XEN cell maintenance. Taken together, our genetic data suggest that FGF4 is required for establishment and maintenance of the Developmental Biology, Sloan-Kettering Institute, New York, NY, USA extraembryonic endoderm lineage, and that the FGF signaling levels must be Mammal preimplantation development is mainly devoted to the produc- finely regulated to generate correct numbers of PrE vs. EPI cells within the tion of extraembryonic tissues, the trophectoderm and primitive endoderm ICM of the blastocyst. (PrE), essential to embryo survival and patterning. While the mechanisms of

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Poster Board Number: 2327 this lineage barrier could be a major step to understand cell fate specification of the early embryo. CELL POLARITY AND TROPHECTODERM SEGREGATION IN EARLY MOUSE EMBRYOS Alarcon, Vernadeth B., Tamashiro, Dana Ann A. HEMATOPOIETIC STEM CELLS Institute for Biogenesis Research, University of Hawaii, Honolulu, HI, USA Poster Board Number: 2331 Trophectoderm (TE) is the first lineage to segregate in early embryos of CONDITIONING WITH BUSULFAN CONFERS placental mammals. As an extraembryonic epithelium, it is a barrier between the external environment and internal pluripotent tissue of inner cell mass ENGRAFTMENT POTENTIAL ON HUMAN (ICM) in the blastocyst stage of preimplantation development. TE dif- HEMATOPOIETIC STEM CELLS COMPARABLE ferentiates into invasive trophoblasts at implantation and contributes to placenta formation. Cell polarity in the outer cells of early embryos closely TO HOXB4 TRANSDUCTION IN SHEEP IN coincides with TE specification, suggesting that cell polarity establishment UTERO TRANSPLANTATION is linked with mechanisms of TE differentiation. We previously showed that cell polarity regulator PARD6B is essential for TE-specific features, namely Abe, Tomoyuki, Masuda, Shigeo, Nitta, Suguru, Hayashi, Satoshi, epithelialization, blastocyst cavity formation, and upregulation of transcrip- Tanaka, Yujiro, Ban, Hiroshi, Inoue, Makoto, Hasegawa, Mamoru, tion factor CDX2 in the outer cells. Pard6b knockdown (KD) disrupted cell Nagao, Yoshikazu, Hanazono, Yutaka polarity, as shown by loss of apical domain marker aPKCzeta, suggesting Division of Regenerative Medicine, Center for Molecular Medicine, Jichi that PARD6B functions upstream of aPKC. Here, we tested the possibility Medical University, Tochigi, Japan, Department of Agriculture, Utsunomiya of whether PARD6B and aPKC are interdependent. Double KD of aPKCzeta University, Tochigi, Japan, Division of Fetal Medicine, Department of and aPKCiota/lambda was performed by injecting plasmids encoding shRNA Maternal-Fetal and Neonatal Medicine, National Center for Child Health specific to each isoform in 1-cell embryos. When embryos injected with non- and Development, Tokyo, Japan, DNAVEC Corporation, Ibaraki, Japan, target or GFP shRNA plasmid formed blastocysts, double KD embryos failed Division of Biological Production Science, United Graduate School of to form a cavity and resembled Pard6b KD embryos. Absence of the cavity Agricultural Science, Tokyo University of Agriculture and Technology, is consistent with the double KD embryos having disrupted epithelium, due Tokyo, Japan to discontinuous localizations of tight junction component ZO-1. PARD6B Background: In utero transplantation (IUT) of hematopoietic stem cells protein maintained its characteristic apical domain localization, although im- (HSCs) has been pursued as a treatment for congenital hematologic and munostaining levels appeared diminished. These results implicate that aPKC genetic disorders. Although the engraftment of HSCs following IUT has regulates the level, but not the localization, of PARD6B. Next, we examined been achieved, the levels of engraftment have been generally low. In order whether PARD6B is necessary for trophoblast differentiation in vitro, by to achieve clinically relevant levels of HSC engraftment, there are many po- performing the outgrowth assay. The results were that differentiation of tro- tential ways including the HSC expansion via HoxB4 transduction, and the phoblast giant cells was highly attenuated in KD embryos, while markers of niche expansion either by co-transplantation with mesenchymal stem cells ICM derivatives were upregulated. Thus, PARD6B is essential for TE epitheli- (MSCs) or by myeloablation using busulfan (BU). BU is an alkylating agent alization and CDX2 upregulation, and loss of PARD6B in the preimplantation and has application as an alternative to total body irradiation to create open embryo interferes with trophoblast differentiation. niches for HSC transplantation. However, conditioning with BU in IUT has Poster Board Number: 2329 not been reported. Here, we compared the efficacy of BU conditioning with that of HoxB4 transduction or of co-transplantation with MSCs in the con- DISSECTING EARLY LINEAGE SPECIFICATION BY text of sheep IUT. Methods: To determine the optimal dose of BU in sheep CONVERSION OF MURINE EXTRAEMBRYONIC IUT, fetal sheep at 40-47 gestation days (full term, 147 days) were treated with BU at 0, 3, or 7.5 mg/kg (calculated by maternal body weight) via the TROPHOBLAST STEM CELLS TO PLURIPOTENT maternal vein. In the fetuses receiving 3 mg/kg of BU, the fetal hematopoi- STEM CELLS esis was significantly suppressed without adverse events. In the fetuses re- ceiving 7.5 mg/kg of BU, they died of severe suppression of hematopoiesis. Kuckenberg, Peter, Kubaczka, Caroline, Peitz, Michael, Bruestle, We therefore determined that the optimal dose of BU for sheep IUT was Oliver, Schorle, Hubert 3 mg/kg. Next, to examine the appropriate timing of BU treatment in IUT, Department of Developmental Pathology, University of Bonn Medical BU at 3 mg/kg was administered via the maternal vein at 2 or 6 days prior School, Bonn, Germany, Institute of Reconstructive Neurobiology, to IUT of human cord blood (CB) CD34+ cells into the fetal liver (6-day- University of Bonn Medical School, Bonn, Germany interval group [BU-6], n = 4; 2-day-interval group [BU-2], n = 4; control group, n = 6). We also performed IUT of human CB CD34+ cells together In mammals, life starts with a single totipotent cell, followed by several with human MSCs in addition to BU-treatment at 6 days prior to IUT (BU-6 cleavages and specification of the earliest cell lineages at the blastocyst + MSC co-transplantation group, n = 4). In addition, we performed IUT stage. The trophectoderm (TE) represents the extraembryonic trophoblast of human CB CD34+ cells transduced with HoxB4 by Sendai virus vector lineage and envelops the inner cell mass (ICM), which gives rise to the (HoxB4 group, n = 4). After birth, the engraftment of human hematopoietic embryonic tissues and the primitive endoderm. Embryonic stem cells (ESCs) cells in the lambs was quantitatively evaluated by colony PCR of the bone derived from the ICM are pluripotent because they differentiate into all marrow. Results & Discussion: The BU-6 group showed increased levels of somatic tissues including the germline but have lost the ability to form engraftment compared to the control group (p < 0.01), although the BU-2 trophoblast derivatives. Likewise, trophoblast stem cells (TSCs) derived from group did not show increased levels of engraftment. Thus, the engraftment the TE-layer exclusively recapitulate the developmental potency of their was improved by BU-treatment at 6 days, not at 2 days (that is, not at the parental lineage and contribute to all trophoblast cell types of the concep- regular clinical setting), prior to IUT. In the BU-2 group, BU may have still tus. Thus, prior to implantation, transcriptional networks and epigenetic remained at the time of IUT due to the immature metabolism of drugs in modifications separate the extraembryonic and embryonic fate irrevocably. sheep fetuses, resulting in the failure to improve engraftment. Notably, the Here, we report that extraembryonic trophoblast stem cell lines could be conditioning with BU resulted in improved engraftment to similar levels as converted to induced pluripotent stem cells by forced expression of Oct4, the HoxB4 transduction (i.e. HoxB4 group). Regarding the co-transplan- Sox2, Klf4 and cMyc. Dissecting the molecular mechanisms that overcome

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tation of MSCs (i.e. BU-6 + MSC co-transplantation group), there was no Poster Board Number: 2335 additional effect of MSCs on the engraftment, presumably because he- matopoietic niches had already restored at the time of IUT (that is, at 6 days MODULATION OF NOTCH SIGNALLING after the administration of BU). Conclusion: BU-treatment at 3 mg/kg via IS REQUIRED DURING IN VITRO T CELL the maternal vein 6 days prior to transplant can enhance the engraftment of human HSCs in sheep IUT, the efficacy of which would be comparable to DIFFERENTIATION OF HUMAN ADULT AND that of HoxB4 transduction of human HSCs. In the context of IUT, we sug- CORD BLOOD-DERIVED HAEMATOPOIETIC gest that non cell-autonomous enhancement of human HSC engraftment by STEM CELLS treatment with BU would be as effective as cell-autonomous enhancement such as HoxB4 transduction. The synergistic effects by both methods remain Khoo, Melissa L.M., Carlin, Stephen M., Herrmann, Bethany R., to be elucidated. Ma, David D.F., Moore, John J. Poster Board Number: 2333 Blood Stem Cells and Cancer Research, St Vincent’s Centre for Applied Medical Research, Sydney, Australia GENERATION OF MYELOID PROGENITORS AND T cell development occurs within the thymus following a series of stage-spe- EVALUATION OF THEIR OSTEOCLASTOGENIC cific differentiation events that are governed by multiple signal transduction POTENTIAL FROM HUMAN PLURIPOTENT pathways. Notch signalling has been shown to regulate key lineage decisions leading to the T cell fate, and was employed to promote the generation of STEM CELLS T-progenitors and T-lineage cells in the Notch ligand Delta-like 1 (DL1)- Root, Sierra H., Aguila, Héctor L. expressing OP9 stromal-co-culture system. However, while Notch signalling appears necessary for early stages of T cell differentiation, it remains unclear Genetics and Developmental Biology, University of Connecticut Health whether continuous Notch activation is required in subsequent stages. In Center, Farmington, CT, USA, Immunology, University of Connecticut clinical transplantation, hematopoietic stem cells (HSCs) are routinely used Health Center, Farmington, CT, USA from adult mobilized peripheral blood (mPB) and cord blood (CB), although Hematopoietic progenitors have been successfully transplanted for blood clear differences exist in the T-lineage potential of these cells, with reduced replacement therapies. Still, one of the main problems is the rejection as- T cell generation observed from adult mPB. To address these issues, we have sociated to genetic differences between donor and host. For this reason studied the effect of Notch signal removal on cell surface phenotype and the development of efficient protocols to derive transplantable hematopoi- gene expression of human adult mPB- and CB-derived (CD34+Lin-) HSCs etic progenitors from human pluripotent stem cells is important. We have during differentiation in OP9-DL1 co-culture. Following OP9-DL1 co-culture identified monocyte progenitors able to generate macrophages, dendritic (28-84 days), Notch signal was removed for 7 days by transferring cells to cells and osteoclasts. All these cells are critical on the maintenance of tissue OP9-Control co-culture or through addition of gamma-secretase inhibitor homeostasis and control of immune responses. We hypothesize that these DAPT. CB cells differentiated to DP phenotype earlier (28 days) in OP9-DL1 progenitors could be transplantable contributing to multiple physiologi- co-culture than adult mPB cells (42 days), and also with greater frequency cal processes. We have focused on the development of osteoclasts, which (CB 10% vs Adult mPB 2%, at 70 days). In addition, CB co-cultures declined are specialized cells that develop as terminally differentiated bone resorb- to low cell numbers beyond 70 days, while adult mPB co-cultures continued ing cells in the context of bone endosteum. We propose that this feature to differentiate for up to 90 days. Interruption of Notch signalling in devel- makes them optimal vectors to deliver bioactive molecules to modify bone oping T cells resulted in increased expression of CD4 mRNA and protein, marrow microenvironments. This could include molecules able to increase and promoted the generation of DP phenotypes in both CB- and adult mPB- engraftment and progression of progenitors after bone marrow transplan- derived cells. Notch inhibition provided the greatest benefits at different tation, molecules that could improve bone repair processes or molecules time points for CB and adult mPB, 28-42 days and 70-84 days respectively. able to eliminate or reduce bone tumor metastases. We could accomplish Differences in CD3 expression were also observed, with moderate expres- this by developing monocyte progenitors from genetically modified human sion in CB-derived DP cells (13%), but only marginal expression in adult pluripotent stem cells expressing the gene(s) of interest. We have optimized mPB-derived (1%) DP cells at 70 days. We investigated the expression of two methods for developing functional osteoclasts from hESCs. The first is genes related to T cell development by real-time RT-PCR, and found TCF7, based on cocultures between hESCs and mouse stroma cell lines and the GATA3, HES1, DTX1, PTCRA, and CD8B to be initiated by Notch signalling second on the development of embryoid bodies under hematopoietic induc- (OP9-DL1 co-culture), with a subset of these genes (TCF7, PTCRA, GATA3 ing conditions. These methods can reproducibly generate derivatives with and CD8B) increasing with time in OP9-DL1 co-culture; while, IKZF1 and hematopoietic characteristics. These early hematopoietic derivatives can be MAML1 were expressed in developing HSCs regardless of Notch signal- isolated by flow cytometry, expanded in early hematopoietic cytokines and ling. For CB co-cultures, Notch removal yielded significant increases in CD4, further cultured in osteoclastogenic conditions generating osteoclasts identi- IKZF1, MAML1, and CDKN1B expression, along with slight increases in fied as TRAP positive multinucleated cells with the ability to resorb mineral GATA3, TCF7, and PTCRA; while expression of Notch signalling markers, matrix when developed over bone slices. Using antibodies against cell HES1 and DTX1, were decreased, as expected. The effect of Notch removal surface markers expressed in early monocytic lineage, we have been able to on gene expression in adult mPB co-cultures was markedly different with identify intermediate stages that could represent myeloid progenitors with few changes observed following DAPT addition, suggesting the existence of osteoclastogenic potential. These include cells with the phenotypes CD45+ molecular differences between adult mPB and CB HSCs that may account CD34+ CD38- CD45RA+ CD115+, and CD45+ CD14+ CD115+ CD117+. for the observed differences in T cell generation. Our results reveal that We are currently evaluating the individual potential of these populations to while Notch signalling is essential for establishing the T-lineage in humans, it generate osteoclasts in vitro and we are performing clonal studies to define appears to restrict differentiation in later stages of T cell development, with their precursor frequencies for their ability to generate cells with character- removal of Notch activity allowing for more efficient DP generation. istics of osteoclasts, macrophages and dendritic cells. In addition we will test the ability of these progenitors engraft and form osteoclasts in vivo upon transfer into immunodeficient animal models. Supported by NIH/NHLBI Grant. 1RC1HL100569-01 to H.L.A

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Poster Board Number: 2337 used source of CD34+ cells for allogenic HSCT are cells from Bone Mar- row or Peripheral Blood (PB) after mobilisation. Unfortunately we showed CHEMOTACTIC AND ADHESION PROPERTIES that those cells present a less marked tendency to differentiate towards the OF HUMAN POSTNATAL LYMPHOID T-lymphoid compartment. In our experiments we found that the majority of PB stem cells cultured in presence of notch-ligand rapidly loose CD34 PROGENITORS SEEDING THE THYMUS marker and express CD7 marker much less than cultured CB cells: conversely Andre-Schmutz, Isabelle, Rouiller, Julien, Luce, Sonia, Bruneau, the culture leads to the formation of a huge CD34- CD7- population that Julie, Schiavo, Andrea, Reimann, Christian, Cavazzana-Calvo, express myeloid markers. We are currently adopting two strategies. Enrich the CD34+ cells in HSC by a 4 days culture in a medium containing IL-7, Marina SCF, TPO, IGFBP2, FGF-1 and Angptl5; indeed exposure of HSC to notch- U768, INSERM, Paris, France ligand has been shown previously to inhibit their myeloid differentiation. The production of T cells involves the generation of a lymphoid progenitor A second approach uses a Fc-R blocking agent that showed the ability to in the bone marrow that can migrate into the thymus to differentiate into block the myeloid differentiation in culture leaving the pre-T population mature T cells. This process occurs throughout the life of an individual. In unchanged; expand the initial and more immature stem cell pool “pre-acti- situations of T-cell immune deficiencies, whether primary or secondary in the vating” cells in a medium with a different cytokines cocktail () or use a Fc-R context of for partially incompatible HLA the hematopoietic stem cell trans- blocking agent that showed the ability to block the myeloid differentiation in plantation, knowing the early stages of lymphopoiesis would help to identify culture leaving the pre-T population unchanged. Finally we also explore the new therapeutic strategies. We have recently characterized in human bone possibility to use other protocols of mobilisation such as G-CSF in combina- marrow, a lymphoid progenitor population that can generate in vitro B, T tion with AMD3100 because it may influence the potential of T-lymphoid and NK lymphocytes, and dendritic cells. A very similar population charac- reconstitution. terized by a Lin-CD10 + CD34 + CD24-CD7- phenotype is detected in the Poster Board Number: 2341 blood and the thymus and could be one of the missing links between bone marrow and thymus after birth. Immunofluorescence on thymic sections TRACKING THE SCID REPOPULATING ACTIVITY revealed the presence of rare and small clusters of CD34+CD7- progeni- tors near the junction between cortex and medulla. Transcriptome analysis, OF HUMAN HEMATOPOIETIC CELLS DURING combined with flow cytometry, revealed the expression of some chemokine EX VIVO CULTURE receptors (CXCR4, CCR7) and adhesion molecules (integrins, CD44, PSGL1), Baudet, Aurelie, Karlsson, Christine, Larsson, Jonas which may be involved in the migration of this population from the bone marrow to the thymus. Chemotactic assays in vitro indicate a possible role Molecular Medicine and Gene Therapy, Lund University, Lund, Sweden of SDF1 and CCL21 in this migration process. Human hematopoietic stem cells (HSC) are defined by their ability to Poster Board Number: 2339 reconstitute hematopoiesis in immunocompromised mouse models such as the NOD/SCID strain and its derivatives. Identification of so-called SCID INDUCTION OF HUMAN T-CELL DEVELOPMENT repopulating cells (SRC) in cultures of hematopoietic stem- and progeni- tor cells (HSPC) is critical in order to study stem cell regulatory pathways FROM CD34+ PROGENITORS BY EXPOSURE TO and to further establish protocols for ex vivo expansion of HSCs. However, IMMOBILIZED NOTCH LIGAND DELTA-LIKE-4 prediction of SRC activity in cultured HSPCs by cell surface marker expres- sion would greatly facilitate such efforts. While the markers CD34 and Schiavo, Andrea Alex, Reimann, Christian, Rouiller, Julien, CD133 are commonly used to track primitive cells in culture, they define Caccavelli, Laure, Beldjord, Kheïra, Amsellem, Sophie, Dal Cortivo, a heterogeneous population with both stem- and progenitor cell activity. Liliane, Cavazzana Calvo, Marina, André-Schmutz, Isabelle In freshly isolated human hematopoietic cells, SRCs are highly enriched University of Padua - Université Paris Descartes, Paris, France, U, INSERM, in the Lin-CD34+CD38-CD90+CD45RA- compartment as assayed by Paris, France, Biothérapie, Hôpital Necker, Paris, France, Hôpital Necker, serial transplantations. Consequently we evaluated whether these same Paris, France, Biologie et de Pathologie Médicales, Institut Gustave markers could be used to enrich for SRCs in cultured HSPCs. Cord blood ROUSSY, Villejuif, France derived CD34+38-90+45RA- cells were cultured for 10 days in serum- and stroma-free conditions that support maintenance of SRC activity. The Allogenic Hematopoietic Stem Cell Transplantation (HSCT) is used to treat CD34hi population from these cultures was then separated and evaluated primary immunodeficiencies and some hemopathies. During the last years, for expression of CD38, CD45RA and CD90. We found that both CD38 many progresses have been made in this field but patients that undergo and CD45RA were downregulated in a generalized manner in the cultured this procedure are often subject to lethal infections because of the slow cells and therefore are of limited value as negative markers. CD90, on the reconstitution of the T-lymphoid compartment. Our goal is to establish a other hand, showed distinct expression in a small subset of the CD34hi cells method to generate in vitro large amount of T-cell precursors that could be (10-20%). We assayed the SRC content of CD34hiCD90+ and CD34hi90- injected into patients to speed up T-cell reconstitution after allogenic HSCT. cells by transplanting limiting numbers of each population to sublethally Exposure of hematopoietic stem cells to notch-ligand molecules trigger their irradiated NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) recipients. Trans- proliferation and differentiation towards the T-cell lineage. Notch signal- plantation of 2000 CD34hiCD90+ cells resulted in robust reconstitution of ling recreate in vitro the same kind of stimuli present in the thymus during human cells (20±5%) after 16 weeks. By contrast, 30-fold more cells (60 thymopoiesis. Co-culture of CD34+ cells with murine stromal lines express- 000) cells had to be transplanted to reach similar reconstitution levels from ing notch-ligands (i.e. delta-like-1 or delta-like-4) leads to a complete T-cell the CD34hiCD90- fraction (13±13%). Thus, the CD34hiCD90+ fraction of differentiation but, considering the murine origin and the genetic modifica- cultured HSPCs is highly enriched in SRCs. We next evaluated the same frac- tion of the stromal cells, our ligand-based strategy seems more suitable for tions for their ability to form mature hematopoietic colonies (CFCs). Striking- human application. Our strategy is to expose CD34+ cells to the ligand ly, we found that the CD34hiCD90+ cells had barely detectable CFC activity only in a medium containing a cocktail of cytokines (IL-7, SCF, Flt3-ligand while the CD34hiCD90- cells efficiently generated CFCs of multiple lineage and TPO) known to be important in human haematopoiesis. CD34+ Cord types. This shows that CD90 expression in cultured human hematopoietic Blood cells proliferated and after some days of culture begin to express CD7 cells uncouples the most primitive cells that have SRC activity from more marker. We demonstrated that the CD34+/- CD7+ population contains committed progenitors with CFC activity. We conclude that CD90 is a useful cells able to reconstitute the T-lymphoid compartment when injected into surrogate stem cell marker for in vitro studies of human HSPCs. irradiated NOD/SCID/γcnull mice (within 2 months). Nowadays the most

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Poster Board Number: 2343 dynamic interplay between these regulatory axis remain poorly understood. Simultaneous tracking of the concentration of 30+ factors in hematopoietic THE ROLE OF THE PKC ISOFORM δ IN LINEAGE cell culture, including TGF-b1, MIP-1b, and MCP-1, demonstrated that COMMITMENT OF HUMAN HEMATOPOIETIC computer controlled media supplementation could be used to regulate para- crine interactions. Maintaining endogenous factors at specified levels using STEM CELLS a fed-batch delivery strategy yielded significant improvements in expansions Berger, André, Hamdorf, Matthias, Schüle, Silke, Reinhardt, Jens, of total cell numbers (TNC, ~180x), colony forming cells (CFCs, ~64x), long- Flory, Egbert term culture-initiating cells (LTC-ICs, ~28x), and NOD/SCID/IL-2Rgc-null (NSG) repopulating cells (~11x, detected at limiting dilution after 16 weeks). Medical Biotechnology, Paul-Ehrlich-Institute, Langen, Germany Unexpectedly, supplementation of the transcription factor, HOXB4, to fed- Stem cells and their differentiation into a variety of specialized cells and batch and control cultures yielded the expected boost in progenitor expan- tissues set these cells in the focus of regenerative medicine. However, a well- sion only in “sub-optimal” control conditions. Hypothesizing that HOXB4 controlled characterization of cells is one of the current challenges during action may be dependent on the skewing of supportive vs. non-supportive translation into clinical applications. Since surface marker expression may not cells, and the consequent impact of the cell produced paracrine factors, we reflect the cell-specific differentiation status, identification and directed con- performed systematic kinetic tracking of 20 hematopoietic cell types and trol of the mechanisms that drive cell fate and lineage commitment of these secreted factor concentrations, during several supportive (fed-batch, HOXB4 multi-potent cells might add a further tool for cell characterization. In this supplemented, Notch ligand Delta1 supplemented) vs. non-supportive study, we focused on the intracellular processes that occur during differen- (control) cultures. Meta-analysis of this data revealed a non-autonomous tiation of human hematopoietic cells (HSCs) towards myeloid dendritic cells link between HOXB4, increased megakaryocyte production, and stimulated (mDCs). This differentiation is induced by the cytokines GM-CSF and IL-4 stem cell proliferation, as well as between Notch ligand, decreased myeloid and led to activation of the ERK, PKC and JAK/STAT but not the SAPK/JNK cell production, and a decrease in stem cell inhibition. These predictions have and p38 MAPK signaling pathways. Only the ERK and PKC pathway were been directly experimentally tested using sorted purified HSCs and CD41+ found critical for differentiation into mDCs. The transcription factor PU.1, megakaryocytes and CD14+ monocytes. Our results identified complex which is described as one of the key factors for myeloid lineage commit- non-linearities in the system dynamics and we are using a combination of ment, was phosphorylated by the PKC isoform δ but not by ERK. Moreover, experimental and mathematical tools to understand and further manipulate we identified for the first time that the transactivation domain of PU.1 is cell fate decisions. Collectively, these studies support a dominant role for phosphorylated by PKC δ. Accordingly the PU.1 transactivation activity was non-stem cell autonomous feedback inhibition in the regulation of blood regulated by PKC δ, whereas the DNA binding activity of the transcription stem cell self-renewal, especially in adult stem cell cultures where heteroge- factor was not affected. Supporting our findings, inhibition of PKC- and neity ultimately emerges. Overcoming cell non-autonomous control of HSC ERK1/2-specific signaling by small molecules prevented differentiation self-renewal will enable novel strategies to enhance endogenous stem cell towards mDCs as well as phosphorylation-mediated transactivation activity growth, enabling more effective strategies to treat hematologic disease and of PU.1. Our data provide new insights into the molecular mechanisms increase the therapeutic potential of umbilical cord blood cells. promoting the differentiation process of HSCs towards mDCs and introduce Poster Board Number: 2347 the PKC isoform δ as critical mediator. In addition, our approach underlines the feasibility and importance of uncovering the intracellular processes in EXPRESSION OF INHIBITORY RECEPTOR ILT3 BY differentiation-competent cells in order to support their characterization as well as to provide a rationale for directed, small-molecule-driven control for HUMAN ADULT HEMATOPOIETIC STEM CELLS (re-)programming. AND MYELOID PROGENITORS Poster Board Number: 2345 Dobrowolska, Hanna M., Schwartz, Joseph, Li, Qing, Serban, Geo, Suciu-Foca, Nicole, Colovai, Adriana I. PREDICTIVE CONTROL OF HUMAN Pathology and Cell Biology, Columbia University Medical Center, New HEMATOPOIETIC STEM CELL SELF-RENEWAL York, NY, USA BY MANIPULATING NON-STEM CELL The mechanisms which control the function of mature immune cells inte- ASSOCIATED ENDOGENOUS SIGNALLING grate activating and inhibitory signals. The inhibitory receptor ILT3 is a mem- ENABLES A GREATER THAN 10-FOLD ber of the immunoglobulin-like transcript family and is expressed by mono- cytes and dendritic cells. Expression of ILT3 increases the activation threshold EXPANSION OF LONG TERM NOD/SCID of antigen presenting cells and inhibits T cell responses, such as proliferation REPOPULATING CELLS IN A CLINICALLY and cytokine release. Since little is known about the expression of this inhibi- tory receptor during the differentiation of human hematopoietic stem cells RELEVANT CLOSED SYSTEM BIOREACTOR (HSC), we studied ILT3 expression on normal and leukemic hematopoietic Csaszar, Elizabeth, Kirouac, Daniel C., Yu, Mei, Zandstra, Peter W. progenitors. In vitro differentiation of human HSC obtained from peripheral University of Toronto, Toronto, ON, Canada blood of allogeneic healthy donors was induced using interleukin-3 (IL-3) and granulocytes-monocytes colony stimulating factor (GM-CSF). Culture Intercellular (between cell) communication networks are an important was maintained for 20 days. Flow cytometry analysis of CD34 positive hu- component of the stem cell niche. These networks maintain homeostasis man hematopoietic stem cells indicated lack of cell surface expression of the and coordinate regenerative and developmental cues in multicellular organ- inhibitory receptor ILT3. However, on day 10 of culture, ILT3 was detected isms. Despite the importance of intercellular networks in stem cell biology, on a cell population co-expressing CD13, CD33, HLA-DR, CD11c (+/-), and their rules, structure, and molecular components are poorly understood. dim cMPO. These cells were negative for CD34, CD117 and CD14. This The hematopoietic system provides a powerful platform to study cell-cell phenotype is most consistent with that of pro-monocytes. To study ILT3 interactions due to our ability to characterize and prospectively control cell expression on the leukemic counterparts of human myeloid precursors, we types, secreted factors, and their ratios, using clinically relevant human analyzed bone marrow cells obtained from 49 patients with acute myeloid blood progenitors. Although evidence suggests that both cell autonomous leukemia (AML). We found that ILT3 was present on the surface of leukemic (stem cell-associated transcription factors, such as HOXB4) and cell non- cells from all patients with AML displaying monocytic differentiation, but not autonomous (the stem cell niche) mechanisms regulate stem cell fate, the on leukemic cells from patients with other types of AML. Our data indicate

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Thursday Poster Abstracts that expression of ILT3 is not restricted to mature monocytes and that ILT3 miR-451 binding sequences), there were decreased late erythroid (CD34- expression is acquired by normal myeloid precursors at the pro-monocyte CD71hiCD235ahi) cells, as compared to control vector-transduced cells, stage. ILT3 is also expressed by the leukemic counterparts of monocyte after EPO-induced erythroid differentiation. In TF1 cells transduced with the precursors and therefore, it may represent a useful marker for the diagnosis miR-144 trap, no effect was detected. In TF1 or CD34+ cells transduced of AML with monocytic differentiation. Although the function of ILT3 on the with the miR-144 overexpression lentivirus, no effect was detected; how- pro-monocytes is not yet known, our findings suggest that ILT3 may partici- ever, in TF1 or CD34+ cells transduced with the overexpression lentivirus pate in the regulation of HSC maturation and malignant transformation. containing the genomic miR-144~451 cluster, the late erythroid cell popula- tion was decreased. The decreased erythropoiesis observed in cells trans- Poster Board Number: 2349 duced to overexpress the miR-144~451 cluster was unexpected, because it MODELLING TEL/AML1+ ACUTE was recently reported that miR-144~451 cluster overexpression in a mouse erythroleukemia (MEL) cell line promoted late erythroid differentiation (as LYMPHOBLASTIC LEUKEMIA USING HUMAN measured by benzidine staining). Explanations for these contradictory results EMBRYONIC STEM CELLS include (a) technical factors, e.g. resulting in overexpression level differ- ences; and/or (b) miR processing or functional differences between primary Berks, Richard, Coles, Mark human cells and a mouse leukemia cell line model. Centre for Immunology and Infection, Department of Biology, University of York, York, United Kingdom Poster Board Number: 2353 The TEL/AML1 fusion protein is created by the pre-natal chromosome trans- L4, AN IMPORTANT REGULATOR FOR location t(12;21)(p13;q22), and is the most common gene mutation in child- HEMATOPOIESIS DURING MOUSE EMBRYONIC hood cancer, responsible for 20% of all cases of pre-B cell Acute Lympho- blastic Leukaemia (ALL). However, the cellular and molecular mechanisms DEVELOPMENT by which TEL/AML1 causes the disease are unclear, and mouse models Yu, Hongyao, Jin, Ying do not fully replicate the human disease. In addition, the frequency of the Shanghai Institutes for Biological Sciences, Institute of Health Sciences, translocation amongst the leukaemia-free population is still debated, and the Shanghai, China role of common secondary mutations (such as the deletion of the remaining untranslocated TEL allele) is not fully understood. Human embryonic stem Our previous study demonstrated that L4, a putative serine-threonine (hES) cells present a unique opportunity for modelling diseases by virtue kinase, is a downstream target of transcription factor Oct4. L4 promotes dif- of their pluripotency and ability to be expanded in culture and genetically ferentiation of mouse embryonic stem cells towards extraembryonic endo- manipulated in the lab. As part of our research into TEL/AML1+ leukae- derm lineages through activation of the MAPK (Erk1/2) pathway. However, mia, we have generated several hES cell lines expressing the TEL/AML1 the physiological role of L4 in mouse embryonic development is absolutely fusion protein. We intend to differentiate these cell lines to investigate the unknown. To address this question, we generated L4 knockout mice through effect of TEL/AML1 on haematopoiesis and B cell lymphopoiesis, using homogenous recombination. Interestingly, L4-null embryos can survive both embryoid body and co-culture differentiation techniques. We will also until birth, but die soon within 12 hours neonatally for pulmonary atelecta- investigate the role of TEL allele deletion in this disease by shRNA-mediated sis. Through blood cell counting, embryos lacking L4 display fetal anemia knockdown of TEL mRNA in haematopoietic progenitors derived from these symptom of fewer erythrocytes, lower hematocrit and hemoglobin content. hES cell lines. This will complement our work with the overexpression and The mutants have a severe reduction of hematopoietic organ size, including knockdown of TEL in patient-derived leukaemia cell lines. smaller fetal liver, spleen and bone marrow. In E14.5 fetal liver, a predomi- nant hematopoietic organ during embryonic development, the percentage Poster Board Number: 2351 and amount of granulocytes in KO fetal liver is significantly increased, which ENFORCED EXPRESSION OF THE MIR-144~451 is consistent with fetal spleen differential lineage at later stage. Around birth, bone marrow starts and then sustains a life-long hematopoiesis. In the fetal CLUSTER DECREASED HUMAN ERYTHROID bone marrow at E18.5, by colony forming cell assay, it is of note that the DIFFERENTIATION percentage of granulocyte/myeloid progenitors is also increased. Interest- ingly, in zebrafish, Mpo, a granulopoiesis marker, is also strikingly increased Kim, MinJung, Heiser, Diane, Civin, Curt I. by L4 knockdown with morpholino oligonucleotides. These findings suggest Pediatrics and Physiology, University of Maryland Baltimore, Baltimore, that L4 may regulate the lineage commitment of myeloid progenitors and MD, USA it may be an important regulator for fetal granulopoiesis and evolutionarily conserved. Further investigation will focus on uncovering the phenotypic MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression details and underlying mechanisms through reconstitution and in vitro dif- by inhibiting target gene translation and/or by degrading target mRNAs. ferentiation. The miR-144~451 cluster contains these two miRs, which are expressed from a single precursor transcript. Expression of both miR-144 and miR-451 Poster Board Number: 2355 are upregulated during zebrafish, mouse, and human erythroid differentia- tion. Several mouse and zebrafish studies have knocked down miR-451 ENGRAFTMENT OF EMBRYONIC MURINE expression and observed decreased late erythroid cells. In contrast, miR-144 HEMATOPOIETIC STEM CELLS IN THE MORE knock down had no or less obvious effects, either in zebrafish or mouse erythropoiesis, and there is no study of the function of this cluster in primary PERMISSIVE NEONATAL HEMATOPOIETIC human erythropoiesis. To study this miR cluster in human erythropoiesis, MICROENVIRONMENT we first tested the TF1 human erythroleukemia cell line, as a model, and then primary human CD34+ hematopoietic stem-progenitor cells (HSPCs) Arora, Natasha, McKinney-Freeman, Shannon, Heffner, Garrett, to determine the effects of lentiviral inhibition and overexpression of miR- Jang, Il-Ho, Wenzel, Pamela, Daley, George Q. 144 and/or miR-451. When either TF1 or CD34+ cells are cultured with Harvard Medical School, Boston, MA, USA, St. Jude Children’s Research erythropoietin (EPO), the expression of CD71 and CD235a erythroid mark- Hospital, Memphis, TN, USA, Children’s Hospital Boston, Boston, MA, USA ers increases and the expression of CD34 hematopoietic stem-progenitor Yolk sac from embryonic day 9.0 (E9.0) of mouse development lacks defini- cell (HSPC) marker decreases. In TF1 or CD34+ cells transduced with the tive hematopoietic stem cells (HSC) capable of engrafting irradiated adult miR-451 trap (using a lentivirus containing GFP and a series of 8 tandem

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recipients, but long-term multi-lineage reconstitution is robust in neonatal to adopt the stress erythroid fate and it also plays a role in the expansion of recipients, indicating that the neonatal hematopoietic microenvironment is stress erythroid stem cells. more permissive for engraftment of embryonic HSCs. The more permissive microenvironment may allow us to reconcile limiting dilution studies with Poster Board Number: 2359 adult recipients and recent live imaging studies, which describe emerging ENDOTHELIAL AND PERIVASCULAR STROMAL HSCs in the aorta-gonad-mesonephros (AGM). We sought to characterize engraftment of neonatal recipients with hematopoietic populations dissected CELLS PROMOTE MURINE HEMATOPOIETIC from early intraembryonic stages of definitive hematopoietic development STEM CELL MAINTENANCE IN THE BONE in the AGM region. We dissected whole AGM from E11.5 embryos and in- jected cell dilutions from 2 embryo equivalents (ee) to 0.25 ee into the facial MARROW BY SECRETING STEM CELL FACTOR vein of day 1-2 neonatal recipients that had received sublethal conditioning (SCF) with 350 rad irradiation. In the neonatal recipient we detected robust, long- Ding, Lei, Morrison, Sean J. term, multi-lineage hematopoietic engraftment from as little as 0.25 ee. From less than 1 ee of whole AGM, the engraftment chimerism ranged from Howard Hughes Medical Institute, Center for Stem Cell Biology, Life 5-20%. With 2 ee, chimerism was as high as 70%. Most animals showed Sciences Institute, University of Michigan, Ann Arbor, MI, USA balanced donor derived myeloid and lymphoid contribution by 10 weeks Although various cell types have been proposed to create niches for he- post-transplant. However, some animals had predominantly myeloid recon- matopoietic stem cells (HSCs) in the bone marrow, no factor that is geneti- stitution for as long as 14 weeks but ultimately evolved balanced myeloid cally necessary for HSC maintenance has ever been conditionally deleted and lymphoid contribution by 18 weeks. A few animals remained pre- from any cell type in the bone marrow. Absent such data, it is unknown dominantly myeloid even at 18 weeks, suggesting the presence of a novel which bone marrow cells are physiologically important sources of the factors long-term, myeloid-restricted, embryonic HSC. Our data indicate that the that promote HSC maintenance. Stem cell factor (SCF) is required for the neonate harbors a more permissive hematopoietic microenvironment that maintenance of normal numbers of HSCs but it is unknown which bone enables engraftment of early embryonic hematopoietic populations, thereby marrow cells secrete this critical niche factor. To address this question, we allowing us to identify potentially novel classes of embryonic hematopoietic have generated an Scf gfp knockin allele and used it to systematically exam- progenitors. We are currently exploring the neonatal engraftment potential ine the expression pattern of Scf throughout the bone marrow. Perivascular of E9.5 and E10.5 embryonic populations, FACS-purified populations, and and endothelial cells were the two major sources of Scf expression in the hematopoietic populations derived from pluripotent stem cells in vitro. bone marrow while we were unable to detect Scf expression by osteoblasts. Poster Board Number: 2357 To assess SCF function, we generated a Scf fl allele and globally deleted Scf in adult mice using Ubc-CreER. We found that HSC frequency was signifi- HEDGEHOG SIGNALING SPECIFIES THE cantly reduced after global Scf deletion, genetically confirming that SCF is required for the maintenance of normal numbers of adult HSCs. When Scf STRESS ERYTHROID PROGENITOR CELL FATE was conditionally deleted from endothelial cells or perivascular cells, HSC AND PROMOTES THE EXPANSION OF SELF frequency (analyzed by flow cytometry) and function (analyzed by competi- RENEWING STRESS ERYTHROID PROGENITORS tive reconstitution assays) was significantly reduced. In contrast, when Scf was conditionally deleted from osteoblasts, we did not observe significant IN THE MURINE SPLEEN effects on HSC frequency or function. These results demonstrate that en- Wu, Dai-Chen, Paulson, Robert F. dothelial and perivascular cells are two critical components of the HSC niche that promote HSC maintenance by secreting SCF. It remains possible that Pennsylvania State University, University Park, PA, USA osteoblasts secrete other factors that regulate HSCs maintenance and that During fetal development and in response to acute anemia in adults, stress they also contribute, directly or indirectly, to the niche. Together with our erythropoiesis rapidly produces large numbers of new erythrocytes by a previously published data demonstrating that most HSCs localize adjacent to mechanism that is distinct from bone marrow homeostatic erythropoiesis. sinusoidal blood vessels in the bone marrow, our data directly demonstrate Our laboratory has demonstrated that BMP4, SCF, and hypoxia act in that there is a perivascular niche for HSCs in which multiple distinct types of concert to regulate the expansion and differentiation of stress erythroid perivascular cells secrete factors that promote HSC maintenance. progenitors that are resident in spleen in response to acute anemia. In addi- tion to these three signals, we have also identified that Hedgehog signaling Poster Board Number: 2361 is required for the maintenance of the BMP4 dependent stress erythropoiesis CHRONIC KIDNEY DISEASE IMPAIRS THE BONE pathway. Following recovery from acute anemia, bone marrow cells migrate into the spleen. Once in the spleen, hedgehog and BMP4 dependent signal- MARROW CELLS CAPACITY TO RECONSTITUTE ing cause the bone marrow cells to adopt the stress erythroid progenitor A COMPLETE HEMATOPOIETIC SYSTEM IN fate. In this present research, we studied the role of Hedgehog signaling in the generation of erythrocytes immediately following bone marrow MICE transplantation into lethally irradiated mice, which is referred to as erythroid Monge, Matthieu, van Pel, Melissa, Slot, Edith M., Siebelt, Michiel, short-term radioprotection. Pharmacological and genetic approaches were van Solingen, Coen, Koekkoek, Karin, Rabelink, Ton J., Fibbe, utilized. The donor bone marrow cells that are inhibited or mutant in the Willem E., van Zonneveld, Anton Jan Hedgehog signaling pathway are unable to provide erythroid short-term radioprotection. The recovery of the erythroid lineage was significantly Nephrology, Leiden University Medical Center, Leiden, Netherlands, delayed in the surviving mice. Conversely, transplanting donor bone marrow Immunohematobiology and Blood Transfusion, Leiden University Medical cells where hedgehog signaling is activated resulted in enhanced erythroid Center, Leiden, Netherlands, Orthopaedics, Erasmus Medical Center, recovery when compared to mice transplanted with control donor cells. To Rotterdam, Netherlands dissect the cause of defective hematopoietic regeneration when Hedge- Hematopoietic stem cells (HSC) reside in the HSC niche. In this microen- hog signaling is inhibited and to investigate roles of Hedgehog in stress vironment, the bone-lining osteoblasts tightly regulate the HSC through erythropoiesis, we also examined differentiation of erythroid progenitors and various molecular interactions, including N-cadherin, Jagged1 and SDF1. hematopoietic stem cells by blocking Hedgehog signaling. Our research re- Chronic kidney disease-mineral bone disease (CKD-MBD) is a complication sults demonstrate that Hedgehog signaling is essential for bone marrow cells of CKD, notably characterized by increased osteoblast activity under high

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Thursday Poster Abstracts circulating parathyroid hormone (PTH) levels, leading to bone structural excessive inflammation or autoimmunity against stem cells through multiple changes. We developed a mouse model for CKD with features of CKD-MBD local immune suppressive mechanisms (IP mechanisms). Historically Peter to investigate the effects of the bone changes on hematopoiesis. Female Medawar demonstrated these IP sites more than 60 years ago. More recent C57bl/6-Ly5.1 underwent a surgical procedure to induce renal failure stem cell research has led to the discovery of somatic stem cells and their (thermocauterization of the right kidney cortex followed by a contralateral niches, and yet an immunological question whether somatic stem cell niches nephrectomy, CKD group) and was compared to sex- and age-matched more broadly are IP sites where stem cells are shielded from immune attack sham-operated controls (control group). 12 weeks after CKD induction, has been overlooked. Uncovering IP mechanisms of somatic stem cell niches all mice show features of CKD, including elevated levels of serum urea, will help explain how malignant cells derived from stem cells in the niche increased PTH levels and subsequent alteration of the long bones struc- can escape from immune escape, how the development of the autoimmune ture (increased trabecular volume, decreased cortical thickness, decreased disorders against stem cells is prevented, and how the rejection of allogeneic span incurvation). Bone marrow cells (BMC) were obtained and assessed stem cells following stem cell transplantation is prevented. To this end, we phenotypically by FACS analysis for the presence of HSC and hematopoietic herein examined if one of the best-defined somatic stem cell niches, the progenitor cells (HPC). Their respective long- and short-term repopulating hematopoietic stem/progenitor cell (HSPC) niche in the bone marrow (BM), capacities were tested in vitro by the cobblestone area forming cell (CAFC) is an IP site. Using intravital fluorescence microscopy recently developed for assay and in vivo by radioprotection experiments. Osteoblasts were ob- tracking fluorescently labeled cells in the mouse calvarium BM, we demon- tained for gene expression analysis. Phenotypical analysis of BMC obtained strated unexpectedly prolonged survival (longer than 30 days) of trans- from CKD mice show a decreased frequency of HSC and unchanged fre- planted allogeneic HSPCs in non-irradiated immune competent hosts, with quency of HPC compared to control mice. In contrast, in the CAFC analysis, the same survival frequency compared to syngeneic HSPCs, while allogeneic both HSC and HPC frequencies are decreased. To investigate the in vivo differentiated cells are rejected by the host immunity within 7 days. This is relevance of these findings, 250E3, 100E3 and 50E3 BMC obtained from surprising and appears contrary to the well-defined clinical experience that CKD mice or control mice were transplanted into lethally irradiated (9.5 Gy) indicates strong immune suppressive therapy and careful donor selection are C57Bl/6-Ly5.2 recipients (n=5 to 6). Survival rate and granulocyte chimerism required to prevent rejection in allogeneic BM transplantation. Prolonged levels were monitored. Nine weeks after transplantation, recipients of 250E3 survival of allogeneic HSPCs without immune suppression is consistent and 50E3 BMCs obtained from the CKD donors showed similar survival with Peter Medawar’s observation of prolonged survival of transplanted rates compared to recipients of the control donors (250E3: 80% vs 80%, allogeneic/xenogeneic grafts in other IP sites, supporting our hypothesis that p=NS; and 50E3: 20% vs 20%, p=NS). In contrast, survival was decreased the HSPC niche also has IP mechanisms. These HSPCs were lost after the among recipients of 100E3 BMCs obtained from CKD donors (50% vs 20%, depletion of FoxP3 regulatory T cells (Tregs). Notably, Tregs accumulated on p=NS). The granulocyte chimerism levels at 3 and 9 weeks after transplanta- the vascularised endosteal surface, made clusters with transplanted HSPCs. tion is reduced in recipients of 250E3 CKD-BMC (week 3: 98.9±0.6% vs Time-lapse in vivo imaging revealed active Treg movement around HSPCs, 92.9±2.2%, p<0.05; week 9: 93.0±10.6% vs 83.3±18.3%, p=NS), 100E3 suggesting immune-surveillance activity. Allogeneic HSPC transplantation CKD-BMC (week 3: 99.3±0.6% vs 82.9±16.2%, p=NS; week 9: 99.4±0.0% increased IL-10 production of BM Tregs. In vivo knockout of IL-10 in Tregs vs 98.6%), and 50E3 CKD-BMC (week 3: 99.72% vs 96.85%; week 9: resulted in pronounced rejection of allogeneic HSPCs. Together Tregs appear 96.7% vs 1.1%). To investigate the relationship between the CKD-MBD to participate in creating HSPC niche where Tregs provides IP via an IL-10 and the observed changes in the hematopoietic compartment, osteoblasts dependent mechanism, shielding allogeneic HSPCs from host immunity. In expression of genes involved in bone metabolism and interactions with the addition to processes supporting stem cell function, the somatic stem cell HSC niche was measured by qRT-PCR. In CKD-osteoblasts, RANK-L, M-CSF niche will provide a relative sanctuary from immune attack. and N-Cadherin gene expression increased respectively 2.4-, 2.9-, and 1.6-fold compared to control osteoblasts. SDF1 and PTH1R gene expres- Poster Board Number: 2365 sion was respectively 2- and 5-fold decreased; and Jagged1 gene expression BONE MARROW-DERIVED STEM CELL remained similar. In conclusion, we show in the CKD mice 1) functional in vitro and in vivo impairment of the HSC and HPC, and 2) differential TRANSPLANTATION IN A MOUSE MODEL OF expression of genes involved in both bone metabolism and HSC homeostasis RETINAL DEGENERATION expression in osteoblast. Taken together, our results suggest a functional defect in the HSC niche in CKD due to impaired osteoblast function. Enzmann, Volker, Lecaudé, Stéphanie, Pepin, Andrea, Tschopp, Markus, Wolf, Sebastian Poster Board Number: 2363 Ophthalmology, University of Bern, Bern, Switzerland IN VIVO IMAGING OF REGULATORY T CELLS As retinal degeneration is one of the most relevant causes of blindness in the PROVIDING IMMUNE PRIVILEGE TO ADULT industrialized world the development of regenerative therapies is crucial. In the current study, we have therefore investigated integration, differentiation MOUSE HEMATOPOIETIC STEM CELL NICHE and potential to rescue vision of bone marrow-derived stem cells (BMSC) Fujisaki, Joji, Sykes, Megan, Strom, Terry B., Scadden, David T., Lin, after transplantation into the subretinal space of mice with pharmacologi- Charles P. cally induced retinal degeneration. Adult C57BL/6 mice (6-8 weeks) were systemically treated with sodium iodate (NaIO3, 15 mg/ kg) to induce reti- Massachusetts General Hospital, Boston, MA, USA, Beth Israel Deaconess nal degeneration. Seven days later GFP+ BMSC or GFP+ fibroblasts (60’000 Medical Center, Boston, MA, USA cells in 4 ul BSS) were transplanted subretinally using a transscleral approach. Stem cells reside in a specialized microenvironment or niche, where stem BMSC were prepared immediately before transplantation from the bone cells are regulated and protected from environmental insults. Despite numer- marrow of GFP+ mice (C57BL/6-Tg(UBC-GFP)30Scha/J) using paramag- ous new discoveries made in recent years about the molecular and cellular netic beads for lineage depletion as well as for negative CD45 selection. mechanisms governing adult stem cell niches, the question regarding to the Fibroblasts were prepared from GFP+ mice (E13) and cultured under normal immunological aspect of the stem cell niche has been overlooked and largely culture conditions. Visual acuity was measured using the optokinetic reflex unexplored. Notably, the testis, feto-placental unit and hair follicle are loca- (OKR) at baseline, after NaIO3 injection as well as 7, 14, 21, and 28 days tions of stem cells that are all strong immune suppressive environments, after transplantation. Furthermore, electroretinograms (ERG) were recorded called immune privileged sites (IP sites) where foreign allografts can survive under scotopic and photopic light conditions at day 28 post transplantation. without immune suppression. The critical role of these organs or tissues for Morphometric measurements and immunohistochemistry (IHC) for retina- the host or species survival will provide a biological imperative to prevent specific markers (RPE65, GFAP, MAP-2, beta III tubulin) were performed on

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paraffin sections (7 um) of the eye at the latest time point. OKR measure- direct the differentiation of stem/progenitor and cells of other lineages to an ments showed higher visual acuity after inoculation of Lin-CD45- BMSC erythroid cell fate. compared to fibroblast-transplanted controls (e.g., 13% at day 21) but not the contralateral eye at all-time points after surgery. ERG measurements Poster Board Number: 2369 revealed higher amplitudes of a-/b- wave under scotopic (163/223 uV vs. A FETAL LIVER-DERIVED CONDITIONED 76/213 uV) and a-/b-/c-wave at photopic (9/84/207 uV vs. 2/43/77 uV) conditions in animals that received BMSC transplants compared to con- MEDIUM ENHANCES THE GROWTH FACTOR- trol animals receiving fibroblasts. Retinal thickness and number of rows of DEPENDENT PROLIFERATION AND SURVIVAL photoreceptor nuclei in the outer nuclear layer showed no significant differ- ences between the transplantation area (32±9 um; 5±2) and the level of the OF ADULT MOUSE BONE MARROW optic nerve head (25±5 um; 6±1). IHC revealed expression of glial markers ERYTHROID PROGENITORS BY MODULATING (GFAP) by transplanted BMSC in the subretinal space. Lin-CD45- BMSC THE DYNAMICS OF ERK SIGNALING integrate into the retinal structure and express glial marker after transplanta- tion into the damaged subretinal space. The procedure led to improvement Wang, WeiJia, Akbarian, Vahe, Mohammed, Noor, Audet, Julie of visual function in individual cases but not in the general population. Institute of Biomaterials and Biomedical Engineering, University of Toronto, Thereby, these cells might represent a new source for regeneration therapy Toronto, ON, Canada in retinal degenerative diseases. Stem and progenitor cell fate (self-renewal, proliferation, survival, differenti- Poster Board Number: 2367 ation) is tightly controlled by niche factors and the interplay of these factors is particularly important to comprehend for the development of stem cell SINGLE LINEAGE TRANSCRIPTOME ANALYSIS therapies. During erythropoiesis, stem cell factor (SCF) and erythropoietin REVEALS KEY MOLECULAR CHANGES (EPO) jointly control the fate of erythroid progenitors at the colony forming unit-erythroid (CFU-E)/proerythroblast stage. In the present study, we first IN ERYTHROID PROGENITORS IN LATE observed that SCF (200ng/mL) and EPO (10U/mL) together induced a syn- GASTRULATION MOUSE EMBRYOS ergistic cell growth from mouse bone-marrow c-Kit+CD71highTer119- cells [a population composed of 70±4% (±SD) CFU-Es] over a 3-day serum-free Baron, Margaret H., Isern, Joan, He, Zhiyong, Fraser, Stuart, Schulz, culture period. By 6 hours, EdU incorporation and Annexin-V apoptosis Vincent, Tuck, David, Gallagher, Patrick G. assays showed that SCF+EPO significantly increased the proliferation and Medicine and Tisch Cancer Institute, Mount Sinai School of Medicine, New survival of these cells compared to SCF or EPO alone. We then used a York, NY, USA, Pediatrics, Yale University School of Medicine, New Haven, multiparameter phospho-specific flow cytometry assay to quantitatively CT, USA, Pathology, Yale University School of Medicine, New Haven, CT, measure the activation dynamics of three signaling cascades [the mitogen- USA activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/Akt, Primitive erythroblasts (EryP) are the first cell type specified from mesoder- and Jak/signal transducer and activator of transcription 5 (STAT5) pathway] mal progenitors toward the end of gastrulation in the mammalian embryo. at the single-cell level. The data showed that SCF and EPO jointly modu- EryP progenitors arise and are present in the mouse yolk sac from embryonic lated the duration of extracellular signal-regulated kinase (ERK) signaling in day (E) ~E7.5-8.5, then begin to differentiate to erythroblasts that enter the c-Kit+CD71highTer119- cells: SCF+EPO induced sustained activation of ERK circulation and continue to mature in a stepwise, synchronous fashion until while SCF or EPO alone activated ERK in a transient manner. By modulat- their enucleation several days later. We have purified these first hematopoi- ing the duration of ERK activation using a specific MEK inhibitor, U0126, etic-committed progenitors from staged embryos based on the expression of results from real-time PCR suggested that the sustained ERK activation was a nuclear GFP transgene that is expressed specifically within the EryP lineage required for the prolonged downregulation of Bim (a proapoptotic Bcl2 fam- as early as E7.5. Genome-wide expression profiling allowed us to define the ily member) and upregulation of cyclin D2 (a positive cell-cycle progression transcriptome from each stage of development and revealed highly dynamic regulator). Next, we examined the effects of a conditioned medium (CM) changes during the progression from progenitor to maturing erythroblast. derived from mouse fetal livers and found that it could further enhance the We have focused on the emergence of EryP progenitors in the yolk sac survival and proliferation of c-Kit+CD71highTer119- cells when combined and on the transition to circulation stage, when progenitor activity is lost with SCF+EPO. Data from Annexin-V apoptosis assays showed that the CM and a peak is observed in the number of genes whose expression changes. increased the percentage of live cells (Annexin-V-7-AAD-) by 50±0.5% after TRANSFAC analysis of promoters of differentially expressed genes allowed 24 hours compared to cells that were cultured with SCF+EPO only. In the us to identify candidate transcriptional regulators, some of which have not EdU incorporation experiment, at 24 hours, 56% of c-Kit+CD71highTer119- previously been implicated in erythroid development (e.g. Nkx3.1, known cells incorporated EdU when cultured in CM+SCF+EPO compared with previously as a regulator of prostate stem cells). We designed experiments to 30% in SCF+EPO only. Interestingly, nearly all the cells underwent apoptosis test predictions from our microarray analysis and found that EryP progenitor by 24 hours when cultured in CM only, which was similar to the level of numbers are regulated by TGF-beta1 and hypoxia. In most mammalian cells, apoptosis measured when cells were cultured without cytokines. Finally, we the response to hypoxia is mediated by the transcription factor HIF-1. Hif-1 examined the dynamics of ERK activation in c-Kit+CD71highTer119- cells is apparently not expressed in EryP. Howver, Hif3a/Ipas, a Hif-1 target gene upon the stimulation with the CM. Exposure to CM+SCF+EPO not only that encodes a dominant negative regulator of HIFs and that is thought increased the amplitude of ERK activation by 120±28% compared with to function as a feedback regulator in response to hypoxia, is expressed in SCF+EPO (at saturating concentrations), but the duration as well. At 60 EryP as early as E7.5 and is upregulated as the cells mature. These findings minutes, the ERK activation was significantly higher than that induced by suggest that the response to hypoxia by EryP may involve a pathway that SCF+EPO. CM alone did not activate ERK. Mass spectrometry experiments is distinct from that of most other cells. EryP progenitors express genes as- are currently underway to identify new erythropoiesis-stimulating agents in sociated with aerobic glucose metabolism (the Warburg effect), a phenotype the CM. Our study has shed light on the mechanism by which combinatorial characteristic of cancer and other rapidly proliferating cells. Whether this factors control erythroid progenitor cell fate through modulating the dynam- glycolytic profile reflects the energy needs of these cells or a more unique ics of ERK signaling. feature of primitive erythropoiesis is under investigation. This study is the first lineage specific transcription profiling of a differentiating cell type in the early mouse embryo and will provide a strong basis for future work on normal erythropoiesis throughout ontogeny. It may also help guide efforts to

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Thursday Poster Abstracts

Poster Board Number: 2371 in Shb knockout bone marrow (wild-type 12 ± 1,7 percent of K+L-S+ vs 7,3 ± 0,9 for knockout). Additionally, Shb null LT-HSCs and multipotent progeni- SCL GENE DOSAGE SUSTAINS THE tors (MPPs; CD34+Slam-K+L-S+) display reduced proliferative rates (percent QUIESCENCE AND MAINTENANCE OF MOUSE BrdU 2,3 ± 0,8 in wild-type vs 0,2 ± 0,2 in knockout for LT-HSC and 3,5 ± 1,8 in wild-type vs 0,4 ± 0,1 in knockout for MPP) and Shb knockout MPPs ADULT HEMATOPOIETIC STEM CELLS also show a slower passage through the cell cycle. However, when subjected Rojas-Sutterlin, Shanti, Lacombe, Julie, Herblot, Sabine, Haman, to hematopoietic stress in the form of 5-fluorouracil (5-FU) treatment, Shb André, Hoang, Trang knockout LT-HSCs exhibit signs of reduced susceptibility to this drug. In competitive transplantation experiments of LT-HSCs and MPPs, both cell Institute for Research in Immunology and Cancer - Montreal University, types engrafted equally well, regardless of whether they were of wild-type Montreal, QC, Canada or Shb knockout origin. However, expansion (LT-HSCs at 16 weeks post In the adult, the majority of hematopoietic stem cells (HSCs) are in a transplantation 30,3 ± 3,1 percent myeloid cells of wild-type donor origin vs quiescent state whereas progenitors are more cycling. Stem cells have to 17,7 ± 2,2 for knockout) or maintenance (MPPs at 6 weeks post transplan- properly regulate their entry into and exit from the cell cycle in order to tation 14 ± 4,6 percent myeloid cells of wild-type origin vs 3,0 ± 0,7 for respond to physiological demands in blood cells and avoid the exhaustion knockout) of the engrafted cells was reduced in cells of Shb-deficient origin. of the stem cell pool. Very few genes regulating quiescence are actually Collectively, our data suggest a role for Shb as a regulator of LT-HSC and known, mainly due to experimental limitations. We have observed that the HPC proliferation and cell cycle progression. These studies implicate Shb Scl (stem cell leukemia) gene encoding a basic helix-loop-helix transcription signaling as a regulator of the HSC cell cycle and provide insights that could factor is more highly expressed in quiescent HSCs than in dividing HSCs contribute to the development of new stem cell therapies and improved and progenitors. Furthermore, we show that Scl gene dosage controls stem treatments for stem cell derived leukemia. cell activity when HSCs are transplanted in limiting conditions, requiring Poster Board Number: 2375 extensive proliferation. Indeed, Scl+/- HSCs are less competitive in trans- plantation assays and have reduced mean stem cell activity. Furthermore, WNT/BETA-CATENIN ACTIVITY IS REQUIRED these Scl+/- HSCs are more in G1 compared to their wild type counterparts that are mostly in G0, as assessed by Hoechst/Pyronin Y labelling of the FOR HEMATOPOIETIC STEM CELL Lin-Kit-Sca+CD150+CD48- population. Finally, we describe an optimized DEVELOPMENT IN THE MOUSE EMBRYO experimental procedure using 5-fluorouracil (5-FU) to evaluate the propor- tion of quiescent HSCs when Scl gene dosage is decreased. We show that Ruiz-Herguido, Cristina, Espinosa, Lluis, Bigas, Anna 10 days following 5-FU treatment >60% of Sca-1+ cells are killed in Scl Institut Municipal d’Investigació Mèdica (IMIM-Hospital del Mar), heterozygous mice compare to <20% in the control condition, indicating Barcelona, Spain that these cells were in cycle. Accordingly, we found a two-fold expansion The role of Wnt in the generation of hematopoietic stem cells (HSC) in the of Scl-deficient Sca-1+ cells within 20 days after the nadir post 5-FU, while mammalian embryo has not yet been addressed. We have now identified in control cells the expansion is not significant. At this time point, most of several Wnt molecules that are expressed in the Aorta/Gonad/Mesonephros control HSCs have returned to their normal G0 state as only 15% are found (AGM) region of the midgestation mouse embryo. In addition, we have in G1SG2M compared to 35% of HSCs from Scl+/- mice, indicative of a de- characterized an endothelial-like population residing at the base of the ficient G0 exit from the cell cycle. We therefore conclude that Scl regulates emerging hematopoietic clusters in the AGM region, which expresses CD31 HSC quiescence in steady state and in stress conditions by controlling the but is yet negative for c-kit or CD45 and it is able to generate hematopoietic G0-G1 transition. cells. This rare embryonic endothelial population contains nuclear beta- Poster Board Number: 2373 catenin and requires beta-catenin activity to produce hematopoietic cells, since this capacity is enhanced by Wnt3a and abolished in the presence of THE ADAPTOR PROTEIN SHB REGULATES Wnt inhibitors. Most important, conditional deletion of beta-catenin in the CELL CYCLE PROGRESSION IN ADULT MOUSE endothelial cells of the mouse embryo (by VEC-cre) impairs the contribution of these cells to the adult hematopoiesis, whereas deletion of this gene in HEMATOPOIETIC STEM CELLS committed hematopoietic stem cells (by vav-cre) does not affect their he- Gustafsson, Karin, Heffner, Garrett, Wenzel, Pamela L., Curran, matopoietic contribution. These results indicate that beta-catenin is required in the endothelial precursor before the determination to hematopoietic Matthew, Grawé, Jan, McKinney-Freeman, Shannon L., Daley, stem cells. Finally, we will discuss the effects of constitutive versus transient George Q., Welsh, Michael activation of beta-catenin in de novo generated HSC. Dept of Medical Cell Biology, Uppsala University, Uppsala, Sweden, HHMI, Children’s Hospital Boston, Harvard Medical School, Boston, MA, USA, Deptt of Genetics and Pathology, Uppsala University, Uppsala, Sweden, Dept of Hematology, St. Jude Children’s Research Hospital, Memphis, TN, USA Adult hematopoietic stem cells (HSCs) constitute a rare population of cells that reside primarily in the adult bone marrow. Despite the scarcity of these cells, this cell type supports the continuous production of peripheral blood cells throughout life. Multi-lineage potential as well as a considerable proliferative capacity are therefore essential HSC qualities. Shb is a widely expressed, Src homology 2(SH2) domain containing adaptor protein, com- monly found in downstream signaling of activated tyrosine kinase receptors such as the vascular endothelial growth factor receptor-2 (VEGFR-2) and the T cell receptor (TCR). Our recent findings implicating Shb in angiogen- esis and T cell function prompted us to examine HSC and hematopoietic progenitor cell function in the Shb knockout mouse. We report modestly reduced relative numbers of long-term HSCs (LT-HSCs; CD34-Slam+K+L-S+)

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Thursday Poster Abstracts

Poster Board Number: 2377 Poster Board Number: 2379 BCR-ABL IMPAIRS T CELL DEVELOPMENT THE ROLE OF ALCAM MEDIATED CELLULAR FROM MURINE INDUCED PLURIPOTENT STEM INTERACTION IN THE REGULATION OF CELLS; A POSSIBLE EXPLANATION FOR T CELL HEMATOPOIESIS ESCAPE FROM LEUKEMIC CLONE IN CHRONIC Jeannet, Robin MYELOID LEUKEMIA City of Hope, Duarte, CA, USA Chanda, Bidisha, Izawa, Kiyoko, Harnprasopwat, Ratanakanit, Alcam, which encodes the activated leukocyte cell adhesion molecule Takahashi, Keisuke, Kobayashi, Seiichiro, Kanegae, Yumi, Saito, (CD166), is a cell surface immunoglobulin superfamily member mediating Izumu, Tojo, Arinobu homophilic adhesion as well as heterotypic interactions with CD6. It has recently been shown that Alcam+ endosteal subset in the bone marrow con- Molecular Therapy, Advanced Clinical Research Center, Institute of Medical tain hematopoietic niche cells able to support hematopoietic stem cell (HSC) Science, University of Tokyo, Tokyo, Japan, Molecular Therapy, Advanced activity. We examined Alcam mRNA levels and cell surface expression by Clinical Research Center, Institute of Medical Science, University of Tokyo, quantitative RT-PCR and flow cytometry in various hematopoietic stem and Tokyo, Japan, Laboratory of Molecular Genetics, Institute of Medical progenitor subsets. We found that Alcam is highly expressed in long-term Science, University of Tokyo, Tokyo, Japan, Laboratory of Molecular repopulating HSC (LT-HSC), multipotent progenitors (MPP), and granulo- Genetics, Institute of Medical Science, University of Tokyo, Tokyo, Japan cyte/macrophage progenitors (GMP). To assess the function of Alcam in Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder homeostatic and stress hematopoiesis, we took advantage of an Alcam null generally believed to originate from a hematopoietic stem cell carrying the mouse allele. We show that Alcam-deficient mice exhibit normal homeo- BCR-ABL fusion gene, which encodes 210kD and 190kD constitutively static hematopoiesis. In competitive repopulation transplantation, Alcam- active tyrosine kinases termed as p210 and p190, respectively. In spite of deficient cells show an enhanced early stage engraftment compare to wild the stem cell origin and the competence for differentiation even toward type cells in primary recipients. The self-renewal capacity of Alcam-deficient mature B cells, there is a longstanding consensus that CML never involves HSC, however, seems to be impaired based on lower engraftment efficien- the T cell lineage at least in chronic phase. To gain insight into this apparent cies in secondary transplantation. We show that there is a lower frequency conflict, we used in vitro T cell differentiation model from murine induced of long-term repopulating cells in Alcam-deficient mice by limiting-dilution pluripotent stem (iPS) cells. C57BL/6 mouse embryonic fibroblasts (MEF) transplantation. Preliminary data suggest that the engraftment capacity of were reprogrammed using a polycistronic, self-inactivating (SIN) lentiviral phenotypically purified Alcam-deficient LT-HSCs is reduced compared to Tet-On vector encoding human Oct4, Sox2 and Klf4, which were tandemly wild type LT-HSC. These data suggest that Alcam mediated cellular interac- linked via porcine teschovirus-1 2A peptides, together with another lentiviral tion may be important for regulation of hematopoietic repopulation upon vector expressing rtTA driven by the EF-1α promoter. To delete almost all transplantation. We found that Alcam-deficient cells as home as efficiently the vector sequences including the transgenes after derivation of iPS cells, a to the bone marrow cavity, suggesting that the engraftment defect may not loxP site was inserted into the truncated 3’LTR of each vector. Doxycycline be due to defective bone marrow homing. Clonal differentiation assay of (DOX)-inducible iPS colonies were picked up after 23days of culture on a defined phenotypic LT-HSC reveals that Alcam-deficiency leads to a bias in MEF feeder layer. Pluripotency of these cells were confirmed by their expres- lineage differentiation potential. Studies are ongoing to further analyze its sion of embryonic stem cell signatures as well as formation of teratomas role in proliferation, cell cycle kinetics, and chemotaxis. These studies impli- containing all three germ layer-derived tissues in NOD-SCID mice. A clone cate Alcam mediated cell-cell interaction in the regulation of hematopoietic of MEF-3FiPS cells were further transduced with p190ΔccER, a ligand- transplantation and recovery. controllable p190-estrogen receptor fusion protein, by infection of murine stem cell virus (MSCV)-based retroviral vector. Finally, adenovirus-mediated Poster Board Number: 2381 expression of Cre recombinase successfully excised the lentiviral vector THE SHELTERIN PROTEIN ACD/TPP1 PLAYS components and left only remnant 291-bp SIN LTRs containing a single loxP site. There is some evidence that ligand-free p190ΔccER may be rapidly A CRITICAL ROLE IN THE MAINTENANCE OF degraded via ubiquitin-proteasome pathway, and its stability and tyrosine HEMATOPOIETIC STEM CELLS INDEPENDENT kinase activity is absolutely dependent on a ligand, 4-hydroxytamoxyfen OF TELOMERE LENGTH (4-HT). For T cell lineage differentiation, MEF-3FiPS/p190ΔccER cells were recovered from a feeder-free culture supplemented with LIF and plated onto Jones, Morgan, Osawa, Gail A., Friedman, Ann, Luo, Wylie, a subconfluent OP9-DL1 monolayer in the presence of murine Flt3 ligand Keegan, Catherine E., Maillard, Ivan and murine IL7 with or without 0.5 μM 4-HT. After three weeks of T cell lin- Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA eage differentiation, iPS cell-derived blood cells were collected and subjected to FACS analysis for their lineage confirmation. Approximately 70% of Telomere homeostasis is intimately linked to stem cell biology. Maintenance lymphocyte-like cells from the 4-HT(-) culture expressed CD3, but only 20% of telomere length through the enzymatic activity of telomerase is essential of counterparts from the 4-HT(+) culture expressed CD3, suggesting that for the long-term preservation of somatic stem cell populations. In addition, in this culture system Bcr-Abl impairs T cell development possibly through stem cells must be protected from inappropriate DNA damage response interfering with Notch signaling. The precise mechanism underlying impaired activation by exposed telomeric ends, a function fulfilled by the shelterin T lymphopoiesis by Bcr-Abl is under investigation. protein complex. The shelterin complex consists of six proteins that bind to the telomere. Tpp1, encoded by the gene Acd, plays a central role in com- plex organization as it links elements that bind the double-stranded portion of the telomere to those that bind the single-stranded overhang. We report an essential function for the shelterin complex and specifically for Tpp1 in the maintenance of hematopoietic stem cells (HSCs). Utilizing adrenocortical dysplasia (acd), a spontaneous autosomal recessive mouse mutation causing profound hypomorphism in Acd expression, we identified phenotypic and functional abnormalities in fetal hematopoietic progenitors. Specifically, fetal HSCs were larger, more granular, and expressed higher levels of the cell

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Thursday Poster Abstracts surface protein Sca-1, as compared to wildtype counterparts. Quantification isolated from GFP+ transgenic mice were then injected. Three months later, of phenotypically defined fetal liver HSCs (CD150+CD48-Lin-Sca-1hicKithi the degree of chimerism was the same in chemotherapy-treated and irradi- cells) revealed a significant reduction in stem cell numbers compared to ated mice, both in the circulation and in peripheral organs. However, GFP+ littermates. Cell cycle analysis utilizing BrdU incorporation demonstrated cells could not be detected in the brain of chemotherapy-treated mice safe abnormal cell cycle status in acd hematopoietic progenitors, including an for the structures not protected by the BBB. Taken together, these results accumulation of cells in G2/M phases of the cell cycle. This phenotype could demonstrate that myeloablation and peripheral chimerism is not sufficient be related to DNA damage response activation and subsequent cell cycle to induce the entry of BMDC into the brain and that irradiation is a needed arrest. p53 deficiency rescued the HSC depletion phenotype, but induced treatment for allowing such recruitments. Understanding how bone marrow- a paradoxical exacerbation of the increased Sca-1 expression and cell size derived cells enter the CNS is critical for the development of therapy of abnormalities. These findings suggest p53-dependent and independent brain-related disorders using hematopoietic stem cells. This study provides elements of the acd HSC phenotype. When assessed functionally, acd the first elements for understanding the necessary mechanisms to permit the HSCs failed to provide tri-lineage hematopoietic reconstitution of lethally entry of BMDC into the brain. irradiated recipients in competitive repopulation assays. To further study the effects of Acd-deficiency on HSCs, we utilized an Acd allele in which exons Poster Board Number: 2385 3-8 were flanked by loxP sites, allowing conditional inactivation following VASOPRESSIN AND OXYTOCIN REGULATE Mx1-Cre activation via poly(I:C) injection. To determine if the HSC require- ment for Acd was exclusively cell-autonomous, we transplanted Acdfl/- HEMATOPOIESIS THROUGH THE WNT AND Mx1-Cre+ bone marrow into lethally irradiated recipients. We then allowed AKT PATHWAYS hematopoietic reconstitution for 6 weeks prior to administering poly(I:C) by intraperitoneal injection. In this setting, Acd inactivation resulted in the Mayer, Balazs, Nemeth, Krisztian, Young, W Scott, Lee, Heon-Jin, depletion of HSCs within 2 weeks of the initiation of poly(I:C) injections. Nemeth, Michael, Hsieh, Matthew, Tisdale, John, Maric, Dragan, Furthermore, in competitive repopulation assays, Acd inactivation resulted Parmelee, Alissa, Holmbeck, Kenn, Mezey, Eva in a rapid failure of tri-lineage hematopoiesis in animals reconstituted with Craniofacial and Skeletal Diseases Branch, National Institutes of Health, Acd-deficient bone marrow. Together, these data indicate that HSCs have an National Institute of Dental and Craniofacial Research, Bethesda, MD, USA, exquisite, cell-autonomous requirement for Acd. Because HSC depletion was National Institutes of Health, National Institute of Mental Health, Bethesda, observed extremely rapidly after Acd inactivation, it could not be explained MD, USA, Rosewell Park Cancer Institute, Buffalo, NY, USA, National by telomere shortening. These findings represent the first report of an essen- Institutes of Health, National Heart, Lung and Blood Institute, Bethesda, tial role for Acd/Tpp1 and the shelterin complex in HSC maintenance and MD, USA, National Institutes of Health, National Institute of Neurological hematopoietic homeostasis, independently of telomerase function. Disorders and Stroke, Bethesda, MD, USA Poster Board Number: 2383 Vasopressin (also known as arginine-vasopressin, AVP) and oxytocin (OXT) are members of a peptide family that is highly conserved in vertebrates. MECHANISMS OF BONE MARROW DERIVED Their genes and the corresponding peptides are closely related. The vast CELLS ENTRY TO THE BRAIN majority of AVP and OXT are synthesized within the magnocellular neurons of the hypothalamic supraoptic and paraventricular nuclei and transported Lampron, Antoine, Lessard, Martine, Rivest, Serge along their axons to the posterior pituitary where they are stored and Department of Molecular Medicine, Faculty of Medicine, Université Laval, ultimately released into the blood stream. Once released, AVP regulates salt Laboratory of Endocrinology and Genomics, CHUL Research Centre, and water homeostasis and OXT regulates parturition and lactation. Brattle- Québec, QC, Canada boro rats, which naturally lack AVP, are less resistant to hemorrhage than The clinical potential of hematopoietic stem cells has been demonstrated in wildtype animals and need longer time to recover their original blood cell humans and mouse models for a variety of diseases of the central nervous numbers. Based on this phenomenon, we hypothesized that AVP not only system (CNS), including Alzheimer’s disease, multiple sclerosis, Parkinson’s controls blood pressure and enables water reabsorption in the kidneys but disease, amyotrophic lateral sclerosis and others. These therapies focus on also may have a role in hematopoiesis. We had supporting data obtained the beneficial effects of bone marrow-derived cells (BMDC) in the CNS, from our earlier G-protein coupled receptor array that showed that human mostly microglia, the resident macrophages of the brain. Even though they hematopoietic stem cells (CD34+, CD38- and CD34+, CD38+ cells) express show a great clinical potential, bone marrow derived cells populate the CNS vasopressin receptors (AVPR) AVPR1B and AVPR2 and the oxytocin receptor by poorly understood mechanisms. As lethal doses of whole-body irradiation (OXTR). In this study, we confirmed the presence of AVPR1A, AVPR1B, appear to be the only treatment inducing the entry of BMDC to the CNS, AVPR2 and OXTR in murine and human hematopoietic cells (lineage-, we investigated the effects of irradiation on the brain of wild type C57/Bl6 c-kit+, Sca-1+ cells and CD34+ cells respectively) using immunocytochem- mice and set to understand if myeloablation was sufficient to induce the istry and RT-PCR. Murine cells, treated with AVP and OXT in vitro, had entry of GFP+ cells into the brain. Minor damages were found in few re- an increased proliferation rate compared to that of control cells. In colony gions of the brain 6-12 hours following irradiation by fluorojade staining and forming unit assays, human CD34+ cells produced more colonies when cleaved caspase-3 immunohistochemistry. Such damages were not detected cultured in the presence of AVP or OXT. This increase was eliminated when 1-7d after irradiation. A weak expression of few neuroinflammatory markers a Wnt or an Akt pathway inhibitor was used suggesting that these signaling was also detected immediately after irradiation, but not at time beyond 3 pathways are involved in the effect, respectively. We demonstrated using days. These localized inflammatory loci did not influence the brain entry of immunocytochemistry and RT-PCR that human and murine bone marrow BMDC, because the infiltration was similar in mice that were transplanted stromal cells (also referred to as mesenchymal stem cells), that are known 6h, 12h, 24h or 7d following irradiation. A concern regarding irradiation to support hematopoiesis, produced AVP and OXT. We also showed the is that it induces severe damages to the blood-brain barrier and that these presence of AVP and OXT receptors on these cells. Using radioimmunoassay, damages are responsible for the entry of BMDC. However, we failed to both spontaneous and angiotensin II induced release of AVP and OXT from detect significant alteration of the BBB. These results demonstrate that irra- bone marrow stromal cells into the cell culture medium were detected. We diation does not induce major damages to brain cells, but it clear predispose hypothesize that the increased serum AVP concentration after hemorrhage it for allowing recruitment of BMDCs. We performed myeloablation in wild in concert with the locally released AVP from bone marrow stromal cells may type mice by adapting a busulfan/cyclophosphamide chemotherapy regimen regulate hematopoiesis. Due to their proximity to HSCs, the stromal cells commonly used in bone marrow transplants in humans. Bone marrow cells might act as local amplifiers (i.e., release their AVP in response to circulating AVP) to increase efficiency.

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Thursday Poster Abstracts

Poster Board Number: 2387 Poster Board Number: 2389 DIRECT INVOLVEMENT OF BMP4 IN ADULT ERYTHROID DIFFERENTIATION OF CD133 HEMATOPOIETIC STEM CELL HOMING STEM CELLS OF UMBILICAL CORD BLOOD Khurana, Satish, Buckley, Shannon, Schouteden, Sarah, Verfaillie, BY UP REGULATION OF MIR 451 AND DOWN Catherine REGULATION OF MIR 150 Interdepartmental Stem Cell Institute, Leuven, Belgium Kouhkan Kahangi, Fatemeh, Soleimani, Masoud, Daliri, Morteza, Bone morphogenetic protein 4 (BMP4) has been implicated in mesoderm Behmanesh, Mehrdad, Mossahebi Mohammadi, Majid, Alizadeh, patterning and differentiation of hemogenic endothelium. Its role in regulat- Shaban, Galadari, Helia, Mohammadi, Shahin ing adult hematopoiesis has not been clearly defined, mostly due to embry- Tarbiat Modares University, Tehran, Islamic Republic of Iran, Hematology onic lethality resulting from disturbing BMP4 signaling. Recent findings dem- and Blood Banking, Tarbiat Modares University, Tehran, Islamic Republic onstrated that BMP4 is an important component of the hematopoietic stem of Iran, National Institute Genetic Engineering and Biotechnology, Tehran, cell (HSC) niche. We here aimed to determine the effect of BMP4 on en- Islamic Republic of Iran,Tehran Medical University, Tehran, Islamic Republic graftment of HSC. Adult murine bone marrow (BM) derived c-kit+Lineage- of Iran Sca-1+ (KLS) cells were cultured with SCF and Tpo with or without BMP4 in serum free medium. Alternatively, the BMP4 inhibitors chordin (Chd) and The hematopoietic stem cells (HSC), with self-renewing and differentia- twisted gastrulation (TSG) were added to the culture. Cells were transplant- tion potential, are necessary for the lifelong sustenance of distinct lineage ed in lethally irradiated mice and donor derived chimerism was analyzed. of blood cells. Lineage specification of HSC is a multi-step process that Significantly higher chimerism was seen when BMP4 treated cells were used controlled by complex network of growth factors, transcription factors and as graft, both in primary as well as in secondary recipients, whereas Chd and tiny regulatory RNA molecules known as miRNAs. Micro RNAs are a novel TSG decreased chimerism. As no effect on the cell cycle status of these cells class of small regulatory RNA molecules that participate in gene regulation was found, in vitro migration and adhesion experiments were performed, and cell fate controlling by inhibiting protein translation or by degrading of which demonstrated that the BMP4 treated cells adhered significantly better target mRNA. Recent studies have demonstrated that specific miRNAs (such to the BM stromal cells. We further demonstrated that BMP4 treatment as miR-451 and miR-150) have key roles in erythropoiesis. To date, growth increased Integrin α4 (Inta4) expression, which a part of heterodimer VLA4, factors are unique effectors for in vitro lineage differentiation of HSC to known to play an important role in HSC adhesion and homing. In contrast, erythropoiesis. But these factors are most expensive for clinical application Chd and TSG decreased adhesion and expression of Inta4. To determine the and therapeutic strategies. So, the aim of this study is to investigate whether mechanisms underlying BMP4-mediated changes in Inta4 expression, we specific miRNAs could be considered as a substitute to growth factors for evaluated Smad-dependent and -independent pathways. Upon exposure HSC differentiation into erythroid cell progenitors. For this reason, miR-451 to BMP4, Smad1/5/8 was phosphorylated and translocated to the nucleus gene was cloned in retroviruses plasmid. New retroviral particles which are in KLS cells. However, no change in expression of Smad target genes was included miR-451 produced in packaging cell line HEK 293T .CD133+ cells found. When the cells were incubated with Dorsomorphin, which inhibits were isolated from mononuclear cells (MNCs) of umbilical cord blood by Smad phosphorylation, we continued to see increased Inta4 expression. MACS method. The study was performed in three different groups: First, a On the other hand, when the p38 inhibitor SB203580 was added, Inta4 control group, which didn’t receive any treatment; second, a group which expression was suppressed, suggesting that BMP4 affects Inta4 expression transduced by produced retroviruses and a third one, which transfected by via Smad independent mechanism. The transcription factor MiTF, known to miR-150 antisense. Erythroid differentiation of CD133+ cells were evaluated positively regulate Inta4 expression, was activated in response to BMP4, but after up-regulation of miR-451 and down-regulation of miR-150 using qRT- was inhibited by the p38 inhibitor SB203580. Using shRNA, we knocked PCR by specific primers and flowcytometry analysis. Collected data indicated down the expression of MiTF gene, which led to decreased expression of that hemoglobin genes (α, β, γ, δ and θ) were over expressed in genetically Inta4 in BM derived lin- cells. More importantly, BMP4 could no longer modified CD133+ cells in comparison to control group of CD 133+ cells. induce expression of Inta4 in cells wherein MiTF was knocked down. Finally, Flowcytometry assay demonstrated that genetically modified CD133+ cells as BMP4 increased Inta4 expression and adhesion to BM stroma in vitro, we differentiated to erythroid cells efficiently. Our results suggest that overex- tested if BMP4 improves engraftment via improving in vivo homing. The pression of miR-451 and down regulation of miR-150 could be cost effective homing potential of the cells was assayed by detecting donor derived cells substitute to growth factors for CD133+ differentiation to erythroid cell in the recipient BM after 16 h of transplantation through the intra venous progenitors. It is a hope that, this study could open new doors to production route. These studies clearly showed improved homing of KLS cells follow- of artificial blood. ing BMP4 treatment, which could be inhibited by either the p38 inhibitor Poster Board Number: 2391 or knockdown of MiTF. Overall, our results present the first evidence for a direct role of BMP4 in regulating expression of Inta4, mediated by p38 THE BASIC PROTEIN G0S2 ACTIVATES phosphorylation, which in turn activated MiTF, leading to improved homing, and hence engraftment. QUIESCENCE IN HEMATOPOIETIC STEM CELLS BY INTERACTING WITH NUCLEOLIN Lacorazza, Daniel,, Yamada, Takeshi, Bae, Leon, Park, Chun Shik Department of Pathology and Immunology, Baylor College of Medicine, Texas Children’s Hospital, Houston, TX, USA, Department of Pediatrics, Baylor College of Medicine, Texas Children’s Hospital, Houston, TX, USA The G0/G1 switch gene 2 (G0S2) is an early response gene identified in hematopoietic cells with potential function as a gatekeeper of the G0 to G1 transition. Even though quiescence of hematopoietic stem cells is critical for bone marrow transplantation and maintenance of normal and leuke- mic stem cells, it is not clear its regulation by cell intrinsic factors. By using gain- and loss-of-function models, we report that G0S2 inhibits proliferation

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Thursday Poster Abstracts of lineage- Sca-1+ c-kit+ CD150+ cells at steady state. Therefore, ectopic EETs as novel HSC self-renewal modulators potentially through regulating G0S2 expression in bone marrow cells impaired hematologic reconstitution PI3K pathway. This discovery may have clinical application in marrow or in competitive transplantation even though G0S2-expressing bone mar- cord blood transplantation. row cells outcompeted wild type donors in bone marrow. Treatment with 5-azacytidine or retroviral expression of G0S2 reduces proliferation of K562 Poster Board Number: 2395 cells, suggesting a tumor suppressor function in hematopoietic malignancies. GENE CORRECTION BY DIRECT PROTEIN Co-immunoprecipitation and mass spectrometry identified nucleolin as G0S2 protein partner in hematopoietic cells. Our results establish a novel function DELIVERY for G0S2 as activator of quiescence in normal and leukemic hematopoietic Li, Shuyi, Goyal, Deepika, Li, Zhentian, Chen, Zhong, Wade, cells. Marlene, Meiler, Steffen E., Porteus, Matthew, Dynan, William Poster Board Number: 2393 Institute of Molecular Medicine and Genetics, Georgia Health Sciences University, Augusta, GA, USA, Department of Anesthesiology & EPOXYEICOSATRIENOIC ACIDS REGULATE Perioperative Medicine, Georgia Health Sciences University, Augusta, GA, HEMATOPOIETIC STEM CELL SELF-RENEWAL USA, Department of Pediatrics-Cancer Biology, Stanford University, Palo DURING STRESS RESPONSE AND EMBRYONIC Alto, CA, USA HEMATOPOIESIS Zinc finger nucleases (ZFNs) facilitate targeted editing of the genome by creating DNA double-strand breaks at user-specified locations. Although Li, Pulin, Pugach, Emily K., Riley, Elizabeth B., Panigrahy, Dipak, ZFN-mediated gene correction rests on established principles, current Bowman, Teresa V., Heffner, Garrett C., McKinney-Freeman, methods are inefficient, and the use of viral vectors for ZFN expression is Shannon, Schlaeger, Thorsten M., Daley, George Q., Zeldin, Darryl clinically problematic. To address these concerns, we have explored methods C., Zon, Leonard I. for direct delivery of ZFNs as proteins to the target cells. As first step to test feasibility of this strategy, we are using a model of a generic recessive Division of Hematology/Oncology, Children’s Hospital Boston, Boston, MA, disease based on correction of a defective GFP transgene. We use one cell USA, Vascular Biology Program, Children’s Hospital Boston, Boston, MA, line derived from a rosa26 knock-in mouse and another derived from a hu- USA, Department of Hematology, St. Jude Children’s Research Hospital, man tumor cell line (3T3 GFP* and 293/258D, respectively). A pair of highly Memphis, TN, USA, Division of Intramural Research, National Institute of specific ZFNs incise the GFP transgene near the mutation site, and donor Environmental Health Sciences, Research Triangle Park, NC, USA template allows correction by homologous recombination repair. We first During bone marrow transplantation, hematopoietic stem/progenitor cells developed methods for producing high purity, concentrated ZFN protein. (HSPCs) are taken out of their native niche, exposed to various foreign We then explored three strategies for introducing this protein into cells: (1) signals and undergo rapid proliferation and differentiation. To explore how microinjection of protein and GFP donor template DNA into cell nucleus, (2) the fate decisions are made under such stress conditions, we developed a polyplex method based on formation of a complex between transferrin- a novel imaging-based competitive marrow transplantation in zebrafish. polyethyleneimine, DNA, and ZFN proteins, and (3) electroporation using GFP+ marrow cells were ex vivo treated with various chemicals for 4 hours, protein and donor DNA. All three methods show promise based on efficient mixed with untreated DsRed2+ marrow cells and injected retro-orbitally into transfer of proteins to cells. We are currently evaluating correction efficien- transparent adult zebrafish. The feasibility of handling hundreds of zebrafish cies. We plan to test these methods in hematopoietic stem cells, with the for transplantation per day and the ease of assessing transplant outcomes in ultimate goal of correcting the sickle globin allele, which is the single most over one hundred zebrafish per hour allowed us to screen a library of 480 common disease-causing mutation in humans worldwide. Toward this end, small molecules with known bioactivity, aimed at identifying new drugs and we plan to adapt these approaches for high-throughput transfer of ZFN pathways regulating HSPC engraftment. Two structurally related eico- proteins directly to hematopoietic stem/progenitor cells (HSPCs). sanoids, 11,12-epoxyeicosatrienoic acid (11,12-EET) and 14,15 epoxyei- cosatrienoic acid (14,15-EET), were able to enhance GFP+ marrow engraft- Poster Board Number: 2397 ment compared to DsRed2+ engraftment in zebrafish. Similarly, in mice, ex DELETION OF TET2 IN MICE LEADS TO vivo treatment of CD45.1+ whole bone marrow with 11,12-EET for 4 hours resulted in a 2-fold increase of long-term repopulating units by 32 weeks in DYSREGULATED HEMATOPOIETIC STEM a limiting dilution competitive transplantation assay. Sorted CD45.1+ bone CELLS AND SUBSEQUENT DEVELOPMENT OF marrow cells from the primary recipients in the 11,12-EET group still had a competitive advantage in the secondary transplantation. Epoxyeicosa- MYELOID MALIGNANCIES trienoic acids are naturally synthesized from arachidonic acid by cytochrome Li, Zhe, Wang, Jiapeng, Cai, Chenleng, petersen, bruce, Yang, P450 enzymes, especially 2C and 2J families (CYP2C and CYP2J) in human. FengChun, Xu, Mingjiang Microarray data had previously shown that murine CYP2J6 are enriched in Hematology/Oncology, Mount Sinai School of Medicine, New York, NY, quiescent long-term HSCs in mice. 60,000 Tg(Tie2:CYP2C8) mouse bone USA, Mount Sinai School of Medicine, New York, NY, USA, Department of marrow cells can multi-lineage engraft lethally irradiated recipients more Pediatrics, Indiana University School of Medicine, Indianapolis, IN, USA efficiently (6/7) than an equal number of wild type marrow cells (2/6). Our findings on the activity of EETs on adult HSCs elicited the study of Recently, several groups have reported alterations of a novel gene, Ten- EET in embryonic hematopoiesis. Treating zebrafish embryos with 11,12- Eleven-Translocation-2 (TET2), in a variety of myeloid malignancies including EET increased the RNA level of HSPC marker runx1. We blocked potential myelodysplasic syndrome (MDS, ~30% of cases), myeloproliferative neo- signaling pathways downstream of EETs by treating zebrafish embryos with plasms (MPN, ~20%), chronic myelomonocytic leukemia (CMML, ~42%; chemical inhibitors and 11,12-EET together, and found that the PI3K inhibi- classified by the WHO as a myelodysplastic/myeloproliferative neoplasm), tor, LY 294002, could block upregulation of runx1 by 11,12-EET. Western and acute myeloid leukemia (AML, ~20%). TET2 encodes a methylcytosine blotting confirmed that the PI3K/AKT pathway is rapidly upregulated by dioxygenase that may contribute to epigenetic regulations via catalyzing 11,12-EET in human leukemia cell line U937. We hypothesize that EETs the conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine regulate stem cell stress response and embryonic hematopoiesis by modulat- (5-hmC), which could epigenetically regulate gene expression by altering ing PI3K pathway activity. In conclusion, we performed the first competitive methylation-driven gene silencing. However, little is known regarding the marrow transplantation-based chemical screen, leading to the discovery of biological function of Tet2 and its role in the pathogenesis of myeloid ma-

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lignancies. To study the function of Tet2 in vivo, we generated Tet2:nlacZ/ and differentiation. Mechanistic studies elucidating the impact of HIF-α on nGFP knock-in mice, in which the targeted allele is Tet2-null and nlacZ/ the long-term repopulation ability of HSC and on maturation steps driving nGFP is expressed under the endogenous transcription regulatory elements HSC commitment are necessary to understand the precise role of HIF-1α of the Tet2 gene. Using the heterozygous Tet2:nGFP mice, we demonstrated and HIF-2α in bone marrow, especially in the hypoxic niche area. that Tet2 is expressed in hematopoietic stem cells (HSC) at its highest levels and downregulated upon commitment to differentiation. The Tet2-/- mice Poster Board Number: 2401 contained an increased frequency of Lin-Sca-1+c-Kit+ (LSK) cell population THE HIPPO SIGNALING PATHWAY REGULATES prior to the development of myeloid malignancies. An in vivo competi- tive reconstitution assay revealed that Tet2-/- LSK cells have an increased HEMATOPOIETIC STEM-LIKE PROGENITORS IN capacity to regenerate both short-term and long-term hematopoiesis in DROSOPHILA MELANOGASTER vivo and have a preferential differentiation into granulocytic/monocytic lineages. Tet2-/- mice displayed a phenotype resembling many characteris- Ferguson, Gabriel B., Martinez-Agosto, Julian tics of CMML at 2-3 months of age, including hepatomegaly, splenomegaly, Molecular Biology Institute, University of California, Los Angeles, Los increased WBC counts with a disproportionate number of monocytes, and Angeles, CA, USA, Human Genetics, University of California, Los Angeles, increased cellularity in bone marrow (BM). Approximately 30% of the Tet2- Los Angeles, CA, USA /- mice died within 10 months of age due to the development of erythroleu- Hematopoiesis in Drosophila melanogaster takes place in two stage-specific kemia and/or myeloid leukemia. Furthermore, following Tet2-/- BM, but not waves of development, one embryonic and the other during larval stages. wild-type (WT) or Tet2+/- BM transplantation, 2 of the 7 WT recipient mice Definitive hematopoiesis takes place in the larval lymph gland, an organ that exhibited increased WBC counts in the peripheral blood with monocytosis contains a distinct population of slow cycling, niche-dependent stem-like and splenomegaly. Our data indicate that Tet2-deficient mice phenotypically progenitors that give rise to all mature hematopoietic cell types. Mainte- recapitulate the patients with myeloid malignancies, demonstrating that Tet2 nance of these progenitors is dependent on highly conserved signaling path- functions as a tumor suppressor gene to maintain hematopoietic stem cell ways such as Notch, Wingless/Wnt, and Hedgehog. We have also identified homeostasis. the recently emerging Hippo signaling pathway as a novel candidate for Poster Board Number: 2399 maintaining this population of cells. In mammals, Hippo signaling has been shown to help maintain pluripotency in mESCs, regulate organ size, and act LONG-TERM RECONSTITUTION ABILITIES as a tumor suppressor pathway as it negatively regulates the oncoprotein AND DIFFERENTIATION OF HUMAN YAP-1 via phosphorylation. In Drosophila this pathway is highly conserved and acts by inhibiting the homolog of YAP-1, the transactivator protein HEMATOPOIETIC STEM CELLS REQUIRE Yorkie (Yki). Here, we show that Yki and its binding partner, the TEAD HYPOXIA-INDUCIBLE FACTOR-1 ALPHA AND -2 homolog Scalloped (Sd), are developmentally regulated in the lymph gland. ALPHA EXPRESSIONS Yki and Sd expression is observed throughout the progenitor cell popula- tion while these cells undergo rapid expansion. However, as the progenitors Mazurier, Frédéric, Rouault-Pierre, Kevin, Rezvani, Hamid Reza, become quiescent in later stages of development, Yki is limited to a small, Lamrissi-Garcia, Isabelle, Ivanovic, Zoran, de Verneuil, Hubert scattered population of cells. To further elucidate the role of Hippo in regu- U1035, INSERM - Université Bordeaux Segalen, Bordeaux, France, lating Drosophila hematopoiesis, we used RNA interference and overexpres- Établissement Français du Sang-Aquitaine Limousin - CNRS UMR 5164, sion constructs targeted to specific populations of cells in the lymph gland. Bordeaux, France Progenitor specific RNA interference directed against Warts which directly inhibits Yki, results in a significant expansion of hematopoietic progenitors Physiological oxygen level is tightly regulated in mammalian tissues. More- at the expense of mature hemocytes. Furthermore, depletion of Yki or Sd over, oxygenation differs from one tissue to the other, as well as within a among the progenitor population leads to increased differentiation and single tissue, due to vasculature modeling. Oxygen level has been shown loss of progenitors. Interestingly, Sd also appears to have a very important to be a key stimulus of cell fate during embryogenesis and cancer progres- role in regulating differentiation in the lymph gland. Removal of Sd in cells sion. It is now widely admitted that stem cells fate follows oxygen gradients that appear to be “intermediate progenitors” leads to rapid and complete with low oxygen levels (hypoxia) promoting an undifferentiated state. differentiation of the lymph gland. In contrast, overexpression of Sd leads to Hematopoietic stem cells (HSCs) reside in bone marrow niches in which a massive expansion of these cells. Our results demonstrate a novel role for oxygen availability is low, even absent. Hypoxia-inducible factors (HIFs) the Hippo pathway in the maintenance of hematopoietic stem-like progeni- are the main factors regulating the cellular response to oxygen variation. tors in the Drosophila lymph gland. Studies on mouse models reveal that HIF-1 expression in HSC supports their quiescence, whereas HIF-2 participates in HSC maintenance through Poster Board Number: 2403 microenvironment properties. However, knowledge of the HIFs functions in human HSCs is poorly documented. Here, we have investigated the DISEASE SPECIFIC LENTIVIRAL TARGET SITE potential function of HIF-1α and HIF-2α within human HSCs. Knockdown SELECTION IN PROGENITORS OF FANCONI of both factors was obtained by lentivirus-mediated short-hairpin RNA ANEMIA PATIENTS strategy. Transduction of CD34-positive cord blood cells has revealed that HIF-1α knockdown affected neither progenitor cell differentiation nor Nowrouzi, Ali, Gonzalez-Murillo, Africa, Molina, Javier, Weber, short-term reconstitution ability in immune-deficient NOD-scid-IL2Rγnull Christian, Paruzynski, Anna, Guenechea, Guillermo, von Kalle, mice, but that HIF-1α is involved in long-term repopulation and T-lymphoid Christof, Bueren, Juan, Schmidt, Manfred differentiation. HIF-2α expression is required for both in vitro and in vivo NCT Heidelberg/DKFZ, Heidelberg, Germany, Hematopoietic and Gene erythroid differentiation. In contrast to HIF-1α, HIF-2α expression is required Therapy Division, CIEMAT||CIBERer, Madrid, Spain, Hematopoietic and for both short- and long-term reconstitution in NOD-scid-IL2Rγnull mice, Gene Therapy Division, CIEMAT||CIBERer, Heidelberg, Spain, Translational with a more pronounced deficiency in the long term observed for HIF-2α Oncology, NCT Heidelberg/DKFZ, Heidelberg, Germany compared to HIF-1α. The discrepancy observed between the two factors might be explained by differences in the kinetics of expression and/or in the The fundamental role of the Fanconi Anemia (FA) pathway for DNA repair target genes. Overall, our data strongly support the existence of multiple but and maintenance of genetic integrity supports profound impacts on the preponderant functions of HIF-α subunits in human stem cell maintenance transcriptional state of FA derived stem cells from FA patients. Clinically, FA

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Thursday Poster Abstracts patients are characterized by congenital malformations, progressive bone Poster Board Number: 2407 marrow (BM) failure, and predisposition to acute myeloid leukemia and other malignancies. Although the therapy of choice for FA patients is the AUTOLOGOUS TRANSPLANTATION OF BONE hematopoietic transplantation from HLA-identical siblings, gene therapy MARROW MONONUCLEAR CELLS IN A using lentiviral vectors (LV) may constitute a good option for patients without a compatible sibling. Previous studies have shown efficacy of LVs to COMPARTMENT SYNDROME SALINE MODEL correct the phenotype of FA hematopoietic progenitors and stem cells (HP/ IN SINCLAIR MINI-SWINE: A PILOT STUDY HSCs). We hypothesised that disease specific transcriptional states influence distribution of LV preferentially tagging genes, gene classes and biochemical Zheng, Bo, Wu, Ping-Cheng, Gregory, Cynthia, Gregory, Kenton pathways relevant for the disease specific phenotype. Optimized DNA isola- Providence St. Vincent Medical Center, Orgon Medical Laser Center, tion procedures for minimal cell numbers, suitable for large scale molecular Portland, OR, USA screening of hematopoietic CFC-colonies by linear amplification-mediated Compartment syndrome (CS) is an acute injury caused by trauma-related PCR (LAM-PCR) and massive parallel pyrosequencing (454 GS Flx system; swelling and increased pressure within tissue compartments leading to isch- Roche) were used to analyze the insertional inventory of 890 EGFP-positive emia, infarction, and permanent disability from muscle and nerve damage. colonies generated by transduced FA BMCs from three patients, identify- A large animal model of CS using plasma infusion to increase anterior tibialis ing 599 unique mappable vector insertion sites (ISs). For comparison of LV muscle compartment pressure was developed. A pilot study of autologous target site distribution in normal HPs, we also characterized the insertional bone marrow progenitor cell (BM-MNC) therapy of this injury demonstrated inventory of 660 colonies derived from healthy individuals, detecting 358 a surprisingly large number of engrafted BM-MNC’s distributed throughout unique mappable vector ISs. LV vectors showed high integration frequency the muscle to an extent not seen in published studies. A concerned was this into genes (80%) and enrichment near to CpG islands in FA derived HP/ robust cell engraftment was an artifactual consequence of the CS model HSCs. Comparison of LV target site distribution in FA derived stem/progeni- due to retained plasma in the muscle. The aim of this study was to assess tor cells with healthy samples revealed that gene classes involved in cell the possibility of using saline-a more physiologic edema-mimicking fluid for cycle progression, p53 regulated apoptotic cell signaling cascades and DNA infusion to create CS pressure, ischemia and infarction and determine the replication and repair were significantly (p<0,001) favored by the LV inte- effect on BM-MNC engraftment 3 months after injury. Unilateral CS was gration machinery in FA progenitors. We are currently investigating in a FA created in the anterolateral muscle compartment of the hind limbs of three serial transplantation mouse model the clinical safety and clonal dynamics of Sinclair mini-swine by saline infusion into the compartment for six hours at FA corrected HSC in vivo using a therapeutically applicable LV based vector. a pressure greater than 120 mmHg. Following infusion, a fasciotomy was Our findings indicate disease specific differences of LV target site distribution performed with a single mid-line incision through the fascia surrounding the in FA HP/HSCs influenced by an altered transcriptional state of FA derived muscle. Bone marrow was collected one week after injury and transferred to stem cells. an automated cell separator system (SEPAX, BioSafe) for BM-MNC isolation. Poster Board Number: 2405 BM-MNC marker profile was assessed by flow cytometry. CM-DiI-labeled BM-MNCs were injected intra-muscularly throughout the whole anterior DIRECT DIFFERENTIATION OF HEMATOPOIETIC tibial muscle. Muscle and nerve function data using ankle torque and motor STEM CELLS INTO MONOCYTIC LINEAGE BY nerve conduction, were acquired at six time points: pre- and post-injury, BM-MNC injection day, and six and twelve weeks post-injury. Gait analysis INDUCTION OF MIR-424 EXPRESSION was performed pre-injury, days one, two, and three post-injury, and at Soleimani, Masoud, Rahimian, Ali, Kaviani, Saeid weekly intervals up to six weeks. Immunofluorescent staining was performed on tissue harvested at four or twelve weeks post-injury to detect CM-DiI- Hematology Dept, Tarbiat Modares University, Tehran, Islamic Republic of labeled BM-MNCs and to assess myogenesis (desmin, dystrophin, α-actinin Iran and Phalloidin), angiogenesis (CD31 and vWF) and neurogenesis (GFAP). Differentiation is a multi step process which occurs in a complicated and In all pigs, normal muscle function returned by four weeks post-injury, while precise manner. There are two classes of epigenetic elements, contributing nerve function recovered by eight weeks post-injury. These results suggest in the process: The transcription factors and the micro RNAs. Transcription that saline is an inadequate infusate to create realistic severe CS muscle in- factors are proteins that bind to specific DNA sequences, thereby control- jury with permanent disability. The BM-MNCs were heterogeneous for their ling the transfer of genetic information from DNA to mRNA. Alternatively, expression of: (1) mesenchymal stromal cells markers CD29 (27.3±2.21%), microRNAs are a brand of small RNAs that control the expression of genes CD90 (46.15±9.75%) and CD44 (56.55±11.76%); (2) the pan-hematopoi- at the post transcriptional level. These non-coding RNAs perform by miss etic lineage marker CD45 (86.84±0.25%); (3) the endothelial cell markers match pairing to the 3’-UTR of their target mRNA and block the transla- CD31 (15.9±2.65%), as well as perivascular cell (pericyte) markers CD146 tion. The effect of these epigenetic elements in major cellular processes has (1.27±0.26%) and CD105 (1.27±0.38%); (4) and primitive stem cell and widely been shown in different types of cells including the hematopoietic myogenic cell markers cKit (3.21±0.91%), Sca-1 (1.42±0.88%), CXCR4 lineage differentiation and miR-424 has introduced as an effective factor in (1.70±1.19) and CD56 (0.86±0.52%). These results suggest that adult bone monocyte/macrophage differentiation. In present study, we demonstrated marrow is a potential multi-potent cell source for injured tissue repair. CM- that over expression of the miR-424 in CD133+ hematopoietic stem cells DiI labeled cells were detectable for up to eleven weeks post-cell injection, of cord blood will cause their differentiation into cells showing monocytic demonstrating that CM-DiI is a reliable method for long-term cell tracking characteristics. Stable over expression of miR-424 were stablished using a within muscle tissue. CM-DiI labeled BM-MNCs located in proximity to retroviral vector construct containing the precursor of miR-424 sequence. newly regenerated myofibers, capillary and peripheral nerve in vivo. These Tracking the differentiation process were taken place using quantitative RT- findings suggest that autologous BM-MNCs injected into injured muscle PCR, and flow cytometry for monocyte markers such as CD11b and CD14. have a robust, long term engraftment within the injured tissue where they The cells showed monocytic characteristics 7 days after transduction, CD11b may promote myogenesis, angiogenesis and neurogenesis. and CD14 expression were significantly increased, and the cells became adherent to the substrate with pseudo pods and flatten macrophage-like morphology. We concluded that according to the current experiment and previous studies, miR-424 is responsible for monocyte/macrophage differen- tiation of human cord blood CD133+ hematopoietic stem cells and has the ability of directing them to express monocytic characteristics.

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Poster Board Number: 2409 of inflammatory challenge on HSC proliferation using lipopolysaccharide (LPS) injections. Analysis revealed that 2-4 time and ≥5x divided LKS at 3 LIGHT REGULATE HEMATOPOIETIC STEM CELL weeks from LPS-treated mice reconstituted multi-lineage blood cells whereas DIFFERENTIATION both fractions from control mice failed to engraft. This data provide in vivo evidence that naturally occurring hematoimmunological challenges, as Zou, Gang-Ming, Chen, Weika gram-negative bacterial infection induces HSC proliferation and self-renew- Shanghai Cancer Institute, Shanghai Jiaotong University, Shanghai, China al. These findings demonstrates dynamic and demand-adapted HSC cycling kinetics, leading to similar turnover of entire HSC pool at end of life, and Cytokine LIGHT is a member of TNF superfamily. It is expressed primarily on might represent a biological principle that could hold true for other somatic activated T lymphocyte, and detectable on monocyte, granulocyte, and im- stem cell-sustained organ-systems. mune dendritic cells. It mainly plays the role in immune regulation including T cell activation and dendritic cell maturation. We recently reported its role Poster Board Number: 2413 as an inducer in embryonic stem cell differentiation, but its role in regula- tion of adult stem cell is yet defined. In the present study, we examined the SHEEP YOLK SAC AS A SOURCE OF expression of LIGHT receptor in HSC. We found that HSC did not produce HEMANGIOBLASTIC CELLS cytokine LIGHT but express HVEM, a LIGHT receptor, on its surface. We fur- ther found its novel role in promoting myeloid differentiation of hematopoi- Pessolato, Alicia Greyce G., Martins, Daniele S., Fontes, Aparecida etic stem cells driven by GM-CSF. Compared to the control group, CFU-GM M., Oliveira, Maria E., Alberto, Miryan L., Rosa, Nathália G., colony formation rate is increased when LIGHT is added in the culture. Magalhães, Danielle A., Freitas, Marcela C., Fernandes, Andrielle C., However, LIGHT down-regulate erythroid differentiation driven by EPO. Mello, Fernanda U., Vicente, Wilter R., Covas, Dimas T.2, Miglino, Further examination showed that LIGHT enhances GM-CSFR expression in Maria A.1 mouse HSCs. The data suggest LIGHT promote myeloid differentiation of Department of Surgery, Faculty of Veterinary Medicine and Animal hematopoietic stem cells. These studies identified a novel role of LIGHT in Sciences, São Paulo, Brazil, Institute of Science and Technology in Stem Cell hematopoietic stem cell differentiation regulation. Therapy, Ribeirão Preto, Brazil, Faculty of Animal Sciences and Veterinary, Poster Board Number: 2411 Jaboticabal, Brazil DYNAMIC AND DEMAND-ADAPTED CELL The yolk sac (YS) is an embryonic attach present in all vertebrate species and plays important roles, among them the rise of multipotent stem cells, CYCLE REGULATION IN HEMATOPOIETIC STEM termed hemangioblasts. In YS, the hemangioblasts are arranged in clusters, CELLS named blood islands. The central hemangioblasts of the islands form the first hematopoietic stem cells (HSC) and the peripheral differentiate into Takizawa, Hitoshi, Regoes, Roland R., Bonhoeffer, Sebastian, Manz, angioblasts, the endothelial precursors cells (EPC). To characterize the cells Markus G. with hemangioblastic features, we submitted the YS of different embryonic University Hospital Zurich, Zurich, Switzerland, Institute of Integrative ages to light and electron microscopy techniques to study the development Biology, ETH Zurich, Zurich, Switzerland of the blood islands. Subsequently, we isolated adherent and non-adherent cells from YS of sheep embryos and performed in vitro expansion and High-throughput and lifelong blood production is maintained by a small characterization on days D+0, D+3, D+6 and D+9, under different aspects: fraction of hematopoietic stem cells (HSCs). Evaluation of in vivo steady- morphological and gene expression by real time PCR. After cultivation the state HSC cycling kinetics showed equal division rate and contribution to YS presented adherent cells to the plastic surface with endothelial morphol- blood formation in all HSCs, while transgenic mouse models suggested ogy and non-adherent cells with spherical morphology. The results of light two HSC pools, fast-dividing HSCs with limited self-renewal and contribu- and electron microscopy demonstrated that at 15 days of age there were tion to hematopoiesis and quiescent HSCs with lifelong self-renewal but several blood islands with a small amount of blood cells inside. Between 21st very low hematopoietic contribution. Thus, HSC division and contribution and 23rd days, the islands had larger size and large amount of blood nucle- to steady-state hematopoiesis, the relationship between divisional history ated primitive cells in the mesenchymal region of the YS. At 25 days the and cell cycle status, and the impact of naturally-occurring hematopoietic islands showed fusion and apoptosis. The analysis of gene expression D+0 challenges on HSC self-renewal and differentiation remain to be determined. showed that from the 23rd to 25th days of age there was an overexpression To address these directly, we established a high resolution non-invasive in of genes CD150, Annexin-5, LMO, Gata-3, VEGF, SCF, Flk-1 and Runx-1, vivo HSC divisional tracking system with CFSE (carboxyfluorescein diacetate which are expressed by the hemangioblasts. This expression pattern sug- succinimidyl ester). I.v. transfer of Lin-c-kit+Sca-1+ cells (LKS) into non- gests that probably the primary hematopoietic profile was being established conditioned congenic recipient mice allows us to evaluate 0-7 divisions in in vivo at these ages, and the precursor population is preserved to maintain steady-state recipient bone marrow over at least 20 weeks. Single cell and the hematopoiesis. In the embryonic period from the 27th to 32nd days the limiting dilution transplantation showed that both 0- and ≥5x divided LKS at expression of CD150 gene, expressed by HSC, decreased and the expression ≥7 weeks after chase contain multi-lineage repopulating cells with fluctuat- of the genes SCF LMO, Runx-1, Flk-1 and Gata-3, expressed by the heman- ing, lifelong hematopoietic contribution. Furthermore, steady-state serial gioblasts and EPC increased again. This occurs because over the course of transplantation with fast cycling HSC revealed that they can slow down, embryonic development the cellular demand increases in the YS, with the suggesting dynamically changing cycling activity of HSC over time. Math- need angiogenic and vasculogenic. However, after 3 days in culture, non- ematical modeling based on limiting dilution transplantation, revealed no adherent cells of the YS overexpressed the genes Flk-1 and CD41, and the evidence for a dichotomy of biologically defined HSCs in different groups. adherent cell portion began to overexpress the gene Runx-1, which is also These data demonstrate that although there is cycling heterogeneity in HSC expressed by the hemangioblast, but at a secondary stage of differentiation, at any given time, HSC are not permanently split into two populations with suggesting an initial differentiation of the cell population due to cultivation. different cycling kinetics. Transfer of aged cells or cells that underwent in Gene expression D+6 of adherent cell portion showed a high expression of vivo expansion after transplantation, i.e. cells with extensive proliferative Annexin-5 gene and SCF, and the analysis of adherent cells D+9 showed a history, into steady-state young recipient mice showed increased frequency low expression of SCF gene, which probably indicates that this primitive cell of dormant cells compared with young donor cells, indicating existence of population and precursor of hematopoiesis differentiates in a short period of an intrinsic drive for quiescence, which would protect cells from cancer- time in vitro or needs to migrate to other secondary sites of hematopoiesis ous genetic errors associated with DNA replication. We tested the effects to proceed to a populating and a definitive hematopoietic establishment.

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Thursday Poster Abstracts

Moreover, the high expression of Annexin-5 indicates an important function development in the placenta and in other places of the hematopoietic devel- of YS as placental anticoagulant, but also indicates that the apoptosis occurs opment at different stages may provide effective tools to enhance protocols very early in this organ, because in ovine specie the YS is persistent only at for HSC harvesting, ex-vivo expansion, and generation from pluripotent cells embryonic period. In conclusion, our results indicate that a microscopic char- or extraembryonics membranes like yolk sac. acterization and isolation of this precursor cell type from the YS enables the knowledge about the mechanisms of hematopoietic/endothelial differentia- tion and the influence of culture on these cells, making this organ a source of stem cells relevant to cell therapy. MESENCHYMAL CELL LINEAGE ANALYSIS Poster Board Number: 2415 Poster Board Number: 2419 CHARACTERIZATION OF SHEEP FGFR2 MUTATION CONFERS LESS FUNCTIONAL MESENCHYMAL STEM CELLS FROM DAMAGE IN ADULT HUMAN MESENCHYMAL HEMATOPOIETIC PLACENTAL TISSUES AND STEM CELLS AS COMPARED TO FIBROBLASTS EMBRYONIC ORGANS Yeh, Erika, Atique, Rodrigo Ft, Alonso, Nivaldo, Matushita, Hamilton, Passos-Bueno, Maria Rita Pessolato, Alicia Greyce G., Fernandes, Andrielle C., Martins, Daniele S., Fontes, Aparecida M., Oliveira, Maria E., Vicente, Wilter Department of Genetics and Evolutive Biology, University of Sao Paulo, Sao Paulo, Brazil, School of Medicine, University of Sao Paulo, Sao Paulo, Brazil R., Ambrósio, Carlos E., Covas, Dimas T., Miglino, Maria A. Institute of Science and Technology in Stem Cell Therapy, Ribeirão Preto, Fibroblast growth factor signaling pathway is involved in multiple functions, Brazil, Faculty of Animal Sciences and Veterinary, Jaboticabal, Brazil from early development to homeostasis of the adult organism, with a wide variety of effects: from mitogenic, regulatory, morphological, to endocrine The placenta is composed of two main tissues: the trophoblast and the effects. Gain-of-function mutations in FGFR2 are causative of several con- mesoderm. Trophoblasts comprise the most abundant cell type in the pla- genital skeletal disorders, including Apert Syndrome (AS). Using adult hu- centa and line the maternal blood spaces, whereas the mesoderm-derived man mesenchymal stem cells (hMSC) harboring AS mutation (p.Ser252Trp) allantois generates the feto-placental vasculature. The allantois grows from obtained from the periosteum, we evaluated the roles of FGF signaling in the posterior primitive streak into the exocoelomic cavity and fuses with the proliferation and osteogenic differentiation and compared to fibroblasts ob- chorion, inducing the extensive branching of the fetal vasculature into the tained from the same individuals. Though during three days wild-type (WT) trophoblast layer, a communication between mother and conceptus. During fibroblasts and hMSCs had similar growth, AS hMSC grew half the number the embryogenesis, the placenta has potential to generate multipotential of WT hMSCs after 72 hours (p=0.0018) and AS fibroblasts grew twice as myelo-lymphoid hematopoietic stem cells (HSC). Due to fetal blood circula- fast as WT fibroblasts in the same period (p=0.0002). During osteoblastic tion been directed from the dorsal aorta through the placenta to the fetal osteogenesis, we performed alkaline phosphatase assay at the 9th day and liver, most HSC generated in the aorta-gonad-mesonephros (AGM) region alizarin red staining at the end of 3 weeks. Alkaline phosphatase was 3.5 traverse the placenta along their journey towards the liver and may pause times higher in AS than in WT hMSC (p=0.0002) and 6.8 times elevated in placental niches for expansion, thereby contributing to the total pla- in AS than in WT fibroblasts (p<0.0001). Accordingly, alizarin red staining cental HSC count. The placenta as a major organ producer and maintainer absorbance was 50% higher in AS than in WT hMSC (p=0.0002) and 70% of HSC in an undifferentiated state provides a valuable model to further elevated in AS than in WT fibroblasts (p<0.0001). In conclusion, excessive elucidate regulatory mechanisms governing HSC emergence and expansion FGF signaling due to FGFR2Ser252Trp had opposite effect on AS hMSC during the embryo development. However, the manner in which the MSC and AS fibroblasts and enhanced osteogenic potential of both AS fibroblasts population behaves in hematopoietic support and the relation of this cell compared to WT fibroblasts and AS hMSCs compared ro WT hMSCs, yet in type with the initial blood development still need to be better understood. a milder way in mutant hMSCs. All in all, hMSC seemed to be less affected Therefore, we hypothesized that the MSC extracted from sheep placental to the pathogenic effects of the mutation than in fibroblasts. Further studies membranes (chorion, allantois and chorioallantois) and embryonic organs in order to better understand the molecular mechanisms underneath these (hepatic bud, AGM and dorsal aorta) with high hematopoietic potential, findings are required. Funding: FAPESP-CNPq which give stromal support to development and establishment hematopoi- etic can suffer or show alterations depending on hematopoietic phase of the Poster Board Number: 2421 embryonic development. Due this, we isolated MSC from many placental and embryonic tissues and performed in vitro expansion and characteriza- CHARACTERISTICS OF MESENCHYMAL tion, under different aspects: morphological, phenotypical by flow cytometry STROMAL CELLS IN SYNOVIAL FLUID OF and gene expression, differentiation potential and tumorigenic potential. The CHILDREN WITH JUVENILE IDIOPATHIC results demonstrated that this cell population exhibits typical characteristics of MSC. After adherence to the plastic surface they showed fibroblasts typi- ARTHRITIS AND CHILDREN WITHOUT JOINT cal morphology. Immunophenotype characterization demonstrated that the INFLAMMATION cell population of all tissues was positive for the sheep MSC surface marker CD44, negative for the hematopoietic markers CD45, CD41/CD61, CD38, Bernotiene, Eiva, Miskinyte, Giedre, Unguryte, Ausra, Panaviene, CD48 and the endothelial marker CD31. The gene expression demonstrated Violeta, Jarmalaite, Sonata, Mackiewicz, Zygmunt, Daniunaite, also that all tissues expressed MSC genes like VEGF, ColA-1, MMP-2 and Kristina, Bagdonas, Edvardas, Astrauskiene, Danute Linute FGF-b, but the dorsal aorta showed a exponential expression of Vimen- Department of Experimental and Clinical Medicine, State Research Institute tin and Leptin. When cultured in defined medium, the cells derived from Centre for Innovative Medicine, Vilnius, Lithuania, Clinic of Children`s all tissues were capable of differentiating into osteocytes and adipocytes. Diseases, Faculty of Medicine, Vilnius University, Vilnius, Lithuania, Also, the placental-derived MSC do not possess tumorigenic potential after Cathedral of Botanics and Genetics, Faculty of Natural Sciences, Vilnius infusion into NOD/SCID mice. In conclusion, our results indicate that was University, Vilnius, Lithuania possible to isolate MSC from niches of hematopoietic development like placental membranes and embryonic organs. Moreover, improved under- Background. Due to superior chondrogenic differentiation properties, as standing of the molecular and cellular mechanisms of MSC that govern HSC compared to bone marrow-derived mesenchymal stem cells (MSC), synovi- um might appear the source of choice for MSC for the regenerative therapy

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of damaged cartilage in arthritis, articular trauma and some orthopedic dis- confluence in 24-well plate and very low potential if they didn’t manage to eases. However, juvenile idiopathic arthritis (JIA) can present in a very young cover well in 24-well plate. MMSC clones with high PP from 2 donors were age, even in the first years of life, and getting samples of synovial biopsy analysed by means of ligation-mediated polymerase chain reaction (LM- becomes an unrealizable task. Therefore, the employment of easily obtain- PCR). DNA was isolated from those clones and fragmented by frequently able source for MSC with similar potential, such as synovial fluid (SF)-MSC, cutting Sse9 I endonuclease. Restricted DNA was enriched with fragments deserves particular attention. Presence of MSC in SF of human adults has containing vector and nearby genomic content using primer complementary been recently demonstrated, reporting numbers of 2 CFU/ml in rheuma- to vector sequence bound to biotin and paramagnetic particles covered by toid arthritis, 37/ml in osteoarthritis and 14/ml - in healthy controls. Data streptavidin. A short adapter was ligated to the enriched DNA fragments fol- on SF-MSC in children are lacking. Aim of our study was to characterize lowing 2 stages of nested PCR. Then the pattern of PCR products obtained SF-MSC in children with JIA and children without autoimmune inflammation from each clone was compared by electrophoresis with subsequent sequenc- in the joints. Methods and Results. 34 JIA patients and 6 children that were ing of PCR products. The obtained sequences were compared with human investigated for orthopedic pathology, and served as controls, were enrolled genome and vector integration sites were determined. It was shown that the into the study. Age of patients was 3-17 years. SF-MSC were successfully proportion of MMSC clones with high PP was 3,7 ± 0,6% at 1st passage, isolated from all tested samples of synovial fluid. Flow cytometry analysis reached maximum of 5,0 ± 1,4% at 3d passage and then declined. Clones of SF-MSC revealed typical for MSCs immunophenotype, and differentia- with high PP were not detected at 7th passage. The proportion of MMSC tion capacities into adipogenic, osteogenic and chondrogenic lineages were clones with medium PP reached maximum of 6 ± 2% by 2nd -3d passage demonstrated. Colony forming units (CFU) were established in relation to and then also declined. Such clones were not detected at 7th passage as 1 ml of punctured SF, and to total punctured SF. High variation of SF-MSC well. The maximum proportion of clones with low PP was 23 ± 12% at 3d numbers was observed between individuals of either group. At average, passage and then declined to 7% at 7th passage. The majority of MMSC 24.4±20.4 CFU/ml were determined in SF of control children. Numbers clones possessed very low PP and their mean proportion within passages of MSC at the initial stages of JIA, within first month, were similar to the was 82 ± 4%. The vector integration site analysis of 37 MMSC clones with control levels and had no relation to the age of patients. However, longer high PP and 7 clones with medium PP showed that each analyzed till now disease duration, more than 4 months, was associated with increased rates clone contained unique integration sites. Clones with the same integration of MSC, 103 ± 132.1 CFU/ml, p=0.045. Whereas, in patients with long and site were not revealed in passages. It means that every analyzed clone with aggressive course of JIA, that were administered basal therapy, substantially high and medium PP was a progeny of distinct parental MMSC. The data lower numbers of SF-MSC, 8.6±10.6 CFU/ml, p=0.000, were observed, as suggest that not a single mesenchymal stem cell with the ability to self- compared to the patients with long and light course of the disease. Relative renewal was detected by LM-PCR analysis. Our results show that MMSCs epigenetic stability was revealed by analysis of DNA methylation of genome represent a heterogeneous population of cells with different but limited PP. stability regulation- and cell cycle control- associated gene promoters in The probability of the presence of mesenchymal stem cells in the population SF-MSC. Conclusion. The presence of SF-MSC with typical trilineage dif- of MMSCs seems to be very low. ferentiation potential was determined in SF of all tested children with JIA or without autoimmune inflammation in the joints. In JIA, numbers of SF-MSC Poster Board Number: 2425 are modulated by longer duration and higher aggressiveness of the course ACCELERATING THE CELL CYCLE OF HUMAN of the disease. SF could appear an easily accessible source of joint-derived MSC for research and, possibly, diagnostic or therapeutic purposes. ADIPOSE TISSUE STEM CELLS ELEVATES THEIR Poster Board Number: 2423 DIFFERENTIATION PLASTICITY TOWARDS THE MESODERM AND ECTODERM LINEAGES HETEROGENEITY OF HUMAN MULTIPOTENT Boquest, Andrew C., Boulland, Jean-Luc, Noer, Agate, Perreault, MESENCHYMAL STROMAL CELLS Marie-Claude, Glover, Joel C., Collas, Philippe Bigildeev, Alexey, Zhiromkina, Oxana, Sats, Natalya, Shipounova, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway Irina, Drize, Nina In contrast to adult stem cells which have an extended G1-phase, pluripo- Laboratory of Physiology of Hematopoiesis, National Hematological tent ESCs predominately exist in the S-phase of the cell cycle. We therefore Research Centre, Moscow, Russian Federation speculate that the open chromatin structure conferred during S-phase con- Multipotent mesenchymal stromal cells (MMSCs) participate in the regen- tributes to elevated stem cell plasticity. Here, we test this hypothesis in adult eration of different tissues. The aim of this study was to investigate the stem cells by markedly increasing the proliferation dynamic of human adi- proliferative potential (PP) and clonal composition of bone marrow (BM) pose stem cells (hASCs) prior to differentiation along either the mesoderm derived MMSCs. MMSCs from 6 donors (4 males and 2 females) 15-43 or ectoderm lineages. We exposed hASCs to regular MSC culture medium years old were used for this experiment. 3x106 BM nucleated cells were (CTL; DMEM/F12+10% FCS) spiked with EGF (20ng/ml), bFGF (20ng/) plated in 25 cm2 flask inα -MEM with 10% of fetal calf serum. The cells and B27 (STIM treatment) which we have previously shown to markedly in- were passaged at the density 105 cells per flask after they reached confluent crease percentages of S-phase cells. We find, on average, a 27-fold increase monolayer. MMSCs were infected with self-inactivating 4th-generation in cell numbers in STIM cultures compared with a 5-fold increase in CTL lentivirus vector bearing enhanced green fluorescent protein. 107 virus cultures after 14 days. Compared to CTL cells, STIM cells appear smaller, particles were added to MMSCs for 6 hours with 8 μg/ml of polybrene 24 elongated, uniform in shape and less sensitive to contact growth inhibition. hours after passage 0. This vector integrates into DNA in random manner STIM treatment does not affect surface protein expression of MSC-related thus uniquely marking each MMSC and its progeny. At each passage the antigens (CD90, CD44, CD105 and CD13). However, it increases transcripts proportion of marked by lentivector (green) and viable cells was measured of cell cycle-related genes (CCN A2 and CCND1) and some plasticity-related and MMSCs were cloned 1 cell per well in 96-well plate in standard medium genes (DNMT3B and NESTIN), but not those associated with pluripotency supplemented with 5 ng/ml of basic fibroblast growth factor. Clones were (SOX2, KLF4, NANOG and OCT4B). It also dramatically reduces transcripts transferred to 24-well plate after reaching confluent monolayer and then for E-cadherin, a hallmark of the epithelial to mesenchymal transition. STIM to 6-well plate and finally to 25 cm2 flask. The ability of MMSC clones to treatment also substantially improves osteogenic differentiation as well adipose and osteogeneic differentiation was assessed at this stage. Clones adipocyte formation, though the latter effect was less dramatic compared were considered to have high PP if they reached confluent monolayer in 25 to that of forming bone. Chondrogenesis, however, appears unaffected by cm2 flask, medium PP if they grew to confluence in 6-well plate, low PP - STIM treatment. To assess the influence of STIM treatment on ectoderm development, we plated cells on matrigel and add a novel induction medium

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Thursday Poster Abstracts

(serum-free high glucose DMEM with IBMX, DEX, forskolin, 8-CPT cAMP) controversial vascular niche. We have analyzed agrin-deficient mice and which elevates intracellular cAMP, leading to cell cycle arrest and formation found that they are characterized by hypoplasia of hematopoietic tissues. of neurogenic-like cells. 24 h after neurogenic induction, the majority of We have found that CD34+ CD135- LSK population is reduced because of STIM cultures demonstrate neurogenic-like features such as interconnecting in vivo apoptosis, and this is not due to an intrinsic defect of agrin-deficient multipolar cells with phase-bright somata, multi-branched processes and HSCs, since they are able to proliferate and differentiate in transplanted neurofilament immunoreactivity. Only few cells with these characteristics are wild-type recipients. Moreover we have demonstrated that LSK cells express present in neurogenic induced CTL cultures. Addition of the HDAC inhibi- α-dystroglycan, the receptor of non-neuronal agrin. To further confirm the tor valproic acid with FCS and B27 supplement maintains the neurogenic role of α-dystroglycan in this system, we have shown that wild-type CD34+ phenotype, including MAP2 and NeuN immunoreactivity, in long-term (> CD135- LSK population undergoes apoptosis in vivo when α-dystroglycan- 3 weeks) cultures. Global methylation profiling using MeDIP reveals that agrin interactions are blocked. Agrin-mediated cell-cell contacts between neurogenic-specific genes are relatively unmethylated in ASCs, supporting hematopoietic stem cells and endosteal osteoblasts in the BM are therefore an underlying neurogenic potential. We are currently using electrophysiol- crucial for CD34+ CD135- LSK cell survival and expansion. We are con- ogy to determine whether such cells possess neuronal functionality. We vinced that these results will contribute to achieve a better understanding conclude that the dramatic acceleration of the cell cycle in hASCs installs an of the crucial cellular interactions occurring at the HSC niches and thus pave elevated level of plasticity which favors differentiation into both mesodermal the way for an optimized manipulation in the field of regenerative medicine. and ectodermal lineages. Poster Board Number: 2431 Poster Board Number: 2427 MESENCHYMAL STROMAL CELLS ISOLATED HETEROTOPIC OSSIFICATION IS MEDIATED FROM MULTIPLE SCLEROSIS PATIENTS INHIBIT BY BMP-RESPONSIVE PROGENITORS IN THE LYMPHOCYTE PROLIFERATION AND INDUCE SKELETAL MUSCLE INTERSTITIUM REGULATORY T CELLS GENERATION Wosczyna, Michael N., Biswas, Arpita, Cogswell, Cathy, Oliveira, Gislane L. V., Malmegrim, Kelen C.R., Colombini, Amanda Goldhamer, David J. M., Covas, Dimas T., Voltarelli, Julio C. Center for Regenerative Biology, UConn Stem Cell Institute, Department of School of Medicine, National Institute of Science and Technology in Stem Molecular and Cell Biology, University of Connecticut, Storrs, CT, USA Cells and Cell Therapy, Ribeirão Preto, Brazil Heterotopic ossification is a debilitating condition that can result from Introduction: Multipotent mesenchymal stromal cells (MSCs) are being cur- traumatic injury, surgery or genetic disease. We investigated the cellular rently considered as a potential cell source for therapy of autoimmune dis- origins of heterotopic bone in the mouse using Cre/loxP lineage analysis eases (AD), due to their immunomodulatory properties. MSCs have shown and bioassays of heterotopic ossification based on intramuscular injection ability to suppress lymphocyte proliferation in vitro and exert regulatory of bone morphogenetic protein 2 (BMP2). We demonstrate that osteogenic functions in other immune cells. Several mechanisms have been involved in progenitors that express the receptor tyrosine kinase, Tie2, pre-exist in the the immunosuppressive effects of MSCs, including direct cell-to-cell contact skeletal muscle interstitium and are not derived from the endothelium or and secretion of soluble factors. Furthermore, MSCs have been reported from hematopoietic lineages. Prospectively isolated Tie2+ progenitors, which to induce the production of IL-10 and the generation of regulatory T cells. were purified to apparent homogeneity by FACS, robustly contributed to These properties of MSCs suggest a potential role of these cells in the induc- BMP2-dependent heterotopic ossification after cell transplantation. Clonal tion of tolerance in AD and also provide a rational basis for their application analysis of FACS-fractionated cells revealed that these progenitors are bipo- in the treatment of multiple sclerosis (MS). Aim: To evaluate the antiprolif- tent, exhibiting the capacity for both spontaneous adipogenic differentiation erative capacity of MSCs derived from MS patients on phytohemagglutinin and BMP-dependent osteogenic differentiation. Identifying the cells-of-or- (PHA)-induced lymphocyte proliferation and the ability of these MSCs to igin responsible for heterotopic ossification provides a potential therapeutic induce regulatory T cells in vitro. Subjects and Methods: MSCs cultures were target to treat, mitigate or prevent this disabling condition. derived from bone marrow aspirates from MS patients (N=8). Mononuclear Poster Board Number: 2429 cells were separated by Ficoll-Hypaque and MSCs were isolated by plastic adherence. In the inhibition assays, peripheral blood mononuclear cells (PB- AGRIN IS A NOVEL NON-REDUNDANT PLAYER MCs) isolated from healthy donors were labeled with CFSE and co-cultivated OF THE MOUSE HEMATOPOIETIC STEM CELL with patients’ MSCs at several ratios (1:2; 1:5;1:10;1:20;1:50 and 1:100) in the presence of PHA at 37°C in a 5% CO2. In the immunoregulation assays, NICHE PBMCs non-labeled were co-cultivated with patients’ MSCs at 1:5 ratio, Mazzon, Cristina, Anselmo, Achille, Cibella, Javier, Soldani, also in the presence of PHA. After 5 days of coculture, CD3+-lymphocyte proliferation was assessed by CFSE dilution method and the percentages Cristiana, Kim, Natalie, Burden, Steven, Dustin, Michael, Sarukhan, of subpopulations of CD4+CD25hiFoxp3+, CD4+CD25hiCTLA-4+ and Adelaida, Viola, Antonella CD4+CD25hiGITR+ were assessed by flow cytometry. The results were Humanitas Clinical Institute, Rozzano, Italy, Skirball Institute of analysed by Oneway analysis of variance (ANOVA) with Dunnett’s multiple Biomolecular Medicine, New York University School of Medicine,, New comparison post test and Mann-Whitney t test. This study was approved York, NY, USA, Skirball Institute of Biomolecular Medicine, New York by the Clinical Hospital of Ribeirão Preto ethics committee. Results: We University School of Medicine, New York, NY, USA observed that MSCs isolated from MS patients efficiently inhibited PHA- While the concept of niche as a specialized microenvironment housing induced lymphocyte proliferation in a dose-dependent manner. There stem cells is currently accepted, the cellular and molecular interactions that were significant differences (P=0.037) in proliferation mean percentage (P) control the balance between maintenance of stem cell number and he- when comparing PBMCs plus PHA (P: 67.20 ± 5.16%) with patients’ MSCs matopoiesis remain only partially defined. In this study, we have discovered plus PBMCs at ratios 1:2 (P: 32.96 ± 1.75%) and 1:5 (P: 41.73 ± 9.09%). a novel, non-redundant and unexpected role of agrin -- a critical regulator The percentage of proliferation inhibition (PI) from MSCs at 1:2 ratio was of the neuromuscolar junction -- in hematopoiesis. We found that agrin 49.89% and there were no significant differences in proliferation inhibi- is expressed by MSCs and osteoblasts at the endosteal niche, whereas no tion at several tested ratios (P>0.05). We observed significant differences expression was detected in reticular and endothelial component of the (P=0.03) in CD4+CD25hiFoxp3+ T cells percentages recovered after 5 days of coculture with patients’ MSCs (9.65 ± 4.82%) when compared to

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controls (only PBMCs: 1,76 ± 1,24%). There were no significant differences Poster Board Number: 2435 in CD4+CD25hiCTLA-4+ and CD4+CD25hiGITR+ subpopulations of T cells recovered after cocultures with patients’ MSCs (P>0.05). Conclusions: The SPECIFIC MIGRATION OF MESENCHYMAL MSCs isolated from bone marrow from MS patients efficiently inhibited STROMAL CELLS TO CHEMOKINES IN VIVO lymphocyte proliferation in a dose-dependent manner and were able to induce regulatory CD4+CD25hiFoxp3+ T cells in vitro. These data suggest Kollar, Katarina, Cernat, Laura, Marusciac, Laura, Roeder, Olga, that patients’ MSCs release soluble factors with immunosuppressive capacity Seifried, Erhard, Henschler, Reinhard and ability to induce regulatory T cells generation. So, autologous MSCs Institute of Transfusion Medicine and Hematology, Frankfurt, Germany could be a potential cell source for therapy of MS. However, more studies should be performed before considering the use of MSCs from MS patients Mesenchymal stem/stromal cells (MSC) are adult-derived cells that have in clinical settings. multipotent lineage potential, an immunoprivileged status and therapeutic potential for repair of tissue injury. Bone marrow derived MSC will migrate Poster Board Number: 2433 to sites of inflammation in preference to bone marrow, however, the mo- lecular mechanisms of migration are still unknown. Here we utilise an acute MESENCHYMAL STROMAL CELLS SHOW model of skin inflammation in mouse which expresses chemokines including ENHANCED MIGRATION TO HEART AFTER CCL4, CCL5, CCL7, CCL12, CCL19, CXCL1, CXCL4, CXCL13, CXCL14 and PRE-TREATMENT WITH TNF- AFTER ACUTE CXCL16 to study the specific migratory pathways utilised by human bone α marrow derived MSC. We next analysed mRNA and protein expression of MYOCARDIAL INFARCT hMSC chemokine receptors (CRs). As has been previously found, hMSC had Kollar, Katarina, Blair, Chris, Ahmed, Ishtiaq M., Cook, Matthew, low levels of CR expression, both at the mRNA level and surface protein lev- el. However, it was noted that the majority of chemokine receptors studied Feneley, Michael P., Atkinson, Kerry M., Brooke, Gary P. could be detected as proteins at an intracellular level. Therefore, although, Mater Medical Research Institute, Brisbane, Australia, Victor Chang Cardiac hMSC express specific chemokine receptors for chemokines released after Research Institute, Sydney, Australia acute skin inflammation, they appear to be tightly regulated at both the Mesenchymal stem/stromal cells (MSC) are adult-derived cells that have mRNA and at the cell surface level. Furthermore, hMSC expressed chemok- multipotent lineage potential, an immunoprivileged status and therapeutic ines CCL2, CCL7, CCL19 and CXCL12 at the mRNA and protein level which potential for repair of tissue injury. We have previously shown that in both could have functional consequences for their migration. In a In vivo matrigel healthy (unperturbed) and inflammatory (myocardial infarct model) settings plug assay, hMSC were shown to migrate specifically to chemokines CCL5, only small numbers of murine MSC show specific migration and engraft- CCL19 and CXCL12 suggesting enhanced migration strategy for cellular ment after intravenous injection. To determine if this was due to an inability therapy. to respond to chemotactic stimuli, chemokine expression was analysed at Poster Board Number: 2437 various time points by qRT-PCR in mice after acute myocardial infarction (AMI). Many chemokines were up-regulated after AMI, with the highest EXPANSION OF MESENCHYMAL STEM CELLS at 24 hrs being CXCL12 and CCL7. We next analysed mRNA and protein expression of MSC chemokine receptors (CRs). As we have previously found CAUSES DNA-METHYLATION CHANGES WHICH with human MSC, murine MSC had low levels of CR expression, both at CORRELATE WITH REPRESSIVE HISTONE the mRNA level and surface protein level. However, it was noted that the MARKS majority of chemokine receptors studied could be detected as proteins at an intracellular level. Therefore, although, MSC express specific chemokine Schellenberg, Anne, Lin, Qiong, Koch, Carmen M., Schüler, Herdit, receptors for chemokines released after AMI, they appear to be tightly Joussen, Sylvia, Denecke, Bernd, Bokermann, Gudrun, Zenke, regulated at both the mRNA and at the cell surface level. This seems to have Martin, Wagner, Wolfgang functional consequences as MSC migration towards CXCL12 and CCL7 in Stem Cell Biology and Cellular Engineering, Helmholtz-Institute for vitro showed low migration efficiency. Using UpCellTM tissue culture plastic Biomedical Engineering, Aachen, Germany, Cell Biology, RWTH Aachen for gentle dissociation of MSC and by pre-treating MSC with pro-inflamma- Medical School, Aachen, Germany, Institute of Human Genetics, RWTH tory cytokines, we found enhanced surface expression and up-regulation of Aachen Medical School, Aachen, Germany, Interdisciplinary Centre for mRNA for chemokine receptors CXCR4, CCR1, CCR2, CCR3 and CCR5. The Clinical Research, RWTH Aachen Medical School, Aachen, Germany chemotactic response of MSC to CXCL12 and CCL7 was significantly higher in cells pre-treated with TNF-α, IFN-γ, IL-1α and IL-6, and to CCL7 was sig- Fifty years ago, Leonhard Hayflick discovered that the number of cell divi- nificantly higher in cells pre-treated with IFN-γ, IL-1α and IL-6. In vivo, MSC sions is limited for cells in culture -- after about 40 to 60 divisions the pro- pre-treated with TNF-α and IFN-γ showed significantly increased migration liferation rate decays until the cells ultimately enter a senescent state. This to site of skin wound injury at 24 hrs (p=0.0006) compared to untreated phenomenon applies to all somatic cells in culture which are not immortal- MSC. In the AMI model, MSC pre-treated with TNF-α showed significantly ized and it is of specific relevance for cellular therapy. In this study, we have increased migration to the infarcted myocardium at 3 hrs (p=0.05) and 24 expanded mesenchymal stromal cells (MSC) from human adipose tissue and hrs (p=0.001) compared to untreated MSC, suggesting pro-inflammatory analyzed genetic and epigenetic sequels. Analysis of subsequent passages treatment strategy for enhanced migratory efficiency to AMI. revealed, that the subpopulation of cells which gives rise to fibroblastoid colony-forming units (CFU-F) decreases already during the exponential growth-phase. Therefore, the progeny of MSC is based on a subset of highly proliferative cells which undergoes a higher average number of cell divisions than estimated by the conventional long-term growth curves based on the ratio of cells seeded versus cells harvested per passage. Adipogenic and osteogenic differentiation potential decreased within the initial passages, too. Relevant chromosomal aberrations were not detected by karyotyping and SNP-microarrays and this is in line with various recent reports indicating that the genome of human MSC is relatively stable. Subsequently, we have compared DNA-methylation profiles of early and late passages with the Infinium Human Methylation27 Bead Array. DNA-methylation profiles dif-

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Thursday Poster Abstracts fered markedly in comparison to MSC from bone marrow indicating that the The goal of this study was to compare the transfection efficiency of GFP in epigenetic makeup of MSC preparations is highly dependent on the tissue of human MSC derived from the umbilical cord using nucleofection (Amaxa), origin. Furthermore, there were consistent senescence-associated modifica- lipofection (manufacturer) and Neon transfection (Invitrogen). We measured tions at specific CpG sites and these revealed overlapping hyper-methylation the gene transfection efficiency using flow cytometry, fluorescence micros- or hypo-methylation in MSC from bone marrow, MSC from adipose tissue copy and live/dead staining. The transfection efficiency was evaluated as a and dermal fibroblasts. DNA-methylation changes in these regions were function of the number of transfected cells, number of dead cells post-trans- also related to age-associated changes in MSC from bone marrow of young fection, proliferation index and if the integration in the genome is transient and elderly donors. Notably, senescence-associated DNA-methylation were or stable. In our preliminary results, lipofectamine and Neon transfection had highly significantly enriched in regions with have been specified for repres- a minimal impact in cells viability and proliferation, and the gene transfec- sive histone marks such as trimethylation of H3K9, H3K27 and EZH2 targets. tion efficiency was low. In contrast, nucleofection had more impact on cell These results support the notion, that cellular aging is not just a random ac- viability and proliferation, and it had the highest transfection efficiency, too. cumulation of cellular defects, but that it is precisely regulated by epigenetic Our preliminary data indicate that nucleofection may be a more efficient means in the course of culture expansion. method to incorporate foreign DNA in Wharton’s jelly derived MSC Poster Board Number: 2439 Poster Board Number: 2443 A MSC POPULATION WITH ENHANCED ISOLATION AND IDENTIFICATION OF STEM RESPONSE TOWARDS TUMOR STROMA CELLS FROM DOG ADIPOSE TISSUE Bolontrade, Marcela F., Sganga, Leonardo, Ciolfi, Federico, Garcia, Ceh, Katerina, Zorko, Bojan, Zupanc, Matej, Majdic, Gregor Mariana, Piaggio, Eduardo, Sorrentino, Miguel Angel, Robinson, Veterinary Faculty of Ljubljana, Ljubljana, Slovenia, Sentjur Veterinary Anibal, Mazzolini, Guillermo, Podhajcer, Osvaldo Hospital, Sentjur, Slovenia Fundacion Instituto Leloir Cell Therapy Area, Buenos Aires, Argentina, In recent years there was a fast development in the field of regenerative Universidad Austral, Pilar, Argentina, Hospital Naval Pedro Mallo, Buenos veterinary medicine. Autologous canine adipose tissue-derived mesenchy- Aires, Argentina, Universidad Austral, Buenos Aires, Argentina mal stem cell (cAD-MSC) therapy has been introduced as a therapy for Mesenchymal stromal Cells (MSCs) are a heterogeneous population with treatment of osteoarthritis, bone fractures and ligament damage in dogs. the ability to migrate to tissues under remodelation, including tumors. The Adipose tissue is especially suitable as a source of stem cells as it is very heterogeneity of MSCs preparations may undermine potential clinical ap- rich in mesenchymal stem cells, the harvesting of adipose tissue is not very plications since not all the isolated cells may be responsive to the chemot- demanding and procedure for patient is minimally invasive. The aim of our actic and homing signals from the tissue under remodelation. Even more, work is to isolate and identify mesenchymal stem cells from canine adipose chemotactic signals from different tissues may compete, thus reducing the tissue. The tissue was collected from subcutaneous withers region of dogs, amount of MSCs arriving into a site of clinical interest. We isolated adult using standard surgical procedure. After digestion of collected adipose tis- MSCs from voluntary donors for allogeneic transplants and selected a popu- sue, cells were cultured in a special DMEM based medium. Cells readily grew lation of MSCs with enhanced chemotactic and adhesive responses. These to confluency. Interestingly, among fibroblasts like cells we also observed properties included a higher migration response towards various chemotactic colonies of small round cells that morphologically resemble embryonic stimuli including conditioned medium from tumor cell lines, as well as a stem cells. Identification of these cells is currently underway using immu- stronger adhesive response towards extracellular matrix components and nocytochemistry and quantitative RT PCR to detect expression of stemness human endothelium. These biological properties may be predictive of an markers such as Nanog, Oct4 and others. We are also trying to induce chon- in vivo enhanced ability to reach a tumor. In vivo migration assays showed drogenesis in these cells using previously published methods. The results of that this MSC population showed an enhanced ability to reach a tumor mass our study show that mixed population of dispersed cells derived from dog in a mouse orthotopic tumor model. Thus, the heterogeneity of MSCs could adipose tissue could be successfully cultured and unlike in human adipose be an advantage that allows for selection of a population with the desired derived cells, cells resembling embryonic stem cells are commonly growing biological properties. We were able to identify a MSC population with under suitable conditions. enhanced chemotactic and adhesive responses that may be useful for tumor targeting strategies. Poster Board Number: 2441 MESENCHYMAL STEM CELL DIFFERENTIATION OPTIMIZING GFP TRANSFECTION EFFICIENCY Poster Board Number: 2445 OF WHARTON’S JELLY- MESENCHYMAL HUMAN UMBILICAL CORD-DERIVED STROMAL CELLS MESENCHYMAL STROMAL CELLS AS A Lopez Rodriguez, Yelica V., Trevino, Elizabeth, Weiss, Mark, SOURCE OF TREATMENT FOR NEUROPATHIC Detamore, Michael, Mellott, AJ PAIN AFTER SPINAL CORD INJURY Kansas State University, Manhattan, KS, USA, Kansas University, Takikawa, Sachiko, Yamamoto, Akihito, Sakai, Kiyoshi, Shohara, Manhattan, KS, USA Ryutaro, Matsubara, Hiroki, Fujio, Masato, Ikeno, Masayuki, Mesenchymal stromal cells (MSCs) are an attractive therapeutic resource Yamagata, Mari, Ando, Yuji, Iwase, Akira, Kikkawa, Fumitaka, due to their regenerative and inmunoregulatory capacities. For preclinical Ueda, Minoru testing, it would be important to determine biodistribution of the MSCs. Obstetrics and Gynecology, Nagoya University, Nagoya, Japan, Oral and Many laboratories use fluorescent reporter gene expression in order to Maxillofacial Surgery, Nagoya University, Nagoya, Japan follow the distribution of these cells in vivo, in situ and in real time. Previ- ous work revealed very low transfection efficiency in MSC. Therefore, we Purpose: Spinal cord injury (SCI) results in a severe impairment of motor proposed to develop optimized protocols that yield a high-efficiency and and sensory function below the level of the lesion. Recent studies have stable transfection. Green fluorescent protein (GFP) is an important reporter demonstrated that transplantation of stem cells in experimental SCI models gene and widely used for these studies, gene transfer and gene expression.

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would support functional recovery. The cellular sources of those stem cells human bone marrow MSCs were expanded and used for neural induction. are human embryonic stem cells, induced pluripotent stem cells, fetal neural Neural induction was performed by the method described recently. Briefly, stem cells and adult mesenchymal stem cells from bone marrow. For clini- hMSCs were grown on poly-ornithine and laminin coated plates or on cal use, however, there are significant issues including ethics, safety and Corning Synthemax Surface, synthetic xeno-free replacement for biologi- invasive procedure for stem cell isolation. Thus an ideal stem cell source for cal coatings, in the medium of NeuroCult/N2 supplemented with 200nM SCI treatment is still an elusive subject. This study investigates the potential trichostatin A (TSA) (inhibitor of histone deacetylases), 3uM RG-108 (DNA of human umbilical cord-derived mesenchymal stromal cells (hUCMSCs) in methyltransferase inhibitor), 300uM 8-BrcAMP (highly stable, biologically the treatment of SCI. Materials and Methods: We obtained umbilical cords active form of cAMP), 1uM Rolipram (inhibitor of phosphodiesterases), and upon Caesarian sections with permission of the ethical committee. The 20ng bFGF. This treatment induced gradual neural changes in morphology stroma of umbilical cords, the Wharton’s jelly, was treated with collagenase and the gene expression profile. After two weeks of treatment cells were for 6 hours, then centrifuged and incubated. We examined expressions of used for transplantation. The NYU Impactor was used to produce a con- neural cell lineages markers in hUCMSCs by FACS and quantitative RT-PCR. sistent, uniformly moderate injury. One week after spinal cord injury, adult Immediately after the transection of the spinal cord at the 9-10th thoracic female Sprague Dawley rats were randomized to receive NI-hMSCs, hMSCs vertebral levels of SD rats, total 1x106 hUCMSCs were transplanted into or PBS. Ten animals from each group were for behavioral studies and ten the rostral and caudal stumps. Eight weeks after transplantation, we evalu- for stereological and immunohistochemical studies. Locomotor function was ated motor and sensory functions of the lower extremities. Furthermore, evaluated using the BBB Locomotor Recovery Scale for open-field walking. histological analysis was performed to investigate the role of hUCMSCs in The response to thermal stimulation was measured by latency of forelimb the recovery of SCI. Results: hUCMSCs showed adipogenic, osteogenic and and hindlimb paw withdrawal to radiant heat of 55°C. For immunohis- chondrogenic differentiation. Over 90% of hUCMSCs expressed cell mark- tochemistry and histological assessment after 24h and 1, 2, 4, and 12 weeks ers of neural stem and progenitor cells. The same population also secreted of post-injury animals were perfused intracardially with 0.9% PBS followed GABA, and increased gene expressions associated with GABA production by 4% paraformaldehyde in PBS. Spinal cords were dissected, post-fixed in (Mash1, Helt, and Ptf1a), compared with bone marrow-derived mesen- 4% paraformaldehyde/PBS at 4°C for 1 h and cryoprotected in 30% su- chymal stem cells. In the suspension culture system, hUCMSCs increased crose/PBS. Cryostat cross-sectioned samples were used for immunostaining expression of transcription factors of neural stem cell lineages (Sox2, and stereological studies. Results demonstrated that transplanted NI-hMSCs NeuroD1, neurogenin1, and neurogenin2). After hUCMSCs transplantation, survived, differentiated and significantly improved locomotor recovery of SCI model rats started movements in 3 joints of the lower limb. They also ISC rats. Transplantation also reduced the volume of cavity and promotes showed improvements in sensory hypersensitivity. Upon histological analysis, white matter sparing as compared to the controls. Findings from this study the frozen tissue sections of exhibited differentiation of engrafted hUCM- prove the importance of neural induction as a necessary preliminary step SCs into neural cells. Additionally, apoptosis of neural cells were reduced in before transplantation of MSCs into injured or diseased CNS. Thus, hMSCs hUCMSCs transplantation group. Conclusion: hUCMSCs possess both the neurally modified by this methodology may provide an alternative source of characteristics of mesenchymal stem cells and neural stem cells, in particular autologous adult stem cells that could be useful for injured or diseased CNS GABAergic neurons. Transplantation of hUCMSCs supports improvement regeneration. of neural function through neural differentiation and suppression of cell apoptosis. Furthermore, transplantation of hUCMSCs acted as GABAergic Poster Board Number: 2449 neurons attenuated hypersenalgesia following SCI. These results suggest ENHANCED DIFFERENTIATION OF HUMAN hUCMSCs as a valuable source to play an important role in widening the possibility of therapeutic applications for SCI. BONE MARROW-DERIVED MESENCHYMAL Poster Board Number: 2447 STEM CELLS INTO FUNCTIONAL NEURAL CELLS BY DTOPV-MODIFIED SURFACES TRANSPLANTED NEURALLY MODIFIED HUMAN Heo, June Seok, You, Jungmok, Ko, Si-Hwan, Kim, Eunkyoung, BONE MARROW DERIVED MESENCHYMAL Kim, Hyun Ok, Kim, Han-Soo STEM CELLS REDUCE THE VOLUME OF Cell Therapy Center, Yonsei University College of Medicine, Seoul, CAVITY, PROMOTE WHITE MATTER SPARING Republic of Korea, Department of Chemical and Biomolecular AND SIGNIFICANTLY IMPROVE LOCOMOTOR Engineering, Yonsei University, Seoul, Republic of Korea, Department of Microbiology, Yonsei University College of Medicine, Seoul, Republic RECOVERY IN SPINAL CORD INJURED RATS of Korea, Department of Chemical and Biomedical Engineering, Yonsei Alexanian, Arshak R., Fehlings, Michael G., Zhang, Zhiying, University, Seoul, Republic of Korea, Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea, Dept Maiman, Dennis J. of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Dept of Neurosurgery, Medical College of Wisconsin, Milwaukee, WI, USA, Republic of Korea Dept of Neurosurgery, University of Toronto, Toronto, ON, Canada Recently, stem cell-based therapy has emerged for use in novel therapeutics During the last two decades an overwhelming amount of basic and pre- for various incurable diseases, including neurodegenerative disorders. For clinical research has been accumulated that demonstrate the therapeutic successful recovery from such neurologic diseases, the most pivotal factor usefulness of MSCs in the treatment of several diseases including neurologi- is directed neural cell differentiation from precursors. It has been reported cal disorders and injuries. Recent studies suggest that neural induction of that mesenchymal stem cells (MSCs), highly abundant and easily accessible the MSC prior to transplantation could provide a significant advantage over adult stem cells, are capable of differentiating into many cell types including naive hMSC in the treatment of several animal models of CNS disorders. neural lineage cells and this ability of MSCs holds considerable clinical prom- Recently, we developed a new method for efficient generation of neural ise. In this present study, we investigated the effect of modified surfaces in cells from human BM-derived MSCs (hMSC). Neural induction was achieved inducing MSCs differentiation into neural cells. MSC-derived neurospheres by exposing cells simultaneously to inhibitors of DNA methylation, histone were successfully generated by culturing them for 7 days on DTOPV with a deacetylation and pharmacological agents that increased cAMP levels. The hydrophobic surface. Subsequently, the cells were induced to differentiate aim of this study was to determine whether transplanted neurally induced into neural cells with neurotrophic factors. Results of RT-PCR analysis and hMSCs (NI-hMSCs) will survive, differentiate, and promote tissue protec- immunostaining demonstrated that these cells expressed markers for neural tion and functional recovery in injured spinal cord (ISC) of rats. To this end, cells. Furthermore, such cells showed that the transition of immature states

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Thursday Poster Abstracts to more mature states with functionality on micro-patterned DTOPV. Taken Poster Board Number: 2453 together, surface modifications using DTOPV supported enhanced neural cell differentiation from MSCs. Our data suggest that surface modifications STAT3 ACTIVITY IS CORRELATED WITH can be an effective method for guided differentiation of MSCs that would THE NEUROGENIC POTENTIAL OF HUMAN improve its therapeutic action in neurological diseases and other incurable diseases. (This research is supported by grant SC-2130 from Stem Cell Re- UMBILICAL CORD-DERIVED MESENCHYMAL search Center of the 21st Century Frontier Research Program funded by the STEM CELLS Ministry of Education, Science and Technology, Republic of Korea.) Perruisseau-Carrier, Claire, Jurga, Marcin, Degoul, Olivier, Forraz, Poster Board Number: 2451 Nico, McGuckin, Colin NEURAL GENE EXPRESION OF HUMAN CTI-Lyon, Lyon, France UMBILICAL CORD BLOOD DERIVED MULTI- Umbilical cord mesenchymal stem cells (UC MSC) open new perspecives for regeneration of injured brain. UC MSC can be easily isolated from umbilical LINEAGE PROGENITOR CELL (MLPC) AFTER cord with good cellular expansion capacities. Their immunomodulating prop- BEEN CULTURED IN NEURAL DIFFERENTIATION erties confer them with an advantage for clinical transplantation compare MEDIA to other stem cells. In addition, UC MSC derived from Wharton’s jelly have been shown to express both mesenchymal (CD90, CD73, CD44, CD105) Long, Xiaoxia, Kletzel, Morris and neuroepithelial lineage markers (Nestin, TrkA, TrkB, NeuroD1, Pax6) Division of Pediatrics/Hematology/Oncology/Transplantation, Children’s that make them good candidates for neuronal progenitors commitment and Memorial Hospital, Chicago, IL, USA, Division of Pediatrics/Hematology/ maturation into functional neurons. Cytokines are major players in the regu- Oncology/Transplantation, Children’s Memorial Hospital, Northwestern lation of cell proliferation, apoptosis, immune response and differentiation University Feinberg School, Chicago, IL, USA that activates the JAK/STAT pathway among others. STAT3, one member of this cascade, mediates the expression of growth-promoting gene products Our previous study showed that when (MLPC™) cell lines (BioE, MN) and plays a role in neuronal development, survival and immunomodula- were cultured with a cytokine neural differentiation media that contained tion. Activation of STAT3 pathway was shown to inhibit neuronal terminal dbcAMP, IBMX, BDNF, hEGF, NGF, FGF-8 and bFGF in NEUROBASAL media differentiation and to promote differentiation of neural precursors toward plus B27 supplement, these cells will differentiated into neural like cells. glial lineage. We found that STAT3, as well as its co-factor CREB, is highly Neural markers, such as Tuj-1, GFAP, Nestin, NeuN and GalC could be de- expressed in UC MSC during expansion in vitro. Moreover, by comparing tected on the cell surface by immunofluorescent staining. Our current study serum-free commercial media versus xeno-serum containing culture condi- is to detect their neural gene expressions by Real-Time-PCR. Material and tions (10% FBS), we revealed that STAT3 is almost exclusively expressed in method: MLPC™ cells were cultured in mesenchymal stromal cell growth the nucleus of the cells in absence of serum (up to 92%) when it remains medium (MSCGM, Cambrex BioScience) until 40-50% were confluent. preferentially in the cytoplasm in presence of serum (60%). This nuclear lo- Cells were then detached by 0.1% EGTA/PBS and transfer to 75cm2 tissue calization was then validated by immunostaining of phosphorylated STAT3, culture flasks that were coated with poly-D-lysine and mouse Laminin-I. suggesting an active form of STAT3. In addition to this, immunostainings Cells were then seeded at 3x104/cm2 with MSCGM. After 24 hours of of the cellular proliferation marker Ki67 revealed a higher proliferation level incubation, the MSCGM media was replaced by our neural differentiation in serum-free than in serum-containing conditions. UC MSC maintenance media (NDM, a combination of dbcAMP, IBMX, BDNF, hEGF, NGF, FGF-8 and proliferation seems to be promoted in serum-free culture by activating and bFGF in NEUROBASAL media plus B27 supplement). NDM media molecular pathways which down regulate their differentiation into neuronal was changed on day 3. Cells were collected on day 6. RNA was extracted lineage. and purified by using QIAGEN RNeasy Plus Mini Kit. High Capacity cDNA Reverse Transcription Kit (Applied Biosystem, CA) was used to get cDNA to Poster Board Number: 2455 use as templates in Real-Time-PCR. The gene expressions of glial cell marker glial fibrillary acidic protein (GFAP), neuronal cell marker tyrosine hydroxy- GLYCOSAMINOGLYCAN-RICH MATRIX lase (TH) and early neural cell marker Nestin were detected on 7500 Real- PRODUCTION AT CHONDROGENIC CULTURE OF Time-PCR System (Applied Biosystem, CA) by using TaqMan® Gene express HUMAN MARROW-DERIVED MESENCHYMAL Assay Kits (Applied Biosystem, CA). A Relative Quantification Study program was used to analyze our data. MLPC™ cells of the same cell line, grew in STEM CELLS TREATED WITH GSK3 INHIBITORS regular MSCGM was used as a reference sample. Human GAPTH was used Baghaban Eslaminejad, Mohamadreza, Karimi, Negar, Shahosseini, as endogenous control. A neuralblastoma cell line NGP was used as positive Maryam control. Results: On day 6, MLPCs in neural differentiation media showed neural cell morphology. Our Real-time-PCR result showed that, compared Stem Cell Department, Royan Institute, Tehran, Islamic Republic of Iran, with MLPCs cultured in regular MSCGM, cells grew in NDM had a 32 times Department of Developmental Biology, University of Science and Culture, increase on the expression of GFAP gene and a 128 times increase on the Tehran, Islamic Republic of Iran, Genetic Department, Royan Institute, expression of TH gene. The expression of Nestin was the same. Conclusion: Tehran, Islamic Republic of Iran After been cultured in our neural differentiation media, Umbilical Cord Blood Background: Cartilage-like tissue produced following mesenchymal stem Derived (MLPC™) cell lines demonstrated an increase gene expression of cell (MSC) in vitro differentiation would be a suitable material for regenera- GFAP and TH. Nestin gene expression does not change, but the protein tion of hyaline cartilage defects in near future hence many attempts have expression can be detected on cell surface after the differentiation. been made to optimize the differentiation conditions toward creating high quality tissue constructs. One important structural component of cartilage is its glycosaminoglycan (GAG) content which imparts to the tissue the unique property of functioning as biochemical spring during weight bearing process. On the other hand there is some evidences that activating wnt signaling pathway in MSC chondrogenic culture is associated with up regulation of some cartilage-specific gene expression. The specific objective of this study was to investigate the effect of Lithium Chloride and SB216763 (two well-

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known GSK3 inhibitors) on GAG production at human marrow-derived MSC onstrated that TSP-2 expressed from hUCB-MSCs have positive correlation chondrogenic culture. Methods: The cells that were used in this study were with chondrogenic potential of hUCB-MSCs. These results suggested that the surplus of marrow MSCs derived from the patients who were volun- TSP-2 could be the marker for selection of hUCB-MSCs with high chondro- teer for resurfacing of articular cartilage at Royan Institute’s cell therapy genic potential in cartilage regeneration. center. Before establishing chondrogenic culture the optimal concentrations of Lithium chloride and SB216763 were determined based on the yield of Poster Board Number: 2459 viable cell number at MSC culture treated with varying concentrations of DECELLULARIZED MATRICES MODULATE either reagent. Passaged-3 MSCs were then centrifuged into small aggrega- tion and provided with routine chondrogenic medium supplemented with HUMAN SYNOVIUM-DERIVED STEM CELL either lithium or SB216763 reagent at a concentration determined in the CHONDROGENESIS previous experiment. Chondrogenic culture without GSK3 inhibitors was taken as control. Three weeks after culture initiation GAG contents of the Li, Jingting, Pei, Ming micro mass cultures were quantified and statistically compared using acidic Orthopaedics, West Virginia University, Morgantown, WV, USA mucopolysaccarid quantification kit. Furthermore at the end of culture pe- Objective: Application of cell-based therapy in cartilage defect repair riod the expression of beta catenin (a key molecule in canonical wnt signal- remains a great challenge due to the shortage of tissue-specific stem cells ing pathway), Sox 9, Aggreacan and Coll II genes were compared using real and their limited regeneration capacities. Our goal was to assess whether time RT-PCR analysis. Results: According to our data, the cultures treated extracellular matrix (ECM) deposited by human synovium-derived stem cells with 5 mM Lithium and 1 µM SB216763 tended to have comparatively (hSDSCs) instead of human adipose stem cells (hASCs) and human dermal more viable cells; therefore these concentrations were used in the differen- fibroblasts (hDFs) can provide a tissue-specific stem cell microenvironment tiation experiments. The addition of either SB216763 or lithium to chondro- to enhance hSDSC proliferation and chondrogenic potential. Methods: genic cultures tended to significantly enhance cartilage matrix production. Passage 3 hSDSCs, hASCs, and hDFs were used to prepare decellularized In this regard at SB216763 and Lithium-treated cultures the average GAG ECMs, referred to as SECM, AECM, and DECM. hSDSCs were expanded on concentrations were 6.17 ±0.7 and 6.12 ±1.1 µg/ml respectively compared the above three ECMs or conventional plastic flasks (Plastic) for one pas- to 2.00 ±0.3 µg/ml at control culture (P<0.05). This effect seemed to be sage. The expanded cells were evaluated for their chondrogenic potential mediated through activation of Wnt pathway since at both treated culture in a pellet culture system with TGF-β3-containing serum-free chondrogenic beta catenin, Sox9, Aggrecan and Coll II genes expression were significantly medium using gross morphology, histology, immunostaining, biochemical upregulated compared to those of the control. Conclusion: Addition of analysis, real-time PCR, and biomechanical testing. ECM expanded hSDSCs either SB216763 or Lithium to human marrow MSC chondrogenic culture were further evaluated for their osteogenic potential using Alizarin Red upregulate canonical wnt signaling beta catenin and cartilage-specific gene S staining and alkaline phosphatase activity assay and for their adipo- expression and enhance GAG production at the culture. genic potential using Oil Red O staining. Results: ECM expanded hSDSCs Poster Board Number: 2457 exhibited an enhanced proliferation capacity and a robust chondrogenic potential compared to those grown on Plastic. Surprisingly, AECM and A MOLECULAR MARKER FOR SELECTION OF DECM pretreated hSDSCs yielded pellets with the largest size, the highest UMBILICAL CORD BLOOD-DERIVED HUMAN cell number and chondrogenic index (GAG/DNA), and the highest level of chondrogenic marker genes (collagen II, aggrecan, and Sox9) compared to MESENCHYMAL STEM CELLS (HUCB-MSCS) in hSDSCs expanded on SECM. Histology (Safranin O staining for sulfated WITH HIGH CHONDROGENIC CAPACITY glycosaminoglycans) and immunostaining data (collagens I, II, and X) were consistent with the above biochemistry and real-time PCR data. Our biome- Jeong, Sang Young, Choi, Soo Jin, Oh, Wonil, Yang, Yoon Sun, chanical testing data showed that hSDSCs expanded on ECMs yielded pel- Jeon, Hong Bae lets with higher stiffness and Young’s Modulus compared to those grown on Biomedical Research Institute, MEDIPOST Co., Ltd, Seoul, Republic of Plastic, despite there being no statistically significant difference. Intriguingly, Korea there was no concomitant enhancement of osteogenesis and adipogenesis of ECM-pretreated hSDSCs. Conclusion: Decellularized matrix can provide Human mesenchymal stem cells (hMSCs) are being recognized as a useful a stem cell microenvironment, enhancing embedded hSDSC expansion and cell source for tissue regeneration in cartilage. Among hMSCs from diverse chondrogenic potential. The mechanism underlying the greatest chondro- tissues, umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) genic potential in hSDSCs expanded on AECM and DECM remains unknown have been little applied in cartilage regeneration. Although therapeutic uses and needs to be further investigated. of hUCB-MSCs in cartilage regeneration are steadily attempting, it is difficult to maintain therapeutic outcome due to donor dependent chondrogenic dif- Poster Board Number: 2461 ferentiation potential of hUCB-MSCs. To overcome this difficulty, it requires a selection of high potent hUCB-MSCs to form cartilaginous tissue. Based HUMAN ADIPOSE-DERIVED STEM CELL on our previous study, we chose thrombospondin-2 (TSP-2) as a candidate CHARACTERIZATION AND COMPARATIVE GENE for marker protein associated with chondrogenic differentiation potential of hUCB-MSCs. To verify the correlation between TSP-2 expression and chon- EXPRESSION PROFILING drogenic potential of hUCB-MSCs, degree of chondrogenic differentiation Boucher, Shayne E., Dadi, Srividya, Chen, Caifu, Sherlock, Jon, Au- and TSP-2 expression level of eight hUCB-MSCs originated from different Young, Janice individual were compared to each other by safranin-O staining and enzyme- linked immunosorbent assay (ELISA). As a consequence, TSP-2 expression Primary & Stem Cell Systems, Life Technologies, Frederick, MD, USA, had increased in highly differentiated group, not in lower group during Genomic Assays, Life Technologies, Foster City, CA, USA chondrogenic differentiation. These results show that TSP-2 expression posi- We have developed a rapid qPCR-based characterization system for profiling tively correlates with chondrogenic differentiation of hUCB-MSCs. Further- gene expression pattern in cultured adipose-derived stem cells (ADSCs). more, the raise of TSP-2 in hUCB-MSCs enlarges size of chondrogenic pellet Similar to bone marrow-derived mesenchymal stem cells (MSCs), ADSCs and increases expression of chondrogenic markers including type II collagen, can differentiate into adipocytes, osteoblasts or chondrocytes. Using ADSCs aggrecan and sox-9. On the other hand, suppression of TSP-2 expression isolated from excess human adipose tissue, we expanded these cells and dif- using small interfering RNA (siRNA) results in down-sizing of chondrogenic ferentiated them into multiple cell types under optimized culture conditions. pellet and decreased expression of chondrogenic markers. Overall, we dem-

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Expressed transcripts specific for adipocytes, osteoblasts and chondrocytes Poster Board Number: 2465 were detected in distinct lineages using select TaqMan qPCR targets. The differential expression of targets important in early, mid- and late adipocyte- FLUORESCENCE MICROSCOPY TO ASSESS differentiation (FABP4, HP, ADIPOQ, CD36, LEP and LPL) were detected in DIFFERENTIATION OF HUMAN ADIPOSE- adipocyte lineage. Increased expression of adipocyte markers was correlated with a relative decrease in osteocyte-specific markers BGLAP, BMP2 and DERIVED STEM CELLS AND SKELETAL MUSCLE COL1A1 that are likely due to signaling induced by adipogenesis differentia- PRECURSORS TOWARDS BROWN ADIPOCYTES tion media. In parallel, expression of osteogenic and chondrogenic markers were observed with a decrease in adipocyte marker expression in osteoblasts Drowley, Lauren, O’Shea, Patrick, Peng, Xiao-Rong, Sullivan, Mike and immature chondrocytes. The ability to track and modulate expression AstraZeneca, Loughborough, United Kingdom, AstraZeneca, Gothenburg, of MSC-related differentiation genes has the potential for understanding Sweden mechanisms important in MSC biology and disorders. Introduction: Due to the increasing epidemic of obesity, there has been Poster Board Number: 2463 significant interest in adipose tissue. There are two types of fat cells within adipose tissue, each with distinct properties. White fat cells store energy CD10 POSITIVE CELLS FROM HUMAN whereas brown fat cells use energy in a process known as thermogenesis. ADIPOSE TISSUE AS A POTENTIAL SOURCE OF Thermogenesis and brown fat play an essential role in maintaining tissue ho- meostasis. Uncoupling protein 1 (UCP-1) is expressed in the mitochondrial LUMINAL MAMMARY EPITHELIAL CELLS membranes of brown fat and UCP-1 is critical in non-shivering thermogen- Chapellier, Marion, Bachelard-Cascales, Elodie, Pochon, Gaetan, esis and provides a major means of heat generation. Brown adipose tissue is more prevalent in infants, though recently studies have shown brown fat Delay, Emmanuel, Maguer-Satta, Veronique depots in adults, mainly located in the interscapular region. There are two Cancer Research Center of Lyon-CRCL, Inserm U1052-CNRS5286, Lyon, different types of brown fat cells which appear to arise from different de- France velopmental origins. One subset of brown fat is derived from an adipogenic The mammary gland is composed of two cell types, luminal and myoepi- precursor and is responsive to β-adrenergic stimuli. The other type of cell thelial cells, derived from a common progenitor. It is surrounded by an arises from a myf5+ myogenic precursor. These cells appear to be dependent adipose micro-environment that regulates mammary glands development on the transcriptional regulator PRDM16. Methods: Adipose-derived stem and differentiation by cell-contact and soluble factors. Furthermore, the cells (ADSCs) were obtained and expanded according to manufacturer’s adipose tissue is partly composed of multipotent cells that have been largely instructions. Primary myoblasts were isolated according to a modified proto- described for their ability to differentiate towards several lineages such as col. Briefly, the muscle is minced and digested with collagenase and trypsin neural, cardiac, mesenchymal, muscles, and bones. A study has showed the and plated in skeletal muscle growth media in gelatin-coated flasks. After ability of adipose immature cells to express the epithelial marker keratin18 in passage, the cells are assessed for expression of CD56, a marker of myogen- specific culture condition and the transdifferentiation of adult adipose cells ic lineage commitment. For differentiation, ADSCs or myoblasts were plated into milk secreting luminal cells has been demonstrated in pregnant mice. in 96-well plates and grown until confluence. They were then maintained In this context, we wonder if immature cells contained in human adipose in differentiation media in the presence or absence of the thiazolidinedione tissue might be able to differentiate into mammary epithelial cells. As we PPARγ-agonist pioglitazone. Throughout the differentiation protocol, media previously described the CD10 cell surface antigen as an easy-to-use tool to and compounds were replenished every 2-3 days. Fluorescence microscopy enrich sphere-forming cells, we used this property to isolate immature cells was used to assess differentiation after 1, 2, and 3 weeks. The cells were from normal mammary gland and from adipose tissue obtained from lipo- stained with adiponectin, a general adipose marker, and UCP-1, a marker aspirates. We then compared sorted CD10+ cells from different tissue origin found specifically in brown adipose tissue, with cell nuclei counterstained on their phenotypic, molecular and functional properties. Sorted CD10+ cells with Hoescht 33342. Results: In ADSCs, we demonstrated a significant in- from adipose and mammary gland present some common properties like crease in UCP-1 expression in the presence of pioglitazone compared to cells enrichment in sphere-forming cells and the expression of genes considered treated with differentiation media alone. In contrast, ADSCs not exposed to as stem cells markers. However, our results also showed that CD10+ cells differentiation media showed no increase in UCP-1 expression. In skeletal populations from these different tissues displayed distinct phenotypic and myoblasts, pioglitazone also induced expression of UCP-1, though not to molecular profiles on differentiation markers of mammary progenitors like the same levels as in ADSCs. Discussion: Increasing differentiation towards the integrins CD29 and CD49f or the myoeptihelial markers DeltaNp63 and brown fat is a potential target of therapeutic treatments to decrease obesity Smooth Muscle Actin. Following cell sorting, while mammary CD10+ cells and metabolic diseases. However, as it has recently been shown that brown generate luminal epithelial colonies, adipose CD10+ cells do not generate fat can arise from two distinct developmental origins, it is important to any sort of epithelial colonies. Despite this, when adipose CD10+ cells are examine the differences between the cell populations in their differentiation grown as spheres in a medium previously conditioned by mammary CD10+ capacity and function. cells, we observed by qPCR an induced expression of the epithelial keratin Poster Board Number: 2467 18 and the Claudine 4, recognized as luminal markers. These results sug- gest that sphere-conditioned media allows adipose CD10+ cells to engage MICROARRAY ANALYSIS OF IN-VITRO toward epithelial lineage. We are currently testing a number of mammary soluble factors including the environment estrogens for their ability to fully OSTEOGENESIS IN HUMAN ADIPOSE DERIVED commit these primed cells toward mammary luminal progenitors able to MESENCHYMAL STEM CELLS generate luminal colonies. Taking in consideration our own results on mam- Elcin, A. Eser, Ozdag, Hilal, Ibsirlioglu, Tulin, Elcin, Y. Murat mary stem cells regulation and the work of others, we suggest that CD10+ cells from adipose tissue might be able to differentiate and express markers Ankara University Stem Cell Institute, Biotechnology Institute and Faculty specific of the luminal mammary epithelium when placed in an appropriate of Science, TEBNL; Gazi University, GEF, Biology Division, Ankara, Turkey environment. In this context, the adipose tissue appears not only as a physi- Adipose tissue contains a rich source of mesenchymal stem cells (AMSCs) cal support for the mammary gland but might also constitute a stem cell with the ability to differentiate into adipogenic, myogenic, chondrogenic, reservoir for this tissue. and osteogenic lineages, as a matter of fact presenting therapeutic potential for tissue engineering and cellular therapies. Here, we evaluated the expres- sion profile of the human transcriptome during in-vitro osteogenesis of

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human (h) AMSCs, with focus on osteogenic differentiation at Day 14 and BMP4. The SMC phenotype of adipose-derived cultures was found to be Day 28. Methods: Abdominal adipose tissue of adult patients was collected quite similar to that of hUC-SMCs. Conclusion: Findings support the notion from elective operations under ethical approval. hAMSCs were isolated from that, human adipose mesenchymal stem cells can be differentiated into vas- the adipose tissue and expanded. The phenotypical marker profile of hAM- cular smooth muscle-like cells. Ongoing work is being performed to further SCs was determined using flow cytometry and immunofluorescent analysis, delineate the pathway in which AMSCs commit to functional SMCs, under histo/immunohistochemistry and characteristic growth in culture. These cells flow conditions mimicking the hemodynamics. were cultured either in DMEM:F12 supplemented with 10% FBS (EM), or in EM supplemented with osteogenic inducers, namely ascorbic acid, sodium Poster Board Number: 2471 β-glycerophosphate and dexamethasone (OM). Expression profile of the COMPARISON OF ADIPOGENIC human transcriptome was evaluated by using Affymetrix HG-U133 Plus2 microarrays. After the determination of gene expression profiles, selected DIFFERENTIATION OF HUMAN MESENCHYMAL differentially expressed genes were confirmed by quantitative reverse STEM CELL LINES FROM DIFFERENT TISSUES transcription-PCR. Results: AMSCs isolated from human subcutaneous fat were expanded and characterized by their CD90 (%98.4), CD73 (%91.9), OF ORIGIN CD105 (%96), CD29 (%95.4), HLA-ABC (%89.9), CD44 (%98.3), CD34 Hasskamp, Joanne H., Haskins, Jeffrey R. (%5.9), CD45 (%5.6), HLA-DR (%4.9), CD133 (%2.6), CD3 (%3.0), CD31 Thermo Fisher Scientific, Pittsburgh, PA, USA (%4.0) and CD146 (%6.0) immunophenotype and tri-lineage differentiation potential. hAMSCs cultured under osteogenic medium conditions synthe- Introduction: Tissue of origin of human mesenchymal stem cells (hMSC) sized bone-like organic matrix, expressed an osteogenic phenotype based on influences differentiation efficiency. Epigenetic memory is thought to explain Alizarin red and von Kossa stainings, especially after 28 days of osteoinduc- at least in part the observed differentiation differences between hMSC tion. Findings from gene expression profile analysis indicated the upregula- isolated from different tissues. Adipose, amniotic and bone marrow hMSC tion (≥four-fold) of gene transcripts, largely involved in skeletal tissues and cell lines are commercially available for research purposes. A comparison of bone growth, but some others with bone inhibitory or other functions, adipogenic differentiation of hMSC from different tissues of origin using Hy- namely TGF-β1, TGF-β2, ALPL, RUNX2, MGP, SOX9, COL1, and POSTN. clone differentiation media was performed and assessed using a quantitative Conclusion: We have described the transcriptional profile of hAMSCs imaging system. Results illustrate tissue of origin differences and exemplify osteogenesis under osteogenic culture conditions. Findings may facilitate a the potential benefit of selecting tissue of origin based on the intended use broader understanding of the osteogenic commitment of the adipose MSCs. of the hMSC. Materials and Methods: Human adipose, amniotic and bone marrow mes- Poster Board Number: 2469 enchymal stem cells were purchased from Thermo Scientific and cultured using the Hyclone AdvanceSTEM mesenchymal stem cell expansion kit DIFFERENTIATION OF HUMAN ADIPOSE (Thermo Scientific). Cells were harvested with 0.25% trypsin, manually MESENCHYMAL STEM CELLS INTO SMOOTH counted in a hemacytometer and seeded at 10,000 viable cells per well of MUSCLE-LIKE CELLS: A POTENTIAL CELL 96 well plates. Hyclone AdvanceSTEM adipogenic differentiation media was used to differentiate hMSC cell lines from the three tissues of origin above. SOURCE FOR VASCULAR TISSUE ENGINEERING Control wells were maintained in mesenchymal stem cell expansion media. Arslan, Y. Emre, Elcin, A. Eser, Elcin, Y. Murat Differentiations continued for 2 to 4 weeks, feeding the cultures 2 to 3 times per week by replacing the majority of the spent medium. Adipogenic differ- Ankara University Stem Cell Institute and Faculty of Science, TEBNL; Gazi entiation was assessed using Oil Red O staining or immunostaining for fatty University, GEF, Biology Division, Ankara, Turkey acid binding protein 4 (FABP4). Images acquired on the ArrayScan® VTI In recent years, adipose-derived mesenchymal stem cells (AMSCs) have imaging system were analyzed for relative fluorescence intensity of FABP4 drawn attention as an alternative potential cell source to bone marrow in the cytoplasm. Results: The adipose hMSC cell line differentiated into MSCs, for tissue engineering and regenerative medicine applications. adipocytes most readily, exhibiting visible lipid droplets and relative FABP4 While the differentiation of AMSCs into smooth muscle-like cells (SMCs) fluorescence intensity that was at least five-fold above background and two- has previously been reported, there exists a need for the optimization of fold that of bone marrow hMSC. The bone marrow hMSC cell line showed the induction conditions. Methods: hAMSCs were isolated from human li- a lesser extent of adipogenic differentiation in the same time frame, tending poaspirates collected under ethical approval; then cultured and characterized to have FABP4 fluorescence intensity at least two-fold above background. immunophenotypically by FACS, as well as by their tri-lineage differentiation The amniotic hMSC cell line showed little to no adipocyte phenotype after potential. Human umbilical cord artery smooth muscle cells (hUC-SMCs) the 4 weeks of differentiation with the Hyclone adipogenic media. Conclu- were isolated at our laboratory and used as positive control. Cells were sion: hMSC cell lines differentiated with adipogenic differentiation media expanded and the confluent cultures between passages 2-5 were used. show differences in differentiation efficiency according to tissue of origin. Different combinations and induction periods of the signalling molecules The comparison of adipogenic differentiation of adipose, amniotic and bone were employed to induce differentiation of AMSCs into SMC-like cells; i.e. marrow hMSC cell lines showed most efficient adipogenic differentiation transforming growth factor-beta1 (TGFβ1), bone morphogenetic protein 4 using adipose hMSC. (BMP4), angiotensin II (AngII), TGFβ1/BMP4, TGFβ1/Ang II, and BMP4/ Ang II were applied for a period 7 and 14 days. Differentiation of AMSCs was evaluated by immunocytochemistry using antibodies against five SMC proteins, and by quantitative RT-PCR. Results: Human AMSCs had CD 90+, CD105+, CD73+, CD29+, CD44+, CD34-, CD45-, and CD133- immuno- phenotype. Results demonstrated varying levels of differentiation towards the SMC phenotype, with regard to employed growth factor combinations and induction periods. Light microscopy revealed that differentiated SM-like cells highly expressed alpha smooth muscle actin (α-SMA), but lesser levels of myosin heavy chain (MY-HC), calponin (CAL), caldesmon L (CADL), caldesmon H (CADH) at Day 7. Over-all, the expression of SMC-specific proteins significantly increased after 14 days, in all induction groups. How- ever, the most effective induction combination was found to be the TGFβ1/

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Thursday Poster Abstracts

Poster Board Number: 2473 Poster Board Number: 2475 HUMAN ADIPOSE TISSUE-DERIVED SSEA-4 NON-ADHERENT MESENCHYMAL SUBPOPULATION CAN BE DIFFERENTIATED PROGENITORS ARE PRESENT IN THE STROMAL TOWARDS THE ENDOTHELIAL AND VASCULAR FRACTION OF FRESHLY ISOLATED OSTEOGENIC LINEAGES HUMAN ADIPOSE TISSUE AND ARE ABLE Mihaila, Silvia M., Frias, Ana M., Pirraco, Rogério P., Rada, TO SELF-RENEW IN SUSPENSION WHEN Tommaso, Reis, Rui L., Gomes, Manuela E., Marques, Alexandra P. CULTURED ON THEIR NICHE 3B’s Research Group Biomaterials, Biodegradables and Biomimetics, Mehrkens, Arne, Di Maggio, Nunzia, Gueven, Sinan, Scherberich, University of Minho, Caldas das Taipas, Guimaraes, Portugal Arnaud, Banfi, Andrea, Martin, Ivan Promoting functional vascularization of bone engineered constructs is Orthopaedic Department, Basel University Hospital, Basel, Switzerland, critical for supporting cell organization and for guaranteeing its survival by ICFS, Basel University Hospital, Basel, Switzerland providing an efficient nutrient and oxygen supply. Human adipose tissue might consists in a promising cell poll for tissue engineering applications as it Introduction: Recently, the presence of multipotent mesenchymal cells in the can be harvested in large amounts with low site morbidity and because the non-adherent fraction of human bone marrow (BM) cultures has been re- developmental plasticity of adipose derived stem cells (hASCs) was already ported. Mesenchymal progenitors cells have also been found in the stromal proven both in vitro and in vivo. Additionally, hASCs comprise different cell vascular fraction (SVF) of adipose tissue and in a variety of other tissues. subpopulations with distinct differentiation potential Since hASCs express This led to the hypothesis that multipotent mesenchymal progenitors are SSEA-4, an early stem cell marker and considering the pluripotency character present in all tissues of the body, representing a common reservoir of regen- of the SSEA-4+cells, it was hypothesized that the SSEA-4+hASCs subpopu- erative cells. We have shown that non-adherent progenitors of BM stroma lation exhibited features relevant for bone tissue engineering applications, (BM-NAMP) represent the most primitive compartment of MSC, capable of demonstrated by its differentiation potential towards the endothelial and os- self-renewing without loss of proliferation and differentiation capacity when teogenic lineages. SSEA-4+hASCs were isolated using an immunomagnetic cultured with the initially adhering BM fraction. Based on these results, we sorting technique and were cultured either in basal medium, in EGM-2 MV sought to determine whether the NAMP class of progenitors can also be (endothelial growth medium) or osteogenic medium. Cells cultured in EGM- found in the SVF of adipose tissue (AT-NAMP). Methods: Adipose tissue was 2 MV formed endothelial-like colonies characterized by a typical endothelial obtained from 12 healthy donors (21-69 years old). All experiments com- cobblestone morphology and expressing endothelial markers (CD31, CD34, menced with a ,,Plate0“: Nucleated cells of freshly isolated human adipose CD105, von Willebrand factor) as confirmed by real time RT-PCR, immu- tissue were plated at clonal density (9 cells/cm2) at standard culture condi- nocytochemistry and flow cytometry. Moreover, the endothelial character tions: a-MEM, 10% serum, 5ng FGF-2/ml. A) Cells were plated (Plate0). Af- of these cells was also confirmed by their ability to incorporate acetylated ter 3 days the non-adherent fraction was resuspended in fresh medium and low-density lipoprotein and to form of capillary-like structures when seeded replated in a new dish (Plate1). B) To investigate whether AT-NAMP could on Matrigel, demonstrating their in vitro functional competence. In addi- regenerate themselves as non-adherent progenitors, serial replating experi- tion, SSEA-4+hASCs maintained consistent endothelial phenotype along ments were performed. Cells were plated (Plate0). After 3 days the non-ad- passages, when cultured in EGM-2 MV. Cells with these characteristics were herent fraction was replated in a new dish (Plate1). At day 7, the non-ad- not observed in the adipose tissue stromal vascular fraction cultured under herent fraction was replated in a new dish (Plate2) and so on until Plate4 at the same conditions. SSEA-4+hASCs cultured in basal medium displayed a fi- day14. Colonies were fixed 14 days after plating, respectively. C) To deter- broblastic-like morphology and exhibited a mesenchymal phenotype (>90% mine whether the AT-NAMP could proliferate if cultured with the initially ad- CD90+, CD73+ and CD105+), which shifted to osteogenic when these cells hering SVF, cells were plated and the non-adherent fraction was resuspend- were cultured in osteogenic differentiation medium. The expression of os- ed in fresh medium and replated into Plate0 at each medium change. After teogenic-related markers (osteopontin and osteocalcin) was observed both 7 or 14 days, the non-adherent fraction was plated into a new dish and the at molecular level and protein levels, and matrix mineralization was detected colonies were fixed after 14 days. Results: A) At 14 days, Plate1 contained by Alizarin Red staining confirming the successful osteogenic differentiation 21.3±7.5% (n=8) of the colonies present in Plate 0. B) Colonies decreased in of the SSEA-4+ hASCs. In summary, it was established a successful protocol number as compared to the initial CFU-f (Plate0=100%, Plate1=16.0±9.0%, for the isolation and differentiation of SSEA-4+hASCs towards the endothe- Plate2=8.4±9.7%, Plate3=1.6±2.9%, Plate4=0.1±0.3%, n=1) but increased lial and osteogenic lineages. This work demonstrated that from a single in size (Plate0=4.3±0.5mm, Plate1=6.1±0. 7mm, Plate 2=5.9±1.4mm, cell source and by selecting the appropriate subpopulation, it is possible to Plate3=5.9±0.8mm, Plate4=9.5±0.7mm). C) When kept in the original dish obtain relevant types of cells envisioning tissue engineering applications to for 7 (Plate2*) or 14 days (Plate4*), AT-NAMP generated a higher number obtain a vascularized bone tissue substitutes. of colonies as compared to when they underwent a serial replating for the same time (Plate2=12.8±13.2%, Plate2*=56.3±46.5%; Plate4=0.1±0.3%, Plate4*=129.1±66.0%, n=4, p<0.01). There was no significant decrease in the size of the colonies (Plate0=4.3±0.8mm, Plate2*=5.4±1.1mm, Plate4*=6.1±1.8 mm, n=4, p=n.s). Discussion: The results show that a population of non-adherent mesenchymal progenitors is also present in hu- man adipose tissue SVF cultures. Similarly to BM-NAMP, AT-NAMP did not simply display delayed adherence, but were stably non-adherent. When kept in contact with the initially forming niche, AT-NAMP could expand in sus- pension while preserving their proliferation capacity, suggesting they were able to undergo self-renewing divisions in these conditions. In conclusion, the presence of NAMP may be a property of the mesenchymal progenitor compartment of different tissues and not specific to BM stroma alone.

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Poster Board Number: 2477 corneal/limbal injury in vivo. Material and Methods: Eight-week-old male BALB/c mouse cornea was covered by filtering paper with 0.5N NaOH for SYSTEMIC HUMAN ORBITAL FAT-DERIVED 30 seconds, and then, corneal as well as limbal epithelium was removed STEM CELL TRANSPLANTATION AMELIORATES by surgical knife. One drop with 200,000 OFSCs in 5 ul PBS or 5 ul of PBS was treated twice a day. Image of corneal clearance and corneal wound MACROPHAGE-MEDIATED ACUTE stained with fluorescein strip were recorded every day. Mice were sacrificed INFLAMMATION IN LIPOPOLYSACCHARIDE- for histopathological exam, immunohistochemistry staining, immunofluo- INDUCED ACUTE LUNG INJURY rescence staining, and western blot analysis at the end of third day after corneal injury. Results: Compared with PBS treatment, topical treatment with Ho, Jennifer Hui-Chun, Chien, Ming-Hsien, Ku, Chia-Chi, Chang, OFSCs promoted corneal re-epithelialization in vivo evidenced by signifi- Yun-Chuang, Pao, Hsiang-Yin, Chen, Chi-Long, Bien, Mauo-Ying cantly decreased corneal opacity and would area. Pathological examination Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan, Taipei demonstrated that OFSCs ameliorated acute alkali injury on cornea including Medical University, Taipei, Taiwan diminishing corneal edema and decreasing stromal infiltration. In OFSCs treated cornea, human β2-microglobulin or human IgG expressing cells Introduction: Acute lung injury (ALI) results in acute respiratory distress could be detected on mouse corneal epithelium, especially at limbal area syndrome (ARDS). Facilitating tissue repair without fibrotic change as and central basal layer. Some of transplanted cells expressed cytokeratin-3 well as attenuating local inflammatory reaction is the goal in a successful illustrating their corneal epithelial differentiation. Conclusions: Topical OFSCs treatment of ALI/ARDS. Except supportive care, cell-based therapy has a treatment was effective at promoting corneal wound healing. The therapeu- great potential in treating ALI. The aim of this study is to investigate the tic effect was achieved by both direct differentiation into corneal epithelial effects and the mechanisms of systemic human orbital fat-derived stem cells cells and inflammation inhibition by OFSCs. (OFSCs) transplantation on lipopolysaccharide (LPS)-induced ALI. Material and Methods: Twenty-five μg LPS in 50 μl sterile saline or 50 μl of sterile Poster Board Number: 2481 saline was delivered into 8-week-old male BALB/c mice via intra-tracheal injection. Twenty minutes later, animals were further randomized into CONTROLLING HUMAN ADULT STEM CELL subgroups receiving tail vein injection of 3×105 OFSCs (or 5-chloromethyl- FATE BY COMBINATORIAL BIOPHYSICAL fluorescein diacetate-labeled OFSCs) in 50 μl of phosphate buffer solution STIMULI (PBS) or 50 μl of PBS. Three days after intra-tracheal injection, animals were sacrificed and lung tissues were examined by histopathological, immunohis- Choi, Yu Suk, Vincent, Ludovic, Engler, Adam tochemical or immunofluorescence staining and Western blot analysis. Blood Bioengineering, University of California, San Diego, La Jolla, CA, USA samples were collected and measured by flow cytometry, ELISA, and pro- inflammatory cytokine array. Results: Systemic OFSCs transplantation did Stem cell differentiation is regulated by many local niche cues, recently not trigger immune response in BALB/c mice. OFSCs significantly reduced including extracellular matrix properties, which control how individual cells LPS-induced pulmonary inflammation evidenced by decrease in neutrophil spread and ‘feel’ their surroundings. It is not clear how these stimuli signal and macrophage (CD68 expression cells) infiltration, inhibition of CD14, to stem cells collectively as occurs in vivo. Here we demonstrate how these properties regulate the fate of human adult stem cell clusters versus isolated inducible nitric oxide synthase (iNOS), and transforming growth factor-β cells of varying shape. Microwells were fabricated from silicone mold with (TGF-β) protein expression. Besides, OFSCs reduced the circulation level of macrophage (CD11b expression cells) as well as macrophage-released 50 - 80 μm tall features made by photolithography. Using microwells to pro-inflammatory chemokines- B-lymphocyte chemoattractant (BLC) and achieve single cell shape control, clusters of bone marrow-derived stem cells interleukin-12 (IL-12), and subsequent decreased circulation level of helper (BMSCs) or adipose-derived stem cells (ASCs) were cultured having defined T cell (CD4 expression cells) triggered by LPS. Moreover, few human OFSCs cell-cell junction points. BMSCs or ASCs recognized, stretched, or migrated could be detectable in the recipient lung after acute inflammation subsided. towards each other to make cell-to-cell contacts in fibronectin or type I Conclusions: Systemic OFSCs transplantation was effective at modulating collagen coated microwells with stiffness ranging between soft (0.1 kPa) the inflammation during acute lung injury. The therapeutic effect was attrib- and rigid (34 kPa). Cell-cell junction components were found to regulate uted to inhibit macrophage-mediated inflammatory response. the transmission of mechanical tension from one cell to influence differen- tiation of another cell when cultured as doublets or multi-cell clusters (1:1, Poster Board Number: 2479 1:2, amd 1:4 ratio) in the presence of other differentiation niche cues, e.g. appropriate muscle stiffness or growth factors. By blocking specific cadherins HUMAN ORBITAL FAT-DERIVED STEM CELLS and pharmacologically inhibiting BMSC or ASC tension, underlying cell-cell PROMOTE CORNEAL RE-EPITHELIALIZATION and cell-matrix sensing mechanisms indicate hierarchical signaling patterns. These studies demonstrate that niche-regulated changes in cell shape and IN VIVO matrix stiffness for one stem cell can influence adjacent cells in a manner Ho, Jennifer H., Pao, Hsiang-Yin, Chang, Yun-Chuang, Cheng, not previously appreciated in single cell analyses or those that employ only Chieh-Feng growth factors. Ophthalmology, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan, Center for Stem Cell Research, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan Introduction: Extensive loss of limbal epithelial cells results in recurrent corneal ulceration and corneal opacity. Replacement of stem cells by limbal transplantation is the only way for ocular surface reconstruction. However, destruction of the healthy fellow limbus tissue is inevitable while allogenic limbal graft for bilateral diseases is restricted by tissue rejection and unex- pected inflammation. We have demonstrated that human orbital fat-derived stem cells (OFSCs) possess the corneal epithelial cell differentiation ability upon mix-cultured with human corneal epithelial cells. The aim of this study is to investigate the therapeutic effect and mechanisms of OFSCs on acute

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Thursday Poster Abstracts

Poster Board Number: 2483 by CFU-F assays. Our data revealed a two-fold higher CFU enrichment in the CD271brightSSEA-3-CD56+ subset compared to the CD271brightSSEA- IN VITRO POTENTIAL OF HUMAN BONE 3+CD56- population. Analysis of the differentiation capacity revealed that MARROW STROMAL CELLS TO DIFFERENTIATE CD271brightSSEA-3+CD56- MSC gave rise to osteoblasts and adipocytes, but not to chondrocytes. In contrast, CD271brightSSEA-3-CD56+ cells were INTO INSULIN PRODUCING CELLS IN CO- able to differentiate into chondrocytes but not into adipocytes. Collectively, CULTURE WITH PANCREATIC STROMAL CELLS SSEA-3 is a suitable and selective marker for the isolation of osteoblast and adipocyte precursors, whereas CD56 is the more appropriate target for the Khoshchehreh, Reyhaneh, Ebrahimi, Marzieh, Baharvand, Hossein, isolation of chondrocyte precursors. The phenotypic and functional proper- Bagheban Islami Nejad, Mohamad Reza, Aghdami, Nasser, Samani, ties of the described subsets may contribute to isolate promising starting Fazel populations for applications in the field of regenerative medicine. Department of Stem Cells and Developmental Biology, Royan Institute, Poster Board Number: 2487 Tehran, Islamic Republic of Iran Mesenchymal stem cells (MSCs) derived from bone marrow are multipotent HUMAN STEM CELLS FROM ELEVATOR PALATE cells that have the capacity to trans-differentiate into a variety of cell types MUSCLE TO BONE TISSUE ENGINEERING FOR including insulin islet cells. However the efficacy is low. It has been reported that pancreatic niche including stromal, epithelial and endothelial cells is im- CRANIOFACIAL DISEASES portant in pancreatic organogenesis. The aim of this study is to explore the Bueno, Daniela F., Kobayashi, Gerson S., Almada, Bruno Vinicius potential of marrow and umbilical cord vein MSC to differentiate into func- Pimenta, Martins, Marilia T., Jazedje, Tatiana, Vieira, Natassaia, tional islet like cells in co-culture with pancreatic mesenchymal cells. BM- Aguena, Meire, Fanganiello, Roberto Dalto, Tanikawa, Daniela, MSCs and UC-MSCs were obtained from healthy donors and were cultured. Raposo-Amaral, Cassio Eduardo, Franco, Diogo, Alonso, Nivaldo, MSCs with high CD90, CD73, CD105, CD44 and very low CD34 and CD45 expression were differentiate into islet-like cells under defined conditions Passos Bueno, Maria Rita and in presence of pancreatic MSCs . Insulin and c-peptide positive cells Genetic and Evolutive Biology, University of São Paulo, São Paulo, Brazil, were evaluated with immunoflourescence and insulin release after glucose SOBRAPAR Hospital, Campinas, Brazil, Plastic Surgery, Federal University challenge was tested by ELISA. QRT-PCR was done to detect expression of of Rio de Janeiro, Rio de Janeiro, Brazil Insulin, Glut2, Nkx6.1 and Nkx2.2 at mRNA level. Our results showed that Bone reconstruction in craniofacial diseases, which affects about 4% of new- only BM-MSC can differentiated to insulin secreting cells. About 15.8%±2.6 borns, has been the focus of intensive research. We have recently reported and 13.5%±5.5 of cells were positive for insulin and c-peptide, respectively. that orbicularis oris muscle fragments, which are discarded during cheilo- However they were not functional when treated by different concentration plasty of cleft lip and palate (CL/P) patients, are a rich source of human of glucose. Our results revealed that expression of insulin and Glut2 upregu- mesenchymal stem cells (hMSCs). We previously reported that these cells lated by pancreatic MSCs only at mRNA level, and significant differences may be useful to enhance the speed of bone regeneration “ïn vivo” when were not between cells which co-cultured with pancreatic mesenchyme and associated with a collagen biomaterial. This prompted us to investigate if it is cells which cultured alone. Our results showed that 1. Human BM-MSCs in possible to obtain hMSCs from elevator palate muscle fragments, discarded compare with umbilical cord vein MSCs are able to differentiate into insulin during palatoplasty of CL/P patients, and if these cells, associated with producing cells in vitro and 2. Pancreatic mesenchyme may increase β pre- CellCeran (60% hydroxyapatite/40% ß-tricalciumphosphate) could be use- cursors; however it needs to be studied more. ful in the reconstruction of large bone defects. This biomaterial is designed Poster Board Number: 2485 to synthetically replicate the hard calcium phosphate extracellular matrix of the bone and display good porosity for cell infiltration and adhesion, which CHARACTERIZATION OF DISTINCT HUMAN may increase bone regeneration efficiency. Elevator palate muscle fragments BONE MARROW DERIVED MESENCHYMAL of 5 CL/P patients were used in this study. The cells obtained from this tissue were characterized by flow cytometry and reacted positively for mesenchy- STEM/STROMAL CELL SUBSETS mal (CD73, CD166) and adhesion (CD29, CD90) markers, and negatively Sivasubramaniyan, Kavitha, Harichandan, Abhishek, Grimm, for hematopoietic (CD45) and endothelial (CD31) markers. Furthermore, we observed in vitro multipotenciality of these cells to osteogenic, chondrogen- Sabrina, Bühring, Hans-Jörg ic, myogenic and adipogenic differentiations. These hMSCs were called El- Department of Internal Medicine II, University Clinic of Tuebingen, evator Palate Muscle-Derived Stem Cells (EPMDSC). To evaluate the in vivo Eberhard-Karls University, Tuebingen, Germany osteogenic capacity of EPMDSC, we used the xenotransplantation model. We have recently shown that SSEA-3 is a selective marker for a subset of Two symmetric full-thickness cranial defects (12.56 mm2) on each parietal human bone marrow (BM) derived CD56- mesenchymal stem/stromal cells region of 5 non-immunosuppressed Wistar rats were performed. The left (MSCs). To analyze the phenotypic characteristics of SSEA-3+ and SSEA-3- side was covered with CellCeram only and the right side with CellCeram BM cells in more detail, BM cells were stained with CD271, CD56, and se- and EPMDSC (105cells). Animals were euthanized at 30 days postopera- lected markers of known or antibody-defined identity. Our data demonstrat- tively and cranial tissue samples were taken for histological analysis. No ed that CD13, CD29, CD73, CD140b, and CD200 were expressed at similar animals died of infection or any other complication. Histological examination levels in both subsets. Other markers such as CD56 and CD166 (ALCAM) revealed higher deposition of lamellar bone islands on the right side. These were exclusively found on CD271brightSSEA-3- cells, whereas CD26 (DPP4) islands were filled with woven bone intermixed with granulation tissue and CD146 (MCAM) were expressed only on cells of the CD271brightS- with remnants of the biomaterial. On the other hand, the side containing SEA-3+ subset. The majority of the analyzed markers found on primary CellCeram alone had the defect filled with loose connective tissue exhibiting MSCs were also expressed on cultured MSCs. However, the expression of chronic in inflammatory infiltrate, intermingled with remnants of the bioma- CD271, SSEA-3 and CD56 on primary MSCs disappeared after plating the terial. We also observed positive staining for human nuclei through immuno- cells into culture. To analyze the clonogenic and differentiation capacity of histochemical analysis only in the new bone on the right side. Our results SSEA-3+ and SSEA-3- MSCs, BM cells were stained with antibodies against show, for the first time, that EPMDSC have hMSC properties. Furthermore, CD271, CD56 and SSEA-3 and gated on CD271brightSSEA-3+CD56- and it can be successfully used with CellCeran to enhance bone regeneration in CD271brightSSEA-3-CD56+ cells. The clonogenic capacity was determined vivo, opening a new field for future bone tissue engineering to rehabilitate patients with craniofacial diseases using hMSCs and CellCeran. Supported

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by FAPESP-CEPID, MCT, INCT and CNPq. subpopulation of (trans)-differentiating cells. To address this question, we have differentiated hMSC into osteoblast and adipocytes, isolated RNA and Poster Board Number: 2489 analyzed the transcriptome in a high temporal resolution using Illumina IN VITRO TRANSFORMATION OF microarrays. mRNA expression changed very rapidly (within 30 min after induction of differentiation) and rigorously increased up to 2 days of dif- HUMAN MESENCHYMAL STEM CELL TO ferentiation. From the data, we were able to identified 77 genes that were HEPATOCELLULAR CARCINOMA DURING activated within 4 days of osteogenic differentiation and were unresponsive to adipogenic differentiation. Some of these genes were already activated OSTEOGENIC DIFFERENTIATION within 24 hrs after initiation of differentiation and remain stably expressed. Ahmadbeigi, Naser, Soleimani, Masoud, Seyedjafari, Ehsan, Additionally, we identified around 130 cell surface molecules that were Gheisari, Yousof, Amanpour, Saeid, Atashi, Amir, Omidkhoda, higher expressed during osteogenic than adipogenic differentiation. Cur- Azadeh, Saltanatpour, Manijeh, Vasei, Mohammad rently, we are characterizing some of the selected cell surface molecules for their osteogenic potential. Preliminary FACS analyses of one of the cell Hematology, Tarbiat Modares University, Tehran, Islamic Republic of Iran, surface molecules indicated that it is expressed by 8% of the bone marrow Biotechnology, Tehran University, Tehran, Islamic Republic of Iran, Stem derived MSC and increased up to 50% upon osteogenic differentiation while Cell Technology Research Center, Tehran, Islamic Republic of Iran, Cancer being unchanged in adipogenic differentiation. Osteogenic differentiation Research Center of the Cancer Institute, Tehran University of Medical of the positive and negative sorted populations resulted in a morphological Sciences, Tehran, Islamic Republic of Iran, Pathology, Tehran University of change during differentiation. Currently we are investigating the in vitro and Medical Sciences, Tehran, Islamic Republic of Iran in vivo bone formation capacity of the different populations. In conclusion, Human mesenchymal stem cells (hMSC) have been widely used in cell we have identified MSC lineage specific marker genes that can be used to therapy and are expected to be a potential source for transplantation. monitor osteogenic as well as adipogenic differentiation and are ideal tools Although most of the reports indicate their safety in ex vivo expansion, the to unravel stem cell plasticity as well as hold the potential to be used for in potential risk of tumorgenicity of hMSC during expansion still limits its clini- vivo tracking of cells. cal application. We have observed spontaneous tumor transformation during Poster Board Number: 2493 expansion and differentiation in one out of 97 samples of hMSCs which had been retrieved from bone marrow. The transformed cells were round to NANOSTRUCTURES INHIBIT MESENCHYMAL polygonal and lost their spindle shape morphology and contact inhibition. They showed decrease in CD90, CD105 and CD106, and expressed α-feto DIFFERENTIATION OF HUMAN INDUCED protein, albumin, alkaline phosphatase, and CXCR4 in flow cytometry. Cyto- PLURIPOTENT STEM CELLS genic analysis revealed hypertriploidy with complex chromosomal transloca- tions. Soft agar transformation assay showed their ability to form multiple Hammerick, Kyle E., Kasper, F. Kurtis, Mikos, Antonios G. colonies. Large tumoral masses were developed in 30 days after subcuta- Bioengineering Department, Rice University, Houston, TX, USA neous injection into nude mice. In immunohistochemistry, the tumor cells Nanostructures can approximate the structural topography surrounding cells were strongly positive for Hepar-1, pan CK, EMA, α-feto protein, placental in their native environments. Therefore, nanostructures have shown promise alkaline phosphatase, CD10, vimentin, CK8 and were negative for CEA, and in their ability to alter cellular behavior, maintaining and influencing the CD34, consistent with a poorly differentiated hepatocellular carcinoma. This differentiation of stem cells. The incorporation of nanostructures with micro- study indicates that hepatocellular carcinoma can aberrantly develop from structures can also help recreate the hierarchical length scales witnessed in hMSCs during in vitro culture and support this theory that bone marrow bone. Different types of cells have demonstrated a wide variety of responses hMSC can be a source of some solid organ cancer such as hepatocellular to nanostructures ranging from increased spreading to differentiation. We carcinoma in human. hypothesize that nanostructured electrospun polymers can influence the dif- Poster Board Number: 2491 ferentiation of human induced pluripotent stem cells (hiPSCs) by altering the distribution of focal adhesions and the ability of cells to spread on substrates. IDENTIFICATION OF EARLY LINEAGE SPECIFIC This will cause changes in cytoskeletal tension and control downstream MARKER GENES OF DIFFERENTIATING HUMAN signaling altering expression of genes related to mechanoregulation. We employed electrospun microstructured and nanostructured polycaprolactone MESENCHYMAL STROMAL CELLS (PCL) polymer scaffolds to direct differentiation of hiPSCs toward mesenchy- van de Peppel, Jeroen, Koedam, Marijke, Schoordijk, Wenda, mal cell fates. Cells attached to all PCL surfaces, however, cells spread more on flat and microstructured PCL scaffolds than nanostructured scaffolds. Schaaf, Gerben, Eijken, Marco, van Leeuwen, Hans HiPSCs maintained morphology and expression profiles of embryonic mark- Internal Medicine, ErasmusMC, Rotterdam, Netherlands, 2Internal ers Oct-4, SSEA-4, and alkaline phosphatase longer under differentiating Medicine, Transplantation, ErasmusMC, Rotterdam, Netherlands, 3Cell conditions on nanostructures than on either flat or microstructured surfaces. Biology, ErasmusMC, Rotterdam, Netherlands Microstructured scaffolds promoted the expression of key mesenchymal cell Mesenchymal Stromal Cells (MSCs) can be isolated from various tissues markers CD73, CD90, CD105, and CD166. These data suggest that nano- including bone marrow, adipose tissue, skeletal muscle and umbilical cord. topographies may be a useful tool in feeder free culture and maintenance of Since the discovery in the late 90s of the multi-lineage potential of these hiPSCs, while microstructures can be used to direct hiPS cell spreading and cells only little progress is made to define specific marker genes that identify consequently mesenchymal differentiation. MSCs and early lineage-specific progenitors. As a consequence, most MSC isolations are heterogenic and can respond differently in different experi- ments. Our previous analyses indicated that “classical” osteogenic markers (ALPL, RUNX2 and COL1A1) are not only expressed during differentiation of hMSC into osteoblast but also at similar levels in fat tissue and differen- tiating MSC into adipocytes. This shows that these genes are not optimally suited as predictors for early osteoblast development and to track cell fate. Identification of specific marker genes for development of osteoprogeni- tors from MSC cultures is necessary and will enable us to follow individual

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Poster Board Number: 2495 Poster Board Number: 2497 OSTEOGENIC DIFFERENTIATION OF HUMAN CHARACTERIZATION OF CARTILAGE INDUCED PLURIPOTENT STEM CELLS: IN GENERATED FROM HUMAN MESENCHYMAL VITRO AND IN VIVO STEM CELLS ON NANOFIBER SCAFFOLDS: Phillips, Matthew, Kuznetsov, Sergei A., Cherman, Natasha, Park, A COMPARATIVE STUDY USING HUMAN Kye-Yoon, Chen, Kevin G., McClendon, Britney N., Hamilton, CARTILAGE SAMPLES AS THE BENCHMARK Rebecca, McKay, Ronald, Chenoweth, Josh, Mallon, Barbara, Caballero, Montserrat, Dahl, John P., Pappa, Andrew K., van Aalst, Robey, Pamela G. John A. CSDB, NIDCR/NIH/DHHS, Bethesda, MD, USA, SCU, NINDS/NIH/DHHS, Surgery: Plastic & Reconstructive Surgery, University of North Carolina, Bethesda, MD, USA, Lieber Institute for Brain Development, Baltimore, Chapel Hill, NC, USA, Department of Otolaryngology: Head & Neck MD, USA, LMB, NINDS/NIH/DHHS, Bethesda, MD, USA Surgery, University of North Carolina, Chapel Hill, NC, USA Derivation of osteogenic cells from induced pluripotent stem cells (iPSCs) for Introduction: Our laboratory is interested in the generation of a nanofiber- use in bone regeneration in fracture non-union, and to treat large segmen- supported tissue engineered elastic cartilage implant that can be used tal defects, would be a welcome alternative to the use of adult stem cells for pediatric ear reconstruction. The aim of this work is to study in vitro- that have a limited capacity to generate large number of cells needed for generated cartilage in parallel to human cartilage samples, in order to extensive bone repair; e.g., to treat large segmental defects. Additionally, guide the development of such an implant. Methods: The in vitro tissue human iPSCs are advantageous over human embryonic stem cells (hESCs) engineered cartilage was generated using mesenchymal stem cells derived because they can be autologous, thereby avoiding immunological rejection. from umbilical cord (hUCMSC) culture on either polycaprolactone (PCL) or Previously, we devised a method for differentiating hESCs into osteogenic D, L-lactide-co-glycolic acid (PLGA) nanofiber scaffolds under chondrogenic cells by growing them with ceramic particles to form a cohesive “carpet”- conditions. After 21 days of differentiation, the cells were harvested for like structure. Upon transplantation into immunocompromised mice, bona histological, biochemical, and quantitative PCR analysis. Control cells were fide bone was formed with osteoblasts and osteocytes of human origin, al- grown under both chondrogenic and non-chondrogenic conditions in the though non-descript connective tissue was also present. We applied a similar absence of nanofiber scaffolds. In parallel, the expression of various extracel- technique to iPSCs generated from human skin fibroblasts that have been lular matrix components was analyzed in cartilage samples obtained from previously well characterized. Undifferentiated colonies were mechanically discarded adult conchal bowl for nasal surgery, and from pediatric patients passaged into KO DMEM with 10% FBS for 7 days, plus either: Medium A with preauricular remnants and microtia. Results: Analysis of the human (10-8 M dexamethasone, 10-4 M L-ascorbic acid 2-phosphate); or Medium samples showed similar levels and distribution of collagen I, X, and elastin. B (10-6 M retinoic acid for 13 days); or Medium C (10-9 M rapamycin for 6 Collagen II was significantly less expressed in the microtia samples compared days); or Medium D (10 days each, 6 ng/ml bFGF, then 10 ng/ml BMP-2). to adult conchal bowl and preauricular remnants. The hUCMSCs cultures Cultures in B, C and D were subsequently switched into A. After 16-37 days, differentiated on nanofiber scaffolds were positive for glycosaminosglycans cells were enzymatically passaged 3 times in A prior to in vitro osteogenic and sulfated proteoglycans. Compared to control cells, hUCMSCs grown on differentiation (A plus 5 or 10 mM beta-glycerol phosphate) or in vivo trans- PLGA nanofiber scaffolds had more extensive glycosaminoglycan produc- plantation with ceramic particles. In some cases, the last passage was seeded tion (p≤0.03), a higher ratio of collagen II/collagen I (differentiation index) onto ceramic particles to form a carpet prior to transplantation. In vitro, cells (p≤0.02), and higher levels of collagen X mRNA expression (p≤0.005). grown in A and B, followed by growth in osteogenic differentiation medium, Discussion: These data provide information regarding the composition of formed nodular structures that stained with alizarin red, indicative of calcium endogenous human ear cartilage, which is the benchmark for any attempts accumulation. However, at six weeks following in vivo transplantation at tissue engineered cartilage. In addition, our in vitro results suggest that of cells from all the conditions, none of the transplants contained bone, hUCMSCs grown on PLGA nanofiber scaffolds may represent an optimal although osteoid-like material rimmed with cuboidal cells was apparent in starting material for the development of a nanofiber-based cartilage implant many of the small pores of the ceramic particles, suggesting the need for for use in pediatric ear reconstruction. longer time points. To determine if the tissue source of iPSCs can influence subsequent osteogenic differentiation, we also reprogrammed human bone Poster Board Number: 2499 marrow stromal cells (BMSCs, well-known osteogenic precursors), using a Cre-excisable constitutive polycistronic (OKSM) lentivirus. Transduced cells SHEAR IS A REQUIREMENT FOR were initially plated in KSR-containing medium on MEFs. Most clones were MECHANICALLY INDUCED CHONDROGENESIS then isolated under the same conditions but some were successfully isolated in mTeSR medium on MatrigelTM, with one clone being subsequently OF HUMAN BONE MARROW DERIVED STEM transitioned back to growth on MEFs. By FACS analysis, all were negative CELLS for SSEA-1 and positive for SSEA-4, Tra-1-60 and Tra-1-81, identical to H9 (WA09) hESCs used for comparison. By immunohistochemistry and quan- Schätti, Oliver, Grad, Sibylle, Goldhan, Jörg, Alini, Mauro, Stoddart, titative RT-PCR, POU5F1 and NANOG were observed at levels comparable Martin J. to hESCs. Two clones grown on BD MatrigelTM were then expanded in an AO Research Institute Davos, Davos Platz, Switzerland, ETH Zürich, Zürich, undifferentiated state using a novel monolayer culture method. Studies are Switzerland underway to verify that these clones form teratomas in vivo, and to exam- Introduction: Bone marrow derived mesenchymal stem cells (MSCs) offer ine their osteogenic differentiation potential. In conclusion, while hESCs and great promise in the repair of defects of the musculoskeletal system. Increas- iPSCs share many characteristics, differentiation of iPSCs into an osteogenic ingly mechanical force is being shown to regulate the phenotypic fate deci- phenotype may require different conditions than for hESCs. Studies in prog- sions in these cells. While a number of studies have shown a beneficial effect ress will determine if BMSC-derived iPSCs are more inclined to osteogenic of load for chondrogenesis of MSCs there is some controversy as other stud- differentiation than those derived from skin fibroblasts. ies have indicated an inhibitory effect. The present study aimed to test the theory that shear is required for mechanically induced in vitro chondrogene- sis of human MSC-laden scaffolds. Methods: P3 human bone marrow MSCs were seeded into 3D fibrin-polyurethane scaffolds (8mm×4mm) at a density

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of 4×106 per scaffold. Cell-scaffold constructs were cultured in ITS+DMEM high levels of vimentin and collagen I, and were negative for pluripotency (with no TGF-β added). They were pre-cultured for 2 days, then loaded 1 markers (Oct4, Nanog, SSEA4, Tra-1-60). Osteogenic and chondrogenic hour daily for 3 weeks. Cyclic axial compression (10-20% of scaffold height, differentiation was induced in vitro in derived MSC, although adipogenic sinusoidal strain, 1Hz) was performed by a ceramic hip ball 32 mm in di- differentiation was limited, as observed for primitive fetal MSC. In summary, ameter. For groups involving shear, oscillation of the ball over the construct we report a rapid and potentially-scalable method to produce MSCs from surface was applied (±25°, 1 Hz). Samples were divided into 4 groups: 1 hiPSCs, which are comparable to standard EB-derived and somatic MSC. unloaded control group and 3 loaded groups with different load regimens (compression alone, shear alone, compression and shear). Measurements Poster Board Number: 2503 included DNA, glycosaminoglycan, and histology. mRNA expression of DIFFERENTIATION OF HUMAN PLURIPOTENT chondrogenic markers (AGG, COL 2, SOX9, COMP) osteogenic markers (COL 1, ALP) and the hypertrophic marker COL 10 was also performed. STEM CELLS INTO MESENCHYMAL STEM Results: Mechanical load led to an increase in all the chondrogenic genes CELLS BY MODULATING INTRACELLULAR investigated. However, the magnitude varied greatly depending on whether shear was incorporated into the loading protocol. At the mRNA level Sox9, SIGNALING PATHWAYS COMP, AGG, COL2 and COL10 were all up-regulated to a greater extent by Tran, Tung N., Mi-Jin, Jang, Han, Yong Mahn shear alone than by compression alone and a combination led to the great- Biological Sciences, KAIST, Daejeon, Republic of Korea est increase. Compression combined with shear up-regulated the 2 cartilage specific matrix proteins by 2 (AGG) or 3 (COL2) orders of magnitude when Human pluripotent stem cell-derived mesenchymal stem cells (hPSC-MSCs) compared to compression alone. This was reflected in matrix synthesis as are one promising alternative cell source for MSCs-based therapies. Here, seen in histological preparations. Only compression and shear led to a matrix we developed an efficient protocol to generate hPSC-MSCs on the feeder- rich in glycosaminoglycan staining. Quantitative analysis also determined free culture system by step-wisely modulating key signaling pathways that the inclusion of shear led to a greater synthesis, and a greater retention such as TGF-β, WNT, BMP, and FGF. Mesoderm-lineage cells were first of total synthesized glycosaminoglycans, within the tissue engineered con- induced from hPSCs (hESCs and hiPSCs) by treatments with Activin A, struct. Conclusions: These data would suggest that shear is a critical compo- BIO (6-bromoindirubin-3’-oxime), and BMP4 for 3d and further cultured nent when inducing chondrogenesis in human mesenchymal stem cells by in the medium containing bFGF for 10d. Proportion of CD105+ cells was mechanical means. Uniaxial load does not appear to be sufficient under the approximately 20% by MACS sorting using CD105 antibody. The CD105+ conditions applied here. This may play a role in natural healing within the cells were positive for MSC-specific markers such as CD29, CD44, CD73, musculoskeletal system, whereby the same cell type (MSCs) initiates bone CD90, and HLA-ABC, but negative for non-MSC markers such as CD34, repair (uniaxial load) and cartilage repair (multiaxial load) resulting in differ- CD45, CD31, and HLA-DR. Furthermore, CD105+ cells had differentiation ent tissues from different mechanical cues. This knowledge could be used to potentials into adipocytes, osteoblasts, and chondrocytes in vitro. The results develop more optimal rehabilitation protocols to be used after implantation demonstrate that functional mesenchymal stem cells could be efficiently of MSCs for cartilage repair. generated from human pluripotent stem cells by modulating signaling pathways. This research was supported by a grant (SC-2210) from Stem Cell Poster Board Number: 2501 Research Center of the 21st Century Frontier Research Program funded by SMALL MOLECULE MESENGENIC INDUCTION the MEST, Republic of Korea. OF HUMAN IPS FOR MESENCHYMAL STEM Poster Board Number: 2505 CELL GENERATION RESVERATROL PROMOTES OSTEOGENESIS OF Chen, Yen-Shun, Ellis, Rebecca, Pelekanos, Rebecca, Rorne, Rachel, HUMAN MESENCHYMAL STEM CELLS BY UP- Ryan, Jennifer, Reggatt, Liza, Wolvetage, Ernst, Fisk, Nicholas M. REGULATING RUNX2 GENE EXPRESSION VIA University of Queensland Centre for Clinical Research, Brisbane, Australia, SIRT1/FOXO3A AXIS Australian Institute for Bioengineering and Nanotechnology, Brisbane, Australia Tseng, Pei-chi, Chen, Ruey-Jien, Peng, Hsiao-Wen, Kuo, Min-Liang, Hou, Sheng-Mou, Yen, B.Linju, Yen, Men-luh Mesenchymal stem cells (MSCs) have been limited in their translational po- tential by their rarity in somatic organs, heterogeneity, and need for harvest Dept of Ob/Gyn, National Taiwan University College of Medicine, Taipei, by invasive procedures. Generation of MSCs from human induced pluri- Taiwan, Institute of Toxicology, National Taiwan University College of potent stem cells (hiPSCs) would offer a potentially robust, scalable source Medicine, Taipei, Taiwan, Dept of Orthopedic Surgery, Shin Kong Wo for cell therapy, and obviate the ethical issues associated with ESC or early Ho-Su Memorial Hospital, Taipei, Taiwan, Institute of Cellular and System fetal sources. Attempts to date to derive MSC from pluripotent cells have Medicine, National Health Research Institutes, Zhunan, Taiwan used cumbersome or untranslatable techniques such as coculture, physical Reports of the bone-protective effects of resveratrol, a naturally occuring manipulation, sorting or viral transduction. We devised a single-step small phytoestrogen and agonist for the longevity gene SIRT1, have highlighted molecule method to induce rapid differentiation of human embryonic stem this compound as a candidate for therapy of osteoporosis. Moreover, cells (hESCs) and hiPSCs into MSCs. First, hESC/hiPSC-derived mesodermal/ SIRT1 antagonism enhances adipogenesis. There has been speculation that ectodermal-like monolayer cells were generated by culturing hESC/hiPSC resveratrol can promote osteogenesis through SIRT1 but the mechanism in serum-free medium (KOSR) with the TGF-β receptor kinase inhibitor, remains unclear. In this study, we investigated the molecular mechanism of SB431542. After 10 days treatment, qRT-PCR arrays of hiPSC demonstrated how resveratrol can modulate the lineage commitment of human mesen- that mesodermal, MSX2, NCAM, HOXA2 and ectodermal, PAX6 genes were chymal stem cells to osteogenesis other than adipogenesis. We found that up-regulated (> 3 fold) and pluripotency genes, OCT4 (-271 fold), Nanog resveratrol promoted spontaneous osteogenesis but prevented adipogenesis (-6 fold) down-regulated. Next, the monolayer cells were differentiated in human embryonic stem cell-derived mesenchymal progenitors. Res- toward fibroblast-like MSC morphology by inducing epithelial-mesenchymal veratrol up-regulated the expression of osteo-lineage transcription factor transition (EMT) machinery, with SNAI2 (14 fold) and TWIST2 (7 fold) RUNX2 and OSTEOCALCIN while simultaneously suppressing PPARγ mRNA up-regulation in conventional MSC culture media (DMEM-HG + 10% FBS). expression. Using specific inhibitors, we found that the osteogenic effect of hESC- and hiPSC-derived MSC both manifest a typical MSC immunophe- resveratrol was mainly mediated through SIRT1 but not estrogen receptor notype (CD29+, CD73+, CD105+, CD90+, CD45-, CD31-), expressed signaling. Furthermore, resveratrol activated SIRT1 activity without altering

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SIRT1 protein level, and enhanced FOXO3A protein expression, a known of MSCs compared with ES cells. target of SIRT1. As a result, resveratrol increased the amount of the SIRT1/ FOXO3A complex and enhanced FOXO3A-dependent transcriptional activ- Poster Board Number: 2509 ity. Ectopic overexpression of SIRT1/FOXO3A enhanced RUNX2 promoter NANOPARTICLE LABELING OF BONE activity, suggesting an important role for the SIRT1/FOXO3A complex in regulating resveratrol-induced RUNX2 gene transcription. Further muta- MARROW-DERIVED RAT MESENCHYMAL STEM tional RUNX2 promoter analysis and chromatin immunoprecipitation assay CELLS AND THEIR USE IN DIFFERENTIATION revealed that resveratrol-induced SIRT1/FOXO3A complex bound to a distal FOXO response element (-1269/-1263), an action which transactivated AND TRACKING RUNX2 promoter activity in vivo. Taken together, our results describe a Akhan, Ece, Ibrahimova, Vusala, Tuncel, Donus, Akcali, Kamil C., novel molecular action of resveratrol on promoting osteogenesis of human Bugdayci, Emre mesenchymal stem cells by up-regulating RUNX2 gene expression via the Molecular Biology and Genetics, Bilkent University, Ankara, Turkey, SIRT1/FOXO3A axis. Chemistry, Bilkent University, Ankara, Turkey Poster Board Number: 2507 Multipotent Mesenchymal stem cells (MSCs) are capable of self-renewal HUMAN UMBILICAL CORD PERIVASCULAR and differentiate into multiple lineages including hepatocytes. They are promising candidate for cell-based therapies due to their ability to migrate CELLS (HUCPVCS) VERSUS BONE MARROW- in vivo to promote regeneration of damaged tissue, treat inflammation, DERIVED MESENCHYMAL STROMAL CELLS and promote angiogenesis without inducing immune reaction. However it is difficult to track the injected MSCs and their differentiation efficacy (BM-MSCS): POTENTIAL ROLE OF EPIGENETICS in the host. Conjugated polymer based water-dispersible nanoparticles ON MULTIPOTENT CELL DIFFERENTIATION represent a new class of probes for cell imaging and tracking because they CAPACITY offer high brightness, improved photostability, high fluorescent quantum yield and non-cytotoxicity comparing to conventional dyes and quantum Yannarelli, Gustavo, Pacienza, Natalia, Cuniberti, Luis, Medin, dots. In this study we developed a novel approach by using fluorescein Jeffrey A., Keating, Armand emitting nanoparticle (NP) to label and follow the fate of MSCs in the host. Ontario Cancer Institute, University Health Network, Toronto, ON, Canada, Copolymer of fluorene-benzothiodiazole derivatives (PFBt) containing cross- Department of Physiology, Favaloro University, CONICET, Buenos Aires, linkable allyl groups attached to fluorene monomer has been converted Argentina into water-dispersible green-emitting NPs (emission wavelength is at 521 nm) using reprecipitation method. Our in vitro experiments revealed that Human umbilical cord perivascular cells (HUCPVCs) are a readily available approximately 70% of MSCs have uptaken NP to their cytoplasm upon 12 alternative source of mesenchymal stromal cells (MSCs) for cell therapy hours incubation. After NP treatment significant number of cells, 68% of the and may overcome some of the limitations of bone marrow (BM)-derived labeled cells, were found viable as revealed by MTT (3-(4,5-Dimethylthiazol- MSCs, such as a decline in function with donor age. The molecular char- 2-yl)-2,5-diphenyltetrazolium bromide) assay. In addition, we also showed acteristics of HUCPVCs however, have not been previously studied. We that the NP-labeled MSCs retained the fluorescent signal after they were hypothesized that the enhanced properties of HUCPVCs including higher differentiated into adipocytes and osteocytes. We also examined the fate of stem cell frequency and greater multipotency, compared with BM-MSCs, NP-labeled MSCs in rat liver regeneration model. Our in vivo results showed result from their neonatal tissue origin. We determined telomerase activity, that the NP-MSCs migrated to regenerated rat liver. In conclusion, the utili- telomere length, and expression of the pluripotency factors OCT4, SOX2, zation of NP could be a promising tool for the tracking of MSCs in vivo and and NANOG in both types of MSCs. The proliferation rate of HUCPVCs in vitro and therefore can be a useful tool to understand the biology of stem was significantly higher than for BM-MSCs (population doubling time: 23.6 cells such as differentiation and homing mechanisms. vs 44.4 h, respectively; P<.001). Interestingly, we found that HUCPVCs exhibited significantly longer telomeres (2.0-fold difference) and 2.2-fold Poster Board Number: 2511 higher telomerase activity than BM-MSCs. These results are consistent with a MSC population that has higher proliferative capacity and contains more EFFECT OF ESTROGEN ON THE stem cells. While we found no difference in OCT4 gene expression, SOX2 DIFFERENTIATION OF BONE MARROW- and NANOG genes were down-regulated in HUCPVCs compared with DERIVED RAT MESENCHYMAL STEM CELLS BM-MSCs. Protein expression of these sequences, however, was significantly higher in HUCPVCs, not only with respect to the frequency of positive cells INTO ADIPOCYTES AND OSTEOCYTES (OCT4: 92.7% vs 67.1%; SOX2: 99.1% vs 71.3%; NANOG: 46.6% vs Bitirim, Verda C., Ayaloglu Butun, Fatma, Akcali, K. Can 5.0%) but also in relative content per cell (OCT4: 2.5 vs 1.2 MFI; SOX2: 4.0 vs 0.8 MFI; NANOG: 1.2 vs 0.1 MFI) as assessed by flow cytometry. Molecular Biology and Genetics, Bilkent University, Ankara, Turkey HUCPVCs and BM-MSCs showed similar OCT4 (51% vs 48%, P=.590) and Mesenchymal stem cells (MSCs) have the potential to differentiate into NANOG (17% vs 27%, P=.056) promoter methylation, indicating that the different cells, and have a high capacity to proliferate. In addition to their differences in expression of these pluripotency factors between the MSCs capacity to differentiate into bone, muscle, fat, cartilage, nerve and liver was not associated with epigenetic changes. It is noteworthy that the degree cells, not encountering any rejection problem by the host make them a hope of methylation at these loci in MSCs is greater compared with embryonic in cell based treatment of many chronic diseases. Recent studies have shown stem cells (<5%) but less than that for fibroblasts (>80%), inferring that the that, differentiation is accomplished by the activation of specific transcrip- multipotentiality of MSCs may be epigenetically restricted. This mecha- tion factors. Our aim in this study is to reveal the effect of estrogen on the nism may explain the lower differentiation capacity, and possibly, the very differentiation of rat MSCs into adipocytes and osteocytes. To accomplish low risk of tumor formation of MSCs compared with embryonic stem (ES) this aim, we investigated the role of estrogen on the adipocyte, osteocyte cells. In conclusion, we showed that HUCPVCs exhibit enhanced expres- and chondrocyte lineage specific transcription factors as well as histone sion of telomerase and pluripotency factors compared with BM-MSCs and modifying enzymes. We isolated MSCs from female and ovariectomized demonstrated that these differences are not epigenetically regulated. Our rats and cultured in the presence and absence of 10-7M estrogen. Estrogen results also suggest that the methylation of OCT4 and NANOG promoters is treatment caused a decrease in the expression of adipocyte lineage specific a potentially important mechanism for restricting the differentiation capacity transcription factors such as Ppar γ, C/EBPa, FABP in the MSCs isolated from

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normal female animal but not from ovariectomized animals. On the other Poster Board Number: 2515 hand the expression of Runx2, an osteocyte lineage transcription factor, increased upon estrogen treatment in MSCs isolated from bone marrow of TRANSGENIC ADULT MURINE MESENCHYMAL both normal and ovariectomized rats. Similarly estrogen addition increased STEM CELLS CONDITIONALLY the expression of chondrocyte lineage specific transcription factors, Sox9 and ATF3 but not MEF2C. Our results also revealed that estrogen differen- OVEREXPRESSING SDF-1β ENHANCE NEW tially regulates the expression of histone modifying enzymes. In conclusion, BONE FORMATION IN BOTH IN VITRO AND IN estrogen affects the differentiation of rat MSCs which may lead to new VIVO MODEL SYSTEMS horizons in the treatment of diseases that require bone and cartilage repair as well as obesity characterized with excessive fat storage. This work was Herberg, Samuel, Fulzele, Sadanand, Yang, Nianlan, Kondrikova, supported by the Scientific and Technological Research Council of Turkey Galina, Hussein, Khaled, Elsalanty, Mohammed, Shi, Xingming, (TUBITAK) grant SBAG109S460 to KCA. Hamrick, Mark W., Hill, William D. Poster Board Number: 2513 Cellular Biology & Anatomy, Georgia Health Sciences University, Augusta, GA, USA, Orthopedic Surgery, Georgia Health Sciences University, Augusta, TRACKING OF MOUSE MESENCHYMAL STEM GA, USA, Institute of Molecular Medicine and Genetics, Georgia Health CELLS USING SPECIFIC REPORTER GENES Sciences University, Augusta, GA, USA, Oral Biology, Georgia Health Sciences University, Augusta, GA, USA Fuegemann, Christopher J., Hesse, Michael, Fleischmann, Bernd K., Background: Bone marrow-derived mesenchymal stem cell (BMSC) dif- Breitbach, Martin ferentiation is regulated through the multifunctional chemokine stromal Institute of Physiology I, University of Bonn, Bonn, Germany cell-derived factor-1 (SDF-1). Interaction with its cellular receptor CXCR4 Mesenchymal stem cells (MSCs) are multipotent, can be easily isolated from is known to direct the migration of stem cells associated with injury repair bone marrow and other tissues and expanded ex vivo for clinical use. So far, in different tissues. In vitro pre-conditioning of BMSCs with SDF-1 prior MSCs are characterized by their adherent properties, immunophenotype, to transplantation has been shown to significantly enhance cell engraft- and their differentiation potential in vitro. Because of a lack of distinctive ment and new tissue formation in different animal models. However, this markers for MSCs, the current understanding of their endogenous localiza- approach does not allow for direct modulation of SDF-1 levels in vivo. The tion and functions in vivo remain elusive. However, the identification of tetracycline-inducible gene regulation system provides for tight regulation the MSC niche is important in order to better understand the cell biological of transgene expression in vitro and in vivo. Recently, we described the characteristics of these cells and their physiological role. We therefore aim to development of a RetroX-Tet-Off-SDF-1β transgenic stem cell line condi- genetically label mouse MSCs using specific marker genes to track develop- tionally overexpressing SDF-1β. SDF-1β, which is identical to SDF-1α except ment and fate in vitro by use of the embryonic stem cell (ESC) system and for 4 additional amino acids at the C-terminus, is about twice as potent as in vivo by generating transgenic mice. We identified potential mouse MSC SDF-1α due to its GAG-dependent stabilization on cell surfaces. The current marker genes using comparative gene expression profiling and confirmed study evaluated the regenerative potential of this novel cell line using both their specificity by PCR and immunostainings on established MSC lines and in vitro and in vivo analyses. Materials and Methods: RetroX-Tet-Off-SDF- control cells. Three genes were chosen based on their high and exclusive ex- 1β BMSCs conditionally overexpressing SDF-1β were generated as described pression in MSCs compared to other cell types. Reporter vectors expressing previously. In vitro techniques used to assess the osteogenic differentiation EGFP under control of the respective promoters were generated from bacte- capacity included ELISA for osteocalcin (OCN), Alizarin red S staining, and rial artificial chromosomes (BACs). Transfection of the vectors into estab- qRT-PCR analysis (OCN, Runx2, Col1α) after culturing cells in differentia- lished MSC lines gave rise to EGFP positive cells with the ability to differenti- tion medium containing 0.25 mM ascorbic acid, 1 µM dexamethasone, ate into the osteogenic and adipogenic lineage, proving expression of the and 10 mM β-glycerophosphate ± 100 ng/ml rhBMP-2 for 21 d. 100 ng/ constructs in multipotent MSCs. Next, we generated transgenic ESC lines ml doxycycline (Dox) was added to suppress SDF-1β transgene expression using the MSC reporter vectors. Differentiation of ESCs into MSCs was dem- in controls. For in vivo studies, 20 lethally irradiated 6-month-old C57BL/6 onstrated by colony forming assays and expression of specific MSC markers male mice were divided into two groups and given intra-medullary tibial using PCR and flow cytometry. In accordance with these results EGFP posi- transplants (left tibia) with RetroX-Tet-Off-SDF-1β BMSCs (9.24x105 tive cells could be detected starting from day eight of ESC differentiation. cells/70 µl saline + 1% diprotin A). Right tibias received saline for vehicle Finally, transgenic mice are currently generated by diploid aggregation using control. Control mice had access to 25 µg/ml Dox in drinking water + 5% transgenic ESC clones in order to investigate the in vivo distribution of MSCs glucose ad libitum; experimental mice received 5% glucose only. After 4 and to gain new insights in the biology of MSCs. wks, animals were euthanized and tibias were collected for µCT scanning to analyze standard histomorphometric parameters, including bone volume fraction (BV/TV). Results: ELISA of RetroX-Tet-Off-SDF-1β BMSCs (- Dox) revealed significantly increased OCN production relative to controls (+ Dox) in presence of BMP-2 at 14 d. Transgene overexpression of SDF-1β acceler- ated and enhanced osteogenic differentiation relative to controls as detected by Alizarin red S staining at 14 and 21 d, even in absence of BMP-2. qRT- PCR confirmed that transgene overexpression of SDF-1β further enhanced BMP-2-induced upregulation of osteogenic markers in a temporal fashion (e.g., OCN: 55.9-fold/- Dox; 22.2-fold/+ Dox after 7 d). Tibial µCT analyses showed significant augmentation of intra-medullary new bone formation in both experimental and control transplants relative to vehicle controls; transplantation with RetroX-Tet-Off-SDF-1β BMSCs (- Dox) resulted in greater bone formation compared to controls (+ Dox) (BV/TV: 64.1/- Dox; 51.8/+Dox; 5.8/vehicle control; p<0.01). Conclusion: In vitro and in vivo analyses indicate a critical role of SDF-1β in BMP-2-induced osteogenic differentiation and high regenerative potential of our novel RetroX-Tet- Off-SDF-1β transgenic stem cell line. Future studies will examine the use of these cells in acute and chronic bone injury models.

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Thursday Poster Abstracts

Poster Board Number: 2517 Poster Board Number: 2519 HIGH-THROUGHPUT AND SYSTEMATIC MURINE BONE MARROW DERIVED VERY INVESTIGATION OF MATRIX MECHANICAL AND SMALL EMBRYONIC-LIKE (VSEL) STEM CELLS BIOCHEMICAL CONTROL OF MOUSE BONE GIVE RISE TO MESENCHYMAL STROMAL MARROW STEM CELL DIFFERENTIATION CELLS Chen, Wen Li Kelly, Simmons, Craig A. Heider, Andreas, Cross, Michael, Alt, Rüdiger Institute of Biomaterials and Biomedical Engineering, University of Toronto, Translational Centre for Regenerative Medicine, University of Leipzig, Toronto, ON, Canada Leipzig, Germany, Department of Hematology, University of Leipzig, Leipzig, Germany Stem cell lineage specification is modulated by the biochemical and me- chanical properties of the extracellular matrix (ECM). Given the complexity Introduction: The broad differentiation potential of bone marrow (BM) cells of cellular responses, it is hypothesized that the integrated effects of multiple makes BM an attractive candidate for diverse applications in regenerative extracellular cues as opposed to their individual actions are critical to un- medicine. Clearly, the non-hematopoietic compartment of BM contains derstanding and controlling cell behavior. However, the systematic screen- stem cells of endothelial and mesenchymal origin, but also more primitive ing of large number of input stimuli for stem cell engineering is unfeasible very small embryonic-like (VSEL) stem cells, which have shown an in vitro using conventional cell culture systems. Cell-based arrays have been used differentiation potential into cells of all three germ layers. However, these to study mixtures of biochemical factors, but none is capable of systemati- promising stem cells are extremely rare. We have optimized methods for the cally examining the combinatorial effects of biochemical and mechanical detection, isolation and amplification of VSEL stem cells, as a basis for their matrix signals. To address this need, we have developed an array platform further characterisation and use in regenerative therapies. Furthermore, we to study the contributions of both ECM composition and substrate stiffness have characterized the relationship between VSEL cells and other stem cell to stem cell fate decision. In our proof of concept investigation, permuta- types in murine BM, namely PalphaS cells and mesenchymal stromal cells. tions of Type I collagen, fibronectin, and laminin at different concentrations Materials and Methods: Lin- CD45- Sca-1+ VSEL cells and PDGF-Ralpha+ (50, 100, or 200 μg/mL) were patterned onto polyacrylamide substrates PalphaS stem cells were FACS analysed, purified from C57Bl/6 mouse BM of various elasticities (3, 21, or 144 kPa) according to statistical design of by successive magnetic and FACS sorting, and entered into colony forming experiments. A mouse bone marrow-derived mesenchymal stem cell (MSC) unit fibroblast (CFU-f) assays. Results: It has been reported, that collagenase line (D1) was seeded onto the ECM hydrogel arrays. In order to identify the treatment can increase the recovery of mesenchymal stromal cells (MSC) matrix combinations favorable to MSC osteogenic or adipogenic differentia- from murine BM. We have optimized the enzymatic treatment of crushed tion, the immuno-fluorescence of lineage-specific transcription factors (Y = long bone fragments, and were able to confirm an increase in the number of osterix or PPARγ) was quantified and modeled as polynomial function of the Lin- CD45- Sca-1+ VSEL cells in parallel with an increase in CFU-f activity. input stimuli (x): Y= K+ ∑i=1βixi +∑i=1∑j=1βij xixj +∑i=1∑j=j∑k=1βijk xixjxk This shows that VSEL cells and mesenchymal progenitors are both closely at- +βijkl xixjxkxl+∑i=1βiixii2. Beta coefficients correspond to the estimated tached to bone tissue and may therefore be physically liked to the endosteal factor effects (βi), interactions (βij, βijk, βijkl) and quadratic dose responses stem cell niche. To purify VSEL cells, Lin- were first enriched by magnetic (βii). The extent of osteogenic differentiation, evaluated by osterix level, affinity cell sorting, and then double FACS sorted to obtain pure Lin- CD45- was biphasically modulated by substrate stiffness (βstiffness*stiffness= Sca-1+ VSEL cells. About a quarter of the VSEL population co-expressed -0.23, P<0.0001; βstiffness= 0.071, P=0.015); positively modulated by PDGF-Ralpha. This population is known as the PalphaS population and collagen (βcollagen I = 0.066, P=0.04); and synergistically promoted by the is highly enriched in mesenchymal stromal cells. When plated in standard combined effect of substrate stiffness and laminin (βstiffness*βlaminin= CFU-f assays, >1% of the VSEL cells gave rise to fibroblast colonies. How- 0.067, P=0.059) and the four-way interaction among substrate stiffness, ever, the number and size of detectable primary CFU-f further increased up collagen, fibronectin and laminin β( stiffness*collagen*fibronectin*laminin= to 5-fold under hypoxic conditions (1% pO2). This indicates that primary 0.094, P=0.011). In contrast, adipogenic differentiation, evaluated by PPARγ CFU-f included in the VSEL population are sensitive towards oxidative expression, was negatively regulated by substrate stiffness (βstiffness= stress. Lastly, we compared the frequency of VSEL cells and CFU-f activity -0.34, P<0.0001; βstiffness*stiffness= 0.22, P=0.003), collagen (βcollagen in young and aged mice. Again, VSEL/PalphaS cells frequencies paralleled I = -0.11, P=0.023), and the combination of fibronectin and laminin a marked decrease of CFU-f activity with age. Conclusions: Here we show (βfibronectin*laminin=-0.127, P=0.017). These results our hypothesis that that non-hematopoietic Lin-CD45-Sca-1+ VSEL cells are closely attached to MSC differentiation can be differentially modulated by multiple matrix cues bone tissue and are highly enriched in CFU-f. These results are compatible (including mechanical and biochemical), thereby reiterating the importance with a localisation of VSEL cells in the endosteal stem cell niche, and raise of multifactorial considerations in both biological investigation and cell the question of whether VSEL cells play a functional role in the maintenance engineering applications. In summary, we have successfully developed and of the HSC niche. Both, VSEL cell abundance and CFU-f activity markedly implemented ECM arrays on mechanically tunable substrates to study stem decreased with age, which may be of relevance in respect to a possible sup- cell differentiation. This platform is flexible and can be adapted to study portive function of mesenchymal cells in hematopoiesis. A further investiga- other extracellular factors in any cell type. The combination of this novel tion of the relationship between VSEL cells and mesenchymal CFU-f should screening platform and statistically modeling not only improve fundamental help to better understand the role of VSEL cells in vivo. understanding of matrix control of stem cell lineage specification, but also have practical relevance for rationale design of biomaterials.

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Poster Board Number: 2521 pluripotent stem cells often employ serum based media formulations and utilize coculture systems with animal feeder cells. We now present a method MURINE VERY SMALL EMBRYONIC LIKE STEM for the routine generation of mesenchymal stem cells from multiple human CELLS ARE THE PRIMARY SOURCE OF BONE induced pluripotent stem cells (iPSCs). We started with iPSCs that were grown on Matrigel in serum free media (mTESR), removed them from their MARROW DERIVED LUNG EPITHELIAL CELLS adherent conditions and allowed them to form three dimensional aggregates Kassmer, Susannah H., Bruscia, Emanuela, Zhang, Ping-Xia, Krause, (embryoid bodies, ‘EBs’). After one day as EBs, we exposed the cultures to Diane S. the TGFβ signaling inhibitor SB 431542 for 9 days. After a total of 10 days of differentiation, cells were trypsinized and plated as adherent cultures. Un- Laboratory Medicine, Yale University, New Haven, CT, USA, Department of der these conditions cells could be passaged after an additional 4 to 5 days Pediatrics, Yale University, New Haven, CT, USA and often already showed a complete conversion to an MSC phenotype as Previous studies have demonstrated that bone marrow (BM) derived cells determined by flow cytometric marker analysis for CD73, CD44 and CD56. differentiate into non-hematopoietic cells of multiple tissues. In recent years, We then harvested cells from 3 iPSC lines in 2 different experiments and several studies demonstrated engraftment of BM derived cells as epithe- analyzed their gene expression pattern in comparison to the gene expres- lial cells of the lung. To date, it remains unknown which population(s) of sion of primary human MSCs. This analysis is currently ongoing. In addition, BM cells are responsible for this engraftment. To test the hypothesis that these cells could be efficiently differentiated into the adipocyte lineage as non-hematopoietic stem cells in the BM are the primary source of marrow determined by Oil-Red-O staining and we plann further validation of these derived lung epithelial cells, hematopoietic or non-hematopoietic BM cells cells by immunocytochemistry and gene expression analysis. Furthermore we were transplanted into irradiated surfactant-protein-C (SPC)-null mice. will test their multipotentiality using muscle and chondrocyte assays. Sum- From 2 to 6 months after transplantation, donor-derived, SPC-positive type mary: We established a robust and reproducible method to generate MSCs 2 pneumocytes were predominantly detected in the lungs of mice receiv- from multiple iPSC lines. These MSCs show a typical cell surface marker ing non-hematopoietic cells, and were exceedingly rare in mice receiving expression and can be differentiated into the adipocyte lineage. hematopoietic cells. (7 out of 9 recipients of nonhematopoietic vs. 1 out Poster Board Number: 2525 of 20 recipients of hematopoietic BM cells, p=0.0001). We hypothesized that primitive stem cells contained in the non-hematopoietic fraction of MORE EFFECTIVE REPAIR OF ISCHEMIC the BM are capable of differentiating into lung epithelial cells. A previously identifed population of very small embryonic-like (VSEL) stem cells in adult REGIONS BY ADIPOSE TISSUE-DERIVED MSCS bone marrow is capable of differentiating into all three germ-layer lineages IS ASSOCIATED WITH STRONGER EXPRESSION in vitro. VSELs are contained in the non-hematopoietic compartment of the OF CCL5 bone marrow, and do not express CD45. In order to test directly whether VSELs can become type 2 pneumocytes, VSELs or hematopoietic stem- and Kimura, Kenichi, Nagano, Masumi, Yamashita, Toshiharu, progenitor cells (HSPC) were isolated from H2B-GFP transgenic mice and Salazar, Georgina, Akimoto, Keiko, Mishima, Hajime, Matsushita, transplanted into SPC-KO recipient mice. Donor derived, SPC and H2B-GFP Shonosuke, Ohneda, Osamu positive type 2 pneumocytes were uniformly detected in the lungs of mice Regenerative Medicine and Stem Cell Biology, Graduate School of receiving VSELs, but not in mice receiving HSPC (3 of 3 vs. 0 of 3 mice, Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan, respectively. P=0.019). The levels of engraftment with nonhematopoietic Department of Orthopaedic Surgery, Graduate School of Comprehensive BM cells and VSELs were comparable. With Image Stream technology, the Human Sciences, University of Tsukuba, Tsukuba, Japan, Department most sensitive approach used, approximately 1 in 2400 lung epithelial cells of Cardiovascular Surgery, Graduate School of Comprehensive Human was a donor derived type 2 cell. We conclude that very small embryonic like Sciences, University of Tsukuba, Tsukuba, Japan stem cells contained in the non-hematopoietic fraction of murine BM are the primary source of marrow derived lung epithelial cells, and that VSELs in To better understand the correlation between the expression of mesenchy- adult bone marrow are a potential source of pluripotent stem cells for tissue/ mal stem cells (MSCs) markers and the ability to support tissue regeneration organ regeneration. in vivo, has we isolated MSCs from human adipose tissue, bone marrow and dental pulp and examined their ability to repair bone fracture or improve Poster Board Number: 2523 blood flow in ischemic regions using mouse model. These MSCs showed EFFICIENT GENERATION OF MESENCHYMAL similar cell morphology and expression pattern of cell surface markers analyzed by FACS. In vitro differentiation assays, each type of MSCs dif- STEM CELLS FROM IPSCS AND THEIR USE IN ferentiated into osteogenic, adipogenic, and chondrogenic lineages. Having DRUG DISCOVERY demonstrated that these MSCs had the potential to differentiate into three lineages in vitro, we confirmed the function of those stem cells acting in Nguyen, Leana, Mason, Mike, Strulovici, Berta, Grskovic, Marica, vivo by using a mouse bone fracture model. Bone calcification was signifi- Irion, Stefan cantly higher in mice transplanted with MSCs compared to control mice iPierian, South San Francisco, CA, USA injected with PBS. Furthermore, the degree of calcification at the joint region was higher in mice transplanted with adipose tissue-derived MSCs (AT- The increase in the prevalence and incidence of Type 2 diabetes is a major MSCs) compared to those transplanted with bone marrow-derived MSCs contributor to the rise in healthcare costs on a global scale. Adipocytes play (BM-MSCs) and dental pulp-derived MSCs (DP-MSCs). Histological analysis a crucial role in the etiology of the disease and an attractive therapeutic revealed that there were many more lamellar bones at the joint transplanted approach would be to identify compounds that reduce their resistance to with AT-MSCs. In order to compare the effects of transplantation of MSCs, insulin. However our ability to routinely use adipocytes for drug discovery a vascular occlusion models was prepared by ligating the proximal end of is limited by the heterogeneity between sample sources, which provides a the femoral vessel with the popliteal vessel in mice. Interestingly, the blood significant challenge for the interpretation of results. The ability to routinely flow in the ischemic region rapidly increased in mice injected with AT-MSCs. generate large numbers of adipocytes from a single well characterized donor At day 7 after the transplantation, the relative blood flow was significantly that exhibit a disease specific phenotype, would facilitate the development greater in AT-MSCs recipients compared to that in DP-MSCs and PBS recipi- of screens to identify bioactive molecules that revert the cellular phenotype ent mice. And we observed a significant accumulation of lectin-binding of insulin resistance. Adipocytes can be derived from mesenchymal stem and endothelial cells in the AT-MSCs recipients at the femoral region on day 7 progenitor cells (MSCs); however, current methods of deriving MSCs from

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Thursday Poster Abstracts and 14. The number of CD45-positive cells and F4/80-positive cells such Poster Board Number: 2529 as monocyte and macrophage at the femoral region were also higher in AT-MSCs recipients compared to DP-MSCs. In order to determine how AT- TWIST-1 INTERACTIONS INVOLVED IN MSCs support new vessels formation, the mRNA expressions of angiogenic MEDIATING MESENCHYMAL STROMAL/CELL and migration factors were evaluated by RT-PCR under the normoxic and hypoxic conditions. We also found that the expression of CCL5 mRNA was PROLIFERATION, DIFFERENTIATION AND higher in AT-MSCs than BM-MSCs and DP-MSCs. These results suggest LINEAGE COMMITMENT that AT-MSCs has promising potential for bone tissue engineering and cell therapy applications. Cooper, Lachlan, Isenmann, Sandra, Arthur, Agnieszka, Cakouros, Dimitrios, Hynes, Kim, Zannettino, Andrew C.W., Gronthos, Stan Poster Board Number: 2527 Haematology, The Robinson Institute Centre for Stem Cell Research / IMVS OSTEOPONTIN PLAYS A CRITICAL ROLE IN / Hanson Institute, Adelaide, Australia, Haematology, IMVS / Hanson Institute, Adelaide, Australia, Haematology, SA Pathology / Hanson DIRECTING OSTEOBLASTOGENESIS AND Institute / Cardiac Repair Group, Adelaide, Australia ADIPOGENESIS OF MESENCHYMAL STEM The TWIST family of genes, Twist-1 and Dermo-1, are basic helix-loop- CELLS helix transcription factors that mediate mesodermal tissue development and contribute to correct patterning of the skeleton. We have demonstrated Chen, Qing, Shou, Peishun, Zhang, Liying, Su, Juanjuan, Xu, that enforced expression of Twist-1 and Dermo-1 in human bone marrow Chunliang, Li, Wenzhao, Huang, Yin, Chen, Xiaodong, Hu, derived mesenchymal stem cells (MSC) increases the lifespan of these cells Gangzheng, Denhardt, David T., Shi, Yufang and promotes adipogenic differentiation while inhibiting osteogenesis. The Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai present study identified 350 differentially expressed genes in human MSC Institutes for Biological Sciences, Chinese Academy of Sciences/Shanghai overexpressing Twist-1, using cDNA microarray analysis (Illumina Human Jiao Tong University School of Medicine, Shanghai, China, Department of v8). Confirmatory real-time PCR demonstrated that the Wnt pathway Molecular Genetics, Microbiology and Immunology, University of Medicine genes, Wnt2 and Wnt2b, were upregulated in human MSC following and Dentistry of New Jersey-Robert Wood Johnson Medical School, enforced expression of Twist-1. Both Wnt 2 and 2b were selected for further Piscataway, NJ, USA, Department of Cell Biology and Neuroscience, investigation based on their key roles in mesenchymal cell fate determina- Nelson Biological Laboratories, Rutgers, The State University of New Jersey, tion and lineage commitment. Putative Twist-1 (E-box) binding sites within Piscataway, NJ, USA the promoter regions of the genes of interest were identified at nine sites for Wnt2. The promoter regions of both Wnt2 and 2b, up to 3kb from the Osteopontin (OPN), a primarily secreted protein found in all body fluids, transcriptional start sites, were amplified and cloned into luciferase reporter is a multifunctional cytokine functionally important in bone remodeling, vectors (PGL3-basic, Promega) to demonstrate direct interactions between progression of autoimmune disease, cancer metastasis, and responses to Twist-1 protein molecules and the promoter regions of the genes of interest. stress. In addition, a non-secreted form of OPN has also been implicated CHIP analysis was subsequently used to confirm the putative Twist-1 DNA in a growing number of cellular processes, including migration, fusion, and binding sites on the respective promoters. These studies have identified motility. Since OPN is important in regulating bone formation, we examined Twist-1 as a potential master switch that determines the cell fate of MSC. the role of OPN in the differentiation of mesenchymal stem cells (MSCs) and found that OPN-deficient MSCs have a high tendency to differentiate into Poster Board Number: 2531 adipocytes and a reduced osteoblastogenesis potential under their respective differentiation conditions. Antibodies to OPN or siOPN could also promote IMPROVED CALCIFICATION OF ARTIFICIAL the adipogenic differentiation of wild type MSCs. Furthermore, addition of BONE TISSUE IN 3D ALGINATE-COLLAGEN exogenous recombinant OPN could restore the osteoblastogenesis ability of OPN-deficient MSCs but had no effect on adipogenic differentiation. CAPSULES MADE FROM ULTRA-HIGH- We also found that in absence of OPN expression, c/EBPβ, a key transcrip- VISCOSITY (UHV) ALGINATE tion factor for adipose-specific genes expression, was highly up-regulated. Marcin, Jurga, Ben Azouna, Nesrine, Le Roy, Helene, Schulz, Julia This OPN-mediated c/EBPβ regulation was reproducibly correlated with adipogenesis. Thus, our results suggest that extracellular OPN plays an C., Zimmermann, Heiko, Forraz, Nico, McGuckin, Colin P. important role in osteoblastogenesis, while intracellular OPN is important for Cell Therapy Research Institute, Saint-Priest, France, Division of Biophysics adipogenesis. Our studies revealed a novel mechanism of action of OPN in and Cryotechnology, Fraunhofer Institute for Biomedical Engineering, St. the differentiation of mesenchymal stem cells. Ingbert, Germany, Fraunhofer Institute for Biomedical Engineering; Saarland University, St. Ingbert, Germany Transplantation of bioactive macroporus scaffolds is very effective in regen- eration of complicated bone fractures, orthodontics and reconstitution of bone deficits (e.g. cleft palate in children). Mesenchymal stem cells (MSCs) can support bone regeneration process due to their osteogenic and immuno- modulatory properties. In our study we have used an easy accessible MSCs derived from human umbilical Wharton’s jelly for 3D bone tissue engineer- ing in novel 3D alginate-collagen scaffolds made from ultra-high viscosity (UHV) alginate. We found that artificial bone tissue could be generated in defined serum-free medium containing dexamethasone and BMP2. Calcifi- cation process indicated by OsteoImage assay and Alizarin Red staining was significantly increased when MSCs were encapsulated in alginate-collagen 3D scaffolds in comparison to standard 2D microplate cultures. Moreover, our results have shown that collagen was necessary for MSCs differentiation into osteoblasts and for deposition of extracellular calcium in serum-free culture conditions. Generation of 3D artificial bone implants based on animal

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free bioscaffolds and defined media will be very useful for regenerative Poster Board Number: 2535 medicine application. ISOLATION, IDENTIFICATION AND ADIPOCYTE Poster Board Number: 2533 DIFFERENTIATION OF HYPERBARIC OXYGEN- HOMING OF ALLOGENIC OVINE TREATED EQUINE PERIPHERAL BLOOD- MESENCHYMAL STEM CELLS IN DERIVED MESENCHYMAL STEM CELLS COLLAGENASE INDUCED TENDONITIS OF THE Dhar, Madhu, Neilsen, Nancy, Eaker, Shannon, Adair, Henry, Geiser, ACHILLE’S TENDON Dennis Crovace, Antonio, Lacitignola, Luca, Rossi, Giacomo, Francioso, Department of Large Animal Clinical Sciences, The University of Tennessee, Edda Knoxville, TN, USA, Department of Pathobiology, The University of Tennessee, Knoxville, TN, USA, GE Healthcare, Knoxville, TN, USA D.E.T.O., University of Bari, Valenzano (BA), Italy, Veterinary Science, University of Camerino, Matelica (MC), Italy Little is known about the identification and isolation of mesenchymal stem cells (MSCs) derived from equine peripheral blood. Questions regarding the Objectives: Mesenchymal stem cells (MSCs) clinical utility is due to their proliferation, differentiation and self-renewal of equine MSCs remain to be convenient isolation, their lack of significant immunogenicity permitting elucidated. We set out to isolate MSCs from equine peripheral blood, while allogenic transplantation without immunosuppressive drugs, their lack of performing immunophenotypic analysis, and determining overall growth ethical controversy. The pourpose of this study is to evaluate the efficacy potential in vitro. Although the identification of horse antigens using anti- and the homing of local injection of allogenic MSC marked with Red Fluo- human antibodies remains an ongoing hurdle for equine immunologists, we rescent Protein (RFP) in collagenase induced tendonitis in the ovine Achille’s screened a variety of anti-mouse and anti-human antibodies, including CD tendon. Materials and Methods: Four sheep (2 years old, female, 45 Kg markers know to be present on human MSCs. We also evaluated and as- weight) have been enrolled in the study. The sheep have been investigated sessed the potential of hyperbaric oxygen (HBO2) to mobilize MSCs into the to exclude any previous Achilles’ Tendon lesion. Three weeks before starting blood stream. Although MSCs from Ficoll-separated buffy coat contained of the study one sheep was randomly selected for Bone Marrow harvesting a heterogeneous population of CD90 positive and negative cells, only the for MSC cultivation. The MSC obtained were trasfected with a Lentivirus CD90+ cells expanded and subsequently differentiated into adipocytes. for the integretion of gene for expression of Red Fluorescent Protein (RFP). MSCs remain CD51+ throughout the isolation and expansion processes. After a week the other 3 sheep were injected in both Achilles’ Tendon NANOG and SOX2 mRNA expression was also present in the CD90+ equine with 400 U.I. of Collagenase IA of Cl. hystoliticum. After two weeks the MSC population. Oil-red-O staining and increased mRNA expression of left Achilles’Tendon of each sheep was injected with a solution of 6x106 peroxisome proliferator-activated receptor gamma was used to determine MSCRFP in one ml of Fibrine glue (TISSUCOL, Baxter). The other tendons adipogenesis potential. Interestingly, a 2-8 fold increase in CD90+ cells after were used as negative control and received as a treatment the injection of three HBO2 treatments was observed, possibly exhibiting a mobilization ef- the same volume of saline solution. At 3-4-6 weeks from the treatment the fect of MSC’s into peripheral blood. Three horses, which did not yield viable tendons have been harvested and evaluated for Morphology, Collagen I MSCs prior to HBO2 treatments, produced viable MSCs after exposure to and III expression, presence of CD34+ cells and visualized at fluorescence HBO2, having the ability to expand and differentiate into adipocytes. These microscope to assess RFP expression of grafted MSC. Results: The results of data reveal that peripheral blood-derived MSCs contain a relatively lower these investigations showed RFP -MSC presence in treated tendons respect number of CD90+ cells (compared to the adipose tissue), and can be mo- to the control ones at 3, 4 and 6 weeks after the treatment. Moreover, the bilized into circulation by HBO2 resulting in viable MSCs. Future work will RFP positive tissue showed high expression of Collagen I and low Collagen center around expanding screen of antibodies in the equine MSCs, as well as III with, good morphology compared with the lesions treated with the saline cellular transplantation studies in injured horses. solution. The presence of high expression of collagen I and low collagen III with good morfopholgy, in term of restored tendon architecture, can be re- Poster Board Number: 2537 lated to the presence of the MSCs into tendon lesions, because a large num- ber of cells can home in the site of injection. In MSCRFP treated tendons a SIRNA-MEDIATED KNOCKDOWN OF marked expression of CD34+ was detected at each time period. Conclusion: VINCULIN MODULATES MAPK1 ACTIVATION These results showed that intralocal administration of MSC into the tendon lesion can lead to a good effect of injured tendon. The local infusion delivery AND STIFFNESS-BASED STEM CELL entails injecting MSCs directly into the tissue of interest and guarantees a DIFFERENTIATION higher number of engrafted cells and optimal therapeutic effect. Indeed the presence of high positve RFP cells in treated samples have been evaluated Holle, Andrew W., Vijayraghavan, Deepthi, Del Alamo, Juan Carlos, at 3, 4 and 6 weeks after the treatment. We evaluate also, that quality of Engler, Adam J. tendon healing in MSCRFP treated tendons has been based on a better Bioengineering, University of California San Diego, La Jolla, CA, USA, architecture of collagen fibers and high expression of Collagen I respect to Mechanical and Aerospace Engineering, University of California San Diego, Collagen III, related to the control tendons. Moreover, in control tendons, La Jolla, CA, USA no RFP cells have been detected. This phenomenon shows that even when The stiffness of the extracellular matrix (ECM) of mesenchymal stem cells there is a lesion in another tendon, the cells injected intralocally home in (MSCs) has been shown to influence proliferation, migration, and differ- on the treated area and persist in that zone until 6 weeks from treatment. entiation. Despite serious scientific inquiry, a consensus on the signaling Interestingly we evaluated that in MSCRFP treated tendons there is a high pathways that are necessary and sufficient for this mechanosensitive ability expression of CD34+ cells. These findings can be explained to a chemotactic has yet to be reached. Force-sensitive, membrane-localized channels have effect of the MSC on CD34+ cells, even if their role is still unknown. The been shown to be upregulated on stiff substrates, but blocking stretch- data obtained in this study confirm that MSC allograft have a positive effect activated channels (SACs) did not affect stiffness-induced expression of on tendon healing and that the local injection in the tendon allow the hom- Myogenin D (MyoD), a muscle differentiation marker, although other routes ing of MSC with good engraftment efficiency. for calcium ions into the cell are feasible. Calcium-depleted culture media, which reduced stiffness-induced MyoD expression by 30%, appeared to inhibit Mitogen-Activated Protein Kinase (MAPK) phosphorylation, whose

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Thursday Poster Abstracts pharmacological inhibition also blocked differentiation. Mechanosensitive their use. An unlimited supply of exogenously derived liver cells would MAPK activation could also occur via contractility-induced conformational facilitate development of cell-based therapies for the treatment of life- changes of focal adhesion proteins, namely vinculin, which has been shown threatening liver diseases .Recent evidence have shown that hepatocyte-like to expose a cryptic binding domain for MAPK1 upon force-induced co- cells (HLC) can be generated from non-liver cells including bone marrow, activation with Talin. In situ vinculin siRNA knockdown resulted in an 80% blood monocytes, umbilical cord, amniotic cells, and even skin fibroblasts. decrease in stiffness-induced MyoD after four days on muscle-like 11 kPa The hepatogenic potential of mesenchymal stem cells from different sources hydrogels and reduced MAPK1 activation. The effect of vinculin knockdown being known and with the clinical use of MSC is fast becoming a reality, on focal adhesion assembly, adhesion strength, and cell contractility was HLC differentiated from MSC hold promise for future clinical applications. assessed with by immunofluorescence, spinning disc assay, and 3-D traction Furthermore, umbilical cord has been shown to be a good source of MSCs force cytometry, respectively, indicating vinculin’s importance in converting (UCMSCs) and is currently the best option available to take cell therapy biophysical information into biochemical signals. Together, these data sug- from autologous to allogeneic platform due to its immunoprivileged nature. gest the first in situ evidence that force-sensitive focal adhesion proteins can The basic aim of this study was to explore the potential of UCMSCs to dif- activate stem cell differentiation signals. ferentiate to hepatocyte-like cells and assess the in vitro functionality of the differentiated cells with regards to albumin secretion and urea production, Poster Board Number: 2539 the gold standard for hepatocyte differentiation. Previous study from our MOBILIZATION OF BONE MARROW laboratory have reported the isolation and characterization of MSC from umbilical cord The growth kinetics, immunophenotype and differentiation MONONUCLEAR CELLS BY ADULT NEURAL potential of these UCMSCs have been studied and over 90% of these cells STEM PROGENITORS CELLS INVOLVES A SDF1- exhibited a MSC phenotype of CD73+/CD105+/CD29+/CD44+/SSEA4+/ HLA-ABC+/HLA-DR-/CD45-/CD14-/CD31-/vWF-. These cells upon DEPENDENT MECHANISM sequential treatment to pro hepatic factors like EGF, FGF, HGF, nicotinamide, Lugo-Martinez, Veronica-Haydée, Sii-Felice, Karine, Wade-Gueye, OncostatinM and Dexamethasone showed a distinct change in morphology Marième, Squiban, Claire, Buard, Valérie, Benderitter, Marc, from fibroblastic cells to polygonal cell structure with prominent nucleus, Tamarat, Radia The cells also expressed endodermal lineage specific markers AFP , HNF-3 beta, HNF-4 alpha and Sox-17. On maturation, these differentiated cells Service de Radiobiologie et Epidemiologie, Institut de Radioprotection et expressed CK18, albumin and TDO2, indicative of fully differentiated Sûreté Nucleaire, Fontenay-aux-roses, France hepatocyte with down regulation of undifferentiated MSC markers. The Objective: Recent reports have demonstrated that vasculogenesis and differentiated cells are being currently analyzed for their in vitro functionality neurogenesis share common factors and signaling pathways. However, the using Albumin assay, Urea production assay, PAS staining for glycogen stor- mechanisms involved in these interactions remain undefined. We hypoth- age and LDL uptake analysis. esized that adult neural stem progenitor cells (NSPC) enhance bone marrow Poster Board Number: 2543 mononuclear cells (BM-MNC)-related effects and may participate to wound healing. Methods and Results: NSPC and BMMNC were administrated to EFFECT OF HAZARDOUS CHEMICALS ON THE skin punched wounds of both non irradiated and irradiated mice (20 Gy, lo- cally). At day 3, NSPC/BMMNC co-transplantation promoted dermal wound CORD BLOOD CELLS healing and enhanced wound closure by 1.4- and 1.3-fold, respectively in McKee, Christina, Mirza, Asma, Dailey, William, Ciavattone, both non irradiated and irradiated mice compared to treatment with NSPC Nicholas, Chaudhry, Faisal, Bloda, Martin, Dinda, Sumi, Chaudhry, alone. We also showed that GFP-positive NSPC incorporated into new G. Rasul dermal tissue by their capacity to differentiate into CD31-positive endothe- lial cells and into O4-positive neural cells. Results obtained from co-culture Dept of Biological Sciences, Oakland University, Rochester, MI, USA, of NSPC and BMMNC suggested that NSPC upregulated VEGF levels and School of Health Sciences, Oakland University, Rochester, MI, USA promoted BMMNC proliferation, assessed by Ki67-positive staining, and dif- A large number of chemicals are introduced for industrial and pharmaceuti- ferentiation into cells with endothelial phenotype, double positive for both cal purposes every year. Despite their stringent safety screenings, some DilLDL and BS1lectin. In addition, in vivo, NSPC administration increased chemicals still prove to be hazardous to environmental and human health. plasma levels of nerve growth factor (NGF) and SDF-1. Conclusion: NSPC This is particularly important for those chemical which cause early develop- trigger BMMNC mobilization through a SDF-1-related pathway, and partici- mental defects. These chemicals are classified as teratogens. Another class of pate to dermal wound healing in physiological and pathological conditions. chemicals that causes mutations is called mutagens. However, the study of Poster Board Number: 2541 both these types of chemicals and drugs is difficult due to the lack of model systems needed to investigate their effects on early development. The avail- DIFFERENTIATION OF UMBILICAL CORD ability of embryonic and cord blood stem cells prompted us to hypothesize that they can be used to examine developmental and mutagenic effects of DERIVED STEM CELLS TO HEPATOCYTE-LIKE- hazardous chemicals. We exposed stem cells to several known and unknown CELLS IN VITRO teratogenic and mutagenic agents and analyzed the growth and differentia- tion of cells as well as expression of specific genes. These results indicated Sarang, Shabari, Tillu, Parnita, Ravindran, Geeta, Viswanathan, that 5-fluorouracil and 6-mercaptopurine are highly cytotoxic at 10-8 M Chandra concentration and induced differentiation in both embryonic and cord blood Regenerative Medicine, Reliance Life Sciences Pvt. Ltd., Navi Mumbai, stem cells. 5-Azacytidine was cytotoxic at 10-7 M concentration in stem India cells. Persistent and widely used polychlorinated biphenyls (PCBs) were also Liver Cirrhosis is generally irreversible and treatment usually focuses on found to be highly cytotoxic in the tested cells. Further studies are in prog- preventing progression and complications. In advanced stages of cirrhosis, ress to determine molecular basis of the toxicity of these chemicals. liver transplantation is the main therapeutic procedure at present. Due to a serious shortage of liver donors and the high risk of transplant rejection an alternative therapeutic approach is needed for patients with liver failure. Hepatocyte cell transplantation has been the alternative option for end stage liver failure patients, but low availability of functional hepatocytes restricts

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PRE-CLINICAL AND CLINICAL APPLICATIONS OF Poster Board Number: 2549 A CHEMICALLY DEFINED, SERUM MESENCHYMAL STEM CELLS FREE GROWTH MEDIUM FOR HUMAN Poster Board Number: 2547 MESENCHYMAL STEM CELLS FROM VARIOUS CD105+-MESENCHYMAL STEM CELLS TISSUES MIGRATES FROM PERIPHERICAL BLOOD INTO An, Songzhu (Michael), Zhu, Yanan OSTEOARTHRITIS JOINT: AN ANIMAL MODEL StemRD Inc., Burlingame, CA, USA Human mesenchymal stem cells (hMSCs) hold great promise for the treat- Arufe Gonda, Maria, De la Fuente González, Alexandre, Lesende ment of a wide variety of ailments such as acute myocardial infarction, Rodriguez, Iván Aarón, Fuentes Boquete, Isaac, De Toro, Fco., autoimmune diseases, and bone or cartilage disorders. The hMSCs can be Blanco García, Fco. derived from various tissues, including bone marrow, adipose tissue, and Medicine, University of Coruña, A Coruña, Spain, INIBIC-CHUAC, A umbilical cord and cord blood. Traditionally, expansion of hMSCs has been Coruña, Spain achieved by culturing hMSCs in medium supplemented by fetal bovine serum (FBS). However, concerns have been raised over animal-born patho- Introduction and objectives: There are not clear evidences about the migra- gens, immune reactions and lot-to-lot variations caused by the undefined tion of mesenchymal stem cells from a systemic circulation to another components in FBS. To address these issues, we have developed a chem- niche as an injury place in the organism. The current study treats to answer ically-defined, serum-free and animal-free medium, named “MesenGro” this question. Material and methods: We have injected via intravenous for the expansion of hMSCs. Our studies show that MesenGro is suitable and directly into the monkey joint a CD105 enriched sub-population of for the expansion of hMSCs from various tissues, including bone marrow, mesenchymal stem cells (MSCs) from human synovial membrane, previously adipose tissue, and umbilical cord blood. MesenGro performs better than characterized by flow cytometry revealing that were positive for CD90 and FBS-containing medium in supporting robust expansion of hMSCs. Another CD44 and negatives for CD34 and CD45 indicating that presence of MSCs advantage of MesenGro is the simplification of cell passage procedures. markers. CD105 enriched sub-population of MSCs was labelled with oxac- Most market products of serum-free hMSC media require pre-coating of the arbocyanine -DiO (green) when they were injected via intravenous (IV) and tissue culture surface with an expensive, proprietary formulation in order with octadecyl (C18) indocarbocyanine -DiI (red) when they were injected to achieve optimum attachment of hMSCs. We have tested different cell directly into the knee, intra-articular (IA), to follow their evolutions through culture surfaces for use with MesenGro, and found that certain surfaces can the animals injected by different ways and their localization. The animal support maximum cell attachment without the need for pre-coating. This models used were adult monkeys Macaca Fascicularis which had been unique feature of MesenGro greatly reduces the time and cost involved in injured into the left knee to create an Osteoarthritis (OA) animal model and hMSC passage. Finally, we show that extensive culture in MesenGro main- the right knee was used as control. One group of two animals was injected tains full multi-lineage differentiation potentials of MSCs. In sum, MesenGro twice in the vein with 1x106 CD105+-MSCs labelled with DiO only. Another serves as an ideal substitute to FBS-containing growth medium and offers two animals were injected directly in the knee twice with 1x106 CD105+- the advantages of being completely chemically-defined as well as simplifying MSCs labelled with DiI at the same time these animals were injected twice in the passage procedure over the existing market products. the vein with 1x106 CD105+-MSCs labelled with DiO. The injections were realized into the animals with an interval of one week between them. The Poster Board Number: 2551 animals were sacrificed one month after treatment. Results: Immunohis- tochemistry analysis of the tissues from the animals revealed that CD105+- EFFICIENT CLINICAL-SCALE PRODUCTION MSCs migrated towards the injured knee joint. Neither labelled cells nor OF HUMAN MESENCHYMAL STEM CELLS IN teratomes were found in other vital organs into the animals. The CD105+- MSCs labelled with DiI (red) and with DiO (green) were only located in COMPUTER-CONTROLLED MICROCARRIER the injured knee and they were not found in the normal one. Furthermore BIOREACTORS IN A DEFINED MEDIUM CD105+-MSCs labelled with DiI (red) and with DIO (green) were found in crossed ligaments and knee muscle (vastus medialis), synovial membrane, Panchalingam, Krishna M., Jung, Sunghoon, Paramchuk, Wendy J., both meniscus and cartilage of patello femoral groove all of them only in the Rosenberg, Lawrence, Behie, Leo A. injured joint and not in the normal one. An exhaustive immunofluorescence University of Calgary, Calgary, AB, Canada, McGill University, Montreal, analysis of DiO labelled cells revealed that they were negatives for CD68, QC, Canada which is a macrophage antibody marker, and some of them were positives Human mesenchymal stem cells (hMSCs) have many potential applications for SDF-1, which is a marker of osteoclastogenesis and produces liberation in tissue engineering and regenerative medicine. In fact, much evidence of IL-6. Conclusions: These results seem indicate that: 1) The MSCs injected in the literature makes compelling arguments for their use in cell therapy in a peripherical vein go to injured tissue, in this case injury knee. 2) The treatments for multiple sclerosis (MS) and diabetes. Currently, hMSCs are MSCs injected directly in an injured place keep in the same place. 3) The generated through conventional static adherent cultures in the presence of MSCs could be recruitment by SDF-1. fetal bovine serum (FBS) or human-sourced supplements for clinical applica- tions. However, these methods suffer from variable culture conditions (i.e. ill-defined medium components, heterogeneous culture environment, and limited growth surface area per culture), and thus are not ideal procedures to meet the expected future demand of quality-assured hMSCs for human therapeutic use. Therefore, it has been necessary to develop an effec- tive well-characterized hMSC production protocol, which is controllable, reproducible and scalable. As a first step, we have developed a new defined medium that supported the isolation and expansion of multipotent hMSCs from bone marrow under serum-free conditions. We demonstrated that our defined medium was significantly superior to FBS-containing media for the

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Thursday Poster Abstracts reproducible isolation and expansion of hMSCs, leading to a significantly duced for a minimally manipulated point-of-care cell therapy. Future studies reduced lag phase and an increased growth rate. Moreover, hMSCs cultured will explore injection volume on adipose tissue generation and any diffusion in our medium displayed a higher clonogenic capacity and a superior degree limiting effects. Additionally, biocompatibility and safety/toxicity evaluations of homogeneity in morphology and phenotype, compared to FBS-based in accordance with ISO-1099 will be performed. controls. In follow-up studies, we targeted the design of a scalable biopro- cess under computer control in suspension bioreactors using conventional Poster Board Number: 2555 microcarrier technology. Unlike some published results, our microcarrier pro- THERAPEUTIC POTENTIAL OF HUMAN cedure did not suffer from prolonged lag phases and diminished cell growth rates. Specifically, when hMSCs were inoculated at 2.4 x 104 cells/mL into ADIPOSE TISSUE-DERIVED MESENCHYMAL our suspension bioreactors containing Cytodex 3 microcarriers in our defined STEM CELLS FOR ALZHEIMER’S DISEASE medium, we achieved a maximum cell density was 4.38±0.23 x 105 cells/ mL (i.e. cell-fold expansion of 18.3) within 6 days of culture for a 3.05 cell- Katsuda, Takeshi, Takeshita, Fumitaka, Sakai, Yasuyuki, Ochiya, fold expansion/day. On the other hand, experiments with Cytodex 3 and Takahiro a conventional FBS-containing medium yielded a maximum cell density of Division of Molecular and Cellular Medicine, National Cancer Center 2.08±0.24 x 105 cells/mL on day 11 (i.e. cell-fold expansion of 8.7) giving a Research Institute, Tokyo, Japan, Institute of Industrial Science (IIS), The 0.79 cell-fold expansion/day. These data suggest that the use of computer- University of Tokyo, Tokyo, Japan controlled bioreactor technology together with our new defined serum-free medium represents an efficient method for the generation of clinical-scale Introduction: The pathological hallmark of Alzheimer’s disease (AD) is production of hMSCs in a controllable, reproducible and scalable process. intracerebral accumulation of amyloid-β peptide (Aβ) deposits. Three kinetic factors which determine the steady-state concentration of Aβ in the brain Poster Board Number: 2553 are (i) the rate of Aβ generation from Aβ precursor protein (APP), (ii) the rate of transport of Aβ across the blood-brain barrier and (iii) the rate of Aβ IN VITRO EVALUATION OF ADIPOGENESIS degradation in the brain parenchyma. Neprilysin (CD10) is a rate-limiting FROM HUMAN ADIPOSE DERIVED STEM Aβ-degrading enzyme in the brain, and is, therefore a potential target for treatment of AD. Recently, we have found by microarray analysis that hu- CELLS man adipose tissue-derived mesenchymal stem cells (hAT-MSCs) express Atzet, Sarah, Tandeski, Terry, Patel, Neel, McCollough, Amanda, neprilysin gene. Considering the homing ability of hAT-MSCs to injured sites, Prestwich, Glenn D., Zarembinski, Thomas I. we hypothesized that transplantation of hAT-MSCs to AD model animals would restore the accumulation of Aβ deposits in the brain. Here, we report Glycosan Biosystems, Salt Lake City, UT, USA, University of Utah, Salt Lake the results of in vitro study on therapeutic potential of hAT-MSCs. Materi- City, UT, USA als: We used hAT-MSCs obtained from three donors (hAT-MSCs#1, #3, and Currently there are no permanent solutions for the repair of soft tissue #4). We investigated the expression of neprilysin of these cells at mRNA contour defects due to tumor resection, congenital defects, and trauma. and protein levels by quantitative RT-PCR (qRT-PCR) and western blot- While a few inert materials (i.e. plastics) have been proposed for implanta- ting, respectively. We also examined the neprilysin enzymatic activity in the tion to fill a subcutaneous defect, none can fully restore the lost adipose cell lysates using a fluorogenic peptide substrate. Results: We detected the tissue. In this work we propose to use the stromal vascular fraction (SVF) neprilysin gene expression in all the hAT-MSCs by qRT-PCR. The expression and the inherent adipose derived stem cells (ADSC) in combination with levels of neprilysin were varied by 2.5-fold among donors and it should be HyStem-C, a hyaluronic acid based matrix, to address soft tissue contour addressed that its expression level of the hAT-MSCs was higher than those defects and eventually as a means to replace lost adipose tissue. SVF is of bone marrow-derived MSCs. Accordingly, we detected neprilysin expres- obtained from human lipoaspirates that are routinely discarded. Hystem-C sion at protein level by western blotting. Furthermore, we confirmed that all is a semi-synthetic extracellular matrix (ECM) hydrogel that can be used for the hAT-MSCs exhibited abundant neprilysin enzymatic activity. Conclusion: either 2D or 3D cell cultures or as a minimally invasive delivery matrix for Our results of in vitro study suggested that hAT-MSCs had the capacity cellular therapies. In this work we will (1) evaluate HyStem-C’s functionality to contribute to clearance of Aβ accumulated in the brain. hAT-MSC will as a matrix for ADSC culture (both 2D and 3D), (2) evaluate the effects of provide a novel therapeutic approach against AD. seeding density on cellular growth, (3) examine the qPCR profile for ADSC cultured on HyStem verses tissue culture polystyrene, and (4) the ability to Poster Board Number: 2557 differentiate ADSC to mature adipocytes in vitro on and in HyStem-C. Adi- THE CONVENTIONAL DENSITY GRADIENT pose derived stem (ADSC) cells were isolated from human lipoasperates as previously described. The stromal vascular fraction (SVF), which contains the SEPARATION TECHNIQUE IS NOT AN EFFECTIVE ADSCs, was also isolated. ADSCs or SVF were added to pre-gel HyStem-C METHOD FOR THE ISOLATION OF HUMAN solutions to obtain 3D cell cultures. For 2D culture cells were plated on top of gelled HyStem-C. Various cell seeding densities and the effects of SVF BONE MARROW MESENCHYMAL STEM CELLS were examined. ADSC were cultured on either TCPS or HyStem for 2 weeks Ahmadbeigi, Naser, Babaeijandaghi, Farshad, Mortazavi, Yousef, at which point cells were isolated and gene expressions for the two condi- Gheisari, Yousof, Vasei, Mohammad, Rostami, Shahrbanoo, tions and the initial cell isolation were evaluated. Cultures were allowed to Soleimani, Masoud proliferate to approximately 80% confluency at which point adipogenesis differentiation media was added to the culture. After 14 days, cultures were Department of Hematology, Tarbiat Modares University, Tehran, Islamic fixed and stained using H&E and Oil Red O to identify mature adipocytes. Republic of Iran, Stem Cell Biology Department, Stem Cell Technology Differences in culture conditions were identified by quantitative image Research Center, Tehran, Islamic Republic of Iran, Department of Pathology, analysis. With over 200,000 liposuction procedures each year, subcutaneous Zanjan University of Medical Sciences, Zanjan, Islamic Republic of Iran, adipose tissue is an abundant and readily accessible source for ADSC. The Department of Pathology, Shariati Hospital, Tehran University of Medical stromal vascular fraction (SVF) which contains ADSC as well as circulating Sciences, Tehran, Islamic Republic of Iran, Hematology, Oncology and BMT blood cells, fibroblasts, pericytes, endothelial cells and preadipocytes can be Research Center, Shariati Hospital, Tehran University of Medical Sciences, easily obtained from lipoaspirate samples. In this work, we determined that Tehran, Islamic Republic of Iran the minimally manipulated SVF results in comparable tissue genesis as the Bone marrow (BM) derived mesenchymal stem cells (MSCs) have attracted more purified ADSC. This is significant in that the regulatory hurdles are re- increasing attention in regenerative medicine and tissue engineering. As

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Thursday Poster Abstracts

MSCs represent a small fraction of BM population, density gradient separa- may also be a useful method for enhancing the engraftment and homing of tion technique followed by an in vitro expansion step is commonly used HSCs. to enrich this population for cell therapy applications. Herein, we assessed the efficiency of density gradient separation technique as the conventional Poster Board Number: 2561 initial step in MSC isolation which has not been studied so far. Accordingly, TARGETING HUMAN MESENCHYMAL STEM after centrifuge of 10 mL of BM samples of healthy donor patients (n=10) on Ficoll-Paque gradient, the upper (serum and mononuclear cell layer) CELLS TO MURINE KIDNEYS USING PULSED and lower (Ficoll-Paque medium and RBC pellet) fractions were cultured FOCUSED ULTRASOUND: IMPLICATIONS FOR separately using standard MSC culture and RBC lysis protocols, respectively. Quantification and characterization of the seeded cells after a 10-day culture STEM CELL THERAPY period showed that following density gradient separation of BM samples, Ziadloo, Ali, Burks, Scott R., Chaudhry, Aneeka, Gold, Eric, Dean, 44±25.6% and 56±25.6% of MSCs had been segregated in the upper and Dana, Jordan, Kay, Frankel, Victor, Frank, Joseph A. lower fractions, respectively. We hypothesized that aggregation of MSCs in Radiology and Imaging Sciences, National Institutes of Health, Bethesda, native BM, could simply explain the deposition of MSCs in the lower frac- MD, USA tion. To examine this hypothesis, 10 mL of BM samples (n=10) were filtered through a 40 µm mesh. The filtrate and the residue were cultured separately. Introduction: Stem cell therapy has shown promise in various diseases; The cells were assessed immediately after seeding and also every day for 10 however, the utility of this treatment is limited by inefficient homing of cells days. It was shown that the most portion of MSCs do not filtered through to targeted pathology following intravascular injection. Continuous focused the mesh (59.4±22.8% vs 40.6 ±22.8%). The filtrate and the residue con- ultrasound (FUS) exposures are used clinically to treat uterine fibroids and sisted mainly of MSC aggregates of different size. Close assessment of the ablate a variety of tumor types in cancer treatment. Alternatively, pulsed cell aggregates, we found that some of them are partially composed of fat (pFUS) exposures have non-destructive mechanical effects (acoustic radia- particles which could probably explain those MSCs segregated in the upper tion forces, cavitation) that enhance tissue permeability and increase drug portion following density gradient separation. Therefore, it can be concluded and gene delivery. In this study, we demonstrated that pFUS to a kidney that MSCs are presumed to be present mainly as cell aggregations in native induces local inflammation and enhances migration of superparamagnetic BM. According to the clump nature of MSCs, the colony forming unit fibro- iron oxide nanoparticle (SPION)-labeled mesenchymal stem cells (MSC) blasts (CFU-F) assay as the common method for counting MSCs, does not injected intravenously. MSC migration was monitored by MRI and findings properly assess the MSC yield and underestimates the frequency of these were correlated to histology and expression of proinflammatory cytokines cells in BM at the level of 10-100 MSC per 1x10^6 BM cells, as reported and growth factors in the kidney. Results: In vivo and ex vivo T2*-weighted by some investigators. The gradient separation technique is not an efficient MRI showed migration of SPION-labeled MSCs to the pFUS-treated kidney method for MSC isolation. Instead, using small pore size filters could ef- and not to the contralateral kidney. Histological analyses revealed the pres- fectively isolate MSCs. ence of MSCs in peritubular regions and rarely in glomeruli on days 1 and 3 post-pFUS. No major changes in cytoarchitecture were observed with pFUS, Poster Board Number: 2559 only minor structural changes were noted in the pFUS-treated kidney (tran- A STRATEGY FOR ENHANCING THE sient disorganization of brush borders in proximal tubules) which resolved by day 7 post-pFUS. Furthermore, we characterized the molecular responses of ENGRAFTMENT OF HUMAN HEMATOPOIETIC tissue to pFUS and show it is accompanied by a short-lived upregulation (on STEM CELLS IN NOD/SCID MICE days 0 and 1 post pFUS) of several proinflammatory cytokines and growth factors which are associated with MSC migration and homing (e.g. IL1b, Kim, Dae Seong, Lee, Myoung Woo, Kim, Hye Ryung, Kim, Hye IL2, IL6, IL10, MCP-1, INFg, SDF1a, VEGF). In general, we observed a re- Jin, Yang, Jin Mo, Cheuh, Hee Won, Lee, Soo Hyun, Jung, Hye Lim, turn to baseline levels by day 3 following pFUS. Disscusion: We demonstrate Sung, Ki Woong, Yoo, Keon Hee, Koo, Hong Hoe pFUS exposures induce a degree of local inflammation that is followed by Dept of Peadiatric Hematology, Samsung Medical Center, Sungkyunkwan increased migration of iv-injected MSCs compared to untreated tissue. Using University School of Medicine, Seoul, Republic of Korea pFUS to stimulate local inflammation is an especially attractive technology as it is non-destructive and can be narrowly focused deep within the body To overcome the limitations of allogeneic hematopoietic stem cell transplan- with negligible effects to intervening tissue. Non-destructive pFUS exposures tation (HSCT), such as graft rejection and graft versus host disease (GvHD), essentially allow excellent spatio-temporal control over local inflammatory we conducted a study to identify a strategy for enhancing HSC engraftment responses and gives rise to the possibility of using FUS as a clinically appli- during HSCT. Cotransplantation experiments with mesenchymal stem cells cable, non-invasive tool to target migration of MSCs to tissues. (MSCs) derived from adult human tissues, including bone marrow (BM), adipose tissues (AT), and umbilical cord blood (CB), and intra-bone marrow Poster Board Number: 2563 transplantation (IBMT) of HSCs were conducted. AT-MSCs and CB-MSCs suppressed T-cell proliferation in mixed lymphocyte preparations as ef- ELUCIDATION OF THE HUMAN MESENCHYMAL fectively as BM-MSCs. We showed that AT-MSCs and CB-MSCs enhanced STEM CELL HOMING MECHANISM the engraftment of HSCs as effectively as BM-MSCs in NOD/SCID mice, suggesting that AT-MSCs and CB-MSCs can be used as alternative stem cell Droujinine, Ilia A., Zhao, Weian, Thiriot, Aude, Cui, Hao, Zhang, sources for enhancing the engraftment and homing of HSCs. CB-MSCs de- Liang, LeBlanc, Sarah, Suryaprakash, Shruthi, Sagar, Vinay, Kumar, rived from different donors showed different degrees of efficacy in enhanc- Namit1, Teo, Weisuong1, Sarkar, Debanjan1, von Andrian, Ulrich ing the engraftment of HSCs. The most effective CB-MSCs showed higher H.2, Karp, Jeffrey M.1 proliferation rates and secreted more monocyte chemoattractant protein Center for Regenerative Therapeutics, Department of Medicine, Brigham (MCP)-1, regulated upon activation normal T-cell expressed and secreted and Woman’s Hospital, Harvard-MIT Division of Health Sciences and (RANTES), epidermal growth factor (EGF) and vascular endothelial growth Technology, Harvard Stem Cell Institute, Harvard Medical School, Boston, factor (VEGF). In addition, IBMT with HSCs alone enhanced the engraft- MA, USA, Department of Pathology, Harvard Medical School, Boston, MA, ment of HSCs as effectively as cotransplantation with MSCs. Our results USA suggest that AT-MSCs and CB-MSCs that exhibit immature characteristics based on proliferative capacity and secreted factors could be alternative Mesenchymal stem cell (MSC) therapy possesses significant potential for stem cell sources for cotransplantation in HSCT. Furthermore, IBMT of HSCs treatment of numerous pathologies due to MSC multipotency and secretion

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Thursday Poster Abstracts of pro-angiogenic and immunomodulatory factors. Systemic administration sion on microcarriers using a low (2%) serum-containing medium. Although of MSC is preferred over the more invasive local transplantation. Although very promising, the use of fetal bovine serum poses a regulatory risk for there is evidence that systemically administered MSCs can home to sites of the commercial production of MSCs for an approved cell therapy applica- inflammation, efficiency of homing is low. This may account for the failure tion. We report here the expansion of human MSCs in a microcarrier-based of recent late-stage clinical trials to meet primary endpoints. Hence, eluci- system using commercially available serum-free and xeno-free reagents dating the poorly-defined MSC homing mechanism is critical for enhancing (StemPro® MSC SFM XenoFree, Life Technologies). Spinner flask studies the therapeutic benefit of MSCs and for understanding the basic biology showed the ability of the xeno-free system to support expansion of MSCs of cell homing. We hypothesized that MSC homing is an active process from bone marrow (BM MSCs) and adipose tissue (ADSCs) while retaining governed by molecular mechanisms. To test this, we investigated the inter- the desired phenotype and differentiation potential. After 14 days of culture, action of MSCs with endothelial cells (ECs) under both naive and activated BM MSCs reached a maximum cell density of (2.0±0.2)x105 cells/ml (fold (with TNF-α) conditions in vitro and in vivo. Using a flow chamber assay increase of 18±1), while ADSCs expanded to (1.4±0.5)x105 cells/ml (fold in vitro, we found that a subset of MSCs exhibited leukocyte-like capture, increase 14±7). Moreover, metabolic analyses showed that both type of cells rolling, and attachment to activated ECs at physiologically relevant shear displayed a similar metabolic trend, with higher consumption of glucose and stress. Moreover, we examined the active interaction of MSCs with TNF-α- production of lactate in the first days of culture, followed by lower values of inflamed ear venular endothelium by intravital microscopy, and preliminary specific consumption/production rates for the remaining time of expan- studies suggested that MSC are able to actively adhere to endothelium. sion. To examine the scale-up potential of this system, cell expansion was Results from flow cytometry, in vitro flow chamber assay, and antibody evaluated in 800 ml fully-controlled stirred-tank bioreactors. After 8 days, blocking experiments suggested that MSC homing is regulated by selectins BM MSCs expanded in this xeno-free, bioreactor system reached 1.2x108 and integrins. Specifically, MSC expressed a number of adhesion molecules total cells (fold increase of 12). The expanded cells maintained tri-lineage and were capable of binding to VCAM1 after induction with activating differentiation potential (osteogenesis, chondrogenesis and adipogenesis) antibodies to integrin α4, and to P-selectin. Functional antibody blocking and retained MSC immunophenotypic profile (>90% expression of CD90, data demonstrated contribution of endothelial P-selectin to adhesion, with CD105, and CD73). The data show that a commercially available serum-free the MSC P-selectin ligand likely being a sialyl-Lewis X (SLeX)-containing and xeno-free system can support large-scale MSC expansion in a microcar- protein. Current work investigates the identity of the MSC ligand responsible rier-based bioreactor process. for binding to P-selectin, using flow cytometry, enzymatic digestion, and P- selectin precipitation-mass spectrometry analysis. Overall, our findings sug- Poster Board Number: 2567 gest that a sub-population of MSCs can actively home to sites of inflamma- THE ASSESSMENT OF CONTROLLED tion through a leukocyte-like adhesion cascade, albeit with limited efficiency. Our results provide significant opportunity for rational design of MSC (e.g., CRYOPRESERVATION CONDITIONS OF HUMAN ligand expression) for enhanced delivery to diseased tissues. UMBILICAL CORD STROMAL TISSUE FOR THE Poster Board Number: 2565 POTENTIAL USAGE OF MSCs FOR STEM CELL CLINICAL GRADE EXPANSION OF HUMAN TRANSPLANTATIONS AND BANKING MESENCHYMAL STEM CELLS USING A Balci, Deniz, Can, Alp MICROCARRIER-BASED SYSTEM UNDER Ankara University Biotechnology Institute, Ankara, Turkey, Histology and Embryology, Ankara University School of Medicine, Ankara, Turkey SERUM-FREE AND XENO-FREE CONDITIONS In recent years, human umbilical cord stroma-derived mesenchymal Campbell, Andrew, dos Santos, Francisco, Andrade, Pedro Z., stem cells (hUCS-MSCs) were successfully differentiated into adipocytes, Lobato da Silva, Cláudia, M.S. Cabral, Joaquim, Abecasis, Manuel chondrocytes, osteocytes and myocytes, thus they are now considered M., Gimble, Jeffrey, Boucher, Shayne, Roos, Eric, Kuligowski, as a remarkable and promising stem cell source for cellular therapies. In Sandra, Vemuri, Mohan, Chase, Lucas addition to those mesenchymal lineages they were also differentiated into functional neuron-like cells and hepatocytes. While no graft rejection has Life Technologies, Grand Island, NY, USA, Department of Bioengineering, been reported in the recipient organism even in xenotransplantation studies, Instituto Superior Técnico (IST), Lisboa, Portugal, IPOFG-Instituto Português they were experimentally shown to attenuate tumor cell growth and gene de Oncologia Francisco Gentil, Lisboa, Portugal, Pennington Biomedical transfers. In present study, we demonstrate a reliable and efficient method Research Center, Louisiana State University, Baton Rouge, LA, USA, Life for cryopreserving the umbilical cord tissue pieces as a source of stroma- Technologies, Frederick, MD, USA, Cellular Dynamics International, derived MSCs. Conventional freezing, multi-step slow freezing (MSSF), Madison, WI, USA and vitrification methods were comparatively tested using varying doses of Human mesenchymal stem cells (MSC) are currently being evaluated in non-permeant [trehalose, sucrose, and hydroxyethyl starch (HES) + human numerous clinical trials as therapeutic agents to treat diseases such as myo- serum albumin (HSA)] cryoprotectant agents (CPAs). In all groups, DMSO cardial infarction, GvHD, ischemic stroke, inflammatory bowel disease, and was used as a cell permeant CPA. The efficiency of those three cryopreserva- other autoimmune diseases. Current treatment regimes may require multiple tion techniques was determined by cell viability assays, the expression of cell doses of MSCs at greater than 1x106 cells/kg each. The large cell numbers surface markers, cytoskeletal and extracellular matrix proteins. Conventional required makes the development of a clinically relevant scale-up process freezing and vitrification did not provide any significant success in tissue a necessity. Currently, most MSC culture processes use traditional two di- preservation. MSSF however presented highest cell survival rates comparable mensional systems and employ xenogenic serum-containing media. The use to those obtained from cell cryopreservation protocols reported so far. After of such techniques and reagents are expensive, labor intensive, regulatory determining the ice nucleation temperature for each sample, latent heat unfriendly, and are not readily amenable to scale-up production. Recently, evolution was suppressed during freezing (between 0°C and −40°C at a rate progress has been made in the development of alternative cell culture tech- 0.3-1°C/min) and cooling down to −120°C at a rate of 5-10°C/min). The nologies to support large-scale expansion of MSCs under GMP-compliant cell survival rate was found highest by MSSF with sucrose (0.1 M) at 0.3°C/ conditions. One such strategy involves the use of microcarriers to achieve min freezing rate (82% of the unfrozen samples). In this group no significant large-scale, high density MSC expansion. Moreover, microcarriers allow for a difference was noted before and after the cryopreservation in cell and tissue 3-D microenvironment, which mimics MSC in vivo niches, contrary to tissue morphology and the levels of MSC marker CD90 (Thy1), vimentin, BMP-4 culture plates. Recent work has demonstrated the feasibility of MSC expan- (bone morphogenetic protein), Nanog-a self renewal marker in embryonic

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stem cells, and in situ expression of the contractile proteins; actin, desmin, of brain tumors or for other therapeutic indications. α- smooth muscle actin and cytokeratin. CD29, CD54, CD90 and CD105 were found strongly positivity while CD73 and CD 133 were weak positive; Poster Board Number: 2571 and CD34 and CD45 were found negative in sucrose groups. Second high- IMMUNOSUPPRESSION BY IDO AND B7-H1 IN est cell survival ratio was obtained using trehalose (0.2 M) group at 0.3°C/ min freezing rate (68% of the unfrozen samples). Compared to HES (6%) MESENCHYMAL STEM CELLS DERIVED FROM +HSA group, the number of survived cell was higher although the tissue ADULT HUMAN TISSUES microarchitecture was found more damaged in trehalose group. In addition, desmin and cytokeratin filament organization in isolated hUCS-MSCs was Lee, Myoung Woo, Kim, Dae Seong, Kim, Hye Ryung, Kim, Hye found heavily deteriorated in the trehalose group. In conclusion, controlled- Jin, Yang, Jin Mo, Lee, Soo Hyun, Cheuh, Hee Won, Jung, Hye Lim, rate MSSF for freezing of the umbilical cord tissue strips turned out to be Sung, Ki Woong, Yoo, Keon Hee, Koo, Hong Hoe a valuable technique in preserving the mesenchymal stem cell source by Dept of Peadiatric Hematology, Samsung Medical Center, Sungkyunkwan decreasing the freezing rate to 0.3°C/min preferably in a sucrose+DMSO University School of Medicine, Seoul, Republic of Korea combination. Using this effective and reproducible cryopreservation method might save the umbilical cord tissues for years right after the birth. When Mesenchymal stem cells (MSCs), which evoke only minimal immune necessary, tissue resident MSCs can be successfully thawed and isolated. reactivity, may have anti-inflammatory and immunomodulatory effects. In this study, we conducted a comparative analysis of the immunomodulatory Poster Board Number: 2569 properties of MSCs derived from adult human tissues including bone mar- row (BM), adipose tissues (AT), umbilical cord blood (CB), and cord Whar- MRI TRACKING OF IRON-LABELED ton’s jelly (WJ). AT-MSCs, CB-MSCs, and WJ-MSCs effectively suppressed THERAPEUTIC HUMAN NEURAL STEM phytohemagglutinin-induced T-cell proliferation as effectively as did BM- CELLS IN A MURINE BRAIN TUMOR MODEL: MSCs. Levels of interferon (IFN)-γ secreted from activated T-cells increased over time, but these levels were significantly reduced when cocultured with IMPLICATIONS FOR CLINICAL USE each type of MSCs. In addition, expression of indoleamine 2,3-dioxygenase Gutova, Margarita, Thu, Mia S., Metz, Marianne, Khankaldyyan, (IDO) and B7-H1 molecules increased in MSCs treated with IFN-γ via JAK/ STAT1 signaling pathways. Treatment with anti-IFN- antibodies, JAK1/2 Vazgen, Abramyants, Yelena, Herrmann, Kelsey, Vo, Tien, Najbauer, γ inhibitor or STAT1 siRNA restored PHA-induced T-cell proliferation. Use of Joseph, Annala, Alexander J., Barish, Michael E., Moats, Rex, Frank, an antagonist, 1-methyl-L-tryptophan, also restored PHA-induced T-cell Joseph A., Aboody, Karen S. proliferation, suggesting that IDO contribute to IFN-γ-induced immunosup- Neurosciences, City of Hope National Cancer Center, Duarte, CA, USA, pression in MSCs. In addition, decrease of PHA-induced T-cell proliferation Radiology and Imaging Sciences, National Institute of Health, Bethesda, by MSC recovered by treatment with anti-B7-H1 antibodies, indicating that MD, USA, Radiology, Childrens Hospital Los Angeles/University of B7-H1 molecules involve in process for MSC’s immunosuppressive prop- Southern California, Los Angeles, CA, USA erty. Moreover, infusion of MSCs or MSCs treated with IFN-γ decreased symptoms for human peripheral blood-derived mononuclear cells-induced Therapeutic use of stem cells is currently under investigation for several graft versus host disease in NOD/SCID mice. These data indicate that IFN-γ cancers, with a first-in-human study of neural stem cell (NSC)-mediated produced by activated T-cells is correlated with induction of IDO and B7-H1 treatment of recurrent gliomas currently in Phase I clinical trial. At present, expression by MSCs, which, in turn, suppress T-cell proliferation. Our find- the movements and fates of donor stem cells cannot be tracked in real time ings suggest that MSCs derived from AT, CB, or WJ could be substituted for in human patients, and such information can only be obtained at autopsy. A BM-MSCs for treatment of allogeneic conflicts. particularly promising method for monitoring donor NSCs involves loading the cells with iron nanoparticles just prior to administration, and subsequent- Poster Board Number: 2573 ly using magnetic resonance imaging (MRI) to track their migration and distribution over time. MRI is already used widely as the primary method of CARDIOMYOBLAST-LIKE CELLS assessing therapeutic responses in brain tumor clinical trials. In the context DIFFERENTIATED FROM HUMAN ADIPOSE of stem cell-based therapies, MRI can also be used to non-invasively follow dynamic spatio-temporal patterns of stem cell migration and tumor target- TISSUE-DERIVED MULTILINEAGE PROGENITOR ing, providing valuable information for optimizing treatment regimens and CELLS IMPROVE LEFT VENTRICULAR strategies. We have previously demonstrated that superparamagnetic iron DYSFUNCTION AND SURVIVAL IN A SWINE oxide (SPIO)-labeled NSCs can be visualized by MRI in ex-vivo studies. Moreover, following iron-labeling, NSCs retained their viability, tumor-tro- MYOCARDIAL INFARCTION MODEL pism, and therapeutic transgene expression. Here, we extended our studies Okura, Hanayuki, Saga, Ayami, Soeda, Mayumi, Tani, Junko, through dynamic in vivo MRI monitoring of NSC tumor-tropism at multiple Shudo, Yasuhiro, Miyagawa, Shigeru, Ichinose, Akihiro, Sawa, time-points after intracranial and intravenous administration to glioma- bearing mice. We observed that Small Superparamagnetic Iron Oxide (SPIO- Yoshiki, Tahara, Shinya, Matusyama, Akifumi Feridex) and Ultrasmall Superparamagnetic Iron Oxide (USPIO-Feraheme)- Foundation for Biomedical Research and Innovation, Kobe, Japan, Osaka labeled NSCs localized to experimental orthotopic gliomas as early as 24 h University Graduate School of Medicine, Osaka, Japan, Kobe University after intracranial administration contralateral to the tumor and 15 min after Hospital, Kobe, Japan, Osaka University Graduate School of Medicine, intravenous administration. Iron-labeled NSCs that migrated to tumors were Osaka, Japan detected as hypointense signals by MRI and NSC localization was corre- Adipose tissue-derived multilineage progenitor cells (ADMPCs) are mul- lated with Prussian blue-stained tumor sections. Our data demonstrate that tipotent cells. We have reported that human ADMPCs (hADMPCs) could time-dependent migration and spatial distribution of iron-labeled NSCs can differentiate into cardiomyoblast-like cells (CLCs) by induction with dimeth- be safely monitored by real-time MRI in living mouse brain. Furthermore, ylsulfoxide (DMSO) and the cells have improved left ventricular dysfunc- iron-labeling does not affect the biological characteristics of NSCs, does not tion and survival in a rat myocardial infarction model. Here we examined cause accumulation of iron in other organs and blood and allows real-time whether human CLCs could be utilized to treat cardiac dysfunction using MRI tracking of therapeutic NSCs. This study lays the foundation for use of swine myocardial infarction model. First, we confirmed the induction of iron labeling in the clinical setting to track stem cells used during treatment various cardiac marker-expressions in hADMPCs after DMSO treatment for

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48h, such as a-cardiac actin, cardiac myosin light chain, and myosin heavy in safe per se. Next, we examined hADMPCs single dose administration via chain, none of which were detected in noncommitted hADMPCs. To confirm portal vein toxicity studies with nude rats to find the minimal lethal dose in their beneficial effect on cardiac function, we used chronic swine myocar- conformity with reliability assurance and GLP, hADMPCs were injected via dial infarction (MI) model. 20 swine received first ballooning for 60 min portal vein in dose escalation fashion and the studied indicated that minimal and reperfusion to first diagonal branch (D1; #9) and 1 week later second lethal dose of the cells was over 1.5x107 cells/kg in nude rats. Systematic ballooning for 90 min and reperfusionto left ascending coronary artery (#6). distribution studies were performed using DiI-stained hADMPCs. Almost all Four weeks later second ballooning and reperfusion, only 13 severe chronic the cells transplanted via portal vein were located in the parenchymal of the MI animals (ejection fraction <35% by echocargiogram) could be applied for liver and the cells did not differentiated into osteocytes and adipocytes, and examination. The immunosuppressed chronic MI swine (Tacrolimus 0.1mg no DiI-prestained cells were observed in the other tissues including lung 14 i.m./kg/day) were randomly assigned to receive intracoronal transplanta- days after transplantation. In the studies, no effects were observed on cen- tion of hCLCs (n=8) or placebo lactic Ringer’s solution with heparin (n=5) 4 tral nervous system and ventilation. Here we reported that hADMPCs could weeks after second ballooning and reperfusion. DiI-stained hCLCs distrib- be applied for First-in-Man clinical trials in safe in GLP-adapted non-clinical uted into the scarred myocardial milieu soon after intracoronal transplanta- studies. tion. Echocardiogram was examined at the transplanted day, 4, 8 and 12 weeks after transplantation. Follow-up showed rescue and maintenance of Poster Board Number: 2577 cardiac function in the hCLC transplanted group only, but not in the placebo NON CLINICAL STUDIES (GLP) FOR CLINICAL transplanted animals (ejection fraction; 45% vs 20% at 12 weeks after transplantation, respectively), indicating that coronal transplantation of the APPLICATION OF CARDIOMYOBLAST LIKE hCLC resulted in recovery of cardiac function. Twelve weeks after transplan- CELLS DIFFERENTIATED FROM HUMAN tation, 7 of 8 hCLCs transplanted animals survived, but only 1 of 5 placebo transplanted ones did (Log-rank test: p=0.0147. Hazard ratio=0.108: 95% ADIPOSE TISSUE DERIVED MULTILINEAGE CI 0.013 to 0.625), resulted in improvement of long-term survival rate. His- PROGENITOR CELLS tological studies demonstrated that the pre DiI-stained hCLCs engrafted into Okura, Hanayuki, Saga, Ayami, Soeda, Mayumi, Matsuyama, the scarred myocardium and expressed human specific alpha-cardiac actin 12 weeks after transplantation. Human alpha cardiac actin positive cells also Akifumi expressed cardiac nuclear factors; Nkx2.5 and GATA-4. To regard the in situ Foundation for Biomedical Research and Innovation, Kobe, Japan differentiation of the CLCs into cardiomyocytes, the localization of human Adipose tissue-derived multilineage progenitor cells (ADMPCs) are mul- alpha-cardiac actin-, human cardiac troponin I- and human myosin heavy tipotent cells. We have reported that human ADMPCs (hADMPCs) could chain-positive cells was confirmed. These results indicated that the intracoro- differentiate into human cardiomyoblast-like cells (hCLCs) by induction nal transplantation of hCLC, which could be committed from hADMPCS by with dimethylsulfoxide (DMSO) and we have shown the efficacy in which DMSO, is a potentially effective therapeutic strategy for future cardiac tissue the hCLCs have improved left ventricular dysfunction and survival in a rat regeneration. myocardial infarction model, and the intracoronal transplantation of hCLC Poster Board Number: 2575 into swine chronic severe myocardial infarction models is a potentially effec- tive therapeutic strategy for cardiac tissue regeneration. Here we performed NON CLINICAL STUDIES (GLP) FOR CLINICAL non-clinical Good Laboratory Practice (GLP) studies to examine whether APPLICATION OF HUMAN ADIPOSE TISSUE human hCLCs could be utilized to treat cardiac dysfunction in safe. First, to deny tumorigenicity of DMSO non-treated hADMPCs we examined DERIVED MULTILINEAGE PROGENITOR CELLS chromosomal stability test, soft agar colony formation assay, and tumor FOR THE PATIENTS WITH SEVERE FAMILIAL formation assay in nude mice in conformity with GLP according to WHO Ex- pert Committee on Biological Standardization, Forty-Seventh Report: WHO HYPERCHOLESTEROLEMIA Technical Report Series, 878, 1998. No significant tumorigenic characters Okura, Hanayuki, Saga, Ayami, Soeda, Mayumi, Matsuyama, have been observed in the cells. To confirm the genomic stability in pas- Akifumi saging, comparative genomic hybridization (CGH) assays were performed between passage 3 (P3), passage 5 (P5) and passage 8 (P8) of hDAMPCs. Foundation for Biomedical Research and Innovation, Kobe, Japan There were no significant aberration was occurred between P3 and P5, Adipose tissue-derived multilineage progenitor cells (ADMPCs) are mul- P3 and P8, and P3 and P8. Second, we performed electron micrograph to tipotent cells. We have reported that human ADMPCs (hADMPCs) could deny viral infections in the cells indicating that no viral-like particles were differentiate into hepatocytes in vivo after transplantation via portal vein observed. These results indicated that hADMPCs and hCLCs as DMSO- and we have shown the efficacy in which the hADMPCs have improved liver treated derivatives derived from hADMPCs might be in safe per se. Next, we dysfunction and reduced the serum levels of Watanabe heritable hyper- examined hCLCs single dose administration via left ventricle toxicity studies lipidemic (WHHL) rabbit, an animal model for homozygous FH. Here we with nude rats to find the minimal lethal dose in conformity with reliability performed non-clinical Good Laboratory Practice (GLP) studies to examine assurance and GLP, hCLCs were injected into left ventricle in dose escala- whether human hADMPCs could be utilized to treat the patients with FH tion fashion and the studied indicated that minimal lethal dose of the cells or other liver dysfunction in safe. First, to deny tumorigenicity of hADMPCs was over 1.5x107 cells/kg in nude rats. Systematic distribution studies were we examined chromosomal stability test, soft agar colony formation assay, performed using DiI-stained hADMPCs. Almost all the cells transplanted via and tumor formation assay in nude mice in conformity with GLP according coronary artery in swine were located in the cardiac tissue and the cells did to WHO Expert Committee on Biological Standardization, Forty-Seventh not differentiated into osteocytes and adipocytes in the cardiac milieu, and Report: WHO Technical Report Series, 878, 1998. No significant tumori- no DiI-prestained cells were observed 3 months after transplantation in the genic characters have been observed in the cells. To confirm the genomic other tissues. In the studies, no affects were observed on central nervous stability in passaging, comparative genomic hybridization (CGH) assays system and ventilation. Here we reported that hCLCs could be applied for were performed between passage 3 (P3), passage 5 (P5) and passage 8 (P8) First-in-Man clinical trials in safe in GLP-adapted non-clinical studies. of hDAMPCs. There were no significant aberration was occurred between P3 and P5, P3 and P8, and P3 and P8. Second, we performed electron micrograph to deny viral infections in the cells indicating that no viral-like particles were observed. These results indicated that hADMPCs might be

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Poster Board Number: 2579 treatment for NP-C disease. With the investigation of neurogenesis in the adult brain, the hopes have risen that these neurodegenerative conditions BIODISTRIBUTION AND EFFECTS OF IV- could be overcome, or at least ameliorated, by the generation of new neu- INJECTED MESENCHYMAL BONE MARROW rons. Our previous studies have shown that bone marrow-derived mesen- chymal stem cells (BM-MSCs) contribute to improved neurological function CELLS IN A MOUSE MODEL OF CHAGAS in NP-C mouse brain following intracerebellar transplantation. Therefore, DISEASE we hypothesized that BM-MSCs would be beneficial in NP-C mouse due to promote neurogenesis. We found that NP-C embryonic neural stem cells Jasmin, Fnu, Jelicks, Linda A., Koba, Wade, Tanowitz, Herbert B., (NSCs) exhibit impaired self-renewal ability and decreased rate of neuronal Mendez-Otero, Rosalia, Campos de Carvalho, Antonio C., Spray, differentiation compared to wild-type mice. When we cocultured BM- David C. MSCs with NP-C NSCs using an indirect, the capacity of self-renewal and Albert Einstein College of Medicine, Bronx, NY, USA, Instituto de Biofísica differentiation into neurons or oligodendrocytes was enhanced. BM-MSCs Carlos Chagas Filho, Rio de Janeiro, Brazil transplantation also increased the proliferation and neuronal differentia- tion of NSCs within the subventricular zone (SVZ) in NP-C mouse. Notably, Chagas disease, resulting from infection with a protozoan parasite, is a major cytokine antibody array and enzyme-linked immunosorbent assay (ELISA) cause of cardiomyopathy in Latin America. Treatment options are limited to revealed that chemokine (C-C motif) ligand 2 (CCL2) is released from BM- a small number of drugs that were developed more than four decades ago MSCs that modulate proliferation of endogenous NSCs in NP-C mice. These and which have various drawbacks. Stem cell therapy with bone marrow findings demonstrate that BM-MSCs stimulate regenerative neurogenesis, mesenchymal cells (MSCs) has emerged as a novel therapeutic option for resulting in delayed atrophy of the striatum in NP-C mice (SC-4170). cell death-related heart diseases, but efficacy of MSCs has not been tested in Chagas disease therapy. We have used cell tracking strategies following Poster Board Number: 2583 labeling of MSCs with nanoparticles to investigate migration of transplanted MSCs in a murine model of Chagas disease, and have correlated MSCs TRANSPLANTATION OF BONE MARROW migration with cardiac function in chagasic animals by magnetic resonance STROMAL CELLS PRODUCES LONG-TERM imaging (MRI). We also quantified the expression of metalloproteinase (MMP) using a metalloproteinase fluorescence probe (MMPSense750) in ATTENUATION OF PAIN HYPERSENSITIVITY IN control and chagasic animals treated with MSCs or PBS after 2 months of RATS infection. Mice were intraperitoneally infected with 5 x 104 trypomastigotes Guo, Wei, Wang, Hu, Zou, Shiping, Gu, Ming, Wei, Feng, Dubner, and treated by tail vein injection of MSCs, at 2 months after infection. MSCs were labeled with fluorescent nanoparticles (X-Sight761, Carestream) or su- Ronald, Huang, George T., Ren, Ke perparamagnetic iron oxide nanoparticles (Feridex, Bayer) and were tracked University of Maryland, Baltimore, MD, USA, Boston University, Boston, by optical or magnetic resonance imaging (MRI). Our optical imaging results MA, USA at two days after transplant showed that a small number of cells migrated to In light of the recent development on the therapeutic potential of mes- chagasic hearts in half the analyzed animals, whereas the majority of labeled enchymal stromal cells, we systematically evaluated the effect of bone MSCs migrated to liver, lungs and spleen. We were unable to detect MSCs marrow stromal cells (BMSC) on persistent pain behavior in a rat model. The in the heart by MRI at several tested times (1, 6, 13 days and 1 month) model involves ligation injury of a tendon of the masseter, which produces likely due to low sensitivity of the method as used with Feridex. However, long-lasting and constant orofacial mechanical hypersensitivity of myogenic MRI did show that therapy with MSCs reduced right ventricular dilatation origin. Our results showed that intravenous and local infusion of rat primary that is typical of the chagasic mouse model. Additionally, the optical imag- BMSC at 3 days-4 months post injury produced attenuation of mechanical ing obtained using MMPSense750 showed higher MMP expression in the hypersensitivity that was maintained up to 5 months. The mechanical hyper- whole body, abdomen, heart, spleen and white fat in the infected animals sensitivity was rekindled by naloxone when tested at 1-5 weeks after BMSC (treated with MSCs or PBS) when compared with the control group. MMP infusion. In contrast, naloxone methiodide, a peripherally acting opioid re- expression in the legs and paws was higher in the chagasic animals treated ceptor antagonist, only rekindled hyperalgesia in the first 3 weeks of BMSC with PBS when compared with controls or chagasic animals treated with treatment. Downregulation of mu opioid receptors by RNA interference in MSC. Moreover, proteins previously described as affected by infection, such the rostral ventromedial medulla, a key structure in descending pain modula- as connexin43 was totally recovered after 1 month of cell treatment and tion, reversed the effect of BMSC when RNA interference was introduced this recovery persisted after 2 months of treatment. In addition, laminin γ1 at 5- but not 1-week after BMSC transplantation. Thus, BMSC produced was partially recovered by MSC 1 month after cell therapy. In summary, long-term attenuation of hyperalgesia and this effect involved activation of the beneficial effects observed by cell therapy in chagasic mice is due to an peripheral and central opioid receptors in a time-dependent fashion. The indirect action of the cells in the heart, most likely through secretion of im- findings prompt studies to elucidate cellular mechanisms of BMSC-induced munomodulatory and/or growth factors, rather than the generation of new anti-hyperalgesia and translate these observations into clinical settings. cardiomyocytes or even incorporation of large numbers of stem cells into (This work was supported by National Institutes of Health grants: DE11964, working myocardium. DE018573, NS060735, NS059028 and DE019156.) Poster Board Number: 2581 MSC STIMULATE ENDOGENOUS NEUROGENESIS THROUGH CCL2 IN NP-C DISEASE MICE Lee, Hyun, Bae, Jae-sung, Jin, Hee Kyung Kyungpook National Univ, Daegu, Republic of Korea Niemann-Pick type C (NP-C) disease is neurological degenerative disorder with psychiatric signs and cognitive troubles that results from significant alterations to neuronal and axonal structure. To date, there is no satisfactory

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Poster Board Number: 2585 Poster Board Number: 2587 PERI-OCULAR AND INTRA-ARTICULAR THE EFFECT OF HUMAN ADIPOSE TISSUE- INJECTION OF CANINE MSCS: AN IN VIVO DERIVED MESENCHYMAL STEM CELLS ON SAFETY, IMAGING, MIGRATION, AND AVASCULAR NECROSIS OF THE FEMORAL IMMUNOMODULATION STUDY HEAD IN BEAGLE DOGS Wood, Joshua A., Chung, Dai-Jung, Park, Shin Ae, Reilly, Choi, Hyung Jun, Kwon, Euna, Cho, Ka Hee, Kim, Jong Min, Park, Christopher M., Carrade, Danielle M., Ly, Irene, Zwingenberger, Sang Young, Hyon, Min Kyong, Che, Jeong Hwan, Ra, Jeong Chan, Allison, Hayashi, Kei, Cherry, Simon R., Borjesson, Dori L., Russell, Kang, Byeong Cheol Paul, Murphy, Christopher J. Graduate School of Immunology, Seoul National University, Seoul, Republic Surgical and Radiological Sciences, University of California, Davis, of Korea, Department of Experimental Animal Research, Clinical Research Davis, CA, USA, Pathology, Microbiology, and Immunology, University Institute, Seoul National University Hospital, Seoul, Republic of Korea, of California, Davis, Davis, CA, USA, Center for Molecular and Genomic Stem Cell Research Center, RNL BIO Co., Ltd., Seoul, Republic of Korea Imaging, University of California, Davis, Davis, CA, USA, Surgical and Avascular necrosis of the femoral head (AVN) remains a major unsolved Radiological Sciences & Opthalmology and Vision Science, University of problem in orthopaedic surgery of the hip. Recently, mesenchymal stem cells California, Davis, Davis, CA, USA have been proposed for the repair of damaged tissue including bone and Mesenchymal stem cells (MSCs) continue to show great clinical potential cartilage. In this study, we evaluated the effect of human adipose tissue- in the treatment of ischemic, degenerative, and inflammatory diseases. derived mesenchymal stem cells (hATMSCs) on the treatment of avascular Allogeneic sources of MSCs have been studied in vivo for their potential to necrosis (AVN) of the femoral head in beagle dogs. AVN of the right femoral regenerate bone, treat myocardial infarction, and treat spinal cord injury. In head was surgically induced by vascular-deprivation and freezing methods many of these applications, MSCs are administered intravenously; however, in 1-year-old, 15 female beagle dogs. At 2 weeks after operation, they were localized injection may improve site specific engraftment and residence time. divided into 5 groups randomly and performed the core decompression. In addition, the safety and efficacy of repeated allogeneic MSCs doses has Under fluoroscopic control, saline (Control) or hATMSCs (Group I, 1.65×108 yet to be fully investigated. MSCs were harvested from the adipose tissue of cells/dogs) or scaffold (Group II, β-TCP) or two cell-loading density of six beagles. Following in vitro expansion, allogeneic MSCs were trans- hATMSCs-loaded β-TCP (Group III, 3.3×106 or Group IV, 1.65×108 cells/ planted 6 times over a 9 week trial period. Trans-conjunctival injections of 4 dogs) were transplanted into the lesion of osteonecrosis through transtro- million cells into the regions of the lacrimal gland and the gland of the 3rd chanteric route. The healing response was evaluated by X-ray, MRI at 0, 4, eye lid were performed weekly on the right eye with the identical regions of 8, 13 weeks after transplantation and by gross observation and histologi- the left eye receiving sham injections. Complete ophthalmic examinations cal assessment at sacrifice. To address safety-concerns, we conducted a were performed over the 9 week trial period. In addition to ocular injections, GLP-compliant toxicity study. SEM studies showed that hATMSCs filled the intra-articular injections of 5 million cells were performed on the right stifle pores/surfaces of scaffolds in a cell-loading density-dependent manner. In joint with sham injections in the left stifle joint every other week through- control group, we could observe empty lacunae at epiphysis with pale and out the course of the study. To study residence time, 3 dogs were injected fibrous bone marrow. Through MRI contrast T1 and histology, hATMSCs- with fluorescently labeled MSCs on weeks 1 and 6 of the study into the loaded β-TCP scaffold groups had better osteogenesis ability compared lacrimal and third eye lid regions. The remaining 3 dogs received 66 million with control, group I and II. New bone formation was dose-dependently fluorescently labeled cells intra-articularly into the right stifle region on week increased at metaphysis in hATMSCs-loaded β-TCP scaffold-treated groups. 6. To investigate any immunomodulatory effects, mixed leukocyte reactions In toxicological aspect, no significant changes were found in body weights, (MLRs) were performed 1 week prior to the first injection and 3 weeks after clinical signs, hematological/biochemical values, organ weights, histopatho- the last injection on all 6 animals. In the tenth week, complete necropsies logical findings. These results showed that transplantation of hATMSCs ap- of all dogs were performed and tissue samples from over 60 locations pears to be a safe and could have the potential for the partial improvement were collected to investigate MSC migration and engraftment. No clini- of osteogenesis in the treatment of AVN in dog model. cally significant adverse effects following either peri-ocular or intra-articular Poster Board Number: 2591 injections were observed. In vivo imaging documented that a significant population of MSCs maintained residence in the lacrimal and stifle regions COMPLETE PULP REGENERATION BY for at least 3 weeks following injection. Post-mortem examination revealed significant engraftment and residence for >4weeks after injection in all TRANSPLANTATION OF DENTAL PULP STEM injected regions. Significant migration of MSCs from the stifle region to the CELLS gastrointestinal tract was observed (p<0.05). Finally, stimulated, MLRs dem- onstrated a significant (four-fold) reduction in lymphocyte proliferation after Nakashima, Misako, Iohara, Koichiro, Koga, Takeshi MSC injections when compared to pre-injection levels (p<0.05). The MLR Department of Oral Disease Research, National Center for Geriatrics and results support previously documented immunomodulatory effects of MSCs Gerontology, Research Institute, Obu, Japan and demonstrate that repeated local injections result in systemic immune Dental pulp have many functions in homeostasis of teeth, and regenera- modulation. We have shown for the first time that repeated peri-ocular and tion of pulp following root canal treatment in pulpitis or periapical disease is intra-articular injections of allogeneic MSCs can be safely administered to critical for longevity of teeth and improvement of quality of life. A promising dogs and that MSCs are efficacious in modulating the immune system. The approach is stem cell therapy. Dental pulp tissue-derived CD105+ stem cells data suggest that local delivery of MSCs may be effective in the treatment with high angiogenic and neurogenic potential were transplanted into a root of immune disorders of the eye and joint. canal with a migration factor after whole pulp removal of mature teeth with complete apical closure in dogs. The root canal was filled with regenerated pulp including vasculature and nerves by day 14. The odontoblast-like cells attached to the dentinal wall in the root canal, and produced dentin-like tissue extending their processes into dentin tubules on 35 days. The trans- planted pulp CD105+ cells had high migratory and proliferative effects by

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the migration factor. They highly expressed angiogenic/neurotrophic factors vivo safety and functional efficacy of UCMSCs will be presented. and localized in the vicinity of newly formed capillaries, suggesting their high trophic effects. When unfractionated total pulp cells were transplanted, less Poster Board Number: 2595 tissue was observed compared with CD105+ cells, and matrix formation and UMBILICAL CORD WHARTON’S JELLY: A NEW mineralization was seen in the regenerated tissue on day 35. Two dimen- sional electrophoretic analyses demonstrated that the qualitative and quanti- POTENTIAL CELL SOURCE OF MESENCHYMAL tative protein expression patterns of the regenerated tissue in pulp CD105+ STEM CELLS FOR WOUND HEALING cell transplantation were similar to those of normal pulp. Some pulp tissue marker genes were similarly expressed in pulp CD105+ cell transplantation Shohara, Ryutaro, Takikawa, Sachiko, Yamamoto, Akihito, Hibi, as those in normal pulp. Thus, this novel cell therapy is the first demonstra- Hideharu, Kikkawa, Fumitaka, Ueda, Minoru tion of complete pulp regeneration after pulpectomy, suggesting a potential Department of Oral and Maxillofacial Surgery, Nagoya University Graduate utility of pulp CD105+ cells for endodontic treatment. School of Medicine, Nagoya, Japan, Department of Obstetrics and Poster Board Number: 2593 Gynecology, Nagoya University Graduate School of Medicine, Nagoya, Japan ROLE OF UMBILICAL CORD DERIVED Cleft lip and palate (CLP) is the most common congenital malformation in MESENCHYMAL STEM CELLS IN THE the newborn. Development of new regeneration therapies enabling scarless wound healing and reconstruction of alveolar bone defect still remains a REMISSION OF TUBULAR DEFECTS IN A major challenge for surgeons. Although bone marrow derived mesenchy- PRECLINICAL MODEL mal stem cells (BMMSCs) are major cell resource for skeletal tissue repair, isolation of BMMSCs from newborn is practically impossible. Umbilical Shetty, Prathibha, Thakur, Anirban, Ravindran, Geeta, Viswanathan, cords, which are routinely discarded as medical waste after birth, have been Chandra shown to contain mesenchymal stem cells (UCMSCs) being useful for cell- Regenerative Medicine, Reliance Life Sciences Pvt. Ltd., Navi Mumbai, based regeneration therapies. In this study, we focused on the isolation and India, Laboratory Animal Research Services, Reliance Life Sciences Pvt. Ltd., identification of UCMSCs from Wharton’s jelly of umbilical cord. Expression Navi Mumbai, India of stem cell marker, multi-lineage differentiation activity and BMP2-induced Acute and chronic renal failures are disorders with high rates of morbidity osteogenic phenotypes of UCMSCs were examined. In addition, using and mortality. Current treatment is based upon conventional dialysis to pro- excisional wound model in mice, we demonstrated that engrafted UCM- vide volume regulation and small solute clearance. Kidney transplantation SCs significantly enhanced wound healing. Thus, our findings suggest that still remains the most effective treatment; however, organ availability lags far UCMSCs represent a promising stem cell resource for cell-based therapy in behind demand. Many key kidney functions including gluconeogenesis, am- CLP patients. moniagenesis, metabolism of glutathione, catabolism of important peptide Poster Board Number: 2597 hormones, growth factors, and cytokines critical to multiorgan homeostasis and immunomodulation are provided by renal tubule cells. Recent findings DIFFERENTIATION POTENTIAL AND suggest that the mesenchymal stem cells have potential to differentiate into IMMUNOGENIC CHARACTERIZATION FOR renal tubule cells. The replacement of renal tubule cell function may thus change the current dismal prognosis of patients with these disorders. Fur- PLACENTAL MESENCHYMAL STEM CELLS OF thermore MSCs are an attractive candidate for renal repair, since nephrons MATERNAL ORIGIN are of mesenchymal origin, leading to the differentiation of nephrons and collecting ducts. Moreover, MSCs have advantages over conventional stem Wang, Libin, Liu, Ting, Ma, Xiaona, Jin, Yiran, Zhu, Yongzhao, Fan, cells, such as their ease of harvest, stable genetic background, lower risk of Heng, Ma, Lijun, Yan, Xiurui, Li, Yukui, Wei, Jun tumor formation, and decreased ethical concerns. Although, bone marrow The Affiliated Hospital of Ningxia Medical University, Ningxia Human Stem has been the conventional source for the isolation of multipotent MSCs, Cell Research Institute, Yinchuan, Ningxia, China they have also been isolated from a variety of other human adult tissues, including muscle, connective tissue, skin, adipose tissue, perichondrium, Background and Aims: Mesenchymal Stem Cells (MSCs) have been proved trabecular bone, placenta, umbilical cord blood etc. Recently, umbilical cord applicable to therapies of multiple human diseases. Despite of numerous has been shown to be a good source of MSCs (UCMSCs) and is the best studies having been carried out on bone marrow MSCs, placental MSCs of option available to take cell therapy from autologous to allogeneic platform maternal origin, which are one of the richest sources of MSCs, are poorly due to its immunoprivileged nature. Previous study from our laboratory has understood. The present study was aimed to explore the multipotency and reported the isolation and characterization of MSC from umbilical cord. The immunogenicity of placental MSCs of maternal origin. Methods: Placental growth kinetics, immunophenotype and differentiation potential of these MSCs were isolated by enzyme digestion of fresh placental tissues from the UCMSCs have been studied and over 90% of these cells exhibited a MSC uterus side of normal term placentas. Maternal origin of MSCs was validated phenotype of CD73+/CD105+/CD29+/CD44+/SSEA4+/HLA-ABC+/HLA- by PCR analysis for 4 polymorphic loci that in combination distinguish differ- DR-/CD45-/CD14-/CD31-/vWF-. We have also shown the therapeutic ent genetic origins with confidence of 99.99%. Multipotency/pluripotency effects of UCMSC in neurological and vascular diseases. The objective of of the MSCs was evidenced by induced differentiation towards adipocytes this study was to explore the potential of the expanded and well charac- and osteocytes as well as pancreatic islet cells. Immunogenecity of the terized UCMSCs in reversing the symptoms in preclinical models of renal MSCs was tested by flow cytometry and quantitative PCR for expression of injury. The acute and chronic renal injury models were created by chemical HLA-DR, B7-1(CD80) and B7-2(CD86). Results: From validated maternal injury with cisplatin and doxorubicin respectively. Based on the symptoms, placental MSCs, adipocytes, osteocytes and pancreatic islet cells were suc- biochemical and histological analysis, mice were randomized and grouped cessfully differentiated as evidenced by oil red O staining for adipocytes, into treated and controls for safety and efficacy studies with UCMSCs., At alizarin red staining for osteocytes, and immunohistochemical staining of every assessment the animals were analyzed for their survivability and the GLP-1,PDX-1,C -Peptide expression for pancreatic islet cells, respectively. plasma samples were subjected to biochemical analysis including BUN and Flow cytometry and quantitative PCR showed no detectable expression of expression of the key markers kidney injury molecule-1 (KIM-1) and Insulin HLA-DR, B7-1 and B7-2. Conclusion: Maternal placenta MSCs are capable like growth-1 (IGF-1).This analysis was then further confirmed by subjecting of differentiating towards no only intra-lineage cells, including adipocytes the kidney sections for the histopathological studies. The results on the in and osteocytes, but also trans-lineage cells, such as pancreatic islet cell type,

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Thursday Poster Abstracts indicating that the MSCs have multipotent/pluripotent property. The cells tightly regulated at the transcriptional level. Although the mechanisms of have limited expression of MHC-II antigen and are naïve for co-stimulating HLA-G expression has been studied in the choriocarcinoma cell line JEG-3, factors. These characteristics in whole make the cells good candidates for the JAK/STAT-1 signaling pathway appears to be aberrant in this tumor cell development of stem cell-based therapies. (The research is supported by the and thus cannot stand as a human trophoblastic in vitro model. Therefore, NSFC (National Natural Science Foundation of China) Funding: (Program the molecular mechanism of HLA-G induction by IFN-γ is still unclear. Since No: 30960176).) the isolation and in vitro culture of primary trophoblast cells from the human placenta have numerous limitations, it has been difficult to conduct mecha- Poster Board Number: 2599 nistic studies of this nature with regards to the immunologically important CHARACTERIZATION OF MESENCHYMAL STEM organ. Therefore, we investigated the mechanisms involved in HLA-G gene activation in response to IFN-γ using PDMCs, a primarily generated cell of CELL TRANSMIGRATION human placental origin. IFN-γ induces the expression of IFN regulatory fac- Teo, Grace Sock Leng, Ankrum, James A., Carman, Christopher V., tor 1 (IRF-1) and phosphorylation of STAT-1 in PDMCs. The fold-induction of HLA-G transcripts after IFN- stimulation is significantly higher than Karp, Jeffrey M. γ JEG-3. Thus, we speculate that the transcriptional control of HLA-G in Massachusetts Institute of Technology, Cambridge, MA, USA, Beth Israel PDMCs involved not only the proximal IFN-γ cis-acting regulatory elements Deaconess Medical Center/ Harvard Medical School, Boston, MA, USA, including the IFN-stimulated response element (ISRE) and IFN-γ activation Brigham and Women’s Hospital/Harvard Stem Cell Institute, Cambridge, site (GAS), but also more distal elements which synergistically render the MA, USA HLA-G gene promoter highly responsive to IFN-γ. In addition, enhancement Systemic administration of multi-potent stromal cells or mesenchymal stem of HLA-G mRNA transcription does not correspond to protein expression cells (MSC) is being explored in clinical trials for treatment of inflammatory levels, suggesting that post-transcriptional regulation may also be involved pathologies. MSC exhibit the remarkable ability to engraft preferentially into in HLA-G expression. Taken together, our results indicate that the JAK/STAT- inflamed/ischemic tissues where they suppress inflammation and promote 1 signaling pathway is necessary and sufficient to mediate the up-regulation tissue regeneration. Mechanisms for the inflammation-specific engraft- of HLA-G gene expression by IFN-γ in PDMCs. Further studies are needed ment of MSC remain largely unknown, but are hypothesized to involve to provide a complete understanding of the molecular mechanisms underly- active homing processes analogous to those used by leukocytes. Here, we ing the modulation of HLA-G in PDMCs, which may ultimately allow the show that, like leukocytes, human bone marrow-derived MSC preferentially development of therapeutic strategies. adhere to and migrate across TNF-α-activated endothelium via discrete Poster Board Number: 3003 para- and trans-cellular pathways in association with ‘transmigratory cups.’ Contrasting leukocytes, MSC transmigration was not preceded by lateral HUMAN FIRST TRIMESTER CHORIONIC migration, occurred on the time scale of hours rather than minutes and was associated with unique non-apoptotic membrane blebbing protrusions STEM CELLS HAVE TRANSLATIONALLY rather than lamellipodia and invadosomes. Thus, MSC indeed home actively ADVANTAGEOUS CHARACTERISTICS OVER to inflamed tissues via both leukocyte-like and novel mechanisms. These in- THEIR TERM COUNTERPARTS sights may facilitate improvements of MSC delivery and therapeutic efficacy. Jones, Gemma N., Moschidou, Dafni, Abdulrazzak, Hassan, Fisk, Nicholas M., De Coppi, Paolo, Guillot, Pascale V. OTHER Imperial College London, London, United Kingdom, University of Queensland, Brisbane, Australia, University College London, London, Poster Board Number: 3001 United Kingdom REGULATION OF HUMAN LEUKOCYTE Human fetal mesenchymal stem cells (hfMSC) isolated from fetal organs, such as liver or bone marrow, have advantageous characteristics over ANTIGEN-G EXPRESSION BY INTERFERON-γ- adult MSC and potential to repair damaged tissues. However, hfMSC MEDIATED JAK/STAT-1 SIGNALING PATHWAY require aborted tissue and are not suitable for autologous use. In contrast, IN HUMAN PLACENTA-DERIVED MULTIPOTENT extra-embryonic fetal tissues, such as placenta, are readily accessible with stem cells isolatable from either surplus tissue collected at routine prenatal CELLS diagnostic procedures or at term delivery. We compared the phenotype Chen, Pei-Min, Wei, Chung-Fan, Hsu, Pei-Ju, Yen, Men-Luh, Yen, and therapeutic potential of human fetal stem cells isolated from early first trimester (eCSC) and term chorion (tCSC). Our results show that eCSC B Linju and tCSC share a common phenotype, i.e. negative for CD14, CD34, and Institute of Cellular and Systems Medicine, National Health Research CD45, but express CD29, CD44, CD90, CD105, vimentin and CD73, along Institutes, Zhunan, Taiwan, Departments of Primary Care Medicine and with low level of HLAI and no expression of HLAII. Similarly to amniotic Obstetrics/Gynecology, College of Medicine, National Taiwan University fluid stem cells, eCSC and tCSC also share characteristics of embryonic stem and Hospital, Taipei, Taiwan (ES) cells, expressing OCT4, NANOG, SOX2, KLF4 and REX1, revealing an Placenta-derived multipotent cells (PDMCs) are progenitor cells isolated intermediate phenotype between ES and adult MSC. However, eCSC and from the human term placenta with differentiation capacity for all three tCSC differ in a number of parameters relevant to regenerative applications. germ layers as well as strong immunomodulatory properties. The placenta In particular, compared to tCSC, eCSC had a higher proportion of STRO- is central to maintaining fetomaternal tolerance, and one of the most 1+ cells (77.4% vs 32.8%, P<0.01), are smaller in size (16.5μm ± 1.7 vs. important immunomodulatory molecules involved is the human leukocyte 24.0μm ± 1.6, p<0.01), have higher kinetics (population doubling time of antigen-G (HLA-G). HLA-G is a non-classical major histocompatibility 48.6 hours ± 11.1 vs. 120.7 hours ± 77.0, p<0.05) and differentiate more complex (MHC) class I molecule which exhibits a restricted tissue-specific readily down the osteogenic pathway in vitro. Furthermore, when trans- expression and exerts multiple immunoregulatory functions for maintaining planted into neonatal osteogenesis imperfecta (oim) mice, the percentage of the tolerance of placenta. Recently, we have found that PDMCs can resist mice without long bone fractures at 8 weeks of age was 50% in eCSC- natural killer (NK) cell-mediated cytotoxicity by interferon-γ (IFN-γ)-induced transplanted mice, but only 20% in tCSC-transplanted mice, and 0% in surface HLA-G expression, suggesting that the expression of HLA-G may be non-transplanted mice. The reduction in long bone fracture rate correspond- ed to 56.4% in eCSC treated mice vs. 18.2% in tCSC-treated oim (X2=11.9,

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P<0.001). Altogether, we conclude that whilst cells with similar phenotype Poster Board Number: 3007 could be derived from placenta tissue during pregnancy and at term, they could differ in their potential for engrafting and regeneration. Therefore it HUMAN MESENCHYMAL STEM/STROMAL could be advantageous to use eCSC as an alternative source of fetal stem CELLS INHIBIT THE PROLIFERATION AND cells in cell therapy. PROMOTE THE CELL CYCLE ARREST OF Poster Board Number: 3005 HUMAN LEUKEMIC CELLS RAPID BEDSIDE PROCESSING AND Herault, Olivier, Vignon, Christine, Georget, Marie-Therese, Levern, CHARACTERIZATION OF HUMAN AUTOLOGOUS Yves, Bernard, Marie-Christine, Kerboeuf, Dominique, Domenech, BONE MARROW STEM CELL CONCENTRATE Jorge, Binet, Christian FOR CELL THERAPY CHU - Service d’Hématologie Biologique, Tours, France, UPRES EA3855, Faculty of Medicine of Tours, Tours, France, Plateforme PAIB, INRA, Chung, Meng-Hong, Sorkin, Adam M., Barnes, Brian, Sakthivel, Nouzilly, France Ramasamy Leukemic cells located in the bone marrow (BM) are interacting with a mi- Research and Development, Arteriocyte, Inc., Cleveland, OH, USA croenvironment which plays a crucial role in the development and progres- Introduction: The application of autologous bone marrow concentrate at sion of leukemia. Mesenchymal stromal cells (MSCs) constitute a population wound/surgical site is thought to improve local tissue healing. In this study, of multipotential cells giving rise to the different hematopoietic microenvi- we have utilized a fully automated tabletop rapid bedside cell processor ronmental cells (adipocytes, osteoblasts, chondrocytes, and vascular-smooth to concentrate bone marrow and analyzed the quantity and phenotype of muscle-like hematopoietic supportive stromal cells). The aim of our study the cellular components. The data from this study indicates that there is an was to evaluate the effects of MSC-contact on the cell cycle and prolifera- increase in white blood cell, mesenchymal stem cells (MSC), and hematopoi- tion of human leukemic cells. BM MSCs were obtained from informed and etic stem cells (HSC) populations and significant reduction in red blood consenting patients undergoing orthopedic surgery, following a procedure cells in bone marrow concentrate. There is also several fold increase in total approved by the local ethical committee. Nucleated cells harvested from the nucleated (TNC), mononucleated (MNC) and platelets (PLT). The concen- iliac crest were seeded (5.10exp4 cells per cm2) in α-MEM supplemented tration of CD133+, CD34+, CD73+, and CD105+ cells are also increased with 10% fetal calf serum (FCS), 20 mmol/L L-glutamine, and 100 units/ several fold in bone marrow concentrates. Methods & Results: Fresh human mL penicillin G. Cells were incubated at 37°C in 95% humidified air and bone marrow aspirate obtained from healthy donors were acquired within 5% CO2. When fully confluent, the layer of adherent cells was trypsinized 24 hours of collection (Lonza). About 6mL Bone marrow concentrate was (0.25% trypsin-EDTA), and the cells were resuspended in culture medium prepared from 60mL filtered, anticoagulated bone marrow using Magel- and seeded at 10exp3 cells per cm2 (passage 1 - P1). BM MSCs were lan® Autologous Platelet Separator System (AMS100) and Surgical Tools used at P2 in all experiments. The three-lineage mesenchymal differentia- for Erythrocyte & Marrow Preparation Kit (MAR01, Arteriocyte Medi- tion of the BM MSCs used was systematically checked by culturing cells in cal Systems). The concentrations of cellular components of bone marrow adipogenic, chondrogenic, and osteogenic induction media as previously de- concentrate (N = 14) were quantified using a XE-5000 hematology analyzer scribed. The human KG1a leukemic cell line (FAB M0/M1 CD34+ leukemic (Sysmex). Total nuclear cell (TNC), mononuclear cell (MNC), platelet (PLT), cells) was cultured in RPMI 1640 with 20 mmoL/L L-glutamine supplement- and hematocrit were evaluated in bone marrow concentrate as well as pre- ed with 10% FCS, 100 units/mL penicillin G, and 100 mg/mL streptomycin. processing bone marrow aspirate. The TNC, MNC, PLT concentrations and KG1a cells were seeded at 1.5 10exp5 cells/cm2 and cocultured with MSCs hematocrit in bone marrow concentrate are 92±46 (103cells/μL), 41±19 for 72 h. We have defined two distinct localizations of leukemic cells relative (103cells/μL), 515±264 (103cells/μL), and 5.7±1.4 (%), respectively. The to MSC layer: those in supernatant (non-adherent cells) and cells adhering TNC, MNC, PLT concentrations and hematocrit in un-processed bone mar- on the surface of MSCs. The cell cycle, proliferation and immunophenotype row are 19±4 (103cells/μL), 7±2 (103cells/μL), 87±31 (103cells/μL), and of these two cell fractions were evaluated at day 0 and day 3. The expres- 35.1±3.4 (%), respectively. To characterize presence of MSCs, HSCs, B-cells sion of CDKN1A (p21CIP1) gene was quantified by qRT-PCR (Universal Pro- and T-cells in un-processed bone marrow and bone marrow concentrate (N beLibrary, Roche). SDS-PAGE and western-blot experiments were realized to = 6), samples were stained with anti-CD34, CD133, CD73, CD105, CD19, quantify p21CIP1 expression. Flow cytometry studies were performed: (a) to CD3, CD4 and CD45 antibodies, and then analyzed on a CyFlow® ML analyze the cell cycle by staining with 7-aminoactinomycin D (7AAD), Alexa (Partec). Cells with positive antigen expression were reported as percentage Fluor®488-conjugated anti-human Ki67 and Alexa Fluor®488-conjugated of leukocyte (CD45+) population. There is a significant increase in white anti-phospho-histone H3 (ser10) antibodies ; (b) to track the cell divisions blood cell, MSC, and HSC populations and significant reduction in red blood with carboxyfluorescein diacetate N-succinimidyl ester (CFSE). Supernatant cells in bone marrow concentrate as compared to un-processed bone mar- of MSCs did not modify the cell cycle and the prolifération of leukemic cells. row. Mean densities of cells expressing MSC markers in marrow concen- Conversely, when compared with cells in the supernatant of MSCs, adhering trates were 869 (CD105+)/1,144 (CD73)+ cells/mL, and mean densities of KG1a cells were characterized by cell cycle inhibition: increase in G0 phase, cells expressing HSC markers were 1,866 (CD133+)/ 2,000 (CD34)+ cells/ decrease in S and M phase, overexpression of CDKN1A and p21CIP1 and mL. There is an increase in TNC, (4.9 fold), MNC (5.8 fold) and PLT (6.2 reduced mitotic activity (CFSE). Altogether these findings suggest that the fold) counts in bone marrow concentrates as compared to un-processed bone marrow microenvironment plays a key role in the regulation of the cell bone marrow. Several stem cell counts including CD133+ (5.4 fold,), CD34+ cycle and prolifération of leukemic cells. Targeting of microenvironmental (5.6 fold), CD73+ (6.5 fold), and CD105+ (5.3 fold) were also increased in interactions in leukemia may have clinical relevance and it will be important bone marrow concentrates. Discussion/Conclusion: The data indicates that to verify if these results can be extended in vivo to other models of human the use of concentrated bone marrow aspirate, prepared intra-operatively leukemias. using Magellan centrifuge devices, can be used as an adjunct cell therapy in orthopedic and other ischemic wound care procedures.

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Poster Board Number: 3009 plasm or had an effect on cellular ultra structure. TEM images revealed that CNTs located in the cytoplasm, even nuclear, which might have an impact MONITORING HUMAN STEM CELL on cellular signaling pathways. Furthermore, MTT assay indicated that CNTs DIFFERENTIATION USING MULTI-PARAMETER resulted in an initial decrease in proliferation from the second day at 5 ug/ ml, third day at 1ug/ml and fourth day at 0.1ug/ml. Real-time PCR con- FLOW CYTOMETRY firmed that the attenuated expression of undifferentiation markers Nanog Hsu, Matthew, Weldon, Don, Santos, Mark, Renteria, Roberto, and Oct-4. The mesoderm gene, GSC (Goosecoid) was greatly increased in Whalley, Jason the presence of 1ug/ml CNTs. Additionally, we found that high concentra- tion of CNTs can accumulate and induce apoptosis in hESCs and activate Research and Development, Merck Millipore, Temecula, CA, USA, the tumor suppressor protein p53 within 24hrs of exposure. These results Marketing, Merck Millipore, Temecula, CA, USA suggest that careful scrutiny of the cytotoxicity of nanomaterials is neces- Flow cytometry is a sensitive, robust, and accurate method for the detection sary, like MWCNTs, that have been previously demonstrated to have limited of key cellular events including phenotypic monitoring and pathway profil- or no toxicity at the cellular level. Moreover, CNTs also induce changes in ing. The multi-parametric, single-cell analysis capabilities inherent to flow the self-renewal and differentiation programs of the stem cells. Further cytometry facilitates the detection and enumeration of multiple phenotypes researches are in progress on both sides. simultaneously on thousands of cells per second providing robust, high con- Poster Board Number: 3013 tent, information on a given population of cells. The guava flow cytometer adds the key advantage of providing highly accurate and robust data sets COXSACKIE VIRUS B INFECTION INDUCES on low volume, small sample size cell populations, as is characteristic in stem cell (especially embryonic stem cell) biology. Stem cells are a unique type of APOPTOSIS IN HUMAN EMBRYONIC STEM cells which can be maintained in vitro as pluripotent cells. Stem cells can also CELLS be induced to differentiate into certain cell types by manipulating culture conditions. Therefore, stem cells have the potential of providing an unlim- Romorini, Leonardo, Scassa, María E., Blügermann, Carolina, Videla ited supply of cells for cell-based therapy as well as providing a resource Richardson, Guillermo A., Questa, María, Fernandez Espinosa, Darío to study basic human developmental biology. In order to facilitate rapid D., Sevlever, Gustavo E., Gómez, Ricardo, Miriuka, Santiago G. characterization of stem cells and to monitor lineage-specific differentiation, FLENI, Buenos Aires, Argentina, CONICET-UNLP, La Plata, Argentina we have developed a series of 3 parameter flow cytometry assays that can be used to detect and quantify changes (or lack of changes) in embryonic Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that and adult stem cell populations. We use fluorescently labeled antibodies can differentiate to a wide range of specialized cells. Group B Coxsackie against ES cell markers such as TRA-1-60, SSEA4, SSEA1, OCT4 and a novel viruses (CVBs) produce acute myocarditis and chronic dilated cardiomyo- marker, HESCA1. The data shows that our antibodies are specific to stem pathy. We have previously shown that hESCs and differentiated contractile cell antigens and correlate well with results from conventional methods such embryoid bodies express CVB receptors and that CVBs infection of these as Western Blot and ICC. Flow cytometry provides a faster and more reliable cells causes a cytopathic effect compatible with cell death. The aim of the alternative to obtaining multiple endpoint (phenotypic) information in a present work was to study if CVBs infection induces programmed cell death mixed cell population. in hESCs. hESCs HUES-5 (H5) and WA-01 (H1) and CVBs (1 and 3) were used. Undifferentiated state of hESCs was validated by immunofluorescence Poster Board Number: 3011 and RT-PCR of pluripotent markers (TRA1-60, TRA1-81, SSEA-4, Oct-4 and Nanog). H1 and H5 cell viability (XTT assay) and apoptosis (DAPI staining, CARBON NANOTUBES AND HUMAN TUNEL, PARP cleavage and caspase-3-like activity assays) were measured EMBRYONIC STEM CELLS: INHIBITION at different time points post-infection (pi). Cell viability decreased depend- ing on moi at 24hs CVB1 and CVB3 pi; DAPI and TUNEL positive apoptotic OF PROLIFERATION AND INDUCTION OF nuclei increased at 5 and 8 hours CVB3 pi, caspase-3-like activity peaks at DIFFERENTIATION 1 hour CVB3 pi and PARP cleavage started at 5 hours CVB3 pi. Quantifica- tion of anti-(Bcl-2, Bcl-XL) and pro-(Bax, Bad) apoptotic genes mRNA levels Yue, Fengming, Tomotsune, Daihachiro, Saito, Naoto, Hara, by RT-Real Time PCR showed a decrease in Bcl-XL mRNA levels for both Kazuo, Usui, Yuki, Itoh, Isamu, Endo, Morinobu, Shirasawa, Sakiko, H1 and H5 at 8hs CVB3 pi. However Bcl-2, Bcl-XL and Bax protein levels Yokoyama, Tadayuki, Nagai, Mika, Sasaki, Katsunori did not change at 5, 8, 16 and 24 hours CVB3 pi. Conclusions: Cytopathic Department of Embryology and Histology, Medical School of Shinshu effect observed after hESCs infection with CVBs is compatible with an early University, Matsumoto, Nagano, Japan, Department of Applied Physical induction of apoptosis. Therapy, Shinshu University School of Health Sciences, Matsumoto, Nagano, Japan, Department of Orthopaedic Surgery, Shinshu University School of Medicine, Matsumoto, Nagano, Japan, Faculty of Engineering, Shinshu University, Nagano, Japan, Laboratory for Advanced Health Science, Bourbon Institutes of Health, Bourbon Corporation, Matsumoto, Nagano, Japan Carbon nanotubes (CNTs) have shown an innovative tool in a large variety of applications, ranging from nanocomposite materials to biomedical devices. The synergy of CNTs with embryonic stem cells (ESCs) provides a promising opportunity for novel therapeutic modalities. However, little is known about the effect of CNTs on stem cell behavior. The main aim of this study is to examine the impact of CNTs on human ESCs phenotype, viability, proliferation, and differentiation. Initially, a range of different concentration of multiwalled nanotubes (MWCNTs, diameter, 10-15nm; length, 100- 300nm) was added to Kyoto human ESCs (khES-1). Transmission electron microscopy (TEM) was performed to investigate if the CNTs can enter cyto-

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Poster Board Number: 3015 proliferation and DNA synthesis rate in a concentration-dependent manner. Silencing BKCa or hEAG1 channel with the specific shRNA also inhibited HUMAN ADULT DENTAL PULP STEM CELLS: proliferation. Flowcytometry analysis showed that paxilline and astemizole SURVIVAL, MIGRATION AND DIFFERENTIATION accumulated human MSCs at G0/G1 phase, and decreased cell population of S phase. In addition, spontaneous Ca2+i oscillations exhibited in most hu- FOLLOWING TRANSPLANTATION INTO STROKE man MSCs were inhibited by the IP3R inhibitor aminoethoxydiphenyl borate RAT BRAIN (2-APB) and the SERCA inhibitor cyclopiazonic acid (CPA), while enhanced by cyclic ADP ribose. Interestingly, cell proliferation was suppressed by Leong, Wai K., Henshall, Tanya, Arthur, Agnes, Kremer, Karlea, 2-APB or CPA, whereas promoted by cyclic ADP ribose via decreasing or Helps, Stephen, Manavis, Jim, Lewis, Martin D., Vink, Robert, increasing ERK1/2 phosphorylation. Conclusion: These results demonstrate Gronthos, Stan, Koblar, Simon A. that in addition to BKCa and hEAG1 channels (but not INa), intracellular Centre for Stem Cell Research, The University of Adelaide, Adelaide, Ca2+ signals regulate cell proliferation in human MSCs, indicating that both Australia, Department of Hematology, The Institute of Medical and ion channels and Ca2+ signals most likely play a crucial role in maintaining Veterinary Science, Adelaide, Australia, School of Medical Sciences, The in situ physiological renewal of human bone marrow. University of Adelaide, Adelaide, Australia, Neuropathology Laboratory, Poster Board Number: 3019 The Institute of Medical and Veterinary Science, Adelaide, Australia Human dental pulp stem cells (DPSCs), derived from adult third molar teeth, ROLES OF PEROXISOME PROLIFERATOR- are multipotent and have the capacity to differentiate into neurons under ACTIVATED RECEPTOR GAMMA ON inductive conditions both in vitro and following transplantation into the mesencephalon of the avian embryo. In this study, focal cerebral ischemia PROLIFERATION STATE OF MOUSE EMBRYONIC was induced via reversible intraluminal middle cerebral artery occlusion in a STEM CELLS DUE TO THE ABSENCE OR rodent model, and human DPSCs were transplanted intracerebrally 24 hours PRESENCE OF LEUKEMIA INHIBITORY FACTOR post-stroke into the peri-infarct region at 2 stereotaxic coordinates: half each into the cortex and the striatum. We then evaluated the behavior of the Ghaedi, Kamran, Peymani, Maryam, Ghoochani, Ali, Karamali, engrafted cells. An average of 2.3% of the original 600,000 transplanted Fereshteh, Karbalaei, Khadijeh, Kiani, Abbas, Nasr-Esfahani, DPSCs survived in the rodent brain at 4 weeks after treatment. While the Mohammad Hossein, Baharvand, Hossein majority of the DPSCs demonstrated targeted migration toward the infarct, Department of Cell and Molecular Biology, Department of Biology, Royan some travelled considerable distances into the contralateral hemisphere. En- Institute for Animal Biotechnology & University of Isfahan, Isfahan, Islamic grafted cells around the lesion expressed the mature neuron-specific nuclear Republic of Iran, Department of Cell and Molecular Biology, Royan Institute protein NeuN as well as the astrocytic marker glial fibrillary acidic protein. A for Animal Biotechnology, Isfahan, Islamic Republic of Iran, Department of number of cells also appeared to engraft into the wall of blood vessels with Stem Cells and Developmental Biology and Department of Developmental morphological appearances suggesting differentiation into smooth muscle Biology, Royan Institute for Stem Cell Biology and Technology and and endothelial cells. The present data substantiates the long-range migra- University of Science and Culture, Tehran, Islamic Republic of Iran tion, neural and angiogenic differentiation potential of human DPSCs in the ischemic brain. These engrafted cell behaviors may represent cellular mecha- Embryonic stem cells have drawn particular interest due to their ability to nisms of actions underlying the enhancement of post-stroke functional differentiation into all three embryonic lineages while maintaining their recovery following cell therapy. other characteristic, self-renewal. These characteristics have made them a powerful model for investigating the mechanisms of cell survival and dif- Poster Board Number: 3017 ferentiation in early embryonic development. There are several factors like BOTH ION CHANNELS AND CALCIUM SIGNALS Leukemia inhibitory factor (LIF) which modulate the self-renewal and stem cell differentiation. Peroxisome Proliferator-Activated Receptor γ (PPARγ) REGULATE PROLIFERATION IN HUMAN ADULT is belong to nuclear receptor dependent ligand-activated transcription fac- MESENCHYMAL STEM CELLS FROM BONE tors that regulates expression many genes involvement in metabolism, cell differentiation, adipogenesis, inflammation and apoptosis. In this study we MARROW demonstrated that activation of PPARγ by its agonists (Rosiglitazone and Zhang, Ying-Ying, Tao, Rong, Li, Gui-Rong Ciglitazone) increased mESCs proliferation state in presence of LIF, while in absence of LIF, PPARγ agonist decreased proliferation state of these cells. In Department of Medicine, LKS Faculty of Medicine, The University of Hong both situations, PPARγ antagonist (GW9662) reversed the effects caused by Kong, Pokfulam, Hong Kong, Division of Hematology, Xinhua Hospital, PPARγ agonists. Moreover, data indicated that LIF increased PPARγ expres- Shanghai Jiao Tong University School of Medicine, Shanghai, China sion. In the absence of LIF, PPARγ agonist reduced Nanog expression as Background: It has been recognized that human bone marrow-derived one of the pluripotent markers while had not any effect on Oct4 expression mesenchymal stem cells (MSCs) are present within the bone marrow cavity level. These results suggest that pivotal roles of PPARγ on proliferative state and serve as a reservoir for the continuous renewal of various mesenchymal of mouse embryonic stem cells depend on presence and absence of LIF. tissues. However, their cellular biology is not fully understood, especially on the regulation of their cellular activities by ion channels and cytosolic free calcium ion (Ca2+i). The present study was designed to investigate the potential roles of Ca2+ signals and ion channels in regulating prolifera- tion in human MSCs. Methods: Whole-cell patch voltage-clamp, confocal microscopy, cell proliferation assay, and Western blot analysis were used to determine membrane currents, Ca2+ signals, cell proliferation, and intracel- lular kinase activity, respectively, in cultured human MSCs. Results: Large- conductance Ca2+-activated potassium (BKCa) channel, ether-à-go-go potassium (hEAG1) channel, and voltage-gated sodium channel (INa) were present in human MSCs, and suppressed respectively by paxilline, astemizole and tetrodotoxin (TTX). Paxilline or astemizole, but not TTX, decreased cell

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Poster Board Number: 3023 TER119- cells delivered I.V. 2x/week for 2 weeks. CFA was injected with the first cells injection. Salivary flow rate, total proteins, epidermal growth PREPARATION AND CHARACTERIZATION factor, amylase, and electrolytes concentrations in saliva were measured OF PITUITARY STEM PROGENITOR CELLS IN pre-treatment and at 4, 8, and 12 weeks post-treatment. Salivary tissues of female NOD mice were analyzed for foci of infiltrated lymphocytes (focus MOUSE score) and the presence of aquaporin-5 (water transporter) as well as for the Kanda, Seiji, Ooka, Hisashi, Okazaki, Haruka, Suzuki, Hiroko, Y-chromosome and eGFP. Results: At week 12 post-treatment, only NOD Nishiyama, Toshimasa mice receiving CFA+ cells or just cells had a recovery of salivary flow and were protected from Sjögren’s syndrome. Salivary flow in non-treated mice, Regeneration Research Center for Intractable Diseases, Kansai Medical on the other hand continued to decrease over the course of the experiment. University, Moriguchi, Osaka, Japan, Dept of Otolaryngology, Kansai CD45-/TER119- -treated mice also had their saliva flow restored qualita- Medical University, Moriguchi, Osaka, Japan tively as their salivary total proteins, epidermal growth factor, amylase, and The growth trouble syndrome in the infant are induced by the abnormality electrolytes levels were not significantly different at 12 week post-treatment of the transcription factors at the development and differentiation of the an- when compared to pre-treatment (before development of Sjögren’s disease). terior lobe of hypophysis, and cause the damages to the hormone produc- None of the transplanted cells identified by the presence of the Y chromo- ing cells such as GH producing cells, prolactin producing cells, etc. and the some or eGFP expression differentiated into salivary epithelial cells. Although lack of the hormone secretion. In this research, in order to find out the keys the focus score was not significantly different between groups (P=0.160), of treatments for these patients by using regenerative medicine, we tried the size of the lymphocytic infiltration was smaller in the cell-treated mice. to isolate stem cells from adult pituitary gland and identify the cell char- Two of the 14 cell-treated mice were free of infiltrated lymphocytes (no acterization and gene expressions. 3 days age of mice were injected BrdU signs of inflammation). Conclusion: This study suggests that a cell-based to the hypodermic at 2 times/day for 3 days continuously, and sacrificed therapy prevents Sjögren’s-like syndrome in the NOD mice. Because the after 10 weeks to identify the slow-cycling cells suggested the possibility transplanted cells did not differentiate into salivary epithelia, paracrine of stem cells. The results of immunohistochemistry showed that a few cells effects of CD45-/TER119- bone marrow cells on the endogenous salivary were identified as BrdU+ and Bcrp1+ double positive cells in the section of glands might be the mechanism of action in re-establishing saliva flow. This pituitary gland. Furthermore, we assumed that it was possible to purify stem research was funded by CIHR. cells as side population (SP) cells. Pituitary cells were isolated from 6 weeks Poster Board Number: 3027 age of mice and stained with Hoechst33342. After staining, SP cells were sorted as a negative fraction. The population of SP cells was about 1% of MISIDENTIFICATION AND CROSS CULTURE total pituitary cells. Furthermore, in order to analysis the gene expression of these cells, we used microarray techniques by Mouse oligo Microarray CONTAMINATION OF STEM CELL LINES: A (Agilent technologies). The results showed that the upregulated genes in SP CONTINUING PROBLEM cells were included some specific markers of stem cells, Bcrp1 and Sca-1 etc. These cells also had the sphere forming ability. Currently we are going to Borchardt, Erin R., Finger, Jared M., Meisner, Lorraine F., Johnson, study whether these cells have the multipotential ability as stem cells. Julie A. Cell Line Genetics, LLC, Madison, WI, USA Poster Board Number: 3025 The misidentification and cross-contamination of laboratory cell lines is a MESENCHYMAL PRECURSOR CELLS RESTORE common problem affecting many facets of biomedical research, and stem SALIVARY GLAND FUNCTION IN NON-OBESE cell research is no exception. This problem has been known for over 50 years and has been described as the most compelling quality control issue DIABETIC MICE WITH SJöGREN’S SYNDROME confronting the scientific community. Numerous studies, including the expe- Khalili, Saeed, Liu, Younan, Kodama, Shohta, Faustman, Denise, rience of cell line repositories in the US, Germany, UK, and Japan estimate Peterson, Alan, Tran, Simon that between 18-36% of cultures are misidentified, cross-contaminated with a second cell line, or misclassified by cell type or species of origin. The con- Dentistry, McGill University, Montreal, QC, Canada, Medicine, Fukuoka sequences of using such lines are far reaching and immeasurable. Aside from University, Fukuoka, Japan, Medicine, Harvard University, Boston, MA, wasting millions of dollars of public money, time, and intellectual resources, USA, Medicine, McGill University, Montreal, QC, Canada use of misidentified and cross-contaminated cell lines is a frequent source of Introduction: Non-obese diabetic (NOD) mice develop diabetes type I (high inconsistent, questionable and even erroneous data. DNA fingerprinting us- levels of blood sugar) and Sjögren’s-like syndrome (loss of saliva output). ing short tandem repeat (STR) analysis provides a rapid and inexpensive way Our previous work showed that injections of MHC class I matched normal to monitor cell line identity; however, misidentification continues to be a bone marrow or spleen cells with Complete Freund’s Adjuvant (CFA) severe problem. While this problem has been historically linked to cancer cell restored normal blood sugar levels and saliva flow in NOD mice that had lines, our data based on performing over 1300 STR-based DNA fingerprints already developed diabetes and Sjögren’s-like syndrome (i.e. established and karyotype analysis of 4500 iPSC and ESC lines (including related fibro- disease). In our previous study, we used unfractionated bone marrow and blast lines and other adult stem cell lines) will show that stem cell lines share spleen cell populations. Recently, it was demonstrated that CD45 negative the same problem. Our data will show the number of stem cell lines that (CD45-) spleen cells regenerated and restored pancreatic functions in NOD are misidentified, of a different species, or cross-contaminated with another mice. Here, we investigated the capacity of such CD45- cells, obtained from cell line. Causes of cell line misidentification will be discussed, including a bone marrow (mesenchymal precursor cells) by magnetic-activated cell sort- strategy to guarantee and maintain cell line identity. In order to preserve ing (MACS), to prevent salivary dysfunction when administered to young the value of stem cell lines and to protect their usefulness in the field of asymptomatic NOD mice. Materials and methods: 8 week-old female NOD regenerative medicine, it falls upon the shoulders of stem cell researchers to mice received one of these treatments: insulin (n=5), CFA only (5), CFA + ensure the correct identity of their cell lines. CD45-/TER119- cells (n=9), CD45-/TER119- cells only (n=5). Donor cells were obtained from male transgenic mice bearing a reporter gene encoding enhanced green fluorescence protein (eGFP) and expanded in culture. To track the distribution of the donor cells we used both the presence of the Y- chromosome and expression of eGFP. Cell injections were 1x107 CD45-/

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Poster Board Number: 3029 ated in the medium containing cytochalasin B to isolate the metaphase II spindle and ooplast. The spindle from the “N group” was transferred into SCHWANN CELLS DIFFERENTIATED FROM the ooplast from the “F group” to construct the “N-F oocyte” using a glass ADULT RAT SKIN STEM CELLS (SKPS) ARE micropipette with a piezo-driven micromanipulator system. Moreover, the spindle from the “F group” was transferred into the ooplast from the “N HIGHLY SIMILAR TO SCHWANN CELLS group” to construct the “F-N oocyte.” For the control study, the spindle ISOLATED FROM PERIPHERAL NERVE AND form the “N group” was transferred into the ooplast from the “N group” ARE DIFFERENT FROM SKPS, THEIR CELL OF to construct the “N-N oocyte.” The reconstructed oocytes were fertilized in vitro and cultured to the blastocyst stage. The rate of blastocyst formation ORIGIN from the pronuclear (PN) stage of embryos derived from the “N-F oocyte” Dworski, Shaalee, Jinno, Hiroyuki, Miller, Freda D. (7.6 %, n=119) was significantly lower than that of the “N-N oocyte” (37.3 %, n= 118) (p<0.001). The rate of that of the “F-N oocyte” (21.2 %, n= Institute of Medical Science, University of Toronto, Toronto, ON, Canada, 99) was comparable to that of the “N-N oocyte” (p=0.01). These results Department of Physiology, University of Toronto, Toronto, ON, Canada, indicate that the ooplasm is more sensitive to cryopreservation than the Developmental and Stem Cell Biology Program, The Hospital for Sick spindle in the unfertilized oocytes. In conclusion, development of the freez- Children, Toronto, ON, Canada ing and thawing method with smaller influence on the ooplasmic function is Schwann cells (SC) myelinate axons and participate in nerve repair. They essential for future application of the cryopreserved unfertilized oocytes to have been transplanted into rodent models of demyelination, peripheral regenerative medicine. nerve injury, spinal cord injury and multiple sclerosis to provide myelination Poster Board Number: 3033 and functional recovery. Traditionally, SC are isolated from peripheral nerve (nerve-SC). Skin-derived precursors (SKPs) differentiated into SC (SKP- TGFB SIGNALLING INHIBITS DLK1 EXPRESSION SC) appear to have the same regenerative ability as nerve-SC and are less invasive to isolate. In this study, SKP-SC were compared with nerve-SC and DURING CHONDROGENESIS IN VITRO with undifferentiated SKPs by microarray. SKP-SC were found to be more Harkness, Linda, Taipaleenmaki, Hanna, Säämänen, Anna-Marja, similar to nerve-SC than to SKPs, their cell of origin. In unbiased cluster- Kassem, Moustapha, Abdallah, Basem M. ing methods, SKP-SC clustered closer to nerve-SC than to SKPs. 99% of probesets did not significantly differ between SKP-SC and nerve-SC, while Odense University Hospital/University of Southern Denmark, Odense, only 69% did not differ between SKP-SC and SKPs. Although SKP-SC and Denmark, University of Turku, Turku, Finland nerve-SC were very similar to each other, 1% of genes differed between DLKl/FA1 (delta like-1 homolog/Fetal Antigen-1) is a novel surface marker them, indicating that SKP-SC are not exactly the same as nerve-SC. The for embryonic chondroprogenitor cells undergoing lineage progression from high similarity between SKP-SC and nerve-SC supports the use of SKP-SC proliferation to prehypertrophic stages. However, mechanisms mediating for transplantation treatments currently using nerve-SC. The differentially control of its expression during chondrogenesis are not known. Thus, we expressed genes found in this study may explain the reparative advantages examined the effect of a number of signalling molecules on Dlk1 expression of SKP-SC transplantation over nerve-SC transplantation observed in other during in vitro chondrogenic differentiation in mouse embryonic limb bud studies. mesenchymal micromass cultures and mouse embryonic fibroblast (MEF) Poster Board Number: 3031 pellet cultures. Dlk1 was initially expressed during mesenchymal conden- sation and chondrocyte proliferation, in parallel with expression of Sox9 EFFECTS OF CRYOPRESERVATION ON and Col2a1, and was down-regulated upon the expression of Col10a1 by hypertrophic chondrocytes. Among a number of molecules that affected FUNCTION OF THE OOPLASM AND SPINDLE chondrogenesis, TGF-β signalling regulated Dlk1 expression. TGF-β1- OF THE UNFERTILIZED OOCYTES ANALYZED induced chondrogenesis was associated with decreased Dlk1 expression and BY THE “METAPHASE II SPINDLE INJECTION these effects were abolished by TGF-β signalling inhibitor SB431542 sug- gesting an involvement of DLK1/FA1 in mediating the function of TGF-β1 (MESI)” METHOD signalling in chondrogenesis. In support of this hypothesis, we found that Fukasawa, Hiroko, Hirata, Shuji TGF-β1 enhanced chondrocyte differentiation in Dlk1-/- MEF compared to wild type MEF. In conclusion, our data identified TGF-β1 as an upstream Obstetrics and Gynecology, University of Yamanashi, Yamanashi, Japan negative regulator of Dlk1 expression and function during the early events Nuclear transfer-derived embryonic stem (ntES) cells can be established from of embryonic chondrogenesis. The cross-talk between TGF-β1 and DLK1 cloned embryos generated by the transfer of a somatic cell nucleus into an showed to promote early chondrogenesis during embryonic endochondral enucleated oocyte. ‘Personalized’ ntES cells, generated using an individual’s bone formation process. somatic cells, are expected to be applied toward future regenerative medi- Poster Board Number: 3035 cine and tissue replacement therapy. Because the success rate of generat- ing ntES cells is still low, many unfertilized oocytes are required to establish NUCLEAR AGO2 REGULATES ATSCS SURVIVAL ntES cells, and this is a severe hindrance in the application of ntES cells to regenerative medicine in humans. Cryopreservation of unfertilized oocytes THROUGH DIRECT CONTROL OF MIR10B AND has been proposed to overcome this issue. If such storage were possible, SEPN1 EXPRESSION. we could perform somatic cell nuclear transfer (SCNT) as the need arose. However, developmental potency of the cryopreserved unfertilized oocytes Im, Young Bin, Kim, Bong Sun, Jung, Jin Sun, Jang, Jin Hwa, Kang, is poorer than that of the cryopreserved embryos. In the present study, the Kyung Sun, Kang, Soo Kyung effects of cryopreservation on function of the ooplasm and spindle of the Seoul National University, Seoul, Republic of Korea unfertilized oocytes were separately analyzed by the “MEtaphase II Spindle Argonaute 2 (Ago2) has a leading function in miRNA-induced RNA silenc- Injection (MESI)” method. Oocytes at the metaphase II were retrieved from ing, a conserved gene regulatory mechanism in cells and organisms. miRNAs B6D2F1 female mice (the control group; “N group”). In the frozen-thawed are critical for stem cell self-renewal, development and other functions. group (“F group”), oocytes, frozen by the vitrification method, were thawed Here, we report that nuclear Ago2, by binding to a specific region of after cryopreservation in liquid nitrogen for 2 days. Oocyte was enucle- functional genes, directly controls adipose tissue-derived stem cell (ATSC)

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Thursday Poster Abstracts survival in response to a critical dose of reactive oxygen species (ROS)- Poster Board Number: 3039 mediated oxidative cell damage or senescence. The role of nuclear Ago2 has not been previously reported. Here, we show that human ATSCs in DNA METHYLTRANSFERASE CONTROLS STEM which Ago2 was downregulated underwent apoptosis. Silencing of Ago2 in CELL AGING BY REGULATING BMI1 AND EZH2 ATSCs significantly induces upregulation of miR10b and miR23b expression. These miRNAs directly interfere with ROS scavenging gene expression, such THROUGH MICRORNAS as TXNL1 and GPX3. Upregulation of miR10b and miR23b is sufficient to Lee, Seunghee, So, Ah-Young, Jung, Ji-Won, Kim, Hyung-Sik, Choi, induce ATSC cell apoptosis via p38 MAPK phosphorylation and caspase 3 Soon Won, Rho, Kyoung-Hwan, Kang, Tae Wook, Shin, Tae-Hoon, activation. In addition, Ago2 overexpression or interference by miR10b and Kang, Kyung-Sun miR23b expression in ATSCs partially rescued H(2) O(2) /ROS-mediated apoptotic cell death by upregulating the expression of TXNL2, JUNK, Department of Veterinary Public Health, Adult Stem Cell Research Center, caspase-3, and cytochrome C. Nuclear Ago2-mediated miR10b and miR23b Research Institute for Veterinary Science, College of Veterinary Medicine, downregulation also allows cells to escape senescence, which results in Seoul National University, Seoul, Republic of Korea, National Institute of TERT activation, stemness overexpression, and improved self-renewal and Health, Chung-Won, Republic of Korea differentiation through Wnt5a/β-catenin activation. Ago2 expression is For maintenance of adult stem cell self-renewal, cellular senescence is critical for stem cells to escape senescence by downregulating miR10b and considered an important hurdle to overcome. Epigenetic regulation of gene miR23b. The Ago2-binding gene selenoprotein N1 (SEPN1) was also ef- expression is one of the main mechanisms that control cellular senescence. fectively involved in ATSC survival and self-renewal through ROS-mediated Here we show that inhibition of DNA methyltransferases (DNMTs) with p38 MAPK inactivation. This work was supported by a KOSEF grant funded 5-azacytidine (5-AzaC) or with specific small interfering RNA (siRNA) by the Korean government (MOST, No. M10841000109-08N4100-10910) against DNMT1 and 3b induced the cellular senescence of human umbili- in South Korea. cal cord blood-derived multipotent stem cells (hUCB-MSCs) and increased Poster Board Number: 3037 p16INK4A and p21CIP1/WAF1 expression. DNMT inhibition changed histone marks into the active forms and decreased the methylation of CpG NUCLEAR AGO2 AND HSP60 COOPERATIVELY islands in the p16INK4A and p21CIP1/WAF1 promoter regions. Enrichment of EZH2, the key factor that methylates histone H3 lysine 9 and 27 residues, CONTROL ATSC SELF-RENEWAL AND was decreased on the p16INK4A and p21CIP1/WAF1 promoter regions. DIFFERENTIATION VIA OCT4 We found that DNMT inhibition decreased expression levels of Polycomb- group (PcG) proteins and increased expression of microRNAs (miRNAs), Jang, Jin Hwa, Jung, Jin Sun, Kim, Bong Sun, Jee, Min Ki, Choi, Jee which target PcG proteins. Decreased CpG island methylation and increased In, Kang, Kyung Sun, Kang, Soo Kyung levels of active histone marks at genomic regions encoding miRNAs were Seoul National University, Seoul, Republic of Korea observed after 5-AzaC treatment. Taken together, DNMTs have a critical Argonaute2 (Ago2) plays a fundamental role in microRNA-mediated gene role in regulating the cellular senescence of hUCB-MSCs through controlling regulation through its intrinsic endonuclease activity. Here, we demonstrate not only the DNA methylation status but also active/inactive histone marks novel functions and molecular mechanisms by which nuclear Ago2 directly at genomic regions of PcG-targeting miRNAs and p16INK4A and p21CIP1/ regulates HSP (Heat Shock Protein) 60 expression and stem cell self-renewal. WAF1 promoter regions. HSP60 is a crucial regulator of ROS (Reactive Oxygen Species), senescence Poster Board Number: 3041 and apoptotic cell death in several tissues and cell types. HSP60 is regulated via inactivation of P38/JNK and P53 and binds directly to regulatory regions SISTEMQCTM: A NOVEL, BROADLY-APPLICABLE of the TERT, c-myc, GPx3 and P53 and STAT3 genes. HSP60 Chip-PCR MICRORNA-BASED MONITORING TOOL experiments also showed that HSP60 was directly bound to the Oct4 and Nanog genes, directly regulating Oct4 and other stemness genes involved FOR STEM CELL QUALITY CONTROL AND in Adipose Tissue-derived Stem Cells (ATSCs) differentiation. HSP60 also DIFFERENTIATION MONITORING positively regulates redox-scavenging factors GPx3 and GPx1, which directly modulate cytosolic ROS in aged ATSCs. Moreover, our study showed that Hector, Ralph, Basak, Indranil, O’Brien, Vincent Oct4 regulates HSP60 expression and vice versa to control ATSC survival Sistemic Ltd., Glasgow, United Kingdom and self-renewal after binding to the HSP60 gene. Furthermore, HSP60- Stem cells derived from both embryonic and adult tissue or from repro- mediated regulation of Oct4 contributes to neuronal and endodermal β-cell grammed somatic cells have significant promise for human regenerative differentiation of ATSCs in vitro and in vivo along with downregulation of medicine. However, despite similarities in developmental potential, several mesoderm-specific gene expression. Disruption of HSP60 or Oct4 expression groups have found fundamental differences between stem cell lines that significantly increases apoptotic cell death and loss of differentiation potency could impact on the potency and/or safety of the resultant cell populations into the neural and β-cell lineages. We show that increased levels of Ago2 but which were not predicted using current monitoring procedures based on or HSP60 effectively induce nuclear localization of HSP60, which directly flow cytometry and analysis of panels of mRNAs. There is a requirement for controls Oct, c-Myc, P53, TERT, GPx3, and STAT3 expression and activate reliable tools to monitor cell populations during the processes of stem cell self-renewal, survival, and transdifferentiation programs. Collectively, we line development, directed differentiation and scale-up to safe, therapeu- suggest a novel model in which nuclear Ago2 controls HSP60 in ATSCs. This tically-useful cell populations. Sistemic have developed a novel, reliable, work was supported by a KOSEF grant funded by the Korean government broadly-applicable monitoring tool that provides both a good indication of (MOST, No. M10841000109-08N4100-10910) in South Korea. cell homogeneity and insights into underlying biological effects associated with any observed alterations in microRNA expression profiles therefore permitting an assessment on the likely impact of the observed changes on cell phenotype. This patented approach, SistemQCTM, provides a simple, robust and cost-effective tool to monitor the maintenance of pluripotentcy in stem cell lines across passages, the staging of directed differentiation from embryonic, iPS or direct reprogramming strategies and, post scale-up, an assessment of functional attributes and safety profile of the cells. Data will

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be presented to support these applications of SistemQCTM. elevated levels of serum creatine kinase, often associated with subacute onset and marked muscle inflammation. Inflammatory cells were detected in Poster Board Number: 3043 both MM and LGMD patients, scattered or organized into clusters, around ISOLATION AND CHARACTERIZATION OF necrotic fibers. Inflammatory infiltrates around vessels mainly consisted of macrophages whereas in endomysial infiltrates were CD4+ and CD8+ PROGENITOR CELLS DERIVED FROM EQUINE cells. Four mice models of dysferlinopathies were described: SJL/J, AJ and 2 YOLK SAC Dysf-/- models. The AJ model showed a slower progressive muscle disease compared to both Dysf -/- and SJL strains except for the highly compro- Franciolli, André Luis, Russo, Fabiele, Fernandes, Isabella, Pignatari, mised abdominal muscles. In order to verify the role of lymphocytes and Graciela, Beltrão-Braga, Patricia, Carvalho, Ana Flávia, Miglino, immune cells on this disease, we generated the Scid/A/J transgenic mice and Maria Angélica compared these animals with the the age-matched A/J mice. The absence Surgery, University of São Paulo, São Paulo, Brazil, Morphology, UNIFEOB, of T and B lymphocytes in this animal model of dysferlinopathy resulted in São João da Boa Vista, Brazil an improvement of the muscle regeneration. The Scid/A/J mice showed increased specific force in the MHC 2A-expressing fibers of the diaphragm The yolk sac is the first fetal membrane that develops in eutherian mammals. and abdominal muscles. Limb muscles of both Scid/A/J and A/J mice had The aim of this study is to isolate and characterize cells derived from equine similar specific force. T and B lymphocytes seem to have a role in the muscle yolk sac and verify their plasticity. The yolk sac explants were cultivated damaging immune response. Establishing a link between dysferlin and im- in DMEM-H with 20% FBS Hyclone and 1% penicillin/streptomycin. The mune cells is crucial to the development of targeted therapeutics. culture flasks were maintained at 37ºC in a humidified atmosphere containg 5% of CO2. After expansion, the cells were analyzed by immunocytochem- Poster Board Number: 3047 istry and RT-PCR. The cultures of equine yolk sac cells were composed of numerous undifferentiated cells floating, with circular format at the first 24 DIRECTIONAL AND NONDIRECTIONAL RNA- hours of cell culture. After first five days, culture medium was discarded, SEQ EXPRESSION ANALYSIS ON THE ILLUMINA selecting just adherent cells in culture flask. Among different cell types in PLATFORM culture, we found cells with fibroblast-like morphology, epithelial and, less frequent, oval cells. Fibroblast-like cells morphology were small, fusiform Syed, Fraz with reduced cytoplasm. The epithelial cells were large and rounded, EpiBio, Madison, WI, USA cytoplasm sparse, with a small, centrally located nucleus. Cells isolated from the yolk sac fragments showed a normal number of chromosomes (2n = Advances in next-generation sequencing have led to the development of 64) until the fifth passage. The colony-forming unit assay showed hetero- new library preparation methods compatible with multiple sequencing plat- geneous colonies in size, with large and small colonies of cells. Cell growth forms. Mechanical and enzyme-based methods are predominantly used for control showed a log way of growing until the fifth passage, maintaining a preparing libraries, with each system having its own drawbacks and no one plato, equilibrium between birth and cell death until the tenth passage, with system being able to offer a complete solution. Multistep protocols, sample a progressive decline of cell survive since that point. Immunocytochemistry loss, lack of automation capability, and labor costs are some of the limita- showed positivity to Oct-4, Nanog, SSEA-3, Vimentin and PCNA3 cell mark- tions of current procedures. One of the challenges in RNA-Seq is the ability ers. Some cells were CK18 positive, showing that cell culture was mixed as to create directional libraries. Directionality of a transcript is important for observed before. RT-PCR confirmed vimentin positivity. The other cell mark- correct annotation of novel genes, and also because it provides important ers should be performed. As the cells were strongly positive for vimentin information regarding gene function. Directionality can also help in deter- mesenchymal stem cell marker, we decided took differentiation cell assay, mining gene expression levels. Nextera™ technology, already becoming which is under way. the method of choice for DNA-Seq library preparation, can be adapted for RNA-Seq (cDNA) library preparation as well. Here we describe methods for Poster Board Number: 3045 creating directional and nondirectional cDNA libraries using EPICENTRE’s patented Nextera technology. Deep sequencing of Nextera-generated cDNA ABSENCE OF T AND B LYMPHOCYTES libraries produces accuracy, coverage, and bias comparable to control librar- ENHANCES SKELETAL MUSCLE REGENERATION ies produced by standard methods. AND AMELIORATES DYSTROPHIC PATHOLOGY IN DYSFERLIN DEFICIENT ANIMAL MODEL CHROMATIN IN STEM CELLS Sitzia, Clementina, Farini, Andrea, Navarro, Claire, D’Antona, Giuseppe, Belicchi, Marzia, Parolini, Daniele, Bottinelli, Roberto, Poster Board Number: 3051 Meregalli, Mirella, Torrente, Yvan TRANSCRIPTIONAL PULSING OF RETROVIRAL Neurological Science, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Centro Dino Ferrari,Università degli Studi di Milano, Milan, TRANSGENES IN MOUSE EMBRYONIC STEM Italy, INSERM U910 Génétique Médicale et Génomique Fonctionnelle; CELLS Laboratoire de Génétique Moléculaire, Hôpital d’enfants de la Timone, Lo, Mandy Y., Rival-Gervier, Sylvie, Ellis, James Marseille, France, Marseille, France, Department of Experimental Medicine, Human Physiology Unit, University of Pavia, Pavia, Italy Stem Cell and Developmental Biology, Hospital for Sick Children, Toronto, ON, Canada Limb Girdle Muscular Dystrophies (LGMDs) are a group of muscular diseases characterized by predominant weakness and wasting of muscles of the Retroviral transgenes undergo transcriptional silencing in embryonic stem pelvic and shoulder girdle. LGMD-2B and MM were found to arise from (ES) cells. While a small subset of cells can escape silencing, expression still defects in the dysferlin gene. In LGMD-2B muscle affection predominates fluctuates over time. We hypothesize this fluctuation, or “transcriptional in proximal muscles, whereas in MM it concerns mainly distal muscles. noise” is due to pulses of transcription at irregular interval. To visualize Dysferlin is a 237 KDa protein, produced from a gene containing 55 coding transcriptional pulsing, we employed the MS2 system, which allows the exons. Dysferlin is localized at the muscle cell membrane and associated live imaging of MS2-tagged transcripts by binding of the MS2-GFP fusion with cytoplasmic vesicles. LGMD-2B and MM are characterized by highly protein. We infected J1 mouse ES cells with a retroviral transgene harbour-

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Thursday Poster Abstracts ing an EF1a-mRFP-MS2 cassette and mRFP positive clones were isolated. to active promoters in ESCs. Condensin, like cohesin, interacts with Nipbl, Sites of transcription were detected only in mRFP-positive clones and not which accounts for its loading at these promoters. Once loaded, however, in uninfected J1 cells. We have established and characterized two clonal cohesin and condensin migrate to distinct secondary sites in the genome. lines bearing provirus that have escaped retroviral silencing. LM-PCR Cohesin migrates to sites bound by the insulator binding protein CTCF while (Ligation-Mediated PCR) was performed to identify the proviral integration condensin migrates to sites near the ends of actively transcribed genes. sites. ImmunoFISH using a DNA-FISH probe against the integration sites Condensin II is trapped at promoters with paused RNA polymerase II when confirmed the transcription foci were found on one of the alleles. Real-time transcription elongation is inhibited, but migrates to transcription termina- imaging experiments in both ES lines displayed transcriptional pulsing, but tion regions when inhibition is released. Cohesin plays an important role in with different dynamics. In addition, “doublets” of transcription foci, which transcription whereas condensin II does not, suggesting that condensin II may represent simultaneously transcribing sister chromatids, were also de- simply exploits the transcription apparatus to distribute itself along chromo- tected. This is the first report of transcriptional pulsing occurring at genuine some arms. These and other results indicate that cohesin and condensin are retroviral integration sites. Further studies will give critical insights into the both loaded at active cell-type specific promoters and become distributed transcription and replication dynamics of retroviral transgenes in ES cells and to different secondary sites by distinct mechanisms. These results provide the role of noise in ES cell biology. mechanistic insights into a variety of diseases caused by mutations in media- tor, Nipbl and the SMC complexes. Poster Board Number: 3053 Poster Board Number: 3057 GLOBAL EPIGENETIC LANDSCAPE OF MOUSE INDUCED PLURIPOTENT STEM CELLS GENOME-WIDE INTERACTIONS OF THE NANOG LOCUS IN MOUSE PLURIPOTENT STEM Mattout, Anna, Biran, Alva, Meshorer, Eran Department of Genetics, The Hebrew University of Jerusalem, Jerusalem, CELLS Israel Apostolou, Effie,Ohsumi, Toshiro K., Polo, Jose M., Borowsky, Embryonic stem cells (ESCs) exhibit unique chromatin features including a Mark, Hochedlinger, Konrad permissive transcriptional program and an open and hyperdynamic chro- Howard Hughes Medical Institute at Massachusetts General Hospital, matin state. Induced pluripotent stem cells (iPSCs), which are very similar to Center for Regenerative Medicine, MGH Cancer Center and Harvard ESCs, hold great promise both as therapeutic agents and as basic research Medical School, Boston, MA, USA, Department of Molecular Biology, MGH tools. However, the mechanisms by which reprogramming occurs and the and Harvard Medical School, Boston, MA, USA chromatin organization that underlies the reprogramming process are largely Accumulating evidence suggests that the three-dimensional structure of the unknown. Here we characterize and compare the epigenetic landscapes genome in mammalian cells can have profound effects on gene expression of partially and fully reprogrammed iPS cells to that of MEFs, the cells of patterns. Previous studies have shown that the Nanog promoter in mouse origin of these iPSCs and to that of ESCs, which serve as a gold standard for embryonic stem cells (mESCs) undergoes intra-chromosomal interactions pluripotent cells. Using immunostaining, we have analyzed the expression with adjacent pluripotency genes. These interactions are disrupted in dif- and distribution of a battery of histone modifications (H3ac, H4ac, H4K5ac, ferentiated cells or upon depletion of interacting proteins such as Oct-4. H3K9ac, H3K27ac, H3K4me3, H3K36me2, K3K9me3, H3K27me3 and Whether Nanog also interacts with other pluripotency loci in the genome γH2AX), as well as HP1α and Lamin A. We find that the fully reprogrammed remained unexplored in that report. Here, we have attempted to identify iPSCs are epigenetically identical to ESCs, for each of the markers we have and characterize the genomic interaction partners of the Nanog promoter on tested. In contrast, the partially reprogrammed iPSCs, which went through a genome-wide scale using a modification of the 4C technology, followed by morphological changes but do not express Nanog, are epigenetically similar Illumina sequencing (4C-Seq). We have created libraries from multiple differ- to the differentiated MEF cells, except for H3K9me3 and HP1α distribution ent mESC and induced pluripotent stem cell (iPSC) lines as well as from so- which resemble more the ESCs heterochromatin pattern . In addition, using matic cells in order to establish a genome-wide “chromosomal interactome” time course experiments of reprogramming, we find that heterochromatin of the Nanog locus in pluripotent and differentiated cells. Using bioinfor- reorganization precedes Nanog expression and active histone marking, matic analyses, we are integrating published data on genomic binding of revealing the initial step that can be detected by light microscopy during pluripotency transcription factors and key histone modifications to establish reprogramming. Together, these data delineate the epigenetic state of iPSCs an integrated view of genomic interactions, transcription factor occupancy compared to ESCs and differentiated cells, and indicate that certain epige- and the histone modification/expression state of genes in pluripotent cells. netic changes must occur in order to reach the pluripotent state. To evaluate the functional importance of these interactions, we are depleting Poster Board Number: 3055 cells of proteins known to mediate long-range interactions and will assess their impact on the Nanog interactome. Together, these analyses will yield CONTROL OF GENE EXPRESSION AND important mechanistic insights into the role long-range genomic interactions CHROMOSOME MORPHOLOGY IN MOUSE play in the establishment and maintenance of pluripotency. ESCS BY COHESIN AND CONDENSIN Bilodeau, Steve, Dowen, Jill, Orlando, David A., Lin, Charles Y., Young, Richard A. Whitehead Institute (WIBR), Cambridge, MA, USA Structural Maintenance of Chromosome (SMC) complexes, which include cohesin and condensin, mediate changes in chromosome morphology as- sociated with transcriptional regulation. At transcriptionally active promoters in embryonic stem cells (ESCs), Oct4/Sox2 and other enhancer bound fac- tors recruit the mediator coactivator, which binds the Nipbl cohesin loading factor. Cohesin is loaded at these active promoters, where it contributes to control of gene expression and DNA looping, thus linking gene expression and chromosome architecture. We find that condensin II is also recruited

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Poster Board Number: 3059 not showing any preference for the haematopoietic lineage. Quantitative PCR and RT-PCR analyses confirm silencing to be epigenetic/transcriptional COMPARATIVE EPIGENETIC ANALYSIS OF A in nature, being a cumulative effect of several chromatin modifications. Fully FEW HISTONE MARKS IN HUMAN ESC, IPS established silencing in undifferentiated cells is irreversible, as shown by the inability of DNA and histone-modifying drugs to reactivate silenced provirus- AND DIFFERENTIATED CELLS es. This epigenetic silencing, however, can be overcome using LV containing Shahhoseini, Maryam, Favaedi, Raha, Mollamohammadi, Sepideh, SAR-Insulator cassette. Such vectors can be produced in high titres despite Baharvand, Hossein their relatively large genomic size, and when transduced, they lead to stable and high level transgene expression. This effect is much more dramatic Genetics, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, during differentiation, increasing the number of GFP+ hES-Monocytes from Islamic Republic of Iran, Stem cells, Royan Institute for Stem Cell Biology 25% to 95%, with a 5-fold higher GFP MFI. It is noteworthy that there and Technology, ACECR, Tehran, Islamic Republic of Iran is significant but incomplete prevention of silencing in undifferentiated The dynamic structure of chromatin involves sevral molecular mechanismes pluripotent cells. We postulate it to be due to the presence of functional de such as histone modifications which act as “epigenetic marks” through- novo DNMTs in these cells, indicating that silencing in pluripotent cells is out the whole genome. Some histone marks like acetylation of lysine 9 of in part due to DNA methylation, which is both irreversible and unprevent- histone H3( H3K9ac) is associated with euchromatin and activation of tran- able. On the other hand, differentiation-induced silencing does not involve scription, specially in pluripotent cells like embryonic stem cells (ESC) and in- DNA methylation, and appears to be due to repressive histone modifica- duced pluripotent stem cells (iPS); while, methylation of histone on the same tions (deacetylation and methylation) which are fully preventable by the location (H3 K9 me) is correlated with heterochromatin and reprresion of SAR-Insulator cassette. Furthermore, the ability to obtain nearly 100% GFP+ transcription. Also, there are “bivalent marks” of H3K27me3 and H3K4me3, hES-Monocytes rules out the possibility of presence of silencing-promoting representatives of silent developmental genes which should be activated sequences within the LV backbone (unlike onco-retroviral vectors) that after onset of differentiation in pluripotent cells. In the current study, total could attract trans-acting silencing machinery (TRIM28, KAP1, HP1 etc.), levels of the four described histone modifications were quantitatively i.e. silencing is by cis-acting mechanisms, depending on the site of proviral compared by the use of nucleosome-ELISA technique, in four human cell integration. Thus, we show that SAR/Insulator-containing lentiviral vectors lineages including: 1) dermal fibroblast cells and 2) their IPS derivatives, and are useful in the derivation of stably genetically modified differentiated cells 3) ESCs and 4) their IPS derivatives after spontaneous differentiation. Our from pluripotent stem cells. results clearly showed high levels of H3K9me in differentiated fibroblast cells Poster Board Number: 3063 in comparison to the three pluripotent cell lineages, and high histone H3K9 acethylation in ESCs and their IPS derivatives, but not in IPS cells derived RETT SYNDROME; UNDERSTANDING from fibroblast cells. Epigenetic analysis bivalent marks also showed a sig- nificant decrease of H3K27me level in the IPS cells derived from fibroblasts, THE NEURON-GLIA CONNECTION IN in comparison to their origin. However, tri-methylation of lysine 4 of histone DIFFERENTIATED NEURAL STEM CELLS H3 had no significant difference between the four lineages. So, it seems that this bivalent mark has no significant change through reprogramming of full Zachariah, Robby M., Olson, Carl, Rastegar, Mojgan differentiated cells such as fibroblasts. Also, all together, it can be concluded Biochemistry & Medical Genetics, University of Manitoba, Winnipeg, MB, that hypomethylation of H3K9 and H3K27 has a more effective role than Canada hyperacetylation, in epigenetic regulation of reprogramming and induction One in every 150 individual suffers from autism, a neurological disease with of pluripotency in human cells, the opinion needs more investigation. an early childhood onset. Autism Spectrum Disorders (ASD) refer to different Poster Board Number: 3061 forms of autism that can be diagnosed by 2 years of age and refer to harm- ful changes that take place in the brain as it grows and develops. Rett Syn- OVERCOMING EPIGENETIC SILENCING OF drome (RTT) is the most-studied example of autism and the primary cause LENTIVIRAL VECTORS DURING MYELOID of mental retardation in females. RTT is a progressive neurological disorder and it is caused by mutations in the Methyl CpG binding protein 2 (MECP2) DIFFERENTIATION: NOVEL ROLE OF gene. There is no effective treatment for Rett Syndrome. However, reactiva- INSULATOR AND SCAFFOLD ATTACHMENT tion of the Mecp2 gene after the onset of disease in mouse models rescues RTT phenotype. This raises hope towards RTT therapy prospects either by REGIONS delivering MECP2 into the affected neurons, or through drug treatments Bodla, Ahmed Salman, James, William targeted towards proteins, both of which can compensate for MeCP2 loss in Sir William Dunn School of Pathology, University of Oxford, Oxford, neurons. Two MeCP2 protein variants (isoforms) exist; E1 and E2, which are United Kingdom both widely expressed. We and others have shown that MeCP2 is expressed in both neurons and glia, however, a comprehensive study regarding the Myeloid cells (monocytes, macrophages) are hard to be genetically manipu- neuronal and glial expression, function and targets of MeCP2 isoforms has lated. In this regard, our lab developed protocols to derive such cells from not been undertaken. We have developed advanced retroviral and lentiviral hES and hiPS cells, with the aim of doing transgenesis at the stem cell level vectors for RTT gene therapy and performed functional studies in neural using lentiviral vectors (LV), which are robust transgenesis tools but their stem cells (NSC) of a RTT mice. We have shown significant improvement usefulness is hindered by the gradual loss of their expression in pluripotent in neuronal dendrite branching through retroviral delivery of the MECP2E1 cells and their differentiated progeny. Here, we demonstrate that by isolating isofrom. However, we do not know whether or not MECP2E2 has a similar provirus-containing chromatin areas from flanking genomic loci using DNA or a unique role in neuronal maturation. We are now studying MeCP2E1 elements called Insulator (Ins) and Scaffold Attachment Regions (SAR), such and MeCP2E2 expression, function and targets in differentiated neurons and silencing can be completely prevented during myeloid differentiation. In the glia and will compare the rescue effect of each isoform in Mecp2 deficient absence of SAR-Insulator (SI) cassette, withdrawal of selective antibiotic differentiated NSC. Our scientific approach is a combination of advanced (Puromycin) leads to gradual loss of GFP expression in undifferentiated stem cell biology, neuroscience and gene therapy technologies with the hESC. This silencing is significantly more pronounced on differentiation ultimate goal of developing novel therapeutic strategies for Rett Syndrome. induction (30% hESC vs. 75% hES-Monocytes silent after 4 weeks). Flow This work has been supported by funds from the Scottish Rite Charitable cytometry analysis of haematopoietic markers shows that silencing is global, Foundation of Canada, Natural Sciences and Engineering Research Council

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Thursday Poster Abstracts of Canada (NSERC), Canada Foundation for Innovation-Leaders Opportu- regulators have evolved to coordinate gene activation and repression in nity Fund (CFI-LOF), Manitoba Institute of Child Health (MICH), Manitoba response to developmental cues, the epigenetic changes that lead to lineage Health Research Council (Establishment and Operating), University of Mani- commitment and tissue morphogenesis during early embryogenesis are toba (URGP and Dr. Paul HT Thorlakson Funds). largely unknown. We are particularly interested in elucidating the regulatory networks that govern heart development because inherited congenital heart Poster Board Number: 3065 defects (CHD) is one of the leading causes of death in children. Moreover, VARIATION IN MYOD BINDING MOTIFS the proper regulation of gene expression patterns by transcription factors and epigenetic regulators is known to control cardiac morphogenesis during IS ESSENTIAL FOR DEVELOPMENTAL a defined window of time during early embryogenesis. A precise under- REGULATION OF TARGET GENES standing of these events during mammalian development, however, has been lacking due largely to the inability to study this process temporally in Soleimani, Vahab, Yin, Hang, Kockx, Christel E.M, van IJcken, vivo. Using a defined mouse ESC-based cardiac differentiation system, we Wilfred F.J, Grosveld, Frank, Rudnicki, Michael A. have employed a genome-wide systems approach using high-throughput Ottawa Hospital Research Institute, Ottawa, ON, Canada, Erasmus genomic methods to elucidate how chromatin and transcriptional states University and Medical Centre, Rotterdam, Netherlands change during cardiac morphogenesis. We have generated maps of the regulatory architecture that integrate the key features that control the The modulation of transcription through differentiation is believed to be transition from a pluripotent state to mesoderm and cardiac progenitors to largely mediated by the differential assembly of regulatory protein com- differentiated cardiomyocytes. Further analysis is beginning to reveal the plexes over DNA-bound transcription factors. Recent evidence on pheno- key chromatin changes and the distinct sets of co-regulated coding and typic variation within and between species indicates that sequence variation non-coding genes as well as functional genomic elements that drive changes between DNA-binding motifs affects transcription factor affinity, and in gene expression patterns during this process. Dissecting the changing hence rates of transcription of target genes. By combining genome wide chromatin and transcriptional landscapes during cardiomyocyte differentia- MyoD-binding site analysis and gene expression profiling during myogenic tion will provide a robust framework on which to better design stem cell differentiation, we show that active switching of MyoD binding from low- to based therapies for cardiac related diseases. high-affinity sites is an extensively deployed mechanism to regulate gene expression during muscle differentiation. The E-boxes mediating MyoD- Poster Board Number: 3071 binding are highly conserved, thus motif degeneracy is likely maintained by selection to modulate the transcriptional output of target genes. Therefore, SWI/SNF-MEDIATED EPIGENETIC CONTROL OF MyoD switching binding between motifs containing variable sequences MULTIPOTENT CARDIAC PROGENITOR CELLS represents a fundamental mechanism that establishes a dynamic range of transcriptional output for developmentally regulated genes. Wang, Zhong, Lei, Ienglam, Gao, Xiaolin, Sham, Mai Har Cardiovascular Research Center, Massachusetts General Hospital, Harvard Poster Board Number: 3067 Stem Cell Institute, Boston, MA, USA, Biochemistry, the University of Hong INTEGRATIVE GENOMIC ANALYSIS OF DIRECT Kong, Hong Kong, China CONVERSION OF FIBROBLASTS TO NEURONS Dissecting the mechanisms of multipotent cardiac progenitor cell (CPC) differentiation will be essential for understanding the etiology of congenital Vierbuchen, Thomas, Wapinski, Orly L., Tsai, Miao-Chih, Kareta, heart defects (CHDs) and providing molecular basis for cell-based heart Michael, Qu, Kun, Chang, Howard Y., Wernig, Marius therapies. Much is known about transcription factors in cardiogenesis and Stanford Medical School, Palo Alto, CA, USA CPC development. However, our knowledge of epigenetic mechanisms gov- erning the differentiation of CPCs is rather limited. One most likely critical We recently reported that the forced expression of Ascl1, Brn2 (also called epigenetic mechanism is mediated by ATP-dependent chromatin remodelers, Pou3f2) and Myt1l was sufficient to rapidly and efficiently convert mouse which open up chromatin to modulate transcription factor access to DNA. embryonic and postnatal fibroblasts into functional neurons in vitro. In order Our studies of the SWI/SNF chromatin remodeling complex identified that to gain insight into this reprogramming process, we have determined the BAF250a, a key regulatory subunit of SWI/SNF, plays an essential role in genome-wide distribution of these three transcription factors in fibroblasts CPC differentiation and cardiomyocyte specification. Therefore, deciphering during early phases of reprogramming using ChIP-seq. In order to charac- how BAF250a/SWI/SNF regulates CPC differentiation could have significant terize the functional consequences of these binding events, we generated implications in understanding and treating heart disease. To gain a better matched RNA-seq libraries to monitor global changes in gene expression. understanding of BAF250a in CPC differentiation, we have generated both These studies should provide novel insights into the functions of these tran- pan cardiac and second heart field (SHF) BAF250a conditional knockout scription factors, both in the context of lineage reprogramming and neural (KO) mice as well as embryonic stem cell (ESC) reporter lines for in vitro development. CPC lineage tracing. We discovered that BAF250a is highly expressed in Poster Board Number: 3069 early embryonic heart and cardiac- and SHF-specific BAF250a KO leads to severe and distinct cardiac defects in mice. Our ESC-based in vitro differ- DISSECTING EPIGENETIC REGULATORY entiation assays show that lack of BAF250a in CPCs inhibits cardiomyocyte formation but appears to promote smooth muscle and endothelial cell speci- NETWORKS DURING DEFINED STAGES OF fication. Moreover, BAF250a controls the proper expression of key cardiac CARDIOMYOCYTE DIFFERENTIATION factors MEF2c, Nkx2.5, and Bmp10 in SHF CPCs. Chromatin immunopre- cipitation and DNAse I digestion assays indicate that BAF250a regulates Wamstad, Joseph A., Alexander, Jeffrey M., Li, Fugen, Holloway, gene expression by recruiting Brg1, the catalytic subunit of SWI/SNF, which Alisha K., Levine, Stuart S., Bruneau, Benoit G., Boyer, Laurie A. modulates chromatin accessibility around the promoter regions of BAF250a Biology, Massachusetts Institute of Technology, Cambridge, MA, USA, target genes. We are currently employing genome-wide ChIP-seq and Gladstone Institute of Cardiovascular Disease, San Francisco, CA, USA deep-sequencing technologies to identify BAF250a/SWI/SNF-regulated nu- Spatial and temporal control of gene expression patterns throughout cleosome and histone methylation patterns in CPCs and fully differentiated mammalian development requires precise control over transcription. While cardiomyocytes and elucidate how BAF250a/SWI/SNF-mediated chromatin complex regulatory networks comprising transcription factors and epigenetic modifications control the expression of cardiac transcription factors. These

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studies will establish the mechanism by which BAF250a regulates chromatin matid breaks detected in metaphases 2 hour after 1Gy irradiation in both organization and epigenetic landmarks that control CPC differentiation. type of cells (pluripotent and differentiated). Our data indicate that human pluripotent stem cells can effectively utilize the DNA-PK-dependent NHEJ mechanism for repair of radiation-induced DSBs during the late G2 stage of the cell cycle, prior to entering mitosis. Furthermore, we demonstrate that iPS CELLS DNA-PK is responsible for the excessive misrepair of DSBs observed in hESCs Poster Board Number: 3075 compared to somatic cells. HUMAN INDUCED PLURIPOTENT STEM Poster Board Number: 3079 CELLS FROM PATIENTS WITH ATAXIA EFFICIENT HUMAN IPSC DERIVATION USING TELANGIECTASIA FLUORESCENCE ACTIVATED CELL SORTING Nayler, Samuel P., Kozlov, Sergei, Lavin, Martin F., Wolvetang, Ernst TECHNOLOGY Stem Cell Engineering Group, Australian Institute of Bioengineering and Ahmad, Faizzan S., Ritz, Anita, Hua, Haiqing, Eggan, Kevin, Noggle, Nanotechnology, Brisbane, Australia, Radiation Oncology and Biology, Scott A., Kahler, David J. Queensland Institute of Medical Research, Brisbane, Australia The New York Stem Cell Foundation, New York, NY, USA, Pediatrics and An increasing number of genetically inherited diseases have been mod- Naomi Berrie Diabetes Center, Columbia University, New York, NY, USA, eled using induced pluripotent stem (iPS) cell technology. It remains to be Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, seen whether these current models recapitulate the phenotypes observed USA in these disorders or may have future clinical relevance. Amongst these Induced Pluripotent Stem Cell (iPS) Technology has demonstrated enormous disease models are chromosomal instability syndromes, such as Friedrich’s potential in disease modeling and regenerative medicine. However current Ataxia and Fanconi Aenemia. We present for the first time, generation reprogramming methodologies are inefficient and time-consuming. There- and characterization of a novel iPS model for Ataxia-Telangiectasia (A-T) fore, techniques that increase the efficiency of iPS generation will greatly -- a chromosomal instability syndrome hallmarked by neurodegeneration, facilitate the application of this technology for large-scale iPS cell banking immune deficiency and predisposition to cancer. This study has answered a programs and drug screening assays. It has been previously described that fundamental question about the involvement and potential roadblock of the when human fibroblasts are reprogrammed by forced expression of four Ataxia Telangiectasia Mutated (ATM) protein and its downstream targets transcription factors (Oct4, Klf4, Sox2, cMyc), pluripotent surface mark- with regard to reprogramming cells to pluripotency. We demonstrate that ers (e.g. SSEA4, Tra1-60) are upregulated while fibroblast surface markers unlike certain other chromosomal instability syndromes, reprogramming is (e.g. CD13) are downregulated. Here we describe the utility of Fluores- possible without molecular intervention. Additionally, we show that A-T iPS cence Activated Cell Sorting (FACS) for generating iPSCs from primary cells display aspects of the complex phenotypic defects observed concurrent human adult skin fibroblast cell lines. We reprogrammed two adult and with the syndrome, and existing A-T cell models, such as radiosensitivity and one foreskin fibroblast cell lines for 7-14 days after infection, then isolated cell cycle aberrations. This study demonstrates the underlying promise of iPS and replated enriched populations of CD13NEGSSEA4POSTra-1-60POS cells to translational medicine, offering potential insight into understanding cells isolated by FACS. Based on our analysis at cellular and molecular levels ATM biology in multiple tissue lineages and developmentally specific con- (immunocytochemistry analysis, qPCR, embryoid body differentiations and texts as well as providing drug-screening platforms for next generation drug teratoma production), iPS cells generated by FACS are indistinguishable from discovery and testing technologies. those generated by previously described methods. However, our strategy Poster Board Number: 3077 decreased the time required to manually pick and expand colonies, and improved the efficiency of obtaining fully reprogrammed iPSCs. MISREPAIR OF RADIATION-INDUCED DNA Poster Board Number: 3081 DOUBLE-STRAND BREAKS IN HUMAN PLURIPOTENT CELLS GENERATION OF DOWN SYNDROME IPSC Bogomazova, Alexandra, Lagarkova, Maria, Tskhovrebova, Leyla, MODEL TO STUDY TRISOMY 21 ALTERATIONS Shutova, Maria, Kiselev, Sergey OF HEMATOPOIETIC CELL DEVELOPMENT Laboratory of Stem Cell Technologies, Vavilov Institute of General Genetics, Galat, Vasil V., Malchenko, Sergey, Wang, Min, Elio, Vanin, Galat, Moscow, Russian Federation Yekaterina, Soares, Bento M., Crispino, John The incorrect repair of DNA double-strand breaks (DSBs) is the ultimate Department of Pathology, Children’s Memorial Research Center, cause of the formation of chromosomal aberrations. To evaluate the ac- Northwestern University, Chicago, IL, USA, Cancer Biology and curacy of DSB repair in pluripotent and isogenic somatic cells, we used a Epigenomics, Children’s Memorial Research Center, Northwestern G2-chromosomal radiosensitivity assay. We analyzed radiation-induced University, Chicago, IL, USA, Hematology/Oncology, Northwestern chromosomal aberrations in solid-stained metaphases 2 hours after irradia- University, Chicago, IL, USA tion at dose 1 Gy. Two hESC lines were compared with isogenic fibroblast- Children with Down syndrome (DS) show a spectrum of clinical anomalies. like cell lines, and induced pluripotent stem cell line was compared with The natural history of leukemia in children with DS suggests that trisomy parental endothelial cells. Each of the three pluripotent cell lines exhibited 21 contributes to aberrant expansion of hematopoietic cells in the fetal manifold higher level of chromatid exchanges (up to 10 times) compared liver during gestation. DS children commonly show macrocytosis, abnormal with isogenic somatic cells. The frequency of chromatid exchanges in hESCs platelet counts, and an increased incidence of Transient Myeloprolifera- and differentiated cells exhibited a quadratic dose response. This suggests tive Disease (TMD), Acute Megakaryocytic Leukemia (AMKL) and Acute two-hit mechanism of exchange formation and, therefore, involvement of Lymphoid Leukemia (ALL). We developed iPSCs model of Down syndrome non-homologous end joining (NHEJ). The chemical inhibition of key NHEJ patients to study how trisomy 21 alters hematopoietic cell development in component DNA-dependent protein kinase (DNA-PK) by NU7026 caused humans. We put forward approach to establish altered molecular signature significant decrease of radiation-induced chromatid exchanges in hESCs by comparing gene expression, microRNAs and methylation profiles of iPS but not in somatic cells. Also, NU7026 led to 4-6 fold increase of chro-

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Thursday Poster Abstracts cells and their differentiated progenitors generated from individuals with but not decreasing levels of H3K9 methylation. Furthermore, we show that and without DS. The new iPSC lines were generated by forced expression FMR1 protein (FMRP) can be detected both in FX-iPS and FX-neuronal cells of transcription factors in the fibroblasts of normal and Down syndrome following 5-azaC treatment. Finally, we show that FMR1 gene expression is patients with retroviral transduction. The established iPSC lines were shown maintained even when 5-azaC treatment is withdrawn. This is the first time to express a set of pluripotency markers characteristic for hESC and capable that reactivation of FMR1 is shown in stem cells or neuronal cells of FX pa- to differentiate in vitro. We will present the results of ongoing integra- tients by a pharmacological treatment. These findings may eventually pave tive analysis encompassing high throughput datasets of gene expression, the way to affect the progression of the disease in FX patients. microRNAs and methylation. Poster Board Number: 3087 Poster Board Number: 3083 FULLY-PLURIPOTENT HUMAN IPS CELLS INDUCED PLURIPOTENT STEM CELLS DERIVED AND TRANSMITOCHONDRIAL DERIVATIVES: FROM HUMAN PANCREATIC ISLET BETA CELLS TOWARD PATIENT-SPECIFIC STEM CELL SHOW EPIGENETIC MEMORY AND PREFERRED THERAPY FOR MITOCHONDRIAL DNA DISEASE DIFFERENTIATION INTO INSULIN-PRODUCING Bertolotti, Roger CELLS CNRS, Gene Therapy & Regulation Research, Faculty of Medicine, Bar-Nur, Ori, Russ, Holger A., Efrat, Shimon, Benvenisty, Nissim University of Nice - Sophia Antipolis, Nice, France Genetics, Hebrew University, Jerusalem, Israel, Human Molecular Genetics The stringency of the mouse tetraploid blastocyst complementation assay and Biochemistry, Tel-Aviv University, Tel-Aviv, Israel has been instrumental in the identification of fully pluripotent iPS cells and Human induced pluripotent stem (iPS) cells generated from somatic cells in the demonstration that most of the former deemed bona fide mouse by expression of defined transcription factors appear to be highly similar iPS cells were indeed unable to match embryonic stem (ES) cells. Protocol to human embryonic stem cells. Using two genetic lineage tracing systems improvements validated on mouse cells by tetraploid complementation we demonstrate the generation of iPS cell lines from human pancreatic islet stand thus as the key drives of the unbiased full pluripotency path hampered beta cells. These reprogrammed cells acquired markers of undifferentiated by reprogramming caveats including genomic/epigenomic aberrations. True pluripotent cells and differentiated into cells from the three embryonic germ fully pluripotent human iPS cells will thus most likely substitute patient- layers. However, the beta-cell-derived iPS (BiPS) cells maintained open specific iPS cell-based therapy for current emerging allogeneic human ES chromatin structure at key beta-cell genes. BiPS cells also demonstrated an cell-based therapy. Interestingly, in addition to basic regenerative medicine increased ability to differentiate into insulin-producing cells in differentiation and stem cell nuclear-gene therapy, such fully-pluripotent patient-specific assays in vitro and in vivo, compared with ES cells and isogenic non-beta iPS iPS cells open a double exciting avenue for the treatment of mitochondrial cells. Our results suggest that the epigenetic memory may predispose BiPS DNA (mtDNA) diseases. Fully pluripotent iPS cells are the long-sought drive cells to differentiate more readily into insulin producing cells. These findings of our transmitochondrial stem cell gene therapy approach aimed at tackling demonstrate that human iPS cell phenotype may be influenced by their cell most mtDNA diseases whether of heteroplasmic (mixture of wild-type of origin, and suggest that their skewed differentiation potential may be and mutant mtDNA molecules) or homoplasmic (pure mtDNA population) advantageous for cell replacement therapy. origin. On the other hand, it has been recently shown that iPS cells gener- ated from somatic cells of patients incurring a heteroplasmic mtDNA disease Poster Board Number: 3085 (Person marrow pancreas syndrome) have a propensity to eliminate mutant mtDNA culminating with the segregation of disease-free iPS cells. Should RESTORATION OF FMR1 EXPRESSION this property of iPS cells to eliminate deleterious mtDNA be non-disease- IN FRAGILE-X INDUCED PLURIPOTENT specific, it would result in a true breakthrough for the cure of heteroplasmic mtDNA diseases. Regarding our original unrestricted approach, the genesis STEM CELLS AND NEURONAL CELLS BY A of transmitochondrial iPS cells should be straightforward since 1) the self- PHARMACOLOGICAL TREATMENT renewing and differentiative properties of fully-pluripotent iPS cells match ES cell ones and 2) transmitochondrial ES cells have already been produced for Bar-Nur, Ori, Caspi, Inbal, Benvenisty, Nissim the genesis of mtDNA disease mouse models (Sligh et al, 2000). Transmito- Genetics, Hebrew University, Jerusalem, Israel chondrial ES cells are cybrids resulting from the fusion of a somatic cytoplast Patient specific induced pluripotent stem (iPS) cells are pluripotent stem cells (enucleated cell) with an ES cell that has been cured from its mtDNA content generated from somatic cells of affected individuals by expression of defined by a transient growth period in the presence of rhodamine-6G (R6G). Under transcription factors. These cells withhold a tremendous potential for study- optimized conditions, R6G-treated cells are rescued by cytoplast fusion; such ing disease mechanism and for potential drug screening with cell types not a repopulation of mtDNA-free ES cells by cytoplast mitochondria results available before. Fragile-X (FX) syndrome belongs to the autistic spectrum in homoplasmic transmitochondrial cybrids. Of note, neither the elimina- (AS) disorders, and is the most common cause of inherited mental retarda- tion of mtDNA mediated by R6G nor the fusion with a somatic cytoplast tion in males. It is nearly always caused by silencing of the FMR1 gene due appears to interfere with the pluripotent ES epigenotype, thereby enabling to abnormal CGG repeat expansions in the 5-UTR of the gene, that leads to the genesis of transmitochondrial mice. Cured either by segregative growth abnormal DNA methylation of the gene promoter and consequent transcrip- or by transmitochondrial protocols, disease-free patient-specific iPS cells tional silencing. Recently, we have generated 11 FX-iPS cell lines from 3 dif- can then enter the directed differentiation path of our proposed universal ferent FX patients. We have shown that FMR1 gene expression is silenced in stem cell gene therapy platform and be returned to the patient as tissue- all of these cell lines. Here, we investigated the potential role of a demethy- specific wild-type stem cells. Our approach is thus discussed in light of 1) the lating FDA-approved drug, 5-azacytidine (5-azaC) to restore FMR1 gene long-term and synergistic transient epigenetic/regenerative gene therapy expression in FX-iPS cells and FX-neuronal cells differentiated from FX-iPS arms of our universal platform, 2) current transgene-free reprogramming cells. We show that following 7 days of 5-azaC administration, FMR1 gene protocols aimed at clearing the oncogenic hazard incurred by early iPS cells, expression is restored, reaching up to 45% of normal levels. This reactivation 3) concurrent allotopic gene therapy restricted to a few mtDNA mutants and is coupled with extensive DNA demethylation of the gene promoter as well hampered by random-integration hazards, and 4) the full pluripotency of iPS as increasing levels of histone H3 acetylation and histone H3K4 methylation, cells that could provide the means to overcome the multisystemic prevalence of mtDNA pathologies.

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Poster Board Number: 3089 Poster Board Number: 3091 INITIAL CULTURE CONDITIONS OF HUMAN MULTIPLE TARGETS OF ESCC MIRNAS DERMAL FIBROBLASTS INCREASE EFFICIENCY PROMOTE HUMAN INDUCED PLURIPOTENCY AND PACE OF THEIR REPROGRAMMING Blelloch, Robert, Subramanyam, Deepa, Judson, Robert, Lamouille, Bharathan, Sumitha P., Musheer Aalam, Syed Mohammed, Manian, Samy, Derynck, Rik Kannan V., Srivastava, Alok, Velayudhan, Shaji R. University of California-San Francisco, San Francisco, CA, USA Haematology Department and Centre for Stem Cell Research, Christian The ESCC family of miRNAs, including members of miR-290 and miR-302 Medical College, Vellore, India clusters, enhances the reprogramming of mouse embryonic fibroblasts to Slow reprogramming kinetics (~4-5 weeks) and low induction efficiency induced pluripotent stem cells. However, the downstream targets through (0.01%) limit the use of human Induced Pluripotent Stem Cells (hiPSCs) in which these miRNAs influence reprogramming are unknown. We now biomedical research and regenerative medicine. The reprogramming process find that the human ESCC miRNA orthologs hsa-miR-302b and miR-372 also results in generation of heterogeneous population of cells and isolation enhance human reprogramming. Furthermore, these miRNAs repress of fully reprogrammed iPSCs is tedious as several clones have to be screened multiple target genes. Downregulation of individual targets results in only to identify the truly pluripotent ones. Moreover, the slow reprogramming partial recapitulation of the miRNA effects. Many of these targets regulate a process can cause genetic or epigenetic abnormalities, inhibition of tumor relatively small number of cellular processes including cell cycle, epigenetic suppressors and activation of oncogenic pathways in the cells. Recent stud- regulation, epithelial-mesenchymal transition (EMT). Whereas ESCC miRNAs ies reported to improve efficiency of reprogramming involved additional ge- have a known role in regulating the unique ES cell cycle, we now show netic and signaling pathway manipulations that increase the risk of genomic that they additionally regulate EMT by targeting Rho and TGF-β signaling. alterations and tumorigenicity. Use of small molecules in combination with They increase the kinetics of the mesenchymal-epithelial transition during transcription factors improves the efficiency of reprogramming by almost reprogramming and block TGF-β-induced EMT of human epithelial cells. 100 fold with the colonies appearing in less than two weeks. However, it is Additionally, the ESCC miRNAs regulate DNA methyl binding proteins and not clear whether these early appearing colonies had the properties of iPSCs, protein trafficking enzymes during reprogramming. These results emphasize including silencing of the transgenes. Experiments on downregulation of p53 how the ESCC miRNAs act on multiple downstream pathways to promote expression in fibroblasts that resulted in higher proliferation rate and lower induced pluripotency. senescence suggested that these properties favour higher reprogramming Poster Board Number: 3093 efficiency. We found that culture conditions for human fibroblasts affected proliferation and senescence rates. We, therefore, hypothesized that cells VALIDATING HUMAN IPS CELL-DERIVED grown in different conditions will show difference in efficiency and kinetics MOTOR NEURONS FOR ALS MODELING of reprogramming. We cultured human fibroblasts for four passages in two different media conditions; condition 1, using a commonly used cell culture Boulting, Gabriella L., Kiskinis, Evangelos, Eggan, Kevin medium for fibroblasts and condition 2, using a commercially available Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, medium containing growth factors in which we found higher proliferation USA and less senescence of human fibroblasts. Mouse receptor for retroviruses, Slc7a1, was introduced into fibroblasts using lentiviruses and they were Human induced pluripotent (iPSC) stem cells present exciting opportunities grown in their respective media for additional 7 days. For iPS generation, for creating in vitro models of human disease, such as the late-onset motor these fibroblasts were transduced with SOX2, KLF4, cMYC and OCT4 us- neuron disease Amyotrophic Lateral Sclerosis (ALS). Contrary to reported ing retroviruses along with RFP, which was used for monitoring transgene shortcomings in the behavior of iPSCs, we found that using standardized silencing during reprogramming. Significant difference in efficiency and pace procedures in two independent laboratories, 13 iPSC lines gave rise to func- of reprogramming was observed between the cells preconditioned in two tional spinal motor neurons within a range of efficiencies similar to 5 human different culture conditions. At day 10, in condition 2, around 100 small embryonic stem cell (ESC) lines. Furthermore, our test set of iPSCs including colonies expressed the pluripotency markers, SSEA4, TRA-1-60, TRA-1-81, 16 independent lines from 7 individuals allowed us to evaluate the effect of NANOG and OCT4, and all of them showed transgene silencing (RFP-). varying age, sex, ALS health status, and persistent transgene expression on Condition 1 produced around 40 colonies which expressed pluripotency the functional ability of these lines to generate motor neurons. With these markers, but transgenes were still expressed in more than 90% of them. On fully characterized iPSC lines we can now assay for cell-autonomous pheno- day 13, uniform population of hES like colonies with 200-1000 cells were typic differences in ALS patient-derived motor neurons compared to those found in condition 2 and the efficiency of reprogramming was found to be from healthy control individuals. In unpurified long-term neuronal cultures, 0.5% by alkaline phosphatase staining and 0.35% by TRA-1-81-Nanog the number of ALS-iPS-derived motor neurons does not differ significantly co-expression, illustrating faster kinetics and higher efficiency of reprogram- from that of controls, but differences in survival can be directly tested using ming compared to other protocols described for generation of human iPS purified iPSC-derived motor neurons harboring an hb9::GFP transgene. cells. These colonies could be maintained for 8 passages in culture without the loss of their morphology and expression of pluripotency markers. This study illustrates that initial culture conditions that favour higher proliferation of fibroblasts with less senescence can improve the efficiency and kinetics of reprogramming and induce homogeneous population of iPSC colonies that exhibit hES morphology, co-expression of pluripotency genes and silencing of transgenes.

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Poster Board Number: 3095 contains SAD-associated variants in SORL1. We have screened patients from the UCSD Alzheimer’s disease resource center (ADRC) and found increased HUMAN INDUCED PLURIPOTENT STEM CELLS incidence of these SORL1 risk variants in the genomes of SAD patients. DERIVED FROM A SPORADIC ALZHEIMER’S Quantitative RT-PCR analysis of SORL1 in iPSC-derived neurons from J.C.V and SAD patients indicates a significant loss of SORL1 mRNA expression DISEASE PATIENT compared with controls, confirming that these cells recapitulate this feature Chung, Henry C.Y., Lie, Khun H., Lin, Ruby C.Y., Sachdev, of SAD. Future experiments in iPSC-derived neurons will examine APP Perminder S., Sidhu, Kuldip S. processing and tau phosphorylation as measures of cellular AD phenotypes. Finally, we will pursue Zinc Finger Nuclease genome targeting to correct the Stem Cell Lab, University of New South Wales, Sydney, Australia, SORL1 risk variants in human neurons. Ramaciotti Centre for Gene Function Analysis, University of New South Wales, Sydney, Australia Poster Board Number: 3099 Our current knowledge of disease pathology is largely attributed to animal UNDERSTANDING SPINAL MUSCULAR models, however not all models recapitulate the deficits in human patients. Hence, generation of cellular models is an invaluable tool for the study of ATROPHY MECHANISMS THROUGH disease processes in vitro. A limited number of disease-specific pluripotent HUMAN IPSC MODELS DERIVED FROM cell lines had been generated through genetic engineering of existing hESC HAPLOIDENTICAL SIBLINGS WITH A lines or derivation of new lines from pre-implantation genetic diagnosed (PGD) embryos. With the advent of induced pluripotency technology, mul- DISCORDANT PHENOTYPE OF THE DISEASE tiple disease-specific pluripotent cell lines have been produced for a range Boza, Maria G., Wanisch, Klaus, Tizzano, Eduardo, Yañéz, Rafael of genetic and degenerative disorders. Such cells provide a platform for the discovery of novel biomarkers which may aid early diagnosis, provide a School of Biological Sciences, Royal Holloway-University of London, limitless supply of cells for drug screening and importantly study the mecha- Egham, United Kingdom, Hospital de la Santa Creu i Sant Pau, Barcelona, nisms underlying the pathophysiology of the disease. Here, we describe the Spain generation of human induced pluripotent stem cells (iPSCs) from a sporadic Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenera- female patient with clear clinical symptoms of Alzheimer’s disease (AD). Phe- tive disease caused by mutations in the Survival of Motor Neuron 1 gene notypically, AD-iPSCs are indistinguishable from control iPSCs and hESCs, (SMN1), which result in dramatic decreases in the expression of SMN however genome-wide transcriptomic analysis revealed a small number of protein. Low levels of SMN lead to the specific degeneration of alpha motor genes that were significantly (False discovery rate-corrected p-value < 0.05) neurons (MNs) in the anterior horn of the spinal cord, with variable degrees and differentially (Fold change > 1.5) expressed between control and disease of severity associated to the overall amount of the protein present. The populations. Further analysis into this group of genes revealed biological mechanism of the disease is poorly understood and the research is impeded functions that were related to neurogenesis, neuronal maturation, nervous by the lack of relevant models, given the fact that human MNs cannot be system development etc. as determined by gene ontology (GO) enrichment. obtained from living individuals. Recently, an iPSC-based model of SMA This may suggest that AD-iPSCs may have altered neuro-biological activities showed proof on principle of the validity of the use of the iPSC technol- during their development into neurons. We next differentiated AD-iPSCs ogy to model some aspects of the disease in cell culture. Encouraged by into neurospheres, which were similar to control cells in terms of gene and this pioneering finding we have produced iPSCs from several members of a protein expressions. The generation of AD-iPSCs, which at the pluripotent discordant consanguineous family in which four haploidentical siblings share and/or differentiated states show if any specific neuronal deficits, may serve the same homozygous SMN1 mutation, but show nonetheless different as a potential in vitro model for the study of Alzheimer’s disease. phenotypes of the disease. All sisters have four SMN2 copies and similar Poster Board Number: 3097 Plastin-3 levels, which do not explain the differences in severity. Standard characterization of these iPSCs and differentiation protocols are currently AN IPSC MODEL OF SORL1 VARIANTS IN being carried out. Differentiation into motor neurons will allow us to model and study SMA mechanisms, novel ameliorating factors for SMA symptoms SPORADIC ALZHEIMER’S DISEASE and to test new drugs and therapeutic viral vectors already developed in our Young, Jessica E., Williams, Daniel A., Israel, Mason A., Goldstein, laboratory. Lawrence S.B. Poster Board Number: 3101 Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA, CIRM Stem Cell Internship Program, San Diego State HIGH CONTENT-IMAGING CHARACTERIZATION University, San Diego, CA, USA OF IPS CLONES FOR SPINAL MUSCULAR Alzheimer’s disease (AD) is the most common neurodegenerative disorder ATROPHY (SMA) PROVIDES A CONVENIENT affecting elderly adults. Similar to other neurodegenerative disorders AD is DRUG SCREENING PLATFORM progressive, results in loss of cognitive function, is fatal and has no cure. AD is complex with only a rare number of cases following a clear genetic inheri- Maury, Yves, Lecuyer, Camille, Gauthier, Morgane, Côme, Julien, tance pattern while the majority of disease cases arise sporadically (SAD). Girard, Mathilde, Yates, Frank, Laabi, Yacine, Peschanski, Marc, Despite no clear genetic pathway for SAD, many genes have been linked to Martinat, Cecile the disorder by genome-wide association studies. Our work focuses on the ISTEM/CECS/UEVE U861, Evry, France, INSERM-PARIS 11 UMR935, sortilin-related receptor-1 gene (SORL1), which was linked to SAD in 2007 Villejuif, France and has been shown to play an important role in the amyloid precursor protein (APP) pathway. SORL1 is involved in intracellular sorting of APP Recent advances in stem cell technology have the potential to allow produc- through endocytic pathways and loss of SORL1 expression has been docu- tion of a virtually limitless supply of normal human cells that can be dif- mented in SAD cases. Because SAD is difficult to model in transgenic labora- ferentiated into any specific cell type. Moreover, using induced pluripotent tory animals, patient-specific induced pluripotent stem cells (iPSCs) may help stem cell technology, they can also be generated from patients with specific to elucidate the contribution of individual genomic variants to SAD. Here disease traits, enabling more relevant modelling and drug screens. Since we use iPSCs from J. Craig Venter (J.C.V) whose published diploid genome 2008, the number of disease, for which related human iPS cell lines have

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been generated, has increased incredibly. However, the generation and the Poster Board Number: 3105 characterization of the disease-related hiPS clones are time consuming and lead to the limited number of clones used for further analysis. We describe ISOLATION OF MECP2-NULL RETT SYNDROME here a new approach for the selection of bona fide iPS clones based on an PATIENT HIPS CELLS AND ISOGENIC automated stem cells amplification and high content- imaging characteriza- tion. We developed this approach in the context of the Spinal Muscular CONTROLS THROUGH X-CHROMOSOME Atrophy (SMA). This autosomal recessive genetic neurodegenerative disease INACTIVATION is one of the principal genetic causes of infant death and is characterized by targeted loss of lower motor neurons, leading to muscular wasting and pa- Cheung, Aaron YL, Horvath, Lindsay M., Grafodatskaya, Daria, ralysis. SMA results from diminished expression of the widely expressed sur- Pasceri, Peter, Weksberg, Rosanna, Hotta, Akitsu, Carrel, Laura, vival motor neuron (SMN) protein. Childhood SMA is classically subdivided Ellis, James into three clinical groups (Type I, II and III) on the basis of age of onset and Program in Developmental & Stem Cell Biology, The Hospital for Sick clinical course. We have generated human iPS cells from 3 SMA fibroblast Children, Toronto, ON, Canada, Department of Biochemistry and Molecular cell lines (Type I and II) and 2 controls. Up to 60 individual iPS clones were Biology, Pennsylvania State College of Medicine, Hershey, PA, USA, collected and grown in a 96 well plate format allowing the development of Program in Genetics & Genome Biology, The Hospital for Sick Children, a high content-imaging characterization. Thus, pluripotency was first evalu- Toronto, ON, Canada, Department of Reprogramming Science, Centre for ated by monitoring the expression of OCT4, TRA1-60, SSEA3/4 markers. iPS Cell Research and Application, Kyoto, Japan Proviral silencing was also analyzed permitting to evaluate the fully repro- grammed state. More interestingly, we developed a high content-imaging Rett Syndrome (RTT) is a neurodevelopmental autism spectrum disorder that screening to quantify the decreased expression of SMN protein specifically in affects girls due primarily to mutations in the gene encoding Methyl-CpG SMA hiPS clones. The use of high content-imaging characterization of hiPS Binding Protein 2 (MECP2). The majority of RTT patients carry missense and provides an interesting approach to evaluate the efficiency of reprogram- nonsense mutations leading to a hypomorphic MECP2 while null mutations mation for a larger number of clones as well as the variability from clone to leading to the complete absence of a functional protein are rare. MECP2 clone. These findings may assist in accelerating the development of drug is an X-linked gene subject to random X-chromosome inactivation result- screening for SMA. To a longer extend, our results may also contribute to a ing in mosaic expression of mutant MECP2. The lack of human brain tissue faster characterization of disease-related hiPS. motivates the need for alternative human cellular models to study RTT. Here we report the characterization of a MECP2 mutation in a classic female RTT Poster Board Number: 3103 patient involving rearrangements that remove exons 3 and 4 creating a functionally null mutation. To generate human neuron models of RTT, we ANALYSIS OF THE HETEROGENITY OF X isolated human induced Pluripotent Stem cells (hiPS) from RTT patient fibro- CHROMOSOME INACTIVATION IN HUMAN blasts. RTT-hiPS cells retained the MECP2 mutation, are pluripotent and fully reprogrammed, and retained an inactive X-chromosome in a nonrandom PLURIPOTENT STEM CELLS pattern. Taking advantage of the latter characteristic, we obtained a pair of Bruck, Tal, Benvenisty, Nissim isogenic wild-type and mutant MECP2 expressing RTT-hiPS cell lines that Genetics, Hebrew University of Jerusalem, Jerusalem, Israel retained this MECP2 expression pattern upon differentiation into neurons. Phenotypic analysis of mutant RTT-hiPS cell-derived neurons demonstrated In female mammals, X chromosome inactivation is a process in which one a reduction in soma-size compared to the isogenic control RTT-hiPS cell- of the two X chromosomes is silenced, following Xist expression. Mouse derived neurons from the same RTT patient. Analysis of isogenic control and pluripotent stem cells do not express Xist, and harbor two active X chro- mutant hiPS-derived neurons represents a promising source for understand- mosomes. However, analysis of X inactivation in human embryonic stem ing the pathogenesis of RTT and the role of MECP2 in human neurons. cells (hESCs), mainly based on Xist expression, was not conclusive. Here, we studied X-inactivation in hESCs by meta-analysis of the expression of the Poster Board Number: 3107 entire set of genes on the X chromosome in 21 female hESC lines. Thus, we could divide the ES cell lines into three categories: lines with no X-inactiva- NEURONAL MATURATION DEFECT IN INDUCED tion, lines with full X-inactivation, and lines with partial X-inactivation. The PLURIPOTENT STEM CELLS FROM RETT partial inactivation of the X chromosome always involved the middle of the SYNDROME PATIENTS chromosome, surrounding the Xist transcription site. The status of XCI in some of the cell lines was validated by either allelic specific expression or Kim, Kun-Yong DNA methylation analysis. Interestingly, analysis of 15 female human in- Yale University, New Haven, CT, USA duced pluripotent stem cell (hiPSC) lines demonstrated similar heterogeneity in the inactivation of X chromosome and classification into the same three RTT (Rett Syndrome) is a progressive X-linked neurological disorder that categories detected in hESCs. Thus, we could show that in some hiPSC lines affects 1 in 15,000 female births and one of the most common forms of the X chromosome was activated upon reprogramming. Activation of the X mental retardation in female. Since the identification of causative link be- chromosome upon reprogramming was shown in hiPSC generated in 3 dif- tween MeCP2 and RTT, the investigation of mechanistic studies of RTT has ferent labs and 4 different reprogramming methods, implying no correlation progressed rapidly. Despite the essential information on RTT obtained from between these variables and XCI state. Based on our analysis we propose a murine models, the phenotype of the MeCP2 knock-out mouse is not per- model for the dynamics of XCI in pluripotent cells. In this model, pluripotent fectly mimic the human RTT and it still remains unclear how loss of MeCP2 stem cells show three states of XCI. The variations in XCI might be created function leads to neurological defects. A main limitation in studying human during the reprogramming process, and/or result from epigenetic changes in RTT is lack of the development of the cellular model of human disease with culture. genome predisposed to the given disease. Therefore, here we attempted to generate in vitro human cellular model of RTT using iPS cells. RTT-iPS cells were derived from RTT fibroblasts by overexpressing reprogram- ming factors. RTT-iPS cells showed features of pluripotency. Interestingly, we found that while some iPS cells retained X chromosome inactivation status of fibroblasts, others showed the activation of both X chromosomes. Hence, we obtained the iPS cells expressing only a single active X chromo-

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Thursday Poster Abstracts some expressing either mutant or wild type MeCP2, as well as iPS cells with Poster Board Number: 3111 reactivated X chromosomes, expressing both mutant and wild type MeCP2. Upon neuronal differentiation, mutant RTT-iPS cells dose not display any de- IN VITRO OSTEOGENIC DIFFERENTIATION fects in early stage of neural differentiation. However, during later neuronal OF NOVEL HUMAN EMBRYONIC STEM CELL maturation the monoallelic mutant or biallelelic RTT-iPS cells show a defect in neuronal maturation consistent with RTT phenotypes. In sum, we devel- DERIVATIVES oped the human cellular model of RTT using human iPS cells and this model Das, Shreyasi, Parsons, Stephanie, LaRocca, David, Funk, Walter, will be invaluable tool to investigate the cellular and molecular mechanisms West, Michael D., Snyder, Evan Y. underlying their abnormalities and to test the effects of drugs in rescuing synaptic defects. Del E. Webb Neuroscience, Aging and Stem Cell Research Center, Burnham Institute for Regenerative Medicine, La Jolla, CA, USA, Mandala Poster Board Number: 3109 Biosciences LLC, La Jolla, CA, USA, BioTime, Inc., Alameda, CA, USA, BioTime, Inc., La Jolla, CA, USA EPIGENETIC ABNORMALITIES IN RETT Repairing large bone defects after major trauma is difficult as the availabil- SYNDROME FIBROBLASTS ARE PARTIALLY ity of using the patient’s own bone is often limited. Recently, we obtained CORRECTED BY REPROGRAMMING derived human embryonic progenitor cells lines (ACTCelerate cell lines) that may generate a robust source of osteocytes. To determine the osteogenic Slavin, Ileana, Sheridan, Steven D., Kwok, Showming, Morey, potential of these cells, cell morphology, differentiation and mineralization Robert, Lynch, Candace, Loring, Jeanne F., Sur, Mriganka, Laurent, were analyzed. These cells exhibited a number of osteo-related markers Louise C., Haggarty, Stephen J. including osteoblast-cadherin (CDH11), osteomodulin (OMD), secreted Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA, phosphoprotein 1 (SPP1), osteonectin (SPARC) as examined by microarray. Brain and Cognitive Sciences, Massachusetts Institute of Technology, Additionally, these cells displayed mineralization as detected by Alzarian red Cambridge, MA, USA, Reproductive Medicine, University of California, staining. These results showed that ACTCelerate cell lines maybe useful as a San Diego, San Diego, CA, USA, Reproductive Medicine, University of surrogate model to study osteocyte differentiation and bone mineralization. California, San Diego, San Diego, CA, USA, Center for Human Genetic Poster Board Number: 3113 Research, Massachusetts General Hospital, Boston, MA, USA Rett syndrome is a neurodevelopmental disorder caused by mutations in ENDOGENOUS PROTEIN TAGGING IN HUMAN MECP2, a gene located on the X chromosome. Since males have only one X PLURIPOTENT STEM CELL-DERIVED NEURAL chromosome, affected males carry one mutant copy and no normal copies of the MECP2 gene, and generally die prior to birth. Affected females carry STEM CELLS one mutant copy and one normal copy in each of their cells; however, in any Doerr, Jonas, Schmidt, Johannes, Lutz, Dominique, Hein, Marco given cell, either the mutant copy or the normal copy is active, due to X- Y., Poser, Ina, Hyman, Anthony A., Mann, Matthias, Koch, Philipp, inactivation. Girls with Rett syndrome often appear normal in early infancy, Brüstle, Oliver but experience loss of developmental milestones and onset of stereotypical movements and breathing patterns at 6-18 months. The stochastic nature Institute of Reconstructive Neurobiology, Bonn, Germany, Max Planck of X-inactivation results in a range of clinical severity, depending on the Institute of Biochemistry, Martinsried, Germany, Max Planck Institute for fraction of cells in the brain that have inactivated the mutant versus the Molecular Cell Biology and Genetics, Dresden, Germany wild-type copy of MECP2. In order to develop an in vitro model of Rett Protein interaction studies represent a powerful tool to study cell signaling syndrome where we could study the potential defects in neural differentia- cascades, cell-cell interactions as well as principles of signal transduction tion, function, and survival caused by mutations in MECP2, we generated mechanisms. The majority of the human interactome studies conducted so induced pluripotent stem cells (iPSCs) from dermal fibroblasts from 8 pa- far are based on overexpression paradigms in tumor cell lines. Commonly tients with Rett syndrome and 8 control individuals. Since the MECP2 gene encountered problems in this context are unspecific interactions due to encodes a methyl-CpG-binding protein, we compared the DNA methylation supra-physiological protein expression levels, the use of transformed cells profiles of both dermal fibroblasts and iPSCs from the affected and control and a non-tissue specific proteome. Ideally, protein-protein interaction data subjects. We used a high-resolution genome-wide microarray to interrogate should be generated in the context of a tissue-specific somatic cell express- the methylation status of 450,000 individual cytosines distributed across the ing the protein of interest under physiological and pathological conditions. genome. The DNA methylation profiles of the affected and control fibro- Pluripotent stem cells (PSC) can differentiate into various somatic cell types, blasts were markedly different, while those of the affected and control iPSCs are amenable to genetic modification and thus represent an attractive cell were largely indistinguishable. The reversal of the abnormalities in DNA source for matching these criteria. However, classic methods of gene target- methylation seen in the Rett syndrome fibroblasts with reprogramming to ing have proven inefficient in human PSC. In this study we present two iPSCs suggests that it may be possible to recapitulate the onset of aberrant innovative strategies for protein tagging in PSC-derived human neural stem DNA methylation by performing directed differentiation of these iPSCs to cells, a somatic stem cell type, which exhibits extensive self-renewal, clono- the neural lineage. genicity and stable neurogenesis. The introduction of GFP-tagged proteins via bacterial artificial chromosomes permitted the derivation of polyclonal cell populations (pools) with faithful protein expression in more than 90% of the cells, as judged by Western blotting and subcellular compartmental- ization in immunofluorescence studies. Using this technique, we generated multiple cell lines harboring tagged proteins including murine Proliferation Cellular Nuclear Antigen (mPCNA1), human Jumonji, AT Rich Interactive Domain 1C (hJARID1C), human Ubiquitin-like with PHD and Ring Finger Domains 1 (hUHRF1), the Methyl CpG Binding Protein 2 (MECP2) involved in the pathogenesis of Rett syndrome and the Alzheimer’s disease-associated proteins Nicastrin (hNCSTN) and Valosin-containing protein (hVCP). Protein- protein interaction studies by a label-free, quantitative affinity purification- mass spectrometry approach identified several novel interaction partners

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of these candidates. Alternatively, we employed an adeno-associated viral Poster Board Number: 3117 (AAV) system to introduce a FLAG tag into genes of interest either directly after the initiating methionine or before the stop codon without modifying IINDUCTION OF HEMATOPOIESIS FROM promoter-associated regulatory regions or exonic/intronic sequences. Both HUMAN INDUCED PLURIPOTENT STEM CELLS systems are sufficiently sensitive to report changes in protein expression levels and compartmentalization induced by extrinsic stimuli. Our data sug- (HIPSCS) IN COCULTURE WITH AUTOLOGUS gest that protein tagging in PSC-derived lt-NES® cells represents an efficient HIPSC-DERIVED MESENCHYMAL STEM CELLS approach for studying protein-protein interactions in human neural cells, UNDER ANIMAL SERUM-FREE CONDITIONS particularly in the context of neurodegenerative diseases. Ebihara, Yasuhiro, Ma, Feng, Mochizuki, Shinji, Hanada, Sachiyo, Poster Board Number: 3115 Matsuzaka, Emiko, Nishihama, Natsumi, Hiramoto, Takafumi, Tsuji, THE HUMAN EMBRYONIC STEM CELL INDUCED Kohichiro PLURIPOTENT STEM CELL SHARED RESOURCE Dept. of Pediatric Hematology/Oncology, Research Hospital, IMSUT, Tokyo, Japan, Division of Stem Cell Processing, Center for Stem Cell Biology and FACILITY AT MOUNT SINAI, NEW YORK Regenerative Medicine, IMSUT, Tokyo, Japan Alexeeva, Vera, Ortega, Sandra, D’Souza, Sunita L. Human induced pluripotent stem cells (hiPSCs), which have the abil- Department of Gene and Cell Medicine, Mount Sinai School of Medicine, ity to differentiate into all cell types in the body, hold great promise for New York, NY, USA regenerative medicine. However, the culture inducing differentiation of hiPSC depends on animal feeder cells and/or serum in most cases. Such The maintenance of human embryonic stem cells (hESCs) and their dif- a culture system is a huge obstacle for clinical applications of these stem ferentiation into multiple lineages offers unprecedented opportunities to cells because of xenogeneic pathogen contamination. To solve this issue, investigate and understand the earliest stages of human development. Re- we developed a novel method for inducing hematopoiesis from hiPSCs cent revolutionary developments in the ESC field have led to the discovery in the coculture with autologous hiPSC-derived mesenchymal stem cells that forced expression of defined gene/protein factors in adult somatic cells (MSCs) using autologous human serum instead of animal serum. When results in their transformation into an ESC-like pluripotent stem cell state undifferentiated hiPSCs (line SPH-0103, which we established from skin referred to as “induced pluripotent stem cells (iPSCs).” This technology fibroblasts obtained from a healthy donor with a written informed consent allows the derivation of patient-specific stem cell lines without the onus of by transduction of OCT3/4, SOX2, KLF4 and c-MYC) cultured on murine social, moral and ethical dilemmas associated with the creation of new hESC embryonic fibroblast (MEF) feeder cells were recultured on gelatin-coated lines. Like ESCs, iPSCs can self-renew thus providing us with a potentially culture dishes with autologoous serum-containing media (auto-media) in unlimited source of cells. They can be genetically modified to define gene the absence of MEF feeder cells. Cells were passaged several times with function and differentiated into multiple lineages. Furthermore, derivation auto-media, and adherent stromal cells were induced after 4-6 weeks. The of iPSC lines from individuals prone to various diseases also provides us with stromal cells were spindle-like shaped, revealed a phenotype of CD45-, the opportunity to understand the etiologies of many complex syndromes. CD34-, CD14-, CD105+, CD31-, and SSEA-4+/-, and showed the ability A dedicated hESC/iPSC shared resource facility (SRF), henceforth referred to differentiate into mesenchymal tissues in vitro, indicating these stromal to as the ‘Core,’ can aid principal investigators with pilot and/or continuing cells were MSCs. In the coculture with irradiated hiPSC-derived MSCs in stem cell projects. Our first main objective is to make available the latest the presence of auto-media, undifferentiated hiPSCs were maintained at developments in the field of ESC/iPSC biology to scientists. Currently we are least for four weeks. To induce the differentiation of these undifferentiated exploring different iPSC technologies to aid in the development of trans- hiPSCs into hematopoietic cells, the culture media in the coculture was gene-free iPSC lines and induced pluripotent cancer (iPC) cell lines. However changed to serum-free hematopoiesis-supporting media containing various generating disease-specific iPSCs is just the first step in this process. The cytokines. 10-14 days later, small round cells appeared and formed the next step involves the differentiation of these cells into the diseased tissue- cobblestone regions in the coculture. When hematopoietic colony assay of specific cells in order to recapitulate aspects of the disease. To facilitate the the cocultured cells was conducted using autologus human serum, but not latter, the Core, is involved in generating various reporter systems. These fetal calf serum, hematopoietic colonies including myeloid, erythroid, and reporters will allow us to separate desired populations and establish condi- multi-lineage colonies were formed. Cytospin samples picked-up from these tions necessary to improve the efficiency of generating specific lineages. In colonies revealed that these colonies contained various blood cells, such as addition, these reporter cell lines will also allow siRNA, micro RNA, small erythroid cells, macrophages, neutrophils and megakaryocytes. In immuno- molecule/drug screens in novel cell populations. The second main objec- staining, the induced erythroid cells expressed both α-globin (100%) and tive of the Core is to quality control stem cell lines, reagents. These quality β-globin (90%), indicating that most of the erythroid cells had adult type control services will include karyotyping, mycoplasma testing of hESC, iPSC hemoglobin. These results demonstrated that the present culture system us- and iPC cell lines as well as the supply of these cell lines and differentiated ing autologous hiPSC-derived MSCs and human serum was able to generate lineages to the interested scientific community (MTA permitting). Scientists various hematopoietic/ blood cells without animal feeder cells and serum. It are also provided with tested stem cell reagents at vastly discounted pricing can be useful for the future clinical application of hiPSCs made possible due to the establishment of two stem cell supply centers, bulk purchasing and NYSTEM funding. Last but not the least, the third main ob- jective of the Core is to continue to conduct classes to teach iPSC generation and hESC/iPSC/iPC cell differentiation into the lineage of choice. The goal of this education is to increase in the number of labs that are able to carry out stem cell research independently. Taken together, these services will have a three-fold benefit. Firstly, it will alleviate the quality control burden of individual scientists and allow them to concentrate on important scientific questions. Secondly, it will allow collaborative projects involving hESC / iPSC /iPC cell lines to be initiated with multiple laboratories by removing the prohibitive cost and providing the expertise required to establish and sustain this technology and lastly it will provide the Core with a source of revenue to meet its expenditures.

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Poster Board Number: 3119 the generation and phenotypic analyses of induced pluripotent stem cells (iPSC) from dermal fibroblasts of two normal subjects and two PD patients MONOCLONAL ANTIBODIES RAISED with parkin mutations (one with homozygous exon 3 deletion and the other SPECIFICALLY AGAINST UNDIFFERENTIATED with compound heterozygous deletions of exons 3 and 5). Multiple lines of iPSCs were generated for each subject using tetracycline-inducible lentivi- HUMAN EMBRYONIC STEM (HES) CELLS ruses expressing Oct4, Sox2, Klf4, c-Myc and Nanog. All four representative PROVIDE A NOVEL TOOL TO DETECT AND iPSC lines analyzed (one for each subject) exhibited morphology indistin- ISOLATE EARLY REPROGRAMMED COLONIES guishable from the H9 human embryonic stem cells (hESC) and could be passaged indefinitely on MEF feeders. Pluripotency markers such as Tra-1- DURING HUMAN INDUCED PLURIPOTENT 60, Tra-1-81, SSEA3, SSEA4, Oct4, Sox2, Nanog, AP, etc. were expressed at STEM (HIPS) CELL LINE GENERATION levels similar to H9 hESC, while viral transgenes were strongly silenced. They maintained normal karyotypes and could be differentiated in vitro (through Ellerström, Catharina C., Johannisson, Jenny, Norén, Helena, EB assay) and in vivo (through teratoma assay) to cells of all three germ lay- Caisander, Gunilla, Davidson, Lindsay, Kilmare, Eva, Englund, Mikael ers. Using a directed differentiation protocol, we differentiated the four iPSC C.O., Hyllner, Johan, Strehl, Raimund lines to midbrain DA neurons. These neurons expressed markers such as TH, Cellartis AB, Gothenburg, Sweden, Cellartis AB, Dundee, United Kingdom En1, DAT, VMAT2, AADC, MAP2, synaptophysin, NR1 and PSD95. They formed functional synapses, fired spontaneous action potentials, and had Novel mouse monoclonal antibodies (mAbs) were generated by immuniz- spontaneous excitatory postsynaptic currents, Na+ currents, K+ currents, ing balb/c mice with whole cell suspensions of undifferentiated hES cells. NMDA-gated currents and GABA-gated currents. Dopamine was released The resulting established mABs ES-CellectTM and hES-CellectTM are highly in a calcium- and activity-dependent manner. Selective dopamine uptake selective surface markers for undifferentiated pluripotent stem cells and was robustly seen in neurons derived from the two normal subjects, but not quickly downregulate during cell differentiation. We have evaluated these from the two PD patients. This phenotype suggests that parkin mutations antibodies during the generation and characterization of a number of hIPS abrogate selective dopamine uptake through dopamine transporter and cell lines and have found that the mAbs bind strongly and specifically to the thus disrupt the temporal and spatial precision of dopaminergic transmis- reprogrammed cells. We noted as an interesting observation that putative sion. We also found that the mRNA levels of monoamine oxidases A and B hIPS cell colonies albeit showing definitive stem cell morphology that were were greatly increased in parkin-deficient neurons, compared to the normal negative for hES-CellectTM immunocytochemical staining also turned out neurons. Consequently, the amounts of protein carbonyls induced by dop- not to be truly reprogrammed in more detailed follow up analysis. There- amine treatment were much higher in parkin-deficient neurons, compared fore the novel mABs could potentially provide a unique tool to separate the to normal neurons. These results suggest that parkin controls dopamine- “true” from the “false” reprogrammed cell lines at a very early stage. As a induced oxidative stress by suppressing the transcription of monoamine further advantage, unlike the often used Tra1-60, Tra1-81 and SSEA anti- oxidases. Thus, these iPSC-derived human midbrain DA neurons not only bodies the novel mABs show no cross reactivity to fibroblast cells and other reveal mechanistic insights into the cellular functions of parkin, but also pro- human somatic cells which are usually applied as starting populations for vide novel targets and a screening platform for disease-modifying therapies reprogramming. As the mAbs recognize cell surface epitopes, they can be of PD. used in a non-cytotoxic manner for the detection of living cells without re- quiring fixation or permeabilization. This is an essential advantage in the de- Poster Board Number: 3123 tection and characterization of early reprogrammed cells, as small amounts of cell material is available at that stage. The novel mABs were furthermore LRRK2 AND ALPHA-SYNUCLEIN MUTANT applied for live fluocytometry as well as FACS and magnetic bead based PARKINSON’S DISEASE IPSC-DERIVED sorting of living pluripotent stem cells. We have used the established mAbs DOPAMINERGIC NEURONS DEMONSTRATE in fluocytometry to quantify the ratio of human pluripotent stem cells and human fibroblasts (hFF) in mixed cell suspensions to determine efficiency. DISEASE RELATED PHENOTYPES Magnetic bead separation with hES-CellectTM results in 95-99% purified Byers, Blake Hampton, Cord, Branden J., Nguyen, Ha Nam, Schüle, undifferentiated cells in the positive fraction, whereas the negative fraction Birgitt, Langston, William, Palmer, Theo, Reijo, Renee contains up to 90% of feeder cells. The identification of the surface antigens recognized by the mAbs, which appear to have a specific role in undifferen- Stanford University, Palo Alto, CA, USA, John’s Hopkins, Baltimore, MD, tiated, pluripotent human stem cells is under investigation. USA, Parkinson’s Institute, Sunnyvale, CA, USA Poster Board Number: 3121 Studies of Parkinson’s disease (PD) have been hindered by lack of access to affected human dopaminergic (DA) neurons. Here, we report the genera- PARKIN MUTATIONS INCREASE OXIDATIVE tion of two induced pluripotent stem cell lines with genetic mutations: (1) homozygous p.G2019S mutations (G2019S-iPSCs) in the Leucine-Rich STRESS IN HUMAN MIDBRAIN DOPAMINERGIC Repeat Kinase-2 (LRRK2) gene, the most common PD-related mutation, NEURONS DERIVED FROM PATIENT-SPECIFIC and (2) a triplication of the gene encoding alpha-synuclein, SNCA, on one IPS CELLS allele, resulting in early onset and rapidly progressing PD. These genetic PD iPSC lines were able to differentiate into DA neurons and showed increased Jiang, Houbo, Ren, Yong, Yuen, Eunice Y., Zhong, Ping, Ghaedi, expression of key oxidative stress response genes and α-synuclein protein. Mahboobe, Hu, Zhixing, Azabdaftari, Gissou, Nakaso, Kazuhiro, Moreover, G2019S-mutant DA neurons were more sensitive to caspase-3 Yan, Zhen, Feng, Jian activation, caused by exposure to hydrogen peroxide, MG-132, and Department of Physiology and Biophysics, State University of New York at 6-hydroxydopamine, compared to unaffected DA neurons. These findings Buffalo, Buffalo, NY, USA, Department of Pathology, Roswell Park Cancer suggest that G2019S-iPSC and SNCA-triplicatoin-iPSC derived DA neurons Institute, Buffalo, NY, USA, Department of Neurology, Tottori University, exhibit early phenotypes linked to PD. In conjunction with the high pen- Yonago, Japan etrance of the LRRK2 mutation and the clinical resemblance of both genetic forms of the disease to sporadic PD, these cell lines may provide an in vitro Parkinson’s disease (PD) is characterized by the selective degeneration of PD model and enable the screening of novel pharmacological agents and di- nigral dopaminergic (DA) neurons. Mutations of parkin represent the most agnostics. Further, these results demonstrate that PD neurons endogenously frequent cause of recessively inherited Parkinson’s disease. Here, we report accumulate α-synuclein protein and suggest that PD degeneration is linked

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to oxidative stress. ity genes can perturb glutamatergic neurotransmission through the NMDA receptor pathway. The progression of neural development in induced pluri- Poster Board Number: 3125 potent stem cells derived from human fibroblasts taken from patients with INDUCED PLURIPOTENT STEM CELLS schizophrenia and “control” normal psychiatric evaluated patients may be evaluated for many parameters that may shed insight in neuronal abnormal- DERIVED FROM PARKINSON’S DISEASE ities seen in patients with schizophrenia. Specifically, biomarker candidates PATIENT DIFFERENTIATED INTO MIDBRAIN for schizophrenia can be investigated at various time points from the stem cell stage through neuronal differentiation to mature functional neurons. If DOPAMINERGIC NEURONS a reliable biomarker is found that can be linked to the pathogenesis of the Kikuchi, Tetsuhiro, Morizane, Asuka, Okita, Keisuke, Inoue, disease, such as a protein involved in the NMDA receptor hypofunction Haruhisa, Takahashi, Jun hypothesis, one may be able to prevent or control the disease by using phar- macological agents or minimizing environmental factors that can contribute Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan, to the onset of the disease. In addition, this in vitro model of schizophrenia Center for iPS Cell Research and Application, Kyoto University, Kyoto, could not only help in studying the etiology of the disease and discovering Japan valuable biomarkers, but it could also be used as a platform to test a variety Induced pluripotent stem cells (iPSCs) are promising source for cell replace- of molecular therapies. ment therapies. However, several problems remain to be solved before they can be used in clinical settings. Such problems include the use of Poster Board Number: 3129 xeno-materials such as feeder cells, immune rejection by the host, and re- XENO-TRANSPLANTATION OF IPSC DERIVED activation of virally transfected genes. As for immune rejection, autologous transplantation of iPSCs derived from somatic cells of patients can be a solu- NEURAL STEM CELLS FROM A PATIENT WITH tion. A recent study showed that iPSCs derived from patients with sporadic LYSOSOMAL STORAGE DISEASE Parkinson’s disease (PD) could differentiate into dopaminergic (DA) neurons. Upon transplantation into PD model rats, they successfully reduced amphet- Griffin, Tagan,Wolfe, John H. amine- and apomorphine-induced rotational movements, indicating their Cell and Molecular Biology, University of Pennsylvania, Philadelphia, PA, contribution to functional recovery. In this study, we show that iPSCs derived USA, Children’s Hospital of Philadelphia, Philadelphia, PA, USA from patients with sporadic PD can differentiate into dopaminergic neurons Approximately 1 in 7000 people worldwide has a lysosomal storage disease. using our feeder-free floating culture method. First we generated iPSCs from The majority of these diseases are caused by mutations in genes encod- the dermal fibroblasts of these patients by reprogramming with episomal ing one of many lysosomal hydrolases in the cell. The resulting pathologi- vectors. Polymerase chain reaction (PCR) confirmed that resulting iPSCs had cal changes affect a number of organ systems, notably the CNS. Enzyme no genomic integration of those vectors. On the day 0 of neural induction, replacement by way of gene or cell therapy has been shown to prevent and pluripotent iPSCs were seeded onto 96-well plates in GMEM supplemented correct a number of these changes. We therefore sought to demonstrate the with 8% KSR. With the addition of Nodal and BMP inhibitors from day 0 to feasibility of using patient derived neural stem cells as a treatment. We used day 5, most of the cells in the floating culture were positive for nestin, an the well-characterized lysosomal storage disease mucopolysaccharidosis type early neural marker, at day 12. From day 12 onwards, the medium was re- VII (MPS VII). The enzyme β-glucuronidase is non-functional in patients placed with Neurobasal medium supplemented with B-27, and DA neurons with MPS VII, resulting in a buildup of the enzyme’s glycan substrates. were induced by the addition of brain-derived neurotrophic factor (BDNF), The downstream effects of this buildup result in complex pathology often glial cell line-derived neurotrophic factor (GDNF), dibutyryl cyclic AMP, and including mental retardation. We began by generating induced pluripotent Ascorbic acid. Immunohistochemistry and quantitative PCR indicated mid- stem cells (iPSCs) from the fibroblasts of an MPS VII patient using retroviral brain DA neuronal identity of the generated neurons. So far, iPSCs derived vectors expressing Oct4, Sox2, Klf4, and c-Myc. The resulting iPSC colonies from the PD patients with known abnormal genes have not been shown to express markers of pluripotency and form teratomas in vivo. The iPSCs differentiate into DA neurons. Therefore, we also evaluated if iPSCs derived were then differentiated into a homogenous population of nestin+ neural from the PD patients with known genetic background can be differentiated stem cells. Upon withdrawal of growth factors, these cells differentiate into into midbrain DA neurons via the same floating culture method. astrocytes and neurons, the majority of which are GABAergic. iPSC derived Poster Board Number: 3127 neural stem cells were injected bilaterally in the ventricles of neonatal SCID mice. One month post-transplantation the neural stem cells survived primar- USING HUMAN INDUCED PLURIPOTENT STEM ily in clumps located throughout the ventricular system. After four months, CELL TECHNOLOGY TO STUDY NEURONAL the cells had left the ventricles and migrated throughout white matter tracts in the brain. At this time point, most cells retain expression of nestin. These PHENOTYPIC DIFFERENCES IN SCHIZOPHRENIA experiments demonstrate a strategy for personalized neural cell transplanta- Hernandez, Kristina, Moore, Jennifer C., Sun, Nawei, Sheldon, tion as a possible treatment for lysosomal storage disease. (Supported by Michael, Tischfield, Jay A., Hart, Ronald P., Brzustowicz, Linda M., NIH grants R01-NS056243 and T32-DK007748.) Firestein, Bonnie L. Cell Biology and Neuroscience, Rutgers, The State University of New Jersey, Piscataway, NJ, USA, Genetics, Rutgers, The State University of New Jersey, Piscataway, NJ, USA Schizophrenia is a complex neurological disease that is caused by a combina- tion of genetic and environmental factors. Many large-scale genetic studies have identified multiple genes that increase susceptibility to schizophrenia, but on their own are neither necessary nor sufficient to cause the disorder. In schizophrenia, it may be of more importance to understand the molecu- lar pathways that these genes are involved in and how a combination of their perturbations can increase a person’s susceptibility to the disease. In particular, it has been shown that many of the schizophrenia susceptibil-

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Thursday Poster Abstracts

Poster Board Number: 3131 Poster Board Number: 3133 CYTOGENETIC ABERRATIONS IN HUMAN GENERATION OF HUMAN INDUCED ESC AND IPSC LINES SHOW SIGNIFICANT PLURIPOTENT STEM CELLS USING THE DIFFERENCES: A COMPARISON OF EXTRACELLULAR MATRIX OF HUMAN CHROMOSOME CHANGES IN 1500 IPS AND DECIDUA-DERIVED MESENCHYMAL CELLS 1500 ES CELL LINES Fukusumi, Hayato, Shofuda, Tomoko, Kanematsu, Daisuke, Finger, Jared M., Panella, James, Wells, William, Kositzke, Beth, Yamamoto, Atsuyo, Suemizu, Hiroshi, Nakamura, Masato, Lezama, Lindsey, Hazelbauer, Stephanie, Johnson, Julie A., Meisner, Yamasaki, Mami, Sasai, Yoshiki, Kanemura, Yonehiro Lorraine F. Department of Regenerative Medicine, Institute for Clinical Research, Cell Line Genetics, LLC, Madison, WI, USA Osaka National Hospital, National Hospital Organization, Osaka, Japan, Department of Stem Cell Research, Institute for Clinical Research, We report cytogenetic findings based on the study of 1448 human ESC lines Osaka National Hospital, National Hospital Organization, Osaka, Japan, and 1421 human iPSC lines performed over a five year period (2006-2010). Molecular Analysis laboratory, Central Institute for Experimental Animals, Although both types of cell lines demonstrated a similar chromosome aber- Kawasaki, Japan, Department of Pathology and Regenerative Medicine, ration rate (28% ESC, 22% iPSC), the types of aberrations were significantly Tokai University School of Medicine, Isehara, Japan, Department of different, suggesting that they arose by different mechanisms related to their Molecular Medicine, Institute for Clinical Research, Osaka National respective methods of derivation. Under optimal conditions, ESC lines are Hospital, National Hospital Organization, Osaka, Japan, Organogenesis derived from the inner cell mass of karyotypically normal embryos, while and Neurogenesis Group, Center for Developmental Biology, RIKEN, Kobe, iPSC lines are derived from adult cells exposed to various viral transduction Japan schemes that, in addition to inducing pluripotency, can also induce chromo- some breakage and rearrangement. Although ESC lines initially have normal Human induced pluripotent stem (hiPS) cells are generated by introducing karyotypes, it has been shown that many develop numerical chromosome defined sets of transcription factors into somatic cells. The properties of hiPS aberrations (predominantly trisomy 12 and 17) that result from mitotic cells are similar to those of human embryonic stem (hES) cells. Therefore, non-disjunction followed by selection over time in culture. These specific hiPS cells, like hES cells, are considered promising sources for future regen- trisomies demonstrate a proliferative advantage that enables the trisomic erative medicine. In general, hiPS/hES cells are generated and maintained on clones to overgrow a coexisting karyotypically normal clone in relatively feeder cells such as mouse embryonic fibroblasts (MEFs) or on feeder-free few passages. Our data will show that half the karyotypically abnormal ESC culture systems like Matrigel, which is the extracellular matrix produced by lines demonstrated numerical chromosome aberrations and slightly less than a mouse tumor cell line. However, for clinical applications, quality-controlled half showed structural chromosome aberrations (the majority of which were xenobiotic-free culture systems are required to minimize health risks from unbalanced translocations). In contrast to ESC lines, iPSC lines demonstrated animal-derived pathogens and immunogens. To avoid using animal-derived few numerical aberrations, with the majority of the aberrations consisting of materials, human-derived primary cells, like foreskin fibroblasts, have been structural rearrangements, half of which were balanced translocations. In- used as feeder cells for the generation and maintenance of hES cells. It terestingly the majority of the balanced translocations detected in iPSC lines was recently found that hiPS cells derived from human dermal fibroblasts were observed at early passages without a coexisting second clone, while could be generated and maintained on their own parental cells, which are balanced translocations in ESC lines were rare. The fact that the structural isogenic and autologous. However, not every fibroblast line tested was able aberrations (including balanced translocations) detected in iPSC lines involve to function as feeder cells. Furthermore, it is easier to perform quality control different chromosomes in different cell lines and were detected at an early in a feeder free system, such as Matrigel. A defined matrix composed of fi- passage, suggests that they result from double strand DNA breakage and bronectin is able to maintain hiPS/hES cells, but its activity is generally lower repair occurring during the genomic instability associated with the repro- than that of Matrigel, depending on the culture conditions. We previously gramming process. This is supported by the fact that the majority of the reported that Peri Cellular Matrix of Decidua-derived Mesenchymal cells aberrations in the clonally-derived iPSC lines are detected in all of the cells (PCM-DM) is an effective human-derived material for maintaining hiPS/hES and are not found coexisting with a karyotypically normal clone as is often cells. The maintenance activity of PCM-DM is similar to that of Matrigel, observed with the emergence of trisomy 12 and 17 in hESC lines. Although, and the PCM-DM preparation is reproducible, because the decidua-derived the karyotypes of the iPSC lines with balanced translocations tend to remain mesenchymal cells (DMCs) can be prepared and expanded in large quanti- stable over many passages, in ESC lines the presence of numerical aber- ties. In this study, we examined whether PCM-DM can be used for generat- rations tends to increase chromosome instability and promote karyotypic ing hiPS cells from DMCs. DMCs grown on PCM-DM were reprogrammed evolution. Data will be presented demonstrating the different types and by the retroviral transduction of Yamanaka factors (Oct4, Klf4, Sox2, frequencies of chromosome aberrations in the ESC and iPSC lines to support c-Myc). Thirty days after the transduction, hES-like colonies appeared and these claims. were picked up and cultured on PCM-DM. These established hiPS clones expressed high alkaline phosphatase activity and hES-cell specific genes and surface markers, which are expressed at low or undetectable levels in native DMCs. These cells formed embryoid bodies in vitro and teratomas in vivo that contained tissues derived from all three germ layers. These characteris- tics along with genome integrity were maintained for at least 30 passages. These findings indicated that PCM-DM could maintain and support the gen- eration of iPS cells. We suggest that PCM-DM is a practical, human-derived substrate that can be used, not just for the maintenance of hiPS cells, but also for their generation.

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Poster Board Number: 3135 be considered as a new approach to rapidly generating of iPSCs. Clinical Relevance: The immunological characters of the hAMSCs-derived iPSCs will EEFFICIENT GENERATION OF INTEGRATION be investigated in our future works, which will provide a significant improve- FREE PATIENT-SPECIFIC PLURIPOTENT STEM ment for transplantation of iPSCs in regenerative medicine. CELLS USING TEMPERATURE SENSITIVE Poster Board Number: 3139 SENDAI VIRUS VECTORS IPS CELL BASED IN VITRO MODELLING OF Fusaki, Noemi, Ban, Hiroshi, Iida, Akihiro, Hasegawa, Mamoru, Ihn, METACHROMATIC LEUKODYSTROPHY Hironobu, Era, Takumi Erwes, Kim, Quandel, Tamara, Lünskens, Raphaela, Fischer, Julia, DNAVEC Corporation, Tsukuba, Ibaraki, Japan, Dermatology and Plastic Brandt, Matthias, Peitz, Michael, Gerlach, Debora, Böckenhoff, Surgery, Kumamoto University, School of Medicine, Kumamoto, Japan, Department of Cell Modulation Institute of Molecular Embryology and Annika, Matzner, Ulrich, Gieselmann, Volkmar, Brüstle, Oliver Genetics (IMEG), Kumamoto University, Kumamoto, Japan Institute of Reconstructive Neurobiology Life & Brain Center, University of Bonn, Bonn, Germany, Institute of Biochemistry and Molecular Biology, Patient-specific induced pluripotent stem cells (iPSCs) provide novel and University of Bonn, Bonn, Germany considerable opportunities not only in the basic and pharmaceutical studies of the diseases but also in development of regenerative medicine in the near Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused future. A major limitation of current reprogramming strategies is the integra- by a deficiency of the enzyme arylsulfatase A (ASA), which in turn leads tion of vectors used to transduce the reprogramming factors. As a result, it to an intralysosomal accumulation of the sphingolipid cerebroside sulfate is necessary to select ‘elite’ iPSCs with silenced foreign genes of low copy (sulfatide). In the nervous system of MLD patients, sulfatide accumulates number from numerous iPSC colonies. Here we show that Sendai virus (SeV) mainly in myelin-forming oligodendrocytes and Schwann cells, resulting in vector, an RNA replicon vector designated from SeV that carries no risk of demyelination and severe neurological symptoms. The precise pathomecha- integrating into the host genome, is most likely the best solution to the ef- nisms leading to cell degeneration are still largely unknown. A human in ficient generation of non-integrated patient-specific human iPSCs. We have vitro model based on disease-specific oligodendrocytes could provide a established 33 patient-derived iPSCs in the program for 48 intractable dis- tool for modeling the pathogenesis of the disease. In this study, iPS cells eases including inherited (neural, muscular, metabolic and other types), skin, derived from MLD-specific fibroblasts were converted into multipotent radial bone and connective tissue-derived diseases and disorder of chromosomes glia-like neural stem cells (NSC) with strong gliogenic potential (glioNSC) under the grant of Ministry of Labor and Health Japan. Established iPSCs expressing nestin, Pax6, Sox2, musashi, BLBP, RC2, 3CB2 and vimentin. ASA from all of the fibroblasts showed gene expression of human ES markers and enzyme activity was significantly decreased in MLD-glioNSC compared to viral/factor-free nature. Importantly, clonal variation among iPSCs from each healthy control cells derived from wild-type iPSC, confirming a disease-relat- patient was analyzed by the expression of specific genes and revealed that it ed phenotype. Immunofluorescence using an antibody to sulfatide and lipid was little in contrast to retrovially generated iPSCs. extraction and subsequent thin layer chromatography revealed accumula- tion of sulfatide in MLD-glioNSC-derived oligodendrocytes. We expect this Poster Board Number: 3137 iPSC-based model system to provide insight in the molecular pathogenesis ROBUST GENERATION OF INDUCED of MLD and to facilitate the exploration of novel therapeutic regimens. PLURIPOTENT STEM CELLS FROM HUMAN Poster Board Number: 3141 AMNIOTIC MESENCHYMAL STEM CELLS CHARACTERIZATION OF IPSC-BASED Ge, Xiaohu, Toma, Ildiko, Wang, I-Ning E., Sebastiano, Vittorio, DISEASE MODELS DERIVED FROM PATIENTS Reijo-Pera, Rene, Yang, Phillip WITH TWO MAJOR TYPES OF X-LINKED Division of Cardiovacular Medicine, Stanford University, Palo Alto, CA, ADRENOLEUKODYSTROPHY USA, Institute for Stem Cell Biology & Regenerative Medicine, Stanford University, Palo Alto, CA, USA Jang, Jiho, Huh, Yong Jun, Kim, Ji Young, Yoo, Jeong-Eun, Lee, Jeong-Ah, Lim, Boyoung, Kang, Hoon-Chul, Kim, Dong-Wook Background: Robust reprogramming of human placenta-derived stem cells (hPSCs) holds a tremendous potential to understand cell-specific mechanism Department of Physiology, Yonsei University College of Medicine, Seoul, of pluripotency and differentiation. Placental tissue is readily available and Republic of Korea, Department of Pediatric Neurology, Yonsei University easily procured without invasive procedures, and its use does not elicit ethi- College of Medicine, Seoul, Republic of Korea cal debate. Oct4 and Nanog are strongly expressed in mesenchymal stem X-linked adrenoleukodystrophy (X-ALD) is caused by ABCD1 gene muta- cells, which are benefit for iPSCs reprogramming. We hypothesize that high tions leading to accumulation of very long chain fatty acids (VLCFA) in the reprogramming efficiencies of partially pluripotent hAMSCs for iPSCs gen- nervous system, adrenal gland and testis. We established induced pluripo- eration and hAMSC-derived iPSCs will retain the immunoprivileged charac- tent stem cells (iPSCs) from the two major types of X-ALD patients with ter of hAMSC. Methods and Results: The c-kit (+) subpopulation of human childhood cerebral ALD (CCALD) and adrenomyeloneuropathy (AMN). Both amniotic mesenchymal stem cells (hAMSCs) was sorted by fluorescence- CCALD- and AMN-iPSCs normally differentiated into oligodendrocytes, the activated cell sorting (FACS). Induced pluripotent stem cells (iPSCs) were cell type primarily affected in the X-ALD brain, indicating no developmental generated from the hAMSCs with immunomodulatory phenotype through defect due to the ABCD1 mutations. Although low in X-ALD-iPSCs, VLCFA single integration polycistronic vectors containing 4 Yamanaka factors at day level was significantly increased after oligodendrocyte differentiation. Most 8 of tissue culture. Immunostaining and RT-PCR results revealed that hAM- strikingly, VLCFA accumulation was much higher in CCALD-oligoden- SCs-derived iPSCs expressed stem cell surface markers and pluripotency- drocytes than AMN-oligodendrocytes but was not significantly different specific genes, in particularly, SOX2 and REX1 were detected that no seen in between CCALD- and AMN-neurons, reflecting the typical phenotypes of the c-kit (+) subpopulation of hAMSCs. Conclusion: Our results demonstrate the CCALD type brain. We also demonstrated reversal of VLCFA accumula- hAMSCs are able to generate expeditiously a robust population of iPSCs tion by pharmacological induction of compensatory ABCD2 gene. Our study which express stem cell surface markers and pluripotency-specific genes. indicates X-ALD-iPSCs would provide a unique cellular model for studying Data from our experiments suggest us reprogramming of hAMSCs should etiopathophysiology and development of therapeutics for X-ALD. This work

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Thursday Poster Abstracts was supported by grants from the Stem Cell Research Center of the 21st To evaluate the quality of human induced pluripotent stem (hiPS) cells is Century Frontier Research Program funded by the Ministry of Education, required for future drug screening. First, we compared the characteris- Science and Technology (MEST) (SC1110), from National Research Founda- tics of gene expression in hiPS cells with those in hES, EC, MSC and also tion, MEST (2010-0020353), and from Korean Health Technology R&D neural tumor cell lines by using the PCR array for gene expression profiling Project, Ministry for Health, Welfare & Family Affairs (A100694). in pluripotency, growth factor, oncogenes and tumor suppressor genes. Next, spontaneous differentiation tendency of hiPS cells were determined Poster Board Number: 3143 by analyzing gene expression pattern in embryoid bodies of hiPS/ES cells. IMMUNOHISTOCHEMICAL ANALYSIS OF THE Third, hiPS/ES cells were treated with a definitive endoderm differentiation protocol in a multi-well plate, and the ratio of Foxa2-positive cell population HUMAN IPS CELL DERIVED TERATOMAS was determined by an automatically scanned fluorescent image analyzer. Higuchi, Yuichiro, Kawai, Shinji, Hashimoto, Haruo, Nakamura, For these analysis, we have utilized cell lines in the lists of JCRB Cell bank of National Institute of Biomedical Innovation (http://www.jhsf.or.jp/bank/ Masato, Suemizu, Hiroshi Category-Index. html ), and also hES/ iPS cell lines from Kyoto University Biomedical, Central Institute for Experimental Animals, Kanagawa, Japan, (Japan), Osaka National Hospital National Hospital Organization (Japan), Pathology, Central Institute for Experimental Animals, Kanagawa, Japan, and the National stem cell bank (USA). Bioinformatic analysis of our results Molecular Oncology Pathology, Tokai University, Kanagawa, Japan indicated that hES/iPS/EC cell lines clustered together. Their gene expression Induced pluripotent stem (iPS) cells have unlimited proliferative capacity and pattern was more similar to each other than to any of neural tumor cells, pluripotency as with an embryonic stem (ES) cells. Since the use of iPS cells or MSC in the analysis. And also, differences in differentiation ability were would overcome the transplant rejection and ethical problems, iPS cells were determined among the hES/iPS cell lines. expected as a source of surrogate cells for regenerative medicine. Many Poster Board Number: 3147 groups reported the establishing methods of the human iPS cells. However, it is also suggested that there are many difference in the proliferative capac- EXPANSION OF HUMAN IPS CELLS ON XENO- ity or pluripotency among the established iPS cell clones. To apply the iPS cells for the clinical or basic research, the establishment of “standardized” FREE, SYNTHETIC SURFACE IN DEFINED iPS cell had become very important issue. Characteristics of the iPS cells are MEDIUM evaluated by analyzing the expression of stem cell marker genes, methyla- Jin, Sha, Yao, Huantong, Melkounian, Zara, Ye, Kaiming tion pattern of the epigenome, karyotype or whole genome sequence. Pluripotency of human iPS cell was generally evaluated by in vitro embryoid Biomedical Engineering Program, University of Arkansas, Fayetteville, AR, body formation and/or in vivo teratoma formation. Pluripotent stem cells USA, Corning Life Sciences, Corning, New York, NY, USA (including ES or iPS cells) injected into immunodeficient mouse form tera- The ability to establish and stably propagate human induced pluripotent toma. Since it consists of the derivatives of all three germ layers, teratoma stem cells (hiPSCs) in vitro raised hopes for treating various incurable dis- formation is regarded as a landmark test for pluripotency. We performed the eases. Similarly to human embryonic stem cells (hESCs), the expansion and teratoma formation of human iPS cells by using a NOD/Shi-scid IL2Rgnull maintenance of hiPSCs requires special niches, such as extracellular matrix (NOG) mouse, and determined the immunohistochemical profiles of various (ECM) for cell attachment and proliferation. This can be achieved by grow- organ-specific markers in the teratoma. We then found discrepancy between ing hiPSCs on mouse embryonic fibroblast feeder layers (MEFs) or Matrigel, expression pattern of immunohistochemaical markers and morphological ECM extract from mouse tumor. However, these surfaces have several characteristics in the teratoma. For example, typical immunohistochemical drawbacks. First, the preparation of MEFs is a time- and labor-consuming marker for mesenchymal cells, vimentin was also observed in morpholog- process. Second, the use of animal-derived biological materials brings in- ically-characterized epithelial cells. These results indicated that it was not consistency to the culture system and poses a risk for viral transmission and sufficient to evaluate the maturity of teratomas only by morphological fea- pathogen contamination. One solution is to develop stem cell inspired sub- tures. We then aimed to establish a new index of the immunohistochemical strates by chemical modification of cell culture surface with ECM-derived cell analysis of human iPS cell-derived teratoma. Our studies would provide the adhesion-promoting peptides. Recently, Corning Life Sciences has developed novel criteria for analyzing a pluripotency of iPS cells. synthetic, xeno-free, peptide-functionalized cell culture surface, Corning® Poster Board Number: 3145 Sythemax™ Surface, that supports hES cell self-renewal for >20 passages in xeno-free, defined medium. The goal of this study was to examine adhesion COMPARATIVE ANALYSIS OF and growth of IMR90 hiPS cells on Synthemax Surface relative to commonly used Matrigel coating in defined mTeSR1 medium. Our results demonstrate CHARACTERISTICS AMONG HUMAN IPS, ES shorter doubling time for cells cultured on Synthemax surface (36.7 hrs) AND NEUROBLASTOMA CELL LINES compared to Matrigel (43.6 hrs), suggesting faster proliferation rate. To understand the molecular mechanisms behind improved iPSC growth on Hirata, Mitsuhi, Hayashida, Midori, Tateyama, Daiki, Ozawa, Synthemax surface, we performed global gene profiling to study the expres- Yutaka, Matsumura, Hiroko, Iemura, Masashi, Shofuda, Tomoko, sion of genes that are involved in cell attachment and proliferation. We Kanemura, Yonehiro, Kohara, Arihiro, Kawabata, Kenji, Mizuguchi, found that thirty four genes were up-regulated when hiPSCs were cultured Hiroyuki, Furue, Miho Kusuda on Synthemax surface relative to Matrigel. Particularly, the expression levels Department of Disease Bioresources, National Institute of Biomedical of collagen type I, IV, VI, XXI, and XXII increased significantly for Synthe- Innovation, Osaka, Japan, Institute for Clinical Research, National Hospital max condition. The expression levels of catenin family genes (catenin-β1 Organization Osaka National Hospital, Osaka, Japan, Laboratory of Stem (CTNNB1), catenin-δ1 (CTNND1), and catenin-δ2 (CTNND2) were also Cell Regulation, National Institute of Biomedical Innovation, Osaka, Japan, enhanced. Furthermore, a number of ECM receptors, including integrins Department of Biochemistry and Molecular Biology, Graduate School of (integrin α2 (ITGA2), integrin α3 (ITGA3), integrin α5 (ITGA5), integrin αV Pharmaceutical Sciences, Osaka University, Osaka, Japan (ITGAV), integrin β1 (ITGB1), integrin β5 (ITGB5), laminin β1 (LAMB1), and laminin γ1 (LAMC1) were considerably up-regulated. Our results suggest Human induced pluripotent stem derived hepatocytes would be useful for that the physicochemical signals provided by Synthemax surface activate biomedical research, regenerative medicine, and drug discovery. They are Wnt- and integrin-mediated signaling pathways, promoting efficient hiPSC particularly applicable to drug screenings, such as for the determination of attachment and proliferation. In combination with defined medium, Synthe- metabolic and toxicological properties of drug compounds in vitro models. max surface provides fully defined culture system for efficient expansion of

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hiPS cells. Poster Board Number: 3151 Poster Board Number: 3149 REPROGRAMMING OF HUMAN DENTAL PULP WHERE THE TRANSCRIPTION FACTORS STEM CELLS TO STUDYING THE BIOLOGICAL INTEGRATE IN THE HUMAN GENOME DURING MECHANISM INVOLVED IN AUTISM SPECTRUM THE REPROGRAMMING IN THE GENERATION DISORDER (ASD) OF HUMAN INDUCED PLURIPOTENT STEM Pignatari, Graciela C., Russo, Fabiele, Fernandes, Isabella, Bordini, (iPS) CELLS? Daniela, Miglino, Maria Angélica, Mercadante, Marcos, Muotri, Alysson, Beltrão-Braga, Patricia C B Junqueira-Reis, Luiza C., Picanço-Castro, Virgínia, Covas, Dimas University of São Paulo, São Paulo, Brazil, Federal University of São Paulo, Tadeu, Russo-Carbolante, Elisa Maria de Sousa São Paulo, Brazil, University of California San Diego, San Diego, CA, USA Faculty of Pharmaceutical Sciences of Ribeirao Preto-USP, Ribeirao Preto, Brazil, Center for Cell Therapy and Regional Blood Center, Ribeirao Preto, Introduction: Recently, Yamanaka and colleagues demonstrated a method Brazil, Faculty of Medicine of Ribeirao Preto-USP, Ribeirao Preto, Brazil of cellular reprogramming, called induced pluripotent stem cells (iPS), where genes expressed at the embryonic stage are again expressed in adult cells. Introduction: In the search of an alternative for Embryonic Stem (ES) cells, The application of this technique in human cells seems very promising for the Induced Pluripotent Stem (iPS) cells came with the promise of circum- drug trials, cell therapy protocols (because they are autologous) or genetic vent some of limitations of ES cells, like some important concerns related to diseases studies. Our group is interested in better study Autism Spectrum ethical issues, safety, immune compatibility and availability, which preclude Disorder patients (ASD). ASD is a disease still without genetic causes well their use in basic or clinical research. The iPS cells, as well as ES cells, can elucidated and represents a pediatric major concern, whose incidence has differentiate in all types of somatic and germ cells and have the ability to increased at epidemic levels (currently estimated that 1% of all school-age grow indefinitely in culture while maintaining pluripotency. These cells can children). This disorders affects the psycho neurological development and be generated from somatic cells of normal individuals or from patients with the person’s ability to communicate, understand and speak, affecting their some genetic disease, making them an important tool for drug screening, social life. In this work, our goal is produce iPS using human dental pulp construction of disease models and toxicological trials. Great advances have stem cells obtained from deciduous teeth spontaneously exfoliated from au- happened in reprogramming differentiated cells into iPS cells through the tism spectrum disorders (ASD) patients. The patients were selected by a pe- forced exogenous expression of transcription factors (TF) with the use of vi- diatric team of psychiatrists from UPIA-UNIFESP. iPS cells will be differentiate ral vectors, but the lentivirus integration in human genome and its influence into neuronal cells and then characterized and studied, constituting an ASD in reprogramming is little studied and known. In this work, we are study- in-vitro model. Methods: Human dental pulp stem cells were reprogrammed ing the integration target of lentivirus vectors containing TF, which were using the four Yamanaka’s factors carried by retroviral vectors. The iPS were used to generate human iPS cells. Material and Methods: The iPS cells were derived and cultured under feeder-free conditions. In vitro analyses (im- generated from fibroblasts transduced by lentiviral vectors containing the TF munocytochemistry, real-time and RT-PCR for endogenous and exogenous SOX2, TCL-1A and C-MYC, and characterized by our group. The integra- genes, embryonic bodies production) and in vivo (teratoma formation) tion pattern in different colonies is being evaluated in the reprogrammed were done. Results: At this time, only dental pulp stem cells from non ASD cells from six isolated colonies of iPS cells, cultivated in MatrigelTM (BD individuals were subjected to analysis of reprogramming and pluripotency. Biosciences) with mTeSR® medium (STEMCELL Technologies). The DNA of In vitro and in vivo analyses confirmed cellular reprogramming. Conclusion: reprogrammed cells were extracted and digested with restriction enzyme In spite of the wait for the fall of exfoliated deciduous teeth, the cells from to create a ligation site to a synthetic linker. The integration sites were dental pulp from healthy patients were successfully reprogrammed, thus can determined by LM-PCR technique, performed with a specific primer for the be good candidates for cellular reprogramming of patients with ASD. LTR and another for the linker. The PCR products were cloned in TOPO® TA Cloning™ vector (Invitrogen) and used to transform competent bacteria, Poster Board Number: 3153 resulting in 32 bacterial colonies with sequences of six different colonies of iPS cells. We performed PCR in appropriate conditions of amplification EPITHELIAL CELLS FROM HUMAN ORAL for sequencing. The sequences obtained were analyzed with the program MUCOSA AS A CELLULAR MODEL FOR BLAST and GTSG to identify sites of homology with the human genome, STUDYING GENETIC DISEASES in which chromosome, position, genic region the TF integrated, among others analysis. Results: Were found 22 sequences of six iPS colonies with Russo, Fabiele, Fernandes, Isabella, Miglino, Maria Angelica, homology with the human genome, and these, 11 occurred in genic regions: Pignatari, Graciela, Beltrão-Braga, Patricia VIT (chromosome 2), SLIT2 (chromosome 4), C6orf142 (chromosome 6), University of São Paulo, São Paulo, Brazil ADAM32 (chromosome 8), ANKRD19 (chromosome 9), PCDH9 (chromo- some 13), GPHN (chromosome 14), THSD4 (chromosome 15), AC106037.2 Introduction: Stem cells obtained from biopsies of oral mucosa have been (chromosome 18) e CCNB3 (chromosome X), all within introns. In total, identified as a new source of mesenchymal stem cells. These cells have been integrations were identified in chromosomes 1, 2, 4, 6, 8, 10, 11, 13, 14, 15, used to treat serious ocular diseases thus showing a great therapeutic poten- 18, 19 and X. We also analyze the integration distances of the transcription tial. As the biopsy is an invasive procedure in this project we aim to establish start site and integrations near CpG islands, and the proportion of integra- a culture of epithelial oral mucosal (EOM) cells from scraping the palate, tions within ± 10Kb of them is low. Conclusion: The number of sequences which is a noninvasive procedure with a minimal discomfort to patients. It is analyzed is still small, but the results suggest a variable pattern of lentiviral worth mentioning that the oral mucosa is composed of stratified squamous integration, that there is no preference for a particular chromosome or non keratinized epithelial cells, which are juxtaposed, with little extracel- chromosomal location, which may suggest that the reprogramming does lular matrix. The establishment of EOM cell culture could be very useful not depend of the insertion site, but further analysis must be performed. as a model for obtaining big amounts of cells in order to studying genetic This analysis is important for evaluate if the integration of lentiviral vector diseases. Moreover, these cells could be used as a model of cells for cellular may affect the generation of iPS cells, to then be possible characterize and reprogramming protocols, thus avoiding invasive procedures to obtain stem determine the safety in use of this cells. cell from EOM and obtaining pluripotent stem cells instead. Methods: This project was approved by committee on ethics of human research. The oral

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Thursday Poster Abstracts mucosa was evaluated and volunteers with a history of smoking, chewing Poster Board Number: 3157 tobacco and infections or inflammations in the oral mucosa were discarded. The cultures were made by scraping the oral mucosa using a sterile dispos- A LARGE NUMBER OF POINT MUTATIONS IN able brush, after oral hygiene. The brush was then immersed in a tube con- MOUSE IPS CELLS taining cell culture medium with antibiotics. After culturing, cells were evalu- ated for their proliferation, morphology and expression of cell markers. The Abe, Masumi, Hoki-Fujimori, Yuko, Jincho, Yuko, Kasama, Yasuji, protocol details are omitted under patent process rules. Results: EOM cells Ando, Shunsuke, Sunayama, Misato, Uda, Masahiro, Nakamura, were successfully cultured. Morphological characterization was performed Miki, Araki, Ryoko by light microscopy using an inverted microscope, observing epithelial cell Transcriptome Research Group, National Institute of Radiological Sciences, characteristic morphology, also using HE, papanicolaou and toluidine blue Chiba, Japan staining techniques. Immunocytochemistry assays using anti-cytokeratin pan, anti-cytokeratin 18, anti-PCNA3, anti-nanog and anti-Oct4 were also In order to understand the genetic integrity of iPSCs, we conducted a performed being positives for cytokeratins and PCNA3. RT-PCR showed genome-wide sequencing study of the iPSCs with a shot gun procedure. positivity for cytokeratins 4 and 13, conexina 43, vimentin and GAPDH. Due to a large number of SNPs, it has been quite hard to perform a com- Cells were negative for cytokeratin 12 (marker of corneal epithelium), Oct4 prehensive point mutations study thus far. Here, we generated genome and nanog as expected; since these cells were collect very superficially with integration-free iPSCs from the MEF of C57BL/6J mouse strain that is the minimum invasion of mucosa oral tissue. Conclusion: The EOM cell culture only strain of which whole genome sequences have been determined. We was established with ease, is painless and minimally invasive. examined three lines, two of 4F(OSKC) iPSC lines, of which the ability for germline transmission was verified, and one of 3F(OSK) line. Approximately Poster Board Number: 3155 3 x 107 bp were sequenced for each line that correspond to 1/100 entire genome for comparing them with the C57BL/6J genome sequences depos- EXTRAVILLOUS TROPHOBLAST STEM CELLS ited in public data base to obtain point mutation candidates. We successfully (EVT) FROM HUMAN TERM PLACENTA ARE identified point mutations in all three lines. All mutations were heterozygous, NOT GOOD CANDIDATES FOR USING IN CELL and while most of them reside in intergenic region, several point mutations were detected at very close to or just in known genes loci. Importantly, cell THERAPY PROTOCOLS line-specific reduction of the transcripts that were expressed from the gene Fernandes, Isabella, Russo, Fabiele, Pignatari, Graciela, Borbely, locus in which point mutation occurred, was observed. Furthermore, most Alexandre, Sandri, Silvana, Prado, Karen, Miglino, Maria Angélica, of them, 75%, occurred at G/C sites. Considering that G/C content of mammalian genomes is 42%, the results indicate a presence of strong bias Bevilacqua, Estela, Beltrão-Braga, Patricia for G/C nucleotides in the occurrence of point mutations. In addition, our University of São Paulo, São Paulo, Brazil analysis of genome integration-free 3F iPSC lines revealed that the point Introduction: The placenta is an embryonic tissue that has attracted great mutations occurred at very early stages of iPSC generation, not during their interest as a source of stem cells for regenerative medicine due to pheno- culture passage. Sister iPSC clones that exhibited identical pattern of point typic plasticity of some of the various cell types isolated from this tissue. mutations were established by chance, and in addition they have equal Extravillous trophoblast stem cells (EVT) that are the subject of study for this amount of wild and mutant alleles, 1:1 ratio. Thus the observation not only project are involved in maintaining the tolerance of the fetus by the mother, indicated that the mutations occurred prior to their colonies isolation from because it contains cells that have immunomodulatory properties, inducing culture dish, but also that the mutations already existed at their converting immunotolerance. In this project we cultured and characterized EVT cells. step from somatic cells. Here we found that even genome integration-free Methods: This project was approved by the ethics committee on human iPSCs of which full developmental potential was verified, have a significant research. Pieces of tissue were fixed in paraformaldehyde 4% for histological number of point mutations in their genomes. Our results suggested an characterization of placental tissue and determine the exact place for collect accumulation of a large number of point mutations, at least 200 - 1,100, in EVT cells. The EVT cell culture was made using cotyledon fragments from iPSCs. We must note that our results do not necessarily preclude their use human term placenta (38-40 weeks of gestation). The tissue was subjected in medical applications. However, iPSCs must be assessed very carefully and to enzimatic digestion and the cells were submitted to density gradient entire genome sequencing will be essential for their evaluation. centrifugation. The cultured EVT cells were characterized to cytokeratin 7, Poster Board Number: 3159 human leukocyte antigen-G (HLA-G), Nanog, Oct4 and vimentin. Results: EVT cells were positive for cytokeratin 7, HLA-G Nanog and Oct4, confirm- CONSTITUTIVE HETEROCHROMATIN ing the trophoblastic origin and characteristic of stem cell. However, culture of these cells showed that the EVT cells have low rate of cell proliferation in REORGANIZATION DURING MOUSE SOMATIC our results of MTT assay, a characteristic that limits their use in cell therapy CELL REPROGRAMMING protocols. The placental tissue was observed positive staining for HLA-G, Djuric, Ugljesa, Fussner, Eden, Hotta, Akitsu, Perez-Iratxeta, cytokeratin 7, Nanog and Oct4 on region of EVT cells. Conclusion: Taking into account our findings, we conclude that EVT cells are difficult to culture Carolina, Lanner, Fredrik, Dilworth, F. Jeffrey, Bazett-Jones, David P., and low proliferation. Therefore, we believe it would be interesting to repro- Ellis, James gramming (iPS) of these cells to check that they will be able to increase their Stem Cell and Developmental Biology, SickKids Hospital, Toronto, ON, proliferative capacity and still maintain your profile immunomodulation. Canada, Genetics and Genome Biology, SickKids Hospital, Toronto, ON, Canada, Centre for iPS Cell Research and Application, Kyoto, Japan, Regenerative Medicine Program, Sprott Center for Stem Cell Research, Ottawa, ON, Canada Induced pluripotent stem (iPS) cell reprogramming is a gradual epigenetic process that reactivates the pluripotent transcriptional network by eras- ing and establishing repressive epigenetic marks. In contrast to loci-specific epigenetic changes, heterochromatin domains undergo epigenetic resetting during the reprogramming process but the effect on the heterochromatin ultrastructure is not known. Here, we characterize the physical structure of

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heterochromatin domains in reprogrammed iPS cells using Electron Spectro- and APD was determined for each cell by linear regression analysis. This scopic Imaging. Retroviral reprogramming factors are expressed in partial iPS yielded a flat slope (-2.5 ± 1.4 ms/s (APD/period), n =8) in wild-type, but cells and here we demonstrate that retroviral transgenes take on a repressive a steeply positive slope (9.9 ± 3.7 ms/s, n =11, p<0.05) in Scn5aΔ/+ car- epigenetic structure in full iPS cells, marked by hypermethylated DNA and diomyocytes. Furthermore, early after-depolarizations (EAD) were observed hypoacetylated histone H3 of retroviral LTR promoters. As this transgene in Scn5aDelta/+ (53.8%) but not in wild-type (0%) cardiomyocytes. These silencing occurs around the time of activation of endogenous pluripotency- results are very similar to those obtained from the LQTS 3 mouse and also related genes, this epigenetic hallmark can be used to investigate the timing from patients carrying this mutation. Thus, cardiomyocytes generated from of heterochromatin domain reorganization during the reprogramming LQTS 3-specific iPS cells do preserve the pathophysiological hallmarks of the process. In somatic and partial iPS cells, constitutive heterochromatin marked disease in vitro. These results encourage patient-specific pharmacological by H3K9me3 is highly compartmentalized into chromocentre structures screening approaches of human LQTS 3 using iPS-derived cardiomyocytes. of densely packed chromatin fibres. In contrast, chromocentre boundaries are poorly defined in pluripotent embryonic stem and full iPS cells, and are Poster Board Number: 3163 characterized by unusually dispersed 10 nm heterochromatin fibres in high REPROGRAMMING EFFICIENCIES OF PEI Nanog-expressing cells, including pluripotent cells of the mouse blastocyst prior to differentiation. This heterochromatin reorganization accompanies TRANSFECTION IN MURINE INDUCED retroviral silencing during promotion of full reprogramming in partial iPS cells PLURIPOTENT STEM CELL GENERATION by Mek/Gsk3 2i inhibitor treatment. Thus, constitutive heterochromatin is compacted in partial iPS cells but reorganizes into dispersed 10 nm chroma- Lee, Won I. tin fibres as the fully reprogrammed iPS cell state is acquired. Academy for the Advancement of Science and Technology, Bergen County Academies, Hackensack, NJ, USA Poster Board Number: 3161 The expression of four defined transcription factors, Sox2, c-Myc, Oct3/4, CARDIOMYOCYTES OBTAINED FROM MURINE and Klf4, has been demonstrated to be sufficient for the reprogramming of INDUCED PLURIPOTENT STEM CELLS WITH mammalian somatic cells into pluripotent stem cells. These induced pluri- potent stem cells (iPSCs) have immense potential for regenerative medicine LONG QT SYNDROME 3 RECAPITULATE and tissue repair. Yet current strategies for the reprogramming process entail TYPICAL DISEASE-SPECIFIC FEATURES IN the use of retroviral, lentiviral, or adenoviral transfection for transgene deliv- VITRO ery. The utilization of a viral vector raises questions regarding the safety of the iPSCs generated, especially in regards to regenerative medicine, as viral Friedrichs, Stephanie, Malan, Daniela, Fleischmann, Bernd K., reactivation of transgenes has been shown to result in tumorigenesis. Thus, Sasse, Philipp an analysis of the efficiency of new transfection methods for the repro- Physiology I, University of Bonn, Bonn, Germany gramming of somatic cells is necessary. Here we explore a novel method of reprogramming through the use of polyethyleneimine (PEI) transfection and Induced pluripotent stem (iPS) cells can be derived from somatic cells using demonstrate that it is a viable alternative to conventional techniques. This overexpression of pluripotency factors and then be differentiated into all cell study also investigates the efficiencies of reprogramming and differentiation types including cardiomyocytes. Therefore, patient specific iPS cells could be in two disparate murine cell lines, examining the effects of prior differential employed for the investigation of cardiac diseases in vitro. To proof the fea- memory on iPSCs. Because the method is host-factor independent and does sibility of this concept we generated iPS cells from a mouse model with long not involve any viral components, it is predicted that it may serve as a safe QT 3 syndrome (LQTS 3) carrying the human DeltaKPQ mutation in the and effective method of iPSC generation for future cell-based regenerative cardiac SCN5A Na+ channel (Scn5aDelta/+). Cardiomyocytes obtained from therapies. iPS cells were investigated to search for the electrophysiological hallmarks of LQTS 3 in vitro. Generation of iPS cells was achieved by retroviral transduc- Poster Board Number: 3165 tion of murine embryonic fibroblasts from Scn5aDelta/+ and wild-type mice either with the three factors Oct4, Sox-2, and Klf4, or additionally with the RETROVIRAL SILENCING AND PLURIPOTENCY fourth factor c-Myc. Successful reprogramming was confirmed by posi- IN MOUSE IPS CLONES tive staining for the pluripotent markers Oct4 and SSEA1 and by teratoma Manian, Kannan V., Musheer Aalam, Syed Mohammed, Chettri, formation in SCID mice. In vitro differentiation of iPS cells was obtained via embryoid body’s (EB) formation using the hanging drop method and we Pranav, Bharathan, Sumitha P., Srivastava, Alok, Velayudhan, Shaji observed generation of cells of all three germ layers including spontane- R. ously beating cardiomyocytes. Gene expression analysis revealed a reduced Department of Haematology and Centre for Stem Cell Research, Christian expression of Nanog and Oct4 during differentiation whereas the level of Medical College, Vellore, India cardiac structural and functional genes (MYL2, MYL7, MYH6, MYH7, HCN4 Retrovirus mediated overexpression of transcription factors has the highest and SCN5a) increased in both wild type and Scn5aDelta/+ cells. Single efficiency in somatic cell reprogramming (0.01%). However, only a few cardiomyocytes of wild-type and Scn5aDelta/+ iPS cells were obtained clones will be established as stable iPS cell lines with the expression of by dissociation of beating areas of EBs. We found similar SCN5a sodium pluripotency genes. One of the major causes for this extreme low efficiency channel distribution patterns using immunostainings as well as similar peak of generation of induced pluripotent stem cells (iPS) is the lack of retroviral sodium currents using patch clamp. In contrast, biophysical characterization silencing. Exogenous gene expression is required only in the initial phase of of the sodium current yielded a significantly faster recovery from inactiva- reprogramming and to achieve pluripotency, they should be silenced as the tion in Scn5aDelta/+ (tau = 3.9 ± 0.9 ms, n =7) compared to wild-type endogenous genes start their expression. The molecular and cellular events (tau = 8.2 ± 1.6 ms, n =7) cardiomyocytes. The TTX-sensitive late sodium that occur in the transgene expressing cells in long term culture and whether current was also significantly larger in Scn5aΔ/+ (3.8 ± 0.7 pA/pF, n =8) these cells attain transgene silencing and pluripotency finally have not been than in wild-type (1.4 ± 0.5 pA/pF, n =7) cardiomyocytes. These differences clearly documented. This information is important for calculating the ef- are very similar to earlier reports on DeltaKPQ sodium channels. Analysis ficiency of iPS generation using retroviral protocols. Using retroviruses we of action potential (AP) changes at various pacing frequencies showed a transduced mouse embryonic fibroblasts (MEFs) to express the pluripotency prolonged AP duration (APD) in Scn5aDelta/+ cardiomyocytes at low pacing inducing transcription factors, Sox2, Klf4, Oct ¾ and cMyc and red fluo- rates (<0.5 Hz). For quantitation, the relationship between pacing period rescence protein (RFP) which was useful for monitoring retroviral silencing

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Thursday Poster Abstracts when the iPS clones are generated. Twenty one colonies with the morphol- protein complex (DAPC). Corrected cells were then differentiated into a pro- ogy of mouse embryonic stem (mES) cells were isolated for propagation and liferating population of skeletal myogenic precursors by transient expression we could establish 11 clones after three passages. Out of these, one clone of Pax3. These progenitors express myogenic regulatory factors (MRFs) as was RFP- suggestive of retroviral silencing and all others were RFP+. We well as surface markers characteristic of satellite/progenitor cells, including analyzed these clones for their endogenous expression of Nanog, Oct3/4 M-cadherin, CXCR4, and syndecan-4. When subjected to differentiation and Sox2. The RFP- clone (clone c1) expressed these pluripotency genes culture conditions and dox withdrawal, myogenic progenitors differentiated in levels comparable to mES cells. To understand the dynamics of retroviral efficiently into MyHC+ myotubes. We then performed in vivo studies of silencing in long term culture, three clones were selected. A clone which was these corrected myogenic progenitors. Upon intramuscular transplantation RFP- and had high Nanog expression (clone C1), another which was RFP+ into dKO mice, corrected myogenic precursors gave rise to a significant and had significant Nanog expression (clone C2) and one that was RFP+ number of utrophin+ myofibers. These results represent an important step with low Nanog expression (clone C3) were selected. These clones were forward for the future autologous cell-based therapeutic approach to treat- cultured for 10 passages. Clone C1 maintained morphology and gene ex- ing muscular dystrophy using genetically corrected iPS cells. pression throughout. The colonies formed by clone C2 showed progressive retroviral silencing with gradual loss of RFP and the RFP+ cells of this clone Poster Board Number: 3169 lost their mES cell like morphology and resembled MEFs. We sorted SSEA1+ PATTERNED DIFFERENTIATION IN 3D FORMAT and RFP- cells and cultured them. The colonies formed showed significantly increased expression of Nanog, Sox2 and Oct ¾ and several fold decrease USING MOUSE IPS CELLS IN A MICROFLUIDICS in exogenous gene expression. Clone C2 also exhibited pluripotency by DEVICE embryoid body formation and in vitro differentiation into three germ layers. Bisulfite sequencing showed that the Nanog promoter was hypomethylated HE, Xiaoming, Kawada, Jiro, Kimura, Hiroshi, Kinoshita, Haruyuki, in these cells. However, clone C3 remained RFP+ for several passages and Fujii, Teruo the expression of pluripotency genes remained low throughout. This data Institute of Industrial Science, The University of Tokyo, Tokyo, Japan suggest that mouse iPS clones that do not exhibit retroviral silencing in the initial passages, but express Nanog, can shut down exogenous genes and Embryonic stem (ES) or induced pluripotent stem (iPS) cells are widely ap- attain pluripotency after further passages. This study also illustrates that plied in both fundamental research and biomedical fields because of their incorporation of RFP with the transcription factors by retroviruses will help in ability of self-renewal and differentiation into many derivatives. How to identification and isolation of transgene silenced clones that appear at early induce and regulate over 2 kinds of differentiation simultaneously in vitro and late passages. Our current strategy for generation of mouse iPS involves (i.e. mimic in vivo differentiation) is apparently necessary and important at isolation of colonies with mES like morphology, analyzing their Nanog present stage. However, differentiation in vivo is dynamic in a small scale; expression by quantitative real time PCR and subsequent culturing of the furthermore, gradient distribution of growth factors in vivo played an clones through several passages till transgenes are silenced. important role in directing differentiation. Some researchers had studied how to culture and collect stem cells, and even differentiation using microfluidic Poster Board Number: 3167 devices. However, physical stress produced by flow might give negative influence on proliferation and differentiation of ES, and it is difficult to AN EX VIVO GENE THERAPY APPROACH TO maintain continuously the distribution of specific chemicals spatially. Our TREAT MUSCULAR DYSTROPHY USING MOUSE group has been successful in controlling 2D differentiation using iPS model by precisely controlling chemical distribution. It is expected to study further IPS CELLS to control differentiation in a 3D format mimicking in vivo differentiation Filareto, Antonio, Parker, Sarah, Darabi, Radbod, Schaaf, Tory, spatiotemporally. For the purpose, a microfluidic device was designed to Borges, Luciene, Chamberlain, Jeffrey S., Ervasti, James M., McIvor, control differentiation in 3D, which was able to reduce the influence of Scott R., Kyba, Michael, Perlingeiro, Rita C.R. physical stress, and to keep the chemical distribution simultaneously. Our microfluidic device is composed of two flowing channels and a culture Department of Medicine, University of Minnesota, Minneapolis, MN, USA, chamber located between the channels. The walls of the chamber consist of Department of Neurology, University of Washington, Seattle, WA, USA, PDMS pillars. Flowing velocity would decrease greatly when liquid is flowing Department of Biochemistry, Molecular Biology, and Biophysics, University into the culture chamber, the hypothesis was testified by theoretical simula- of Minnesota, Minneapolis, MN, USA, Department of of Genetics, Cell tion using COMSOL software and practical measurement by perfusing a Biology and Development, University of Minnesota, Minneapolis, MN, USA suspension of fluorescent particles into the microfluidic device. Experiment Mouse induced pluripotent stem (iPS) cells have been shown to be function- results indicated that flowing velocity within the chamber was below one ally equivalent to mouse ES cells. However before iPS cells can be applied for tenth of that in the channel. The theoretical simulation also showed that therapeutic applications, it is necessary to assess their ability to differentiate the gradient distribution of chemical substances can be formed and kept in towards the desired cell type. In terms of skeletal myogenic regeneration, the culture chamber, which was verified by measuring fluorescent intensity we have evidence that differentiating mouse ES and iPS cells have similar within the chamber after a fluorescent substance was perfused into the de- potential. Here we performed proof-of principle experiments to assess vice. After seeding embryonic body (EB) of mouse nanog-GFP iPS cells into whether dystrophic iPS cells can be genetically corrected using a non-viral the culture chamber, the device was connected to syringe pumps, prolifera- gene therapy approach, and evaluated whether their transplantation back tion and differentiation of EB was studied by perfusing a medium containing into dystrophic mice will ameliorate the muscle wasting phenotype. By using FBS (20%) and/or a medium containing leukemia inhibitory factor (LIF) the reported cocktail of reprogramming transcription factors, Oct4, Sox2 through the two channels respectively. After 4 days of culture, it was found and Klf4, we have generated iPS cells from tail tip fibroblasts (TTFs) of dKO that dynamic culture in microfluidic device promoted not only proliferation mice (double knockout for dystrophin and utrophin), a model of Duchenne but also differentiation. It was also observed that patterned differentiation muscular dystrophy. Pluripotency of selected iPS clones was confirmed in was induced in one EB if 2 kinds of media were perfused simultaneously, vitro, by the expression of alkaline phosphatase, Nanog, Oct4, and staining i.e., half of the EB derivatives was differentiated into cardiac tissue, the other for SSEA-1, and in vivo, by their ability to form teratomas upon injection half still maintained undifferentiated. Confocal microphotographs showed into immunodeficient mice. We then corrected the phenotype of these that height of the undifferentiated part gradually lowered at the interface dystrophic iPS cells using a Sleeping Beauty transposon carrying the micro- between undifferentiated and differentiated parts, which suggested that the utrophin gene. This gene has the key structural domains of utrophin, and gradient distribution of differentiation factors were kept continuously within can substitute for loss of dystrophin or utrophin in the dystrophin-associated the chamber. This device shows its advantages in reducing the physical stress

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and controlling the patterned differentiation in microscale and can be a use- 3, 6 and 12 post-transplantation. Results: Mesenchymal progenitors were ful tool in future biomedical research. able to express mesenchymal markers including CD-105, CD-73, CD-29, CD-90 and CD-44 but no hematopoietic markers (CD-34 and CD-45) and Poster Board Number: 3171 pluripotency markers (Oct-4 and Nanog) and also capable to differentiate PRODUCTION OF IPS CELL-CHIMERAS FROM to osteo-adipogenic lineages. All animals were survived by transplantation of hiPSCs-MPs and hBM-MSCs. Transplanted cells were traced in host livers. AGED MICE Cell transplantation significantly prevented expressive alteration in serum Lee, Hyo-Sang, Masterson, Keith, Kang, Eun-Ju, Wolff, Erin, Lactate dehydrogenase and bilirubin but had no significant effects on serum albumin, also reduced oxidative stress. Real time RT-PCR demonstrated Ramsey, Cathy, Ma, Hong, Tachibana, Masahito, Mitalipov, transplanted livers had significantly reduced collagen, TIMP-2 and MMP-9, Shoukhrat gene expression and significantly increased MMP-13, MMP14, MMP-2 and Division of Reproductive Sciences, Oregon Health & Science University, TIMP-1 in day 3 post-transplantation. Necrosis score were significantly re- Beaverton, OR, USA, Division of Reproductive Sciences, Oregon Stem duced by cell transplantation. However fibrosis had no significant difference. Cell Center and Departments of Obstetrics & Gynecology and Molecular Conclusion: Transplantation of hiPSCs-MPs like hBM-MSCs can effectively & Medical Genetics, Oregon Health & Science University, Beaverton, OR, rescue experimental liver failure which can offer a potentially alternative cell USA source to cell therapy for treatment of liver diseases by producing safe iPSCs. Direct reprogramming to induced pluripotent stem (iPS) cells can re-set the Poster Board Number: 3175 developmental potential of differentiated somatic cells. When introduced into host embryos, mouse iPS cells can generate chimeric offspring dem- MICRORNA MIR15A/16 DEFECT REMAINS IN onstrating their restored pluripotency. To date, chimeric mice were gener- NEW ZEALAND BLACK DERIVED MOUSE “IPS- ated only from iPS cells derived by reprogramming of somatic cells from embryonic, fetal of juvenile mice. We sought to investigate if iPS cells from LIKE” CELLS: POTENTIAL USEFULNESS FOR aged mice can produce viable chimeras. We generated iPS cells from skin IDENTIFICATION OF CLL CANCER STEM CELLS fibroblasts isolated from an 18 month old mouse using inducible lentiviral vectors carrying Oct-4, Klf-4, Sox-2, and c-Myc. In addition, we produced Underbayev, Chingiz, Schneider, Joel Solomon, Kasar, Siddha, pluripotent cells by somatic cell nuclear transfer (ntES) using the same Yehia, Ghassan, Fraidenraich, Diego, Raveche, Elizabeth Scott parental skin fibroblasts. Initially, pluripotency of both iPS and ntES cells Pathology and Laboratory Medicine, University of Medicine and Dentistry, was validated using teratoma assay. Both stem cell types induced tumors New Jersey, Newark, NJ, USA, Cell Biology and Molecular Medicine, containing various derivatives of the three germ layers. Next, we injected University of Medicine and Dentistry, New Jersey, Newark, NJ, USA, iPS and ntES cells into CD1 diploid host blastocysts and transferred chimeric Transgenic Core Facility, University of Medicine and Dentistry, New Jersey, embryos to recipients. Thirty-two live pups were born from both cell lines. Newark, NJ, USA Analysis of the coat color revealed that 11 out of 16 pups born after iPS cell injection were chimeric while only 1 out of 16 pups in the ntES cell group The usefulness of pluripotent stem cells such as embryonic stem (ES) cell was a chimera. The degree of stem cell contribution in live mice determined and induced pluripotent stem (iPS) cells is partly due to their amenability to by the coat color chimerism ranged from 10% to 90%. No abnormalities efficient gene manipulation. Homologous recombination between genomic in growth and development of chimeric mice were observed. These results and the exogenous DNA is a very inefficient event, but it takes place in ES/ suggest that direct reprogramming can restore full developmental potential iPS cells with relatively higher efficiency than it does in other cell types. of somatic cells from aged mice to the level seen for somatic cell nuclear In this way mice with a variety of modifications such as null and point transfer-based reprogramming. mutations, chromosomal rearrangements and large deletions have been generated. New Zealand Black (NZB) mouse is a de-novo model of chronic Poster Board Number: 3173 lymphoid leukemia (CLL) that has been studied extensively as a model to investigate both autoimmune diseases, such as systemic lupus erythemato- TREATMENT OF ACUTE HEPATIC FAILURE sus (SLE), as well as B-cell lymphoroliferative disorders. Unfortunately, NZB IN MICE BY TRANSPLANTATION OF HUMAN mouse appeared to be refractory to true pluripotent stem cells derivation. Here we report an induction of induced pluripotent stem-like cells from NZB INDUCED PELURIPOTENT STEM CELLS- spleen primary culture fibroblasts by means of lentiviral delivery of three DERIVED MESENCHYMAL PROGENITORS factors: Oct4, Sox2 and Klf4 in a single polycistronic vector. After 30 days of transduction followed by small molecules (GSK-3 and MEK inhibitors, 2i) Moslem, Mohsen, Valojerdi, Mojtaba Rezazadeh, Baharvand, β supplementation we were able to see ES-like colonies which stained positive Hossein for alkaline phosphatase and SSEA-1 surface antigen. The colonies have Anatomical Sciences, Tarbiat Modares University, Tehran, Islamic Republic been picked and expanded for further analysis. RT-PCR showed the expres- of Iran, Department of Stem Cells and Developmental Biology, Royan sion of basic endogenous pluripotency genes such as Oct4, Sox2, Klf4, cMyc Institute for Stem Cell Biology and Technology, Tehran, Islamic Republic of and Nanog. The NZB iPS cells we derived from NZB mice had decreased Iran levels of miR15a/16 when compared to wild-type ES cells (from non-NZB). Introduction: here we investigate whether human induced pluripotent These results indicate that the NZB derived iPS cells maintain the feature of stem cells-derived mesenchymal progenitors (hiPSCs-MPs) could promote decreased miR 15a/16 expression since this is due to a germline mutation survival, histopathology and molecular changes in CCL4-induced acute liver in NZB. We have lentiviral constructs to deliver miR15a/16 to the iPS cells. toxicity. Methods: Characterization of hiPSCs-MPs was investigated by flow The ability of NZB derived iPS cells with and without exogenous miR15a/16 cytometry and osteo-adipogenic differentiation. Lethal hepatic failure was to differentiate in vitro into B lineage cells will be studied to determine if induced by CCL4 (0.6 ml/kg) gavage in cyclosporine (20mg/kg) treated levels of miR15a/16 correlate with B-1 cell generation (the precursor cell for mice. Human bone marrow mesenchymal stem cells (hBM-MSCs) and hiP- CLL). In the future, the pluripotency potential will be analyzed by functional SCs-MPs (2×106 cells) were transplanted via tail vein 24 hours after CCL4 assays such as teratoma formation, chimera contribution and germ-line administration. Serologic tests, gene expression of collagen, matrix metallo- transmission. The NZB iPS cells will help uncover the unknown mechanisms proteinases (MMPs) and their inhibitors (TIMPs) by real time qRT-PCR, lipid of CLL development and shed the light on potential cancer stem cell pheno- peroxidation and histopatological examinations were performed on days 1, type in this disease.

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Poster Board Number: 3177 Poster Board Number: 3179 CHIMERIC PIGS PRODUCED FROM INDUCED INCREASING THE EFFICIENCY OF GENERATING PLURIPOTENT STEM CELLS DEMONSTRATE PORCINE INDUCED PLURIPOTENT STEM CELLS NORMAL DEVELOPMENT AND LACK TUMOR WITH MICRORNA FORMATION Song, Guangqi, Zhang, Yang, Tang, Lina, Zhang, Peng, Fan, Anran, West, Franklin D., Uhl, Elizabeth W., Stowe, Heather, Gallegos Tang, Bo, Li, Ziyi Cardenas, Amalia, Liu, Yubing, Yu, Ping, Pratt, Scott L., Stice, Jilin University, Changchun, China Steven L. Porcine embryonic stem (ES) cell lines would be useful for biomedical Animal and Dairy Science, University of Georgia, Athens, GA, USA, research and transgenic breeding. Nevertheless, numerous attempts have Department of Pathology, University of Georgia, Athens, GA, USA, Animal been made to generate porcine ES cells via various means without success. and Veterinary Sciences, Clemson University, Clemson, SC, USA, Animal It is exciting to know that a new type of pluripotent stem cells, known as and Veterinary Sciences, Clemson University, Clemson, SC, USA induced pluripotent stem (iPS) cells, have been demonstrated that have the full development potential of ES cells in murine and human. Different tissue Pigs are a desirable large animal model species to study stem cell therapies cells can be induced to iPS cells by defined transcription factors, which will and have been an important animal model for diabetes, atherosclerosis and provide an extraordinary supply in plenteous applications. While four por- traumatic brain injury to name a few. However, porcine stem cells derived cine iPS cell lines have been reported, there are still several problems need from the inner cell mass of developing blastocysts have proven to have to be solved, such as low efficiency. Here, we suppose that pluripotency limited pluripotency. These cells typically possess embryonic stem cell (ESC) relevant microRNAs (miRNAs) could improve the efficiency of generat- like morphology and express a limited number of markers, yet fail more ing porcine iPS cells. To test whether these miRNAs promote the induction stringent pluripotency tests such as contribution to chimeric animals. Our lab of pluripotency, we selected seven types of miRNAs, including miR-302a, has recently developed porcine induced pluripotent stem cells (piPSCs) by miR-302d, miR-371, miR-372, miR-200c, miR-294, and miR-17, which overexpressing the human POU5F1, SOX2, NANOG, LIN28, KLF4 and C- were introduced with retroviruses expressing Oct4, Sox2, Klf4 and C-myc MYC genes. piPSCs showed ESC morphology, immunoreactivity and for the into porcine embryonic fibroblasts (PEFs). MiRNAs were introduced on days first time contributed to tissues representing all 3 germ layers and potently 0, 4 and 8 post-infection by transfection of synthesized double-stranded the germline in chimeric offspring. In the mouse, numerous tumors were RNAs that mimics their mature endogenous counterparts. To maintain a formed in chimeric animals derived from iPSCs resulting in health problems high levels of mimics, 32nM miRNA mimics was transfected into PEFs on and high mortality rates. This has raised significant questions pertaining to days 0, 4 and 8. MiRNAs were divided into 5 combinations: (1) miR-302a & the tumorigenicity of iPSCs and whether data in the mouse will translate miR-302d; (2) miR-371 & miR-372; (3) miR-200c; (4) miR-294; (5) miR-17. to other species and eventually human iPSC therapy. To begin to address Results showed that miR-302a & miR302d group increased the efficiency this question we performed necropsies and histological analysis of collected from 0.05-0.1% to 0.2-0.3% of transduced PEFs, and reduced the induced tissues from 11 chimeric pigs at 2 (n=4), 7 (n=4) and 9 (n=3) months of age days from 18 to 14. We chose 5 colonies of each line that exhibited typical to determine potential aberrant organ development and tumor formation undifferentiated human ES-cell-like morphology. Selected colonies were that may have resulted from the incorporation of piPSCs. Necropsy results expanded and further characterized. Expanded colonies retained the original revealed that animals demonstrated normal organ development and lacked morphology, displayed high nuclear/cytoplasmic ratio, and were AP positive. tumor formation in all 11 animals tested. Tissues representing all 3 germ lay- Semiquantitative reverse-transcription PCR (RT-PCR) demonstrated the ers ectoderm, endoderm, mesoderm and gonad were tested for the presents integration of four transgenes into the genome of all tested iPS cell lines, of human POU5F1 by PCR analysis. Animals proved to be highly chimeric and expressions of endogenous Oct4, Nanog, Lin28 and Sox2 robustly with most individuals having multiple tissues positive for human POU5F1. increased. Porcine iPS colonies were stained and showed positive for human Histological analysis of positive brain, lung and heart samples showed stage-specific embryonic antigen of SSEA4 and transcription factors of Oct4, normal morphology and development. Semen quality of male chimeras was Nanog and Rex1 (Zfp42). Bisulfite genomic sequencing analysis of Oct4 and also tested to determine if chimeric animals were capable of generating Nanog promoters showed that it was highly unmethylated, whereas CpG normal sperm and evaluate the potential ability of transgenes to be passed dinucleotides in these regions were highly methylated in parental PEFs. Fur- on to future offspring. Ejaculates from male chimeras showed an average thermore, the cells were able to form embryoid bodies after 3 days in hang- semen concentration of 253x106 cells/ml, motility of 75.2% and normal ing drop culture. RT-PCR analysis confirmed expressions of AFP, alphaAT, morphology in 57.5% of sperm. These results indicate that chimeric animals BMP4, Enolase, GFAP and Neurod genes, which were marker genes of three are likely to be fertile, however have a high percentage of abnormal sperm. germ layers. In vivo testing of iPS cell lines for their developmental potential The overall lack of abnormal development and tumor formation in chimeric (teratoma and chimera assay) is currently underway. Our data indicate that animals suggests a low tumorgenecity level in piPSCs. The development miRNAs can boost the dedifferentiation of porcine somatic cells into iPS of piPSCs that do not cause tumors presents an attractive and power- cells. This study provides a new insight to generate porcine iPS cells without ful translational model to study the safety and potentially efficacy of iPSC exogenous gene integration and more visible merits in biomedical and therapies. The fact that piPSCs that overexpress multiple reprogramming breeding research. This work was supported by the National Basic Research factors including C-MYC contradicts several studies in the mouse suggesting Program of China (2009CB941001). that animals more physiologically similar to humans should be incorporated in future iPSC safety studies.

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Poster Board Number: 3181 cells, including questions about 1) specific routes of delivery of differentiated cells derived from stem cells to specific organ targets, 2) the ability of these CREATING BOVINE IPSCS: LENTIVIRAL cells to persist in situ once delivered, and/or to proliferate, 3) the ability of INFECTION EFFICIENCY OF FETAL BOVINE these cells to express genes associated with the desired differentiated cell type, 4) the ability of these cells to mitigate the targeted disease or debilita- CELLS tion, and 5) assurance that delivery of these cells will not elicit undesirable Sparks, Wendy O., Powell, Anne M., Donovan, David M. secondary conditions such as cancer. Nonhuman primates (NHPs) afford the most clinically relevant model systems for preclinical studies of stem cell Animal Biosciences and Biotechnology Laboratory, ANRI, ARS, USDA, based therapeutic approaches to optimize efficacy and safety. Baboons are Beltsville, MD, USA similar in size and anatomy to humans, possess a genome that is 92% iden- The absence of embryonic stem cells for domestic farm animals has impeded tical to that of humans and are the most utilized NHP species for transplan- the ability to explore their functional genomics via site-directed genomic tation studies. Here, we describe the first derivation of baboon iPSCs based changes in these economically important breeds. Strategies to reprogram on retroviral transduction of fetal baboon fibroblasts with human genes cells into induced pluripotent stem cells (iPSCs) are thus highly appealing. encoding OCT4, SOX2, KLF4 and c-MYC. Colonies exhibiting typical iPSC Establishment of stable bovine iPSCs is expected to: 1) provide an im- morphology were selected, cloned and expanded, yielding eight iPSC-like mense tool to further our understanding of bovine functional genomics, lines, of which three exhibited normal morphology and growth behavior. Ex- e.g. increase our genomic understanding of traits of economic and social pression of pluripotency genes in these cells, including OCT4 and NANOG, importance including nutrition, growth, and bioefficiency, 2) allow us to was confirmed by RT-PCR and immunostaining. Karyotyping, teratoma and preserve genetic information of elite livestock, and 3) reduce breeding times in vitro differentiation studies are underway. This represents the initial step required to transfer favorable traits into existing breeds. Several labs have re- in our long-term goal to develop an NHP resource that will be made avail- ported that reprogramming with the Yamanaka factors Oct3/4(O), Sox-2(S), able to researchers for model studies of protocols involving patient-specific Klf-4(K), and c-Myc(M) has failed to produce stable lines of bovine iPSCs. In stem cells. order to verify lentiviral infection levels predictive of bovine cell reprogram- ming, we examined the infection efficiency of an eGFP vector in several Poster Board Number: 3185 lines of bovine tissues of differing sources (bovine fetal fibroblasts (BFF) INTRACEREBRAL TRANSPLANTATION OF and mixed epithelial/fibroblast cultures from both the amniotic fluid and Wharton’s jelly of the umbilical cord). To examine the effect of multiplicity of NEURONS DIFFERENTIATED FROM INDUCED infection we infected the bovine tissues using serial dilutions of a lentivirus PLURIPOTENT STEM CELLS INTO A RODENT containing a constitutively expressed eGFP at concentrations of 20X, 4X, 2X and 1X. Mouse fetal fibroblasts (MFF) and porcine fetal fibroblasts (PFF) MODEL OF MIDDLE CEREBRAL ARTERY which can both be successfully reprogrammed into iPSCs, were used as a OCCLUSION positive control. Cells were examined at three times points post infection Chang, Da-Jeong, Lee, Nayeon, Choi, Cheonggab, Jeon, Iksoo, using flow cytometry and protein quantification. Analysis of initial infec- tion efficiency showed a significant difference between target tissues. To Kwon, Jihye, Park, In-Hyun, Lee, Hyun-Seung, Hong, Kwan Soo, examine how lentiviral infection affected growth rates of these cultures Daley, George Q., Lindvall, Olle, Song, Jihwan when exposed to reprogramming factors, tissues were infected with serial CHA Stem Cell Institute, CHA University, Seoul, Republic of Korea, dilutions (4X, 2X, and 1X) of lentiviruses encoding OS, MK, Nanog, and Department of Genetics, Yale University School of Medicine, New Haven, the constitutive eGFP lentivirus. These cells were examined at 3 time points CT, USA, MRI Team, Korea Basic Science Institute, Ochang, Republic of post infection: 3 days post infection (dpi), 5 dpi, and 7 dpi. After 7 days in Korea, Department of Biological Chemistry and Molecular Pharmacology, ES media (10 dpi) both the MFF and PFF exhibited iPSC-like colonies, while Harvard Medical School, Boston, MA, USA, Wallenberg Neuroscience the four bovine tissues did not. Overall, both the eGFP alone, and the mixed Center, Lund University, Lund, Sweden 5-factor/eGFP infected cells showed a slight decrease in expansion relative Induced pluripotent stem cells (iPSCs) generated from somatic cells of to uninfected controls at day 3. In addition, the mixed 5-factor/eGFP in- patients can be used to create models of human diseases. Furthermore, they fected cells showed decreased expansion at day 3 relative to the eGFP alone. may serve as potential sources of transplantable cells that can be used in Our results suggest a decreased rate of infection of bovine cells compared to novel cell therapies. Ischemic stroke mainly caused by middle cerebral artery MFF. Higher viral MOI, an increase in the number of target cells, and the use occlusion (MCAo) represents the major type of stroke; however, there are of additional transcription factor genes or small molecules will be tested for still very limited therapeutic options for the stroke-damaged patients. Neural the ability to counteract these reduced infection efficiencies. stem cells (NSC) or neural precursor cells (NPC) are known to survive and Poster Board Number: 3183 ameliorate neurological deficits when they are engrafted in animal models of various neurological diseases. In this study, we differentiated a human iPSC ISOLATION AND INITIAL CHARACTERIZATION line, Detroit 551-iPS8 into NPC and evaluated their therapeutic potentials OF BABOON INDUCED PLURIPOTENT STEM following transplantation into a rodent model of MCAo. iPSC-derived NPC were transplanted into the contra-lateral side of striatum at 7 days after CELLS MCAo, and the transplanted animals were monitored up to 8 weeks using Hornecker, Jacey L., Navara, Christopher S., Ichida, Justin K., various behavioral tests and animal MRI before they were sacrificed for Eggan, Kevin, Hornsby, Peter J., McCarrey, John R. immunohistochemical analysis. Transplanted animals exhibited significant behavioral improvements in stepping, rotarod and modified neurological Department of Biology, University of Texas at San Antonio, San Antonio, severity score (mNSS) tests. We also found that the transplanted human TX, USA, Harvard Stem Cell Institute, Harvard University, Cambridge, MA, cells were co-localized with Nestin, DCX, MAP2, NeuN, DARPP-32, TH, USA, Department of Physiology, University of Texas Health Science Center GAD65/67-positive cells, of which results can be correlated with neu- at San Antonio, San Antonio, TX, USA ral regeneration and behavioral recovery in the transplanted animals. In The derivation of induced pluripotent stem cells (iPSCs) through the process addition, we found that the transplanted iPSC-derived NPC gave rise to of reprogramming somatic cells has engendered new potential for patient- significant reduction of gliogenic and inflammatory responses in the ischemic specific regenerative medicine. Nevertheless, significant challenges remain region, judged by decreased expression of markers for gliogenesis (GFAP) regarding the practical implementation of clinical protocols involving these and inflammation (Iba-1 and ED1). More importantly, we observed that the

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Thursday Poster Abstracts extent of endogenous neurogenesis, represented by BrdU+ cells was signifi- Poster Board Number: 3189 cantly increased, and the subsequent migration (DCX+ cells) and neuronal differentiation (DARPP-32+ cells) were also increased in the transplanted QUAIL INDUCED PLURIPOTENT STEM CELLS animals. Taken together, these results strongly suggest that iPSC, when DERIVED USING HUMAN REPROGRAMMING transplanted into stroke animal models, do not only form neural tissues by themselves, but also provide milieu to protect the stroke-damaged ischemic FACTORS area, as well as to promote the proliferation, migration and differentiation of Lu, Yangqing,, West, Franklin D., Jordan, Brian J., Mumaw, Jennifer stroke-induced endogenous stem cells. This work was supported by grants L., Jordan, Erin T., Gallegos-Cardenas, Amalia, Beckstead, Robert B., from the National Research Foundation (2010-0008719), Republic of Korea, Stice, Steven L. and Korea Food & Drug Administration (S-11-04-2-SJV-993-0-H), Republic of Korea to J.S. Animal Reproduction Institute, Guangxi University, Nanning, China; Animal and Dairy Science, University of Georgia, Athens, GA, USA, Animal and Poster Board Number: 3187 Dairy Science, University of Georgia, Athens, GA, USA, Poultry Science, University of Georgia, Athens, GA, USA GENERATION OF INDUCED PLURIPOTENT For decades the quail (Coturnix japonica) has been a popular animal model STEM CELLS FROM ADULT LIVER CELLS IN in numerous research fields including neural development and disease. As COMMON MARMOSET a developmental model, quail-chicken chimeras have lead to the elucida- tion of key facets in developmental patterning and cell fate partially due Tomioka, Ikuo, Igarashi, Hiroshi, Sasaki, Erika to the ease of access to and the ability to manipulation the avian embryo. Department of Applied Developmental Biology, Central Institute for Avian pluripotent stem cells and derived committed cell lines would offer Experimental Animals, Animal Experimentation Center, Kawasaki, Japan a cell source which could recapitulate normal development in vitro and Embryonic stem cell (ESC)-like induced pluripotent stem (iPS) cells now in vivo when transplanted into embryos or altered development through promise the development of regenerative medicine and therapeutic applica- genetic manipulation of the stem cells. However, currently there are no tion for humans. Although we successfully generated marmoset iPS cells established quail embryonic stem cell lines that can be maintained in long from fetal liver cells last year, the iPS cells from fetal cells are not suitable term culture without loss of developmental potential and no report of avian for preclinical model of regenerative medicine. In this study, to establish induced pluripotent stem cells. Here we demonstrate for the first time in a the preclinical model in non-human primates closely related to human non-mammalian species the successful generation of induced pluripotent species through autogenic transplantation, we generated marmoset iPS stem cell in quail (qiPSC). Quail embryonic fibroblast were transduced with cells from adult liver cells. We introduce the six human transcription fac- six human pluripotency genes, OCT4, NANOG, SOX2, LIN28, C-MYC and tors Oct4, Sox2, Klf4, c-Myc, Nanog and Lin28 into liver cells from adult KLF4 in individual lentiviral vectors driven by the elongation factor 1-alpha female marmoset. Seven days after introduction, the cells were plated onto (EF1-α) promoter (viPS kit, Thermo Scientific). Twenty four hours after trans- murine embryonic fibroblasts (MEFs) at 2 × 105 cells per 10-cm dish. At duction, cells were replated onto mitotically inactivated mouse embryonic the same time, DMEM containing 10% FBS was replaced with medium for fibroblast (MEF) feeder in 20% KSR stem cell media. Likely qiPSCs, were ESC culture (knockout DMEM containing 10% knockout serum replace- observed around 17 days after transduction growing as compact colonies. ment, 1 mM l-glutamine, 0.1 mM MEM nonessential amino acids, 0.1 mM Prospective qiPSC colonies were mechanically picked and cultured in feeder β-mercaptoethanol, and 10 ng/ml LIF). After 3-4 weeks on MEFs, several free conditions using mTeSR1 (Stem Cell Technologies). qiPSCs derived in colonies resembling ESC were observed and picked for further expansion in the present study meet the criteria that define a pluripotent stem cell line. ESC medium. Two cell lines were established, followed by validation analyses They showed high telomerase activity, rapid proliferation and have been of monkey iPS cells. First, we tested the expression of undifferentiated ESC maintained for more than 50 passages without loss of pluripotent pheno- marker genes using immunocytochemistry and RT-PCR. We detected alka- type. They are highly positive for AP, PAS, transcriptional factor OCT4 and line phosphatase activity and the expression of ESC-specific surface markers, SOX2 and cell surface cell surface markers SSEA1 and EMA1. Under in vitro including SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. RT-PCR revealed embryoid body differentiation conditions, qiPSC formed all three germ layers that these iPS cells expressed endogenous Oct4, Sox2, Klf4, c-Myc, Nanog ectoderm, mesoderm and endoderm. Ultimately these cells were capable and Lin28 genes, while all transgenes were silenced. Moreover, karyotype of generating chimeric animals once introduced into stage X embryos. Cells analysis revealed that these iPS cells retained a normal karyotype (46, XX) efficiently incorporated into tissues from all 3 germ layers, extraembryonic after a 2-month culture. Teratomas were formed after transplantation of iPS tissues and potentially the germ line. The formation of qiPSC chimeras that cells into the kidney of immunodeficient mice. RT-PCR analysis of embryoid show contribution to all 3 germ lineages demonstrated the robust and de- bodies demonstrated that marmoset iPS cells had the developmental poten- finitive pluripotency of the derived qiPSCs and that reprogramming factors tial to give rise to differentiated derivatives of all three primary germ layers. are highly conserved among species. In summary, we generated marmoset iPS cells via the retrovirus-mediated Poster Board Number: 3191 transduction of six transcription factors, Oct4, Sox2, Klf4, c-Myc, and Lin28 into adult liver cells, and this will dramatically accelerate the development of NUCLEAR DELIVERY OF IPS TRANSCRIPTION preclinical studies for regenerative medicine in the future. FACTORS USING PROTEIN NANOCAPSULES Biswas, Anuradha, Liu, Jing, Liu, Ying, Gu, Zhen, Fan, Guoping, Tang, Yi Chemical and Biomolecular Engineering, University of California at Los Angeles, Los Angeles, CA, USA, Human Genetics, University of California at Los Angeles, Los Angeles, CA, USA Stem cell biology has grown dramatically after the groundbreaking discovery that somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells from both mouse embryonic and adult fibroblasts upon the expres- sion of a defined set of transcription factors, Oct4, Sox2, Klf4 and c-Myc (OSKM). These findings suggested the possibility of disease and patient-

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specific regenerative medicine therapies as well as a powerful means to detect the cells undergoing reprogramming at much earlier time point than create disease models in culture and to screen the efficacy and safety of OCT4-GFP reporter expression. Thus, this new tool will allow us to study drugs. However, safety concerns of delivering these transcription factors us- the early stage events of the cell destiny changes from somatic cell to iPSC. ing genetic methods remain widespread due to the potential for unexpected We further expanded the scope of the work into different adult stem cells, genetic modifications by the exogenous sequences in target cells. Protein- including HSC, NSC and cancer stem cells. For each case, we discovered based therapeutic methods are safer alternatives to gene therapies because promising hit compounds, and the target protein identificatoin and practical no random or permanent genetic changes are involved, and only transient separation/characterization are in progress. In this talk, the general concept actions of proteins are required for the desired effects. However, most native and applications of DOFLA will be fully discussed with various case studies. proteins are unable to penetrate the cell membrane and often suffer from loss of function due to proteolysis or aggregation in serum. To combat these Poster Board Number: 3195 issues, we have developed a polymeric biomimetic delivery vehicle which MIR-34A MICRO RNA PROVIDES A BARRIER can be degraded by a ubiquitous intracellular protease after cellular entry. Nuclear proteins were encapsulated in nanocapsules (NCs) constructed FOR SOMATIC CELL REPROGRAMMING with monomers and a synthesized bisacryolated peptide crosslinker which Choi, Yong Jin, Lin, Chao-po, Ho, Jaclyn, Zhong, Yingchao, Kim, is recognized and cleaved by the protease. Release of encapsulated protein Sang Yong, Bennett, Margaux, He, Xingyue, Hannon, Greg, He, Lin was confirmed in a cell-free system upon proteolytic degradation of NCs. In vitro cell culture studies demonstrated successful intracellular delivery of Dept of Molecular & Cell Biology, University of California Berkeley, protein NCs and elucidated the importance of the recognizable crosslinker Berkeley, CA, USA, Cold Spring Harbor, Cold Spring Harbor, NY, USA and presence of intracellular protease for observed nuclear localization of Enforced expression of defined transcription factors induces somatic cells protein. We have prepared degradable NCs encapsulating OSKM transcrip- to generate pluripotent stem cells. However, reprogramming driven by tion factors and delivered these 10-20 nm NCs to various cell lines including these transcription factors is a low efficiency process with slow kinetics, human fibroblasts, human fetal neural precursor/progenitor cells (NPCs) indicating that additional mechanisms of gene regulation may be required and amniotic fluid derived cells (AFDCs). We demonstrate that OSKM for generating induced pluripotent stem cells (iPSCs). Recent studies have proteins delivered via NCs show enhanced nuclear localization compared to demonstrated the important role of c-myc and p53 in promoting and sup- proteins delivered intracellularly via protein transduction domains. We have pressing reprogramming, respectively. Although c-myc overexpression or also established a reporter luciferase assay for each protein by develop- p53 deficiency significantly increases reprogramming efficiency, coincident ing constructs containing specific OSKM binding elements to enhance the oncogenic effects may limit their application in iPSC generation. Interest- expression of a luciferase reporter. Using this effective functional assay, we ingly, the miR-34 miRNAs are identified as key effectors of both c-myc and demonstrate that these engineered NCs are able to deliver active proteins p53. c-myc represses miR-34 transcription while promoting iPSC generation; to the nuclei of various cell lines. This proteolytically triggered intracellular p53 activates miR-34 transcription as it suppresses somatic cell reprogram- delivery system may be able to effectively deliver OSKM transcription factors ming. To investigate the role of miR-34 miRNAs in somatic cell reprogram- to safely reprogram various cell lines into pluripotency. ming, we generated miR-34-deficient mouse embryonic fibroblasts and Poster Board Number: 3193 observed a significant increase in reprogramming efficiency and kinetics either with or without c-myc transduction. More importantly, unlike loss of STEM CELL DETECTION AND ISOLATION USING p53, which enhances reprogramming efficiency at an expense of iPSC pluri- potency, genetic ablation of miR-34a significantly promotes iPSC reprogram- DIVERSITY ORIENTED FLUORESCENCE LIBRARY ming without compromising their ability to self-renew and differentiate. APPROACH (DOFLA) Suppression of iPSC reprogramming by miR-34a was due, at least in part, to its repression of Nanog, Sox2 and N-Myc, suggesting post-transcriptional Chang, Young-Tae, Yun, Seong-Wook, Kang, Nam-Young, Park, regulation of pluripotency genes as a novel mechanism to enhance repro- Sung-Jin, Leong, Cheryl, Chandran, Yogeswari, Lee, Sung Chan, gramming efficiency. Taken together, our findings reveal the essential role of Kim, Jun Young, Vendrell, Marc, Ha, Hyung-Ho miR-34 miRNAs as a restraint on somatic cell reprogramming; thus transient Chemistry, National University of Singapore, Singapore, Singapore, manipulation of miR-34 abundance may be a viable strategy for generating Laboratory of Bioimaging Probe Development, Singapore Bioimaging iPSCs. Consortium, Singapore, Singapore Poster Board Number: 3197 There has been increasing interest in the use of ESC and iPSC in biomedical research and clinical therapy. The recent successes of iPSC generation from MICRORNAS AS INDICATORS OF somatic cells of patients with specific diseases and their differentiaion into PLURIPOTENCY STATUS various cell types exemplify how stem cells can be used for the treatment of patients with complex diseases. However, despite the general enthusi- Oni, Eileen, Zhao, Li, Moore, Jennifer C., Hart, Ronald P. asm about the multiple applications of stem cells, their practical use both in Rutgers University, Piscataway, NJ, USA research and disease therapy has been hampered by the heterogeneity of stem cells and their unpredictable proliferation and differentiation. There- MicroRNAs are both required for and are indicative of pluripotency. A large fore, the development of tools and technologies that may facilitate the number of microRNAs have been found to be expressed preferentially in isolation, identification and characterization of stem cells is one of the most human embryonic stem cells (hESC) and induced pluripotent cells (iPS). demanding requisites in the field of stem cell research and applications. We However, differences have been noted in expression patterns between hESC have employed combinatorial chemistry to develop several diversity oriented and iPS. We previously identified novel hESC-enriched microRNAs by deep fluorescence libraries (DOFL) and successfully applied them for the discovery sequencing and we found that some of these novel microRNAs are differen- of bioimaging probes for a number of targets such as beta-amyloid plaque, tially expressed in iPS. Our goal is to identify both known and novel microR- DNA, RNA, GTP, human serum albumin, chymotrypsin, glutathione, and NAs that can be used to screen and validate iPS cell lines. Novel microRNAs heparin. Recently, this approach was applied to develop small molecule were screened by qPCR and assessed using a luciferase bioassay. TaqMan probes which detect specific types of living cells such as alpha cell and qPCR assays for a selected set of known microRNAs identify the shift muscle cells.Using this technique, we have discovered a novel fluorescent from differentiated phenotype to pluripotency and also indicate different compound, CDy1, that selectively stain ESC and iPSC. In further charac- expression patterns among several iPS lines. MicroRNA expression patterns terization of the fluorescent probe in iPSC, we found that our probe can likely indicate a combination of epigenetic status and transcription factor oc-

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Thursday Poster Abstracts cupancy within microRNA-encoding genes. A small set of microRNAs could Poster Board Number: 3201 be used to characterize hESC and iPS, both to validate pluripotency and to distinguish iPS cultures with variable phenotypes. CELLULAR REPROGRAMMING OF Poster Board Number: 3199 ENDANGERED SPECIES FOR CONSERVATION AND RESEARCH ESTABLISHMENT OF AN INDUCED PLURIPOTENT STEM CELL BIOBANK AT Friedrich Ben-Nun, Inbar, Montague, Susanne C., Houck, Marlys L., Charter, Suellen J., Laurent, Louise C., Ryder, Oliver A., Loring, CORIELL INSTITUTE FOR MEDICAL RESEARCH Jeanne F. Fecenko-Tacka, Karen, Schmidlen, Tara J., Allen, Randall, Panckeri, Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA, Kimberly, O’Rourke, Matthew, MacMillan, Annette, Tang, Zhenya, San Diego Zoo Institute for Conservation Reserach, Escondido, CA, USA, Berlin, Dorit S., Keller, Margaret A. San Diego Zoo Institute for Conservation Research, Escondido, CA, USA, Department of Reproductive Medicine, University of California, San Diego, Coriell Institute for Medical Research, Camden, NJ, USA San Diego, CA, USA Induced pluripotent stem cells (iPSCs) represent a powerful new tool for For some highly endangered species, there are too few reproductively ca- studying human disease at the cellular level. These cells, first established pable animals to maintain adequate genetic diversity in the population, and using viral transformation of fibroblasts, have also been derived from other extraordinary measures may be necessary to save them from extinction. It cell types. More recently, cellular reprogramming has been accomplished has been widely appreciated that the ability to generate induced pluripotent using episomal vectors, mRNA and small molecules. A significant number of stem cells (iPSCs) from adult differentiated cells holds tremendous promise the first iPSC lines described were derived from fibroblasts in the National in many applications relevant to human health, including drug development Institute of General Medical Sciences (NIGMS) Human Genetic Cell Reposi- and cell therapy. Here, we suggest using this method to conserve species tory and the National Institute on Aging (NIA) Cell Repository collections at on the edge of extinction. With only eight living individuals, the northern Coriell. Many of these iPSCs have been submitted to Coriell for distribution. white rhinoceros, Ceratotherium simum cottoni, is a critically endangered Coriell has established a Stem Cell Biobank to expand iPSCs submitted by species. We report here on the successful generation of iPSCs from this the research community and to generate new iPSC lines in-house. Coriell species. These northern white rhinoceros iPSCs express the known pluripo- aims to make large numbers of iPSC lines available for research purposes tency-associated transcription factors POU5F1/OCT4, SOX2 and NANOG. through the Coriell Cell Repositories web catalog (ccr.coriell.org). Biobank- Using the in vitro embryoid body assay, we have shown that they can be ing of iPSCs requires a systematic work flow with extensive molecular and differentiated into cells from all three germ layers: ectoderm, mesoderm and cellular characterization and quality control testing. Expansion of the iPSCs endoderm. By using karyotyping, we have confirmed that these iPSCs have for creation of a distribution lot is performed, along with Coriell’s standard the same normal complement of 82 chromosomes as the source fibroblasts. quality control measures. These measures include viability testing, microbial These data indicate that the northern white rhinoceros iPSCs are normal, contamination testing (including PCR-based mycoplasma screening) and stable pluripotent stem cells, comparable to iPSCs from humans and labora- DNA fingerprint analysis. In addition, chromosomal integrity is assessed tory animals. Northern white rhinoceros iPSCs may be useful for regenera- using G-banded karyotype analysis as well as genotyping via Affymetrix 6.0 tive medicine to help maintain the health of the remaining animals, and GeneChip. Pluripotency testing involves extensive molecular and cellular eventually might be used to develop germ cells to enable re-introduction of characterization. Surface expression of pluripotency markers is measured via genetic material from individuals that are otherwise not reproductively viable flow cytometry. In addition, lines are tested for embryoid body formation into breeding populations. and directed differentiation toward lineages representative of the three germ layers. A panel of qPCR assays has been assembled to allow for efficient Poster Board Number: 3203 screening of steady-state mRNA expression of the cells in the undifferenti- ated state, after embryoid body formation and after directed differentia- DIRECT REPROGRAMMING OF FIBROBLASTS tion. In some cases, teratoma formation assays are performed to assess in INTO EPIBLAST STEM CELLS vivo pluripotency. Cell lines will be made available for research purposes to academic, non-profit and industry investigators after completion of a Han, Dong Wook, Greber, Boris, Wu, Guangming, Tapia, Natalia, material transfer agreement. Customers will be supplied with a Certificate Arauzo Bravo, Marcos, Bernemann, Christof, Schöler, Hans R. of Analysis for each cell line, culturing protocols and troubleshooting tips. Cell and Developmental Biology, Max Planck Institute, Muenster, Germany These processes developed and implemented by Coriell Cell Repositories will aid in establishing best practices for iPSC biobanking and distribution. We Epiblast stem cells (EpiSCs) derived from epiblast tissue of post-implantation present our processes for iPSC banking along with representative data from embryos are pluripotent and can give rise to all three germ layers in terato- lines available through the NIGMS Human Genetic Cell Repository. ma assays. Introduction of the 4 transcription factors Oct4/Sox2/Klf4/c-Myc into somatic cells has been shown to generate induced pluripotent stem cells (iPSCs), which are very similar to embryonic stem cells (ESCs) in a number of characteristics. However, generation of EpiSCs by the direct reprogram- ming of somatic cells using these transcription factors has not been shown to date. Here we show that Yamanaka’s 4 transcription factors can be used to directly generate induced EpiSCs (iEpiSCs) under EpiSC culture conditions. iEpiSCs resemble EpiSCs in morphology, gene expression pattern, epigenetic status, and chimera forming capability, demonstrating that the culture envi- ronment in transcription factor-mediated reprogramming determines the cell fate of the reprogrammed cell. We also show that introducing EpiSC-specific factors with Oct4/Sox2/c-Myc but without Klf4 could also directly generate iEpiSCs. To our knowledge, this is the first report describing the direct con- version of fibroblasts, by transcription factors, to a stem cell other than ESCs.

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Poster Board Number: 3205 growth in the capsules, the cells leaked out of the alginate beads without the outer shells, whereas such leakage was completely blocked by the outer ZFX ENHANCES THE GENERATION OF INDUCED shells. Minimum cell density existed for successful growth in these types of PLURIPOTENT STEM CELLS microcapsules. At an initial cell density of 7.00 x 105 cells/cm3-gel (50 cells/ capsule), cell growth in the capsules was hardly observed. At an initial cell Heng, Dominic Jian-Chien, Gonzales, Kevin Andrew Uy, Ng, Huck- density higher than 2.45 x 106 cells/cm3-gel (160 cells/capsule), however, Hui cellular growth was observed in both types of capsules. In addition, for the NUS Graduate School/Genome Institute of Singapore, Singapore, initial cell density of 1.22 x 107 cells/cm3-gel (800 cells/capsule), a few Singapore, Genome Institute of Singapore, Singapore, Singapore aggregates were formed in each hollow capsule. In both cases, aggregates finally become around 300 micrometers in diameter on Day 8. We could The transcription factor quartet of Oct4, Sox2, Klf4 and c-Myc when over- observe green fluorescence of nanog-GFP of iPSCs incubated in the capsules expressed, can reproducibly convert somatic cells into embryonic stem cell until Day 8 with an initial cell density of 2.45 x 106 cells/mL. Through these (ESC)-like cells also known as induced pluripotent stem cells (iPSCs). Other experiments, we concluded that alginate beads without outer shells are intrinsic factors, especially transcription factors, also play pivotal roles in this not suitable for mass culture of iPSCs because of the cellular leakage from de-differentiation process more commonly referred to as reprogramming. alginate beads and that hollow capsules are effective for making uniform For instance, UTF1, Nr5a2 and Tbx3 have been reported to enhance the ef- EB-like aggregate. We are now investigating the differentiation tendency of ficiency of reprogramming to varying degrees. By identifying more transcrip- iPSCs in these microcapsules with an expectation that such microencapsula- tion factors, we can further dissect the intricate mechanism of reprogram- tion should enhance the local concentrations of autocrine/paracrine factors ming and also translate it to the better understanding of the ESC state. Here, secreted by the cells. we identify the zinc finger protein, Zfx, which had been previously shown to be important for the self-renewal of both embryonic and hematopoietic stem cells, to be able to augment the generation of murine iPSCs. In addi- Poster Board Number: 3209 tion, we demonstrate that Zfx can enhance the kinetics of reprogramming. Next, to better elucidate the mechanistic action of Zfx in reprogramming GENETIC EVOLUTION AND MOSAICISM we analysed both the ChIP-seq data of Zfx in mouse ESCs as well as the DURING REPROGRAMMING TO PLURIPOTENCY microarray data of Zfx-deficient mouse ESCs. We found that Zfx binds and regulates many important proliferative and metabolic genes in mouse ESCs. Hussein, Samer M., Batada, Nizar N., Vuoristo, Sanna, Ching, Interestingly, we found that genes that are negatively regulated by Zfx are Reagan W., Ng, Siemon, Sourour, Michel, Hämäläinen, Riikka, in fact positively regulated by the tumor suppressor p53. Hence, this might Olsson, Cia, Bazett-Jones, David P., Alitalo, Kari, Lahesmaa, Riitta, in part explain the enhancement of reprogramming efficiency with the Otonkoski, Timo, Nagy, Andras overexpression of Zfx, an effect similarly obtained with the inhibition of p53. Samuel Lunenfeld Research Institute, Toronto, ON, Canada, Ontario Altogether, we have uncovered yet another transcription factor, Zfx, which is Institute for Cancer Research, Toronto, ON, Canada, Biomedicum Stem one the architects of the reprogramming process. Cell Center, Helsinki, Finland, The Hospital for Sick Children, Toronto, ON, Poster Board Number: 3207 Canada, Research Program Unit, Molecular Neurology, Helsinki, Finland, Molecular Cancer Biology Laboratory, Helsinki, Finland, Turku Centre for FEASIBILITY OF HYDROGEL-BASED Biotechnology, Turku, Finland MICROENCAPSULATION FOR PROPAGATION Somatic cell reprogramming to induced pluripotent stem cells (iPS) is AND DIFFERENTIATION OF INDUCED achieved by introduction of a defined set of transcription factors. The low induction efficiency associated with reprogramming has been attributed to PLURIPOTENT STEM CELLS IN SCALABLE p53 induced cell cycle arrest, apoptosis and senescence. However, it remains SUSPENSION CULTURE unknown if the reprogramming process itself induces DNA damage that can compromise genomic integrity and the efficiency of establishing iPS lines. Horiguchi, Ikki, Chowdhury, Mohammad Mahfuz, Sakai, Yasuyuki Using a high resolution SNP array, we compared copy number variations of Institute of Industrial Science, The University of Tokyo, Tokyo, Japan early and intermediate passage human iPS cells to their respective parental fibroblast cell origins and to human Embryonic Stem (hES) cells. We show Application of induced pluripotent stem cells (iPSCs) in regenerative medi- that much higher levels of mutation in early compared to intermediate cine requires the development of scalable culture system to obtain a large passage hiPS cells, fibroblast and hES cells is detected. We also demonstrate number of cells for relevant therapies. Generally, stirring suspension bioreac- that most of the mutations are de novo generated. Moreover, a subset of tors are widely used for bioprocess for mass production of mammalian cells. these de novo mutations were identified that place these early passage lines However, direct use of these bioreactors for iPSC culture usually results in under a selective disadvantage. A high degree of genetic mosaicism and cellular damage by shear stress or extensive and inhomogeneous cell aggre- frequent mutations in fragile genomic regions was observed within early gation that causes uncontrollable differentiation. Meanwhile, some research- passage lines, suggesting replication dependent DNA damage occurs during ers reported that the encapsulation of cells could solve these problems. reprogramming. Remarkably, however, during culture, the mutation level Therefore, we are evaluating various types of capsules for iPSC mass pro- of newly established hiPS lines decreases significantly and undergoes high duction and differentiation. We evaluated three types of capsules to culture selection pressure, driving the cells to a genetic state resembling that of hES mouse iPSCs: 1) alginate beads (alginate-gel capsule without outer shell), 2) cells. core-shell capsule (alginate-gel capsule with outer shell of alginate-PLL poly- ion-complex), and 3) hollow capsule (alginate-gel from inside was removed by EDTA.) All types of capsules diameter was 500 - 600 micrometers. Mouse iPSC line (iPS-MEF-Ng-20D-17) that has an enhanced green fluorescent protein (EGFP) under the control of Nanog promoter (obtained from Riken BRC Cell Bank) was cultured in the capsules. Microencapsulated cells were cultured under a continuous shaking condition. The cells grew in aggregate morphologies in all types of capsules. Multiple aggregates of iPSCs were formed in alginate beads and core-shell capsule. However, in each hollow capsule, iPSCs got together and formed single aggregate. After extensive

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Poster Board Number: 3211 differentiation into neuroectodermal cells. Upon induction of differentiation using 10-7 M all-trans retinoic acid (ATRA), all cell lines shared the temporal SINGLE CELL ANALYSIS OF GENETIC expression patterns for Nestin, Neuropilin II, LaminA, Oct4 and Nanog. MOSAICISM IN STEM CELL-DERIVED However, using flow cytometry, we observed in ATRA treated iPS cells a distinct acceleration in the differentiation tempo as measured by the loss of GENOMES GFP fluorescence as an indicator for Oct4 promoter silencing. We further McConnell, Michael J.1, Brennand, Kristen J.2, Voet, Thierry3, Piper, compare the tempo of Oct4 promoter silencing with the kinetics of SSEA1 Julia1, Vermeesch, Joris3, Gage, Fred H.2 loss and acquisition of ectodermal markers. We propose that the kinetics of differentiation as analyzed by flow cytometry provides a simple yet powerful 1 Crick-Jacobs Center for Theoretical and Computational Biology, Salk approach to determine the pluripotency and propensity for precocious dif- 2 Institute for Biological Studies, La Jolla, CA, USA, Laboratory of Genetics, ferentiation of reprogrammed stem cells. Salk Institute for Biological Studies, La Jolla, CA, USA, 3Center for Human Genetics, KU Leuven, Leuven, Belgium Poster Board Number: 3215 Genetic mosaicism occurs when a single founder cell or clonal found- PROBING EARLY GENE REGULATORY EVENTS ing cell population gives rise to genomically diverse progeny. This arises during normal human brain development and when human stem cells are IN REPROGRAMMING BY COMBINATORIAL maintained in vitro. We sought to examine genetic mosaicism among hu- BAYESIAN MODELLING man induced pluripotent stem cell (hiPSC)-derived neurons. To do this we employed single cell approaches because routine analyses -- using bulk DNA Vasquez, Louella J., Mukherjee, Sach or metaphase spreads -- report only the most abundant genomic changes Institute of Advanced Study, Centre for Complexity Science and from a heterogeneous genetic mosaic. Both neuron-to-neuron mosaicism Department of Statistics, University of Warwick, Coventry, United Kingdom, in human brains and cell-to-cell mosaicism in human embryonic stem cells Centre for Complexity Science and Department of Statistics, University of are brought about, in part, by retrotransposition of endogenous L1 mobile Warwick, Coventry, United Kingdom elements. However, the diversity and prevalence of unique genomes in these It is well established that induction of certain combinations of transcription settings is unknown. Moreover, the relationship between retrotransposi- factors (TFs) can induce pluripotency in somatic cells. However, we remain tion and other genomic changes (e.g. aneuploidy, copy number variation) is limited in our understanding of the regulatory events by which TF induction unexplored; limited by access to human neurons and the necessity of single brings about epigenetic reprogramming. Here, we integrate gene expres- genome analysis. We circumvented these limitations, and leveraged the sion data, obtained after 4 days under combinatorial expression of the four clonality inherent in hiPSC derivation, by amplifying single hiPSC-derived factors Oct4, Sox2, Klf4 and c-Myc, with ChIP-on-chip data, to shed light neuronal genomes then using qPCR and microarray assays to identify ge- on early gene regulatory events in reprogramming of mouse fibroblasts. By nomic differences in individual human neurons. Most of the hiPSC-derived doing so, we seek to highlight genes that are likely binding targets of the neurons analyzed here had unique genomes. factors and at the same time show quantitative evidence of dependence Poster Board Number: 3213 on one or more of the factors (singly, or in a combinatorial manner). To this end, we exploit ideas from computational Bayesian statistics to develop a COMPARATIVE ANALYSIS OF model that combines both expression and TF binding calls. Specifically, we REPROGRAMMING BY IPS, FMR AND NORMAL model gene expression as a function of the presence/absence of the factors. We include additional higher-order terms to account for possible (direct REPRODUCTION or indirect) combinatorial effects. Bayesian model averaging was used to Krueger, Winfried H., Tanasijevic, Borko, Rasmussen, Theodore P. explore the large space of possible patterns of dependence and thereby rank genes by probability scores. Our approach also integrated information Department of Pharmaceutical Sciences, University of Connecticut, Storrs, over possible models to give a summary measure of the influence of each CT, USA transcription factor on each gene. The methodology scales to thousands of Methods for reprogramming of somatic cells include fusion with ES cells genes, and could be extended to additional, complementary data. Further, (fusion mediated reprogramming or FMR), reprogramming using induced this methodology could be used to study other epigenetic reprogramming expression of the transcription factors Oct4, Sox2, Klf4 and c-myc (induced events, such as those reported for induced neuron and induced cardiomyo- pluripotency or iPS) and somatic cell nuclear transfer (SCNT). The pluripo- cyte formation. The approach we propose offers a way to highlight, on tency of both iPS and SCNT-derived stem cells has been demonstrated by quantitative grounds, genes that may be important players in the critical, chimeric contribution, yet epigenetic analyses in the last year have shown early stages of reprogramming. Identification of early regulatory hubs will incomplete reprogramming of a number of genomic loci when compared help to generate new hypotheses that can be tested experimentally. Fur- to embryonic stem cells (ESCs). We observed that iPS cells display a greater thermore, our results may aid in the development of dynamic models that propensity for spontaneous differentiation when compared to ESCs. We probe, in greater mechanistic detail, regulatory events that take place further therefore reasoned that these cells retained some form of epigenetic downstream and later in reprogramming. memory despite expressing numerous pluripotency markers. To assess the Poster Board Number: 3217 physiological relevance of the retained epigenetic memory we compared cell cycle, levels of pluripotency markers and differentiation kinetics of mouse INVESTIGATING THE ROLE OF ASTROCYTES IN ESCs, iPSCs and FMR derived pluripotent stem cells that harbor the same GFP transgene under the control of the Oct4 promoter. While the cell cycle PLASTICITY AND DISEASE kinetics of the cells compared did not display any significant changes, differ- Rooney, Gemma E., Rauen, Katherine A., Ullian, Erik M. ences were detected in the levels of GFP expression when these cells were Dept. of Opthalmology, University of California, San Francisco, San grown under culture conditions promoting self renewal. In order to further Francisco, CA, USA, Pediatrics Genetics, University of California, San assess the degree of pluripotency, iPSCs, FMR cells and ESCs were differenti- Francisco, San Francisco, CA, USA ated into embryoid bodies. All cell lines were capable of forming embryoid bodies which displayed highly similar expression patterns for numerous dif- Astrocytes outnumber neurons in the human brain at a ratio of approxi- ferentiation markers although undifferentiated iPSCs also expressed low lev- mately 10:1. Despite their abundance, the role of astrocytes in human els of the endoderm marker Sox17. We also assessed retinoic acid induced disease is still poorly understood. To address this gap in our understanding,

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we have begun to investigate the role of astrocytes in human neurological Poster Board Number: 3221 diseases such as Costello syndrome, a rare developmental disorder caused by mutations in genes that encode protein components of the Ras/MAPK CHILDREN’S HOSPITAL OF PHILADELPHIA pathway. Costello syndrome is characterized by autistic behaviors, mental INDUCED PLURIPOTENT STEM CELL CORE retardation, enlarged ventricles and frontal atrophy. Our first objective is to ask whether astrocytes, derived from somatic cells of patients with Costello FACILITY syndrome, secrete altered levels of synaptogenic factors. Astrocytic proper- Mills, Jason A., Lu, Lin, Kotton, Darrell, Mostoslavsky, Gustavo, ties will be assessed using induced pluripotent stem cell (iPSC) technology High, Katherine, Weiss, Mitchell J., Paul, Gadue, French, Deborah L. and microfluidic chambers developed to rapidly identify factors that are released in response to the glial-stimulants, acetylcholine (ACh) or norepi- Center for Cellular and Molecular Therapeutics, Children’s Hospital of nephrine (NE). We have isolated fibroblasts from selected patient popula- Philadelphia, Philadelphia, PA, USA, Center for Regenerative Medicine tions and optimized iPSC generation using retroviral vectors. Optimization of (CReM), Boston University School of Medicine, Boston, MA, USA, Center neuroectodermal differentiation is also being performed. These studies have for Cellular and Molecular Therapeutics, Howard Hughes Medical Institute, the potential to uncover the role of human astrocytes both in regulating Children’s Hospital of Philadelphia, Philadelphia, PA, USA, Division of normal plasticity and in disease states. Hematology, Pediatrics, University of Pennsylvania, Children’s Hospital of Philadelphia, Philadelphia, PA, USA, Center for Cellular and Molecular Poster Board Number: 3219 Therapeutics, Department of Pathology, University of Pennsylvania, Children’s Hospital of Philadelphia, Philadelphia, PA, USA PROTEIN-BASED REPROGRAMMING: VIRUS Patient-specific induced pluripotent stem cells (iPSCs) can be used to model VERSUS PROTEIN APPROACH disease and provide starting material to manufacture tissues and their Lee, Jieun, Sayed, Nazish, Hunter, Arwen, Yang, William, Reijo Pera, derivatives for applications in regenerative medicine. Numerous methodolo- Renee, Swartz, James, Cooke, John gies are in place to generate iPSC lines, but the process is labor intensive and requires defined standard operating procedures to maintain consistency and Stanford University, Palo Alto, CA, USA reproducibility. In 2008, a hESC/iPSC core facility was established within the Introduction: The generation of induced pluripotential cells (iPSCs) pro- Center for Cellular and Molecular Therapeutics at The Children’s Hospital vides for patient-specific stem cells. However, viral transduction strategies of Philadelphia. One of the goals in our mission is to provide expertise and are beset by biosafety concerns, including concerns of oncogenesis and quality-control reagents for the generation and differentiation of iPSCs disruption of endogenous genes. A protein-based approach obviates these to the CHOP and University of Pennsylvania academic communities. Our concerns and may provide for finer control of dose, duration and sequence methodologies for generating iPSCs have already evolved to coincide with of exogenous reprogramming factors. It is not known what differences in the fast technological advancements of the field. We are currently using a transcriptional control may exist between viral and protein-based strategies polycistronic lentiviral reprogramming construct (STEMCAA) that can be of iPSC induction. Methods: We assessed the functionality of cell-permeant excised from the cells by Cre-mediated recombination. This has the advan- peptide (CPP) SOX2 and OCT3/4 using competitive DNA-binding analy- tage of limiting transgene-induced mutagenesis by enabling the selection sis and also observed translocation of CPP across the plasma and nuclear of single integrants. By using cells derived from fetal liver, peripheral blood, membranes. To further assess the biological function of CPP-SOX2, we and skin biopsies; we have generated normal and disease-specific iPSC lines; performed shRNA gene silencing against Sox2 in hiPSCs. The transcriptional including hematological (Hemophilia B, Down syndrome, Diamond-Blackfan effects of CPP-SOX2 and CPP-OCT3/4 were compared to retroviral factors anemia, Dyskeratosis Congenita), genetic (Mitochondrial Heteroplasmy), (RF) pMX-Sox2 in human fibroblasts focusing on a set of genes regulated by neurological (Cornelia de Lange syndrome), and musculoskeletal (Juvenile Sox2 (i.e. Jarid2, bMyb, Zic2, Sox2, Oct4 and Nanog) and by Oct3/4 (i.e. Idiopathic Arthritis) diseases. We characterize our iPSC lines by morphol- Tcf4, GAP43, SSBP2, Oct 3/4, Sox2 and Nanog) using real-time qRT-PCR ogy, cell surface markers, expression of ESC pluripotency markers such as (n=4). To compare RF versus CPP-based reprogramming in gene regulatory DMNT3B, ABCG2, REX1, NANOG, OCT4, and SOX2; teratoma formation, networks, single-cell analysis using Fluidigm technology and DNA microar- karyotype analysis, and their capacity to differentiate. We maintain our ray analysis were also performed focusing on genes involved in transcription, lines for greater than 20 passages and show that normal fibroblast-derived chromatin remodeling, differentiation, mitochondrial regulation and cell iPSCs are comparable to embryonic stem cells following differentiation into cycle. Results: Intriguing differences between CPP versus RF were observed hematopoietic lineages as shown by cell surface markers, QRT-PCR gene in the magnitude and timing of differential expression of genes highly regu- expression, and progenitor colony forming assays. As a core facility, we work lated by Sox2 and Oct3/4 (n=4, P< 0.045). We confirmed the maintenance with investigators to design new protocols tailored for their individualized of pluripotency by the replacement of CPP-SOX2 in Sox2 knockdown research and are dedicated to incorporating the latest technologies and hiPSCs using qRT-PCR and FACS analysis for Sox2, Oct4, and Nanog. To protocols into our facility infrastructure. determine if these differences could be observed at the single-cell level using Fluidigm technology and DNA microarray in a broader set of genes involved in cell fate, we analyzed a larger gene regulatory network. Conclusion: We observed intriguing differences in the magnitude and temporal pattern of gene expression induced by protein compared with viral approaches to iPSC induction. Different signaling pathways appear to be involved in protein- vs. viral-induced reprogramming.

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REGENERATION MECHANISMS Poster Board Number: 3225 Poster Board Number: 3223 SECRETOME OF HUMAN EMBRYONIC AND MESENCHYMAL STEM CELLS INCREASES COMBINATORIAL HUMAN PROGENITOR CELL MATRIX METALLPPROTEINASE-2 EXPRESSION TRANSPLANTATION OPTIMIZES ENDOGENOUS AND PROMOTES HOST TISSUE REGENERATION BETA CELL REGENERATION THROUGH IN A CHRONIC MODEL OF LIVER INJURY SECRETION OF REGENERATIVE FACTORS Jang, Yu Jin, Han, Ji You, An, Su Yeon, Woo, Dong-Hun, Kim, Bell, Gillian I., Meschino, Michael, Hughes-Large, Jennifer, Jong-Hyun, Kim, Hyo-Jin, Kim, Jong-Hoon Broughton, Heather, Xenocostas, Anargyros, Hess, David A. Biotechnology, Korea University, Seoul, Republic of Korea Physiology and Pharmacology, University of Western Ontario, London, ON, We previously reported that purified hepatocyte-like cells derived from hu- Canada, Department of Oncology, University of Western Ontario, London, man embryonic stem cell (hESC) promoted the liver tissue recovery not only ON, Canada by cell replacement, but also by delivering proteins (secretome) that enhance Transplanted murine bone marrow (BM) progenitor cells engraft dam- endogenous host liver regeneration. In this study, we investigated possible aged islets, stimulate endogenous beta cell proliferation and improve islet therapeutic effects of secretomes obtained from undifferentiated hESC and function. To enrich for analogous human progenitor subtypes that promote mesenchymal stem cell (hMSC), and explored the underlying mechanism beta cell regeneration, we FACS-purified human BM or umbilical cord in a mouse model of chronic liver injury. Mice pre-intoxicated with dim- blood (UCB) cells using high aldehyde dehydrogenase-activity (ALDHhi), a ethylnitrosamine (DMN) were treated with single intraperitoneal injection conserved function in multiple progenitor lineages. Compared to ALDHlo of secretomes or medium used to support the growth of hESCs or hMSCs. cells, ALDHhi cells from UCB and BM are enriched for hematopoietic and Both hESC- and MSC-secretomes induced robust host liver regeneration, endothelial colony forming cells in vitro. While BM ALDHhi cells are also as determined by biochemical and histological analyses. The expression of enriched for multipotent mesenchymal stromal cells (MSC), UCB ALDH- MMP2 was significantly increased in the liver that received hESC- or hMSC- purified populations are devoid of MSC capacity, a cell type previously secretome, compared to control groups. In contrast, expression of α-SMA, implicated in beta cell regeneration. To address beta cell regeneration in a hallmark of activated hepatic stellate cells, was profoundly decreased after vivo, STZ-treated (days 1-5) NOD/SCID mice were tail vein injected with administration of both secretomes. These results suggest that hESCs and human UCB-derived ALDHlo or ALDHhi cells, or human BM-derived ex- MSCs may release soluble factors that support the host tissue regeneration vivo expanded ALDHlo or ALDHhi MSC on day 10, and blood glucose was of chronically injured liver. monitored for >30 days. Compared to PBS-injected controls, UCB-derived ALDHhi cells induced a transient reduction of systemic blood glucose 7-18 Poster Board Number: 3227 days post-transplantation, whereas BM ALDHhi cell transplantation induced CELL THERAPY IN ACUTE LUNG INJURY more lasting hyperglycemia reduction, presumably due to MSC content. Although MSC derived from independent BM samples showed variable Ling, Thai-Yen, Peng, Fu-Chou, Chen, Yao-Chang capacity to regenerate islets, and prolonged MSC expansion diminished the Institute of Pharmacology, Taipei, Taiwan, Institute of Toxicology, Taipei, ability to improve hyperglycemia, transplantation of ALDH-purified MSC Taiwan, Department of Laboratory Medicine, Taipei, Taiwan stably reduced hyperglycemia, and increased serum insulin compared to PBS controls. The mechanisms involved in endogenous beta cell regeneration Paraquat, the commonly used herbicide in Taiwan, is a potent pneumo- differ depending on the population of regeneration initiating cells adminis- toxicant. It was well know that paraquat was accumulated in the lung via tered. Improved hyperglycemia following UCB ALDHhi cell transplantation polyamine-uptake system and underwent a process of redox-cycling leading was due to increased beta cell and endothelial cell proliferation leading to to reactive oxygen species (ROS) production. In clinical, paraquat intoxica- increased islet size and vascularization, while improved hyperglycemia fol- tion evoked sudden lung dysfunction and causes patients acute lung injury/ lowing transplantation of ALDH-purified MSC was due to the formation of acute respiratory distress syndrome (ALI/ARDS) with high mortality. From small islets associated with the ductal epithelium. To further identify novel 2001 to 2002, there were 43 paraquat intoxication cases report in Taiwan targets produced by regenerative MSC, we performed Affymetrix microarray and the mortality was 72%. Unfortunately, so far there were no effective comparing global mRNA expression between 1.) regenerative vs non-regen- therapies for the paraquat intoxication patients. In order to develop a novel erative MSC, and 2.) regenerative MSC at early (P3) vs late (P8) passage. therapy for the patients, ICR mice were used for an animal model. The mice Using this combined strategy we identified 55 mRNA products >2-fold were orally administered with different dosages of paraquat to induce ALI/ upregulated in regenerative MSC at early passage (double hits). Highlighted ARDS. The survival rates of mice were evaluated as well as histopathological in these analyses were pro-angiogenic growth factors (HGF), developmen- analysis. In the studies, respiratory mechanics assessment revealed signifi- tal factors (BMP-2), and members of the EGF superfamily (AREG, EREG). cant difference in lung function; and the autopsy of the lung tissues showed To investigate whether both endogenous regenerative pathways could be pulmonary hemorrhage, thickening of alveolar septum and inflammatory stimulated to optimize islet regeneration, hyperglycemic NOD/SCID mice cells infiltration in lung. The survival rate in high dose of paraquat intoxica- were transplanted with MSC at day 10 to promote new islet formation, tion mice at day-6 were only around 8%. Now, we have successfully gener- followed by transplantation of UCB ALDHlo or ALDHhi cells at day 17 to ated human placenta derived mesenchymal stem cells in serum-free culture increase growth and vascularization of newly formed islets. Compared to condition for translational medicine studies. The cells were used to treat PBS controls, MSC + UCB ALDHhi cell transplantation lead to significant and acute paraquat intoxication animal for cell therapy, and the mechanism for maintained reductions in blood glucose. Collectively these data suggest that the therapeutic effects were also evaluated. After cells therapy, the results combinatorial transplantation of human MSC and UCB ALDHhi cells opti- showed that the survival rate for the mice increased from 8% to 35% at mize endogenous islet regeneration through paracrine secretion of regenera- day-6, however, the other therapies, such as treatment with dexamethasone tive molecules. Further mechanistic understanding of cellular communication and the other cells, did not increase the survival rate. The results indicated with the pancreatic regenerative niche is required. that placenta derived mesenchymal stem cells significantly restored lung tis- sue biological function, decreased the infiltration of neutrophils into the lung tissues and alveolar spaces, and the cytokines associated with neutrophil recruitment and activation were also suppressed, such as GCSF, IL-6, MCP-1

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and KC. Taken together, the cell therapy could attenuate the lung damages, Poster Board Number: 3231 restore the pulmonary functions and recover the survival rate via suppressing neutrophils infiltration for acute paraquat poisoning. This novel cell therapy REGENERATION THERAPY IN ALZHEIMER’S might be useful for clinical application. DISEASE Poster Board Number: 3229 Marutle, Amelia, Wicklund, Linn, Lilja, Anna M., Hovatta, Outi, HUMAN UMBILICAL CORD BLOOD-DERIVED Nordberg, Agneta Alzheimer Neurobiology Center, NVS, Karolinska Institutet, Stockholm, MESENCHYMAL STEM CELLS MIGRATE IN Sweden, Department of Clinical Science, Intervention and Technology, RESPONSE TO COMPLEMENT Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden, Alzheimer Neurobiology Center, NVS, Department of Geriatric Marquez-Curtis, Leah A., Qiu, Yuanyuan, Janowska-Wieczorek, Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Anna Stockholm, Sweden Research & Development, Canadian Blood Services, Edmonton, AB, Canada, Dept of Medicine, University of Alberta, Edmonton, AB, Canada An exciting avenue in regenerative medicine is the great potential that stem cells offer in regards to stimulating brain repair and cell-based replacement Mesenchymal stem cells (MSC) migrate preferentially to sites of inflam- therapies to treat neurodegenerative diseases, such as Alzheimer’s disease mation and tissue injury, but the molecular signals that direct them to their (AD) for which no cure exists. AD is characterized by an early accumulation target microenvironment remain to be elucidated. Recently, we showed that of β-amyloid (Aβ) peptides that deposit into plaques in primarily cortical complement component 1 subcomponent q (C1q), the initiator of the clas- brain regions. It is suggested that neurons laden with intracellular tau- sical pathway of complement activation, enhances the homing-related re- neurofibrillary tangles die as a consequence of increases in βA and declin- sponses of hematopoietic stem/progenitor cells in a manner similar to com- ing levels of neurotrophic support. In AD, the basal forebrain cholinergic plement cleavage fragment C3a as previously reported by us. In this study, neurons are particularly affected. Pluripotent human embryonic stem (hES) we investigated whether C1q elicits directional signals that influence the cells represent a rich source of expandable cells that can be used for gen- recruitment of MSC to damaged tissue/organs and examined the expression erating various cell populations, including neurons and for screening novel in MSC of two putative C1q receptors (cC1qR, which binds to the collagen- candidate drugs. However, enthusiasm for the therapeutic value of these like tail and gC1qR/p33, which binds to the globular head). Moreover, as we stem cells in AD is hampered by the need to first identify the conditions previously demonstrated that matrix metalloproteinases (MMPs), especially under which these cells differentiate into a specific lineage in vitro and their MMP-2 and membrane type 1-(MT1)-MMP, regulate the migration of MSC, behavior in vivo, especially during disease. We have recently developed here we studied the effects of C1q on MMP-2 secretion and MT1-MMP ex- a reliable and reproducible protocol that efficiently produces functionally pression in MSC, and determined whether C1q triggers the phosphorylation mature cholinergic neurons derived from hES cells. Cholinergic neuronal of ERK and whether this pathway is involved in the migration of MSC as it is differentiation is further enhanced in the presence of neurotrophins such as for C3a. Human MSC were isolated from umbilical cord blood, maintained NGF and BDNF. We have also conducted studies, analysing how various Aβ for 3-6 passages, and characterized by adipocyte and osteoblast differentia- forms affect neurogenesis both in human neural progenitors in culture and tion. Migration assays across the reconstituted basement membrane Matri- in animal models of AD. Our findings suggest that neuroprogenitors display gel and gelatin; RT-PCR, Western blot and flow cytometry (to examine the a high degree of synaptic plasticity, and that efforts to reduce amyloid levels expression of cC1qR and gC1qR); zymography (to evaluate the secretion in the brain as well as protect neurons by treatment with neuroprotective of MMP-2); and Western blot (to examine the expression of MT1-MMP cholinergic drugs may significantly promote regenerative processes in the Al- and the phosphorylation of ERK1/2) were used. We found that MSC were zheimer brain. Future studies with molecular imaging of β-amyloid processes chemoattracted by a C1q gradient in a dose-dependent manner and ex- in AD animal models to monitor the effects of functional neurogenesis dur- pressed transcripts and protein for cC1qR and gC1qR. However, only gC1qR ing neurodegeneration will provide important knowledge for the develop- was detected on the surface of MSC, and a monoclonal antibody that blocks ment of stem-cell therapeutic strategies for Alzheimers disease. the C1q/gC1qR interaction significantly inhibited the chemoinvasion of MSC across Matrigel and gelatin towards C1q. Moreover, stimulation of MSC Poster Board Number: 3233 with C1q: (i) enhanced/primed MSC chemotaxis towards a low gradient (20 ng/mL) of the chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) to DOES HUMAN MOTOR NEURON STEM CELL- a level equivalent to that towards a high gradient (100 ng/mL); (ii) increased DERIVED NEUROTROPHIC SUPPORT ENHANCE MMP-2 secretion; and (iii) induced the phosphorylation of ERK1/2. The THE DEVELOPMENT OF THE NMJ IN SPINAL ERK1/2 inhibitor PD98059 decreased the chemoinvasion of MSC towards C1q. In conclusion, this study indicates that human MSC are chemoattracted MUSCULAR ATROPHY? by C1q and that C1q enhances the chemotaxis of MSC in response to SDF- Wyatt, Tanya J., Nistor, Gabriel, Keirstead, Hans S. 1. Our findings provide additional evidence for a broader array of molecules, Anatomy & Neurobiology, University of California, Irvine, Irvine, CA, USA, including the complement proteins, which serve as directional signals for California Stem Cell, Irvine, CA, USA MSC, and suggest a new mechanism for the migration of MSC which may be important in tissue repair or regeneration. Infantile spinal muscular atrophy (SMA) is the most common and se- vere hereditary neurological disease in childhood, and is characterized by motor neuron loss. Children diagnosed with SMA demonstrate severe muscle weakness that makes it difficult for them to breathe, eat, and move, and >95% die within 2 years. Delayed maturation of the neuro- muscular junction (NMJ) is considered to be one of the earliest detectible pathologies in SMA mice. Neurotrophins play a major role in normal NMJ development and thus may be a key factor in the pathogenesis of NMJs in SMA. Currently, we are looking at the potential of hESC-MNPs to promote the maturation of the pre- and post-synaptic regions at the NMJ of SMNdelta7+/+;SMN2+/+;Smn-/- mice in vitro, through the secretion of neurotrophic factors. Further research into the signaling mechanisms

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Thursday Poster Abstracts involved in NMJ maturation may identify additional mechanisms by which Post Mortem Neural Precursor Cells (PM-NPCs). Differentiation yields transplanted cells may be of therapeutic benefit. mostly neurons (about 30-40%) compared to regular NPCs. Also the cho- linergic yield is higher. PM-NPCs are particularly sensitive to mTOR activity. Poster Board Number: 3235 The average dose of rapamycin normally used to inhibit mTOR is 5 ng/mL WHAT DETERMINES WHETHER MURINE but we observed that the effect was significantly evident with a dose 100 fold lower. The higher ERK activation observed in undifferentiated PM-NPCs EPENDYMAL STEM CELLS ARE ACTIVATED is also involved in their differentiation process, since the exposure to ERK DURING PATHOLOGIES OF THE ADULT SPINAL inhibitor PD98059, downregulates significantly the extent of neuronal differ- entiation. PM-NPCs but not classical NPCs synthesize EPO that it is known CORD? to be active as a signalling molecule promoting stem cell-derived neurogen- Beaudoin, Stéfanny, Hamilton, Laura, Dugas, Christian, Aumont, esis and neuronal differentiation, the effect is not observed in astrocytes. Anne, Pineau, Isabelle, Lacroix, Steve, Fernandes, Karl J.L. Blocking the EPO pathway by means of monoclonal antibodies anti-EPO or anti-EPOR markedly inhibits the differentiation of PM-NPCs towards the Pathology and Cellular Biology, GRSNC, CENUM, Université de Montréal, neuronal phenotype. Differently the exposure of regular NPCs to exogenous Montréal, QC, Canada, Université de Montréal, Montréal, QC, Canada, EPO increases their differentiation ability close to level of PM-NPCs. The Molecular Medicine, Université Laval, Québec, QC, Canada potential of PM-PCs in terms of replacement therapy was investigated in Recent work has shown that a subpopulation of ependymal cells in the adult a mouse model of spinal cord injury. 1x 106 of PKH 26 labelled PM-PCs, spinal cord, while normally quiescent, displays neural stem cell proper- kept from animal 6 hours after death (T6), were administered intravenously ties post-traumatic injury. This makes ependymal stem cells a potentially within 2 hours after the traumatic injury of the cord. The improvement of attractive cell population to target for promoting spinal cord repair. In the animal functional recovery and the transplanted cells fate were studied. present study, our goal is to establish under what circumstances, and in 30 days after transplantation animals treated with T6 PM-NPCs show a response to what types of spinal cord pathologies, ependymal stem cells remarkable improvement of the rate of hind limb function evaluated by become activated. To address this question, we have studied the temporal Basso Mouse Scale compared with animals treated with placebo. PM-NPCs and spatial parameters of ependymal stem cell activation in response to migrate predominantly at the injury site, survive and differentiate predomi- three models of spinal cord pathologies: i) Spinal cord contusion injury. Here nantly into cholinergic neurons, reconstitute a rich axonal and dendritic we are using the Infinite Horizon Impactor on minimal setting to preserve network and promote a marked axonal regeneration across the injury site the central canal. This injury model is clinically the most relevant model of of monoaminergic fibers. Moreover the molecular analysis of the lesion site traumatic injury. ii) EAE, the multi-focal demyelinating model of Multiple shows that PM-NPCs induce a remodulation of inflammatory response and Sclerosis. This produces autoimmune-directed demyelination in the CNS of release of neurotrophic factors. Pro-inflammatory cytokines (IL-6, MIP-2 affected mice and undergoes both demyelinating and remyelinating phases. and TNF-alpha) levels significantly decrease after 48 hours from spinal cord iii) LPC-induced focal demyelination, which results in chemically induced injury and PM-NPCs transplantation, while after 7 days we observe a small demyelination of local axons. This model is used as an intermediate between increase of IL-6 and TNF alpha. In conclusion, we purified a new class of contusion injuries and EAE in order to draw better conclusions regarding the neural precursors able to survive after a powerful ischemic insult (PM-NPCs). two main different types of injuries (traumatic vs non-traumatic). Preliminary Their neuronal differentiation requires the activity of mTOR and MAPK and results reveal that these three experimental models display substantial differ- is prevented by exposure to anti-EPO and anti-EPOR antibodies. Moreover, ences in overall patterns of ependymal stem cell activation. The magnitude, these cells represent a liable source for cellular therapy in neurodegenerative spatial extent, and kinetics of stem cell proliferation as well as expression of disorders, especially on spinal cord injury. stem cell markers appear to be regulated according to an injury-type specific manner. Further, they suggest that factors such as central canal disruption, Poster Board Number: 3239 demyelination, and inflammation are likely to be key determinants of the ENHANCING NEUROREPAIR MECHANISMS: acquisition/expression of neural stem cell properties by ependymal cells. In ongoing experiments, we are further defining the molecular mechanisms CLINICALLY RELEVANT ELECTRIC FIELDS involved in the process of ependymal stem cell activation during spinal cord PROMOTE ADULT MOUSE NEURAL pathologies. Eventually, modulation of these mechanisms may enable us to harness endogenous ependymal stem cells to promote spinal cord repair, PRECURSOR CELL MIGRATION either by enhancing tissue preservation or by replacing damaged spinal cord Babona-Pilipos, Rob, Droujinine, Ilia, Popovic, Milos R., Morshead, cell types. Cindi M. Poster Board Number: 3237 Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada, University of Waterloo, Waterloo, ON, Canada, ADULT MOUSE POST MORTEM NEURAL Department of Surgery, University of Toronto, Toronto, ON, Canada PRECURSORS REGENERATE NEURONAL TISSUE Recent studies have shown that neural explants from the embryonic brain AND PROMOTE FUNCTIONAL RECOVERY contain a population of cells that undergo directed migration in response to physiologically relevant electric fields (EFs). The adult mammalian brain AFTER TRANSPLANTATION IN A SPINAL CORD contains a population of neural precursor cells (NPCs) that have been INJURY MODEL shown to migrate to sites of injury and differentiate into neural cell types, albeit with limited efficacy. The ability to direct and enhance the migratory Carelli, Stephana, Merli, Devide, Marfia, Giovanni, Raspa, Andrea, capacity of endogenous NPCs would undoubtedly have profound impact on Messaggio, Fanuel, Madaschi, Laura, Di Giulio, Anna Maria, Gorio, tissue regeneration. We hypothesized that directed migration was a general Alfredo feature of precursor cells and herein report the effects of externally applied Medicine, Surgery and Dentistry, University of Milan, Milan, Italy EFs on pure populations of adult derived NPCs. NPCs isolated from the adult mouse brain were plated on specially designed chambers and placed within Neural stem cells from the subventricular zone of the forebrain, because of an externally applied EF (250mV/mm). Undifferentiated NPCs exhibited their proliferation and differentiation characteristics, are a good tool for tis- electrically-induced directed migration - a phenomenon called galvanotaxis - sue replacement therapies. We recently isolated a subclass of neural progeni- toward the cathodal terminal of the chamber at a rate of 1.09 +/- 0.15 μm/ tors, capable of surviving a powerful ischemia insult: these cells were named min, with a mean directedness of 0.96 +/- 0.01, and importantly, only for as

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long as the EF was present. This phenomenon was shown to be galvanotac- Poster Board Number: 3243 tic, and not chemotactic, as the migratory behavior of the NPCs persisted in the presence of continuous media cross-perfusion, which was introduced to SIGNALLING MECHANISMS UNDERLYING eradicate any chemical gradient artifacts that may arise within the galvano- NEUROTROPHIC ACTIVITY OF RAT taxis chambers. In contrast, NPCs plated in conditions that promote differen- tiation into mature neural cells exhibited smaller overall displacements in the MESENCHYMAL STEM CELLS EF, but more pronounced morphological changes as they extended processes Tse, Kai-Hei, Novikov, Lev N., Wiberg, Mikael2, Kingham, Paul J.1 that aligned perpendicular to the direction of the EF. Early investigations us- Integrative Medical Biology, Umeå University, Umeå, Sweden, Integrative ing the ependymal growth factor receptor (EGFR) blocker Erlotinib suggest Medical Biology & Surgical and Perioperative Sciences, Umeå University, EGFR plays a role in NPC galvanotaxis; NPCs exposed to Erlotinib exhibit Umeå, Sweden a significant reduction in velocity and directedness of migration. This data suggests that neural precursor cells are amenable to galvanotaxis and this Mesenchymal stem cells (MSCs) derived from adipose tissue (ADSC) and may potentially be utilized for directed cell therapy. Ex vivo brain slice stud- bone marrow (BMSC) possess potent neurotrophic potential to mediate ies are currently underway to investigate NPC migration in an environment axon regeneration. However, the mechanisms responsible for the transcrip- that provides the cellular milieu and better mimics the 3D construct of neural tion, translation and secretion of neurotrophins from stem cells have not tissue been examined in detail. Previously we showed that over a period of two weeks, ADSC and BMSC treated with a mixture of glial growth factors, up- Poster Board Number: 3241 regulated expression of glial cell markers including GFAP and S100. These THE INFLUENCE OF CHEMOKINES ON THE cells when co-cultured with neurons also significantly enhanced neurite outgrowth compared with untreated stem cells. In this current study we DIFFERENTIATION OF NEURAL PROGENITOR have investigated if similar neurotrophic activities could be rapidly induced CELLS FROM THE YOUNG ADULT AND AGED in MSCs thereby eliminating the need for long term treatment with glial growth factors. Furthermore we have begun to elucidate the signalling RAT BRAIN pathways underlying these effects. MSCs were isolated from rat visceral fat Gordon, Renee J., Mehrabi, Nasim, Maucksch, Christof, Connor, (ADSC) and bone marrow (BMSC). Both cell types were characterised as Bronwen CD14-, CD45-, CD54+, fibronectin+, collagen I+, integrinβ 1+. The stem cells were directly stimulated with glial growth factors for 24h and then the Pharmacology, University of Auckland, Auckland, New Zealand conditioned media were added to cultures of either primary rat dorsal root We previously examined whether chemoattractant cytokines (chemokines) ganglia neurons or the motor neuron-like NG108-15 cell line. Stimulated are involved in regulating the migration of subventricular (SVZ)-derived MSCs evoked greater neurite outgrowth than untreated cells and these adult neural progenitor cells to areas of cell loss in the adult rat brain. This effects were similar in magnitude to MSCs which had undergone the two research provided unique evidence demonstrating a role for the chemokines week treatment protocol. Having established that neurotrophic activity could MCP-1, MIP-1α and GRO-α in directing adult SVZ-derived neural progeni- be rapidly induced in MSCs, we assessed the contents of the conditioned tor cell migration towards the site of cell death. Prolonging the expression media using ELISA. Both ADSC and BMSC showed enhanced levels of brain of the chemokines MCP-1, MIP-1α and GRO-α may provide a strategy by derived neurotrophic factor (BDNF) protein following 24h stimulation. We which to increase the extent of progenitor cell recruitment from the SVZ also determined the effects of stimulation on Bdnf gene expression. Numer- into the damaged striatum. However the influence of MCP-1, MIP-1α and ous different Bdnf transcripts have been reported, arising as the result of GRO-α on the differentiation of adult neural progenitor cells has not previ- multiple promoter and alternative splicing activities. The common protein ously been investigated. Furthermore, as the majority of neurological disor- coding exon IX was up-regulated in MSCs treated with glial growth factors. ders and injuries occur in the aging brain, it is important to also investigate Furthermore, exon IV, which plays a major role in neuronal activity depen- the effect of chemokines on adult neural progenitor cell cultures obtained dent Bdnf expression in the CNS, was found to be rapidly up-regulated with from the aged brain. This study therefore examined the effect of chemok- stimulation. Since the cyclic-AMP responsive element binding (CREB) protein ines on neural progenitor cell differentiation, and assessed whether progeni- is associated with activation of exon IV expression, we investigated whether tor cells from the aged rat brain (2 months old) follow the same differential phosphorylation of CREB occurred in MSCs exposed to glial growth factors. profile as neural progenitor cells obtained from the young adult rat brain Western blotting and immunocytochemistry showed increased pCREB- (13 months old). Neural progenitor cells were isolated from the SVZ region ser133 in both 24h and two week treated MSCs. By using small molecule of the brain and cultured in vitro for 14-21 days to generate neurospheres. inhibitors against protein kinase A and calmodulin dependent protein kinase Neurospheres were replated in the presence of one of the chemokine pro- we are now investigating the association between CREB regulated path- teins MCP-1, MIP-1 α or GRO-α and allowed to differentiate into mature ways and BDNF expression in MSCs and their overall role for MSCs evoked neural cells over a 3 week period. Following this time we used immunocy- neurite outgrowth. Uncovering these molecular mechanisms in MSCs could tochemistry to identify whether the progenitor cells had differentiated into establish whether the neurotrophic potential of MSCs gives rise to physi- neurons. We found that MIP-1α did not influence neuronal differentiation ological neuron-glia interactions or is merely an in vitro artefact of stress of cultured progenitor cells from either the young adult or aged rat brain. as suggested by some researchers. Furthermore, by understanding the Conversely, GRO-α was found to significantly increase the number of MAP- regulation of neurotrophic factor expression of MSCs, we hope to identify a 2+ neurons in a concentration dependent manner in both young adult and clinically available agent to augment and sustain the neurotrophic effects of aged cultures. Interestingly MCP-1 had no effect on neuronal differentiation MSCs for treatment of nerve injuries. of young adult neural progenitor cells, but increased the number of MAP-2+ neurons in aged progenitor cell cultures. The results from this project further extend our knowledge regarding the role chemokines play in the regulation of adult neurogenesis and provide novel data regarding the response of ag- ing adult neural progenitor cell to micro-environmental cues.

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Poster Board Number: 3245 potential of these cells to participate in various aspects of the regenerative process has been demonstrated broadly; however, organ association of THE MECHANICAL COUPLING OF RAT ADULT secretory and developmental markers to specific peri-organ adipose niches MARROW STROMAL STEM CELLS DURING has not been investigated. To characterize this topographical association, we explored the potential of cells isolated from the stromal vascular fraction CARDIAC REGENERATION EXPLORED IN A 2-D (SVF) of renal-derived adipose to express key renal associated factors. We CO-CULTURE MODEL report that renal adipose SVF derived cells demonstrate hypoxia regulated expression of EPO transcript/protein and VEGF transcripts. Using isoelectric Valarmathi, Mani T., Fuseler, John W., Goodwin, Richard L., Davis, focusing, we demonstrate that renal and non-renal adipose show unique Jeffrey M., Potts, Jay D. patterns of EPO protein post-translational modification, consistent with the Cell Biology and Anatomy, University of South Carolina, Columbia, SC, idea that renal and non-renal sources represent functionally distinct adipose USA depots. In addition, renal adipose-SVF cells specifically express the key renal developmental transcription factors WT1 & PAX2 and are associated with Cardiovascular disease is a leading cause of significant morbidity and mortal- zonally compartmentalized localization of the stem cell related markers ity globally. Postnatal cardiomyocytes undergo terminal differentiation and a CD146, CD117, CD24, c-myc, PODXL and SNAIL2. Taken together, these restricted number of human cardiomyocytes retain the ability to divide and data are consistent with the idea that renal adipose derived cells may be regenerate in response to ischemic injury. However, whether these neo- primed to recreate a regenerative micro-environment within the kidney. cardiomyocytes are derived from endogenous population of resident cardiac These findings open the possibility of isolating solid-organ associated adi- stem cells or from the exogenous double assurance population of resident / pose derived cell populations for therapeutic applications in organ-specific or circulating bone marrow-derived stem cells that populate the damaged regenerative medicine products. myocardium is unresolved and under intense investigation. The vital chal- lenge is to ameliorate and/or regenerate the damaged myocardium. This can Poster Board Number: 3249 be achieved by stimulating proliferation of native quiescent cardiomyocytes and/or cardiac stem cell, or by recruiting exogenous autologous or alloge- A SMALL MOLECULE WNT INHIBITOR neic cells such as fetal or embryonic cardiomyocyte progenitors (ECMs) or INCREASES ENGAFTMENT AND INHIBITS bone marrow-derived stromal stem cells (BMSCs). The prerequisites are that these neo-cardiomyocytes must have the ability to integrate well within the LINEAGE COMMITTMENT OF MESENCHYMAL native myocardium and must exhibit functional synchronization. Adult BM- STEM CELLS SCs have been shown to differentiate into cardiomyocyte-like cells both in vitro and in vivo. As a result, BMSCs may potentially play an essential role in Young, Pampee P., Saraswati, Sarika cardiac repair and regeneration, but this concept requires further validation. Pathology, Vanderbilt University Medical Center, Nashville, TN, USA In this report, we have provided compelling evidence that functioning car- Wnt signaling plays an important role in stem cell maintenance and self diac tissue can be generated by the interaction of multipotent BMSCs with renewal. Pyrvinium is a FDA-approved drug identified as a Wnt inhibitor ECMs in two-dimensional (2-D) co-cultures. The differentiating BMSCs were in a chemical screen for small molecules that stabilize β-catenin and inhibit induced to undergo cardiomyogenic differentiation pathway and were able Axin degradation by activating casein kinase. Studies performed in our to express unequivocal electromechanical coupling and functional synchro- laboratory identified that temporary inhibition of Wnt signaling through a nization with ECMs. Moreover, this is the first ever documentation of intra small molecule Wnt inhibitor, pyrvinium, induces cellular proliferation within cellular calcium oscillations in the case of co-differentiating adult stem cell, wound tissue and reduces adverse cardiac remodeling in a myocardial infarc- BMSCs, mechanically coupled to ECMs. The remodeling and reorganization tion mouse model by direct affects on host tissue. Recently, we have also of cytoarchitectural features of both BMSCs and ECMs in this co-culture shown that pyrvinium can inhibit the Wnt pathway in mesenchymal stem condition suggest that both BMSCs and ECMs have the plasticity to repro- cells (MSCs) in vitro. In the present study we have evaluated pyrvinium’s ef- gramme in synchrony towards cardiomyogenic lineage and commitment fect on the clinical efficacy of MSC therapy. Thus far, Wnt inhibition has not and, essentially validate our previous findings observed in our 3-D co-culture been used as a therapeutic strategy to enhance stem cell function in vivo. system. This phenomenon of partial dedifferentiation followed by rediffer- Using an in vivo model of granulation tissue formation, we used a quantita- entiation of in vitro cardiac myocytes in the vicinity of adult bone marrow- tive assay to demonstrate that pyrvinium enhanced long-term MSC engraft- derived stem cells may probably explain most of the discrepancies seen in ment. These results were supported by pyrvinium mediated increase in MSC the case of in vivo preclinical and clinical trails. Finally, our 2-D co-culture proliferation in vitro. Pyrvinium also inhibited osteogenic and chondrogenic system provides a useful in vitro model and a prospect to elucidate various lineage commitment of MSCs by directly inhibiting β-catenin. Although molecular mechanisms underpinning the integration and orderly maturation MSCs are a promising target for cell therapy, there is an ultimate need to and differentiation of BMSCs into neo-cardiomyocytes during myocardial develop strategies to enhance their survival and maintain their stemcellness repair and regeneration. in the wounded area. Priming MSCs with a small molecule Wnt inhibitor Poster Board Number: 3247 augments MSC engraftment and delays their lineage commitment in vivo, leading to greater wound granulation tissue with less ectopic differentiation. ORGAN ASSOCIATED DEVELOPMENTAL This study highlights the potential of an effective Wnt inhibitor to enhance MARKERS IN PERI-ORGAN ADIPOSE: KIDNEY the therapeutic efficacy of stem cells in wound repair and regeneration. Basu, Joydeep, Genheimer, Christopher, Sangha, Namrata, Guthrie, Kelly, Kelley, Rusty, Ilagan, Roger, Spencer, Thomas, Presnell, Sharon, Bertram, Tim, Jain, Deepak, Ludlow, John W. Tengion Inc., Winston-salem, NC, USA Adult stem and progenitor cells are currently understood to promote regen- eration by various mechanisms such as release of cytokines and site specific engraftment and directed differentiation. Constitutive cellular popula- tions undoubtedly participate in the regenerative process. Adipose tissue represents a source of both progenitor and committed cell populations. The

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Poster Board Number: 3251 Poster Board Number: 3253 FIBRO OF THE FIBRE - THE CRITICAL ROLE OF REGENERATION OF THE ADULT ZEBRAFISH FIBRO-ADIPOGENIC PROGENITORS IN MUSCLE BRAIN AFTER TRAUMATIC LESION: REGENERATION AND MYOPATHIC FIBROSIS NEUROGENIC RADIAL GLIA-TYPE Zhang, Regan-Heng, Natarajan, Anuradha, So, Leslie, Rossi, Fabio PROGENITORS MAKE NEW NEURONS M. V. Brand, Michael, Kroehne, Volker, Freudenreich, Dorian, Hans, The Biomedical Research Centre, The University of British Columbia, Stefan, Kaslin, Jan Vancouver, BC, Canada BIOTEC - CRTD, TU Dresden, Dresden, Germany Adult mammalian skeletal muscle has a great capacity to grow and regener- Severe traumatic injury to the adult mammalian central nervous system ate after damage or exercise, which is a promising property in developing (CNS) leads to life-long loss of function and neuronal regeneration is not treatments for muscular dystrophies. However, cell therapy using myogenic occurring. In contrast, several non-mammalian vertebrate species, includ- progenitors (MP), namely satellite cells, is challenging. Previously, our group ing adult zebrafish, have a remarkable ability to regenerate injured organs, has identified another muscle-resident population of progenitor cells, the including the CNS. However, the cellular and molecular mechanisms that fibro-adipogenic progenitors (FAP), which have been shown to possess enable or prevent CNS regeneration are largely unknown. To study brain fibrogenic and adipogenic potentials, but are not myogenic. These cells regeneration mechanisms in adult zebrafish, we developed a traumatic le- respond rapidly to tissue damage by proliferation, but do not differentiate sion assay, analyzed cellular reactions to injury and show that adult zebrafish in normal physiology due to inhibition by satellite cells undergoing myo- can efficiently regenerate brain lesions and lack permanent glial scarring. genesis. In return, FAPs have been suggested to produce signals that induce Using Cre-loxP-based genetic lineage tracing, we demonstrate that her4.1- primary myoblast differentiation and serve an intercellular regulatory role positive ventricular radial glial progenitor cells react to injury, proliferate and to myogenesis. On the other hand, there has been substantial fatty fibrotic generate neuroblasts that migrate to the lesion site. The newly generated infiltrate observed in many myopathies. Therefore, we hypothesized that, neurons survive for at least 3 months, are decorated with synaptic contacts 1) a balance between the FAP population and the MP population is crucial and express mature neuronal markers. Thus, regeneration after traumatic le- for normal muscle regeneration, and 2) FAPs are the main source of fibrosis sion of the adult zebrafish brain occurs efficiently from radial glia-type stem/ and adipogenesis in diseased muscle, when they actually differentiate. Our progenitor cells. By combining the brain lesion assay with FACS-sorting of finding of a growth regulatory gene, hypermethylated in cancer 1 (HIC1), to neural stem cells and genome-wide analysis, we study the molecular mecha- be specifically expressed in FAPs in muscle, is key to elucidating the mecha- nisms controlling the ability to regenerate neurons and glia. nisms underlying the FAP’s behaviour in efficient muscle regeneration. HIC1 has been reported in previous expression profile studies as a potential gene Poster Board Number: 3255 for cell quiescence. Knowing the dynamics of FAPs after tissue damage, which undergo an initial proliferation phase and a subsequent decrease in THE ROLE OF THE RHOA/RHO KINASE survival, we find that the gene expression pattern of HIC1 is in close resem- SIGNALING PATHWAY IN GLIAL blance to those dynamics. We aim to delineate the role of HIC1-regulated FAP in fibrosis and regeneration by cell tracing and ablation of FAPs in an DIFFERENTIATION OF MESOANGIOBLASTS acute damage model in vivo. First, we have developed a simple technique to Chun, Ju Lan, Wang, Lei, Kamath, Anant, Frye, Janie, Berry, specifically induce gene expression in the FAP population in vivo. Adeno- Suzanne E. associated virus serotype 5 (AAV5) showed infective specificity to FAPs, Univeristy of Illinois at Urbana-Champaign, Urbana, IL, USA, Department through its transduction receptor, platelet-derived growth factor receptor of Comparative Biosciences, University of Illinois at Urbana-Champaign, α (PDGFRα), which is expressed only by FAPs in damaged muscle. Since Urbana, IL, USA, Chief of Operating Officer, Cellular Engineering genetic knockouts of PDGFRα animals are not viable, we use this method Technologies, Coralville, IA, USA, Department of Pathology, University of to deliver Cre and drive the expression of a diphtheria toxin receptor for Illinois at Urbana-Champaign, Urbana, IL, USA cell ablation, a YFP reporter for cell tracing, and deletion of the HIC1 gene. The strength of FAP targeting is shown in vivo by the ability of AAV5-Cre Duchenne muscular dystrophy (DMD) is a lethal muscle wasting disease, to induce 35% of FAPs in the tibialis anterior muscle of adult mouse, with affecting both skeletal and cardiac muscle. Aorta-derived mesoangioblasts minimal effects on the MP population. This is confirmed in vitro with higher (ADM) are myogenic and regenerate skeletal muscle in animal models of efficiency. Secondly, we have established another FAP targeting system in DMD. In addition, ADM differentiate into Schwann cells in peripheral nerve which an inducible Cre construct is under the control of the endogenous bundles of dystrophic skeletal muscle. ADM are detected in approximately PDGFRα promoter. We are currently employing both of these methods to 51% of peripheral nerve bundles of injected muscle, and are linked to ablate this population in normal physiology, which is currently the best way a 1.7-fold increase in numbers of peripheral nerve bundles in stem cell- to understand the precise physiological role and mechanism of FAPs in the injected gastrocnemius in comparison to the contralateral gastrocnemius regenerative process. This cell type may be a crucial component for consid- injected with saline solution. Innervation of muscle is important for muscle eration in future designs of therapies for muscular dystrophies. Besides that, fiber health and survival. Increased regeneration of nerves in ADM-injected PDGFRα+ fibroblastic and adipogenic progenitors have also been found in dystrophic muscle may therefore lead indirectly to improved survival of other tissues, such as fat depots, lung, and heart; the full understanding of muscle fibers. This may, in part, be the mechanism by which ADM injection FAPs in muscle could address the relationship of fibrosis and tissue regenera- corresponds to a global decrease in degenerating muscle in skeletal muscle tion in other areas. of a mouse model for DMD. To test this, it is necessary to manipulate ADM differentiation into myelinating glial cells. To rule out the possibility that ADM simply fused with host Schwann cells rather than differentiating into the glial lineage, we have studied the glial potential of ADM in the brain, and also in vitro. Following injection into the brain, ADM migrate to regions of white matter and express markers of oligodendrocytes. In vitro, we have cultured mesoangioblasts with factors to stimulate or inhibit signal- ing pathways known to be involved in oligodendrocyte or Schwann cell

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Thursday Poster Abstracts differentiation. Partial inhibition of Rho kinase (ROCK) phosphorylation of Poster Board Number: 3259 myosin light chain, implicated in neural and glial differentiation of neural precursor cells from the CNS, promotes process extension and expression SUBSTANCE P STIMULATES TISSUE REPAIR of markers of oligodendrocyte precursor cells by ADM. We are currently ASSOCIATED WITH INDUCTION OF IL-10 AND disrupting and stimulating ROCK signaling in ADM to determine whether promoting or inhibiting differentiation into myelinating glial cells is important ACTIVATION OF M2 MACROPHAGE AFTER for ADM-mediated muscle regeneration and muscle fiber survival in animal SPINAL CORD INJURY models for DMD. Jiang, Mei Hua, Chi, Guang Fan, Ahn, Woosung, Lim, Ji Eun, Hong, Poster Board Number: 3257 Hyun Sook, Kim, Dae Wook, Choi, Hyeongwon, Kim, Jiyoung, THE FLATWORM MACROSTOMUM LIGNANO: Chung, Eunkyung, Son, Youngsook Genetic Engineering, Kyung Hee University, Yong In, Republic of Korea GENOMICS, TRANSGENICS AND NEOBLAST The neuropeptide substance P (SP) plays an important role in various MARKERS wound-healing processes. In this study, we explored the effects of exog- Simanov, Daniil, Arindrarto, Wibowo, de Mulder, Katrien, enous SP on early inflammatory responses and subsequent tissue repair after Demircan, Turan, van Zon, Patrick, Canela, Andres, Hannon, spinal cord injury (SCI) in rats. SP was injected intravenously immediately Gregory J., Vizoso, Dita B., Schärer, Lukas, Ladurner, Peter, and 24 and 48 hours after inducing spinal cord contusion. RT-PCR analysis, cytokine arrays, and immunohistochemistry revealed that this treatment Berezikov, Eugene markedly increased IL-10 expression, presumably in CD11b+ microglia or Hubrecht Institute, Utrecht, Netherlands, University Medical Center macrophages at the injury site. IL-6 and IL-10 levels 1 day after SCI were Utrecht, Utrecht, Netherlands, Cold Spring Harbor Laboratory, Cold Spring approximately 9 fold and 10 fold higher than those in controls, respectively. Harbor, NY, USA, University of Basel, Basel, Switzerland, University of At 5 days after SCI, tissue from the treated rats showed far fewer ED1-posi- Innsbruck, Innsbruck, Austria tive cells and a preponderance of the alternatively activated (M2) phenotype Macrostomum lignano is a free-living flatworm with high regeneration microglia or macrophages at the injury site, confirmed by induction of the capacity facilitated by neoblasts, the stem cell system of the worm. Due M2-specific markers arginase-1 and CD206 proteins and reduction of M1- to its small size, short generation time, amenability to genetic manipula- specific markers iNOS and CD86. Further, there was reduced activation of tion and easy maintenance in laboratory conditions, M. lignano is a potent caspase-3 and decreased TUNEL-positive neurons and oligodendrocytes at invertebrate experimental model for regeneration studies. Since M. lignano 1 and 5 days after the SCI, respectively. This early suppression of the pro- has entered the research landscape only recently, limited molecular biology inflammatory response and induction of anti-inflammatory cytokines and resources and tools are currently available for this emerging model organism. M2-phenotype macrophages facilitated neural outgrowth and tissue repair We are convinced, however, that with due technological developments, M. at 2 weeks after the SCI, resulting in improved Basso-Beattie-Bresnahan lignano could become the “C. elegans of stem cell research.” Towards this (BBB) locomotor open-field behavioral scores in the rats. These data indicate end we are sequencing and annotating the M. lignano genome and estab- that SP modulates the inflammatory responses to SCI, resulting in reduced lishing transgenesis, as well as forward and reverse genetics techniques. We apoptosis and improved tissue repair, possibly via an enhanced anti-inflam- are using hybrid 454 and Solexa/Illumina approach for sequencing the 600 matory response and mitigation of injury-mediated cell death. SP, therefore, Mb M. lignano genome. The draft genome assembly with N50 scaffold size is a potential anti-inflammatory modulator for treating inflammatory CNS of 14.5 kb and the de novo transcriptome assembly based on 454 sequenc- disorders. Acknowledgement: This work was supported by the future based ing are already publicly available at http://www.macgenome.org. Since technology development program of the National Research Foundation X-ray irradiation is known to specifically deplete neoblast populations in (NRF) (2010-0020405) awarded to Professor Youngsook Son. flatworms, we utilized an RNA-seq approach to compare transcriptomes of Poster Board Number: 3261 irradiated (stem cell free) and non-irradiated control worms. Using de novo transcriptome assembly as a reference, we identified more than 100 candi- SIGNIFICANCE OF REMYELINATION BY date transcripts significantly depleted in irradiated worms, and confirmed by in situ hybridization the neoblast-specific expression for several candidates, GRAFTED NEURAL STEM/PROGENITOR CELLS including a putative HMG box gene. The property of M. lignano to produce INTO THE INJURED SPINAL CORD substantial amounts of embryos on a daily basis helped us to obtained Yasuda, Akimasa, Tsuji, Osahiko, Shibata, Shinsuke, Nori, Satoshi, proof-of-principle for generating transgenic animals, and currently we have several stable GFP-expressing transgenic lines obtained by simple injection Kobayashi, Yoshiomi, Takano, Morito, Takahashi, Yuichiro, Fujiyoshi, of DNA constructs into single cell stage embryos. Of particular interest, Kanehiro, Toyama, Yoshiaki, Okano, Hideyuki, Nakamura, Masaya the HUB1 line with GFP under promoter of the elongation factor alpha has Department of Orthopaedics, Keio University, Tokyo, Japan, Department of ubiquitous expression, including neoblasts, which can be used to perform Physiology, Keio University, Tokyo, Japan neoblast transplantation and lineage tracing experiments. The experimen- Purpose: Previous studies demonstrated that delayed transplantation of tal potential of M. lignano as an invertebrate model for stem cell research neural stem/progenitor cells (NS/PCs) into the injured spinal cord promoted and the progress on the development of genomic and genetic tools for this functional recovery in rodents and non-human primates. However, the model organism will be presented. mechanism of functional recovery has remained unclear. There are several possible explanations for the observed functional improvement as follows: 1) synapse formation by graft-derived neurons, 2) re-myelination by graft-de- rived oligodendrocytes, 3) trophic effects. In the present study, we focused on re-myelination by grafted NS/PCs-derived oligodendrocytes to elucidate the mechanism of functional recovery after NS/PCs- transplantation. Meth- ods: NS/PCs were obtained from striatum of E14.5 myelin-deficient shiverer mutant mice embryos (shi-NS/PCs) and wild type mice (wt-NS/PCs), and in vitro proliferation assay as well as differentiation assay was performed. shi- NS/PCs or wt-NS/PCs were grafted into the injured spinal cord of wild-type

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mice 9 days after injury and motor function was evaluated by BMS scoring scales for 7 weeks. Survival rate of transplanted NS/PCs was also evalu- REPROGRAMMING ated by bioluminescence imaging (BLI) followed by electrophysiological and Poster Board Number: 3265 immunohistological analyses. Results: shi-NS/PCs differentiated into Tuj1+ neurons, GFAP+ astrocytes, and CNPase+ oligodendrocytes, but not into AN INTEGRATION FREE REPROGRAMMING MBP+ oligodendrocytes in vitro. Proliferation analysis revealed that there was no significant difference in the doubling time between shi-NS/PCs and APPROACH USING HERPES SIMPLEX VIRUS wt-NS/PCs. Both grafted NS/PCs showed similar survival, which was con- (HSV)-1 AMPLICON SYSTEM firmed by BLI analysis. Consistent with in vitro differentiation assay, shi-NS/ PCs differentiated into Hu+ neurons, GFAP+ astrocytes, and APC+ oligoden- Khoja, Suhail, Moralli, Daniela, Mandegar, Mohammad A., Cowley, drocytes but not into MBP+ oligodendrocytes in vivo. ImmuneImmuno-EM Sally, Monaco, Zoia L. analysis showed that shi-NS/PC-derived oligodendrocytes could form myelin The Wellcome Trust Centre for Human Genetics, University of Oxford, sheathes, which were much thinner than those of wt-NS/PC-derived ones. Oxford, United Kingdom, Sir William Dunn School of Pathology, University Compared to wt-NS/PCs, shi-NS/PCs showed less therapeutic potential for of Oxford, Oxford, United Kingdom SCI in terms of electrophysiological and motor function recovery. Conclu- Current methods to generate human induced pluripotent stem cells (hiPS) sion: Re-myelination is one of the main mechanisms of the locomotor suffer from either integration in the genome or have limited reprogram- functional recovery after NS/PCs-transplantation to SCI. ming efficiencies, and in some cases rely on multiple rounds of treatment to Poster Board Number: 3263 avoid rapid depletion of reprogramming factors. Here, we present a safe and integration-free method to generate hiPS cells using Herpes Simplex Virus MECHANISTIC STUDY OF THE GLYCOME (HSV)-1 amplicon system that requires a single transduction. The HSV-1 DURING VASCULAR REGENERATION amplicon system not only allows infecting a wide range of mammalian cell types including dividing and non-dividing cells, and avoids cytotoxic effects, Harfouche, Rania, Dadwal, Ushashi, Kopparam, Jawahar, Basu, but also enables efficient delivery of large DNA fragments (~152kb) into Sudipta, Sengutpa, Shiladitya cells distinguishing it from other DNA delivery systems. The head-to-tail, Harvard- MIT Division of Health Sciences and Technology/Brigham and rolling-circle DNA replication mechanism of HSV-1 also allows amplification Women’s Hospital, Cambridge, MA, USA of amplicon plasmid as concatemers leading to increased copy number of re- programming factors and consequently their expression. We have used the Impaired vascularization underlies multiple pathologies, including ischemic HSV-1 amplicon system to successfully reprogram human embryonic fibro- heart disease (IHD), which remains one of the main killers in industrialized blasts, differentiated from HUES-10 cells, to iPS cells. The oriP and EBNA1 countries, affecting 7 million Americans per year. The new paradigm to treat genes were introduced to the HSV-1 amplicon genome to prevent its dilu- IHD entails therapeutic neovascularization, whether by angiogenesis or tion in proliferating cells thus allowing maintenance and segregation of the vasculogenesis. Although multiple factors have been implicated in regulat- amplicon genome as episome. The episome can subsequently be removed ing these tightly controlled processes, limited success has been achieved in through subcloning and screening to derive iPS cells that are completely clinics, creating an unmet need for innovative revascularization approaches. free of vector and transgenes. Overall, the HSV-1 amplicon system provides An emerging concept towards this end is to harness the differentiation an easy way for cellular reprogramming and generation of integration-free potential of embryonic stem cells (ES) to form new blood vessels. Although hiPS cells, and is an important advance towards drug discovery and patient- several clinical trials have been established towards that end, limited success specific cell therapy. has been achieved, mainly due to low yield of functional endothelium. A reason for this lacuna remains an incomplete understanding of the underly- Poster Board Number: 3267 ing mechanisms that direct ES fate cells into vasculature. Indeed, most areas of research focus on the roles of growth factors and cytokines in mediating ENHANCED NEURONAL DIFFERENTIATION these processes. We previously demonstrated that novel mechanisms direct BY EXOGENOUS EXPRESSION OF SPECIFIC ES fate into vasculature, namely the sulfated glycosaminoglycans (HSGAGs) TRANSCRIPTION FACTORS niche. Glycosylation is one of the most common post-translational modifica- tions in eukaryotes, affecting more than half of all known proteins as well as Hart, Ronald P., Ricupero, Christopher L., D’Ecclessis, Michael, many lipids. These modifications are imparted by selective classes of glyco- Toro-Ramos, Alana J., Gupta, Bhavik, Nazar, Ely, Plummer, Mark, synthetic enzymes present at the Golgi apparatus, the rate-limiting of which Grumet, Martin, Moore, Jennifer C. is N-deacetylase/N-sulfotransferase (NDST). We previously demonstrated Rutgers University, Piscataway, NJ, USA for the first time that knockdown of this enzyme inhibits vessel formation in both ES and Zebrafish developmental models, and this effect is rescued To produce defined types of differentiated neurons, we transfected plasmids by adding exogenous HSGAGs. However, our preliminary studies indicate expressing specific transcription factors associated with a desired neuronal that more subtle sulfation permutations significantly impact vasculogenesis, phenotype into rodent fetal precursor cell clones or human embryonic stem indicating that a more detailed understanding of the structure-function rela- cell (hESC)-derived neural stem cells (NSC). Similar to previous studies tionship of HSGAG during vascularization would uncover novel mechanisms demonstrating the reprogramming of rodent non-neuronal cells, transfection to harness in order to direct vascularization de novo. As such, we will focus of only an Ascl1 (MASH1)-expressing plasmid into human NSC increased on sulfotransferases operating after NDST1, namely the 2O-, 3O- and 6-O the number of TuJ1-positive cells in the absence of neuronal differentia- sulfotransferases (OSTs). Using gene expression analysis, we will characterize tion medium. In previous studies, we identified specific members of the Dlx their transcriptomal regulation of OSTs during ES differentiation and dissect homeobox transcription factor family that were transiently induced upon their physiological roles in modulating differentiation using a reverse genetic differentiation of a rodent neural precursor clone producing electrically- approach, while investigating their downstream mechanisms of action dur- active, GABAergic inhibitory interneurons. Exogenous expression of these ing this process. We will use a novel high through-put model of Zebrafish Dlx factors along with Ascl1 altered the phenotype of a different clone of vascular regeneration to confirm the roles of OSTs in vivo. Our long-term precursors that normally produces a mixture of cell types. This transfec- goal is to harness these novel mechanisms to engineer chemically-defined tion increased the numbers of TuJ1-positive cells. Many transfected cells bio-matrices for the directed differentiation of ES into functional vascula- exhibited distinctive neuronal features including an extensive dendritic arbor, tures, which can then be translated into clinical settings for IHD. expression of glutamatergic and GABAergic markers, and more mature

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Thursday Poster Abstracts electrophysiological characteristics as compared to cells transfected with a ile, closed environment. Results show that laser-mediated colony purifica- control plasmid. Exogenous transcription factors are expected to interact tion was used to efficiently derive human iPSC lines (LEAP lines) in 96-well with genomic regulatory sequences to shift patterns of expressed genes, plates. Selection of reprogrammed colonies was guided by brightfield imag- causing a stable change in phenotype. We believe that transient expres- ing and analysis of colony outgrowth over time, resulting in the efficient sion of specific transcription factors will produce stable cultures of distinct isolation of fully reprogrammed, expandable iPSC lines. Data show that this neuronal phenotype that may be useful therapeutically. selection approach is more effective and reproducible than current standard procedures at determining the “best” iPSC lines earlier in the reprogram- Poster Board Number: 3269 ming process. This reduces time and cost of iPSC generation, since less DIRECT CONVERSION OF PERICYTE-LIKE colonies need to be isolated in order to create the desired number of lines. After isolation, iPSC colonies were propagated by laser-mediated sectioning CELLS OF THE ADULT HUMAN BRAIN INTO in 96-well plates while undergoing characterization, again decreasing the FUNCTIONAL NEURONS time and cost needed to care for new iPSC lines. After full characterization, chosen iPSC lines were expanded into larger stocks. Importantly, all LEAP Berninger, Benedikt, Sánchez, Rodrigo, Schichor, Christian, iPSC lines created by laser-mediated derivation and propagation are free of Masserdotti, Giacomo, Lie, D. Chichung, Goldbrunner, Roland, manual and enzymatic disruption. These lines had a normal stem cell mor- Götz, Magdalena phology, expressed high levels of stem cell-associated genes and proteins, Physiological Genomics, Ludwig Maximilians University Munich, differentiated into cells from all three germ layers, and were genetically Munich, Germany, Tumor Biology Lab, Neurosurgical Clinic, Klinikum normal as analyzed by aCGH. While more efficient reprogramming protocols der Universität München, Munich, Germany, Institute of Developmental are still needed to make this process truly scalable, these data demonstrate Genetics, Research Group/Adult Neural Stem Cells and Neurogenesis, that iPSC lines could be generated in every well of a 96-well plate, therefore HelmholtzZentrum München, Munich, Germany, Center for Neurosurgery, enabling efficient, high-throughput iPSC generation. University Hospital of Cologne, Munich, Germany Poster Board Number: 3273 Reprogramming of somatic cells into neurons may provide a new approach towards cell-based therapy of neurodegenerative diseases. Previous stud- PURIFICATION OF HUMAN IPSC-DERIVED ies have shown that astroglia cultured from young postnatal mice can be ENDODERMAL AND RETINAL PROGENITORS directly converted into functional neurons by forced expression of single AND SPECIALIZED CELLS neurogenic transcription factors and the synergistic action of three transcrip- tion factors can induce neurogenesis even from embryonic tip-tail fibro- Hohenstein Elliott, Kristi A., Peterson, Cory, Winquist, Alicia M., blasts. However, major challenges lying ahead for the translation of direct Kan, Natalia, Mercola, Mark, Kamme, Fredrik neuronal reprogramming of somatic cells into therapy concern the question Cyntellect Inc., San Diego, CA, USA, Sanford-Burnham Medical Research whether it can be achieved from cells of adult tissues and of human origin. Institute, La Jolla, CA, USA This is of special importance for tissues difficult to access for transplanta- tion, such as the brain. Here we show that cells expressing pericyte markers, The use of human pluripotent SCs as a source of specialized cells for use such as PDGF receptor-β, from the adult cerebral cortex of mice and human in drug toxicity screening and drug discovery will require efficient differ- patients can be reprogrammed in vitro into βIII tubulin-positive neurons entiation protocols in combination with robust methods for purification of by retrovirus-mediated combined expression of the basic helix-loop-helix SC-derived cell types. Differentiated cells derived from human ESCs and transcription factor Mash1 (mammalian homologue of achaete-schute-1) iPSCs are notorious for growing as tight cell clusters and colonies, making and the SRY-related HMG box protein Sox2, but not by either factor traditional methods of cell purification, such as flow sorting and magnetic alone. These neurons exhibit electrical properties of true neurons such as bead separation, challenging. We have developed a novel approach for au- tetrodotoxin-sensitive action potential firing. Our results indicate that neu- tomated in situ isolation of cell clusters and colonies from surrounding, con- ronal reprogramming is not restricted to early postnatal stages, but can be taminating cells on the LEAPTM Cell Processing Workstation and used it to achieved from somatic cells of adult brain tissue including of human origin. purify SC-derived progenitors and specialized cells. The LEAP uses brightfield Moreover, the fact that pericyte-like cells can be successfully converted into (BF) and fluorescent (FL) imaging combined with image analysis to target neurons may provide a basis for direct reprogramming of cells endogenous colonies for laser-mediated isolation within a sterile, closed environment. to the brain as a viable alternative to transplantation for cell-based therapies Importantly, cells are purified directly within multi-well plates (384-well to of neurodegenerative diseases. 6-well), enabling iterative elimination of immature cells in a step-wise man- ner during differentiation. To demonstrate the utility of LEAP for purification Poster Board Number: 3271 of cell clusters, we used live FL imaging in combination with laser-mediated colony isolation to enrich hESC-derived cardiomyocytes and hiPSC-derived EFFICIENT, HIGH-THROUGHPUT HUMAN IPS pancreatic progenitors to ~100% purity with high viability. In addition laser- CELL DERIVATION mediated colony isolation was used to purify a variety of differentiating cells derived from hiPSCs based on BF imaging alone, including neural rosettes, Hohenstein Elliott, Kristi A., Peterson, Cory, Winquist, Alicia M., retinal pigment epithelial (RPE) cells and hepatocyte-like cells. These data Kamme, Fredrik demonstrate a novel approach for the step-wise purification and isolation of Cyntellect Inc., San Diego, CA, USA SC-derived cell types, including pancreatic and neural progenitors, cardio- Human iPSC lines derived from patient-specific and disease-specific cells are myocytes, hepatocyte-like cells and RPE cells, providing a robust method for currently used as tools to study reprogramming and differentiation pathways the production of specialized cells in multi-well plates for drug discovery. and as models of human disease. These studies have generated interest in large-scale derivation of iPSC lines that may be utilized in drug screening and regenerative medicine. We have developed innovative methodology for automated isolation of individual stem cell colonies and enzyme-free, high- throughput propagation of stem cell lines using the LEAPTM Cell Processing Workstation, thus allowing hundreds of iPSC lines to be derived rapidly. The LEAP uses brightfield and/or fluorescent imaging, combined with image analysis, to target specific colonies for laser-mediated isolation within a ster-

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Poster Board Number: 3275 Our results suggest that there is a previously unappreciated barrier to suc- cessful human nuclear transfer, and that future studies should focus on the IDENTIFICATION OF NANOG2, A NOVEL requirements for somatic genome activation. TRANSCRIPT IN PLURIPOTENT HUMAN STEM Poster Board Number: 3279 CELL POPULATIONS A VALIDATED MRNA REPROGRAMMING Eberle, Irina, Henschler, Reinhard, Cantz, Tobias PROTOCOL FOR THE REPRODUCIBLE Stem Cell Biology, DRK-Institute of Transfusion Medicine, Frankfurt (Main), Germany, Stem Cell Biology, Medical School Hannover, Hannover, GENERATION OF INTEGRATION-FREE HUMAN Germany IPS CELLS The transcription factor NANOG is one of the core components within the Luo, Chenmei, Mahon, Kerry, Yi, Kevin, Zhai, Shuya, Cianci, Amelia, transcriptional network of pluripotent stem cells such as embryonic stem Hamilton, Brad cells (ESCs) and induced pluripotent stem cells (iPSCs). Recent studies Stemgent, Cambridge, MA, USA revealed the existence of various NANOG pseudogenes (NANOGP2-P11) and, more importantly, of a gene duplicate termed as NANOG2. The latter To date, the broad implementation of induced pluripotent stem (iPS) cells was initially described as NANOG pseudogene 1 (NANOGP1), before it in regenerative medicine and drug screening applications has been limited became evident that NANOG2 is a real gene with an own promoter and by the inability to efficiently derive iPS cell lines that are free from genomic regulatory elements. When we investigated several NANOG transcripts in integration. Past publications have highlighted the use of reprogramming human carcinoma cells, hematopoietic stem cells and leukemia cells, the technologies that are: 1) directly integrating into the target cell genome, detected NANOG transcripts were identified as NANOG2. Moreover a 2) directly integrating into the target cell genome, but can be excised, 3) 5´RACE analysis revealed novel 5´-exons and novel promoter regions for minimally integrating into the target cell genome. More recent works have both the NANOG and NANOG2 gene. Thus, the alternative promoter acti- utilized recombinant protein or mRNA to derive iPS cell lines that are inher- vation results in complex splice variant formation leading to the expression ently free from any genomic integration. However, these methods have of two putative NANOG and three putative NANOG2 protein variants. In been plagued by low efficiency and/or the lack of reproducibility. Here, we our study, we addressed the question, which NANOG variants are expressed demonstrate a validated protocol using mRNA for the reproducible repro- in ESC or iPSC and are relevant for induction and maintenance of pluripo- gramming of human fibroblasts to iPS cells. This process is dependent on a tency. RT-PCR experiments showed transcript variants of NANOG in human novel, defined media that is essential for complete conversion to iPS. In ad- ES cells, which contain the novel 5’-exons. The same transcripts were also dition, a highly efficient RNA transfection reagent was developed to ensure investigated in mesenchymal stem cells (MSCs) derived- and fibroblast that mRNA can be delivered to a range of cell types, including fibroblasts derived-iPSCs. In comparison to MSCs, where no NANOG expression but and lymphocytes, with titratable control over expression. This methodology strong NANOG2 expression is detectable, we could detect weak, but signifi- provides a reproducible, non-integrating method for generating iPS cells cant levels of NANOG2 in ESC and iPS cells. Our results revealed that other which has the potential to be expanded to multiple cell types. NANOG or NANOG2 splice variants are also transcribed in these stem cell populations and might support or even overtake the function of NANOG. Poster Board Number: 3281 The latter aspect is currently being investigated in iPSC generation experi- IN VIVO TRANSITION OF MOUSE HEPATOCYTES ments. In conclusion, we demonstrate the existence of mRNA transcripts from different NANOG variants in human pluripotent stem cells and provide TOWARDS A BETA CELL PHENOTYPE BY preliminary evidence that the recently described variant NANOG2 might DELIVERY OF BETA CELL TRNSCRIPTION contribute to the pluripotency-related transcriptional network. FACTORS IN MOUSE Poster Board Number: 3277 Banga, Anannya, Dutton, James, Greder, Lucas, Akinci, Ersin, INABILITY TO PROPERLY INITIATE EMBRYONIC Klindworth, Amber, Slack, Jonathan M.W, TRANSCRIPTION PREVENTS DEVELOPMENT Genetics Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA FOLLOWING HUMAN NUCLEAR TRANSFER Formation of new β-cells for the cell therapy of severe type 1 diabetes is an Egli, Dieter, Chen, Alice, Saphier, Genevieve, Alper, Michael, Alper, important challenge for regenerative medicine. Since the liver and pancreas Michael, Go, Kathryn, Goland, Robin, Leibel, Rudolph, Melton, are formed by very similar developmental mechanisms we consider that he- Douglas, Eggan, Kevin patocytes represent a favorable cell type for direct reprogramming to insulin New York Stem Cell Foundation, New York, NY, USA, Harvard University, producing beta cells. Intravenous delivery of a recombinant adenovirus, en- Cambridge, MA, USA, BIVF, Waltham, MA, USA, RSC, Lexington, MA, coding the three β-cell transcription factors Pdx1, Ngn3 and MafA, into STZ USA, Columbia University, New York, NY, USA induced diabetic mice gave rise to insulin-positive cells scattered throughout the liver and led to a normalization of blood glucose levels. The effectiveness Unfertilized and fertilized eggs of many mammalian species can reprogram of the treatment is increased about tenfold by concurrent treatment with somatic cells to an embryonic state. Somatic cell nuclear transfer could PPAR-agonists, which produce a transient hepatic hyperplasia. Analysis for therefore be a useful approach for producing patient-derived embryonic molecular markers characteristic of β-cells showed the expression of genes stem cells. However, human nuclear transfer embryos commonly arrest dur- essential for β-cell endocrine function including the glucose transporter 2 ing early development for unknown reasons, preventing stem cell derivation. (Glut 2), the potassium channel components Kir6.2 and SUR1 and the key We performed nuclear transfer into donated human oocytes and zygotes β-cell transcription factors NeuroD, Nkx2.2 and Pax4. Investigation of the to investigate the causes of these arrests. We found there was a failure to origin of the new β-like cells, by co-staining of the induced insulin-positive activate embryonic transcription in the transferred nuclei despite develop- cells for albumin (a major hepatocyte protein) revealed an overlap of most ment beyond the stage where zygotic genome activation should normally of the of the insulin-positive cells with albumin (97.6%), indicating a mixed occur. To determine whether these transcriptional failures were sufficient to phenotype intermediate between hepatocyte and β-cell. Hepatocytes cause the observed developmental blockade, we inhibited transcription in isolated from the mice by collagenase perfusion showed glucose-sensitive fertilized control zygotes and found that they arrested at an identical stage. insulin release, a key property of β-cells. The blood glucose normalization

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Thursday Poster Abstracts is lost over a period of approximately 6 to 8 weeks, concurrent with loss of garded as a relatively easy and safe technique when compared with previous most of the insulin-positive cells. We are currently investigating whether this gene transfer methods using repeated transfer or prolonged exposure to po- is actual cell loss or reversion of cell type due to the reprogramming event tentially harmful gene carriers. However, the epigenetic characterization of not resulting in a new stable state. A very small percentage (0.1%) of the iPSCs was not undertaken at genome-wide level. Here, we globally analyzed Sox9 expressing progenitor cell population in the liver in the portal vein re- chromatin signatures such as histone H3 K4me3 and K27me3 as well as gion were found to be insulin-positive by 1 week of adenoviral infection and gene expression profiles in two different mouse iPSCs derived from cardiac increased numbers were seen by 4-6 weeks encircling the portal vein region. and skin fibroblasts and ESCs. Our data indicate that chromatin states of However this small insulin-positive cell population was not found sufficient somatic cells were dramatically changed during de-differentiation and thus to restore the blood glucose level to normal levels. gene expression profiles of two types of iPSCs were almost identical to that of ESC even though iPSCs were generated from two different somatic cells. Poster Board Number: 3283 In addition, the alterations in chromatin structure during reprogramming THE CRITICAL ROLE OF PRDM14-KLF2 IN process were tightly correlated with the changes in gene expression. In sum- mary, the epigenetic reprogramming should be faithfully accompanied to REPROGRAMMING MOUSE EPIBLAST STEM change the cell fate from somatic cells to ESC-like iPSCs. CELLS TO NAÏVE PLURIPOTENCY Poster Board Number: 3287 Gillich, Astrid, Bao, Siqin, Hayashi, Katsuhiko, Vincent, Pasque, Grabole, Nils, Magnúsdóttir, Erna, Trotter, Matthew, Surani, Azim EPIGENTIC REGULATION OF NEURAL GENE University of Cambridge, Cambridge, United Kingdom, Kyoto University, EXPRESSION IN MOUSE MESENCHYMAL STEM Kyoto, Japan CELLS Prdm14, a PR-SET domain containing transcription factor, is a key deter- Termglinchan, Vittavat, Ingrungruanglert, Praewphan, Israsena, minant of primordial germ cell (PGC) specification, and plays a vital role in Nipan genome-wide epigenetic reprogramming of early germ cells. Its alternative Chulalongkorn University, Bangkok, Thailand role in acquisition and maintenance of pluripotency in mouse embryonic stem cells, however, is less clear. Here, we show that Prdm14 promotes Klf2- During the past few years, there have been a number of reports showing mediated reprogramming of mouse epiblast stem cells to naïve pluripotency. that cells with some neural characteristics, including neuron and neural pro- Prdm14 accelerates and enhances global reprogramming of the genome genitors, can be generated from MSCs. However, it is still debatable whether including X-chromosome reactivation in the presence of Klf2 during LIF/ these MSC-derived neuron-like cells can truly perform physiologic function. STAT3 signalling induced reversion of epiblast stem cells to embryonic stem To further our understanding of the neural potential of MSCs and find suit- cell-like cells. Prdm14 may pre-set the epigenome to allow fast and efficient able candidates for enhancing neural transdifferentiation, we studied pattern recruitment of Klf2 to key target loci, since Prdm14 alone has little effect of neural gene expression and epigenetic status of key neural development on reprogramming epiblast stem cells. Reprogramming by Prdm14 and Klf2 genes in MSCs. Here, we demonstrate that while FGF2 and retinoic acid occurs in the absence of Prdm1/Blimp1, another key determinant of PGC (RA) can promotes some morphological changes of MSCs, it is insufficient to specification, suggesting a germline independent reprogramming route. We trigger complete neural transdifferentiation. Although shRNA against RE-1 conclude that Prdm14, which plays a vital role in PGC specification, can also silencing factor (REST), key regulator of epigenetic status of various neural have a significant general role in genomic reprogramming. Since Prdm14 genes by itself was insufficient to enhance expression of most neural genes and Klf2 are both upregulated early during primordial germ cell specification, tested, shREST further enhancement of Ngn2, Nav1.2, and GAD1 expres- our observations indicate that these two factors may also promote epige- sion when MSCs were treated with FGF2 and RA. Overexpression of NRSE netic reprogramming in early germ cells. dsRNA in MSCs was capable of sensitizing subset of REST target genes to subsequent activation. Overexpression of transcription factors Mash1, Brn2, Poster Board Number: 3285 and Myt1l, which recently has been shown to be sufficient to reprogram SYSTEMATIC ANALYSIS OF EPIGENETIC embryonic fibroblast into functional neuron, while could promote some neu- ral characteristic in MSCs, were insufficient to trigger silencing of mesen- REPROGRAMMING PROCESS ACCOMPANIED chyme-specific genes. Epigenetic studies suggest that althogh many neural BY GENERATION OF MOUSE INDUCED genes in MSCs are less tightly suppressed than in adult fibroblast, many early neural developmental genes are more tightly suppressed compared to PLURIPOTENT STEM CELL mouse embryonic fibroblasts. Park, Jihwan, Kwon, Yoo-Wook, Cho, Hyun-Jai, Kim, Hyo-Soo, Poster Board Number: 3289 Park, Young-Bae, Roh, Tae-Young Div. of Molecular and Life Science, POSTECH, Pohang, Republic of OPTIMIZING THE EFFICIENCY AND QUALITY OF Korea, National Research Lab. for Cardiovascular Stem Cell & Innovative IPS CELL GENERATION Research Institute for Cell Therapy, Seoul National University, Seoul, Republic of Korea, National Research Lab for Cardiovascular Stem Tremml, Gabi, Li, Ming, Zhu, John, Chu, Vi Cell, IRICT, Cardiovascular Center & Dept. of Internal Medicine, Seoul Research & Development Stem Cells, EMD/Millipore, Phillipsburg, NJ, National University, Seoul, Republic of Korea, National Research Lab for USA, Research & Development Stem Cells, EMD/Millipore, Temecula, CA, Cardiovascular Stem Cell, IRICT, Cardiovascular Center. & Dept. of Internal USA Medicine, Molecular Medicine and Biopharmaceutical Sciences, Seoul National University, Seoul, Republic of Korea, Div. of Molecular and Life Reprogramming is currently thought to be a stochastic process by which Science, Div. of Integrative Biosciences and Biotechnology, POSTECH, cells revert to a pre-iPS cell intermediate before reaching the naïve ground Pohang, Republic of Korea state. Despite recent advances, reprogramming is still a relatively inefficient process. Naïve mESCs are characterized by expressions of pluripotency Several methods to reprogram somatic cells into induced pluripotent stem marker genes, LIF responsiveness and hypomethylation at key loci. In this cells (iPSCs) have been reported so far. Recently we reported that a single study we sought to identify small molecules that would i) increase the ef- transfer of protein extract from mouse embryonic stem cells (ESCs) could ficiency of reprogramming using a lentivirus polycistronic cassette expressing reprogram adult somatic cells into iPSCs. This protein-based method is re- the four transcription factors, Oct4, Klf4, Sox2 and c-Myc and ii) allow re-

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programmed cells to reach and maintain the naïve ground state of pluripo- mock-transfected control. Our results demonstrate that transfection with tency. In serum containing medium with LIF, a cocktail containing 3 small modified mRNA triggers a potent innate immune response in human fibro- molecule modulators of specific signaling pathways was able to increase the blasts, and that the reduction in immunogenicity achieved by incorporating efficiency of 4 factor-(OKSM) and 3 factor-(OKS) reprogramming of primary modified nucleotides is negligible in the context of frequent transfection. mouse embryonic fibroblasts (MEF) by ten fold. The resulting mouse iPS We previously demonstrated that suppressing the innate immune response colonies possessed proliferative capacity, expressed ES cell-like morphology of cells to exogenous RNA enables frequent transfection with unmodified and immunochemical profile of pluripotent cells and endogenous nanog mRNA. We now extend our findings to show that innate immune suppres- expression similar to a mouse ESC control, except that the viral transgenes sion is also required for frequent transfection with modified mRNA. were not silenced, raising the question whether these cells may be pre-iPS cell intermediates. Mouse ES cells attain the naïve ground state by exposure Poster Board Number: 3293 to defined serum-free media containing LIF and two small molecule inhibi- MECHANISTIC INSIGHTS INTO DIRECTED tors of Mek1/2 and GSK3b, that remove the differentiation inducing stimuli (ESGRO-2i medium). Interestingly, rat iPS cells generated in ESGRO-2i REPROGRAMMING USING CELL FUSION media had increased expression of endogenous nanog transcripts, while the Bhutani, Nidhi, Brady, Jennifer J., Blau, Helen M. viral transgenes were silenced, suggesting that the cells have attained the naïve ground state. We thus asked whether exposure to ESGRO-2i media Microbiology and Immunology, Stanford University, Palo Alto, CA, USA, would similarly aid in the epigenetic reversion of mouse pre-iPS cell interme- Microbiology and Immunology, Baxter Laboratory in Stem Cell Biology, diates to a naïve pluripotent state. After four passages in ESGRO-2i media, Stanford University, Palo Alto, CA, USA all pre-iPS clones displayed significant increases in the transcript levels of One approach to nuclear reprogramming is to use subsets of known factors. endogenous nanog. In several of the clones, a concomitant decrease in By using cell fusion in stable heterokaryons, reprogramming can be studied viral transgene expressions was also observed, suggesting that these clones systematically and factors identified based on their expression in the hu- may be further advanced to the naive full reprogrammed state. With time man fibroblast to which the differentiated mouse cell is fused. Interspecies course experiments, we are investigating whether extended passaging of heterokaryons are the stable fusion products of mouse embryonic stem cells mouse iPS clones in ESGRO-2i would lead to an eventual acquisition of the and human fibroblasts, in which reprogramming is initiated rapidly (1 day) naïve pluripotent state. The naïve state will be assessed by cell morphology, and efficiently (70%) in the absence of cell division or DNA replication. We pluripotency markers, dependency upon LIF/Stat3 signaling, viral silencing, identified Activation-Induced Deaminase (AID) mediated DNA demethyla- and epigenetic effects at key loci, including theDlk-Dio3 gene cluster. In tion as essential for the reprogramming of somatic cell nuclei to pluripotency summary, we propose the following two step reprogramming procedure to in heterokaryons. By siRNA-mediated knockdown, we found that AID is maximize efficiency and quality of iPS cells generated: In the first step, pre- critical for previously silent Oct4 and Nanog promoter demethylation and iPS cell intermediates can be efficiently generated using lentiviral transduc- expression in human fibroblasts. Since DNA demethylation is a bottleneck tion of OKSM factors with 3 small molecule modulators in serum-and LIF to reprogramming in iPS cells, we tested whether AID can overcome it to supplemented medium. In the second step, the resulting pre-iPS cell inter- enhance the efficiency of iPS generation. We have investigated the potential mediates can be directly cultured in ESGRO-2i media to effect the transition role of AID in reprogramming to iPS in loss and gain of function studies, to the naïve pluripotent state. and are identifying additional novel factors with a role in reprogramming to Poster Board Number: 3291 pluripotency and to specialized cell fates. Poster Board Number: 3295 MODIFIED MRNA IS HIGHLY IMMUNOGENIC: IMPLICATIONS FOR DIRECTING CELL FATE NEUROGENIN3 PROMOTES PROLIFERATION Angel, Matthew, Yanik, Mehmet F. OF TRANSDIFFERENTIATED BETA CELLS Massachusetts Institute of Technology, Cambridge, MA, USA Fang-Pei Chang,, Ruei-Ren Wu, Huey-Kang Sytwu and Chia-Ning Shen,, In vitro-transcribed (ivT) mRNA transfection is a powerful method for ex- pressing high levels of proteins both in vitro and in vivo that avoids the risk Stem Cell Program, Genomics Research Center, Academia Sinica, Taipei; of mutation associated with DNA-based methods. However, ivT mRNA in- Graduate Institute of Life Sciences, National Defense Medical Center, duces a potent innate immune response, and frequent transfection with ivT Taipei; Institute of Biotechnology in Medicine, National Yang-Ming mRNA, which is required to sustain high-level protein expression, causes cell University, Taipei, Taiwan death. We previously reported that suppressing the innate immune response Islet transplantation has been recongized as an efficient approach for treat- of cells to exogenous RNA enables frequent transfection with ivT mRNA, ing for type I diabetes patients. However, shortages of donor-supply results and we recently used this technique to achieve sustained, high-level expres- in there are more than 200 million diabetic patients in the world that are sion of reprogramming proteins in primary human fibroblasts. However, as unable to receive the transplantation treatment. To expand the supply of an alternative approach, the incorporation of certain modified nucleotides insulin-producing beta cells to meet future needs, over the last few years, a has been suggested as a method for reducing the immunogenicity of ivT few groups had revealed that hepatocytes are capable to become insulin- mRNA. We demonstrate that a single transfection with modified mRNA producing beta cells by introducing certain pancreatic transcriptional factors containing complete substitution of pseudouridine and 5-methylcytidine for such as Pdx1 to liver cells. However, no reports had demonstrated these uridine and cytidine, respectively, triggers a potent innate immune response transdifferentiated beta cells can be expanded. In the current work, we car- in primary human fibroblasts. This response is characterized by >100-fold ried out a direct conversion approach by introducing two pancreatic specific upregulation of several interferon-stimulated genes including IFIT1,2, and transcriptional factors to adult mouse liver cell line-AML12 or primary 3 and >50-fold upregulation of the receptors of exogenous RNA, TLR3 hepatocytes isolated from Non-obese diabetic (NOD) mice. Mouse has two and RIG-I. Subsequent daily transfections result in further upregulation of insulin genes-ins1 and ins2. Initially, we demonstrated that insulin2 can be immune-related genes, elimination of encoded-protein expression, and mas- induced to express when overexpressing single transcriptional factor, Pdx1, sive cell death. Supplementation of the culture medium with a potent and in mouse hepatocytes. However, induction of insulin1 expression in hepato- specific inhibitor of type I-interferon signaling results in dramatically reduced cytes required simultaneous overexpression of Ngn3 and Pdx1 at the same upregulation of immune-related genes, sustained, high-level expression of cells. And overexpression of Pdx1 and Ngn3 was sufficient to downregulate the encoded protein, and proliferation at a rate indistinguishable from the hepatic phenotypes as judged by reduced expression of transferrin and albu-

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Thursday Poster Abstracts min. Most interestingly, we further demonstrated combination of Pdx1 and Poster Board Number: 3299 Ngn3 can not only induced conversion of hepatocytes to insulin-producing cells but also enhanced de-methylation of insulin-promoter accompanied by MYC DETERMINE THE PARTIAL increasing proliferation of the transdifferentiated cells. Addition of prolifera- REPROGRAMMED CELL FATE tion inhibitors could counteract the effect of Ngn3. Furthermore, we also confirmed these transdifferentiated beta cells can secret insulin and express Chen, Jiekai, Pei, Duanqing beta-cell specific genes such as Glut2 and Kir6.2 which are known to be Guangzhou Institutes of Biomedicine & Health, Chinese Academy of involved in glucose-sensing function in mature beta cells. In summary, Sciences, Guangzhou, China the current work indicates conversion of hepatocytes to insulin-producing beta cells can be achieved by Pdx1 and Ngn3 via introducing cell division The mechanism of somatic cell reprogramming induced by defined factors is together with DNA demtheylation. And the current findings may lead to not clear yet. Partial reprogrammed cells, also called pre-iPSCs, were widely development of new therapeutics for diabetes. used in mechanistic research as an intermediated stage of reprogramming. Here we showed that partial reprogramming was not an essential stage Poster Board Number: 3297 during reprogramming. c-Myc, which is dispensable for reprogramming, plays important role in the generation of partial reprogrammed cells. In the CONVERSION OF FIBROBLASTS INTO absence of c-Myc, partial reprogrammed cells were disappeared. We also FUNCTIONAL SPINAL MOTOR NEURONS showed that serum cooperated with c-Myc to repress the reprogramming at pre-iPSCs stage. Our results showed that the reprogramming approaches Son, Esther Y., Ichida, Justin K., Wainger, Brian J., Woolf, Clifford J., were factorial context-dependent. Eggan, Kevin Poster Board Number: 3301 Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA, The Program in Neurobiology and F.M. Kirby Neurobiology Center, AKT RELATES TO C-MYC FOR PROMOTION OF Harvard Medical School, Longwood, MA, USA REPROGRAMMING The mammalian nervous system is composed of a multitude of distinct neu- ronal subtypes, each with its own phenotype and differential sensitivity to Takahashi, Kazutoshi, Tanabe, Koji, Okada, Aki, Yamanaka, Shinya degenerative disease. Although specific neuronal types can be isolated from CiRA, Kyoto, Japan intact rodent embryos or engineered from stem cells for translational studies, Phosphatidyl-inositol 3’-OH kinase (PI3K) pathway plays important roles transcription factor-mediated reprogramming might provide a more direct of growth and survival in various cell types. One of its targets, AKT, is well- route to their generation. So far, neuronal cells with an unknown develop- known molecule which can phosphorylate the downstream substrates such mental ontogeny have been induced from fibroblasts, but for this approach as GSK3 and MDM2. In undifferentiated cells such as embryonic stem cells to be useful in regenerative medicine, it must generate well-defined types and primordial germ cells, the important roles of PI3K/AKT pathway have of neurons that possess the correct phenotypic properties. To this end, we been demonstrated. However, their roles during reprogramming from so- sought to transdifferentiate fibroblasts into motor neurons, the cell type that matic cells to pluripotent state are still uncovered. To confirm whether PI3K is lost in neurodegenerative diseases such as amyotrophic lateral sclerosis and AKT can affect the reprogramming efficiencies or not, we introduced (ALS). We selected eight candidate factors that participate in motor neuron each of them along with the set of reprogramming factors consisted of development, and supplemented this set with three factors that have been OCT3/4, SOX2, KLF4 with or without c-MYC into human dermal fibro- found to convert fibroblasts into induced neurons (iNs). When we intro- blasts. As results, constitutive active mutant of PI3K catalytic subunit p110 duced these factors into Hb9::GFP-transgenic mouse embryonic or adult (p110-CaaX) but not of AKT (Myr-AKT) could increase the number of fibroblasts, neuronal-looking cells emerged that had turned on the motor induced Pluripotent Stem (iPS) cell colonies in the presence of c-MYC trans- neuron reporter. These induced motor neurons (iMNs) were morphologically gene. In contrast, AKT effectively enhanced the number of iPS cell colonies indistinguishable from their embryonic counterparts and stained positively in the absence of exogenous c-MYC. These data suggest that PI3K can for a range of pan-neuronal and motor neuron markers. Global gene expres- contribute the increase of reprogramming efficiencies with AKT-independent sion analysis indicated that iMNs were highly similar to both embryonic and ways. And the MYC dependency of AKT roles during reprogramming is also stem cell-derived motor neurons but distinct from fibroblasts. Whole-cell suggested. In this presentation, we will discuss the relationship between AKT patch clamp recordings showed that iMNs are excitable, generate action and c-MYC during reprogramming. potentials and respond to both inhibitory and excitatory neurotransmitters similar to embryonic motor neurons. Strikingly, C2C12 myotubes co-cultured Poster Board Number: 3303 with iMNs exhibited regular contractions which could be stopped by curare, a selective inhibitor of acetylcholine receptors; thus, iMNs are capable of GENE-TARGETED OSTEOGENESIS IMPERFECTA forming functional synapses with muscle, a defining characteristic of motor IPSCS EXPRESS NORMAL COLLAGEN AND neurons. In addition, iMNs showed sensitivity to motor neuron-selective degenerative stimuli when co-cultured with glial cells from the SOD1G93A PRODUCE BONE AFTER MESENCHYMAL mouse model of ALS, demonstrating their potential for in vitro disease DIFFERENTIATION modeling. Finally, the same cocktail of transcription factors could produce Hb9::GFP+ cells with a neuronal morphology from human embryonic and Deyle, David R., Khan, Iram F., Ren, Gaoying, Wang, Peirong, Kho, postnatal fibroblasts. This suggests that it may be possible to generate Jordan, Schwarze, Ulrike, Byers, Peter H., Russell, David W. patient-specific human iMNs by transdifferentiation. Taken together, our Medicine, University of Washington, Seattle, WA, USA, Program in findings demonstrate that fibroblasts can be converted directly into a specific Developmental Biology, Baylor College of Medicine, Houston, TX, USA, neural subtype, the spinal motor neuron. This is an efficient process in which Pathology, University of Washington, Seattle, WA, USA over 10% of the starting fibroblasts are routinely reprogrammed under Osteogenesis Imperfecta (OI) is a common genetic disorder caused by domi- optimized conditions. As iMNs show molecular and functional signatures nant negative mutations in type I collagen genes, COL1A1 and COL1A2. In of bona fide motor neurons and recapitulate the ALS disease phenotype in principle, OI could be treated by transplanting patient-specific, mesenchy- vitro, they may have immediate utility in the study of motor neuron biology mal stem cells (MSCs) that do not express the mutant gene. We previously and degeneration. showed that an AAV vector can disrupt the dominant-negative mutant alleles in MSCs from individuals with severe OI by targeted insertion of a

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floxed IRES-Neo selection cassette and that the floxed IRES-neo cassette that loss of Rb improves the reprogramming efficiency and accelerates iPS could be removed by Cre-mediated excision. However, MSCs in long-term cell generation. In particular, we found that loss of Rb enhances the earlier culture have reduced adipogenic potential and proliferative capacity and stages of the reprogramming process. Importantly, our data suggest that they eventually senesce, limiting their clinical utility. Here we show that ad- loss or Rb alone does not interfere with proliferation in reprogramming eno-associated virus (AAV)-mediated gene targeting vectors can inactivate cells. These observations led us to hypothesize that Rb may normally block the mutant collagen gene in MSCs from OI patients and that these targeted dedifferentiation by regulating key transcriptional targets beyond its classical MSCs can be reprogrammed into induced pluripotent stem cells (iPSCs) that cell cycle targets. Consistent with this idea, we observe that the transcription can be expanded and differentiated into mesenchymal stem cells (iMSCs). levels of pluripotency factors are perturbed upon Rb loss in MEFs. We con- To produce gene-targeted iPSCs, MSCs from OI patients were infected with clude that the tumor suppressor functions of Rb in mammalian cells include AAV gene-targeting vectors, selected in G418, and pooled into polyclonal regulating checkpoints at both the G1/S transition and against dedifferentia- populations. G418-resistant MSCs were transduced with either 4 lentiviral tion pathways. reprogramming vectors or a floxed foamy viral (FV) reprogramming vector, and subsequent iPSC colonies were isolated and expanded. Based on South- Poster Board Number: 3307 ern blot analysis, 3 of 4 iPSC clones were targeted at the COL1A1 locus INDUCED PLURIPOTENT STEM CELLS AS and 16 of 21 iPSC clones were targeted at the COL1A2 locus. Sequencing of cDNA isolated from iPSC-derived embryoid bodies determined that both A TOOL TO MODEL WILLIAMS-BEUREN the mutant and wild-type allele had been targeted and karyotype analysis SYNDROME showed that 2 of the 4 lines analyzed had normal cells. Gene-targeted iPSC lines expressed pluripotency genes and two lines were shown to have trilin- Khattak, Shahryar, Thompson, Tadeo, Mital, Seema, Ellis, James eage developmental potential during teratoma formation. iPSCs targeted at Ontario Human Induced Pluripotent Stem Cell Facility and Developmental the mutant allele were differentiated in vitro to generate iMSCs that pro- & Stem Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada, duced normal collagen. Differentiation studies showed that targeted iMSCs Ontario Human Induced Pluripotent Stem Cell Facility, Toronto, ON, could form bone in vitro and in vivo. Cellular senescence and proliferation Canada, Genetics and Genomics Program, The Hospital for Sick Children, assays demonstrated that iMSCs were less senescent than gene-targeted Toronto, ON, Canada MSCs. Also, iPSC clones generated using the floxed FV vector that were Williams-Beuren Syndrome (WBS) is a rare genetic disorder (1/10,000 transiently exposed to Cre recombinase underwent removal of the IRES-neo persons) caused by chromosomal microdeletion of 1.5 -1.8 million base pairs targeting cassette and integrated FV genome leaving only the deleted LTR at 7q11.23 comprising 26-28 genes. This autosomal dominant syndrome is sequence behind, thereby creating transgene-free, gene-targeted iPSCs. typically characterized by supravalvular aortic stenosis, dysmorphic facies, Here we show that that the combination of gene targeting and iPSC deriva- smaller brain size, developmental delay, learning disabilities, and visuospatial tion can be used to produce a potentially therapeutic population of patient- impairment. In vivo experiments have been done in primate models, cats specific cells for transplantation. This approach not only could be applicable and human brain autopsies to study the neuronal size and neuronal-packing to OI, but to other genetic disorders as well. density in primary visual cortex of WBS. We have generated WBS patient Poster Board Number: 3305 specific induced pluripotent stem (iPS) cell lines to study WBS syndrome in vitro. These iPS cells express the pluripotency markers and have a normal INVESTIGATING THE ROLE OF RB DURING human karyotype. Furthermore, these lines can give rise to all three embry- CELLULAR REPROGRAMMING onic germ layers via in vitro differentiation assays and the WBS specific dele- tion was confirmed using fluorescence in situ hybridization. These WBS iPS Kareta, Michael S., Hafeez, Sana, Gorges, Laura, Briggs, Sharon, cells will allow us to study both the cardiac and neural disease phenotypes. Sage, Julien, Wernig, Marius The iPS cells have undergone successful cardiac differentiation to generate Institute for Stem Cell Biology and Regenerative Medicine and Department smooth muscle cells for further characterization. We will generate neural of Pediatrics, Stanford University, Stanford, CA, USA, Institute for Stem precursor cells from iPS and further differentiate them into neurons. We are Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, interested in studying the properties of neurons that are affected by WBS. USA, Department of Pediatrics, Stanford University, Stanford, CA, USA e.g. neural morphology, dendritic spine morphology, synapse formation/ maturation, gap junctions. The second phase of project would be to identify During tumorigenesis, mutant cells, by an incompletely understood mecha- small molecules that revert or protect the cellular phenotype. nism, are able to gain characteristics common to stem cells. In fact many cancers are described, at least in part, by cells which have lost differentiated Poster Board Number: 3309 characteristics (“dedifferentiated”) to acquire stem cell-like characteris- tics. The recent discovery of induced pluripotent stem (iPS) cells, where COMPONENTS THAT TRANSDUCE fibroblasts and terminally differentiated adult cells can be reprogrammed EXTRACELLULAR MATRIX (ECM) SIGNALS into an embryonic stem cell-like state is a well suited model for the study of ARE REQUIRED FOR DEVELOPMENTAL dedifferentiation during tumorigenesis. Indeed the processes behind tum- origenesis and reprogramming, while not identical, do share many similari- COMMITMENT OF C. ELEGANS EMBRYONIC ties, including dedifferentiation, gaining a self-renewal potential, chromatin PROGENITOR CELLS remodeling, and deregulation of the cell cycle machinery. Further linking these two processes is the observation that activation of the Myc oncogene Riddle, Misty R., Djabrayan, Nareg J., Rothman, Joel H. and inactivation of the p53 tumor suppressor both promote reprogramming. University of California, Santa Barbara, Santa Barbara, CA, USA It then follows that other tumor suppressor genes may antagonistically The embryonic cells of Caenorhabditis elegans remain in a pluripotent regulate dedifferentiation. One such candidate is retinoblastoma (Rb) and state until the end of gastrulation as demonstrated by their ability to adopt the related Rb family members, p107 and p130, which have both overlap- endodermal, mesodermal, or ectodermal fates when presented with the ping and distinct functions. Rb is mutated or functionally inactivated in most appropriate tissue-promoting transcription factor; following this period of human cancers, and has been implicated in the maintenance of quiescence developmental plasticity, they become refractory to reprogramming. We in progenitor cells as well as differentiation and cell cycle exit. To test for a found that when the intracellular effector of cell-matrix interactions, PAT-4 potential role for Rb in cellular reprogramming into iPS cells, we used Rb integrin linked kinase (ILK), is knocked down via RNA interference, cells that knock-out and knock-down mouse embryonic fibroblasts (MEFs). We found are past the stage of gastrulation and normally committed to their develop-

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Thursday Poster Abstracts mental pathway are able to adopt endodermal or mesodermal fates when Poster Board Number: 3313 confronted by ectopic expression of end-3 (endoderm-promoting GATA transcription factor) or hlh-1 (the myogenic factor MYOD) respectively. We OVERCOMING BARRIERS THAT HINDER HIGH have also found that other components known to associate with PAT-4, like THROUGHPUT SCREENING FOR FACTORS THAT alpha-parvin (PAT-6), mitogen inducible protein (UNC-112), and alpha inte- grin (INA-1) have a similar role in inhibiting developmental potential of cells. MEDIATE CELLULAR REPROGRAMMING In C. elegans, PAT-4 is thought to function strictly as an adaptor molecule Tonge, Peter, Corso, Andrew, Woltjen, Knut, Nagy, Andras within integrin adhesion complexes because animals carrying mutations in Samuel Lunenfeld Research Institute, Toronto, ON, Canada, University the PAT-4 kinase domain appear wild-type. Here, we will discuss our current of Toronto, Toronto, ON, Canada, Center for iPS Cell Research and efforts to examine developmental potential of cells in embryos that carry Application (CiRA), Kyoto University, Kyoto, Japan kinase dead PAT-4. These experiments may reveal that either PAT-4 kinase activity does have a function in cell fate “lock down” or alternatively, simply The high frequency at which Piggybac transposons stably integrate in to the anchoring cells to the ECM via PAT-4 is required to maintain a differentiated genome, facilitates the efficient production of iPS cells from somatic cells. state. We will also present our progress in investigating how the ECM to cell Through the use of a conditionally active promoter (tet-on system) driving connection leads to nuclear modifications that allow or restrict changes in expression of the reprogramming factors (Oct4, Sox2, KLF4 and C-Myc) it is gene expression. Numerous studies in mammalian systems have suggested possible to generate a secondary reprogramming system, whereby second- the ECM is a critical component both for regulating pluripotency and main- ary fibroblasts stably express the full complement of reprogramming factors taining differentiated cell types and obtaining a basic understanding of the upon exposure to doxycycline. This efficient system provides the means role of the ECM in these processes in vivo using C. elegans as a model may to investigate the reprogramming process. We have generated secondary be informative to the efforts of regenerative medicine. mouse embryonic fibroblasts that conditionally express just three of the four reprogramming factors. Upon exposure to doxycycline these cells fail Poster Board Number: 3311 to reprogram, however, introduction of the fourth factor initiates efficient re- INSIGHTS INTO THE ROLE OF NANOG IN THE programming. Three factor secondary fibroblasts have been utilized in large- scale genomic and chemical screens to identify factors that facilitate the INDUCTION OF NAIVE PLURIPOTENCY reprogramming of somatic cells. The discovery of alternative factors helps Theunissen, Thorold W., Costa, Yael, Radzhisheuskaya, Aliaksandra, elucidate the mechanism(s) driving cellular reprogramming and contributes to the safe production of iPS cells for stem cell-based therapeutics. Silva, José C.R. Department of Biochemistry, Wellcome Trust Centre for Stem Cell Research, Cambridge, United Kingdom Induced pluripotency requires the expression of reprogramming factors EMBRYONIC STEM CELLS CLINICAL APPLICATION and culture conditions that support the self-renewal of embryonic stem Poster Board Number: 3315 (ES) cells. The molecular mechanisms of reprogramming, including the role of endogenous transcriptional regulators, are poorly understood. Using a DERIVATION OF A GMP-GRADE HUMAN loss-of-function approach, we previously showed that the homeodomain- EMBRYONIC STEM CELL LINE WITH THE containing transcription factor Nanog is dispensable for the generation of an intermediate (pre-iPS) cell stage, but strictly required for the establishment DEFINED, LOW PROTEIN AND XENO-FREE of naive pluripotency. This is analogous to early mouse embryogenesis, NUTRISTEMTM MEDIUM where Nanog is required for specification of the naive pluripotent epiblast. Amit, Michal, Shariki, Kohava, Laevsky, Ilana, Miropolsky, Yael, By gain-of-function, we demonstrated that Nanog enhances reprogramming in cooperation with inhibitors of MAP kinase (MEK) and glycogen synthase Peri, Meital, Itskovitz-Eldor, Joseph, kinase 3 (GSK3) or 5-aza-cytidine, an inhibitor of DNA methylation. Nanog Faculty of Medicine, Technion, Haifa, Israel also enables induction of naive pluripotency in minimal conditions that Human embryonic stem cells (hESCs) are pluripotent cells isolated from normally do not support induced pluripotency or the self-renewal of ES cells. blastocysts. Traditionally, these cells have been cultured with a supporting These results highlight the capacity of Nanog to overcome multiple barriers layer in two-dimensional culture, which allows their continuous growth to reprogramming, and reveal a synergy between Nanog and small mol- as undifferentiated cells. However, any future use of hESCs for cell-based ecules that promote reprogramming. However, Nanog is poorly conserved therapy and industrial purposes will require a scalable, reproducible and among eutherian mammals and it is unknown whether naive pluripotency controlled culture system. Employing the ‘whole embryo’ approach, we used is unique to mouse and rat species. We have therefore investigated whether NutriStem™ medium and inactivated human foreskin fibroblasts (HFF) as the capacity to induce naive pluripotency is unique to rodent Nanog. For feeder layer to derive a hESC line in a clean room (Ella GMP facility, Sheaba this purpose, we introduced Nanog orthologs in somatic cells genetically Hospital, Israel). The cell line was passaged mechanically until removed from deficient in endogenous Nanog, and asked whether these orthologs can the GMP-facility, at which point passaging was carried out by collagenase replace the requirement for mouse Nanog during induced pluripotency. This splitting. All materials and disposables were GMP-grade. The HFF feeders system provides a rigorous test for the functional conservation of Nanog, were also derived in the same conditions, and the cells were inactivated us- and allows us to fully characterize any iPS cells that may be generated with ing irradiation. Only one HFF cell line, GF3, was used for derivation, culture Nanog orthologs by transcriptional, epigenetic and developmental criteria. I and banking of the hESCs. The resultant cell line, CL1, demonstrated hESC will discuss the implications of this work for our understanding of the role of features, including typical colony morphology, expression of ESC markers Nanog, and the evolutionary origins of naive pluripotency. and embryoid body and teratoma formation after 25 passages of continuous culture. Using G-banding (200 metaphases) the cells’ karyotype was tested at passage 20 and all were found to be normal 46,XY. These results were also validated using FISH analysis for three different chromosomes (200 cells). Same results were obtained when cell line HFF was employed. Sterility at the master and distribution bank of the cell line was tested by an autho- rized laboratory and the cell line was found negative for bacteria, fungus, micoplasma and viruses. The cell line’s proliferation rates were slightly higher

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as compared to cell lines derived with MEFs and to the same line cultured between hESCs and their differentiated cells in a set of genes, here we pro- with other commonly used ESC media. The cell line was also transferred pose a new technology called methylation-specific castPCR (MeS castPCR) successfully to a dynamic suspension culture, where it was cultured as undif- for accurate and sensitive quantitation of undifferentiated hESCs. This new ferentiated at high scales using spinner flasks and xeno-free medium. CL1 is technology will bridge the gap in hESC product QC and then overcome a hESC line derived using xeno-free and reduced bFGF concentration. CL1 this major obstacle in stem cell therapy. CastPCR combines allele-specific expresses all hESC characteristics, exhibits high proliferation rates and can be TaqMan qPCR with allele-specific minor grove binder (MGB) as blockers to cultured dynamically in suspension, which facilitates mass production. Thus, effectively suppress non-specific amplification from the wild type allele. We cell line CL1 and the presented NutriStem™ medium and culture conditions have successfully designed and validated castPCR assays for ~100 SNPs or can be utilized for clinical and industrial applications. InDels. Results demonstrate that castPCR not only maintains wide dynamic range, high sensitivity, and reproducibility of TaqMan assays but also is able Poster Board Number: 3317 to detect one mutant in 100,000,000 wild-type molecules. Recently, Laurent DIFFERENTIATION OF HUMAN EMBRYONIC et al. and Lister et al. presented a whole-genome comparative view of DNA methylation using bisulfite sequencing of hESCs and various differentiated STEM CELLS TO MATURE DENDRITIC CELLS cells. Based on their findings, we have identified genes and genomic DNA IN A THERAPEUTIC MODEL FOR ALZHEIMER’S regions such as HOXB6 and others which show distinct DNA methylation patterns between hESCs and differentiated cells. These regions could be DISEASE used as methylation markers to distinguish between hESCs and their dif- Begum, Aynun N., Cunha, Celia, Ethell, Doug W. ferentiated (somatic) cells. It is important to note that DNA regions with 0% Molecular Neurobiology Group, Western University of Health Sciences, methylated in hESC and 100% in differentiated cells are mostly desirable Pomona, CA, USA due to incomplete bisulfate conversion. MeS castPCR is aimed to detect converted CpG sites which are used to estimate % of hESC contamination Myeloid Dendritic Cells (DC) are critical for the development of adaptive T in its therapeutic cell products. Our results suggest that MeS castPCR is cell responses to a variety of antigens. Over the past 10 years our group, capable of detecting hESC contamination as little as 0.01% in differentiated and others, have shown that adaptive CD4+ T cells responses to beta-Am- stem cell therapy product during clinical trials. yloid can significantly affect the pathophysiology of Alzheimer’s disease in mouse models. To investigate the prevalence of beta-Amyloid specific T cell Poster Board Number: 3321 responses in clinical blood samples, we have been engineering human DCs to induce highly specific T cell receptor interactions. As a consistent source CLINICAL STRATEGIES FOR CELL of human DCs, we differentiate myeloid dendritic cells (DC) and mature REPLACEMENT THERAPY USING DC from human embryonic stem cells (hESCs), using a method, based on a HUMAN EMBRYONIC STEM CELLS IN Vodyanik and Slukvin protocol. Initial myeloid differentiation was induced by co-culturing hESCs with OP9 stromal cells for 9-10 days, which decreased NEURODEGENERATIVE DISEASE the proportion of CD33+/CD34+ cells and induced the appearance of Cho, Myung Soo, Lee, Haksup, Yoo, Dae-hoon, An, Hwa, Oh, Sun CD45+ cells. Next, myeloid progenitors were expanded with granulocyte Kyung, Ku, Seung-yup, Choi, Young Min, Moon, Shin Yong macrophage-colony stimulating factor (GM-CSF) in a feeder-free culture system for 10 days, increasing the proportion of CD45+ cells from less than R&D center, Jeil Pharmaceutical Co., Ltd., Seoul, Korea, Republic of, Dept 10% to more than 40%. Myeloid morphologies observed included large, of Ophthalmology, Seoul National University Hospital, Seoul, Korea, agranular cells with pumpkin-shaped nuclei. A further 12 days of serum-free Republic of, Institute of Reproductive Medicine and Population, Medical incubation with GM-CSF, and the addition of IL-4, resulted in the appear- Research center, Seoul National University, Seoul, Korea, Republic of ance of immature DCs that were HLA-DR+/CD11c+ /CD80+/CD86+/ ESCs are good cell sources for cell replacement therapy because of their DEC205+/DC-SIGN+. We induced DC maturation with LPS, TNF-alpha pluripotency and ability of infinite proliferation. However, culture environ- or CD40L (for 72 h) and observed mature DC morphologies with long ment with xeno-components, tumorigenicity and insufficiency of differenti- dendrites, and a higher the proportion of cells expressing DC-SIGN. Cells ated cells remain barriers to clinical application. For the purpose of applying differentiated with this protocol are susceptible to infection with lentivirus the ESCs to clinical approach of neurodegenerative disease, we have tried to vector at the myeloid and immature DC stages, proving an opportunity to develop protocols by which ESC-derived neuronal progenitors can maintain generate stable progenitor lines that express recombinant proteins that can and differentiate into neurons without barriers for clinical use. Spherical be used to elicit specific T cell responses from human blood samples. neural masses (SNMs) consists of mostly neuronal progenitors were formed Poster Board Number: 3319 from hESCs which have been created by xeno-free conditions and differenti- ated into mature neurons. SNM reformation method was used to avoid the DETECTION OF RARE HESC IMPURITY BY teratoma formation. For mass production of the reformed SNMs and differ- entiated neurons, we also developed a new method for SNM fragmentation. METHYLATION SPECIFIC CASTPCR These strategies in our study could be helpful for removing some barriers for Chen, Caifu, Li, Kelly, Gioia, Jason, Deng, David clinical application of hESCs. This research was supported by grants (codes: SC3150) from the Stem Cell Research Center of the 21st Century Frontier Genomic Assays R&D, Life Technologies, Foster City, CA, USA Research Program funded by the Ministry of Science and Technology. Human embryonic stem cells (hESCs) retain the remarkable ability to dif- ferentiate into multiple different tissues and to self-renew in vitro. These remarkable abilities make hESCs very attractive to both scientific and clinical communities because they are potentially a renewable source of a wide variety of human tissues which can be used for regenerative medicine, in drug discovery and toxicity testing. However, during the cell type-specific differentiation of hESCs, a small fraction of undifferentiated hESCs frequent- ly remain mixed with the differentiated cells. These undifferentiated hESCs pose serious risk by forming tumors after transplantation into patients. Therefore, it is important to detect & quantify rare undifferentiated hESCs in therapeutic stem cell products. Based on distinct methylation patterns

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Poster Board Number: 3323 karyotyping that can negatively impact their safety profiles. To address this issue, the effects of five different culture methods were examined on the SORTING AND TRANSPLANTATION OF genetic stability and phenotypic properties of hESCs. The culture methods DOPAMINERGIC PROGENITORS DERIVED used were 1- Mouse embryonic fibroblast (MEF) feeders with mechani- cal passaging, 2- MEF feeders with enzymatic passaging, 3- Matrigel with FROM HUMAN PLURIPOTENT STEM CELLS mechanical passaging, 4- Matrigel with enzymatic passaging (StemPro), and Doi, Daisuke, Kikuchi, Tetsuhiro, Morizane, Asuka, Takahashi, Jun 5- Matrigel with enzymatic passaging (NutriStem). The WA09 hESC line was cultured for over 100 passages with six replicates per culture condi- Dept. of Cell Growth and Differentiation, Center for iPS Cell Research and tion. Standard phenotypic assays were performed such as expression of Application, Kyoto, Japan pluripotency-associated genes, differentiation in vitro, proliferation rate, Considering cell therapy with pluripotent stem cells for Parkinson’s dis- telomere length, telomerase activity, apoptosis rate, and teratoma forma- ease into clinical application, the ideal cell graft for cell therapy is: 1) not tion. Molecular assays were also performed, including genome-wide SNP overgrowing, and 2) high purity of dopaminergic cells in the graft. In this genotyping, DNA methylation, gene expression, and lectin microarrays. No report, we examined a FACS-based sorting method using a surface marker significant differences were found in the phenotypic properties of hESCs of neural progenitors, to purify the ideal cell graft for Parkinson’s disease. cultured under the five different methods. The hESCs in all culture condi- We induced neural progenitors from human ES cells (KhES-1) and human tions had similar differentiation potential in vitro and in vivo, expression of iPS cells (253G4, 404C2) by the SDIA (stromal cell-derived inducing activity) pluripotency markers, and self-renewal capacity. Differences were found on method and SFEBq (serum-free embryoid body quick) method with small the gene expression profiles of the hESCs cultured under the five methods. molecules BMP inhibitor and Activin/Nodal inhibitor to induce neural cells We identified differences that were independently attributable to passage efficiently, and we analyzed the expression of a cell surface marker Corin, method (enzymatic or mechanical) and the substrate used to culture the cells by RT-PCR and flow cytometry. By the SDIA method, expression of Corin (MEFs or Matrigel). Gene expression differences were also found between arises from culture day 10 and about 10-15% of living cells were positive on early and late passage samples. The stability of X-inactivation and imprinting day 14. After sorting, purity of Corin positive cells were over 98%, and the marks did not significantly change with culture methods and time in culture, purity of the cells were confirmed by RT-PCR analysis. Corin positive cells but these variables did have effects the general methylation pattern. Copy were also expressed Lmx1a, a transcript for midbrain dopaminergic progeni- number variations were observed at different frequencies depending on pas- tors. Corin positive cells were cultured for 1 to 2 weeks after sorting and sage method, substrate, media type, and time in culture. In fact, partial or 59.79±2.46% of Tuj1 positive neurons were TH-positive, while from Corin full duplications of chromosomes 12, 17, 20, and 14 were detected in some negative cells only 3.77±1.20% of Tuj1 positive cells were positive for TH. culture methods but not in others. In summary, these results suggest that The TH positive cells were also immunoreactive for Nurr1, as a marker for culture methods can significantly influence the genetic stability of hESCs, midbrain dopaminergic cells or progenitors. When grafted into non-lesioned and that the chromosomal duplications found in this study can potentially be NOD-SCID mice on 2 days after cell sorting (differentiation totally for 14-16 used in the future to screen out cell preparations not appropriate for clinical days), no tumor formation was found in the graft of Corin positive cells, but use. graft overgrowth was found in the graft of Corin negative or unsorted cells. When the Corin positive cells were grafted into the 6-OHDA lesioned rats, Poster Board Number: 3327 human cells-derived TH positive cells were found in the graft for 4 weeks THERAPEUTIC INTEGRIN-MEDIATED ECM after transplantation. We succeeded in cell sorting for dopaminergic pro- genitors using a surface marker Corin, and TH positive neurons were found REMODELING PATHWAY BY TRANSPLANTATION in the grafted 6-OHDA rats. Sorting of dopaminergic progenitor cells using a OF VASCULAR ANGIOGENIC PROGENITOR surface marker reduced the risk of graft overgrowth in rodents. We are plan- ning transplantation of the sorted cells to 6-OHDA rats and MPTP-lesioned CELLS DERIVED FROM HESCS ON VASCULAR monkeys, to make a functional evaluation. ISCHEMIC DISEASE Poster Board Number: 3325 Kim, Jumi, Chae, Jung Il, Kim, Hye Eun, Jeon, Young Joo, Lee, Min Ji, Lee, Kyung Il, Chung, Hyung-Min CULTURE METHODS INFLUENCE THE GENETIC CHA Stem Cell Institute, Seoul, Korea, Republic of, Department of Oral STABILITY OF HUMAN EMBRYONIC STEM Pharmacology, Chonbuk National University, Jeonju, Korea, Republic of CELLS Integrin mediated-ECM (extracellular matrix) remodeling is one of the critical Garitaonandia, Ibon, Wambua, Gerald K., Schultheiz, Heather, steps on vascular re-construction in specific pathological condition including ischemic vascular disease, because ECM provides a stable environment for Boscolo, Francesca, Waltz, Shannon, Tran, Ha, Wang, Yu-Chieh, cell growth, differentiation and migration. After massive ischemic injury on Leonardo, Trevor, Altun, Gulsah, Slavin, Ileana, Lynch, Candace, blood vessels, cell transplantation using stem cell source into injured ischemic Loring, Jeanne, Laurent, Louise region can improve vascular function through cell engraftment and forming Chemical Physiology, The Scripps Research Institute, San Diego, CA, USA, neo-vessels on injured site. In this study, we derived vascular angiogenic Department of Reproductive Medicine, University of California, San Diego, progenitor cell population from hESC (hESC-VAPCs) and investigate their San Diego, CA, USA therapeutic effect and unique therapeutic mechanism on hindlimb ischemia disease model. As the result, hESC-VAPCs were retaining representative Human embryonic stem cells (hESCs) and human induced pluripotent stem vascular characteristics in vitro and therapeutic effect on hindlimb ischemic cells (hiPSCs) hold enormous potential for drug screening, toxicology and model mouse. Blood perfusion rate on hESC-VAPCs transplanted group was developmental studies, disease modeling, and cell therapies. However, it is significantly improved than cord bold derived EPCs (CB-EPCs) and vehicle very important for these applications that the cell populations remain stable. medium injection transplantation group. Furthermore, to fine out the thera- A prominent concern for cell therapy in particular is that the cells used are peutic mechanism of hESC-VAPCs, we applied proteomic analysis tool be- safe and do not contain genetic aberrations that could lead to tumorigenic- tween hESC-VAPCs and EPCs. Interestingly for this study, we could confirm ity. In fact, aneuploidies frequently detected through standard karyotyping that main therapeutic behavior was caused by up-regulated participation in hESC cultures, such as trisomies of chromosomes 1, 12, and 17, are also of several ECM and alph-3/beta-1 integrin mediated signaling pathway in commonly found in clinical teratocarcinomas. It is possible that hESC cultures hESC-VAPCs. Together, these data suggest hESC-VAPCs could be a valuable can accumulate subchromosomal genetic changes undetectable by standard

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novel cellular source for therapeutic treatment of vascular ischemic disease Poster Board Number: 3331 due to their participation of up-regulated several ECM molecules and integ- rin mediated pathway. DEFINED CULTURING AND DIFFERENTIATION Poster Board Number: 3329 OF HUMAN INDUCED PLURIPOTENT STEM CELLS INTO ENDODERMAL LINEAGES USING STABILITY OF CGMP HUMAN ES CELLS A FULLY HUMAN RECOMBINANT VITRONECTIN FOLLOWING EXTENDED CULTURE PROTEIN. Ludwig, Tenneille E., Montgomery, Karen D., Ripple, Kyle, Marty, Andrea L., Monsma, Scott A., Garcia II, Bradley H. Witkowski, Marybeth A., Felkner, Daniel J., Johnson, Julie C., Taapken, Seth M., Nisler, Benjamin S., Hei, Derek J. Primorigen Biosciences Inc., Madison, WI, USA WiCell, Madison, WI, USA, Waisman Biomanufacturing, University of In order to establish clinically adaptable methods for therapeutic applications Wisconsin, Madison, WI, USA of stem cells, there is increasing interest in stem cell expansion, propagation, and differentiation under completely defined and xenobiotic-free conditions. Efficient and successful transition of pluripotent stem cell research into Previous work has shown the utility of E-cadherin fusion protein (StemAd- human therapeutics will require high quality, well characterized cell banks here™) as a suitable substrate for growth and expansion of iPS and hES capable of supporting development from research through human clinical while maintaining pluripotency. During early stages of iPS/ES differentia- trials. Pluripotent stem cells, however, pose several unique and significant tion [to lineages of all three germ layers], expression of E-cadherin is lost, manufacturing challenges including control of differentiation and genetic corresponding to the epithelial-mesenchymal transition occurring at the em- stability, properties that may be significantly influenced by cell line charac- bryonic primitive streak. Accordingly, for complete directed differentiation of teristics. For this reason, the availability of stem cell banks that have been adherent cultures, additional adhesion factors are required that can be pro- produced and tested under current Good Manufacturing Practice (cGMP) duced under xeno-free conditions, and for clinical applications, under cGMP guidelines and demonstrate long-term stability is a key component of any conditions. Vitronectin is a well-known component of the extracellular program that intends to advance a stem cell based therapeutic into human matrix that supports cell adhesion and spreading. Vitronectin alone has been clinical trials. To support translational research, WiCell Research Institute shown to support ES cell expansion and iPS cell expansion and differentia- (WiCell) has partnered with Waisman Biomanufacturing (WB) at the Uni- tion, through the cell surface receptor integrin alphaV-beta5. Integrin alphaV versity of Wisconsin to produce a human ES cell Master Cell Bank (MCB) (the vitronectin receptor) is expressed by migrating mesodermal cells during under cGMP guidelines. Wisconsin cell line H9 (WA09) has been expanded gastrulation, in all three germ layers of Xenopus embryos during gastrula- and banked under cGMP conditions using a feeder-independent, defined tion and neurulation, in hES derived definitive endoderm, and in many adult platform for cell culture and cryopreservation (mTeSR1/Matrigel). Full differentiated cell types including astrocytes and keratinocytes [ectodermal], cGMP documentation was developed for the process and extensive quality osteoclasts and lymphoid blood cells [mesodermal], and hepatocytes and control testing was performed on the MCB. In addition to standard testing pancreatic beta-cells [endodermal]. We have developed an engineered ver- for identity, genetic stability, purity, and human ES cell marker expression, sion of human vitronectin, that (alone or in combination with StemAdhere) comprehensive testing for potential adventitious agents, including mu- is capable of supporting pluripotent cell growth and expansion as well as rine adventitious agents, was performed in compliance with FDA and ICH supporting cell adhesion during directed differentiation of iPS cells into en- guidelines. cGMP banking itself, however, is only part of the puzzle. The dodermal lineages such as adult-like hepatocytes and pancreatic beta-cells. long-term stability of the chosen cell line is critical, particularly considering Recombinant vitronectin possesses advantages compared to human-plasma the culture time required to take pluripotent cell lines through differentiation derived vitronectin, including reduced batch-to-batch variability, reduced risk protocols to the final cell product. Concerns regarding stability in extended of adventitious agents, and reduced cost. culture are well founded, as many groups have reported extensive spon- taneous differentiation and karyotypic abnormalities following extended Poster Board Number: 3333 culture, particularly in feeder-free conditions. To address these concerns and further characterize the cell bank, we performed extended culture and ONE-YEAR OBSERVATION OF DOPAMINERGIC assessment of the cGMP H9 bank produced at WB. Cells were thawed into NEURONS DERIVED FROM HUMAN mTeSR1 on Matrigel, and cultured for an additional 50 passages. Karyotype was assessed by g-band every 10 passages through P+50, with STR, marker EMBRYONIC STEM CELLS IN PRIMATE expression (Oct3/4, SSEA3, SSEA4, Tra 1-60, Tra 1-81 and SSEA1), cell MODELS OF PARKINSON’S DISEASE morphology and array comparative genome hybridization (aCGH) assessed Morizane, Asuka, Doi, Daisuke, Kikuchi, Tetsuhiro, Yoshikawa, at early and late passages. cGMP banked cells maintained normal cell mor- phology (photomicrograph), normal karyotype (46, XX by g-band) through Tatsuya, Onoe, Hirotaka, Hayashi, Takuya, Kawasaki, Toshiyuki, 50 passages of culture beyond the MCB. Additionally, late passage material Sasai, Yoshiki, Suemori, Hirofumi, Takahashi, Jun maintains appropriate marker expression (>90% Oct3/4, SSEA3, SSEA4, Tra Cell Growth and Differentiation, Center for iPS Cell Research and 1-60, Tra 1-81 and <5% SSEA1 marker expression by flow cytometry) , and Application, Kyoto University, Kyoto, Japan, Functional Probe Research the aCGH profile is consistent with karyotype reports [46, XX] (by Nimble- Laboratory, RIKEN Center for Molecular Imaging Science, Kobe, Japan, Gen 385K array). Taken together, these results demonstrate the stability Organogenesis and Neurogenesis Group, RIKEN Center for Developmental of the cGMP bank produced by WiCell and WB, and further validate its Biology, Kobe, Japan, Stem Cell Research Center, Institute for Frontier potential usefulness for human clinical applications. Medical Sciences, Kyoto University, Kyoto, Japan Background: Cell therapy for Parkinson’s disease (PD) with human pluri- potent stem cells has attracted much attention. Before the clinical applica- tion, we should estimate its long-term efficacy and safety with non-human primate PD models because they live considerably longer than rodents. Primates also have larger brains compared to rodents, which enables us to inject more cells and to evaluate the results with magnetic resonance imag- ing (MRI) and positron emission tomography (PET). In addition, primate PD models treated with MPTP have pathological and symptomatic similarities to

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Thursday Poster Abstracts human PD patients. Our results here offer a possible usage of stem cell- therapy for Alzheimer’s Methods: In this study we transplanted human ES cell-derived dopamine disease. (DA) progenitors into the putamen of primate PD models. First the monkeys received intravenous injection of MPTP until they developed PD symp- Poster Board Number: 3337 toms. Once their symptoms got stable for at least 6 months, the animals INTERACTIONS BETWEEN MOUSE EMBRYONIC were used for the transplantation study. The human ES cells (KhES-1) were differentiated for 35 or 42 days to generate DA progenitors. We stereot- STEM CELL-DERIVED NEURAL PROGENITORS actically transplanted the cells into the bilateral putamen of PD monkeys AND THE HOST BRAIN (control n=4, d35 n=2, d42 n=5), which received daily immunosupression with FK506 until sacrificed. We monitored the behavioral recovery of the Lassiter, Chelsea, Becker, Sandy, McGill, Sean, Hartman, Nathaniel monkeys with a neurological PD rating scale, a video-based activity assess- W., Grabel, Laura ment system, and a raisin-grasping test, which allows assessment of fine Biology, Wesleyan University, Middletown, CT, USA, Hampshire College, movements. Results: Both histological and image analyses showed that the Amherst, MA, USA, Yale University, New Haven, CT, USA grafted cells could survive at least for one year. The image analyses with MRI and PET revealed that the graft growth stopped by 6 months after Embryonic stem cell (ESC)-derived transplants hold great promise as cell transplantation in most cases. We performed histological analyses of the replacement therapies for neurodegenerative diseases. However, it is surviving graft at one year after transplantation. We observed no teratoma important to first understand the host brain conditions that will lead to a component or overgrowth with mass effect in any animals. All grafts consist- successful transplant. We use a lesion model, transplanting mouse or human ed of neural component with high frequency (≈40%) of NeuN positive cells. ESC-derived neural progenitors (ESNPs) into the hippocampi of mice that Among them, 2-10% of the cells were also positive for tyrosine hydroxylase have been given a kainic acid-induced seizure. Within a week after trans- (TH), a DA neuronal marker. In the grafts we observed areas with high cel- plantation, the ESNPs have migrated posteriorly along the upper blade of lular density in some parts of the grafts. No Oct3/4 positive cell existed but the dentate gyrus (DG), replacing the endogenous cells of the upper blade some scattered Ki67 positive cells (0.2-2.4% per total cells) were detected in that have degenerated due to the fluid injection. After four weeks the trans- those areas. Successful elimination of residual pluripotent stem cells might be planted ESNPs have integrated into the upper blade, and express the granule attributed to the appropriate selection of the hESC line, the efficient protocol cell marker Prox1. Our preliminary data suggest that the transplant recruits for neural induction, and the sufficient differentiation period in vitro. The host endothelial cells from the surrounding hippocampus and that they form animal with the highest number of surviving grafted DA neurons (TH+ cells vessels within a week. The number and size of blood vessels is increased in > 20,000) showed behavioral improvement. Conclusion: Primate model is a and around the DG on the side of the brain receiving a transplant versus the powerful experimental tool with many advantages over rodents especially contralateral control (uninjected) side. We are currently confirming the role in a pre-clinical study. Dopamine neurons derived from hESCs could survive of the transplant in recruiting blood vessels by comparing hippocampi with in the brain of primate PD models without generating tumor. If a sufficient ESNP transplants to those receiving only a fluid injection. In addition we number of TH positive cells survived in the grafts, the functional recovery observe that migrating ESNPs are found in close proximity to endogenous can be expected in the model animals. blood vessels. Our previous data suggest that the posterior migration of the transplanted cells occurs in response to the chemokine CXCL12 (SDF-1), and Poster Board Number: 3335 we now show that this chemokine is present on the host vasculature. Our previous data demonstrated that the CXCL12 receptor CXCR4 is expressed TRANSPLANTATION OF EMBRYONIC STEM on ESNPs. Since the vasculature is coated with laminin, we are investigating CELLS-DERIVED BASAL FOREBRAIN the possible role of the integrin receptor 61 in mediating migration along the blood vessels. We are also using hippocampal brain slice culture as an ex CHOLINERGIC NEURONS IMPROVES THE vivo method for studying this migration and its regulation. Our data so far COGNITIVE IMPAIRMENT OF ALZHEIMER’S suggest a role for blood vessels in supporting the transplant and in providing DISEASE MICE a migratory scaffold. Yue, Wei, Jiang, Man, Zhang, Min, Li, Yuanyuan, Sheng, Nengyin, Poster Board Number: 3339 Qian, Yun, Shu, Yousheng, Jing, Naihe THE AUTISM IPSC BIOREPOSITORY: Institute of Biochemistry & Cell Biology, Shanghai, China, Institute of Neuroscience, Shanghai, China AN INTERNATIONAL RESOURCE FOR INVESTIGATORS STUDYING THE AUSTISM Degeneration of basal forebrain cholinergic neurons (BFCNs) and the result- ed decrease of cholinergic transmission contribute to the cognitive deficits in SPECTRUM DISORDERS Alzheimer’s disease (AD), and clinically the most widely prescribed drugs for Schwartz, Philip H., Brick, David J., Nethercott, Hubert E., Stover, the treatment of AD patients are acetylcholinesterase inhibitors. However, Alexander E., Hagerman, Randi J., Ono, Michele Y., Hessl, David R., the therapeutic strategy of stem cells-derived BFCNs transplantation, which aims at restoring cholinergic function and cognitive impairment, remains Lily, Ngotran, Tassone, Flora elusive. Here, using a serum free differentiation system with sequential Shh Neuroscience, Children’s Hospital of Orange County Research Institute, and BMP9 treatment, cholinergic neurons with basal forebrain identity are Orange, CA, USA, Pediatrics, MIND Institute, University of California, efficiently derived from mouse embryonic stem cells (ESCs) and induced Davis Health System, Sacramento, CA, USA, Psychiatry and Behavioral pluripotent stem cells, as well as human ESCs. Moreover, the matured Sciences, MIND Institute, University of California, Davis Health System, ESCs-derived BFCNs can secrete acetylcholine and form active postsynaptic Sacramento, CA, USA, Biochemistry and Molecular Medicine, University of neuronal network in vitro. After grafted with progenitor cells of the ESCs- California, Davis, Davis, CA, USA derived BCFNs into the nucleus basalis of Meynert (NBM), the cognitive Autism Spectrum Disorders (ASDs) are the fastest growing developmental deficits of two AD mice models are improved significantly. Further analyses disorder in the United States and are, unfortunately, still poorly under- indicate that most of the survival cells are distributed in NBM and character- stood. A critical barrier in autism research is the accessibility to statistically ized with cholinergic neuronal identity. More importantly, the grafted cells relevant numbers of patient-derived tissues, especially neural tissues. The are electrophysiologicaly integrated into NBM cholinergic projection system, derivation of induced pluripotent stem cells (iPSCs) by somatic cell repro- which may contribute to the improvement of cognitive behavior of AD mice. gramming offers the potential for drug screening, toxicology, and basic

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research of patient-specific ASD cell lines. Here, we present the develop- Poster Board Number: 3343 ment of an ASD iPSC resource that consists of patient-derived human skin fibroblasts and iPSCs that are documented by a detailed clinical diagnosis STEMDIFF™ APEL™ IS AN ANIMAL (ADOS, ADI-R). The resource conducts its own research into the causes of COMPONENT-FREE MEDIUM WHICH autism and also provides ASD-specific cells and training to autism research- ers world-wide. Human ASD skin biopsies from well-characterized patient SUPPORTS MULTI-LINEAGE DIFFERENTIATION groups (Autism, PDD-NOS and FMR1 mutations) were obtained at the OF HUMAN PLURIPOTENT STEM CELLS UC Davis MIND Institute. ASD dermal fibroblast cultures were derived and expanded. Fibroblasts were transduced with lentiviruses expressing the Antonchuk, Jennifer, Riedel, Michael J., Fairey, Mark, Hilcove, reprogramming factors: OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG. Simon, Elefanty, Andrew, Ng, Elizabeth, Thomas, Terry E., Eaves, By day 16 post-transduction, the morphology of reprogrammed ASD-iPSCs Allen C., Louis, Sharon A. appeared identical to those of human embryo-derived pluripotent (embry- STEMCELL Technologies Inc, Vancouver, BC, Canada, Embryonic Stem onic) stem cells (hESCs). Classical immunocytochemical staining patterns Cell Differentiation Laboratory, Monash Immunology and Stem Cell (Tra-1-60, Oct-4, and Nanog) for hESCs were also uniformly detected in the Laboratories, Melbourne, Australia, B.C. Cancer Agency and, STEMCELL reprogrammed ASD-iPSCs. Further expansion and neural differentiation is Technologies Inc, Vancouver, BC, Canada accomplished using single-cell passaging and adherent culture, obviating the need for manual passaging and embryoid body formation. Thus, iPSCs can Human pluripotent stem cells (hPSCs), including embryonic stem cells be successfully generated from patients with ASDs. These ASD-iPSCs can be (hESC) and induced pluripotent stem cells (hiPSC), are characterized by their differentiated toward a neural stem cell lineage using methods developed in potential to differentiate into any somatic cell lineage. A diverse set of multi- our laboratory. A comparative analysis of these ASD-specific lines with unaf- step directed differentiation protocols have been described to induce differ- fected controls will allow new insights to be obtained into the cause(s) and entiation to desired cell types, using a variety of media formulations, in em- biology of the ASDs. This cell bank and methodologies supported by this bryoid body (EB)-based or adherent cell-based cultures. We have developed resource are expected to to serve as an international resource for investiga- STEMdiff™ APEL™, a fully defined, animal component-free medium based tors interested in the study of the ASDs. on the publication of Ng et al, which can be used as a base medium for dif- ferentiation of hPSCs to multiple lineages, in EB or adherent cell platforms. Here we demonstrate the utility of STEMdiff™ APEL™ as a base media for cardiomyocyte, hematopoietic or definitive endoderm differentiation using EMBRYONIC STEM CELL DIFFERENTIATION cytokine combinations from the literature. For cardiomyocyte differentiation, EBs of 2,000 cells each were formed and cultured in AggreWell™400 plates Poster Board Number: 3341 in STEMdiff™ APEL™ for 16 days. STEMdiff™ APEL™ was supplemented ANALYSIS OF HCN EXPRESSION IN with Y27632 for the first 24-hours for EB formation, followed by BMP4, Activin A, and Wnt3a for 4 days, and then VEGF and Dkk1 for the remain- HUMAN EMBRYONIC STEM CELL-DERIVED ing 11 days, as published. At the end of the 16-day culture period, beating CARDIOMYOCYTES EBs were counted and cells isolated from the EBs were analyzed by flow cytometry for the cardiomyocyte marker cTnT. The results showed that up Kim, Yoon Young, Ku, Seung-Yup,, Kim, Sung Joon, Oh, Sun to 95% of EBs were synchronously beating (67 ± 45%, mean ± SD; n=3), Kyung,, Kim, Seok Hyun,, Moon, Shin Yong,, Choi, Young Min, and up to 36% of cells were cTnT+ (18 ±16%, mean ± SD; n=3). Secondly, IRMP, Medical Research Center, Seoul National University, Seoul, Korea, STEMdiff™ APEL™ was tested in an adherent cell-based protocol for he- Republic of, IRMP, Medical Research Center, Dept. of Ob&Gyn, Seoul matopoietic differentiation. Undifferentiated hESC aggregates were allowed National University College of Medicine, Seoul, Korea, Republic of, Dept. to attached onto Matrigel™-coated plates in mTeSR™1 for 2 days, and of Physiology, Seoul National University College of Medicine, Seoul, Korea, then the media was replaced with STEMdiff™ APEL™ supplemented with Republic of VEGF, BMP4, SCF, and Activin A for 4 days to induce mesoderm formation, followed by STEMdiff™ APEL™ supplemented with a host of hematopoi- Human embryonic stem cells (hESCs) possess pluripotency that can dif- etic cytokines for a further 9 days. At the end of the 13-day culture period, ferentiation into all kinds of cells in the body. Therefore, differentiation cells were harvested, analyzed by flow cytometry for the hematopoietic into cardiomyocytes (CMs) and analysis of their functionalities are widely marker CD34, and plated in standard colony-forming cell (CFC) assays. This investigated. hESC-derived cells showed expressions of cardiac specific resulted in 34 ± 23% CD34+ cells (mean ± SD; n=6), with a CFC frequency genes in previous reports. In this study, we investigate expression of hy- of 1/5,000 or 1/12,000 cells (n=2). Finally, we tested the application of perpolarization and cyclic nucleotide-activated (HCN) family, which have STEMdiff™ APEL™ in the differentiation of hESCs to definitive endoderm specific roles in autonomic rhythm in heart function. Undifferentiated hESCs in an adherent cell-based protocol. A single cell suspension of H9 hES cells were induced to differentiate with 10 ng/ml of BMP2 for 4 days in RPMI was plated onto Matrigel™-coated plates containing mTeSR™1 to form an medium. After 18 days of differentiation, total RNAs were extracted using adherent culture. The next day media was replaced with STEMdiff™ APEL™ Trizol. cDNA was synthesized using 1 ug of RNA and the expressions of supplemented with Wnt3a, Activin A, and bFGF as published. On day 4, HCN family were analyzed using RT-PCR. Also, the intracellular Ca++ tran- flow cytometry analysis showed that 90 ± 1% (mean ± SD; n=3) of the cells sient in hESC-derived beating cluster was analyzed using calcium-specific expressed the definitive endoderm marker CXCR4. In summary, these results dye, fluo-4. hESC-derived CMs showed regular beating and expression of demonstrate the wide utility of STEMdiff™ APEL™ in a variety of protocols, calcium specific channel-related genes such as HCN-1, -2, -4. In addition, for induction of differentiation to multiple cell lineages. This defined and the increase and decrease of fluorescence were confirmed in contractile robust base medium will facilitate the standardization and development of region. The fluorescence was highest at 18 days of differentiation. In this efficient protocols for directed differentiation, as our understanding of the study, we demonstrated the existence and expression of HCN family that molecular pathways controlling lineage specification and differentiation have roles as pacemaker channel, in hESC-derived cells. Therefore, this continues to progress. result suggested the functional activity of hESC-derived CMs. This work was supported by National Research Foundation of Korea Grant funded by the Korean Government (No. 2010-0016819) and a grant (SC1150) from Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology.

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Poster Board Number: 3345 Poster Board Number: 3347 SUSPENSION CULTURE AND CARDIAC PARTHENOGENETIC HUMAN ES CELLS FORM DIFFERENTIATION OF HUMAN INDUCED FUNCTIONAL NEURONS. PLURIPOTENT STEM CELLS Ahmad, Ruhel, Wolber, Wanja, Koch, Philipp, Eckardt, Sigrid, Geis, Fonoudi, Hananeh, Adhami, Bahareh, Fattahi, Faranak, Ghazizadeh, Christian, Heckmann, Manfred, McLaughlin, K. John, Brüstle, Zaniar, Hosseini Salekdeh, Ghasem, Aghdami, Nasser Oliver, Sirén, Anna-Leena, Müller, Albrecht M. Stem Cells, Royan Institute, Tehran, Iran, Islamic Republic of, Cell Therapy MSZ in the Center for Experimental Molecular Medicine (ZEMM), Center, Royan Institute, Tehran, Iran, Islamic Republic of University of Wuerzburg, Wuerzburg, Germany, Dept of Neurosurgery, University of Wuerzburg, Wuerzburg, Germany, Institute of Reconstructive Human induced pluripotent stem cells (hiPSCs) have the potential to provide Neurobiology, Life and Brain Center, University of Bonn, Bonn, Germany, an unlimited source of cardiomyocytes that offers a precious tool for drug Nationwide Children’s Research Institute, The Ohio State University, discovery and regenerative medicine. However, this application is limited due Columbus, OH, USA, Neurology, University of Wuerzburg, Wuerzburg, to difficulties in large scale expansion of hiPSCs in aherent culture conditions Germany, Physiology, University of Wuerzburg, Wuerzburg, Germany and insufficient efficiency of differentiation protocols. In order to overcome these problems, we present an efficient protocol for expansion and dif- Pluripotent parthenogenetic embryonic stem cells (ESC) from several species ferentiation of hiPSCs in suspension as spheroids. In this study, hiPSCs were including human have been successfully generated and differentiated into treated by Rho-associated kinase (ROCK) inhibitor prior to their dissociation neural and other cell types. In addition, successful gene therapy was recently into single cells and then cultured in suspension in medium conditioned by demonstrated in a mouse model of beta-thalassemia by allele selection using mouse embryonic fibroblasts. After 2 days each single cell forms a spheroid parthenogenetic ESCs. This genetic correction strategy without gene target- which can be passaged every 7 to 9 days. In order to differentiate the hiP- ing is potentially applicable to any dominant disease. Therefore, parthenoge- SCs towards cardiomyocyte, six days after the spheroid formation, they were netic human embryonic stem cells (hpESCs) are promising sources for basic treated one day by Activin A followed by their subsequent treatment with research to study the influence of maternal and paternal genomes on organ BMP4 for four days, the spheroids are then plated on gelatin coated culture development, and potentially for future cell replacement strategies. Here, we dishes. The expression profile of cardiac specific genes was determined by investigated the in vitro differentiation capability of hpESCs into neural stem real-time PCR in every first 15 days of differentiation and Their expression cells (hpNSCs) and further into neural subtypes including midbrain TH-pos- at the protein level were evaluated by immunofluorescence staining and itive neurons. In parallel we examined functional aspects and the imprinting flow cytometry in the final stage of differentiation. Verification of the cardiac status of parthenogenetic cells. hpESCs were in vitro differentiated into cells’ functionality was tested by electrophysiological recordings. In this hpNSCs as previously described for normal, biparental human ESCs. Expres- protocol, beating clusters were observed 5 days post-plating and up to 20% sion analyses showed that hpESC-derived NSCs expressed the neural stem of colonies could beat at the same time, while no beating was observed in cell markers Nestin, Pax6, Sox1, Sox2 and Vimentin and lost expression of the cells differentiated with the same protocol in adherent conditions. Gene the pluripotency markers Oct4 and Nanog both at RNA and protein levels. expression data shows that after addition of Activin A, mesodermal markers Upon spontaneous in vitro differentiation for 28 days hpNSCs generated such as brachury are expressed which then leads to expression of Isl1, a neural subtypes with specific neural morphology and expression of neural/ marker of precardiac mesoderm, after treatment by BMP4. Five days after glial markers such as Tuj1, Map2, Tau, Synapsin and GFAP. hpNSCs were plating, cardiac progenitors are formed which are positive for Nkx2.5, Gata4, also responsive to instructive regionalization cues known to induce regional Tbx5 and Mef2c. In final stage of differentiation cells express Mlc2a/v, MHC phenotypes such as midbrain TH-positive neurons. After 29 days in vitro, and cardiac troponin which are the markers of mature cardiomyocytes. Our hpNSC-derived neurons had a mature resting membrane potential, imma- data also indicates that, more than 80% of the cells expressed Nkx2.5, five ture spike pattern and showed typical neuronal Na+/K+ currents. Expression days after plating and more than 70% of cells expressed connexin 43, myo- analysis of imprinted brain genes in hpESCs and in hpNSCs as compared to sin heavy chain and desmin 10 days later. Electrophysiological recording of biparental human ESCs and NSCs revealed that the maternal-specific im- contractile clusters also verified their functionality. In conclusion, our results printing is by and large maintained upon differentiation. In summary, despite suggest that the combination of culturing hiPSCs in suspension and using the lack of a paternal genome, hpESC-derived NSCs show normal neural the Activin A/BMP4 protocol for differentiation have the ability to form in- differentiation potential and could therefore be therapeutically relevant for creased numbers of functional contractile cardiac cells with higher efficiency future cellular replacement strategies. compared to the previous two-dimensional differentiation methods. Another Poster Board Number: 3349 major advantage of this method is its applicability in large scale culture and differentiation of cells in bioreactors. EFFICIENT PRODUCTION OF NEURAL PROGENITOR CELLS FROM HUMAN PLURIPOTENT STEM CELLS USING STEMdiffTM NEURAL INDUCTION MEDIUM Blak, Alexandra A., Yoshida, Eileen, Antonchuk, Jennifer, Thomas, Terry E., Eaves, Allen C,, Louis, Sharon A. STEMCELL Technologies Inc, Vancouver, BC, Canada, STEMCELL Technologies Inc. and Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada Induction of neuroectoderm from human pluripotent stem cells (hPSCs), which include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), is the first step in differentiation protocols used to produce neural progenitor cells and more specialized cell types of the CNS such as neurons, astrocytes and oligodendrocytes. In many neural induction protocols, the formation of aggregates or so called embryoid

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bodies (EBs) from undifferentiated human pluripotent stem cells is employed embryonic bodies (EBs), with the purpose of promoting neuroectodermal to direct these cells towards a neural fate. However, the routinely used differentiation. As anticipated, this treatment blocked the expression of “scraped” or “spin” EB protocols result in non-uniformly shaped EBs of Nodal and when compared to control cultures, addition of BMP4 resulted varying sizes which can lead to inefficient and uncontrolled differentiation in a significant increase of cells with the ectodermal marker Pax6, as well outputs downstream. In order to improve the consistency of neural induc- as cells with markers for sensory neuronal progenitors Ngn1 and NeuroD. tion from hPSCs, we have developed two novel reagents: 1) STEMdiffTM An additional approach, to enrich for sensory neuron differentiation, was to Neural Induction Medium (NIM) for the rapid and efficient induction of expand EBs in adherent culture using neural induction medium (NIM) for neural progenitor cells (NPCs) from human ESCs and iPSCs, and 2) STEMdif- four (NS4), seven (NS7) or eleven (NS11) days; followed by additional dif- fTM Neural Rosette Selection Reagent (NRSR) for the selective isolation of ferentiation in culture for various time periods. At most, a frequency of 20% “neural rosettes” - an in vitro structure commonly accepted as an indicator sensory neurons was achieved, as indicated by markers of sensory neurons of early NPC induction. In addition, we have optimized protocols, which (peripherin, TrkB and TrkC). Thus, our results reiterate previously described combine the use of these two new reagents with the AggreWell™800, difficulties to selectively obtain highly enriched populations of sensory which is a system to generate uniform sized aggregates in the first step neurons, but indicate possible routes towards this goal. A combination of of the neural induction protocol. Aggregates containing 5000 - 20,000 the above approaches is presently under evaluation. Obtaining higher-grade cells each were formed from hPSCs in STEMdiffTM NIM using the Ag- populations of sensory neuron may have implications for the treatment of greWell™800 procedure and the aggregates were maintained within the sensory deficits such as hearing impairment. AggreWell™800 plates with daily ½ media changes for 5 days. On day 5, aggregates were then harvested from AggreWell™800 plates and sub-cul- Poster Board Number: 3353 tured onto Poly-L-Ornithine/laminin (PLO/L) coated plates in STEMdiffTM NEURONAL DIFFERENTIATION OF HUMAN NIM for a total of 6 days. At day 2 in culture, the colonies were observed for the presence of “neural rosettes” and these were enumerated. On day EMBRYONIC AND INDUCED PLURIPOTENT 6, neural rosette-containing areas were selectively isolated using a combina- STEM CELLS ON 3D SCAFFOLDS: A KEY tion of the STEMdiffTM NRSR and mechanical dissociation. The harvested rosettes were then re-plated onto PLO/L coated plates containing STEM- SOLUTION FOR TRANSPLANTATION THERAPY diffTM NIM to generate adherent cultures. Cells were cultured until they Blumenthal, Jacob, Chen-Konak, Limor, Levenberg, Shulamit reached ~ 90-100% confluence before they were harvested by enzymatic Biomedical Engineering Faculty, Technion - Israel Institute of Technology, dissociation and then either sub-cultured multiple times in a proliferation Haifa, Israel medium (in development) or subjected to differentiation conditions. Our results showed that after 2 days in STEMdiffTM NIM, the attached neural Conventional therapies for many neurological disorders alleviate symptoms, aggregates had formed colonies and 90-100% of the colonies contained with most patients remaining in a state of sustained disability. The trans- “neural rosettes”. Characterization of neural rosette structures by immu- plantation of specific neuronal or glial cells as a disease modifying therapy nocytochemistry revealed that the cells within neural rosettes co-expressed is limited because of the unavailability of the appropriate donor tissue, and the early neural markers, Pax6, Sox1 and Nestin, thus confirming that the the questionability of clinical outcome. Human embryonic stem cells (hESC) first step in the aggregate-based protocol for neural induction using our are an attractive cell source for regenerative medicine applications, as they Neural Induction Medium rapidly yielded early-stage NPCs. We found that possess the potency to differentiate into all mature cell phenotypes including these early-stage NPCs maintained their ability to re-form neural rosettes dopaminergic and motor neurons. Although various neuronal differentiation structures after sub-culture in STEMdiffTM NIM. Finally, the NPCs also protocols are available, the ability to use mature neurons for transplanta- maintained their proliferative and multi-lineage potential as they could be tion is limited, due to the vulnerability of their axons. In order to overcome further passaged up to 3 times in a proliferation medium and differentiated this limit, we examined the feasibility of neuronally differentiating hESC and into mature neurons and astrocytes as determined by the expression of TUJ1 human induced pluripotent stem cells (hiPS) on 3D biodegradable scaffolds. and GFAP, respectively. This work describes a highly efficient system, which These scaffolds can be further used for transplantation into the brain and will increase consistency in protocols for the induction of neural progenitor spinal cord. We differentiated hESC and hiPS cells into spinal motor neurons cells and their progeny from hPSCs. on PLLA/PLGA scaffolds. The first part of the differentiation protocol was carried out on 2D tissue culture plates. When neural rosettes appeared, Poster Board Number: 3351 the cells were lifted and seeded as single cells on 3D scaffolds. Following exposure to retinoic acid and sonic hedgehog the cells acquired a neuronal GENERATION OF PERIPHERAL SENSORY phenotype. The differentiated neurons expressed GFP under the control of NEURONS FROM HUMAN EMBRYONIC STEM the Hb9 motor neuron specific promoter and were co-stained with the neu- CELL-DERIVED NEURAL PROGENITORS ronal antibody βIII-tubulin. In addition, hiPS were also shown to differentiate into astrocytes on the scaffolds, as demonstrated by GFAP staining. Overall, Ali, Rouknuddin Q., Cheng, Yenfu, Kostyszyn, Beata, Ährlund- our preliminary results clearly demonstrate the potential of 3D biodegrad- Richter, Lars, Ulfendahl, Mats able scaffolds seeded with hESC and hiPS differentiated neurons for future Center for Hearing and Communication,, Karolinksa Institution, Stockholm, in-vivo transplantation. Ongoing experiments are currently underway to Sweden, Department of Women and Child Health, Karolinksa Institution, further examine the differentiation process and the ability of various scaf- Stockholm, Sweden folds to support this procedure. Hearing impairment is primarily caused by the degeneration of auditory sensory neurons and hair cells. It has been suggested that a cell therapy approach could offer new means of treatment. Human embryonic stem cells (hESC) have under appropriate conditions been demonstrated to hold the capacity to differentiate into adult neural cell types, including cells with markers for sensory neurons. A number of signalling pathways have been indicated in the maintenance of hESC pluripotency and blocking of these pathways may induce/promote neuroectoderm formation. In the pres- ent study, using a selective inhibitor of Activin receptor like kinase 4/5/7 (SB-431542), we have obstructed Nodal signalling in cultured hESC derived

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Poster Board Number: 3355 DCX- cells transition into the Pax6+/Sox2+/Sox1+/DCX- cells over time, implying that these subpopulations are temporally related. We observed THE SPECIFICATION OF NOCICEPTIVE an upregulation of CD146 (MCAM) in the Pax6+/Sox2+/Sox1+/DCX- cell NEURONS FROM HUMAN EMBRYONIC STEM subpopulation; therefore, CD146 may be useful in identifying and isolating this cell subpopulation for subsequent analysis. We also observed that the CELLS Pax6-/Sox2-/Sox1-/DCX+ cell subpopulation expressed cell surface markers Boisvert, Erin M., Hallowell, Shawn E., Engle, Sandra J., Li, Xue-Jun associated with neurons: CD171 (NCAM L1), CD271 (NGFR) and CD56 (NCAM), and we demonstrate that this subpopulation is highly composed Genetics and Developmental Biology, University of Connecticut Health of postmitotic neurons. These identified cell surface markers and screening Center, Farmington, CT, USA, Genetically Modified Models RCOE, Pfizer tools will permit the isolation and subsequent analysis of cell subpopulations Inc., Groton, CT, USA, Neuroscience, University of Connecticut Health and add to the growing list of markers that can be used to identify neural Center, Farmington, CT, USA cell types derived from pluripotent stem cells. How to direct the differentiation of human embryonic stem cells (hESCs) into functional nociceptor neurons, which are responsible for translating the Poster Board Number: 3359 sensation of pain to the nervous system, remains to be elucidated. Based A NOVEL NODAL ANTAGONIST THAT IS on our successful establishment of a chemically defined system to generate neural lineage cells and spinal progenitors from hESCs, we aim to investi- NECESSARY AND SUFFICIENT FOR HUMAN gate the specification and maturation of nociceptor neurons from hESCs by NERVOUS SYSTEM INDUCTION establishing a chemically defined system. Human ESCs were first differenti- ated into the neural lineage using our system as previously described. The Boles, Nathan, Le, Sheila, Lotz, Steven, Phoenix, Timothy N., addition of retinoic acid and bone morphogen factor 4 (BMP4) at specific Fasano, Christopher A. time points and concentrations yielded a sufficient population of neural crest Neural Stem Cell Institute, Rensselaer, NY, USA, St Jude’s Children Hospital, progenitor cells (AP2a+, P75+). These progenitors were then dissociated and Memphis, TN, USA plated on polyornithine/laminin coated coverslips for terminal differentia- tion in the presence of neurotrophic factors (nerve growth factor, glial cell With the advent of human pluripotent cell technology, the generation of line-derived neurotrophic factor, growth differentiation factor 7). Several neural cell types to study and treat neural disorders has become a realistic weeks after differentiation, nociceptive neurons (TrkA+) were generated in endeavor. With the recent advances in generating neural cell types in a more the cultures. These mature neurons also stained positive for sodium channel defined manner, systematic experiments can now be performed addressing markers (Nav 1.7) as well as P2X3 which plays a role in the peripheral re- how the human embryo induces neural tissue. To date, very little is known sponse to pain. Thus this study provides insights as to some of the important about the molecular events occurring during human neural induction, and factors which may play a significant role in our ability to yield nociceptive critical differences from rodent and human studies in neural development neurons from human embryonic stem cells. The function of these nociceptor have been recently reported. Here we show a secreted protein, is transiently neurons and the mechanisms underlying the generation of these neurons expressed during human development and is necessary and sufficient for are under further investigation. neural induction. Loss of function studies show human embryonic stem cells (hESCs) are significantly impaired in generating neural cells, while gain in Poster Board Number: 3357 function results in robust, spontaneous neural differentiation. Importantly, conditioned media from fibroblasts expressing this protein can induce neural IMMUNOPHENOTYPING CELL differentiation of hESCs. These results are accompanied by a time course SUBPOPULATIONS DEFINED BY EXPRESSION transcriptome analysis and functional assays revealing a role for this protein as a TGF Beta family antagonist, specifically inhibiting the Nodal-CRIPTO OF SOX1, SOX2, PAX6 AND DOUBLECORTIN pathway, a process previously described in lower vertebrates to be essen- IN NEURAL INDUCTION CULTURES OF tial for neural induction. Our study reveals a novel TGF Beta antagonist HUMAN EMBRYONIC STEM CELLS BY FLOW expressed transiently during human development that, by itself, can initiate neural induction in hESCs. These results provide critical insight into how hu- CYTOMETRY man embryos induce a nervous system as well as a novel method for neural Martin, Jody, Vidal, Jason G., Emre, Nil, Carson, Christian T. induction from hESCs by the addition of a single protein. BD Biosciences, La Jolla, CA, USA Poster Board Number: 3361 The differentiation of human pluripotent stem cells toward neural ectoderm DERIVATION OF FUNCTIONAL CRANIAL presents a unique opportunity to study human neurogenesis and neuro- degenerative diseases. Distinct cell subpopulations that represent develop- HUMAN PLACODE CELLS AND SENSORY mental transition points in neural induction cultures have been identified. NEURONS FROM HUMAN EMBRYONIC STEM However, we are just beginning to understand the significance of the dif- ferent cell subpopulations in modeling neural development and disease and CELLS their relevance for therapeutic applications. Moreover, it may be possible Dincer, Zehra, Studer, Lorenz to identify additional uncharacterized cell subpopulations relevant to these Weill Cornell Medical School/Memorial Sloan Kettering Cancer Center, pursuits. We analyzed neural induction cultures by intracellular flow cytom- New York, NY, USA, Developmental Biology, Memorial Sloan Kettering tery with antibodies to the neural stem cell markers Pax6, Sox1 and Sox2 Cancer Center, New York, NY, USA and the neuronal marker doublecortin (DCX), and identified four distinct cell subpopulations: Pax6+/Sox2-/Sox1-/DCX-, Pax6+/Sox2+/Sox1+/DCX-, A major challenge in hES research is to develop efficient differentiation Pax6-/Sox2+/Sox1+/DCX- and Pax6-/Sox2-/Sox1-/DCX+. Subsequently, protocols to derive specific cell types that can be translated directly into we performed an unbiased screen of 242 antibodies to cell surface markers therapeutics. Here, we report for the first time on the in vitro derivation of on neural induction cultures and co-labeled for Pax6, Sox1, and DCX. From cranial placodes from human embryonic stem cells (hESCs) and their further this screen, we identified putative signatures for Pax6+/Sox2-/Sox1-/DCX-, specification into diverse subplacodes, including trigeminal placodes, anterior Pax6+/Sox2+/Sox1+/DCX- and Pax6-/Sox2-/Sox1-/DCX+ cell subpopula- pituitary placodes and lens placodes. Placode cells are the developmental tions. Probability state modeling suggests that the Pax6+/Sox2-/Sox1-/ precursors of sensory organs, including the eye, the nose and the inner ear.

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Thursday Poster Abstracts

To date, relatively little is known about placode development because of the Poster Board Number: 3365 inaccessibility of fetal tissue and the lack of specific markers for the lineage. Most of our knowledge comes from species such as zebrafish, chick, and OPTIMIZED METHOD FOR THE DERIVATION frog and, to a lesser extent, mouse. Our new protocol mimics development, OF MIDBRAIN DOPAMINE NEURONS FROM resulting first in the induction of placode progenitor cells expressing Six1, Eya1, and Dlx3 with a purity of 71%, which can be further differentiated HUMAN EMBRYONIC STEM CELLS IMPROVES into a pure population of sensory neurons expressing Isl1, Brn3a, Tuj1 and SURVIVAL IN VIVO Peripherin. These cells are appropriate for modeling developmental diseases, transplantation and the testing of novel drugs involved in sensory function Kriks, Sonja, Shim, Jae-won, Piao, Jinghua, Ganat, Yosif, Tabar, including pain. Viviane, Studer, Lorenz Developmental Biology, Memorial Sloan-Kettering Cancer Center, New Poster Board Number: 3363 York, NY, USA, Neurosurgery, Memorial Sloan-Kettering Cancer Center, WNT/β-CATENIN SIGNALING PROMOTES New York, NY, USA MIDBRAIN SPECIFICATION OF NEURAL A key characteristic of Parkinson’s disease (PD) is the selective degeneration of midbrain dopamine (mDA) neurons. Currently, only symptomatic treat- PRECURSOR CELLS DERIVED FROM HUMAN ments exist. Due to the selective loss of DA neurons in the substantia nigra EMBRYONIC STEM CELLS of the ventral midbrain, PD is considered one of the diseases most suitable for cell replacement strategies. One possible source of cells for transplanta- Lee, Dongjin R., Kim, Ji Young, Kim, Dae-Sung, Lim, Bo Young, tion are DA neurons derived from human ESCs. Up to now, transplantation Kim, Dong-Wook of DA neurons derived from hESC into rodent PD models has resulted in Brain Korea Project for Medical Science, Yonsei University College of poor in vivo survival after transplantation, most likely due to incomplete Medicine, Seoul, Korea, Republic of, Dept of Physiology, Yonsei University midbrain DA neuron differentiation in vitro. Therefore, it will be crucial to College of Medicine, Seoul, Korea, Republic of address these concerns before hESC-based cell replacement can be proposed as a valid strategy for the treatment of neurological disorders such as PD. Effective differentiation of midbrain dopaminergic (mDA) neurons derived The main goal of this project is to obtain an unlimited source of fully speci- from human embryonic stem cells (hESCs) is crucial for cell replacement fied midbrain DA neurons suitable for pre-clinical applications. Here, we therapy to treat Parkinson’s disease (PD). Therefore, in order to differenti- have developed a protocol for the efficient differentiation of mDA neurons ate hESCs into bona fide mDA neurons in vitro, mechanisms which control from hESC. First, hESC are differentiated to floorplate cells of midbrain development of mDA neurons need to be fully understood. In our study, we origin, which then give rise to mDA neurons. We demonstrate that this show the involvement of Wnt/β-catenin signaling in the anterior-posterior protocol yields hESC progeny with full mDA neuron phenotype including patterning during hESCs-derived DA neuron development and the activa- expression of key transcription factors e.g TH, FoxA2, Lmx1a and Pitx3. tion of Wnt signaling by small molecule glycogen synthase kinase-3 (GSK-3) Transplantation of these hESC derived mDA neurons into immunocompro- inhibitor to obtain the midbrain specific characteristics. We introduced the mised rodent hosts shows good in vivo survival and functional restoration small molecule GSK-3 inhibitor, 6-bromoindirubin-3’-oxime (BIO) to neural of behavioral deficits. Further validation of successful mDA specification in precursor cells (NPCs) derived from hESCs by the simultaneous inhibition of vitro includes testing for DA release and correct electrophysiological proper- BMP and Activin/Nodal signals. Once presented to NPCs, BIO exclusively ties. promoted the expression of midbrain marker engrailed 1 (En1) while reduc- ing the expression of forebrain marker brain factor 1 (BF-1) and hindbrain Poster Board Number: 3367 marker gastrulation brain homeo box 2 (GBX2). Other known specific GSK-3 inhibitors such as 1-Azakenpaullone and lithium chloride (LiCl) DIRECTED DIFFERENTIATION OF HUMAN also showed compatible results to BIO. However, NPCs treated with Wnt EMBRYONIC STEM CELLS TO MIDBRAIN antagonist dickkopf homolog 1 (DKK1) and Frizzled-5 revealed the signifi- cantly reduced expression of En1. Furthermore, a combined treatment of DOPAMINERGIC NEURONS IN A 3D NPCs with BIO and FGF8, a potent factor that promotes midbrain specifica- ENVIRONMENT tion of NPCs, increased the level of En1 expression additively compared with BIO-only group while the decreased expression was found when SU5402, Kim, Jaemin a FGF receptor inhibitor, was added to the combined treatment. This sug- Stem Cell Lab, University of New South Wales, Randwick, Australia gests that the activation of Wnt signal, in the presence of active FGF signal, Parkinson’s disease (PD) is one of the most common neurological disorders can effectively induce NPCs to form midbrain characteristics. Followed by caused by progressive degeneration of dopaminergic (DA) neurons in mid- midbrain specification using GSK-3 inhibitors, NPCs then differentiated into brain. Although many pharmacological and surgical treatments are currently mDA neurons by a serial treatment of sonic hedgehog, FGF8, and ascorbic available, none can cure and all have risks of side effects. Therefore, there acid. Among the differentiated TH-positive neurons, an increased number of has been an extensive effort to replace the degenerating DA neurons by cell cells coexpressed En1 while having a significantly low number of TH-positive transplantation using human embryonic stem cells (hESCs). Although many cells coexpressed GABA. These results show that the activation of the methods have been applied in the differentiation of hESCs to DA neurons, it canonical Wnt signaling pathway of NPCs increases the number of midbrain has been shown that the alginate encapsulation technique promotes better specific neurons within TH-positive neurons. This research was supported by growth, differentiation, maturation or protein secretion of various cell types a grant (SC1110) from the Stem Cell Research Center of the 21th Century including mesenchymal stem cells and hESCs. The entrapment of cells within Frontier Research Program funded by the Ministry of Education, Science and alginate microcapsules allows high-density cell culture and exchange of Technology, Republic of Korea. nutrients, oxygen and stimuli across the membrane, whereas the cells are protected from external threats eg antibodies from the host. Despite the success in many cell types, differentiation studies using alginate encapsula- tion are limited. The aim of this project is to develop an optimal method for induction, amplification and controlled differentiation of hESCs to mid- brain DA neurons in a three dimensional (3D) environment using alginate encapsulation technology. We used encapsulation technology to generate

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Thursday Poster Abstracts a high density of hESC-derived DA neurons by co-culturing with mouse Poster Board Number: 3371 stromal cell line, PA6 cells. The hESCs were treated with Rho-associated coiled-coil kinase (ROCK) inhibitor before and after encapsulation prior to NEURONS IN A SNAP: COMBINED SMALL differentiation to reduce apoptosis. The encapsulated cells were transferred MOLECULE INHIBITION EFFICIENTLY onto monolayer of PA6 cells for 3 weeks in differentiation medium, followed by treatment with sonic hedgehog (SHH) and fibroblast growth factor 8 ACCELERATES HUMAN PLURIPOTENT STEM (FGF8) for a week. The gene and protein expression levels of differentiated CELL DIFFERENTIATION INTO NOCICEPTORS cells from 3D differentiation protocol were compared with a two dimen- sional (2D) non-direct contacted co-culturing system. To further optimize Chambers, Stuart M., Qi, Yuchen, Mica, Yvonne, Lee, Gabsang, the 3D differentiation system, inducing factors, SDF-1, PTN, IGF2, IFGBP4 Niu, Lei, Shi, Songhai, Studer, Lorenz and EFNB1 (SPIE), which are secreted by PA6 cell in culture and have DA Sloan- Kettering Institute, New York, NY, USA neuronal inducing ability, were investigated. In addition, the effect of AMPK Pain is the most visceral human sensations and a frequent symptom in a vast inhibitor (compound C) which inhibits endo- and mesodermal differentiation number of diseases. Modern analgesics (e.g. salicylate, NSAIDs, opioids, was explored to increase the efficiency of differentiation. The differentiating and lidocaine) each have drawbacks such as limited effectiveness, cyto- hESCs were analysed for gene and protein expressions and the data showed toxicity, addiction, and cardiopulmonary depression. To better understand a significant down regulation of pluripotent markers such as Oct4 while up the relationship between pain and disease states and further identify drugs regulation was observed for mature midbrain DA neuronal markers such that can modulate pain, work is needed to find scalable, inexpensive, and as Lmx1b and TH. In comparison to 2D differentiated cells, 3D differenti- efficient methods for deriving pain-sensing neurons, called nociceptors, from ated cells had higher DA neuronal gene and protein expression levels by human pluripotent stem cells (hPSCs). Previously, we have reported a rapid 1000 fold eg TH, Lmx1b suggesting that 3D configuration promoted more and efficient method to generate early neuroectoderm from hPSCs in less efficient DA neuronal generation. Treatment with SPIE factors and with or than 10 days that uses two inhibitors to block SMAD signaling, called dual without compound C for 14 days generated DA neurons with similar ef- SMAD inhibition. In the context of dual SMAD inhibition, a combinatorial ficiency as with PA6 cells co-culture. However, the efficiency of DA neuronal small molecule screen for the rapid conversion of hPSCs into post-mitotic production with SPIE and compound C was lower than 3D co-cultured neurons was performed. We identified a combination of three additional protocol by 100 fold. Our gene and protein expression data suggest that the small molecule inhibitors that act in concert to yield neurons at > 75% 3D environment used for differentiation of hESCs to midbrain DA neurons efficiency in the absence of any recombinant growth factors. The resulting is suitable and provides more efficient DA neuronal generation than 2D dif- bipolar neurons express canonical markers of the nociceptive sensory fate ferentiated cells. However, further optimisation of treatment with SPIE and including NTRK1, BRN3A, ISL1, NEUROG1, Substance P, and CGRP, and compound C protocol and purification of DA neurons by using reporter lines exhibit action potentials consistent with small fiber nociceptors. The most are being investigated. surprising finding is the speed with which stable, mature neuronal cell fates Poster Board Number: 3369 can differentiate from hPSCs using this combined small molecule approach. Taking only 10 days, small molecule based acceleration of neuronal fate ac- FGF AND SMALL MOLECULES EFFICIENTLY quisition occurs in a time frame at least three fold faster compared to normal INDUCE NEURAL DIFFERENTIATION OF in vivo human development. This approach combines two inhibitors to block signaling pathways important to neural stem cell self-renewal and prolifera- HUMAN EMBRYONIC STEM CELLS tion with an inhibitor promoting cell survival and neural patterning. Using a Chen, Seng Mei, Su, Hong-Lin SOX10::GFP bacterial artificial chromosome hPSC reporter line, a SOX10+ neural crest intermediate can be observed emerging by day 8. Detailed Dept of Life Sciences, National Chung Hsing University, Taichung, Taiwan gene expression profiling further supports our findings that nociceptors Human embryonic stem cells (hESCs) have provided a potential source of can be coaxed from hSPSCs in a few days. We anticipate that similar small neural lineages. Previously, the neural induction programs rely on stromal molecule strategies could be employed to compress developmental timing feeder co-culture, dual inhibition of smads signaling, or genetic manipula- and derivation of additional neural and non-neural cell types from hPSCs. tion. Here, we show a short term neural induction program that treating- a Multifactor small molecule screens represent a new generation of directed GSK3β inhibitor Bio greatly improved the neural differentiation of hESCs differentiation strategies in hPSC biology. In addition, the rapid, scalable, in the presence of Activin/nodal inhibitor SB431542 and FGF2.The sox1. and high efficiency derivation of nociceptors offers a unique opportunity to Zic-1 and N-cadherin were expressed and pax6 was detected in the neural study this novel and medically relevant cell type crucial for studies of human rosettes on day7 to day10. On Day 20, Tuj1, glial fibrillar acid protein and pain perception. tyrosine hydroxylase in differentiating hESCs were detected. In addition, mo- tor neurons,confirmed by MNR2 and Olig2 expression, were also generated Poster Board Number: 3373 after treatment of purmorphamine and retinoic acid. The role of Bio will be EXPLORING HUMAN PANCREATIC further explored for establishing an efficient neural differentiation procedure of hESCs. DEVELOPMENT USING CHEMICAL APPROACHES Chen, Shuibing, Lam, Kelvin, Arvanites, Anthony, Rubin, Lee L., Melton, Doug A. Surgery, Weill Cornell Medical College, New York, NY, USA, SCRB, Harvard University, Cambridge, MA, USA Human embryonic stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem (iPS) cells, present unlimited starting mate- rial to generate mature insulin-secreting β cells for transplantation therapy and disease modeling for diabetics, as well as a platform to study human embryonic pancreatic development. By mimicking signals used during embryonic pancreatic development, to the extent that they are known,

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a stepwise protocol is being explored, which involves the generation of Poster Board Number: 3377 definitive endoderm, pancreatic progenitor, endocrine progenitor, and finallyβ -cells. Using the high content screening, we have identified a series EXAMINATION OF CELL SIGNALING PATHWAYS of small molecules and cell lines that promotes the differentiation towards DURING EARLY ENDODERM SPECIFICATION pancreatic progenitors and endocrine progenitors, respectively. We further investigate the mechanism of action of the identified small molecules and FROM HUMAN EMBRYONIC STEM CELLS cell lines and found two signaling pathways, which previously have not been UTILIZING FLUORESCENCE CELL BARCODING reported to be involved in pancreatic development. We are performing ad- AND INTRACELLULAR FLOW CYTOMETRY ditional mechanistic studies to dissect the signaling circuitry that controls the generation, self-renewal and differentiation of pancreatic progenitors and Emre, Nil, Vidal, Jason G., Paramban, Rosanto I., Shah, Khooshbu endocrine progenitors. These studies will not only provide us new reagents K., Apgar, John R., Carson, Christian T. for the directed differentiation, but also provide new insight of human BD Biosciences, San Diego, CA, USA embryonic development. The ability of human embryonic stem cells (hESCs) to differentiate into Poster Board Number: 3375 various cell lineages allows for the study of developmental biology and has applications to the field of regenerative medicine and cellular therapy. To EFFICIENT DIFFERENTIATION OF LIVER AND optimize differentiation protocols and to answer biological questions, it is PANCREATIC PROGENITOR-LIKE CELLS FROM important to understand the signaling pathways that are involved in lineage HUMAN EMBRYONIC STEM CELLS USING specification. We applied a multiparametric flow cytometric screening approach to address this problem in early endoderm differentiation from SMALL MOLECULE INHIBITORS hESCs. Specifically, hESCs were differentiated to early endoderm in the pres- Hasegawa, Kouichi, Pera, Martin F. ence of low serum and Activin A. Our differentiation method was verified using multicolor flow cytometry and image analysis to monitor the down- Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem regulation of pluripotency markers Oct4 and Nanog and the upregulation Cell Research, University of Southern California, Los Angeles, CA, USA of endodermal markers Sox17 and FoxA2. We screened cells at different Understanding the molecular mechanisms that underlie differentiation of time points of endoderm differentiation by flow cytometry for the phos- tissue specific stem/progenitor cells is a prerequisite for many applications in phorylation and activation of different cell signaling pathways. We tested regenerative medicine. However, production of properly functioning stem/ approximately 100 monoclonal antibodies to activated signaling molecules progenitor cells from pluripotent stem cells in culture has been difficult, due and correlated their expression to cells expressing either pluripotency or en- to our limited knowledge of molecular mechanisms in human development, doderm markers to delineate differentiation status. Additionally, to increase and an incomplete understanding of cell lineage relationships in repair or throughput and minimize experimental variability, we utilized fluorescent cell regeneration of adult tissues. Despite several reports demonstrating the barcoding in our screen. This enabled multiple treatment time-points to be differentiation of pancreatic endocrine cells or hepatic cells from human analyzed in a single sample for multicolor staining and analysis. Pathways pluripotent stem cells, transplantation of these cells often failed to rescue investigated included MAPK/ERK, Jak/Stat, PI3/AKT, and Wnt/β-catenin. model animals of disease. It is possible that progenitor cells, rather than These data suggest a comprehensive signaling network that mediates mature end cells, would provide better engraftment. However, the molecular endoderm specification of pluripotent stem cells in culture. This screening mechanisms that govern human hepatic/pancreatic stem cell differentia- methodology can readily be applied to various stem cell populations and tion and self-renewal are still largely unknown. We have developed a novel, their derivatives to explore cell signaling events such as self-renewal, repro- highly efficient and chemically defined method for producing liver/pancreas gramming, and lineage specification. The identification of cellular pathways progenitor-like cells from human ES cells using small molecule inhibitors. The involved in these events will not only aid in the understanding of biology but induced endoderm progenitor cells reached ~80% purity within 3-5 days also enable the use of small molecule inhibitors to replace or modify endog- of induction. The induced cells displayed uniform epithelial morphology, enous cell signaling to increase efficiency of self-renewal or differentiation. and expressed endodermal ductule protein markers (e.g. CK7/19, EpCAM, ECAD). Gene expression analysis revealed that the cells expressed both Poster Board Number: 3379 hepatic and pancreatic markers (e.g. HEX, HNFs, PAX6, PDX1), common IMPROVING THE FIDELITY OF HEPATIC endoderm markers (e.g. SOX17, GATA4 and 6), and weakly expressed pluripotent stem cell markers (e.g. OCT4, NANOG). The marker expression DIFFERENTIATION FROM HUMAN pattern is quite similar to that reported in adult hepatic/pancreatic stem PLURIPOTENT STEM CELLS cells, and that of cells at intermediate phases of liver/pancreatic differentia- tion from pluripotent stem cells. The induced cells have properties consistent Chan, David N. with posterior foregut endoderm, which gives rise to liver and pancreas, but University of California, Los Angeles, Los Angeles, CA, USA is not well characterized in human. The cells can self-renew up to 1 month The technology of induced pluripotent stem cells has generated much in hepatic stem cell culture conditions. These molecular data suggest that promise in regenerative and personalized medicine. Many groups have the induced endodermal cell may be a hepatic/pancreatic stem/progeni- devised strategies for differentiating human embryonic stem cells (hESC) and tor cells. This culture system will provide a platform to study human liver/ induced pluripotent stem cells (hiPSC) to various progeny cell types. Liver pancreatic stem cells in vitro, and for discovery of chemical compounds that cells are one of the cell types highly sought after because liver transplant for can enhance repair of damaged liver and pancreas. The progenitors may terminal liver disease patients is hampered by a chronic shortage of donors. prove to be a suitable cell source of mature hepatocytes for drug testing and Autologous hiPSC-derived hepatocytes provide hope in solving this problem. transplantation. hESC and hiPSC-derived hepatocytes would be invaluable in pharmacologi- cal screenings and could potentially provide a platform for studying the basic biology of hepatic development and diseases. However, unanswered caveats on the safety and fidelity of hESC and hiPSC-derived progeny need to be carefully addressed and this necessitates the purification of hESC and hiPSC-derived progeny. With the help of a liver specific reporter, we are able to differentiate and purify hESC and hiPSC-derived hepatocytes. These

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Thursday Poster Abstracts cells are morphologically and functionally similar to primary hepatocytes Poster Board Number: 3383 and express various hepatic markers. But as many groups have previously reported, we also discovered that these hESC/hiPSC-derivatives have fetal PROMOTING MESODERM AND BLOCKING characteristics and distinct signatures from primary hepatocytes. To decipher ENDODERM: THE ROLE OF BRACHYURY IN if this outcome is due to the early steps of endodermal differentiation, we have characterized the steps of definitive endoderm generation from hESC HUMAN EMBRYONIC STEM CELLS and hiPSC. This study has led to two important observations: first, hESC/ Faial, Tiago, Bernardo, Andreia S., Trotter, Matthew W., Smith, Jim hiPSC-derived hepatocytes continue to express genes normally found only C., Pedersen, Roger A. during early fetal development; and second, that these hepatocytes fail to induce several transcription factors expressed by tissue-derived hepatocytes. Anne McLaren Laboratory for Regenerative Medicine, University of We are currently attempting to silence these early embryonic genes and Cambridge, Cambridge, United Kingdom, Systems Biology, MRC National induce some of these transcription factors, in an attempt to produce hepato- Institute for Medical Research, London, United Kingdom cytes that are more similar to primary somatic hepatocytes. Here we provide The Brachyury gene (T) encodes a T-box transcription factor whose mutant evidence that simply silencing early embryonic genes is not sufficient to phenotype in the mouse shows gross defects throughout the mesoderm. drive hESC/hiPSC-derived hepatocytes to a mature state and outline future The molecular mechanisms responsible for this phenotype remain elusive directions to address this issue. and the genomic targets of T have not been reported for any mammalian Poster Board Number: 3381 species. Here, we used human embryonic stem cells (hESCs) to study the activation of T and to determine its downstream targets. In the primitive PROMININ 1 (CD133) MARKS A POPULATION streak of the mouse, T is expressed at different levels: higher in its posterior side and lower in its anterior side. We established two chemically defined OF MESENDODERMAL CELLS IN HUMAN culture conditions to mimic these cell populations, one containing Activin EMBRYONIC STEM CELLS and the other BMP4. Flow cytometry revealed that cells grown in Activin are Tlow whereas in BMP4 they are Thigh. Importantly, while the Activin- Borchin, Bianca E., Kwek, Joly, Barberi, Tiziano based medium induced the expression of early endoderm genes (SOX17, Australian Regenerative Medicine Institute, Monash University, Clayton, FOXA2, GSC, CER1), the BMP4-based medium induced early mesoderm Australia genes (TBX6, CDX2, KDR, LMO2). We performed ChIP-Seq with hESCs The existence of the mesendoderm, which is a bipotent transient layer grown in these two conditions and identified partially overlapping sets of capable of generating endoderm and mesoderm, has been demonstrated T genomic targets, suggesting that T regulates different genes in opposite during embryogenesis. Although mesendodermal cells have been isolated regions of the primitive streak. To validate these binding events as functional and well characterized in vitro during mouse ESC differentiation, isolation targets of T, we performed microarray transcriptional profiling of wild type from human ESC has not been achieved yet. The pentaspan membrane gly- and T knockdown (shRNA) hESCs lines, grown in Activin- and BMP4-based coprotein prominin 1 (CD133) has been described as a potent stem/progeni- media. Our data indicate that in BMP4-induced cells, high levels of T block tor cell marker with expression detected within an array of embryonic and the expression of its endoderm target genes and up-regulate mesoderm adult tissues, including the hematopoietic and central nervous system (CNS). target genes, whereas in Activin-induced cells, low T levels are permissive for We found that a small transient cell population arising during in vitro hESC an endoderm phenotype and insufficient to up-regulate mesoderm markers. differentiation expresses CD133. At day 12 of hESC differentiation induced Collectively, our data reveal how T has distinct roles in different cell types by the presence of a chemically defined serum free medium (ITS), these cells and reinforce the value of studying the functions of transcription factors in become recognizable for their rounded morphology. They were purified by dissimilar cellular and developmental contexts. Fluorescent Activated Cell Sorting (FACS) for CD133 expression. Sorted cells Poster Board Number: 3385 were then replated and cultured on fibronectin-coated plates in the presence of ITS medium. Spontaneous differentiation revealed the bipotent nature of BRACHYURY AND CDX2 MEDIATE BMP- this population that turned into a mixture of endoderm (Sox17+, FoxA2+ INDUCED DIFFERENTIATION OF HUMAN aSMA-, CD73-) and mesoderm (aSMA+, CD73+, Sox17-, FoxA2-) cells. These prospective mesendodermal cells were obtained by simply differen- EMBRYONIC STEM CELLS INTO MESODERM, tiating hESC colonies seeded at low density in a monolayer culture system. NOT TROPHOBLAST Addition of 40 ng/ml of BMP4 to ITS medium during differentiation greatly enhanced the generation of mesendoderm by inhibiting spontaneous neural Bernardo, Andreia S., Faial, Tiago, Gardner, Lucy, Ortmann, Daniel, differentiation. RT-PCR analysis on CD133+ cells revealed the expression Niakan, Kathy K., Senner, Claire E., Callery, Elizabeth M., Trotter, of genes such as Sox17, T, GSC and Eomes among others, indicative of a Matthew W., Hemberger, Myriam, Smith, James C., Moffett, mesendodermal phenotype. Interestingly, purified mesendodermal cells Ashley, Bardwell, Lee, Pedersen, Roger A. could be maintained undifferentiated in culture by adding BMP4 to the ITS University of Cambridge, Cambridge, United Kingdom, NIMR, London, medium, whereas the addition of 40ng/ml Activin A or 10ng/ml FGF2 and United Kingdom, University of California, Irvine, CA, USA 5ng/mlTGFb3 induced their differentiation into endoderm and mesoderm, respectively. Analysis of single seeded CD133+ clones confirmed their bipo- BMP is thought to induce hESCs differentiation towards multiple lineages tent fate potential. The identification of a representative surface antigen for including mesoderm and trophoblast. The BMP-induced trophoblast phe- the efficient isolation of mesendodermal cells a) facilitates the establishment notype is a paradox in stem cell biology since the potency to generate this of differentiation strategies for the direct development of pure endoderm or lineage is lost prior to hESC derivation. Here we re-addressed BMP function mesoderm derivatives from hESC; and b) provides a tool for the molecular in hESCs and mouse epiblast-derived cells. We found that BMP4 cooperates analysis of tissue specification. with FGF2 to induce mesoderm and circumvent endoderm differentiation. These cells express the mesoderm gene BRACHYURY (BRA). BRA was co- expressed with and necessary for expression of the caudal-homeobox gene CDX2, and both were essential for expression of mesoderm- and tropho- blast-associated genes. While the BMP-induced expression of trophoblast- associated genes increased in the absence of FGF, the resultant cells differed in surface properties and epigenetically from placental trophoblast. More-

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over, these cells co-expressed mesoderm genes. We conclude that BMP Poster Board Number: 3389 acts through BRA and CDX2 to induce hESCs, mEpiSCs and mouse epiblast explants primarily to form embryonic and extraembryonic mesoderm. DIFFERENCES BETWEEN 2D AND 3D Poster Board Number: 3387 PROTOCOL IN HUMAN ESCS TOWARDS PANCREATIC ENDODERM ELUCIDATED. MOLECULAR FEATURES OF TROPHOBLAST DIFFERENTIATION FROM HUMAN EMBRYONIC Cai, Qing, Ordovas, Laura, Kim, Vanuytsel, Park, Yonsil, Mathieu, Chantal, Verfaillie, Catherine AND INDUCED PLURIPOTENT STEM CELLS Interdepartmentaal stamcelinstituut, Leuven, Belgium Seita, Yasunari, Su, Jing, Treff, Nathan, Lu, Chi-Wei According to the World Health Organization (WHO), there will be approxi- OB/GYN, RWJMS/UMDNJ, Piscataway, NJ, USA mately 366 million of people worldwide suffering of diabetes. For patients The integral function of trophoblasts is crucial for embryo implantation with type I diabetes, and to a lesser extent for type II diabetes, the only and development to term. The development of iPS cells for the study of curative therapy is replacement of lost β-cells. Over the last 10 years, many trophoblast differentiation addresses a major limitation of rapid exhaustion labs have attempted to generate functional β-cells from embryonic stem of primary chorionic villi derived culture. ES and iPS cells possess the nature cells (ESCs), as donor pancreata or islets are very scarce. The use of β-cells of limitless replication to derive trophoblasts for comprehensive analysis of derived from human ESCs (hESCs) would provide a renewable source of their functional characteristics, including proliferation, differentiation, and donor tissue. To determine if functional β-cells have been generated from persistence. Moreover, direct differentiation from ES or iPS cells provides a ESC, progeny cells should be transplanted in hyperglycemic mice. However unique system for investigating early stages of trophoblast development, until now, it has remained impossible to reverse the hyperglycemia im- allowing analysis into molecular mechanisms of tissue formation. We set out mediately after transplantation of differentiated hESC. Therefore there is still to test the hypothesis that CDX2 expression plays an essential role of hu- a need to develop better and more reproducible differentiation methods to man trophoblast formation, by observing the dynamic expression of CDX2 generate mature and well-definedβ -cells suitable for cell therapy. This is a in BMP4-treated hES and iPS cells. Transcription profile analysis revealed a critical step for cell therapy applications in diabetes, and will also be useful in largely similar response to BMP4 signaling between human ES and iPS cells. elucidating the developmental steps needed to generate functional β-cells. At day 5 post BMP4 treatment, CDX2 expression peaked to present in about We have tested if aside from adding sequential cytokines to mimic pancreas 50% percent of cells and significantly overlapped with NANOG expres- development, the formation of 3D structures during the pancreas endo- sion. Expression of trophoblast peptidyl hormones and transcription factors derm differentiation with a defined number of cells would aid in generat- further confirms the presence of functional trophoblasts. Increased CDX2 is ing “mature” β-cells. Analyzing the differences in β-cell gene expression detected by both real-time PCR with primers anneal to 3’ coding region and between 2D and 3D differentiation cultures demonstrated that 3D cultures immunostaining using antibody recognizing an epitope of C-terminal DNA from day 12 onwards may indeed increase expression levels of β-cell-specific binding domain. Surprisingly, hybridization-based gene expression microar- genes, including INS, NGN3 and NEUROD1. Currently, we are assessing the ray analysis fails to detect major difference in CDX2 expression compared expression of INS protein and C-PEPTIDE production in vitro and the ability BMP4 induction with undifferentiated ES cells. Exon expression analysis of the 3D cultured ESC progeny to reverse hyperglycemia in vivo. revealed that the first and the second exons of CDX2 are expressed in Poster Board Number: 3391 undifferentiated ES cells, while expression of exon 3 is only stable by BMP4 treatment. The mechanism by which BMP4 induce trophoblast formation in PRODUCTION OF MULTIPOTENT ANTERIOR human pluripotent stem cells appears to involve additional mechanisms than DEFINITIVE ENDODERM FROM HUMAN promoter activation. We further tested the role of CDX2 in trophecotderm differentiation by observing fate of ES and iPS cells ectopically expressing PLURIPOTENT STEM CELLS CDX2. Interestingly, in contrast to known effect of Cdx2 in suppressing Hannan, Nicholas, Cho, Candy H-H, Vallier, Ludovic expressions of pluripotent factors in mouse ES cells, OCT4 remain expressed strongly in CDX2 expressing human ES and iPS cells. Therefore, the mutually University of Cambridge, Cambridge, United Kingdom suppressive nature of Cdx2 and Oct4 in mouse ES Cells is not conserved in Human embryonic stem cells (hESCs) as well as induced pluripotent stem human. Expression of genes indicative of trophoblast cell fates were elevat- cells (IPSCs) represent a unique opportunity to study human development, ed, including transcription factors GCM1, EOMES, HAND1, CSH1, as well as model diseases in vitro, optimise drug screening platforms and most signifi- peptidyl hormones CRH, TRH, ACTH, CG α and β. Only markers of tropho- cantly develop cell replacement therapies for degenerative diseases. One blast are detectable in differentiating CDX2/OCT4 co-expressing cells. The of the main challenges in achieving these clinical promises is the production presence of OCT4 did not promote differentiation toward ectoderm, meso- of populations of progenitor cells that are of high purity, retain their full derm and endoderm, suggesting a dominating effect of CDX2. Unlike BMP4 spectrum of differentiation capacity and are able to expand into numbers induced ES/iPS cell which decade overtime, CDX2 expressing ES and iPS relevant for therapeutic needs. Following a normal path of development cells exhibit behavior of trophoblast stem cells and proliferate for prolonged remains the best approach to generate such cells as well as the develop- period of time, consistent with the presence of telomerase activity. Through ment of a universal differentiation protocol applicable to a wide variety of our analysis, we conclude that trophoblast differentiation from human pluri- pluripotent cell lines. Anterior Definite Endoderm (ADE) is a derivative of the potent stem cells is associated with CDX2 expression, by mechanisms not endoderm germ layer appearing just after gastrulation during mammalian conserved with mouse ES cells. The iPS-derived trophoblasts culture offers development and which gives rise to the ventral foregut. Importantly, key an assay system free of maternal environment conditioning, and can reveal endoderm organs such as the liver and the ventral pancreas originate from phenotypes solely contributed by the genotype of the iPS cells. the foregut. Our group has recently described a method to produce foetal hepatocytes and pancreatic progenitors cells from both hESCs and hIPSCs which importantly, respects key developmental stages in vitro including the differentiation of endoderm into ADE and then the differentiation of ADE cells into ventral foregut. The purity of this initial ADE population has very profound effects on the purity and functionality of the downstream differen- tiated cells types. Therefore, generation of ADE cells represent an essential step of differentiation to generate liver and pancreas cells from hESCs. Here,

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Thursday Poster Abstracts we demonstrate differentiation of hESCs and hIPSCs into ADE expressing endoderm marker GATA-4 were 10- to 20- fold higher in the KDR+/CD31- CXCR4 and HHEX using defined culture system devoid of feeders or serum. population compared to those in KDR-/CD31- cells. Altogether, the real These cells are multipotent giving rise to hepatocytes, pancreatic cells and time PCR data combined with immunostaining analyses indicate that the lung epithelium. Mature cells types can be produced from this ADE popula- CD31-/KDR+ population may represent an early progenitor for the CD31-/ tion expressing albumin, alpha-1-antitrypsin and cytochrome P450 enzymes KDR- hepatic cells or alternatively a supporting cell population that develops for hepatocytes as well as many functional properties such as glycogen stor- concomitantly to the hepatic cells. In summary, using the human ES cell age, LDL uptake and inducible cytochrome P450 activity. Insulin producing system, we have identified some cell components of the developing hepatic pancreatic beta cells express PDX1 and HLXB9 are also release insulin in re- cell niche including an endothelial cell population (KDR+/CD31+) generated sponse to glucose challenge. Lung epithelium expresses Nkx2.1, Aquaporin in association with the hepatic cells (KDR-/CD31-), along with a population and surfactant protein expression and also undergo branching morphogene- (KDR+/CD31-) that may represent an intermediate progenitor for hepatic sis in 3D culture. Furthermore, our approach allows the production of a near cells or supporting cells. homogenous ADE cells expressing CXCR4 which can then be differentiated into cells expressing albumin (90%) in mature liver cells or PDX1 (90%) in Poster Board Number: 3395 mature pancreatic beta-cells. This presents unique advantages for develop- COLLAGEN THIN FILMS AS EXTRACELLULAR ment of drug screening platforms, disease modelling and transplant therapy applications and also represent a first toward the generation of endodermal MATRICES FOR HUMAN PLURIPOTENT STEM cells fully compatible with in vivo applications. CELLS Poster Board Number: 3393 Bhadriraju, Kiran, Elliott, John, Chen, Antony, Peterson, Alexander, DEFINING THE HEPATIC CELL NICHE UPON Plant, Anne University of Maryland, College Park, MD, USA, National Institute of HEPATIC SPECIFICATION OF HUMAN ES CELL- Standards and Technology, Gaithersburg, MD, USA DERIVED ENDODERM The extracellular matrix, along with other components of the cellular mi- Goldman, Orit Rachel, Corneo, Barbara, D’Souza, Sunita, Gouon- croenvironment, provides specific cues for the maintenance of cell phe- Evans, Valerie notype. Particularly in the case of stem cells, absence of a proper culture environment can lead to uncontrolled or aberrant alterations of cell state. An Mount Sinai School of Medicine, New York, NY, USA, NSCI Neural Stem important component of engineering suitable matrices is an understanding Cell Institute, Albany, NY, USA of the specific cues that they provide cells. Matrices of type 1 collagen are of The capacity of embryonic stem (ES) cells to form multiple differentiated especial interest in this context because they provide multiple cues to cells: cell types in culture provides a unique model system for the generation topographical (because it can be varied by varying collagen polymeriza- of transplantable lineages for cell replacement therapies. Endoderm can tion), mechanical (because it can be varied by varying collagen density or be efficiently and reproducibly induced in human ES cell differentiation crosslinking), or receptor-specific cues (because it can be varied by multiple cultures and further differentiated to hepatic cells. However, a robust hepatic aspects of collagen presentation). Traditional bulk gels of collagen as a sub- specification requires a precise identity of the hepatic progenitors and the strate for cell culture have certain disadvantages in terms of poor physical surrounding cellular niche that still remain unclear. Using a human ES cell stability, poor optical properties, and low reproducibility. As an alternative line (HES-2), we have established conditions for an efficient endoderm to bulk gels, we had previously developed thin films of Type 1 collagen, development assessed by the expression of CXCR4 and cKit that have been which ameliorate many of these disadvantages. These submicron-thick films shown to reflect the formation of endoderm in mouse and human ES cell comprised of polymerized collagen fibrils are robust, highly reproducible, cultures. Endoderm induction is very robust, as up to 93% of the cells in have excellent light transmission characteristics, and at the same time elicit day 5 embryoid bodies (EBs) express consistently both cell surface mark- cell responses similar those seen on collagen gels, such as cell spreading and ers. Hepatic specification was induced by plating the dissociated EBs in proliferation. The films can be modified in multiple ways to change their the presence of BMP4 and bFGF. Cell surface markers for endothelial cells properties including mechanical stiffness with or without enzymatic cross- (KDR/Flk-1 and CD31/PECAM) were monitored as endothelial cells have linking, extent of fibril formation, and the presentation of glycosyl groups. been shown to develop in parallel to the ES cell-derived hepatic cells in Using these thin films, we are attempting to understand the specific signals the mouse system. Similarly to the mouse differentiation cultures, human presented to cells by multiple aspects of collagen presentation influence ES cells generated 5 to 17 % of KDR+/CD31+ endothelial cells by day 12 cellular phenotype. In the preliminary studies presented here, we examined of differentiation. As the KDR+/CD31+ endothelial cell emerged, AFP+/ how collagen mechanical stiffness, the presence or absence of polymer- Foxa2+ hepatic endoderm cells developed and by day 17 expressed Albumin ized fibrils, or the presence or absence of glycosylation groups on collagen, indicative of further maturation. The maturation of the hepatic cells was influence the modulation of cell adhesion related signaling. Specifically, we also confirmed by increasing levels of AFP and Albumin transcripts upon examined how these various cues provided by collagen ECM modulate the differentiation. Interestingly, a large population (up to 50%) of cells negative levels of focal adhesion kinase (FAK), and the activation of the collagen for CD31 and AFP but positive for KDR, CK18 and CK8 developed by day receptor DDR2. Our results suggest that different cell responses are affected 12 in hepatic cultures. In order to characterize further the KDR-/CD31- and by distinct cues presented by collagen, and suggest that the collagen thin KDR+/CD31- cell fractions, both populations were isolated by FACS at day9 films are a useful experimental system to systematically develop metrics for and day12 and their RNA processed for gene expression by real time PCR. quantifying pluripotent stem cell differentiation. The endothelial cell identity of the CD31+/KDR+ population was confirmed by high levels for KDR, VWF and CD31 transcripts similar to those found in the human umbilical vein cells (Huvec). Levels of endoderm (Foxa2) and hepatic (AFP and Albumin) markers were 5 to 10 times greater in the KDR-/ CD31- population compared to those in the KDR+/CD31- cells. However, expression of Foxa2 was 10-fold higher in the KDR+/CD31- population compared to those in Huvec cells, and levels of AFP and Albumin were 10- to 100-fold greater in the KDR+/CD31- population compared to those in the day5 CXCR4+/cKit+ endoderm cells. This clearly indicated a hepatic specification occurring in the KDR+/CD31- cells. Furthermore, levels of the

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Poster Board Number: 3397 with the same architecture and bundling seen in collagen fibers. This unique self-assembled multi-scale wrinkle substrate was used to study responses of LENGTHENED G1 PHASE INDICATES hESCs to topography. It has been demonstrated previously that cellular mor- DIFFERENTIATION STATUS ACROSS MULTIPLE phology influenced lineage commitment in human mesenchymal stem cells. Similarly, the self-renewal or differentiation of hESCs was also regulated LINEAGES IN HUMAN EMBRYONIC STEM through interactions with their microenvironment. Results from both f-actin CELLS and nuclear alignment demonstrate that approximately 40% of hESCs align to the wrinkles within 15° of the wrinkle direction after the first 4 hours of Calder, Ashley, Russell, Jennifer, Fazzari, Jennifer, Levadoux-Martin, plating. In addition, the deformation of hESC nuclei due to topography was Marilyne, Collins, Tony, Draper, Jon demonstrated for the first time. The deformed nuclei of hESCs exhibit elon- SCCRI, McMaster University, Hamilton, ON, Canada gated shape and decreased surface area and circularity. The nucleus plays an intricate role in mechanotransduction. Morphological changes in nucleus Human embryonic stem cells (hESC) have potential applications as tools for is often accompanied by downstream changes in molecular pathways and drug screening to identify small molecule regulators of self-renewal or differ- gene expressions. We further studied the effects of such mechanotransduc- entiation. As a model for human development, elucidating the mechanisms tion on protein expression by assessing pluripotent markers. We propose this governing lineage commitment in hESC will allow for efficient derivation forced alignment as a new strategy of mimicking micro-niche cues to induce of specified cell types for clinical use. Recognizing the early steps in loss of differentiation of pluripotent stem cells. pluripotency is key to achieving both of these goals. Here we report the use of a live cell cycle fluorescent reporter in hESC that indicates onset of dif- Poster Board Number: 3401 ferentiation in a lineage unbiased manner. Pluripotent hESC possess a short cell cycle length (24 hours), due primarily to a truncated G1 phase of ap- ADHERENT DIFFERENTIATION OF FUNCTIONAL proximately 3 hours. G1 lengthens (to approximately 8 hours) concomitant NEURONS FROM HUMAN EMBRYONIC STEM with differentiation. Stable hESC lines expressing the live cell cycle reporter exhibit fluorescence only during G1; the fluorescent reporter is actively and CELLS accurately degraded outside of G1. Due to the short length of pluripotent Drury-Stewart, Danielle, Song, Mingke, Mohamad, Osama, Yu, G1 phase, G1 fluorescence is only weakly and transiently detected, however Shan Ping, Wei, Ling it is quickly increased to easily detectable levels upon onset of differentia- tion. This provides a simple visual means of tracking differentiation via G1 Biomedical Engineering, Anesthesiology, Georgia Institute of Technology, length within hESC cultures and individual colonies over time and across Emory University, Atlanta, GA, USA, Anesthesiology, Emory University, treatments. Within colonies, cells with lengthened G1 are typically negative Atlanta, GA, USA for pluripotency markers Oct4 and SSEA-3 following directed differentiation Human embryonic stem (ES) and induced pluripotent stem (iPS) cells contin- towards each of the three germ layers. Differentiated cells with lengthened ue to be of interest in the fields of developmental biology and regenerative G1 also demonstrate increased levels of lineage-specific differentiation mark- medicine, but differentiation in culture is still difficult to optimize. Neuronal ers at both the protein and mRNA level. Automated image analysis of hESC differentiation protocols generally involve the growth of cells in suspension indicates this mutually exclusive relationship between lengthened G1 and culture as embryoid bodies or neurospheres. However, these structures pres- pluripotency exists both on the cellular level and in colonies on a whole. The ent varied microenvironmental cues to the cells, introducing heterogeneity novel application of advanced high resolution time-lapse live imaging, auto- and complicating analysis. They also require expensive recombinant proteins mated image analysis, and use of a live fluorescence cell cycle reporter not over differentiation processes that take weeks to complete. The present in- previously utilized in hESC reveals intriguing spatio-temporal relationships vestigation explores a new adherent neuronal differentiation protocol using and variability of differentiation previously unappreciated in hESC cultures. relatively inexpensive small molecules and common medium supplements. Poster Board Number: 3399 Induction of human pluripotent stem cells to the neuroepithelial stage has been achieved in adherent culture using dual inhibition of SMAD signaling EFFECTS OF FORCED ALIGNMENT ON HUMAN with Noggin, a bone morphogenetic protein (BMP) inhibitor, and SB431542, a transforming growth factor beta (TGF-β) inhibitor. We have investigated EMBRYONIC STEM CELLS the substitution of Dorsomorphin, a relatively inexpensive small molecule Chen, Aaron, Freschauf, Lauren, Lew, Valerie, Sharma, Himanshu, BMP inhibitor for Noggin. For neural progenitor differentiation, human ES Nguyen, Diep, Gopinathan, Ajay, Botvinick, Elliot, Fowlkes, Charless cells were seeded as single cells at 18,000 cells/cm2 and grown to conflu- ence in MEF-conditioned medium. They were then cultured in medium C., Khine, Michelle supplemented with 10μM SB431542 and 3μM Dorsomorphin for five days, Chemical Engineering, University of California, Irvine, Irvine, CA, USA, followed by six days of culture in which SB431542 was withdrawn and the Biomedical Engineering, University of California, Irvine, Irvine, CA, USA, proportion of N2 supplement was incrementally increased. PAX6 and nestin School of Natural Sciences, University of California, Merced, Merced, CA, were used as neural progenitor markers, with greater than 60% of cells USA, Computer Science, University of California, Irvine, Irvine, CA, USA positive for PAX6 and nestin strongly correlating with PAX6. For in vitro Studying of interactions between cells and their microenvironment has be- neuronal differentiation, neuroepithelial cells were dissociated and re-seeded come increasingly important to better understand the fundamentals of stem in medium supplemented with both N2 and B27 supplements. After 3 weeks cell biology and to leverage for tissue engineering applications. Although or more, cells were examined for electrophysiological activity and stained traditional micro or nano fabrication techniques can create the topographi- for neuronal markers. Neurons consistently produced action potentials with cal features for cellular studies, the lack of relevant topographies that can amplitudes of 74.7 +/- 6.9 mV. Repetitive firing was observed in about 1/3 accurately mimic multi-scale physiological cues as well as high cost of capital of the measured neurons, with amplitudes of 60.7 +/- 8.8 mV. Cells formed equipment prevents the widespread use of such techniques in most biologi- networks in vitro that stained for NeuN and β-III Tubulin, both markers cal laboratories. We describe the alignment of human embryonic stem cells of mature neurons. Cells also stained positively for Glutamate Receptor 2 (hESCs) using a simple and inexpensive method to create tunable and robust (GluR2). To test whether this protocol could be applied for transplantation multi-scale unidirectional alignment ‘wrinkles’. The wrinkles are created therapy, adult male mice were trained to remove sticky dots from their paws simply by treating pre-stressed polyethylene film with oxygen plasma, and and subjected to a sensorimotor cortex stroke. Dissociated neural progenitor subsequently shrinking at elevated temperature. The length scale of wrinkles cells were labeled with Hoechst and transplanted into the penumbra seven can be tuned by adjusting plasma treatment time as results in a substrate days after stroke, with 200,000 cells injected per animal. Control animals

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Thursday Poster Abstracts received sham injections. Transplanted animals showed more improvement ability to modulate this activity. Our paradigm offers a novel approach for in the task on the affected side than control counterparts 28 days after the study of disease mechanism in melanocyte associated disorders such as transplantation, suggesting that the transplanted cells facilitated functional CHS and HPS and represents a powerful platform for future applications in recovery of the sensorimotor cortex. In frozen sections, the Hoechst label drug discovery. co-located with MAP2 and NeuN, indicative of neuronal differentiation of transplanted cells. Our investigation provides an efficient and effective in Poster Board Number: 3405 vitro protocol for neuronal differentiation of human ES and iPS cells. This DEVELOPING A HUMAN DRUG DISCOVERY protocol may facilitate further investigation of the mechanisms in neuronal differentiation of human stem cells. More detailed examination of neuronal PLATFORM FOR HUNTINGTON’S DISEASE differentiation and functional assays may prove that it is also useful for Harness, Julie V., Nistor, Gabriel, Keirstead, Hans S. transplantation therapy of brain disorders such as ischemic stroke. University of California, Irvine, Irvine, CA, USA Poster Board Number: 3403 Background and Objective: The objective of this work is to develop a high THE DERIVATION OF MELANOCYTES FROM purity human stem cell based drug discovery platform for Huntington’s Dis- ease (HD). HD is a neurodegenerative genetic disorder that results primar- HUMAN PLURIPOTENT STEM CELLS FOR ily in the loss of medium spiny projection neurons (MSN) of the striatum. DISEASE MODELING Current animal models do not sufficiently recapitulate the complex cascade of neurodegenerative events in the human. Current cellular models are Gruber Mica, Yvonne, Lee, Gabsang, Chambers, Stuart M., complicated by primary extraction and purification methods or if differ- Tomishima, Mark J., Studer, Lorenz entiated from hESC, are confounded by contaminant progenitor popula- Memorial Sloan-Kettering Cancer Center, New York, NY, USA tions. The need exists for high purity MSN and their progenitors (lateral ganglionic eminence progenitors or LGP) at multiple developmental stages Melanocytes are pigment-producing cells found predominantly in the epi- for transplant studies as well as drug discovery and predictive toxicology dermis where they establish a photo-protective barrier against UV-irradiation assays. Methodology and Principal Findings: Participants donated surplus induced DNA damage. Defects in melanocyte biology are associated with genetically abnormal blastocysts carrying HD and healthy blastocysts to numerous pigmentation disorders including albinism, vitiligo, and piebald- the Keirstead Research Group. All participants were consented under UCI ism. Yet, while the developmental biology of melanocytes has been well HSCRO-approved guidelines. Blastocysts were thawed and two hESC lines studied in avian and murine models, the processes underlying melanocyte were derived in animal-free conditions, maintained in animal-free conditions development in the human system remain poorly understood. We hy- and characterized with Cell Line Genetics. hUCI-1 is a genetically normal line pothesized that basic developmental insights can be applied to model the and hUCI-HD1 is a HD line carrying 44 CAG repeats. Both are karyotypi- progressive and selective specification of human pluripotent cell lines along cally normal. Undifferentiated hESC were grown on matrigel-coated flasks to the melanocytic lineage. Melanocytes are known to arise from the neural subconfluence in a feeder-free system. Cultures were then transitioned from crest (NC), a transient, migratory populations of cells unique to vertebrates. conditioned medium to a DMEM-F12 rich medium for neural induction. With the goal of establishing a rapid and defined protocol for the induc- Cells were grown in suspension and passaged routinely in differentiation tion of neural crest in vitro, we adapted a recently developed protocol that medium supplemented with growth factors until day 60, when they were re- supports the highly efficient differentiation of human embryonic stem cells plated and growth factors were withheld for final maturation to MSN by day (hESCs) and induced pluripotent stem cells (iPSCs) into both central nervous 66. Stage-specific immunocytochemistry was performed at day 14 (GSH- system and neural crest precursors. Using a Sox10::GFP BAC hESC reporter 2), day 45 (FoxP1) and day 66 (DARPP-32) to refine growth conditions to line, we have found that optimization of Wnt, BMP, and TGF-β signaling optimize and minimize contamination by other neuronal subtypes. The early within the context of the dual SMAD inhibition protocol greatly enhances stage LGP will be used for studies of striatal development and the MSN will NC induction to 65% of the population. Interestingly, a narrow window of be used for transplant studies, drug discovery and predictive toxicology. Wnt signaling appears to govern neural crest induction within the context of this protocol. We further found that melanocyte-competent progenitors Poster Board Number: 3407 within the NC population could be prospectively identified and enriched based on their co-expression of Sox10::GFP and c-kit. Further optimization THE SIGNALING PROPERTIES OF INOSITOL of the differentiation protocol revealed that induction of this population PHOSPHATES AND PHOSPHOINOSITIDES could be enhanced through treatment with BMP4 and Endothelin-3, two factors implicated in melanocyte specification. A brief period of additional IN HUMAN EMBRYONIC STEM CELLS AND maturation was sufficient to establish a pure and expandable population of HUMAN EMBRYONAL CARCINOMA CELLS pigmented melanocytes. To dovetail this rapid and defined melanocyte dif- ferentiation protocol with iPSC-based disease modeling, we have identified Hoofd, Catherine, Devreker, Fabienne, Deneubourg, Laurence, two melanocyte-associated disorders amenable for in vitro studies. Chediak- Deleu, Sandrine, Nguyen, Thi Mai Uyen, Sermon, Karen, Englert, Higashi Sydnrome (CHS) is a rare, autosomal recessive disease caused by Yvon, Erneux, Christophe mutations in the lysosomal trafficking regulator CHS1/LYST gene. Herman- Laboratoire de Recherche en Reproduction Humaine, Université Libre de sky-Pudlak Syndrome (HPS) encompasses a collection of eight autosomal Bruxelles, Brussels, Belgium, Institut de Recherche Interdisciplinaire en recessive disorders defined by deficiencies in the biogenesis of lysosome-re- Biologie Humaine et Moleculaire, Université Libre de Bruxelles, Brussels, lated organelles, including melanosomes. While in broad terms, HPS appears Belgium, Dept of Embryology and Genetics, Vrii Universiteit Brussels, to be a disorder of vesicle formation and CHS a defect in vesicle trafficking, Brussels, Belgium both disorders result in hypopigmentation. This phenotype can be assessed as a deficiency in melanosome transfer to neighboring keratinocytes. We Introduction and Aims: Human embryonic stem cells (hESCs) and their have therefore developed an assay for qualitatively and quantitatively as- malignant counterparts, human embryonal carcinoma cells (hECCs) sessing melanosome transfer under co-culture conditions with immortalized represent an inestimable source of cells to study early embryonic develop- keratinocytes. We have established iPSC lines from patient samples for each ment and mechanisms involved in tumorigenesis such as cell proliferation of the two disorders as well as a control line, using a Cre-excisable lentiviral and differentiation. To better understand the physiology of hESCs and the system. We currently assess melanosome transfer activity of CHS- and HPS- mechanisms of cellular transformation, we decided to work simultaneously iPSC derived melanocytes and test candidate chemical compounds for their with hESCs lines and a hECCs line. We initiated this work by studying cell

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signalling pathways derived from inositol polyphosphate metabolites in both or iPS cells (n=17 experiments), we observed a highly skewed distribution hESCs and hECCs. The inositol ring offers an energetically and chemically of CFCs. In most experiments, the majority of the EBs contained no CFCs, stable base for the formation of a great diversity of phosphorylated deriva- and the positive EBs contained a highly variable number of CFCs (1-90 per tives: lipid-anchored phosphoinositides (PIPs) or soluble inositol phosphates EB). Pretreatment of hPSCs to promote mesoderm differentiation prior to (IPs). PIPs and IPs show critical second messenger functions in a very large initiation of EB formation and/or addition of hematopoietic growth factors number of cell models in vitro and in vivo. They play major roles in vari- to the EB cultures increased both the proportion of EBs containing CFCs and ous transduction pathways controlling cellular events in cell proliferation, the average number of CFCs per positive EB, but the distribution of CFCs survival and differentiation, membrane motility, adhesion, cytoprotection remained highly skewed. This suggested that the hematopoietic output in and osmoregulation. These properties connect these signalling molecules to the positive EBs was either clonal, due to the generation of limiting numbers tumorigenesis and dysfunctions of these pathways may lead to oncogenic of CFC precursors or supportive cell types, or polyclonal, due to a hetero- transformation. Several enzymes, inositol-kinases and phosphatases, play es- geneous distribution of CFC-generating cells in the EBs, or differentially sential roles in IPs and PIPs signalling pathways. Among the inositol-kinases, determined paracrine effects within different EBs. To distinguish between inositol trisphosphate 3-kinases (Itpks) catalyse the conversion of IP3 to IP4, these possibilities, we lentivirally marked cells with GFP at the time of EB a role also played by the ubiquitous inositol phosphate multikinase (IPMK). formation and subsequently assessed the fluorescence of the individual he- Methods and Results: Different cell lines were used for this study: two hESCs matopoietic colonies derived from CFCs harvested from individual EBs. The lines derived in our lab, one imported from AZ-VUB, and a hECCs cell line. efficiency of stable transduction as measured by expression of GFP in the to- Inositol phosphates formation was analyzed by HPLC and the expression tal cells obtained from pooled EBs when they were harvested for assessment of the different kinases was quantified by Real Time PCR. We studied both was 31±4% (n=12 experiments). We also demonstrated that hematopoietic undifferentiated hESCs and hECCs as well as spontaneously differentiated cells derived from CFCs in these EBs retained the ability to express the GFP hESCs after one or two weeks of culture. Using HPLC, we were able to cDNA transduced ~4 weeks previously. Interestingly, in these experiments separate and highlight the presence of all IPs in our three different hESCs on pooled EBs, between 0-100% of CFCs were marked in different experi- lines and in the hECCs line. For each cell line, the distribution of IPs was ments and this was not strongly correlated with the transduction efficien- equivalent with an abundant IP5 amongst the highly phosphorylated inositol cies, consistent with the concept that only a few cells contribute to the final phosphates. In hESCs spontaneously differentiated after one or two weeks output of hematopoietic progeny. When individual EBs were analysed (142 of culture, we observed that IP5 was repeatedly and significantly decreased EBs in 4 experiments), the majority of positive EBs contained CFC popula- during differentiation. Real time PCR analysis of kinases primordial for IPs tions that were entirely GFP+ or GFP-. However, 37% of EBs containing >1 signalling revealed that some (isoforms A and B) but not all isoforms of CFC did contain both GFP+ and GFP- CFCs. If the GFP- colonies are not the Itpks were increased during spontaneous differentiation of hESCs, but not progeny of CFCs in which a transduced GFP cDNA has been silenced, this IPMK. Considering our undifferentiated cell lines, we showed that the same indicates a multi-precursor origin of the CFCs in some EBs despite the highly isoforms were overexpressed in our cancerous model as compared to our skewed distribution of CFCs among EBs. These findings demonstrate that hESCs lines. To confirm these results, we have assessed their enzymatic that the generation of hematopoietic progenitors is a very rare event in EBs activities in our cells and are in the process to visualize their expression by generated from either human ES or iPS cells, and that lentiviral marking can immunofluorescence. Conclusions: This is the first report of IP contribution offer a powerful strategy to track their developmental origin. to cell signalling in hESCs. We have shown that IPs are present in these cells and are regulated during their differentiation. We also showed that specific Poster Board Number: 3411 enzymes of the inositol phosphates signalling pathway were expressed at DIRECTED DIFFERENTIATION OF HUMAN a higher level in our cancerous counterparts of hESCs and were increased during differentiation of our hESCs. Together, these results suggest that PLURIPOTENT STEM CELLS TO A DEFINITIVE highly phosphorylated inositol phosphates could participate in the control of HEMATOPOIETIC FATE cellular differentiation in a new human model, hESCs. Kennedy, Marion, Sturgeon, Chris, Ditadi, Andrea, Keller, Gordon Poster Board Number: 3409 McEwen Centre for Regenerative Medicine Ontario Cancer Institute, CLONAL TRACKING OF THE GENESIS OF Toronto, ON, Canada HEMATOPOIETIC CELLS FROM HUMAN The hematopoietic system in the early embryo consists of two distinct programs; primitive hematopoiesis and definitive hematopoiesis that can be PLURIPOTENT STEM CELLS DIFFERENTIATING distinguished by the site and timing of development and the spectrum of IN EMBRYOID BODIES lineages generated. Primitive hematopoiesis, the first program to emerge, is restricted to the yolk sac and produces primitive erythrocytes, macrophages Kardel, Melanie D., Eaves, Connie J. and megakaryocytes, but does not give rise to lymphoid cells or hematopoi- Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada etic stem cells (HSCs). The definitive hematopoietic program is induced at Human pluripotent stem cells (hPSCs) represent an attractive source of cells different sites within the embryo following the onset of primitive he- to delineate, manipulate and generate hematopoietic derivatives. When hP- matopoiesis and is characterized by multilineage development including the SCs are induced to aggregate in non-adherent cultures, they form embryoid production of the lymphoid lineage cells and HSCs. To model hematopoietic bodies (EBs) in which hematopoietic cells appear and are produced over the development in human pluripotent stem cell (hPSC) differentiation cultures, following 2-3 weeks. However, the contributions of individual human ES or it is important to develop assays to enable one to distinguish contributions iPS cells to the hematopoietic cell outputs obtained remain undefined, as from the primitive and definitive hematopoietic programs. In this study, we does the identity and frequency of intermediate human cell types that can have used lymphoid potential to monitor the emergence of definitive he- generate hematopoietic derivatives. We hypothesized that if the frequency matopoiesis from human embryonic (hESCs) and induced pluripotent stem of ESC/iPSCs differentiating into hematopoietic cells is high, these would cells (hiPSCs) differentiated as embryoid bodies (EBs) in defined cultures be approximately evenly distributed amongst EBs, whereas, if the frequency in the absence of serum and supportive stromal cells. When induced with of such an event is very low we would observe a skewed distribution, with the combination of Activin A, BMP-4 and bFGF in the absence of serum, some EBs containing no or very few hematopoietic cells and some contain- hPSCs rapidly differentiate and give rise to hematopoietic populations that ing many. From measurements of the hematopoietic colony-forming cell represent the hemangioblast and primitive hematopoiesis. To determine if (CFC) content of 1,725 EBs dissociated and evaluated individually 14-18 the definitive hematopoietic program is also induced under these conditions, days after initiating EB formation from either human ES (n=19 experiments) we assayed different populations for their ability to generate T-lymphocytes

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Thursday Poster Abstracts following co-culture on OP9DL1 cells. With this approach, we identified a Poster Board Number: 3415 progenitor population in hESC-derived EBs that displays the capacity to ef- ficiently differentiate to T-lymphocytes, myeloid cells and erythrocytes. Flow NOVEL DIRECT DIFFERENTIATION METHOD cytometric analyses revealed that these progenitors display a marker profile FOR GETTING HIGHLY UNIFORM NEURAL similar to that of the SCID repopulating cell found in the cord blood. A com- parable progenitor population was detected in hiPSC-derived EBs, indicating PROGENITOR CELLS AND DOPAMINE that the differentiation strategy was broadly applicable to different stem cell NEURONS FROM HUMAN ES CELLS AND IPS lines. These findings indicate that the definitive hematopoietic program is in- CELLS duced in hPSC-differentiation cultures and suggest that the population that displays lymphoid/myeloid/erythroid potential may represent the progenitor Kim, Dohoon, Rhee, Dong-Yoon Rhee, Park, Han-Soo, Moon, of the human HSC. Jung-Il, Won, Chunkil, Sumi, Shoichro, Inoue, Kazutomo Poster Board Number: 3413 Mclean Hospital, Harvard Medical School, Belmont, MA, USA, Dept of Bioscience, Hanyang University, Belmont, Korea, Republic of, Dept of RUNX1C EXPRESSION IDENTIFIES Genetics, Brigham & Women’s Hospital, Harvard Medical School, Boston, HEMATOPOIETIC PROGENITORS IN CD34+ MA, USA, Institute of Life Science, Gyeongsang National University, Jinju, Korea, Republic of, Institute for Frontier Medical Sciences, Kyoto University, CELLS ISOLATED FROM DIFFERENTIATING Kyoto, Japan, Institute for Frontier Medical Science, Kyoto University, RUNX1CGFP/W HUMAN EMBRYONIC STEM Kyoto, Japan CELLS. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can offer a human disease model and promise for autologous cell Elefanty, Andrew G., Azzola, Lisa, Jokubaitis, Vanta J., Vlahos, transplantation in Parkinson’s disease (PD). Here we have examined a novel Katerina, Ng, Elizabeth S., Hirst, Claire, Yu, Qing C., Durnall, direct differentiation method for neural precursor cells (NPCs) and dopamine Jennifer, Williams, Brenda, Haylock, David, Nilsson, Susie, Stanley, (DA) neuron differentiation from hESCs and hiPSCs. Our method showed Edouard G. higher efficiency in NPCs and DA differentiation than it in the other popular Monash Immunology and Stem Cell Laboratories, Monash University, differentiation methods. Our method also induced to yield uniform popula- Clayton, Australia, Molecular and Health Technologies, CSIRO, Clayton, tions of NPCs (over 95% positive) with the midbrain marker expressions, Australia which subsequently differentiated into high proportions of DA neurons within short term (taking only 10days for inducing NPCs from hESCs). The RUNX1 gene plays an essential role in the regulation of blood cell However, NPCs and DA neurons derived from hES cells with other popular formation. Alternate promoters control the generation of two major iso- methods showed less differentiation efficiency and more long duration for forms of RUNX1, which differ at their N-termini. The RUNX1c isoform is differentiation than ours. This study will provide a clinical applicable tool for hypothesized to specifically mark hematopoietic cells. In order to identify getting uniform NP and DA neuron from hES cells. cells expressing RUNX1c during hematopoietic differentiation of human embryonic stem cells (hESCs), we targeted a GFP gene downstream of the Poster Board Number: 3417 RUNX1 distal promoter in hESCs to generate heterozygous RUNX1cGFP/w hESCs and RUNX1cGFP/GFP lines in which both endogenous alleles of the PUTATIVE ROLE OF MICRORNA-21 IN HUMAN RUNX1c gene were deleted. In RUNX1cGFP/w cells, GFP was observed EMBRYONIC STEM CELLS from 10-25 days of differentiation and accurately mirrored expression of the endogenous RUNX1c isoform. GFP was restricted to CD45+ hematopoietic Kim, Yeji, Kim, Nury, Park, Sang-Wook, Han, Yong-Mahn cells and GFP+CD34+ cells were highly enriched for myeloid progenitor cells Biological Sciences, KAIST, Daejeon, Korea, Republic of and displayed an ability to home to the bone marrow of immunocompro- Human embryonic stem cells (hESCs) have unique properties of self- mised mice. Microarray analysis of cDNA synthesized from sorted fractions renewal and pluripotency. Among various cellular regulators, a specific- revealed that GFP+CD34+ cells were enriched for transcripts associated microRNA(miR) plays a salient role in fine-tuning of the gene expression. with hematopoietic stem and progenitor cells. Examination of differentiat- Although miRNAs function as key regulators in the cellular process, roles of ing RUNX1cGFP/GFP cells revealed that RUNX1c was not necessary for the miRNAs are poorly understood in hESCs during differentiation into a special- generation of hematopoietic colony forming cells during the first 14d of ized cell type. Here expression profiles of microRNAs were first investigated hESC differentiation. These results demonstrate that RUNX1c is expressed in in hESCs during differentiation into endothelial cells in vitro by miRNA array hematopoietic progenitor cells and cells with bone marrow homing ability in using 799 human miRNA probes. As results, 27, 56, and 3 miRNAs were differentiating hESCs. enriched in hESCs, hESC-derived CD34 positive cells, and endothelial cells, respectively. Also 34, 1, and 21 miRNAs were depleted in hESCs, hESC- derived CD34 positive cells, and endothelial cells, respectively. Interestingly, expression of miR-21 was not detected in hESCs but gradually increased during differentiation into endothelial cells. SOX2 was screened as a putative target of miR-21 using a target-scan computer program. It was confirmed by a luciferase-reporter assay that miR-21 bound to the 3’ untranslated region of SOX2. Exogenous addition of synthetic miR-21 led to a slight decrease of SOX2 in hESC as compared to non-treated hESCs. Our findings indicate that repression of miR-21 may support to maintain the self-renewal of hESCs.

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Poster Board Number: 3419 Poster Board Number: 3423 PROTEIN KINASE C INDUCES EPITHELIAL TO MECHANICAL STRETCHING ON OSTEOGENESIS MESENCHYMAL TRANSITION IN HUMAN ES OF HUMAN EMBRYONIC STEM CELLS AND IPS CELLS Li, Mingming, Li, Xiao Bing, Cao, Tong Kinehara, Masaki, Kawamura, Suguru, Tateyama, Daiki, Inoue, Dentistry, National University of Singapore, Singapore, Singapore Shin-ich, Matsumura, Hiroko, Hirata, Mitsuhi, Furue, Miho K. Bones function to move and support our body. Bone was formed through Dept of Disease Bioresources, National Institute of Biomedical Innovations, mineralization of osteoid, which is a protein mixture produced by osteoblast Osaka, Japan cells. Many studies have showed in vitro mechano-stimulation is effec- tive in bone cell proliferation and differentiation with controlled delivery of During early embryogenesis, epithelial to mesenchymal transition (EMT) mechanical forces using primary osteoblast cells. In this study, we investi- is required for epiblast cells to facilitate their ingression within the primi- gated the application of mechanical stretching in osteogenesis of human tive streak. We show here that phorbol 12-myristate 13-acetate (PMA), an embryonic stem cells. Human embryonic stem cells differentiation into activator of protein kinase C (PKC) causes EMT in human ES/iPS cells within osteoblast cells have been studied for several years. The basic differentiation 24 hours. After PKC stimulation, morphological changes were observed in components are Dulbecco’s Modified Eagle Medium (DMEM, high glucose) hES/iPS cell colonies; the colonies lacked well-defined edges and surround- media supplemented with 10%FBS, Dexamethason, and Ascorbic acid. ing fibroblast-like cells were increased. We confirmed that the expression of Some protocols also include Vitamine D3 or other small molecule chemicals E-cadherin was down-regulated, and E-cadherin repressor molecules (Twist for osteogenic induction. However, to our knowledge, the role of mechani- and Slug) and vimentin were increased, indicating that PMA induces EMT. cal forces in embryonic osteogenesis is still a new field yet to explore. In our Subsequently, mRNA expression levels of SOX7, CDX2, GATA4, and GATA6 studies, we only used the basic media components for induction to avoid were increased, suggesting that extraembryonic lineage were induced. Our unnecessary complexity. Three experimental groups were set up where results have shown that PKC induced cell differentiation into extraembryonic stretching forces were applied for 7 days intermittent treatment starting lineage by the initiation of EMT in hES/iPS cells. from D7 to D14, D14 to D21 and 14 days treatment from D7 to D21. One Poster Board Number: 3421 group without stretching treatment was set up as control. Induction media were changed to fresh every other day and spent media were collected BALANCE OF PROLIFERATION AND for alkaline phosphatase secretion studies. Over 21 days differentiation, DIFFERENTIATION POTENTIAL COULD BE cells were collected for PCR arrays to screen osteogenic, cytoskeleton and extra-cellular adhesion genes responded to mechanical forces. Immuno- MODULATED BY CULTURE MEDIUM IN HUMAN cytochemical staining was carried out for osteocalcin localization and total EMBRYONIC STEM CELLS collagen assay was studied for collagen deposition. Total cell numbers were counted to compare proliferation status and alizarin red staining was used Lee, Jung Bok, Russell, Daniela Fischer, Graham, Monica, Lee, to observe in-vitro mineralization. The results showed stretching treatment Jong-Hee, Werbowetski-Ogilvie, Tamra E., Hong, Seok-Ho, Bhatia, from D7 to D14 was the most efficient among the three treatment groups Mickie in comparison with control. This treatment group counted the most number McMaster University, Stem Cell and Cancer Research Institute, Hamilton, of cells, which is 3 folds higher than control and 1.5 and 2 folds higher than ON, Canada the other two treatments, which suggests that though stretching may have caused certain level of immediate cell death, cells proliferate faster after- Applications of human stem cells require efficient proliferation, while still re- wards if the treatment is applied early during differentiation. Quantity of taining the ability to generate specialized progeny. Although these combined collagen deposition is the most in this group also, which provide the organic features represent the hallmark properties attributed to human embryonic matrix support for mineralization of osteoid secreted by differentiated stem cells (hESCs), the correlation between proliferation and differentia- osteoblast cells. In conclusion, 7 days intermittent stretching from D7 to D14 tion potential of hESCs controlled by different medium conditions remains was the most effective treatment in comparison with other treatment groups unclear. Here we examined whether the balance of proliferation and dif- studied here and control. ferentiation capacity of hESCs could be modulated permanently by specific media conditions. We demonstrate that semi-defined mTeSR1 media favors Poster Board Number: 3425 self-renewal of hESCs with increased differentiation potential toward neural lineage at the expense of hematopoietic differentiation potential. However, THE EFFECT OF DISTINCT BMP LIGANDS this phenomenon is reversible and can be modulated by the subsequent ON SELF-RENEWAL AND DIFFERENTIATION culture of mTeSR1 treated hESCs in mouse embryonic fibroblast-conditioned medium (MEF-CM), which induces a recovery in hematopoietic differentia- OF HUMAN EMBRYONIC STEM CELLS AND tion potential. Our study suggests that hESCs exist in a range of functional CHORIONIC VILLI CELL-DERIVED HUMAN states spanning the balance from self-renewal to differentiation, all of which INDUCED PLURIPOTENT STEM CELLS can be reversibly modulated by specific media conditions. In summary, we demonstrate that the definition of medium conditions is paramount the Lichtner, Björn, Adjaye, James efficient and controlled modulation of hESC proliferation and differentiation Vertebrate Genomics, Max Planck Institute for Molecular Genetics, Berlin, towards specific lineages. Germany Bone morphogenetic proteins (BMPs) belong to the transforming growth factor beta (TGF-β)-superfamily of biological active cytokines. They consist of approximately twenty members and are present within a broad range of animal species where they are usually highly conserved. BMP ligands are known to have important pleiotropic functions in man. With respect to human embryonic stem cells (hESCs), it has been shown that activation of BMP-specific pathways (via Smad1/5/8) leads to differentiation, whereas activation of TGF-β/Activin/Nodal-specific pathways (via Smad2/3) is

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Thursday Poster Abstracts essential for maintaining the undifferentiated state. Depending on the cell with 14% Insulin+ cells, 2x higher than differentiation entirely at 20% O2. culture conditions, BMPs have shown to induce differentiation of hESC into These cells passively secreted C-peptide but were not glucose responsive. multiple lineages, such as trophectoderm, (extra-embryonic) endoderm Low O2 for extended periods after differentiation was complete drastically and mesoderm. Under specific conditions, distinct BMP ligands have also reduced the amount of residual PSC within, and the tumorigenic potential been described to support differentiation of hESC into vascular progenitor of, differentiated cell populations. Pluripotency marker (Oct4 and Nanog) cells, primordial germ cells and chondrocytes. However, to date, only a few expression and the fraction and number of Oct4+ cells was reduced by up members of the BMP family have been tested for their effects on hESCs and to four orders of magnitude at low compared to high O2. Upon implanta- human induced pluripotent stem cells (hiPSCs), with BMP4 being the best tion into immunocompromised mice, low O2-differentiated cells did not investigated and most commonly used ligand. Our aim is a comparative form tumors or did so slower than high O2-differentiated cells, consistent analyses of members of the BMP family in terms of their role in maintaining with reduced residual PSC. Cell sorting of SSEA-4+ cells after extended low self renewal and pluripotency or induction of differentiation of hESCs and O2 culture of hESC further reduced residual PSC and tumor formation rate. hiPSCs. To this end, we have identified two ligands (BMP5 and BMP13/ These findings establish that culture O2 is important for almost every aspect GDF6) that are inducing differentiation of hESCs, whose nature yet has to of PSC differentiation. We used highly-O2 permeable silicone rubber culture be described. Another of our major aims is to compare the downstream dishes for accurate control of cellular O2 exposure. By modulating O2 dur- signalling effects of these two BMP ligands in hESCs (H1) and hiPSCs. For ing different stages of differentiation, we achieved substantial increases in this purpose, we have successfully induced pluripotency in human chorionic cardiomyocyte and Insulin+ cell yields. Extended culture at low O2 further villi cells - which we name hCViPSC. We are currently investigating the decreased residual PSC by several orders of magnitude. O2 control, alone or nature of the BMP-induced differentiation using microarray-based global combined with other methods could be applied to future cell therapy proto- transcriptome profiling. Furthermore, we are investigating the mechanism cols to generate and increase the safety of differentiated cells. of action of these two BMPs by analyzing which of the several known BMP-downstream targets are activated or repressed. Where applicable, we Poster Board Number: 3429 will also compare the potency between these two and other BMP ligands in NON-INVASIVE CHARACTERIZATION inducing differentiation of hESCs and hCViPSCs. In summary, we envisage that our studies will reveal additional cytokines that could be applicable for OF MOUSE EMBRYONIC STEM CELL lineage specific differentiation of hESC and hiPSC to derive donor cell types DIFFERENTIATION USING RAMAN useful for cellular regenerative therapies in the future. SPECTROSCOPY Poster Board Number: 3427 Knopf, Anne, Koch, Steffen, Schenke-Layland, Katja HYPOXIA MARKEDLY INFLUENCES Dept. of Cell and Tissue Engineering, Fraunhofer IGB Stuttgart, Stuttgart, DIFFERENTIATION OF MOUSE AND HUMAN Germany PLURIPOTENT STEM CELLS Their ability for indefinite self-renewal and potential to differentiate into cells of all three germ layers makes embryonic stem cells (ESCs) an attractive Millman, Jeffrey R., DiIenno, Amanda R., Colton, Clark K. source for tissue engineering and regenerative medicine. When focusing on Chemical Engineering, Massachusetts Institute of Technology, Cambridge, a potential therapeutic application of these cells, a non-invasive and rapid MA, USA method for the characterization the cell’s differentiation is needed. The aim of this study was to determine if Raman spectroscopy could be used as a Efficient differentiation of pluripotent stem cells (PSC) to desired cell types novel, non-contact and label-free method for the in situ identification of for cell replacement therapy remains a challenge, and potential tumor the developmental state of ESCs. A home-built micro-Raman spectrometer, formation by residual PSC in differentiated populations is problematic. Most coupled into an Olympus IX71fluorescence microscope was employed for PSC research is performed in high, non-physiological O2, but cells during all measurements. We used undifferentiated murine ESCs (feeder-free cell embryonic development are exposed to much lower O2. Here we report line CCE), differentiated embryoid body (EB)-derived cells and ESC-derived a wide-ranging study showing that physiological O2 markedly influences cardiovascular cells. Murine embryonic fibroblasts (MEFs) served as controls. differentiation to all three germ layers, with enhanced differentiation to To determine differences in the global biochemical component patterns of cardiomyocytes and insulin-producing cells in particular, and reduces residual the four cell groups, Raman spectra were obtained and analyzed with prin- PSC and tumor formation. We differentiated mouse and human embryonic cipal component analyses (PCA) using the Opus and Unscrambler software. stem cells (mESC and hESC) and mouse induced pluripotent stem cells under For Raman measurements, all cells were transferred to 35 mm glass bottom controlled cellular O2 environments through adhesion culture on highly dishes, incubated for one hour and monitored in suspension. To confirm O2-permeable silicone rubber membranes. Low O2 drastically increased cells differentiation states, immunofluorescence staining was performed for cardiomyocyte differentiation from these PSC. Best results were acquired by the pluripotency markers Nanog, Oct4 and SSEA1, as well as CD31, which differentiation of mESC for 6 d at 5% O2 followed by 20% O2 for 15 d, re- labels cells that are committed to the cardiovascular lineage. After stain- sulting in up to 57% cardiomyocytes and 304 cardiomyocytes generated per ing, Raman spectroscopy was performed on the same cells. We observed initial mESC without purification, factors of 5 and 9 higher, respectively, than significant differences between the global Raman spectra of undifferentiated differentiation entirely at 20% O2. Low O2 culture for the first 6 d increased and differentiated cells. The most striking differences were observed for the by 3x expression of Mesp1/2, which helps restrict mesodermal cells to the Raman peaks 784 cm-1 and 1435 cm-1, which represent a DNA peak and a cardiovascular lineage, but did not increase Brachyury T expression, an early peak related to many cell components including proteins, lipids, amides and mesodermal marker, compared to 20% O2, thereby suggesting that low carbohydrates. If these peaks can therefore be affiliated with pluripotency is O2 acted by restricting the fate of early mesoderm towards cardiomyocytes. currently focus of ongoing studies. This study demonstrates the applicability Low O2 decreased the fraction and number of Nestin+ cells (ectoderm) of Raman spectroscopy for the non-contact in situ characterization of stem by 3x for mESC, but enhanced expression of endodermal genes Sox17, cell differentiation states under physiological conditions. We were able to Foxa2, Hnf4a, and Pdx1, stimulating us to examine differentiation of hESC show that differentiated and undifferentiated cells can be clearly separated to Insulin+ cells using a protocol developed by ViaCyte, Inc (San Diego, using PCA. CA). Differentiation to definitive endoderm (DE) was modestly enhanced by culture at 3-8% O2, but performing differentiation entirely at low O2 was detrimental for producing Insulin+ cells. Differentiation at 5% O2 to produce DE with all subsequent steps at 20% O2 achieved a population

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Poster Board Number: 3431 or small molecule dual inhibition (2i) of mitogen-activated protein kinase (using MEK inhibitor PD0325901) and glycogen synthase kinase (with GSK DIFFERENTIATION OF MOUSE EMBRYONIC inhibitor CHIR99021) reversibly affect levels of Oct4 expression in cells that STEM CELLS INTO CARDIOMYOCYTES are otherwise pluripotent. Effects of this preconditioning are manifested by changes in subsequent differentiation to cardiomyocytes (CM). Precondi- DEPENDS ON EXPRESSION OF CONNEXINS. tioning was performed with a mESC line harboring a GFP reporter driven Wörsdörfer, Philipp, Bosen, Felicitas, Egert, Angela, Schorle, by the distal Oct4 enhancer in self-renewal medium (DMEM with 10% FBS Hubert, Edenhofer, Frank, Willecke, Klaus and Leukemia Inhibitory Factor (LIF)) for up to 24 d at different O2 condi- tions (20%, 5%, 1%) with or without 2i. Differentiation of these cells was Institute of Reconstructive Neurobiology, University of Bonn, Bonn, immediately initiated by embryoid body formation for 2 d in DMEM with Germany, Life and Medical Science Institute (LIMES), University of Bonn, ascorbic acid and without LIF and 2i, followed by attachment culture. Cell Bonn, Germany, Department of Developmental Pathology, University of culture was performed on gelatin- (preconditioning) or fibronectin- (dif- Bonn Medical School, Bonn, Germany ferentiation) coated silicone rubber membranes for accurate control of the Gap junctions are intercellular conduits that allow the diffusional exchange pO2 to which the cells were exposed. mRNA expression was measured with of small molecules (> 1.8 kDa) between neighboring cells. Each gap junction real-time PCR, spontaneously contracting areas were estimated by visualiza- channel consists of two hemichannels (connexons), i.e. hexameric structures tion, Oct4-GFP expression and fraction of MF-20+ (sacromeric myosin formed by protein subunits, termed connexins. Twenty connexin isoforms heavy chain immunostained) cells was measured with flow cytometry, are described in the mouse genome. Mouse embryonic stem (ES) cells ex- and cardiac Troponin-T protein was visualized with immunocytochemistry. press 3 connexin (Cx) proteins (Cx31, Cx43 and Cx45) and are coupled via Preconditioning did not affect fraction of Oct4-GFP+ cells in all conditions. gap junction channels. To elucidate the function of gap junctional commu- However, high O2 (20%) and 2i individually and synergistically operated to nication in embryonic stem cells, we generated Cx43/Cx45 double deficient reversibly increase Oct4 expression in self-renewing mESC. Preconditioning ES cells. These cells show 95% reduction in gap junctional intracellular at 1% O2 without 2i reduced mean Oct4-GFP by 2.5x while 2i and 20% communication (GJIC) as determined by microinjection of neurobiotin. This O2 preconditioning increased mean Oct4-GFP expression by 2.7x compared strong reduction in cell-cell coupling, however, did not alter proliferation to cells preconditioned at 20% O2 without 2i. Initiating differentiation of rate or apoptotic death of ES cells. Besides their expression in embryonic cells with higher Oct4-GFP resulted in a greater fraction and number of stem cells, Cx43 and Cx45 are expressed throughout ES-cell differentia- MF-20+ cells, increased CM markers, and larger spontaneously beating tion into cardiomyocytes and are essential for the proper function of the areas. Cells preconditioned at 20% O2 with 2i produced 26% MF-20+ cells heart. To investigate the role of connexins in early heart development, the compared to only 5% MF-20+ cells differentiated from cells preconditioned Cx43/Cx45 deficient ES-cells were differentiated into cardiomyocytes using at 1% O2 without 2i, despite being differentiated using the same protocols an embryoid-body (EB) based differentiation protocol. The number of EBs at 20% O2. These observations are likely linked as Oct4 dose-dependent displaying spontaneously beating foci was reduced 5-fold in Cx43/Cx45 specification of the cardiac lineage during early differentiation has been deficient EBs. In order to analyze, whether this observation was due to a reported by others. Changes in Oct4-GFP expressions from preconditioning lower number of cardiomyocytes, we performed quantitative PCR analyses were reversed by further self-renewal at 20% O2 (for cells preconditioned to detect marker indicative for cardiac differentiation. We demonstrate that at 5% or 1% O2 without 2i) or removing 2i (for cells preconditioned with cardiomyocyte differentiation was strongly reduced. In further experiments, 2i). Preconditioned cells allowed to recover normal Oct4-GFP expression by we differentiated Cx43/Cx45 deficient ES-cells and wild-type ES cells in such further self-renewal differentiated to produce the same fraction of CM suspension cultures. We observed that wild-type EBs underwent strong mor- as cells that were never preconditioned. These results demonstrate that the phological changes and developed cystic structures which was only rarely manipulation of self-renewing culture conditions can lead to changes in the observed in Connexin deficient EBs. These results suggest that expression of outcomes of defined differentiation protocols, a novel dimension to explore connexins is required for the proper differentiation of ES cells in in vitro EB for directed differentiation of pluripotent stem cells. cultures. Poster Board Number: 3435 Poster Board Number: 3433 DIFFERENTIAL EXPRESSION OF FORMINS LOW OXYGEN CULTURE AND SMALL DURING HEART DEVELOPMENT AND MOUSE MOLECULE INHIBITION OF MITOGEN- EMBRYONIC STEM CELL DIFFERENTIATION TO ACTIVATED PROTEIN KINASE AND GLYCOGEN CARDIOMYOCYTES SYNTHASE KINASE DURING SELF-RENEWAL Maulion, Christopher, Blystone, Scott, Coetzee, William, Morley, REVERSIBLY AFFECT SUBSEQUENT Gregory, Maass, Karen DIFFERENTIATION OF MOUSE EMBRYONIC Cardiology, NYU School of Medicine, New York, NY, USA, Cell and STEM CELLS TO CARDIOMYOCYTES Developmental Biology, SUNY Upstate Medical University, Syracuse, NY, USA, Pediatric Cardiology, NYU School of Medicine, New York, NY, USA Tan, Jit Hin, Millman, Jeffrey R., Colton, Clark K. Cardiac repair by cell replacement therapy is a promising approach for the Dept of Chemical Engineering, Massachusetts Institute of Technology, treatment of end stage heart failure. Pluripotent stem cells can be differ- Cambridge, MA, USA entiated into cells that are excitable and capable of generating mechanical Prior studies on self-renewal culture of embryonic stem cells (ESC) focused force. However, in order to successfully engraft in vivo, individual cells on long term proliferation and pluripotency and were assessed by differ- must exhibit mature electrophysiological properties and integrate into the entiation to derivatives of the three germ layers. These include the discov- mechanical and electrical network of the host myocardium. The actin cy- ery of different states of pluripotency, some achieved by small molecule toskeleton affects both ion channel activity and the formation of mechanical inhibition of signaling pathways. The effects of culture conditions during cell-cell junctions. Formin proteins, a family of 15 genes in mice, dynamically self-renewal on the effectiveness of subsequent differentiation protocols regulate actin microfilament assembly. As in vitro differentiation of mouse have not been examined. Here we show that changes in culture conditions embryonic stem cells (ESC) results in phenotypical immature cardiomyo- during self-renewal (which we term ‘preconditioning’) by low pO2 culture cytes, differences in formin expression might reflect alterations to the actin cytoskeleton of adult cardiomyocytes. Objective: To compare formin expres-

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Thursday Poster Abstracts sion during mouse heart development to the profile of in vitro differentiated of the resting membrane suggesting that the channels are constitutively cardiomyocytes. Methods: Samples: mouse ESC and cardiac tissue from active. Dioctanoylglycerol (DOG), an activator of TRPC channels, caused a embryonic day 14.5, neonatal day 2 or 2 month old mice of the 129P2 Na+ dependent depolarization of the cells, which was blocked by Pyr-2 and strain (heart development); spontaneously contracting cardiomyocytes dif- Pyr-3. The effects of DOG on membrane potential and intracellular Ca2+ ferentiated from 129P2 ESC (in vitro differentiation). Transcript analysis was were most prominent at 5 days of differentiation and at the outer layers performed by quantitative real time PCR analysis (15 mouse formin genes, of the migration zone, correlating with neuronal phenotype as judged by 2 stemness genes, 4 cardiomyocyte differentiation genes). Expression of responses of the cells to kainate, and N-Methyl-D-aspartic acid (NMDA), candidate formin proteins was analyzed using commercially available formin agonists of ionotropic glutamate receptors, and Veratridine, an activator antibodies. Results: qRT-PCR analysis revealed significant changes in formin of sodium channels. These results demonstrate that TRPC channels are expression for 8 of 15 formins during heart development. Four patterns of functionally expressed in NPCs, where they are constitutively active, and can transcript changes were observed: formins expressed in ESC and decreasing be modulated through mGluR5. Our data indicate that coupling to TRPC during development (mDia1, mDia3; Fmnl2); transcripts peeking during fetal channels is low during day 1 of differentiation and increases at the outer heart development (Fhod1, Fmn1); transcripts altered during heart develop- border of the migration zone at day 5. The metabotropic glutamate receptor ment compared to ESC and adult heart (Daam1, Daam2); transcript increas- thus activates some other Ca2+ permeable channel during the initial phases ing during development (Fhod3). In vitro differentiated cardiomyocytes of differentiation but couples to TRPC channels when the cells gain a more revealed significant increases in the expression of embryonic formins (Dia3, neuronal phenotype and migrate further away from the neurosphere. Fmnl2, Daam2) and formins otherwise not differentially expressed during heart development (Dia2, Inf2). Expression of Fhod1 and mDia1 protein was Poster Board Number: 3439 studied in ESC, neonatal cardiomyocytes and in vitro differentiated cardio- IN VITRO DIFFERENTIATION OF FUNCTIONAL myocytes, verifying the observed changes in transcript expression. Conclu- sion: Dynamic changes in formin isoform expression occur during cardiogen- INSULIN-PRODUCING CELLS FROM MOUSE ES esis. The formin profile of in vitro differentiated cardiomyocytes resembles CELLS fetal and not adult cardiomyocytes Modifying formin expression of in vitro differentiated cardiomyocytes could improve functional maturity and there- Saito, Mikako, Shigeto, Hajime, Funabashi, Hisakage, Matsuoka, fore their potential use in cell replacement therapy for cardiac repair. Hideaki Poster Board Number: 3437 Dept Biotechnology & Life Sciences, Tokyo University of Agriculture & Technology, Tokyo, Japan ROLE OF TRANSIENT RECEPTOR POTENTIAL The development of a cell therapy for diabetes would be greatly aided by CANONICAL CHANNELS IN DIFFERENTIATION a renewable supply of insulin-producing cells. Here, we show a newly de- veloped and effective method which induced mouse ES cells to differentiate OF MOUSE EMBRYONIC NEURAL STEM/ into insulin-producing cells. A serum-free system was used in the initial stage PROGENITOR CELLS to induce definitive endoderm differentiation from mouse ES cells. Further, all-trans retinoic acid (RA) was used to promote pancreatic endoderm dif- Jansson, Linda C., Åkerman, Karl E. ferentiation, as indicated by the expression of the early pancreatic transcrip- Physiology, University of Helsinki / Institute for Biomedicine, Helsinki, tion factor Pdx-1. After maturation in medium with nicotinamide and Ex-4, Finland the differentiated cells expressed Insulin1, Insulin2, Kir6.2 and Glut2. These Glutamate has been proposed to regulate NPC (neural stem/progenitor cells could be immunostained by anti-insulin and anti-C-peptide. Intracellu- cell) proliferation through ionotropic and metabotropic glutamate receptors lar insulin content of differentiated ES cells was determined by ELISA. These expressed by NPCs. Both ionotropic and metabotropic glutamate receptors cells secreted insulin, and the glucose-dependent secretion was observed. act by increasing intracellular Ca2+ and different types of cellular functions, Next, the differentiated insulin-producing cells (5X106 cells) were aspirated such as proliferation, differentiation, cell death, secretory responses, are trig- into syringe, and gently placed under the kidney capsule of streptozotocin- gered by local or global rises in intracellular Ca2+. induced diabetic mice. Mice were monitored for blood glucose levels using Group I (mGluR1 and mGluR5) metabotropic receptors act via Ca2+ dis- blood from the tail vein, and the function of transplanted cells was inves- charge from intracellular stores and activation of Ca2+ permeable transient tigated concerning the confirmation of cell survival and glucose (2 g/kg) receptor potential (TRP) channels, in particular members of the canonical tolerance tests after the fasting. When the grafts were examined after 20- transient receptor potential (TRPC) channel subfamily. The TRPC channels day post-transplant, the insulin- and C-peptide-positive cells were observed. are non-selective Na+ and Ca2+ permeable channels and the TRPC sub- However, the function of the blood glucose regulation did not improve. family in particular has been shown to have an important role in neuronal Production of these mouse ES cell-derived cells may represent a critical step survival, differentiation, and synapotogenesis. Our working hypothesis in the development of cells for diabetes cell therapy. is that these channels function as sensors of the microenvironment and Poster Board Number: 3441 transducers of signals by causing local depolarization and elevations of intracellular Ca2+ and that activation of TRPC channels via mGluR5 is of A NOVEL ROLE FOR P38 MAPK IN ENDODERM importance for the differentiation and migration of NPCs. I have shown, using quantitative RT-PCR, that the NPCs we use in our system express FORMATION FROM MOUSE PLURIPOTENT trpc1, trpc3, trpc4 and trpc5 mRNA in their proliferative state and when CELLS differentiation for 1 and 5 days. The mRNA expression generally increased Yap, Charlotte, Goh, Hwee Ngee, Rathjen, Peter David, Rathjen, upon differentiation except in the case of trpc3 where the mRNA expression declined during differentiation. The differentiating cells were then function- Joy ally characterized using Ca2+ imaging. The Ca2+ response to low concen- Zoology, University of Melbourne, Melbourne, Australia trations of (S)-3,5-dihydroxyphenylglycine (DHPG, an agonist of group I The appearance of the primitive streak (PS) visibly marks the beginning of metabotropic glutamate receptors) was blocked by removal of extracel- gastrulation in the mammalian embryo. Primitive ectoderm cells that migrate lular Ca2+, suggesting that a pathway for entry of extracellular Ca2+ was through the PS lose their pluripotency and form PS-like intermediates, a activated by DHPG. The Ca2+ response to DHPG response was also blocked bi-potent progenitor of mesoderm and endoderm. Cells that do not migrate by SKF96365, 2-aminoethoxydiphenyl borate (2-APB), Pyr-2 and Pyr-3, all through the PS form ectoderm. The formation of PS-like intermediates in blockers of TRPC channels. All of these blockers caused a hyperpolarization

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culture recapitulates the complex process of differentiation and provides Poster Board Number: 3445 tools to delineate the molecular mechanisms regulating differentiation towards mesoderm and endoderm, or conversely ectoderm. A comprehen- FIBRILLAR FIBRONECTIN PROMOTES sive understanding of the signalling pathways regulating lineage choice from ENDODERM SPECIFICATION IN MOUSE pluripotent cells will promote the development of optimised differentiation and enrichment protocols for desired cell types. p38 MAPKs are a conserved EMBRYONIC STEM CELLS subfamily of mitogen-activated protein kinases (MAPKs) that have a well Taylor-Weiner, Hermes, Fuhrmann, Alexander, Engler, Adam J. established role in immunity and inflammatory responses in the mammal. Bioengineering, University of California, San Diego, La Jolla, CA, USA Potential roles for this signalling cascade during early gastrulation are not well defined. The p38 MAPK inhibitor SB203580 was used to investigate a While in vitro differentiation protocols often rely exclusively on soluble role for p38 MAPK in the induction of PS-like intermediates from early prim- growth factors to direct ESC fate, the embryonic stem cell niche contains itive ectoderm-like (EPL) cells. EPL cells are a pluripotent analogue of the both soluble factors and fibrillar extracellular matrix (ECM) proteins, includ- mammalian primitive ectoderm, or epiblast, formed from mouse ES cells in ing fibronectin (FN). Endoderm lineage differentiation has traditionally relied culture. EPL cells differentiated in response to serum readily formed PS-like on Activin A and Wnt3a growth factors, though these protocols are limited intermediates. In contrast, cells differentiated in serum supplemented with by low differentiation efficiencies and significant apoptosis. We examined SB203580 formed PS-like intermediates poorly and preferentially formed whether a FN matrix could induce endoderm differentiation in mouse em- neurons. The addition of SB203580 to EPL cells differentiating in response to bryonic stem cells (mESCs) independent of growth factors. mESCs, grown as BMP4, however, did not affect PS-like intermediate formation. This suggests embryoid bodies, showed a spatiotemporal correlation between expression that BMP4 signalling and p38 MAPK activation (via serum) are independent of FN and GATA4, an endoderm gene. When grown on a cell-derived fibril- pathways capable of inducing PS-like intermediates from EPL cells. Analysis lar FN matrix, mESC average endodermal transcript increased 7-fold over of differentiation outcomes suggested that p38 MAPK inhibition impaired undifferentiated mESCs and was significantly higher than transcripts for oth- the ability of serum and BMP4 to induce endoderm, implicating p38 MAPK er lineages or self-renewal. mESCs also responded to changes in fibrillar FN signalling in the formation of endoderm. To confirm this, EPL cells were dif- matrix stiffness by upregulating endoderm markers 60-fold, though thiol- ferentiated in response to serum or BMP4 in the presence of the BMP inhibi- modified scaffolds that stiffen with culture time may even further enhance tor noggin. EPL cells formed PS-like intermediates poorly and differentiated differentiation. In comparison, traditional endoderm induction via growth preferentially to neural lineages when BMP pathways were inhibited in the factors upregulated endoderm genes only 4-fold. Together these data imply presence of serum or BMP4. Endoderm formation, however, was unaffected that fibrillar fibronectin is a novel inducer of endoderm expression. or enriched, supporting a novel role for p38 MAPK in endoderm formation from mouse pluripotent cells. These data suggest that a novel population of Poster Board Number: 3447 endoderm progenitors, distinct from PS-like intermediates may be present EVALUATION OF NEUROTOXICITY OF during mammalian gastrulation. ARSANILIC ACID, DANOFLOXACIN AND Poster Board Number: 3443 MERCURY IN VITRO USING NEURAL WNT-3A INDUCES THE SPECIFICATION OF PROGENITOR CELLS DERIVED FROM MOUSE MOUSE EMBRYONIC STEM CELLS INTO THE EMBRYONIC STEM CELLS VISCERAL ENDODERM LINEAGE Kang, Hwan-Goo, Kim, Eu-Joo, Jeong, Sang-Hee Price, Feodor, Jones, Andrew, Rudnicki, Michael Toxicology and Chemistry, National Veterinary Research and Quarantine University of Ottawa/ OHRI, Ottawa, ON, Canada Service, Anyang, Korea, Republic of, Toxicological Research Center, Hoseo University, Asan, Korea, Republic of During embryonic development the formation of the first three cell types of the blastocyst, namely the trophoblast, epiblast and primitive endoderm Neural progenitor cells (NPCs) have self-renewal ability and can differentiate are specified by a complex process regulated by: morphogens, intracellular into all lineages of neural cell types. This study was performed to establish signaling transduction cascades, the formation of extracellular basement NPCs derived from mouse embryonic stem cells (ES) and to evaluate neu- membranes and cell-cell surface contacts. The Wnt family of secreted rotoxicity of chemicals in differentiation stage into neural progenitor cells. glycoproteins plays diverse and critical roles in the proper formation and spe- Mouse ES cells that were established in our laboratory (NVRQS-ES) were cialization of organs and tissues. Wnts, along with their cell surface receptors cultured in the DMEM/F12 supplemented with N2 and neural basal medium frizzleds, and antagonists (sFRPs, Dkks etc…) are expressed throughout the supplemented with B27. And then the expression of GABA-R, GFAP, Nestin, three cell types of the blastocyst. The primitive endoderm, a cell type that TUJ1, MAP2 and Oct-4 were examined to evaluate the differentiation to abuts the epiblast at E4.5 gives rise to the visceral and parietal endoderm, NPCs from ES at the days of 2, 4, 6, 8, 10, 12 and 14. Cells were treated critical cell types that are involved in patterning the embryo and forming with arsanilic acid (62 μM ~ 4 mM) and danofloxacin (1.25 ~ 80 μM) and the yolk sac. Previously it has been shown that ES cells in which adenoma- mercury (15.13 nM~ 1 μM) in the process of differentiated into the NPC for tous poliposis coli (APC), a negative regulator of canonical Wnt signaling, 8 and 14 d or test chemicals were exposed to 8 d differentiated cell for 6 d. is knocked-out produce only visceral endoderm in teratoma assays. In an Acetylcholine esterase activity was also determined in cells treated by test effort to explore the role of Wnt signaling during early embryonic lineage chemicals for 14 d and cells exposed to test chemicals for 6 d after ES cells commitment we created a stable Wnt3a expressing mouse ES cell line. We were differentiated for 8 d. In differentiation process into NPCs, the expres- demonstrate that high and continuous expression of Wnt-3a causes the for- sion of Oct-4 was significantly decreased from 10 d, and nestin, GABA-R, mation of cystic embryoid bodies and leads to an increase in the production TUJ-1 and MAP2 were increased till 6 day and GFAP till 8 d. The expression of visceral endoderm. This effect can be observed in the undifferentiated of Oct-4 was significantly increased by more than 0.125 mM of arsanilic state as LiCl and BIO, both canonical Wnt activators, are able to induce the acid at day of 8. The expression of nestin and TUJ-1 were decreased by premature expression of key primitive and visceral endoderm genes. Ongo- more than 0.125 mM, and GABA-R, MAP2 and GFAP were by more than ing work is focused on identifying the temporal and mechanistic regulation 0.5 mM, 0.5 mM and 1 mM of arsanilic acid at the days of 8, respectively. of visceral endoderm induction mediated by the Wnt signaling pathway. The expressions of Oct-4 and GABA-R were inhibited by more than 40 μM of danofloxacin and GFAP, TUJ-1 and Nestin, MAP2 were inhibited more than 5 uM, 10 μM and 20 μM of danofloxacin, respectively. The expres-

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Thursday Poster Abstracts sion of Oct-4 was increased by more than 250 μM of mercury. Nestin was Poster Board Number: 3451 decreased by all dose of mercury, and GFAP, TUJ-1 were more than 62.5 nM , and MAP2 was more than 125 nM, and GABA-R was more than 500 nM. DIFFERENTIATING MOUSE EMBRYONIC STEM In the case of cells exposed to chemicals for 6 d after differentiation for 8 d, (ES) CELLS REVEAL A ROLE FOR FATTY ACIDS the expressions of GABA-R and GFAP were decreased by all dose of arsanilic acid and Nestin was more than 0.25 mM, and TUJ1 and MAP2 were more AND SEX STEROID MODIFYING MECHANISMS than 0.5 mM, but Oct-4 was increased more than 0.5 mM of arsanilic acid. IN DETERMINING WHITE ADIPOCYTE The expression of GFAP and TUJ1 were decreased by all dose of danofloxa- LINEAGES. cin, and MAP2 was more than 5 μM, and GABA-R and Nestin were more than 10 μM, but Oct-4 was increased more than 10 μM of danofloxacin. Petrighi Polidori, Gioia, Lomax, Michael A., Docherty, Kevin The expression of GABA-R was decreased by all dose of mercury, and TUJ1 Cell and Developmental Biology, University of Aberdeen, Aberdeen, United was more than 125 nM, and TUJ1 and MAP2 were more than 250 nM, and Kingdom, School of Biosciences, University of Nottingham, Nottingham, GFAP was more than 500 nM, but Oct-4 was increased more than 125 nM United Kingdom of mercury. In the case of cells exposed to test chemicals for 6 d after dif- ferentiation for 8 d, acetylcholine esterase activities were dose-dependently The capacity of adipose tissue to safely store fat is determined in the first decreased by arsanilic acid and those were also decreased by high dose of years of life by mesenchymal regulation of adipogenesis. There is, however, danofloxacin and mercury. We demonstrated that the evaluation of in vitro a dearth of knowledge concerning the mechanisms whereby preadipocytes neuro-toxicity of chemicals using NPCs differentiated from mouse ES estab- differentiate and settle in specific anatomical depots. It is speculated that lished by this study could be used as a critical integrative in vitro model for excess nutrient intake may affect preadipocyte differentiation determining neurotoxicity evaluation of chemicals. According to this study, arsanilic acid not only fat mass during adulthood, but also body fat distribution. There is showed the inhibitory effect on the differentiation into NPCs during differ- also a substantial gender difference in body fat distribution. In females fat is entiation but astrocyte and GABA-R were more affected after differentiation, predominantly distributed in subcutaneous depots with a higher proportion and danofloxacin affects on astrocyte and microtubule during differentiation located in the viscera in males. Visceral fat is associated with a higher risk and after differentiation, and mercury affect on the NPCs, astrocyte and for cardiovascular disease, type II diabetes and metabolic dysfunction. In this microtubule but GABA-R after differentiation. study we used mouse embryonic stem cells to investigate the hypothesis that excess nutrients, in this case long chain fatty acids, can programme dif- Poster Board Number: 3449 ferentiating stem cells towards visceral or subcutaneous fat lineages and that this might be mediated via effects on the expression of sex steroid receptors RAPID AND ROBUST GENERATION OF and modifying enzymes. We show here that treatment of mouse ES cells FUNCTIONAL OLIGODENDROCYTE in chemically defined media with BMP4 to form embryoid bodies (EBs), PROGENITOR CELLS FROM PLURIPOTENT followed by retinoic acid treatment, increases adipogenesis significantly. Several long chain fatty acids were screened for their adipogenic potential. MOUSE EPIBLAST STEM CELLS Among these, the treatment of EBs as monolayers with palmitate signifi- Najm, Fadi J., Zaremba, Anita, Caprariello, Andrew V., Nayak, cantly enhanced the expression of the adipocyte markers CEPBβ, PPARγ, CEBP , AP2, Gpdh, Leptin and Adipsin with a concomitant accumulation of Shreya, Freundt, Eric C., Miller, Robert H., Tesar, Paul J. α lipid droplets as visualised with Bodipy 493/503, and expression of CD36, Genetics, Case Western Reserve University, Cleveland, OH, USA, a fatty acid translocase detected with flow cytometry. Furthermore, the Neurosciences, Case Western Reserve University, Cleveland, OH, USA, functional assessment of differentiation proved that the cells have a lipolytic Microbiology and Immunology, Stanford University, Stanford, CA, USA response to catecholamines. The effects of palmitate were partially mimicked Stem cell biology has garnered much attention due to its potential to impact by its non-metabolisable analogue 2-bromopalmitate suggesting a role for human health through disease modeling and cell replacement therapy. This palmitate as a regulator of differentiation. Palmitate appeared to stimulate is especially pertinent to myelin-related disorders such as multiple sclerosis subcutaneous fat markers and inhibit the expression of visceral adipose and leukodystrophies where restoration of normal oligodendrocyte function markers. At the same time, palmitate had no effect on the expression of could provide an effective treatment. Progress in myelin repair has been androgenising enzymes, whilst increasing the expression of oestrogenising constrained by the difficulty in generating pure populations of oligoden- enzymes. Collectively, these results suggest that palmitate may have feminis- drocyte progenitor cells (OPCs) in sufficient quantities for experimentation. ing effects on preadipocytes with concomitant differentiation towards a Pluripotent stem cells theoretically provide an unlimited source of OPCs but white subcutaneous lineage. significant advances are currently hindered by heterogeneous differentia- tion strategies that lack reproducibility. Here we provide a platform for the directed differentiation of pluripotent epiblast stem cells (EpiSCs) through a defined series of developmental transitions into a pure population of highly expandable OPCs in ten days. These OPCs robustly differentiate into myelinating oligodendrocytes both in vitro and in vivo. Our results demon- strate that pluripotent stem cells can provide a pure population of clinically- relevant, myelinogenic oligodendrocytes and offer a tractable platform for defining the molecular regulation of oligodendrocyte development, drug screening, and potential cell-based remyelinating therapies.

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Poster Board Number: 3453 Poster Board Number: 3455 GENERATION OF TRANSGENIC MURINE INFLUENCE OF DEFINED GROWTH FACTORS ES CELLS OVEREXPRESSING NURR1 AND ON THE DIFFERENTIATION OF MURINE PITX3 AS KEY TRANSCRIPTION FACTORS IN EMBRYONIC STEM CELLS INTO ALVEOLAR DOPAMINERGIC NEURON DEVELOPMENT TYPE II EPITHELIAL CELLS CONCOMITANTLY WITH GLUTATHIONE Schmeckebier, Sabrina, Mauritz, Christina, Schmiedl, Andreas, Lin, PEROXIDASE-1 AS AN ANTIOXIDANT Qiong, Menke, Sandra, Schulze, Wiebke, Ochs, Matthias, Zenke, Martin, Martin, Ulrich Safi, Mojtaba,Tarraf, Pania, Divsalar, Adeleh, Momen Dizghandi, Hassan, Massumi, Mohammad Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Medical School Hannover, Hannover, Germany, Institute for Animal and Marine Biotechnology, National Institute of Genetic Functional and Applied Anatomy, Medical School Hannover, Hannover, Engineering and Biotechnology, Tehran, Iran, Islamic Republic of, Germany, Institute for Biomedical Engineering -Cell Biology, Aachen Department of Biological Sciences, Tarbiat Moallem University, Tehran, University Medical School, RWTH Aachen, Aachen, Germany Iran, Islamic Republic of, Department of Molecular Genetic, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran, Islamic Type II alveolar epithelial cells (AT2 cells) have important functions, including Republic of the production of surfactant and regeneration of lost type I pneumocytes. The ability to produce AT2 cells would offer a variety of new therapeutic Embryonic stem (ES) cells are an in vitro generated pluripotent and immortal options to treat pulmonary injuries and diseases, including genetic surfac- cell type derived from the inner cell mass of the developing blastocyst. tant deficiencies and pulmonary fibrosis. Recently, we demonstrated that ES cells possess ability to differentiate to all three germ layers-derived cell AT2-like cells can be generated from murine embryonic stem cells (mESCs). lineages, like dopaminergic neurons. Parkinson’s disease (PD) is a neuro- However, with respect to the poor efficiency of existing differentiation degenerative disorder, caused by a relativity selective loss of dopaminergic protocols we aimed at the identification of key factors of lineage specifica- (DAergic) neurons in the substantia nigra. Although the main cause of PD tion thereby enabling a more efficient differentiation. Based on published is unknown, but it has been proven that oxidative stress can deteriorate the data demonstrating an effect of keratinocyte growth factor (KGF, FGF-7) on disease. The aim of this study was to establish murine transgenic ES cell lines primary AT2 cells, we investigated the influence of KGF on the differentia- expressing key transcription factors involved in midbrain DAergic neuron tion of mESCs in embryoid bodies. In addition, the effect of dexamethasone, development/differentiation, in addition to an antioxidant -glutathion 8-bromoadenosine-cAMP and isobutylmethylxanthine (DCI) either alone peroxidase-1(GPX-1) to in vitro study of DAergic programming potential. To or together with KGF was evaluated. Effects on the respiratory differentia- end that, we established the Nurr1/GPX-1-, Nurr1/Pitx3- and Nurr1/Pitx3/ tion of ESCs were quantified through qRT-PCR specific for TTF-1, surfactant GPX-1-expressing cell lines from both R1 (a feeder dependent) and CGR8 proteins C (SP-C), B (SP-B) and Clara Cell Specific Protein (CCSP). Microar- (a feeder free) mES cells via leniviral gene transduction. The engineered cells ray analyses have been performed in order to identify molecular pathways were selected by puromycin (800ng/mL) for Nurr1 and Pitx3-expressing of differentiation. Electron microscopy was applied to search for ultrastruc- cells, and GFP expression for GPX-1-Ires2-GFP. The mRNA expression of tural features of respiratory epithelium. Finally, ESC clones expressing SP-C transgenes was confirmed by RT-PCR. The protein expression and nuclear promoter dependent reporter and selection genes were generated to enable localization of Nurr1 and Pitx3 transgenes were confirmed by Immunocy- visualization and enrichment of ESC-derived AT2 cells. Whereas qPCR tochemistry (ICC) and the expression and functionality of ectopic GPX-1 analyses revealed an increase in the expression of SP-B and SP-C after ap- were verified by ICC and enzyme activity assay kit. In general, two DAergic plication of either KGF or DCI, a strong synergistic effect with significantly differentiation protocols are used: co-culture with stromal cells, and EB increased expression of SP-B, SP-C, TTF-1 and CCSP could be shown after formation and using defined factors. We will utilize EB formation protocol combined application of KGF and DCI. Surprisingly, the most profound to differentiate transgenic ES cells to DAergic neurons. The efficiency of effect was detected after early KGF application starting at d0 of differentia- differentiation will be analyzed by Real-time PCR, Immunocytochemistry for tion, suggesting an additional and so far unknown effect of KGF during expression of neuroectermal-, mesencephalic- or DAergic neuron-related early differentiation. Ultrastructural analysis confirmed the presence of AT2- markers, such as Nestin, Pax2, Pax5, Wnt, En, Nurr1 and TH. As a positive like cells with a more mature phenotype than without application of KGF/ control SY5Y, a neuroblastoma dopamine-synthesizing cell line was used for DCI. Finally, microarray analyses revealed upregulation of a series of genes our analyses. The synthesis and secretion of dopamine will be analyzed by known to be associated with lung development. Analysis of microarray data reverse HPLC in supernatant of differentiated cells. In conclusion, this model is currently ongoing in order to identify underlying molecular pathways, and can be used to study of molecular mechanisms underlying of DAergic neu- reporter lines with SP-C promoter dependent expression of selection genes ron development. In the other hand the result of this study may have impact are currently analyzed in detail. In conclusion, were able to demonstrate a on future cell therapy of PD by ES or induced pluripotent stem cells (iPS). synergistic effect of KGF and DCI on the differentiation of mESCs resulting in increased expression of respiratory marker genes and more mature AT2-like cellular ultrastructure. Results of microarray analyses and detailed character- ization of the generated reporter cell lines will be presented at the meeting.

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Thursday Poster Abstracts

Poster Board Number: 3457 Poster Board Number: 3461 THE ROLE OF L-PROLINE AND L-PROLINE PROTEIN KINASE A ACCELERATES EARLY TRANSPORTERS IN THE REGULATION OF PROGRAMMING FROM MOUSE ES CELLS MOUSE ES CELL DIFFERENTIATION IN THROUGH EPIGENETIC INHIBITION OF OCT3/4 CULTURE AND NANOG EXPRESSION Tan, Boon Siang Nicholas, Rathjen, Joy, Rathjen, Peter D. Yamamizu, Kohei, Katayama, Shiori, Tachibana, Makoto, Shinkai, Zoology, University of Melbourne, Melbourne, Australia Yoichi, Yamashita, Jun K. There is an increasing appreciation that amino acids can act as signaling Kyoto University, Kyoto, Japan molecules in the regulation of cellular processes. We have described a role Transcriptional patterning and epigenetic status are dramatically changed for L-proline in the transition of ES cells into primitive ectoderm-like cells, in early differentiation of embryonic stem cells (ESCs). However, molecular EPL cells, in culture. Addition of L-proline to ES cells results in changes in mechanisms of differentiation cues and epigenetic modification during the colony morphology, gene expression and differentiation kinetics consistent programming process still remain unclear. In our studies on mouse ESC dif- with differentiation towards EPL cells. This activity is restricted to L-proline; ferentiation, we found a novel linkage between signaling and epigenetics addition of other amino acids or synthetic analogues of L-proline do not which regulates differentiation behavior of early ESCs. Previously, we dem- induce differentiation. Here we investigate the mechanism of L-proline in ES onstrated that cAMP/protein kinase A (PKA) signaling plays an important cell differentiation. Characterisation of L-proline transport using radioac- role in vascular differentiation. Here, we focused on roles of cAMP/PKA tive uptake assays has shown that the amino acid System A Transporter signaling in early programming from mouse ESCs. We first observed that SNAT2 (SLC38a2) is the major transporter for L -proline transport into ES intracellular cAMP concentration was significantly increased from ESC dif- cells. L-proline uptake is sodium and pH dependent and can be inhibited ferentiation day-2.5. Activation of cAMP signaling with induced expression by the addition of a molar excess of other amino acid substrates of SNAT2. of constitutive active form PKA (CA-PKA), a downstream target of cAMP, Amino acids that block L-proline uptake also block the ability of L-proline to from the initiation of ESC differentiation markedly changed differentiation form EPL cells from ES cells. Several intracellular signalling pathways have kinetics. Quantitative RT-PCR, immunostaining, and FACS analysis showed been shown to be activated in ES cells cultured in the presence of L-proline; that CA-PKA expression induced early disappearance of Oct3/4, Nanog, chemical inhibition of these pathways prevents the differentiation of ES cells and Sox2 expression. In contrast, CA-PKA accelerated appearance of three in response to L-proline. These studies implicate amino acids, amino acid germ layer markers for mesoderm (Flk1, T, Neuropilin1), endoderm (GATA4, transporters and specific signalling pathways in ES cell differentiation and Foxa2, Sox17), and ectoderm (Nestin, Fgf5). Furthermore, teratoma forma- primitive ectoderm formation, and define inexpensive culture supplements tion assay in mouse showed expression of CA-PKA significantly inhibited with potential applications in improving the maintenance, and/or differen- teratoma size and remarkably enhanced three germ layer differentiation. tiation, of pluripotent mouse and human cells in culture. Furthermore, this We focused on and examined epigenetic status during the process. CA-PKA work describes a novel and amenable model to investigate amino acid regu- increased protein expression of G9a, a H3K9 dimethyltransferase, along lation of intracellular signalling, transcriptional activity and cellular function. with H3K9me2 and DNA methylation in Oct3/4 and Nanog gene regula- Poster Board Number: 3459 tory regions in early ESC differentiation. Loss of G9a function using G9a conditional-knockout ES cell line abolished the PKA-elicited acceleration of PROTEOMIC ASSESSMENT OF A CELL MODEL ESC differentiation and epigenetic modification. These results indicate that PKA controls early differentiation via G9a function. Our study demonstrates OF SPINAL MUSCULAR ATROPHY a novel molecular linkage of signaling molecules-epigenetic modifiers-gene Wu, Chia-Yen, Whye, Dosh, Glazewski, Lisa, Choe, Leila, Kerr, expressions-cell behavior. Comprehensive understanding of cell differentia- Douglas, Lee, Kelvin, Mason, Robert W., Wang, Wenlan tion machinery would largely contribute to developmental biology and stem cell technology. Biological Sciences, University of Delaware, Newark, DE, USA, Department of Pediatrics, Columbia University Medical Center, New York, NY, USA, Poster Board Number: 3463 Nemours Biomedical Research, Alfred I. duPont Hospital for Children, Wilmington, DE, USA, Delaware Biotechnology Institute, Newark, DE, USA, THE SRC-FAMILY TYROSINE KINASE Experimental Neurology, Biogen-Idec, Cambridge, MA, USA C-YES SUPPRESSES MOUSE ES CELL Deletion or mutation(s) of the survival motor neuron 1 (SMN1) gene causes DIFFERENTIATION spinal muscular atrophy (SMA), a neuromuscular disease characterized by spinal motor neuron death and muscle paralysis. Complete loss of the SMN Zhang, Xiong, Meyn III, Malcolm A., Smithgall, Thomas E. protein is embryonically lethal, yet reduced levels of this protein result in se- Microbiology and Molecular Genetics, University of Pittsburgh School of lective death of motor neurons. Why motor neurons are specifically targeted Medicine, Pittsburgh, PA, USA by SMN deficiency remains to be determined. In this study, embryonic stem Embryonic stem (ES) cells are characterized by self-renewal, the ability to (ES) cells were derived from a severe SMA mouse model and differentiated multiply indefinitely without differentiation, and pluripotency, the develop- in vitro by addition of retinoic acid and sonic hedgehog agonist. Proteomic mental potential to generate cell lineages derived from all three germ layers. and Western blot analyses were used to probe protein expression alterations Previous work from our group and others has implicated the Src family of in this cell-culture model of SMA that could be relevant to the disease. non-receptor protein-tyrosine kinases in the self-renewal and differentia- tion of mouse ES (mES) cells. Seven members of the Src kinase family are expressed in mES cells, and individual family members play distinct roles in regulating their developmental fate. For example, expression of Hck is rapidly silenced as mES cells differentiate to embryoid bodies (EBs), suggest- ing a role in self-renewal or suppression of differentiation. In contrast, c-Src activity alone is sufficient to induce differentiation of mES cells to primitive ectoderm. Other work has linked c-Yes kinase activity to leukemia inhibitory factor (LIF), the cytokine required to maintain mES cell pluripotency. Knock-

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Thursday Poster Abstracts

down of c-Yes expression induces mES cell differentiation, implying a role mechanism of ESCs differentiation. for this kinase in self-renewal. To better understand the role of c-Yes in mES cell renewal and differentiation, we employed a chemical genetics approach Poster Board Number: 3467 previously applied to c-Src in this system. For these experiments, we used a CDX GENES RESTRICT CARDIOGENIC c-Yes mutant that is resistant to A-419259, a potent ATP-competitive inhibi- tor of Src-family kinase activity (replacement of kinase domain Thr-338 with DEVELOPMENT BY AFFECTING MESODERMAL methionine; TM mutant). A-419259 treatment blocks all Src-family kinase PATTERNING activity in ES cells, preventing differentiation while maintaining pluripotency. Mouse ES cell populations were generated to over-express wild-type c-Yes, Lengerke, Claudia, Wingert, Rebecca A., Beeretz, Michael, Grauer, the TM mutant, an active mutant in which the negative regulatory tail ty- Matthias, Konantz, Martina, Schmidt, Anne G., Bareiss, Petra M., rosine is substituted with phenylalanine (YF mutant), as well as a combined Daley, George Q., Davidson, Alan J. TM-YF mutant. We used an MSCV promoter-based retroviral vector in order Department of Hematology&Oncology, University of Tuebingen, to keep transduced c-Yes expression to less than two times endogenous Tuebingen, Germany, Department of Biological Sciences, University of c-Yes levels. All c-Yes-transduced mES cell populations grew as typical undif- Notre Dame, Notre Dame, IN, USA, Department of Hematology&Oncology, ferentiated colonies in the presence of LIF, and were indistinguishable from Children´s Hospital Boston, Boston, MA, USA, Center for Regenerative cultures of parental mES cells in terms of colony morphology and pluripo- Medicine, Massachusetts General Hospital, Boston, MA, USA tency marker expression as determined by quantitative RT-PCR. Interest- ingly, addition of A-419259 to self-renewing mES cell cultures expressing Cdx transcription factors regulate embryonic positional identities and have the c-Yes-TM mutants induced a dramatic shift to flat, epithelial cell-like crucial roles in anteroposterior patterning (AP) processes of all three germ morphology, suggesting that c-Yes activity alone is not sufficient to support layers. Previously we have shown that the zebrafish homologues cdx1a and self-renewal. Each of the cell populations was then examined for develop- cdx4 redundantly regulate posterior mesodermal derivatives inducing em- mental potential in the EB assay. Low-level overexpression of wild-type c-Yes bryonic blood cell fate specification and patterning of the embryonic kidney. or any of the c-Yes mutants prevented differentiation to EBs, and Q-PCR Here we hypothesize that cdx factors restrict formation of anterior meso- analysis showed that pluripotency marker expression was retained. In addi- dermal derivatives such as cardiac cells by imposing posterior identity to tion to EB formation, we also examined the potential of the c-Yes-expressing developing mesodermal cells. In differentiating murine embryonic stem cells mES cells to form neuroectoderm directly in N2B27 culture medium. While (mESC), Cdx genes are expressed in cardioangioblasts isolated as Flk1+ cells the parental mES cells readily formed neurons, enforced expression of c-Yes at day 4 of embryoid body (EB) development. Using doxycylin-inducible completely blocked differentiation along this pathway. Our findings show mESC lines overexpressing Cdx1, Cdx4 or Hoxa9, conditional overexpres- that c-Yes kinase activity prevents the differentiation of mES cells, and in this sion of these posteriorizing signals, by addition of doxycyclin during the brief way may contribute to the maintenance of pluripotency. window of mesoderm patterning, strongly suppresses cardiac develop- ment, as assesed by functional assays (formation of embryoid bodies (EBs) Poster Board Number: 3465 containing “beating” cell clusters) and gene expression analysis for cardiac markers such as Mesp1, Gata4, Nkx2.5, Isl1, while addition of doxcycyclin CALCINEURIN NFAT AND ERK MAPK to genetically unmodified cells does not have any impact on these processes. PATHWAYS PROMOTE MOUSE In support of these data, loss-of-function studies performed in cdx-deficient EMBYRONIC STEM CELL DIFFERENTIATION zebrafish embryos show a dose-dependent expansion of tbx5a+ anterior- lateral plate mesoderm giving rise to cardiac progenitors. However, further INTERDEPENDENTLY cardiac development of these mesodermal cells requires additional suppres- Zhu, Lili, Jin, Ying sion of the retinoic acid (RA) pathway, possibly due to differential mesoderm responsiveness to inhibitory RA signals in cdx mutants. Under these condi- Shanghai Institutes for Biological Sciences, Institute of Health Sciences, tions, cdx mutants show larger hearts, as assessed by cmlc2 expression at Shanghai, China later time points (e.g. 26 hours after fertilization). Together, our data suggest Embryonic stem cells (ESCs) represent an important research tool and a that cdx proteins affect cardiogenesis by regulating the formation of cardio- potential resource for regenerative medicine. Self-renewal and pluripotency genic mesoderm and together with the RA pathway control the specification are two important properties of ESCs. An important unanswered ques- of cardiac precursor cells. Moreover, the data show that ectopic overexpres- tion is how ESC self-renewal and differentiation are precisely regulated. It sion of AP signals can be used to modulate tissue formation from pluripotent is known that the Erk/MAPK pathway is critical for the induction of ESC stem cells and support the notion that in vitro differentiating pluripotent differentiation. Our previous data showed that the calcineurin/NFAT signal- stem cells can be used as a model to study early mammalian development, ing pathway is also essential and sufficient to trigger ESC transition from offering unique opportunities to study patterning of embryonic fate choices self-renewal to lineage commitment. However, the functional interaction and fate switches in response to external signals. between the Erk/MAPK and the calcineurin/NFAT pathway in the control of Poster Board Number: 3469 ESC properties remains unclear. Here, we addressed this question utilizing inhibitors and genetic approaches. Interestingly, we found that ESCs dis- DERIVATION OF CANINE NEURAL PROGENITOR played similar differentiation phenotypes when either of these two pathways was activated. Moreover, inhibition of the Erk/MAPK pathway attenuated CELLS FROM CANINE INDUCED PLURIPOTENT ESC differentiation induced by calcineurin/NFAT activation. Conversely, STEM CELLS inhibition of calcineurin/NFAT signaling blunted ESCs differentiation driven by the activated Erk/MAPK pathway. Furthermore, we found that these two Luo, Jiesi, Cibelli, Jose pathways could synergistically activate Src kinase expression, which induced Animal Science, Michigan State University, East Lansing, MI, USA ESCs differentiation. Importantly, the blockade of Src activity from the 8-cell For more than thirty years, dog has been used as a valuable model of tissue stage mouse embryo suppressed development of the hypoblast, although it transplantation and regenerative medicine treatment for human diseases, did not impede blastocyst formation. The similar phenomenon was observed due to its similarity in physiology, biochemistry, disease presentation and when the Erk signaling was inhibited. Therefore, these results indicate clinical responses as human. We previously generated canine induced that the Erk/MAPK pathway and calcineurin/NFAT pathway constitute an pluripotent stem cells (ciPSCs) from canine adult fibroblasts by introducing interdependent signaling module in ESCs that triggers the transition from the human genes OCT4, SOX2, c-MYC and KLF4. These ciPSCs expressed self-renewal to differentiation. These findings provide a detailed regulatory

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Thursday Poster Abstracts a number of pluripotency markers and were capable to be differentiated in drug delivery. Ideally suitable and active biological scaffolds will stimulate vitro into cell lineages derived from three-germ layers. Because of the dual- and promote cell differentiation. Self-assembling scaffolds could help repair dependency on leukemia inhibitory factor (LIF) and fibroblast growth factor difficult to regenerate tissues and structures such as spinal cord, bones, (bFGF) of ciPSC, we had to develop novel protocols to obtain neuronal dif- tendon and cartilage. Every year in the United States alone, about 15,000 ferentiation. We removed bFGF from ciPSC culture media and added retinoic people damage their spines. Few recover fully as it is difficult for damaged acid, and then followed by an increase in the concentration of bFGF and N2 nerves to grow across the gap in a severed spinal cord. We have investigated supplement. Without formation of embryoid bodies, individual ciPSCs were several synthetic nanomaterials (such as polycaprolactone, poly (ethylene gradually differentiated into neural lineage. Highly proliferative neural pro- oxide), poly(lactic acid), and poly(lactic acid co-glycolic acid) and biomol- genitor cells, called canine neuronal progenitors (cNPs), were isolated and ecules (such as proteins, peptides, and carbohydrates) for use in developing stably maintained in culture medium with N2 supplement and bFGF. These scaffolds that mimic in vivo microenvironments for 3-D tissue engineering. cells expressed a series of critical neural stem cell markers including nestin, The scaffolds promoted cellular growth of embryonic and cord blood stem PAX-6, A2B5 and PSA-NCAM. Results of qRT-PCR assay demonstrated that cells. Studies are underway to differentiate various stem/progenitor cells the neural lineage genes, such as canine-specific NESTIN, NCAM1, NCAM2, into different cell lineages including osteogenic, chondrogenic, and neural GFAP and MAP2, were highly expressed in cNPs, but the expression of cell types. We have developed nanomaterials that self-assemble to produce mesodermal or endodermal genes was negligible. We conclude that canine scaffolds for generating tissues of various organs such as heart and liver. In pluripotent stem cells can be differentiated to the neural progenitor cell line this study we synthesized thiol-functionalized dextran (Dex-SH, Mn 25K) using an optimized cell culture system. Our results demonstrate an efficient and investigated for in situ hydrogel scaffold formation via Michael type methodology for deriving cNPs, provide a potential method to establish addition using poly(ethylene glycol)tetra-acrylate (PEG-4-Acr). Dex-SH was a model for human central nervous system (CNS) clinical treatment, and prepared by activation of the hydroxyl groups of dextran with 4-nitrophenyl further open the window for the human regenerative medicine research chloroformate and the subsequent reaction with cysteamine. The Dex-SH is and the understanding of the mechanisms of various human and dog CNS highly air sensitive in aqueous solution and must be handled under nitrogen. genetic diseases. We will present our preliminary results for in situ hydrogel formation in the presence of stem cells that support cell proliferation and differentiation. Poster Board Number: 3471 Poster Board Number: 3475 CELLULAR DIVERSITY WITHIN EMBRYONIC STEM CELLS: PLURIPOTENT CLONAL SUBLINES ECAT15-1 AND ECAT15-2 ARE DISPENSABLE SHOW DISTINCT DIFFERENTIATION POTENTIAL FOR EARLY DEVELOPMENT BUT IMPORTANT FOR LATE EMBRYOGENESIS Martinez, Yannick, Dubois-Dauphin, Michel, Krause, Karl-Heinz, Preynat-Seauve, Olivier Nakamura, Tomonori, Nakagawa, Masato, Ichisaka, Tomoko, University of Geneva, Geneva, Switzerland Yamanaka, Shinya Center for iPS Cell Research and Application(CiRA), Kyoto University, Embryonic stem cells (ESC), derived from the early inner cell mass (ICM), are Kyoto, Japan constituted of theoretically homogeneous pluripotent cells. Our study was designed to test this concept, using experimental approaches that allowed ECAT15-1 and ECAT15-2 are highly expressed in undifferentiated embryonic characterization of progenies derived from single parental mouse ESC. Flow stem cells (ESCs), and their expression levels are down-regulated follow- cytometry analysis showed that a fraction of ESC submitted to neural dif- ing differentiation. In vivo, they are expressed in inner cell mass and germ ferentiation generates progenies that escape the desired phenotype. Live cells. ECAT15-1 and ECAT15-2 are located on same chromosome tandemly, imaging of individual cells demonstrated significant variations in the capacity and their coding products share homology and same DNA binding domain. of parental ESC to generate neurons, raising the possibility of clonal diversity According to such a pluripotency associated expression pattern and interest- among ESC. To further substantiate this hypothesis, clonal sublines from ESC ing features, ECAT15-1 and ECAT15-2 were thought to have relationships were generated by limit dilution. Transcriptome analysis of undifferentiated and functions in early embryogenesis and/or fertility. In this poster, in sublines showed marked differences in gene expression despite the fact that order to examine these issues, we generated not only ECAT15-1 single and all clones expressed pluripotency markers. Sublines showed distinct differen- ECAT15-2 single mutant mice but also ECAT15-1/15-2 double mutant mice. tiation potential, both in phenotypic differentiation assays and with respect Surprisingly, we found that all of three mutant mice had respiratory defects, to gene expression in embryoid bodies. Clones generated from another ESC without their expressions, and showed lethality around birth. Moreover, line also showed individualities in their differentiation potential, demonstrat- interestingly, ECAT15-2 single mutant mice showed more sever phenotypes ing the wider applicability of these findings. Taken together, our observa- ECAT15-1/15-2 double mutant mice did. To examine molecular mecha- tions demonstrate that pluripotent ESC consist of individual cell types with nisms, we analyzed ESCs and found that ECAT15-1 and ECAT15-2 showed distinct differentiation potentials. These findings identify novel elements for similar sub-cellular localization and have protein-protein interaction each the biological understanding of ESC and provide new tools with a major other. These results indicated that ECAT15-1 and ECAT15-2 form molecular potential for their future in vitro and in vivo use. complex in early embryo and may regulate later development. Poster Board Number: 3473 SELF ASSEMBLING SCAFFOLDS FOR CULTURING AND DIFFERENTIATING STEM CELLS McKee, Christina, Chavez, Ferman, Pawlak, Piotr, Ciavattone, Nicholas, Dinda, Sumi, Chaudhry, G. Rasul Oakland University, Rochester, MI, USA Self-assembling biocompatible and biodegradable nanomaterials have prom- ising applications in tissue engineering and regenerative medicine as well as

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Poster Board Number: 3477 layers. Methods: PSC were differentiated in vitro, into specific cell types representa- DISSECTING THE MECHANISMS OF PRIMARY tive of the three germ layers without embryoid bodies formation: he- GERM LAYER INDUCTION IN EMBRYONIC STEM matopoietic cells, neural progenitors cells and hepatocyte-like cells. In three independent experiments for each lineage, samples were analyzed at four CELLS time points: undifferentiated hESC cells at days 0, and during differentiation Oron, Efrat, Wu, Jiaqian, Wang, Jing, Zhong, Mei, Snyder, Michael, induction at days 5, 7 and 9. Expression of eight markers of undifferentiated Gerstein, Mark B., Ivanova, Natalia state (SSEA3, SSEA4, TRA160, TRA180, CD24, OCT3/4, nanog and alkaline phosphatase) and markers of specific cell types (hematopoietic, hepatocyte Genetics, Yale University, New Haven, CT, USA, Genetics, Stanford and neuron) were assessed by fluorescence-activated cell sorting (FACS). University, Palo Alto, CA, USA, Computer Science, Yale University, New Results: In all model of differentiation, a similar pattern of loss of pluripo- Haven, CT, USA, Yale Stem Cell Center, Yale University, New Haven, CT, tency marker was observed, particularly for intracellular markers. SSEA3 and USA OCT3/4 were the first markers down regulated and disappeared completely A key feature of embryonic stem cells (ESCs) is their ability to differentiate at day 9. By contrast, the expression of CD24 and NANOG were main- into all three primary germ layers i.e. ectoderm, mesoderm and endoderm. tained during the early stages of induction of specific lineages. Conclusion: Induction of the primary germ layers is a complex process involving multiple the expression of pluripotency markers followed a similar pattern during signaling pathways, many of which are still unknown. Identifying the genes differentiation, independently of specific lineages. Our results suggest that that regulate differentiation is an important task that will contribute to our the monitoring a limited array of pluripotency markers, that may be limited ability to efficiently differentiate ESCs toward any specific fate. Our main to SSEA3 and OCT4, would be sufficient to gauge the loss of pluripotency goal in this study is to identify the genes and gene networks controlling during differentiation of pluripotent stem cells. differentiation. Our underlying hypothesis is that differentiation is induced Poster Board Number: 3481 by temporal signals which lead to transient or definite changes in expression of regulatory genes that in turn re-wire the gene networks to determine the MIRNA REGULATED SUICIDE GENE identity of a cell. Our strategy is to first identify putative differentiation reg- ulating genes. Next we conduct a functional screen to determine how these EXPRESSION TO AVOID TUMOR FORMATION genes affect differentiation. Finally we try to differentiate cis vs. trans effects FOLLOWING ES CELL TRANSPLANTATION among lineages and construct simple gene networks. To identify putative differentiation regulating genes we used RNAseq technology to determine Sachdeva, Rohit, Jönsson, Marie, Nelander, Jenny, Kirkeby, Agnete, the transcriptional profiles of differentiating ESC lines and mouse embryos. Björklund, Anders, Parmar, Malin, Jakobsson, Johan For the embryonic time course we used e3.5, e5.5, e6.5, e7.5, e8.5 and e9.5 Molecular Neurogenetics, Lund University, Lund, Sweden, Neurobiology, CD1 embryos. For the ESC time course we used cce and S1B6a cell lines that Lund University, Lund, Sweden, Developmental Neurobiology, Lund were spontaneously differentiated as EBs and sampled daily for a period of University, Lund, Sweden 11 days. From the numerous interesting groups of differentially expressed We have previously developed a reporter system that exploits the en- genes, we focused on transcription factors and chromatin regulators. 120 dogenous microRNA machinery, to visualize and segregate differentiating genes were chosen for functional analysis. We devised a functional assay neuronal cells in pluripotent cultures. The system is based on a lentiviral based on lentiviral shRNA vectors for stable knock down of candidate genes expressing a fluorescent reporter gene (GFP) regulated by miRNA-292. Since and qPCR with a panel of germ layer-specific markers to follow lineage miRNA-292 is specifically expressed in pluripotent cells it will bind to and induction. 120 stable shRNA-expressing cell lines were generated, differ- degrade the lenti-mRNA hereby preventing GFP expression. However, in entiated as EBs (11 days) and screened for lineage induction. Genes that somatic cell types miRNA-292 is not present and GFP should therefore be showed consistent phenotypes were used for in-situ hybridization of mouse highly expressed. Using this simplistic system it has been possible to track embryos. Our results include phenotypes that suggest involvement of corre- progeny from mouse ES cells, human ES cells and iPS cells as they differenti- sponding genes at various specific stages of lineage specification and reveal ate towards the neural lineage. In addition, we have been able to success- interesting interactions among lineages. fully FACS-purify neuronal progenitors for molecular analysis and transplan- Poster Board Number: 3479 tation. Transplanted GFP enriched cell populations survive and continue to differentiate into mature neurons. By sorting out majority of undifferentiated BENCHMARKING PLURIPOTENT STEM cells from the culture it has been possible to reduce the frequency of tumors. CELL MARKERS DURING DIFFERENTIATION To further reduce the incidence of tumors we have now generated a bidirec- tional lentiviral system expressing HSV-TK regulated by the neuron specific INTO THE THREE GERM LAYERS UNVEILS A miRNA-124, in addition to GFP regulated by miRNA-292. Our approach to STRIKING HETEROGENEITY: ALL MARKERS ARE regulate the suicide gene HSV-TK by a neuron specific miRNA, provides a NOT EQUAL solution to eradicate unwanted cells post transplantation. Ramirez, Jean-Marie, Gerbal-Chaloin, Sabine, Milhavet, Ollivier, Poster Board Number: 3483 Bai, Qiang, Becker, Fabienne, Hamamah, Sammir, De Vos, John EFFICIENT DERIVATION OF NEURAL STEM Institut de Recherche en Biothérapie, Montpellier, France, Institut de CELLS FROM COMMON MARMOSET ES CELLS Génomique Fonctionnelle de Montpellier, Montpellier, France AND iPS CELLS Aim: Pluripotent stem cells (PSC) are defined as cells having the capacity to constitute all cells types of the body. However, the stages and kinetic of the Shimada, Hiroko, Okada, Yohei, Tomioka, Ikuo, Sasaki, Erika, loss of pluripotency is still poorly understood. The analysis of pluripotent Nakamura, Masaya, Okano, Hideyuki markers by flow cytometry assay provides an invaluable tool for monitor- School of Medicine, Keio University, Tokyo, Japan, Central Institute for ing the progress of PSC differentiation and identifies subsets of differenti- Experimental Animals, Kanagawa, Japan ated cells. Here, we compared an array of 8 classic markers of pluripotency comprising SSEA3, SSEA4, TRA160, TRA180, CD24, OCT3/4, NANOG and Common marmoset (Callithrix jacchus) is a small primate, which have short alkaline phosphatase during differentiation into lineages from the three germ life cycle, generate many offspring during their life and easy to breed in a

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Thursday Poster Abstracts laboratory, compared to other types of primate models. Therefore, they are populations we have directed hESC differentiation in vitro towards a neural ideal animal models for pre-clinical studies. We have previously confirmed fate and evaluated the expression of specific molecular markers in p75+ the effectiveness of the transplantation of rodent and human fetal neural and p75+/HNK+ isolated populations. We have found that FACS purifica- stem cells (NSCs) in the locomotor functional recovery of spinal cord injury tion of p75+ and HNK+ cells, isolated at different time points during hESC (SCI) animal models. Because human fetal NSCs are difficult to obtain due to differentiation, identify populations with a gene expression profile distinctive ethical issues, we, recently, have been focusing on NSCs derived from em- of NC cells, pre-placodal region (PPR), neuroectoderm (CNS), and paraxial bryonic stem (ES) cells and induced pluripotent stem (iPS) cells. In fact, we mesoderm. Accordingly, further selection using a neural specific surface found that the transplantation of NSCs derived from mouse and human ES antigen added to FACS purification allowed the identification of a subset of cells and iPS cells are effective for functional recovery of mouse SCI models. p75+/HNK1+ cells with a predominant CNS (as opposite to NC) identity. However, for clinical application, these transplantation studies, using rodent Moreover, p75 expressing cells can be further separated by their different models or xenograft transplantation of human cells into rodent models, are fluorescence-intensity level (bright and dim) and those distinct subpopula- insufficient to evaluate immunological rejections or tumorigenesis of the tion of cells exhibit a diverse expression of NC, CP, CNS or mesoderm associ- transplanted NSCs. Thus, allograft transplantation in primate models, such ated genes, thus revealing the complexity and heterogeneous nature of the as transplantation of marmoset NSCs into marmoset SCI model, is essential. p75+ cell population. Surprisingly enough, we also found that a derivative We have also previously established marmoset ES cells and marmoset iPS of paraxial mesoderm (skeletal myoblasts and myocytes) could be generated cells, and tried to induce neural cells from them. However, simple modi- by p75+ and p75+/HNK-1+ populations after in vitro culture of the sorted fication of previously reported methods for mouse or human ES cells was cells. Our findings underline the fate potential heterogeneity of p75+ and insufficient for the effective induction of neural cells from marmoset ES HNK+ cells generated during in vitro hESC differentiation and therefore their cells. Thus, in the present study, by modifying our own methods for neural inadequate efficacy in defining pure populations of neural crest cells. differentiation from mouse or human ES and iPS cells, we derived NSCs from marmoset ES cells efficiently, which are applicable to the transplantation into Poster Board Number: 3487 SCI model or the study of neural development of primates. We first formed MICRORNAS MIR17, MIR20A, AND MIR106B embryoid bodies (EBs) from three lines of marmoset ES cells (No.40, No.20, CjES001) in the presence of neural inducers, and then formed neurospheres ACT IN CONCERT TO MODULATE E2F ACTIVITY by dissociating EBs and culturing them in suspension in the presence of ON CELL CYCLE ARREST DURING NEURONAL FGF-2. As a result, No. 40 and CjES001 could not form neurospheres at all, but No.20 could form neurospheres efficiently. For the efficient formation LINEAGE DIFFERENTIATION OF USSC of neurospheres from marmoset ES cells, EBs must be cultured for longer Wernet, Peter, Hassane, Abbad, Iwaniuk, Katharina, Hafner, periods than those of mouse ES cells, but could form neurospheres even Markus, Renwick, Neil, Tuschl, Thomas, Schira, Jessica, Müller, when EBs were cultured for shorter periods than those of human ES cells. Hans-Werner, Trompeter, Hans-Ingo These marmoset ES cell-derived neurospheres could be passaged repeatedly and give rise to neurons and astrocytes when cultured adherently without University Medical Center Duesseldorf ITZ, Dusseldorf, Germany, FGF-2. Moreover, by similar culture methods, marmoset iPS cells, three lines Laboratory of RNA Molecular Biology, Howard Hughes Medical Institute, out of seven lines tested, could form neurospheres efficiently, which also Rockefeller University New York, NY, USA, Molecular Neurobiology generate neurons and astrocytes. However, the responses to neural inducers Laboratory, Dept of Neurobiology, University Medical Center Duesseldorf, were distinct among marmoset iPS cell lines. Currently, we are examining Dusseldorf, Germany more detailed characteristics of marmoset ES cell-derived NS/PCs, including Unrestricted somatic stem cells (USSC) from human cord blood constitute a functional properties. In the near future, by the transplantation of marmoset rare CD45-negative population, capable of inducible homogenous in-vitro ES cell and iPS cell-derived neurospheres into marmoset SCI models, we are differentiation into all three germinal layers. Additionally, using a cocktail of going to examine not only functional recoveries, but also the effectiveness growth and differentiation factors (XXL-medium) differentiation of USSC of NSC transplantation in the aspect of immunoglical rejections and tumori- into cells of neuronal lineage (XXL-USSC) expressing neurofilament and so- genicities in primate allograft models. dium channel proteins was obtained. Furthermore, XXL-USSC display certain Poster Board Number: 3485 neurotransmitter phenotypes including expression of GABA, dopamine and tyrosine hydroxylase (TH), the key enzyme of the dopaminergic pathway. HETEROGENEITY OF P75(NGFR)+ CELLS Yet this neuronal lineage differentiation of USSC appears to be limited since patch-clamp analyses failed to detect voltage activated fast inactivating Na+ ISOLATED DURING EARLY IN VITRO HESC current, indicating that XXL-USSC have not yet developed a fully functional DIFFERENTIATION: IMPLICATIONS ON NEURAL neuronal phenotype. However, cultured USSC rapidly stop proliferation CREST-LIKE CELLS PURIFICATION upon addition of XXL-medium and such cell cycle exit events are inher- ently connected to neurogenesis. MicroRNAs are short (~22 nt) non-coding Mengarelli, Isabella, Barberi, Tiziano regulatory RNAs that negatively control gene expression at the post-tran- Australian Regenerative Medicine Institute, Monash University, Clayton, scriptional level. Here the functional impact of microRNAs on cell cycle arrest Australia during neuronal lineage differentiation of USSC was analyzed. Expression profiling revealed downregulation of microRNAs miR-17, -20a, and -106b In a human embryo multipotent and transient structures such as the neural in USSC differentiated into neuronal lineage but not in USSC differentiated crest (NC) and the precursor cells of the cranial placodes (CP) are specified into osteogenic lineage. Transfection experiments using Ki67 immunostain- during the first four weeks of gestation. Because of their early occurrence, ings demonstrated that each of these microRNAs alone was able to promote complexity of formation and scarce availability of human embryos for proliferation of native USSC and to prevent in part cell cycle arrest during experimental analysis, these transient structures remain largely unexplored. neuronal lineage differentiation of USSC. Bioinformatic target gene predic- Investigators are now using in vitro differentiation of hESC to shed light tions followed by experimental target gene validations revealed that miR-17, into such early events. In particular, surface antigens such as the low affinity -20a, and -106b act in a common manner by downregulating a largely nerve growth factor receptor, NGFR (p75) and a carbohydrate moiety- overlapping set of target genes mostly involved in regulation and execution directed antibody (HNK-1) have been used to identify and isolate popula- of G1/S transition. Proproliferative target genes cyclinD1 (CCND1) and E2F1 tions of cells that display NC-like properties. Yet, it has not been clarified the as well as antiproliferative targets CDKN1A (p21), PTEN, RB1, RBL1 (p107), level of purity of isolated NC-like populations as well as their homogeneity RBL2 (p130) were shown as common targets for miR-17, -20a, and -106b. in regard of fate potential. To further investigate the nature of these cell

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Furthermore, these microRNAs also downregulate G2/M transition inhibitor Poster Board Number: 3491 WEE1. Most strikingly, miR-17, -20a, and -106b were found to promote cell proliferation by increasing the intracellular activity of E2F transcription PRIMITIVE ERYTHROPOIESIS IS REGULATED BY factors, a major governor G1/S transition and downstream regulatory target MIR-126 VIA NON-HEMATOPOIETIC VCAM-1+ of the aforementioned pro- and antiproliferative proteins, despite the fact that miR-17, -20a, and -106b directly target this protein family. Summa- CELLS rized, miR-17, -20a, and -106b show a common regulatory impact on a Sturgeon, Christopher M., Chicha, Laurie, Hammond, Scott M., network of cell cycle genes which in turn results in positive regulation of E2F Olson, Eric N., Keller, Gordon transcription factor activity and thus cell proliferation. McEwen Centre for Regenerative Medicine, Toronto, ON, Canada, CNS Poster Board Number: 3489 Discovery Neuroregeneration Group, F. Hoffmann-la Roche Ltd, Basel, Switzerland, Dept of Cell and Molecular Biology, University of North EPIGENETIC ALTERATION OF HOX GENES Carolina, Chapel Hill, NC, USA, Dept of Molecular Biology, University of DURING RETINOIC ACID-INDUCED NEURAL Texas Southwestern Medical Center, Dallas, TX, USA DIFFERENTIATION Primitive erythropoiesis defines the onset of hematopoiesis in the early embryo and is distinguished by the fact that it is restricted to one site, the Taghizadeh, Zeinab, Hatami, Maryam, Baharvand, Hossein, yolk sac, and is active during a narrow developmental window. The dynamic Shahhoseini, Maryam nature of the primitive erythroid program suggests that it is tightly controlled Genetics, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, by mechanisms that regulate both its onset and termination. In this study we Iran, Islamic Republic of, Stem Cells, Royan Institute for Stem Cell Biology have used the mouse embryonic stem (mES) cell model to investigate the and Technology, ACECR, Tehran, Iran, Islamic Republic of role of microRNAs in the regulation of primitive erythropoiesis and provide evidence that miR-126 plays an important role in this process. Expression of Activation of HOX gene cluster is an early event in embryonic develop- miR-126 is induced in Flk-1+ hemangioblast mesoderm prior to the onset ment. One of the major functions of HOX genes seems to be the formation of primitive erythropoiesis by signalling through the Flk-1 receptor. Enforced of body plan during embryonic development; hence HOX gene expression expression of miR-126 results in a prolongation of primitive erythropoiesis, begins during early gastrulation when the principle body axis is established. while inhibition of miR-126 reduces primitive erythroid output. We identify Based on the presence of Retinoic Acid Response Element (RARE) in regula- vascular cell adhesion molecule 1 (Vcam-1) as a relevant target of miR-126 tory region of many of HOX genes, it is deduced that Retinoic Acid (RA) in vascular cells. In a cell non-autonomous manner, silencing of Vcam-1 can influence expression of HOX genes during RA-induced differentiation using shRNA enhances primitive erythropoiesis similar to miR-126 overex- of embryonic model systems. Hence every changes in gene expression is pression. Furthermore, co-culture of Vcam-1+ cells with primitive erythroid consequence of epigenetic changes in their regulatory regions, it is expected progenitors significantly reduced their CFC potential through a Notch- and that epigenetic profile of HOX gene regulatory regions be subjected to canonical-Wnt-independent mechanism. Collectively, these findings uncover changes during development. Embryonic stem cells have the capacity to a novel form of negative regulation of primitive erythropoiesis that may pro- either self renew or differentiate into a wide range of cell types, making vide new insight into the mechanism(s) controlling the developmental switch them an excellent model system to study the mechanisms that initiate HOX from primitive to definitive hematopoiesis. gene expression. In this investigation the expression profile of HOX genes were monitored by quantitative RT-PCR technique, during differentiation of Poster Board Number: 3493 human embryonic stem cells (hESC) to neural precursor cells, in the presence and absence of RA. Furthermore, in order to approve the identity of these ENHANCER DECOMMISSIONING BY THE neural precursor cells, the expression of neural markers such as Nestin and HISTONE DEMETHYLASE LSD1 DURING Sox1 were detected using immunocytostaning and flowcytometery. Our data significantly showed the increased expression of all 1-5 HOX genes EMBRYONIC STEM CELL DIFFERENTIATION of the four A-D clusters in the presence of RA. Epigenetic analysis of the Whyte, Warren A., Bilodeau, Steve, Hoke, Heather, Frampton, HOX gene regulatory regions using chromatin immunoprecipitation (ChIP) Garrett M., Orlando, David A., Young, Richard A. showed a significant decrease in methylation of histone H3K27 parallel to an increase in H3K4 methylation, in RA-induced neural precursor cells. Also, a Biology, Massachusetts Institute of Technology, Cambridge, MA, USA, preferential incorporation of the demethylase UTX rather than JMJD3 was Whitehead Institute for Biomedical Research, Cambridge, MA, USA observed on the promoter of HOX genes. This finding interestingly showed Transcription factors and chromatin modifiers play important roles in pro- the dynamic role of UTX in epigenetic alteration of HOX genes during RA- gramming and reprogramming of cellular states during development. Much induced neural differentiation, the function could not be detectable for the is known about the role of these regulators in gene activation, but relatively demethylase JMJD3, during this developmental process. little is known about the critical process of enhancer silencing during dif- ferentiation. We present evidence that the H3K4/K9 histone demethylase LSD1 plays an essential role in decommissioning active enhancers during differentiation of embryonic stem cells (ESCs). LSD1 occupies enhancers of active genes critical for control of ESC state. However, LSD1 is not essential for maintenance of ESC identity. Instead, ESCs lacking LSD1 activity fail to fully differentiate and ESC-specific enhancers fail to undergo the histone demethylation events associated with differentiation. At enhancers, LSD1 is a component of a NuRD complex containing additional subunits that are necessary for differentiation. We propose that the LSD1-NuRD complex decommissions enhancers of the pluripotency program upon differentia- tion, which is essential for complete shutdown of the ESC gene expression program and the transition to new cell states.

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Poster Board Number: 3495 non-induced ELSC and MMSC groups. These results suggested that there is a population of SSEA-1+ ELSCs in rat marrow. The cells may proliferate and ROLE OF HIF1α IN HESC DIFFERENTIATION form embryonic-like bodies and then differentiate into trilineage cells. Under induction with cytokines, the cells can differentiate toward cardiomyocytes Wisidagama, Dona R., Zhang, Jin, Malone, Cindy S., Teitell, and endothelial cells. After transplantation into the infarcted myocardium, Michael A. preinduced ELSCs differentiate toward cardiomyocytes and repair the Biology, California State University, Northridge, Northridge, CA, USA, myocardium effectively, and the cells are involved in angiogenesis. ELSCs of Broad Stem Cell Research Center, University of California, Los Angeles, Los rats may be applied as a new source of seed cells for exploring regeneration Angeles, CA, USA medicine and tissue-engineering. Human embryonic stem cells (hESCs) have generated enormous interest be- Poster Board Number: 3499 cause of their developmental plasticity and potential for patient-specific ap- plications in regenerative medicine. hESCs originate from the inner cell mass THE RNA-BINDING PROTEIN RBM24 IS of a blastocyst, which is a hypoxic microenvironment (<5% oxygen). In cell REQUIRED FOR CARDIOGENESIS AND HEART culture, hypoxia has been implicated as a controller of hESC pluripotency. We are investigating the role of Hypoxia Inducible Factor 1-alpha (HIF1α), FUNCTION the master regulator of hypoxia signaling, in regulating hESC differentiation. Xu, Xiu Qin, Poon, Kar Lai, Tan, Kar Tong, COLMAN, Alan, Korzh, Over-expressing HIF1 in hESCs increased the expression levels of common α Vladimir developmental genes during retinoic acid-induced differentiation in both normoxic and hypoxic conditions. Our studies also show that HIF1α and Institute of Medical Biology, Singapore, Singapore, Institute of Molecular HIF1β (ARNT) knockdown hESCs have defects in trophoectoderm formation and Cell Biology, Singapore, Singapore and embryoid body formation in both normoxic and hypoxic conditions. Human embryonic stem (hES) cells, with their ability to differentiate into These data suggest that HIF signaling promotes hESC differentiation inde- cardiomyocytes in culture, provide an in vitro model for human heart de- pendent of hypoxic conditions. Further studies on HIF targets are necessary velopment. Through the use of transcriptional profiling, we have identified to elucidate the molecular mechanisms of this new regulatory role for HIF a RNA binding protein, RNA binding motif protein 24 (Rbm24), enriched signaling in differentiation. Acknowledgments: CSUN-UCLA Bridges to Stem in the hESC derived cardiac pure population, and is also found to express in Cell Research Award, CIRM TB1-01183. CIRM Basic Biology I Award, CIRM the early mouse heart, suggesting its role in heart development. Hence, the RB1-01397 function of Rbm24 is studied using zebrafish, a well-established vertebrate Poster Board Number: 3497 model for heart development and disease studies. A functional characteriza- tion of genes by loss-of-function analysis employing morpholino injections ISOLATION OF EMBRYONIC-LIKE STEM CELLS into zebrafish embryos revealed severe cardiovascular defects indicating a critical function of Rbm24 for developmental processes. Our data demon- FROM RAT MARROW AND MYOCARDIAL strate the potential power of integrating a study of hES cell differentiation REPAIRING WITH CELL TRANSPLANTATION in vitro with the analysis of heart development in zebrafish in vivo that provides new insights into the molecular processes of the heart formation in Wu, Jin-hong, Tan, Yu-zhen, Wang, Hai-jie, Li, Zhi-hua vertebrates. Anatomy and Histology and Embryology, Shanghai Medical School of Fudan University, Shanghai, China Poster Board Number: 3501 In recent years, stem cell transplantation has been applied in treating GENERATION OF FUNCTIONAL MELANOCYTE myocardial infarction (MI), diabetes and neurodegenerative diseases. It is important to select desirable models and seed cells for investigating ef- FROM INDUCED PLURIPOTENT STEM CELLS fectiveness of stem cell transplantation. Whether there is a population of Yang, Ruifeng embryonic-like stem cells (ELSCs) in rat marrow is unknown. Therefore, we Department of Pathology and Lab Medicine, University of Pennsylvania, identified ELSCs derived from rat marrow and examined potential of ELSC Philadelphia, PA, USA differentiation toward cardiomyocytes and endothelial cells. SSEA-1+ ELSCs were isolated by FACS. The cells are 4 ~ 5 μm in diameter and have a large Induced pluripotent stem cells (iPSCs) have been generated from different nucleus and less cytoplasm. In immunocytochemistry staining and RT-PCR cell types following ectopic expression of the transcription factors Oct4 and analysis, the cells expressed transcriptional factors Oct-4, Nanog, Rex1, Sox2, combined with either Klf4 and c-Myc or Lin28 and Nanog. iPSCs are Sox2 and Fgf4. The cells proliferated well on feeder of mouse embryonic fi- essentially identical to embryonic stem cells (ESCs) and can be differentiated broblasts and form embryonic-like bodies after treatment with LIF and bFGF into multiple cell types. During differentiation, iPSCs may recapitulate their for two weeks. The cells of the embryonic-like bodies can differentiate into specific commitment and differentiation as characteristics of an individual’s trilineage cells. After induction with cytokines, ELSCs differentiated toward genomic information. As such, iPSCs is an ideal model system for studying cardiomyocytes and endothelial cells in vitro. Potential of ELSC differentia- both normal development and the processes that underlie disease. tion toward cardiomyocytes and endothelial cells in vivo was determined in We generated iPSCs colonies from adult mouse tail-tip fibroblasts (TTFs) us- rat myocardial infarction models. Before transplantation, the cells isolated ing retroviral vectors carrying murine sequences for Sox2, Oct3/4, cMyc and from male rats were induced with BMP-2, IGF-1 and bFGF for 24 hours. Klf4. TTFs-derived iPSCs colonies appeared within 2 weeks post-infection The induced cells were transplanted into hearts of female rat MI models. and were positive for alkaline phosphatase. These iPSCs colonies exhibited Comparing with non-induced ELSC and MMSC (marrow mesenchymal stem the morphology and growth properties of ES cells, expressed ES cell marker cell) groups, cardiac functions were improved and scar area was reduced genes and displayed ES cell pattern Oct4 promoter demethylation. These significantly in the preinduced ELSC group. Y chromosome fluorescence cells formed embryonic bodies and showed pluripotent differentiation in situ hybridization showed that there were more differentiated cells in capacity. Subcutaneous transplantation of iPSCs cells into nude mice resulted the preinduced ELSC group than that in non-induced ELSC and MMSC in terotoma containing a variety of tissues from all three germ layers. groups. Most Y chromosome-positive cells expressed cardiac troponin T and Subsequently, we set up an efficient method for differentiating iPSCs into connexin 43. Connexin 43 expressed between the differentiated cell and a melanocyte population using three growth factors: Wnt3a, endothelin-3, host cardiomyocyte. Density of microvessels in the peri-infarct and infarct and stem cell factor. The iPSCs-derived melanocytes expressed melanocyte regions increased significantly in the preinduced ELSC group compared with markers (such as microphthalmia-associated transcription factor and S100)

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and produced melanin. In addition, we also developed a transposon based early mesendoderm gene expression levels, such as T, EOMES and MIXL1, method to induce iPSCs without the use of viral vectors. In summary, we substantially decreased, while mRNA level of genes repressed by OCT4 have shown for the first time the differentiation of iPSCs into melanocytes. in hESC, such as LEFTY1, GATA4 and GATA6, increased in the absence of This method provides a novel in vitro system for studying the development OCT4. Consistent with disturbed gene expression profile, definitive endo- biology and diseases of melanocytes. Virus free induction of iPSCs alleviates derm differentiation in OCT4 knockdown hESC and iPSC is impaired. Taken the concern of integration mutagenesis and provides iPSCs that are more together, our results demonstrate dynamic transcription factor interplay compatible for cell replacement therapy. orchestrating cell fate decisions after the initiation of differentiation. Poster Board Number: 3503 DEFINITIVE ENDODERM SPECIFICATION IS A EMBRYONIC STEM CELL PLURIPOTENCY CONSEQUENCE OF EXTRA-CELLULAR MATRIX Poster Board Number: 3509 REMODELING DRIVEN BY PI3K SIGNALLING REGULATORY ROLE OF MICRORNAS IN Villegas, S. Nahuel, Barrios Llerena, Martin, Le Bihan, Thierry, Brickman, Joshua M. RESPONSE TO UVC-INDUCED DNA DAMAGE ISCR, University of Edinburgh, Edinburgh, United Kingdom, Centre for IN HUMAN EMBRYONIC STEM CELLS Systems Biology, University of Edinburgh, Edinburgh, United Kingdom Dolezalova, Dasa, Mraz, Marek, Barta, Tomas, Vinarsky, Vladimir, Adhesion of cells to the extra cellular matrix (ECM) has long been associ- Holubcova, Zuzana, Dvorak, Petr, Pospisilova, Sarka, Hampl, Ales ated with homeostasis in adult tissues and enabling cell migration during Dept. of Molecular Embryology, Institute of Experimental Medicine, v.v.i. development and tumour metastasis. However, there is no evidence that and Dept. of Histology and Embryology and Dept. of Biology, Faculty it plays a direct role in lineage specification during embryogenesis. Here of Medicine, Masaryk University and International Clinical Research we use embryonic stem cells (ESC) differentiation to demonstrate a direct Center, St. Anne´s University Hospital, Brno, Czech Republic, Dept of role for ECM in both maintaining cell shape and establishing the anterior- Internal Medicine - Hematooncology, Center of Molecular Biology and posterior axis within the endoderm germ layer. ECM remodelling is driven Gene Therapy, Faculty of Medicine and University Hospital, Brno, Czech by FGF signalling via phosphoinositide 3-kinase (PI3K)/Akt1 and is essential Republic, Center of Molecular Biology and Gene Therapy, Faculty of for anterior endoderm generation in vitro as well as in vivo. Isolated ECM Medicine and University Hospital, Brno, Czech Republic modulates cell shape changes through a temporal restriction of Epithelial to Mesenchymal Transition (EMT) and simultaneously conveying positional Pluripotent human embryonic stem cells (hESCs) are more vulnerable to identity. Based on Mass Spectrometry and gene expression analysis we iden- induction of DNA damage by various means and undergo rapid cell death tified Fibronectin (FN1) and Lamininβ 3 (LAMB3) as determinants of ECM in comparison to slowly proliferating differentiated cell types. Somatic cells activity. Our data imply that ECM composition regulate both polarity and developed various mechanisms how to coop with damaged DNA with identity in the emerging endoderm, linking cellular morphogenesis directly three cell cycle checkpoints (G1/S, intra-S and G2/M) being of the high- to cell fate decisions. est importance. Currently it is thought that “canonical” G1/S checkpoint pathway (which includes proteins p53 and p21) is not functional in hESCs. Poster Board Number: 3505 We have previously shown that in response to UVC irradiation, p53 protein is stabilized and transactivates its downstream genes including p21 but PLURIPOTENT TRANSCRIPTION FACTORS hESCs fail to produce p21 protein. Here we studied the role of small RNA REGULATE MESOENDODERM LINEAGE molecules (microRNAs) in DNA-damage response in hESCs and their role in regulation of G1/S checkpoint pathway. Our microarray analysis identified COMMITMENT DURING DIFFERENTIATION 85 upregulated microRNAs after UVC irradiation in hESCs (>2 fold change) Ying, Lei, Gadue, Paul including miR-193, miR-367, miR-520 and microRNA family miR-302, Dept of Hematology, The Children’s Hospital of Philadelphia, Philadelphia, which are selectively expressed in hESCs. Interestingly, in contrast to regula- PA, USA tion in somatic cell types, the expression of four p21-regulating microRNAs (miR-17, miR-20a, miR-20b and miR-106a) was also upregulated in hESCs Molecular mechanisms involved in early lineage commitment of human em- after DNA damage induction. These data suggest that these G1/S regula- bryonic stem cell (hESC) differentiation are of major interest in development tory microRNAs might contribute to inhibition of p21 protein expression in biology and regenerative medicine. It is known that pluripotency factors are hESCs. Moreover, microRNAs from family miR-302 share a seed sequence present until pre-primitive streak stage. The role that pluripotent transcrip- with p21-regulating microRNAs and were previously shown to directly bind tion factors plays in pluripotent cell differentiation and lineage commitment to mRNA of p21. Importantly we also show that expression of p21 mRNA is still ambiguous. In the present study, we used guided differentiation of is elevated after artificial downregulation of microRNA-processing proteins hESC and inducible pluripotent stem cells (iPSC) to delineate the roles of (Argonaute 2 and Dicer 1) and subsequent DNA damage induction. To- pluripotent transcription factors on the initiation of mesendoderm differen- gether our data suggest that undifferentiated hESCs use microRNAs to block tiation in vitro. Time-course gene expression profiling by Q-RTPCR and flow production of p21 protein and thus reveal important mechanism by which cytometric analysis of protein expression revealed that two pluripotency hESCs prevent canonical G1/S checkpoint pathway from being activated. factors, OCT4 and NANOG, have increased levels after initial differentiation; This study was supported by: CZ.1.05/1.1.00/02.0123, MSM0021622430, while SOX2 expression decreased dramatically. Chromatin immunoprecipi- AV0Z50390512, AV0Z50390703, P301/10/1971, IGAMZCR NT11218- tation (ChIP) analysis of H3K27me3 and H3K4me3 showed progressively 6/2010 resolved bivalent domains in transition from pluripotent status towards mesendoderm differentiation on definitive endoderm genes, such as T, SOX17, FOXA1 and FOXA2, etc. ChIP analysis of SOX2 and SOX17 showed a change in occupancy of SOX2 to SOX17 on regulatory regions of pluripo- tent genes and endoderm-specific genes during differentiation, suggesting a SOX switch important in the induction mesendoderm. By using siRNA to knockdown OCT4 in hESC during differentiation, we demonstrated that

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Poster Board Number: 3511 Poster Board Number: 3515 COMPLEX CDK2/CYCLINB1 AND ITS ROLE IN A GENOME-WIDE RNAI SCREEN IDENTIFIES HUMAN EMBRYONIC STEM CELLS NOVEL REGULATORS IN HUMAN EMBRYONIC Barta, Tomas, Jaros, Josef, Dolezalova, Dasa, Vinarsky, Vladimir, STEM CELLS Sedlackova, Miroslava, Dvorak, Petr, Hampl, Ales Chia, Na-Yu, Bard, Frederic, Ng, Huck-Hui Dept. of Molecular Embryology, Institute of Experimental Medicine, ASCR Genome Institute of Singapore, Singapore, Singapore, Institute of Molecular and Dept. of Biology and Dept. of Histology and Embryology, Faculty of and Cell Biology, Singapore, Singapore Medicine, Masaryk University and International Clinical Research Centre, St. Anne’s University Hospital, Brno, Czech Republic Human embryonic stem cells (hESCs) hold great promise in the area of regenerative medicine. The full potential of hESCs in research and clinical Human embryonic stem (ES) cells must ensure their pluripotent state while applications requires a detailed understanding of the genetic network that propagated in culture. The capability of having undifferentiated status in vit- governs their unique properties. Here, we performed a genome-wide RNA ro is maintained by unique cell cycle regulation. However, little is still known interference screen in search of genes that regulate the self-renewal and about cell cycle regulation and sustaining pluripotency in embryonic stem pluripotentiality in hESCs. Interestingly, 566 genes were identified to be cells. It has been previously shown that cyclin-dependent kinases (CDK), key involved in the maintenance of hESCs, among which interesting complexes molecules involved in cell cycle regulation, provide a link between pluri- such as INO80 chromatin remodeling complex, mediator complex, COP9 potency and self-renewal in human ES cells. Especially, CDK2 emerged as signalosome and TAF complex were implicated for its importance. 200 out central molecule in controlling self-renewal and cell¬-fate decision. Here we of the 566 genes were further validated in a secondary screen assay using show that cell cycle regulator CDK2 in human ES cells physically associates deconvoluted siRNAs and 93 of them could downregulate OCT4 expres- with cyclin B1. This complex is able to phosphorylate histone H1 and RB sion in the different hESC lines. Coupling with higher bioinformatic analysis, in vitro. Maximal activity of CDK2/cyclinB1 has been detected in mitosis, we identified essential pathways that govern hESCs. Transcription factor suggesting that this complex might play a crucial role in mitosis of human ES PRDM14 exemplifies one of the essential regulators in this genome-wide cells. RNA interference screen and we showed that PRDM14 directly regulate the Poster Board Number: 3513 expression of key pluripotency gene POU5F1 through its proximal enhancer. Additionally, PRDM14 is integrated into the core transcriptional regulatory EMBRYONIC CHROMOSOMAL ANEUPLOIDY network wherein our genome-wide location profiling experiments revealed an extensive co-localization of PRDM14 with other key transcription factors RECAPITULATED IN HUMAN EMBRYONIC STEM such as OCT4, NANOG and SOX2. More importantly, in a gain of-function CELL LINES assay, we showed that PRDM14 is able to enhance the efficiency of repro- gramming in human fibroblasts in conjunction with OCT4, SOX2 and KLF4. Biancotti, Juan C., Narwani, Kavita, Mandefro, Berhan, Buehler, Taken together, our study uncovers a wealth of novel determinants that Nicole, Hill, David, Clark, Amander, Lavon, Neta, Benvenisty, Nissim could advance our understanding in hESCs. Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA, ART Reproductive Center, Los Angeles, CA, USA, Dept of Cell, Poster Board Number: 3517 Molecular and Developmental Biology, University of California, Los Angeles, Los Angeles, CA, USA, Dept of Genetics, Hebrew University, THE FIDELITY OF DNA METHYLATION IN Jerusalem, Israel HESCS GROWN IN DEFINED AND XENO-FREE Chromosomal aneuploidies are widely recognized genetic disorders in hu- CULTURE SYSTEMS mans that often lead to spontaneous miscarriage. Pre-implantation genetic Tompkins, Joshua D., Hall, Christine A., Chen, Vincent C., Couture, screening (PGS) is performed for the diagnosis of aneuploidy in early em- Sylvana, Hsu, David, Couture, Larry A., Riggs, Arthur D. bryos. Aneuploid embryos can be used to generate human embryonic stem cell (hESC) lines, which serve as an excellent model for human chromosomal Biology, City of Hope, Duarte, CA, USA, Center for Biomedicine and disorders. We have created a repository of 46 hESC lines from blastocysts Genetics, City of Hope, Duarte, CA, USA diagnosed as aneuploid on day 3 of in vitro development. The hESC lines The use of human embryonic stem cells (hESCs) in clinical therapy will exhibited morphology and expressed markers typical of hESCs. They dem- require the large-scale adaptation of cell lines to fully defined and xeno- onstrated long-term proliferation capacity, and pluripotent differentiation. free cultures. Therefore, it is vital that we first comprehend interactions of The analysis by karyotype indicates that about two thirds of the cell lines hESCs and their microenvironments, as well as the consequences of shifting have an euploid karyotype, while one third remained aneuploid throughout such interactions on a genome-wide scale. In this study, we have analyzed the derivation, resulting in 14 hESC lines carrying either trisomy 12, 13 (Pa- DNA methylation and gene expression profiles of hESCs during adaptation tau syndrome), 16, 17, 20, 21 (Down syndrome), X (Triple X syndrome), or (and reverse adaptation) to various substrate/medium combinations: MEFs/ monosomy X (Turner syndrome). Based on the levels of nucleotide polymor- DMEM-F12 KSR medium, Matrigel/mTeSR1, and CELLstart/STEMPRO. phism heterozygosity in the aneuploid chromosomes, we could determine These experimental conditions range from conventional hESC cultures to that the aneuploidies originated from either meiotic or mitotic chromosomal commercially available fully defined systems. We identify markers of pluri- nondisjunction. The spectrum of aneuploidies in these ES cell lines reflects potency to display consistent DNA methylation and expression patterning the range of common embryonic chromosomal aberrations, and significantly fitting for the pluripotent state in all culture environments. However, culture differs from the spectrum of aneuploid human ES cell lines generated by manipulation commensurately altered the expression of gene sets likely fun- cell adaptation in culture. These results suggest that aneuploid embryos can damental to hESC acclimation. Among observations, differential expression serve as an alternative source for either normal euploid or aneuploid hESC of genes encoding membrane spanning and secreted cell adhesion factors lines, which represent an invaluable tool to study developmental aspects of suggest a substrate-specific membrane reconfiguration. Furthermore, ex- chromosomal abnormalities in humans. pression changes in genes controlling growth factor responses and metabolic pathways appear to closely mirror cellular signaling targeted by medium formulations. Despite hundreds of differentially expressed genes between cultures, we find DNA methylation remains remarkably stable, and the

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overwhelming majority of culture induced methylation events are decidedly down-regulated in the iPS cells. These analyses have identified new potential specific and reversible. However, DNA methylation changes increased both pluripotency factors that are up-regulated and new pathways that may need with culture manipulation and with prolonged passage. Sites of contrasting to be suppressed to maintain the pluripotent state during human stem cell DNA methylation were linked to expression levels of a small number of cen- expansion. tral genes involved in cellular adhesion and in controlling G-protein coupled signaling, of which the latter may be responsible for the wider scope of Poster Board Number: 3521 microenvironment induced expression changes observed during reverse DISTINCT REGULATION OF FOXO ISOFORMS adaptation. Genome-wide, we note correlations between DNA methylation and gene expression including a novel association of gene silencing and IN HUMAN EMBRYONIC AND ADULT MOUSE DNA hypomethylation at promoter distal sites. Observations provide insight HEMATOPOIETIC STEM CELLS into the purpose of DNA methylation in hESCs. Collectively, these results demonstrate that hESC adaptation to defined culture systems is marked by a Ghaffari, Saghi, Zhang, Xin, Rimmele, Pauline, Yalcin, Safak, commensurate response in gene regulation and convey that epigenetic fidel- D’Escamard, Valentina ity should be closely monitored when manipulating hESC cultures, especially Dept Gene & Cell Medicine, Mount Sinai School of Medicine, New York, when considering future therapeutic utility. NY, USA Poster Board Number: 3519 Understanding mechanisms of regulation of stem cell function has important implications for stem cell therapy and regenerative medicine. In the past few IDENTIFICATION OF NEW PLURIPOTENCY years FoxO transcription factors have emerged as critical regulators of stem GENE EXPRESSION PROFILES FOR HUMAN cell activity. The Forkhead Box O (FoxO) members of the Forkhead family of transcription factors comprise four members in mammals (FoxO1, FoxO3, PLURIPOTENT STEM CELLS FoxO4 and FoxO6). These factors are evolutionarily conserved regulators of Kiessling, Ann A., Bletsa, Ritsa, Desmarais, Bryan, Loutradis, longevity and stress response. FoxO are phosphorylated and suppressed by Dimitris the AKT serine threonine protein kinase that mediates their nuclear exclusion in response to growth factors or cytokines. In addition to phosphorylation, Harvard Medical School, Boston, MA, USA, University of Athens, Athens, FoxO proteins are modified by several post-translational modifications such Greece, Bedford Research Foundation, Somerville, MA, USA as acetylation, ubiquitination, methylation and redox modulation whose Pluripotent cells have the capacity to contribute to all the tissues in the integrated combined output determines the FoxO function. It has been body and to form teratomas with cell types from endoderm, mesoderm and suggested that FoxO factors are redundant in their regulation of hematopoi- ectoderm. Gene expression profiles that define pluripotency would both etic stem cell activity and tumor suppressor function. Indeed, these factors facilitate high through-put assessment of the pluripotent state of cultured behave similarly in biochemical studies of cell-based cultures, are known to stem cells, and also provide insights into pathways important to maintaining regulate common target genes, and bind to the same target DNA sequence. an uncommitted, pluripotent state during cell population expansion. Toward However, mouse FoxO knock out exhibit unique phenotypes. In addition, this goal, several reports of gene pathways important to pluripotency have distinct FoxO isoforms are required for the activity of distinct stem cells. appeared. We have combined the gene lists of reported pluripotency factors We have previously reported critical functions for FoxO forkhead transcrip- with PluriNet and with the target genes of the pluripotency transcription tion factors in the regulation of human embryonic (hES) and mouse adult factors, Sox2, Nanog and Pou5f1/Oct4. We have analyzed the compiled hematopoietic stem cell functions. In addition, FoxO3 is required for the list for common pathways and compared it with the target genes of the maintenance of human and mouse leukemic stem cells together suggesting general transcription factor, E2F. We created a sub database of whole overlapping but non-redundant functions of FoxO isoforms in stem cells. genome microarrays of 8Cell human embryos, human embryonic stem Given these properties FoxO have been proposed as valuable candidates (hES) cells, and human fibroblasts before and after induced pluripotency for targeted therapy in cancer and other diseases. In order to investigate (iPS cells). The research was reviewed and approved by the Institutional how FoxO function in stem cells is regulated, we analyzed the subcellular Review Boards of the Bedford Research Foundation and the University of localization (a surrogate assay for FoxO activity), DNA binding and tran- Athens, Alexandra Hospital. Of the 1837 gene elements, 364 (20%) are scriptional activity of the most highly expressed FoxO (FoxO1 and FoxO3) targets of E2F, 1050 (57%) are NANOG targets, 462 (25%) are NANOG in human embryonic and/or mouse hematopoietic stem cells. In addition only, 872 (47%) are SOX2 targets, 254 (14%) are SOX2 only; 18% 459 we compared the same parameters in 293T cells ectopically expressing (25%) are POU5F1(OCT4), 99 (5%) are POU5F1/OCT4 only. Sixteen Flag-FoxO1 or Flag-FoxO3. These analyses showed that FoxO1 and FoxO3 (53%) of the 30 pluripotency factors and 203 (68%) of the 299 PluriNet display overlapping but also distinct DNA binding, transcriptional activity elements were not targets of any of the four transcription factors. Only 47 and response to signaling pathways in these stem cells and in 293T cells. For (16%) of the PluriNet elements and 8 (27%) of the pluripotency factors instance in undifferentiated pluripotent self-renewing hES cells, endogenous were targets of Nanog, Sox2 and/or Pou5F1/Oct4. Only 7 of the pluripo- FoxO1 (and not FoxO3) was mostly nuclear despite abundant presence tency factors were included in PluriNet, only one of which, NANOG, was a of phosphoAKT. Inhibition of phosphoAKT had little impact on subcellular target of Nanog, Sox2 or Pou5F1/Oct4. To identify potential pluripotency localization or DNA binding of FoxO1 but enhanced significantly FoxO3 factors not previously identified as NANOG, SOX2 and POU5F1 targets, we nuclear localization and DNA binding. In particular, inhibition of PI3-kinase screened the 1837 element sub database for elements with both hES and signaling pathway did not impact FoxO1 binding to SOX2 promoter or 8C embryo fluorescence detection greater than 7-fold over fibroblast gene activation of its gene but enhanced significantly the binding of FoxO3 to elements. This identified 66 elements, 38 of which were listed in Plurinet, SOX2 and activation of SOX2 gene. In contrast to human ES cells, in he- eleven of which were E2F only targets, all of which were in PluriNet. One, matopoietic stem cells FoxO3 and not FoxO1 was the most highly expressed BUB1B, was a target of all four transcription factors, five (8%) were targets nuclear FoxO despite highly detectable phosphoAKT. These findings may of Sox2/Pou5f1/Nanog, 57 were targets of Sox2, Pou5f1, or Nanog. Four be due to distinct post-translational modifications of distinct FoxO isoforms. elements over-detected on both the 8Cell embryo and hES cell arrays Altogether our analyses suggest differences in FoxO isoform regulation and were not up-regulated in the iPS cells: GSC, GAD1, GATA6 and NPPB. To function that may have important consequences on targeting these proteins identify genes down-regulated in pluripotent cells, we identified a subset for therapy. of 43 genes detected at 7-fold lower levels in both 8Cell embryos and hES cells; only one was a target of E2F, 11 were targets of Sox2/Pou5f1/Nanog, 31 were targets of Sox2, Pou5f1/Oct4 or Nanog. All 43 genes were also

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Poster Board Number: 3523 tion. 247-B9-reactive antigen was identified as the hnRNP A2/B1 proteins by peptide mass fingerprinting and western blot analysis. Unlike the original ADENOVIRUS EARLY REGION 1B-ASSOCIATED hnRNP A2/B1 proteins, 247-B9-reactive hnRNP A2/B1 was also exposed on PROTEIN 5 REGULATES THE PROLIFERATION the cell surface of various cancer cells and downregulated upon differentia- tion. The present findings demonstrate that 247-B9-reactive hnRNP A2/B1 AND SELF-RENEWAL OF HUMAN EMBRYONIC identified in this study may be a novel cell surface marker for undifferenti- STEM CELLS THROUGH AKT AND ERK ated hESCs and cancer cells, suggesting that it may play as a cell surface- SIGNALING expressed oncofetal antigen. Choi, Hong Seo, Jang, Young Joo, Kim, Cheorl-Ho, Ryu, Chun Jeih Poster Board Number: 3527 Sejong University and Sungkyunkwan University, Seoul, Korea, Republic B-CELL RECEPTOR ASSOCIATED PROTEIN of, Dankook University, Cheonan, Korea, Republic of, Sungkyunkwan University, Suwon, Korea, Republic of, Sejong university, Seoul, Korea, 31 IS EXPRESSED ON THE CELL SURFACE Republic of OF HUMAN EMBRYONIC STEM CELLS AND Human embryonic stem cells (hESCs) are pluripotent cells capable of self- DOWNREGULATED UPON DIFFERENTIATION renewal and differentiation to form cells of all three germ layers. Many Kim, Won-Tae, Choi, Hong Seo, Kim, Cheorl-Ho, Ryu, Chun Jeih studies have attempted to unravel the molecular mechanism that governs hESC pluripotency and self-renewal. Previously, we generated a panel of Sejong University, Seoul, Korea, Republic of, Sungkyunkwan University, murine monoclonal antibodies (MAbs) specific to surface antigens of human Suwon,Gyeonggi-do, Korea, Republic of embryonic stem cells (hESCs) using a modification of the decoy immuniza- B-cell receptor associated protein 31 (BAP31), a resident integral protein of tion strategy. One MAb, 57-C11, recognized a phosphorylated form of the endoplasmic reticulum (ER) membrane, regulates the export of secreted adenovirus early region 1B-associated protein 5 (E1B-AP5) on the surface of membrane protein from the ER to the downstream secretory pathway. It is undifferentiated hESCs. The phosphorylated E1B-AP5 protein was localized also involved in caspase-8 mediated apoptosis. In the previous study, we to SSEA3-, SSEA4-, TRA-1-60-, TRA-1-81-, OCT4-, SOX2-, and NANOG- generated monoclonal antibody 297-D4 against the surface molecule on un- positive hESCs and down-regulated in differentiated hESCs. To understand differentiated human embryonic stem cells (hESCs) by using a modified de- biological function of E1B-AP5 in hESCs, in this study, endogenous E1B-AP5 coy immunization strategy. Of these, MAb 297-D4 bound to human pluri- genes were silenced in H9 cells by small interfering RNAs. The efficiency of potent stem cells H1, H7, H9, CHA-hES4, NT-2/D1, and NCCIT but not to E1B-AP5 knockdown was more than 90%. The expression of undifferenti- human differentiated primary cells, mouse embryonic stem cells, and mouse ated markers SSEA3, SSEA4, TRA-1-60 and TRA-1-81 slightly decreased, embryonic fibroblasts. 297-D4 antigen expression was localized to SSEA3-, but pluripotency markers Oct4 and Sox2 decreased by greater than 50% SSEA4-, TRA-1-60-, TRA-1-81-postive hESCs and downregulated upon dif- in the knockdown cells. E1B-AP5 knockdown reduced the proliferation and ferentiation. 297-D4 antigen, an approximately 30 kDa cell surface protein, clonogenic ability of H9 cells. Morphological changes were also obvious in was identified as the BAP31 by peptide mass fingerprinting and western blot the knockdown cells, showing condensed and smaller colonies with alkaline analysis. 297-D4 binding activity to BAP31 was further confirmed by ectopic phosphatase-negative center. Increased numbers of apoptotic cells of up to expression of BAP31 DNA construct in HeLa cell. The results indicate that 20% were also observed in the knockdown cells. To investigate signaling 297-D4 recognizes BAP31 on the surface of hESCs and suggest that BAP31 pathways in the knockdown cells, the human phospho-MAPK assay was is a putative cell-surface marker for undifferentiated hESCs. performed. As a result, the expression of phospho-AKTs and phospho-ERKs significantly decreased in the E1B-AP5 knockdown cells. The results demon- Poster Board Number: 3529 strate that E1B-AP5 regulates hESC proliferation and self renewal through AKT and ERK signaling. HUMAN ES CELL SPECIFIC MIR-302 CLUSTER ARE INTIMATELY INVOLVED IN COMPLETE Poster Board Number: 3525 NUCLEAR REPROGRAMMING OF IPS CELLS HETEROGENEOUS NUCLEAR THROUGH SUPPRESSION OF MBD2 RIBONUCLEOPROTEIN A2/B1 IS EXPOSED AT Lee, Man Ryul, Prasain, Nutan, Kim, Young-June, Yoder, Mervin C., THE CELL SURFACE OF HUMAN EMBRYONIC Broxmeyer, Hal E. STEM CELLS AND CANCER CELLS, AND Microbiology and Immunology, IUPUI, Indianapolis, IN, USA, Herman B DOWNREGULATED UPON DIFFERENTIATION Wells Center for Pediatric Research, IUPUI, Indianapolis, IN, USA Ryu, Chun Jeih, Choi, Hong Seo, Kim, Kyoung Soo, Kim, Cheorl-Ho Although a number of microRNAs (miRNAs) have been implicated in fine-tuning mRNA translation during early mammalian development, the Sejong University, Seoul, Korea, Republic of, Sejong University and regulatory role of miRNAs involved during reversion of a differentiated cell Sungkyunkwan University, Seoul, Korea, Republic of, Kyung Hee University, to a pluripotent state is incompletely understood. To gain further insight Seoul, Korea, Republic of, Sungkyunkwan University, Suwon, Korea, into the role of miRNAs in nuclear reprogramming we performed miRNA Republic of PCR-array analysis on incompletely (partially) reprogrammed cells and more Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1), a multi- completely reprogrammed human induced pluripotent stem cells (iPSC) that functional RNA-binding protein, is required for maturation of mRNA precur- we obtained during our recent reprogramming experiments with frozen- sors. It is normally found in the nucleus and some in the cytoplasm, and the thawed umbilical cord blood derived CD34+ cells. We found significant changes in its expression and localization are tightly related to cell prolifera- downregulation of hESC-specific miRNAs, miR-302 and 371 clusters, in the tion and carcinogenesis. Previously, we generated monoclonal antibody incompletely reprogrammed cells, which was comparable to that of differ- 247-B9 against the surface molecule on undifferentiated human embryonic entiated cells; i.e. opposite to pluripotent hESC. Therefore, we hypothesized stem cells (hESCs). 247-B9-reactive antigen was expressed on the surface that miR-302 and 371 clusters may promote a transition from partially to of hESCs but not on the surface of the adult primary cells. 247-B9-reactive completely reprogrammed iPSC through regulation of methylation status. To antigen was localized to SSEA3-, SSEA4-, TRA-1-60-, TRA-1-81-, OCT4-, test this hypothesis, we over-expressed miRNA clusters of miR-302 and 371 NANOG-, and SOX2-positive hESCs and downregulated upon differentia- in TRA1-60neg incompletely reprogrammed cells and determined whether

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these miRNAs converted TRA-60neg (incompletely reprogrammed cells) to reduced ABCG2 expression. Our findings suggest that the ABCG2 multidrug TRA1-60pos (completely reprogrammed) cells. Our results indicate that over transporter protein is expressed in iPSCs, and these results indicate that this expression of miR-302 clusters resulted in conversion of more than 10% of protein may have an important protective function in human pluripotent TRA1-60neg cells to TRA1-60pos cells with the same characteristics as com- cells. This work has been supported by STEMKILL, TÁMOP 4.2.2/1, OTKA pletely reprogrammed iPSC, including ability to differentiate into three germ 72057, and KMOP-1.1.2-07/1-2008-0003 layers and acquisition of typical hESC-specific pluripotent markers. We also demonstrated that this more complete reprogramming mechanism func- Poster Board Number: 3533 tioned through miR-302 cluster-targeted suppression of epigenetic regulator, TERATOMA FORMATION BY FEEDER- methyl-DNA binding domain protein 2 (MBD2), which suppresses gene transcription through binding to methylated CpG islands of the target genes. DEPENDENT AND -INDEPENDENT HUMAN While MBD2 expression was unobservable or found at very low levels in EMBRYONIC STEM CELLS hESC, hiPSC, and human embryocarcinoma cells (hECC), MBD2 was highly expressed in TRA1-60neg, incompletely reprogrammed cells as well as in Kunova, Michaela, Matulka, Kamil, Salykin, Anton, Hampl, Ales, human pluripotent stem cells (hPSC)-derived differentiated cells. MBD2 was Dvorak, Petr found to be directly bound to the DNA methylation region of the ES-specific Dept of Biology, Faculty of Medicine, Masaryk University, Brno, Czech gene promoter, as revealed by ChIP analysis of differentiated hPSC and in Republic, Dept of Histology and Embryology, Faculty of Medicine, Masaryk TRA1-60neg incompletely reprogrammed cells. We also found that miR-302 University, Brno, Czech Republic cluster reduced luciferase activity in reporter assays containing wild-type 3’ UTR MBD2 by 50% and suppressed expression of MBD2 in somatic cells. Teratoma formation is being considered to be the most stringent assay Thus, enhanced expression of exogenous miR-302 cluster in incompletely assessing the pluripotency status of human embryonic stem cells (hESCs). reprogrammed cells may play an active role in expression of endogenous However, this assay lacks standards and shows variability that makes any in- ES-specific gene by inhibiting MBD2 expression, thereby promoting more terpretation difficult. The most critical factors influencing teratoma formation complete nuclear reprogramming. Taken together, our results provide strong comprise of the hESC line origin, number of hESCs in the inoculum as well as support for a regulatory role of miR-302 in epigenetic regulation of core presence of feeder cells or Matrigel, site of injection, and strain of the immu- circuitry genes involved in self-renewal and reprogramming of somatic cells nodeficient mouse. In our study, we found that hESCs, which are cultured to a pluripotent state. as high-density monolayers in feeder-free conditions, show significantly increased efficiency of teratoma formation (100% vs. 40% by hESCs grown Poster Board Number: 3531 as low-density colonies in the presence of feeder cells). According to the his- tological analysis, the differentiation capacity is not impaired in any of these ABCG2 MULTIDRUG TRANSPORTER two groups. As survival of hESCs shortly after injection represents one of the EXPRESSION IN HUMAN IPS CELLS DURING key factors influencing the efficiency of teratoma formation, we analyzed the stress response of hESCs maintained either as monolayer or colonies REPROGRAMMING AND DIFFERENTIATION in several suboptimal conditions. We also performed the gene expression Erdei, Zsuzsa, Sebe, Attila, Orbán, Tamás I., Várady, György, Apáti, analysis in both groups of hESCs to identify potential candidates influenc- Ágota, Sarkadi, Balázs ing the efficiency of teratoma formation, particularly focusing on genes related to apoptosis, survival and adhesion. Furthermore, we implemented Membrane Research Group, Hungarian Academy of Science, Budapest, in vitro culture experiments as well as immunohistochemistry to identify Hungary, Dept of Biochemistry and Molecular Biology, University of the remnants of undifferentiated cells within the teratoma. In summary, our Debrecen, Debrecen, Hungary results show that the increased efficiency of teratoma formation by hESCs ABC multidrug transporters form a chemo-immunity network, protecting maintained as high-density monolayer is likely due to the enhanced survival our body against toxic compounds. The human ABCG2 multidrug transport- of those cells shortly after injection into the immunodeficient recipients. er is an ATP-dependent efflux pump, extruding various drugs, xenobiotics and endogenous metabolites from the cells. Currently, there is no consensus Poster Board Number: 3535 about ABCG2 expression and function in human embryonic stem cells; there PROTEOMIC COMPARISON BETWEEN are reports claiming, while others denying the presence of ABCG2 expres- sion in HuESCs. Our group demonstrated previously that ABCG2 is function- HUMAN EMBRYONIC STEM CELLS AND ally expressed in several pluripotent HuES cell lines. In order to collect further THEIR DIFFERENTIATED FIBROBLASTS: evidence for the expression and function of ABCG2 in pluripotent stem cells, and to confirm the connection between ABCG2 expression and the pluripo- IDENTIFICATION OF 206 GENES TARGETED BY tent state, we have investigated the expression of this multidrug transporter HES CELL-SPECIFIC MICRORNAS protein in human iPSCs. Human iPSCs were created from human foreskin Li, Steven SL, Tsai, Zong Yun fibroblasts (HFFs) by introducing the reprogramming factors (c-Myc, Klf4, Oct4, Sox2, +/- Lin28) in a single expression cassette, by using the Sleeping Kaohsiung Medical University Institute of Clinical Medicine, Kaohsiung, Beauty transposon system. After iPSC generation the pluripotent cells were Taiwan grown on mouse embryonic fibroblast (MEF) feeder cells. ABCG2 expression Human embryonic stem T3ES cells were spontaneously differentiated into was examined by using Q-PCR, flow cytometry and immuno-fluorescense autogeneic fibroblast-like T3DF cells as feeder with capacity to support microscopy. Plasma membrane localization of the ABCG2 protein was es- the growth of undifferentiated hES cells. The proteomes of undifferenti- tablished by confocal microscopy. We found that ABCG2 is well measurably ated T3ES cells and their differentiated T3DF fibroblasts were quantitatively expressed in iPS cells, although with a heterogeneous expression pattern. compared. Several heterogeneous nuclear ribonucleoproteins (hnRNPs) and Flow cytometry and RT-QPCR analysis indicated that ABCG2 expression glycolytic enzymes including L-lactate dehydrogenase A (M) were found was linked to the pluripotent state and reprogramming, as HFF cells did not to be abundantly differentially expressed in T3ES. Both miRNA and mRNA express this protein. When the co-expression of the undifferentiated stem profiles from the undifferentiated T3ES cells and their differentiated T3DF cell marker SSEA4 and ABCG2 was assessed, iPSCs were positive for both fibroblasts were determined. 206 genes were found to be targets of four markers, especially in experiments where SSEA4 positivity reached 80%. hES cell-specific miRNAs miR-302d, miR-372, miR-200c and/or miR-367 When iPS cells were spontaneously differentiated to embryonic bodies, by using 2-fold differential expression and inverse expression levels (highly ABCG2 mRNA levels first increased, while further differentiation significantly negative correlations) of miRNAs to their target mRNAs. YWHAZ (14-3-3

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Thursday Poster Abstracts zeta) target of miR-302d and miR-372 was further confirmed by proteomic apoptotic and/or pro-survival signaling cascades that are especially active in comparison between T3ES cells and their differentiated T3DF fibroblasts. Ac- hESCs. So as to determine which cell death pathways might be most active cording to GeneOntology analysis, almost half of these 206 target proteins in hESCs relative to other cell types, we used quantitative PCR to evaluate were located in nucleus and involved in gene transcription.cells and T3DF the basal expression of all members of the BCL-2 family in hESCs (TE06 fibroblasts, respectively. and BG01), hESC-derived neural stem cells, seven human primary cell lines including representatives from each germ layer, and two cancer lines. In Poster Board Number: 3537 addition to BCL-2 family members, we assayed genes that are commonly GENOME-WIDE IDENTIFICATION OF used as markers of pluripotency and germ layers to serve as an internal control. (e.g. Nanog and SOX2 were found to be predominantly expressed MICRORNA TARGETS IN HUMAN ES by hESCs.) Our analysis revealed, surprisingly, that expression levels of the CELLS REVEALS A ROLE FOR MIR-302 IN pro-apoptotic BH3-only BCL-2 family members PUMA, NOXA, and BIM are significantly elevated in hESCs compared to other cell-types. The fact that MODULATING BMP RESPONSE hESCs live while expressing relatively high amounts of the pro-apoptotic Lipchina, Inna, Elkabetz, Yechiel, Hafner, Markus, Sheridan, Robert, molecules NOXA and PUMA was surprising; the fact that both NOXA and Mihailovic, Aleksandra, Tuschl, Thomas, Sander, Chris, Studer, PUMA are regulated by p53 peaked our interest. What benefit, if any, does Lorenz, Betel, Doron basal overexpression of PUMA and NOXA afford hESCs? Is this expression p53-dependent? If so, what signals are p53 responding to? We hope to Memorial Sloan-Kettering Cancer Institute, New York, NY, USA, answer these and other questions in our future work; doing so should offer Developmental Biology, Memorial Sloan-Kettering Cancer Institute, insight into the signaling pathways that govern cell survival - insight that New York, NY, USA, Howard Hughes Medical Institute, Laboratory of undoubtedly will translate to the development of better conditions for both RNA Molecular Biology, Rockefeller University, New York, NY, USA, hESC expansion and promotion of cell survival post-transplantation. Computational Biology Program, Memorial Sloan-Kettering Cancer Institute, New York, NY, USA Poster Board Number: 3541 MicroRNAs are important regulators in many cellular processes including PROTEOMIC ANALYSIS OF HUMAN stem cell self-renewal. Most microRNA studies in pluripotent stem cells were performed in mouse ES cells and there are only few validated targets. There- EMBRYONIC STEM CELL INTEGRIN- fore, it is important to study the function of microRNAs in human ES cells ASSOCIATED COMPLEXES (hESCs) and develop genome-wide strategies to identify their targets. Here, we performed comprehensive microRNA profiling in hESCs and in purified Ajeian, Jila N., Humphries, Jonathan D., Byron, Adam, Askari, Janet neural and mesenchymal derivatives. To systematically identify high-confi- A., Craig, Susan E., Kimber, Susan, Humphries, Martin J. dence microRNA targets, we combined AGO cross-linking and microRNA Faculty of Life Sciences, University of Manchester, Manchester, United perturbation experiments with computational prediction. Application of this Kingdom approach to miR-302/367, the most abundant microRNAs in hESCs, identi- The extracellular matrix (ECM) is one of the key constituents of the stem cell fied new targets from various biological pathways. Functional studies identi- microenvironment mediating cell fate decisions, such as retention of pluri- fied novel roles of miR-302/367 in maintaining pluripotency and regulating potency and differentiation. Adhesion of stem cells to the ECM is primarily TGF-β and BMP signaling in hESCs. This study broadens our understanding mediated by integrins, a family of heterodimeric αβ transmembrane adhe- of microRNA function in hESCs and provides a general strategy for the sion receptors. The extracellular domains of integrins bind to ligands in the genome-wide identification of microRNA targets. ECM whilst the intracellular domains form multi-molecular complexes with Poster Board Number: 3539 cytoplasmic signalling and adaptor proteins that link to the cytoskeleton. In- tegrin signalling plays a significant role in regulating cellular functions, such HUMAN EMBRYONIC STEM CELLS EXPRESS as differentiation and embryonic development in response to the external A UNIQUE REPERTOIRE OF BCL-2 FAMILY environment. Fibronectin (FN), the prototypic α5β1 integrin ligand, has been shown to be an important substrate in embryonic stem cell development. MEMBERS Moreover, FN acts as a critical factor supporting self-renewal in human Madden, David T., Davila-Kruger, Diana, Bredesen, Dale E. embryonic stem cells. In this study a proteomics-based system has been developed to isolate, identify and quantify FN-induced integrin-associated College of Pharmacy, Touro University - California, Vallejo, CA, USA, Buck intracellular signalling proteins in stem cells. Tosyl-activated paramagnetic Institute for Research on Aging, Novato, CA, USA beads were coated with FN, the anti-integrin-β1 antibody (TS2/16) or the Roadblocks to human embryonic stem cell (hESC) based therapeutics include anti-transferrin antibody (OKT-9) as a negative control. Human embryonic (1) the production of defined cell types at a scale required to meet clini- stem cells (hESCs) (5×107) cultured on FN in a fully-defined and serum-free cal demand and (2) the limited survival of cells post-transplantation. Total system were allowed to adhere to ligand-coated beads and subsequently cell numbers are determined by the balance of the rates of division and of lysed with detergent to isolate integrin-associated proteins. Isolated proteins cell death: Strategies to improve growth could include boosting the rate of were analysed by immunoblotting and mass spectrometry (MS). Immuno- division or blunting the rate of cell death. We have focused our studies on blotting showed the enrichment of key proteins, such as integrin-β1, talin, the signals that drive hESCs to programmed cell death with the thought paxillin, integrin-α5, vinculin and FAK, in FN-bound complexes from hESC that understanding these pathways might well extend to preparations of cells compared to OKT9. Mass spectrometry (MS) validated the results post-mitotic cells intended for transplantation (e.g. hESC-derived neu- obtained from immunoblotting, by detecting peptides of adhesion complex rons). The decision to die is regulated by a family of related proteins - the proteins such as integrin-β1, integrin-αV, vinculin, talin, α-actinin-4, zyxin BCL-2 family - that act at the level of the mitochondria. Within this group and fascin in FN-bound complexes. The integral membrane protein transfer- are molecules that either promote or inhibit cell death. Pro-survival BCL-2 rin receptor (CD71) was only enriched in OKT9. These data demonstrate family members include BCL-2, BCL-w, BCL-xL, BCL-B, MCL-1, and A1. that integrin adhesion complexes can be isolated from hESCs using this Pro-apoptotic BCL-2 family members include BAX and BAK as well as a method and also indicate that the method is reproducible and suitable for group of proteins known as the BH3-only BCL-2 family which includes BIM, mass spectrometry. Future work will be directed towards obtaining further BID, PUMA, NOXA, BAD and others. Our working hypothesis is that overly in-depth MS analyses, which followed by bioinformatics evaluation, will abundant BCL-2 family member transcripts in hESCs will point toward pro- implicate molecules of integrin-signaling pathways involved in the regulation

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of stem cell maintenance and differentiation. recently found that cells that meet the pluripotency criteria can emerge from the ES cells cultured in the complete ES medium supplemented with Poster Board Number: 3543 LIF and 50nM atRA for more than a week without passaging. ES cells first FEMALE SEX BIAS IN HUMAN EMBRYONIC lose their pluripotency morphology and become completely flat, subse- quently followed by the emergence of colonies, tentatively called “second- STEM CELL LINES ary colonies,” which resemble pluripotent ES cell colonies. We have found Ben Yosef, Dalit, Amit, Ami, Malcov, Mira, Frumkin, Tsvia, Mei Raz, that the secondary colonies have all the features of pluripotent ES cells such as colony morphology, expression of pluripotency markers, and ability to Nave, Azem, Foad, Altarescu, Gheona, Renbaum, Pinhas, Eldar- differentiate into multiple lineages. Although the maintenance of pluripotent Geva, Talia, Varshaver, Irit, Epsztejn-Litman, Silvina, Levy Lahad, state of ES cells after the retinoids treatment has also been reported previ- Ephrat, Eiges, Rachel ously, the secondary colonies are distinct from these states in terms of the Racine IVF Unit, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, method of retinoids treatment and dynamics of colony formation. To further Tel Aviv, Israel, Medical Genetics Institute, Shaare Zedek Medical Center, characterize these cells, we have compared their gene expression profiles by Jerusalem, Israel, IVF Laboratory, Shaare Zedek Medical Center, Jerusalem, DNA microarray analysis and found that these distinct states of cells repre- Israel, Stem Cell Research Laboratory, Medical Genetics Institute, Shaare sent slight shifts toward different cell lineages (e.g., mesoderm or endoderm) Zedek Medical Center, Jerusalem, Israel while maintaining the pluripotent states. The data suggest that expression of pluripotency markers alone could not indicate the pluripotent state of Introduction: Human embryonic stem cells (HESCs) are derived from spare ES cells. This research was supported entirely by the Intramural Research human blastocyst embryos. Although the factors limiting their rather inef- Program of the NIH, National Institute on Aging. ficient derivation are not fully understood, embryo gender was suggested to be one of them. The aim of this study was to analyze the sex ratio in Poster Board Number: 3547 our disease-bearing-HESC lines in an attempt to identify the origins of sex discrepancy. Methods: We describe a large collection of disease-bearing- LAMININ STIMULATES PROLIFERATION HESC lines, which we established in the Tel Aviv and Shaare Zedek Medical OF MOUSE EMBRYONIC STEM CELLS Centers following preimplantation genetic diagnosis (PGD). The HESC lines were characterized for gender type. The gender of in vitro-derived VIA REDUCTION OF GAP JUNCTIONAL embryos was determined by PGD. A one-sample binomial test was applied INTERCELLULAR COMMUNICATION THROUGH to determine whether the proportion of females significantly deviates from CONNEXIN43 INACTIVATION the 50% expected. Results: Our collection includes 42 different HESC lines. Interestingly, we found that 76% of them were females, a value statistically Suh, Han Na, Lee, Yu Jin, Kim, Mi Ok, Ryu, Jung Min, Ha, Jeong different from the expected 50% (P<0.01), and even more pronounced Won, Han, Ho Jae when taking into account the fact that more male ICMs were plated for Dept of Veterinary Physiology, Biotherapy Human Resources Center (BK), HESC derivation (53% of all blastocysts). To further verify this finding, we College of Veterinary Medicine, Chonnam National University, Gwangju, performed a meta-analysis and integrated our data with the results of all Korea, Republic of 148 PGD-HESC lines on gender distribution published to date worldwide. Similarly, 67% of them were females (P=0.0001). In order to determine Gap junction within extracellular matrix (ECM)-defined boundaries ensures the origin of this gender bias, we took advantage of the PGD for X-linked synchronous cellular activity between cells destined to become functional diseases which enables embryo gender determination prior to their manipu- mediator which regulates cell behaviors. However, the role of ECM on lation for stem cell derivation. No bias towards females was found in day connexin (Cx) function in mouse embryonic stem cells (mESCs) was not 3 embryos (45.9% females), as well as in their development towards the elucidated. Therefore, we examined the role of laminin (Ln) on control of blastocyst stage (P>0.05). Moreover, only 48.0% (96/200) of PGD newborn Cx43 function and its related signal pathways in mESCs. Ln, fibronectin, and babies were females, which is not statistically different from the percentage collagen I induced Cx43 functional changes such as increase of Cx43 phos- of females born following IVF-ICSI (51.3%; P>0.05). Conclusions: The clear- phorylation and decrease of Lucifer yellow diffusion through gap junctional cut tendency for female preponderance in HESC lines could be attributed intercellular communication (GJIC). Next, we examined mediator signal to suboptimal culture conditions rather than from a true gender imbalance molecules between Ln and Cx43. Ln bound integrin β1 and then stimulates in embryos used for derivation of HESC lines. We propose a mechanism in phosphorylation of Caveloin-1 (Cav-1), FAK/Src, and activation of PKC which aberrant X chromosome inactivation and/or overexpression of critical subsequently. Moreover, Ln increased VEGF synthesis and then secreted metabolic X-linked genes might explain this sex dimorphism. VEGF increased PKC activation through VEGFR. Ln induced activation of RhoA, which was blocked by bisindolylmaleimide I (PKC inhibitor; 10-6 M). Poster Board Number: 3545 In sequence, Ln induced Cx43 functional changes were reversed by Y27632 (ROCK inhibitor; 10-6 M). Moreover, Ln decreased F-actin or Drebrin CHARACTERIZATION OF A NOVEL expression levels in plasma membrane followed by decreased colocalization PLURIPOTENT STATE OF MOUSE ES CELLS of Cx43 with F-actin or Drebrin and Drebrin siRNA significantly decreased EMERGING AFTER RETINOIDS TREATMENT Lucifer yellow diffusion. We also found that Ln decreased Cx43 protein level in plasma membrane, which was abolished by monodancylcadaverine (clath- Sharova, Lioudmila, Piao, Yulan, Sharov, Alexei, Ko, Minoru S.H. rin-dependent endocytosis inhibitor; 5 × 10-6 M), chloroquine (lysosomal Laboratory of Genetics, National Institute on Aging, NIH, Baltimore, MD, protease inhibitor; 10-4 M), or lactacystin (proteasome inhibitor; 10-5 M). In USA addition, Ln increased G1/S transition of cell cycle (cyclin E, cyclin D1, p-Rb) and proliferation index in FACS analysis through decrease of Cx43 protein Pluripotent mouse ES cells are characterized by morphology (dome-shaped level in plasma membrane, which was confirmed by 18α-GA. In conclusion, colonies with distinct edge), expression of markers such as alkaline phos- Ln stimulates mESC proliferation through reduction of GJIC via RhoA-medi- phatase (ALP), Pou5f1 (Oct3/4), and Nanog, and ability to differentiate into ated Cx43 phosphorylation and F-actin-mediated Cx43 endocytosis. derivatives of all germ layers in vitro (EB formation and differentiation) or in vivo (teratoma, chimeras with germ line transmission). Maintenance of pluripotent state requires a special culture condition, especially the presence of LIF and replating every 2-3 days. It has been shown that adding retinoids to the culture condition induces the cell differentiation. However, we have

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Poster Board Number: 3549 differentiation of the ICM into epiblast during normal development. GENE REGULATION OF MOUSE EMBRYONIC Poster Board Number: 3553 STEM CELLS BY LOW NITRIC OXIDE METABOLIC CHARACTERIZATION OF MOUSE Tapia Limonchi, Rafael A., Cahuana, Gladys M., Hitos, Ana B., EMBRYONIC STEM CELLS AND EPIBLAST Diaz, Irene, Martin, Franz, Soria, Bernat, Bedoya, Francisco J., DERIVED STEM CELLS Tejedo, Juan R. Zhou, Wenyu, Choi, Michael, Margineantu, Daciana, Nelson, Angel, Cell Therapy and Regenerative Medicine, Centro Andaluz de Biología Hesson, Jennifer, Cavanaugh, Christopher, Hockenbery, David, Molecular y Medicina Regenerativa CABIMER, Sevilla, Spain Ware, Carol, Ruohola-Baker, Hannele Embryonic stem cells have the capability of self renewal and remain pluri- Biology, University of Washington, Seattle, WA, USA, Biochemistry, potent when are cultured under appropriate conditions. The messenger gas, University of Washington, Seattle, WA, USA, Fred Hutchinson Cancer Nitric Oxide (NO) play a relevant role in the maintenance of these pivotal Research Center, Seattle, WA, USA, Comparative Medicine, University of features of ESCs but its impact on the transcriptional machinery and other Washington, Seattle, WA, USA biological functions remain to be explored. Here, we show that when ESCs were cultured with 2 µM of NO donor DETA-NO during five days induced Mouse ES cells and EpiSCs are two distinct pluripotent states representing enhanced epigenetic marks in histones such as: H3K9me3, H3K4me3, cells of the pre-implantation embryo and later post-implantation epiblast. H3K27me3, further H3 and H4 acetylation in accordance with the levels They are thought to maintain their pluripotent state through different signal- of histone deacetylases (HDAC1, 2 and 4, Sirt1). In this context, global ing pathways, which, however, are still largely unexplored. Here we charac- transcriptome analysis reveals that NO modulates signaling pathways such terize mouse ESCs and EpiSCs through metabolic and bioenergetic analysis. as: Apoptosis, cell death, cell cycle or mitochondrial dysfunction. Meanwhile, By characterizing their mitochondrial DNA copy number, cellular ATP levels, significant results for functional ontology of genes regulated by NO in cells oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), cultured in absence of LIF with a fold change > 1.2 is mainly represented by we show that EpiSCs are highly glycolytic, while ESCs exhibit mitochondrial functions such as: Embryonal and organismal development 25%, Cellular respiration in addition to a glycolytic profile. Interestingly, human ESCs are Growth and Development 18%, Metabolism 17%, Gene expression 13%, also highly glycolytic, alike mouse EpiSCs. Through a screen using several in- Cell Death 13% and regulation of cell cycle 8%. These results shed light on hibitors and regulators of the metabolic pathways, we found out that EpiSC the effect of low doses of NO in the regulation of cell survival, promoting a are unable to use mitochondrial respiration to sustain energy demands. To mitochondria-mediated anti-apoptotic function, effect on cell division dur- understand the acquisition of highly glycolytic state in EpiSCs, we identify ing cell cycle, control of gene expression through remodeling of chromatin hypoxia inducible factor 1α (HIF1α) as a key regulator for the metabolic structure, and regulation of cell differentiation through the regulation of switch from ESCs to EpiSCs. We first confirmed that HIF1α target genes, pluripotency and differentiation genes. such as 6-phosphofructo-2-kinase and liver glycogen phosphorylase that are involved in glycolysis, are significantly upregulated in EpiSCs compared Poster Board Number: 3551 to ESCs. We also show that over expression of HIF1α is sufficient to drive ESCs to EpiSC-like cells, as evident in changes of the morphology in culture. WNT PROTEINS ARE ESSENTIAL FOR SELF Cellular gene expression, OCR and ECAR measurements are used to confirm RENEWAL OF MOUSE EMBRYONIC STEM the morphological changes induced by HIF1α. We further show that not CELLS BY PREVENTING DIFFERENTIATION TO only in mouse, but also in human cells, HIF1α over expression is sufficient to induce metabolic changes toward a highly glycolytic state, as observed in EPIBLAST STEM CELLS the OCR and ECAR measurements. Taken together, our studies show for the ten Berge, Derk, Kurek, Dorota, Blauwkamp, Tim, Koole, Wouter, first time the different metabolic and bioenergetic landscapes in two mouse Eroglu, Elif, Nusse, Roel pluripotent stages, and suggest that HIF1α is a critical regulator of metabolic and pluripotent states of stem cells in mice as well as in human. Dept. of Cell Biology, Erasmus MC, Rotterdam, Netherlands, Dept. of Developmental Biology, Stanford University Medical Center, Stanford, CA, Poster Board Number: 3555 USA MAINTENANCE OF GENOMIC INTEGRITY IN Pluripotent stem cells exist in naïve and primed states, epitomized by mouse embryonic stem cells (ESCs) and the developmentally more advanced MOUSE EMBRYONIC STEM CELLS BY POST epiblast stem cells (EpiSCs). The genome of ESCs has an unusual open con- TRANSLATIONAL REGULATION OF PROTEINS formation with a minimum of repressive epigenetic marks, while EpiSCs have INVOLVED IN DNA REPAIR undergone de novo DNA methylation, random X-chromosome inactivation, and other epigenetic changes that prime the genome for differentiation. Tichy, Elisia D., Stambrook, Peter J. An important question in the biology of pluripotent stem cells is the role of Molecular Genetics, University of Cincinnati, Cincinnati, OH, USA external signals in their self-renewal and differentiation. We show here that para- and autocrine Wnt signals are essential self-renewal factors for ESCs, Somatic cells have a frequency of mutation at loci that are heterozygous and that they are required to inhibit the differentiation of ESCs into EpiSCs. approaching 10-4. Such an elevated mutation frequency in germ cells or Furthermore, we demonstrate that Wnt signals together with the cytokine embryonic stem (ES) cells would be unsustainable for a species due to a high LIF support ESC self-renewal in the absence of any undefined factors, and level of fetal lethality and/or congenital deformities and syndromes. There support the derivation of new ESC lines from non-permissive mouse strains. are multiple strategies for preserving genomic strategies. We have previ- We show that these non-permissive ESCs differ from permissive ESCs by ously reported that, in contrast to somatic cells that utilize error-prone non their increased dependence on Wnt signals. Consistent with these find- homologous end joining (NHEJ) to repair double strand DNA breaks, ES cells ings, we show that the Wnt pathway is active in the inner cell mass (ICM) preferentially utilize high fidelity homologous recombination (HR) to repair of pre-implantation embryos, the source of ESCs, but becomes inactive such lesions. The Rad51 protein is intimately involved in HR repair. The upon implantation and subsequent differentiation of the ICM into epiblast, amount of Rad51 protein in mouse ES cells is about 20-fold higher than in the source of EpiSCs. This suggests that the role of Wnt in controlling the isogenic mouse embryo fibroblasts (MEFs). This elevated level cannot be at- ESC-to-EpiSC transition has a physiological counterpart in controlling the tributed to more efficient transcription since the Rad51 mRNA is only 2-fold

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Thursday Poster Abstracts

higher than in MEFs. In contrast, PCNA, a protein required for replication by tetracycline-dependent transient disruption of the Bloom’s syndrome but not for HR repair, is only 3-fold higher in ES cells and its mRNA is about gene (Blm). Deficiency of Blm expression induces recombination between 1.3-fold elevated. The very high elevation of Rad51 in ES cells cannot be ac- homologous chromosomes at the G2 phase of cell cycle that gives rise to counted for by differences in stability, since its half-life is less than two hours homozygous daughter cells after cell division. Since homozygous daugh- and is similar for both cell types. In contrast to Rad51, the half-life of PCNA ter cells constitute a small fraction of the entire cell population, we have is greater than 5 hours. The stability of the two mRNAs is similar, based on devised a drug selection cassette that enriches for homozygous daughter mRNA levels after actinomycin treatment. The most likely hypothesis to cells. Clonal isolation of homozygous cells is also possible by screening the explain the regulation of Rad51 protein and its very high abundance in the drug-resistant clones for SNP homozygosity at telomeric regions distal to ES cells is that there is greater efficacy of Rad51 mRNA translation on a per the vector insertion sites. We have so far generated a collection of ~2,000 molecule basis in the ES cell population. heterozygous and ~200 homozygous mutant clones. Two approaches were used to screen for novel factors involved in differentiation of ES cells. In the Poster Board Number: 3557 first approach, we induced differentiation of clonally isolated homozygous ELUCIDATING FUNCTIONAL PLURIPOTENT mutant ES cells by withdrawing LIF and assessed their resistance to differen- tiation. In the second approach, we pooled heterozygous ES cell clones, and STATES AMONG DIVERSE MOUSE STEM CELL then generated mixtures of homozygous and heterozygous mutant cells by TYPES the above-mentioned bi-allelic mutagenesis method. These mixtures of cells were subjected to differentiation by withdrawing LIF for a certain period of Vanderweide, Stephanie, Forster, Andrew, Ralston, Amy time before transferring to a normal ES cell culture condition. Colonies of University of California, Santa Cruz, Santa Cruz, CA, USA undifferentiated morphology appeared among a vast majority of differenti- ated cells. These colonies were expanded, mutated genes identified, and The existence of several different kinds of pluripotent stem cells, including differentiation-resistance was reassessed. To date, we have identified several embryonic stem cells, epiblast stem cells, embryonal carcinoma cells, and homozygous mutants that are resistant to differentiation cues and are now induced pluripotent stem cells, suggests that diverse routes can lead to pluri- further expanding our screen. Our screening procedure does not involve any potency. Although all of these cell types can differentiate to derivatives of a priori assumption of gene function, and will be useful in unraveling novel all three germ layers or self renew in culture, they all have different origins, regulatory mechanisms associated with the pluripotent state. morphologies, and growth factor requirements. Comparative studies can shed light on the diversity of mechanisms by which stem cells establish and Poster Board Number: 3563 maintain pluripotency. We have systematically compared pluripotency in two of these cell lines using a functional assay to examine their developmental COMPARISON OF GENOME-WIDE SINGLE potential in the context of the lineage specification in the mouse. Specifical- NUCLEOTIDE POLYMORPHISMS (SNP) IN ly, mouse ES cells do not normally form trophoblast, but will do so following overexpression of the transcription factor Cdx2. Surprisingly, teratoma-de- MOUSE STEM CELL-LIKE, COLONY-FORMING rived EC cells were also capable of giving rise to trophoblast upon overex- FIBROBLASTS IN A SPECIFIC CELLULAR pression of Cdx2. This observation supports the proposal that these cell lines ENVIRONMENT are functionally equivalent, in spite of their markedly different origins. We next examined the expression of 18 genes associated with pluripotency in Yum, Kyung Eun, Lim, Jeong Mook, Kim, Boyon, Park, Ae Kyung, these and other pluripotent stem cell types, including epiblast and induced Park, In-hyun, Park, Woong Yang, Ahn, Ji Yeon, Park, Jong Heum pluripotent stem cells. This analysis suggests that ES and EC cells are more College of Agriculture and Life Science, Seoul National University, similar to each other than to many of these cell types, consistent with the Seoul, Korea, Republic of, WCU Biomodulation Program, Seoul National observed similarities in the functional assay. These combined approaches po- University, Seoul, Korea, Republic of, College of Medicine, Seoul National tentially provide a means for detecting previously unrecognized differences University, Seoul, Korea, Republic of, Dept of Genetics, Yale University in the quality of pluripotency, and for predicting stem cell behavior prior to School of Medicine, New Haven, CT, USA subsequent therapeutically-oriented manipulation. We established several lines of stem cells that were derived from normally Poster Board Number: 3561 fertilized embryos (nfESC) or parthenogenetically activated oocytes (pESC). In addition, we have established six lines of stem cell-like, colony-forming USING CONDITIONAL KNOCKOUT OF THE fibroblasts (CFF), which were derived by exposing to a specific cell niche BLOOM’S SYNDROME GENE IN A RECESSIVE without undertaking genetic manipulation. In this study, we compared GENETIC SCREENING FOR NOVEL FACTORS mouse (B6D2F1) SNP genotype of those cell lines (nfESC, pESC and CFF) using a genome-wide SNP microarray that analyzed 623,000 SNP genomic INVOLVED IN DIFFERENTIATION OF MOUSE loci. Somatic fibroblasts from C57Bl/6, DBA/2 and B6D2F1, and induced EMBRYONIC STEM CELLS pluripotent stem (iPS) cells were employed as control cell lines. The results showed that nfESC and iPS cell lines had heterozygotic chromosome recom- Yoshida, Junko, Kokubu, Chikara, Takeda, Junji, Horie, Kyoji bination, which was the same with that of B6D2F1 fibroblasts. On the other Dept of Social & Environmental Medicine, Osaka University Graduate hand, C57Bl/6 and DBA/2 fibroblasts showed homozygotic recombinations. School of Medicine, Suita, Japan Six lines of CFF showed both homozygotic and heterozygotic recombina- Recent studies demonstrated that pluripotency of mouse embryonic stem tion, which was similar to pESC lines. In conclusion, pattern of chromosome (ES) cells involves a fine balance between two opposing factors: one recombination was different between CFFs being derived without genetic involved in promoting pluripotency and the other in inducing differentia- manipulation and genetically manipulated iPS cells. This recombination tion. Elucidation of novel factors affecting this equilibrium will help better pattern observed in CFF was similar to those of pESC, which might suggest understand the pluripotent states of ES cells as well as the induction of iPS parthenogenesis-like cell transformation. cells from somatic cells. Here we present our approach of recessive genetic screening for novel factors involved in differentiation of mouse ES cells using our newly developed bi-allelic mutagenesis method. The bi-allelic muta- genesis method involves the generation of heterozygous mutants by gene trap vector insertion and subsequent derivation of homozygous mutants

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Thursday Poster Abstracts

Poster Board Number: 3565 Poster Board Number: 3567 INVOLVEMENT OF CONNEXIN43 PROTEIN OXYTOCIN STIMULATED CONNEXIN43 EXPRESSION LEVEL IN PROSTAGLANDIN EXPRESSION THROUGH NF-κB/CREB/CBP E2-MEDIATED MAINTENANCE OF MOUSE COMPLEX VIA LIPID RAFT-INDEPENDENT EMBRYONIC STEM CELL UNDIFFETENTIATED OXYTOCIN RECEPTOR-MEDIATED CAMP/PKA STATE IN MOUSE EMBRYONIC STEM CELLS Yun, Seung Pil, Lee, Yu Jin, Park, Jae Hong, Suh, Han Na, Kim, Mi Yun, Seung Pil, Lee, Yu Jin, Suh, Han Na, Park, Su Shin, Jeon, Ji Ok, Ryu, Jung Min, Jang, Min Woo, Park, Su Shin, Seo, Bit Na, Hoon, Han, Ho Jae Jeon, Ji Hoon, Han, Ho Jae College of Veterinary Medicine, Dept of Veterinary Physiology, Biotherapy Dept of Veterinary Physiology, Biotherapy Human Resources Center (BK), Human Resources Center (BK21), College of Veterinary Medicine, College of Veterinary Medicine, Chonnam National University, Gwangju, Chonnam National University, Gwangju, Korea, Republic of, College of Korea, Republic of Veterinary Medicine, Dept of Veterinary Physiology, Biotherapy Human Resources Center (BK21), Gwangju, Korea, Republic of Although many previous reports have examined the function of prosta- glandin E2 (PGE2) on gap junctions and non-differentiated stem cells, its Although oxytocin has an essential role in the regulation of cell junctions in effects on the reciprocal action of connexin43 (Cx43, the major gap junction various cell types such as epithelial cells and cardiomyocytes, its effect on protein) and pluripotency capacity in mouse embryonic stem cells (ESCs) connexin43 (Cx43, the major gap junction protein) regulation is not fully is unclear. Therefore, we investigated the role of PGE2 on Cx43 protein understood in stem cells. Therefore, we invetigated the effect of oxytocin expression levels and its role with regard to maintenance of pluripotency in on connexin43 protein expression levels and its related signaling cascades ESCs. PGE2 (50 μM) increased Cx43 mRNA and protein expression levels, in mouse embryonic stem cells (mESCs). Oxytocin (10-7 M) increased Cx43 and butaprost (10 μM), an E-type prostaglandin receptor 2 (EP2) selective protein expression levels which were inhibited by the oxytocin receptor agonist, also increased Cx43 protein expression levels, which were inhibited inhibitor atosiban (10-5 M). In experiments to examine whether the effect by the EP2 receptor selective antagonist AH 6809 (1 μM). PGE2 activated of oxytocin depends on lipid raft, caveolin-1, caveolin-2, and flotillin-2 were 3’-5’-cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) and detected in lipid raft fraction, but not oxytocin receptor. In addition, colocal- increased phosphorylation of phosphoinositide-3 kinase (PI3K)/Akt. These ization of oxytocin receptor and caveolin-1, 2 was not detected by immuno- PGE2-induced increases were inhibited by PI3K inhibitor LY 294002 (1 precipitation. Moreover, lipid raft disruptor methyl-β-cyclodextrin (MβCD, μM), PI3K inhibitor wortmannin (1 μM), Akt inhibitor (10 μM), adenylyl 10-4 M) did not attenuate oxytocin-induced Cx43 protein expression level. cyclase inhibitor SQ 22536 (10 μM), and PKA inhibitor PKI (0.4 μM). To In experiment to examine its related signaling, oxytocin activated 3’-5’-cy- examine whether the activation of PI3K/Akt was independent of cAMP/ clic adenosine monophosphate/protein kinase A (cAMP/PKA) which were PKA activation, mouse ESCs were incubated with the adenylate cyclase inhibited by adenylyl cyclase inhibitor SQ 22536 (10-5 M) and PKA inhibitor activators forskolin (10 μM) and 8-bromo-cAMP (2 mM). These treatments PKI (4 x 10-5 M). Oxytocin also increased nuclear factor kappa-light-chain- increased Cx43 protein expression levels, but not PI3K/Akt phosphoryla- enhancer of activated B cells (NF-κB) phosphorylation which was inhibited tion. In addition, PGE2 inactivated glycogen synthase kinase 3β (GSK-3β) by PKI. Oxytocin also increased cAMP response element-binding (CREB)/ and promoted nuclear localization and increased accumulation of β-catenin CREB-binding protein (CBP) expression in nucleus and induced formation and active-β-catenin. Furthermore, a chromatin immunoprecipitation (ChIP) of complex with NF-κB, CREB, and CBP as shown by immunoprecipita- assay demonstrated the association of β-catenin with the Cx43 promoter, tion, which was blocked by NF-κB inhibitors SN 50 (2 x 10-5 M) and bay suggesting that β-catenin could regulate Cx43 protein expression at the 11-7082 (10-5 M). Disrupt of this complex by SN 50 and bay 11-7082 also gene transcription level. Downregulation of β-catenin by small interfering decreased oxytocin-induced Cx43 protein expression level. In conclusion, (si)RNA decreased the Cx43 protein expression level. Additionally, down- oxytocin stimulates Cx43 expression through NF-κB/CREB/CBP complex via regulation of the Cx43 protein expression level by the gap junction inhibitor lipid raft-independent oxytocin receptor-mediated cAMP/PKA in mESCs. 18α-glysyrrhetinic acid (10μg/ml) decreased PGE2- maintained levels of pluripotency markers of ESCs, including NANOG, Oct4, Sox2, SSEA1, and Poster Board Number: 3569 FoxD3. In conclusion, these experimental data demonstrate that PGE2 partially stimulates Cx43 expression via GSK-3β/β-catenin via EP2 receptor- CHARACTERIZATION OF THE CNOT COMPLEX dependent cAMP/PKA and PI3K/Akt in mouse ESCs thereby contributing to the maintenance of their pluripotent state. IN MOUSE EMBRYONIC STEM CELL SELF- RENEWAL Zheng, Xiaofeng, Lackford, Brad, Freudenberg, Johannes, Jothi, Raja, Hu, Guang National institute of Environmental Health Sciences, Research Triangle Park, NC, USA Embryonic stem cells (ESCs) are pluripotent cells that can be maintained as stem cells via the process of self-renewal or be induced to differentiate into all cell types in the adult body. They can be used as in vitro models for the study of mammalian embryonic development. They also hold the promise to generate various types of cells for disease modeling, drug discovery, regenerative medicine, toxicity testing, and environmental health studies. To realize these potentials, it is important to understand the molecular basis of the two defining features of ESCs: self-renewal and pluripotency. We and others have previously discovered that components of the Cnot complex play important roles in mouse ESC self-renewal. The objective of this study

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Thursday Poster Abstracts

is to investigate the function of the Cnot complex in mouse ESCs. The Cnot and supports self-renewal of mESCs by two independent mechanisms, one complex is evolutionarily conserved and improtant for the regulation of tran- of which being independent of CDK2 activation. In a last step, we explored scription and mRNA deadenylation, and different members of the complex whether LIF signalling regulates cyclin E:CDK2 kinase. We observed that are known to be associated with different functions. Cnot1, 2, and 3 can stimulation of this pathway results in a transient accumulation of active act as transcription regulators, while Cnot6, Cnot6L, Cnot7, and Cnot8 are cyclin E:CDK2 complexes. It also accelerates the G1-to-S phase transition. deadenylases and play major roles in mRNA deadenylation. We found that Finally, we propose a model in which LIF signalling stimulates the G1-to-S only gene silencing of Cnot1, Cnot2 and Cnot3 led to ESC differentiation. phase transition to shield mESCs from undesired differentiation signals and Therefore, it is likely that the transcription regulatory function, rather than help them to self-renew in the pluripotent state. the deadenylase activity of the Cnot complex, is important for self-renewal. Consistent with this, we found that Cnot1, 2, 3 form a complex and are Poster Board Number: 3573 present in the nucleus of ESCs. They are highly expressed in pluripotent cell SAF-A HAS A ROLE IN TRANSCRIPTIONAL types and down-regulated during ESC differentiation. Importantly, silencing of Cnot1, 2, or 3 do not affect the known self-renew pathways, such as the REGULATION OF THE PLURIPOTENCY GENE LIF, BMP, and MAPK pathways. Gene expression analysis further suggested OCT4 that the genes regulated by Cnot1, 2, 3 are different from those regulated by other known self-renewal transcription factors such as Oct4 and Nanog. Simonsson, Stina Thus, we have identified a novel self-renewal complex Cnot1-2-3 in mouse Clinical Chemistry and Transfusion Medicine, Biomedicine, Goteborg, ESC, and they may function as a unique transcription module to regulate Sweden gene expression in mouse ESCs. To reprogram patient’s own cells into pluripotent stem cells offer possibilities Poster Board Number: 3571 for mapping mechanisms of different diseases and screening for personal- ized therapies and drugs. Oct4 activation has proven essential for success- CYCLIN E SUSTAINS SELF-RENEWAL OF ful reprogramming and pluripotency of embryonic stem (ES) cells, albeit EMBRYONIC STEM CELLS IN THE PLURIPOTENT molecular details of Oct4 activation are not completely understood. Here we report that endogenous SAF-A is involved in regulation of Oct4 expres- STATE sion, binds the Oct4 proximal promoter in ES cells and dissociates from the Coronado, Diana promoter upon early differentiation induced by LIF withdrawal. Depletion of SAF-A decreases Oct4 expression even in the presence of LIF, and human Embryonic Stem Cell and Cortical Development, INSERM France, Bron, SAF-A rescues the knock-down phenotype. France Embryonic stem cells (ESCs) exhibit a very unusual cell cycle structure, char- Poster Board Number: 3575 acterized by a short G1 phase and a high proportion of cells in S-phase. In AN INTEGRATED OCT4 NETWORK FOR the case of mouse ESCs (mESCs), this is associated with a unique mechanism of cell cycle regulation, underpinned by lack of cyclin D and retinoblastoma PLURIPOTENCY OF EMBRYONIC STEM CELLS control, lack of p53-dependent checkpoint in G1, as well as non-oscillatory Wang, Jianlong, Ding, Junjun, Francesco, Faiola, Xu, Huilei, and robust activity of cyclin E and cyclin E:CDK2-associated kinase activity Ma’ayan, Avi throughout the cell cycle. Here, we explored whether and how the regula- tion of the cell cycle actively sustains self-renewal of mESCs. So as to visual- Developmental and Regenerative Biology, Mount Sinai School of Medicine, ize cell cycle progression in live cells, we engineered a mESC line expressing New York, NY, USA, Pharmacology and Systems Therapeutics, Mount Sinai the Fluorescent ubiquitination-based cell cycle indicator (Fucci-mESC). This School of Medicine, New York, NY, USA system enables to distinguish live cells in early G1 (no fluorescence), late G1 Oct4 is a well-known transcription factor that plays fundamental roles in (red fluorescence) from those in S (low green fluorescence) and G2 phases stem cell pluripotency and somatic cell reprogramming. Extensive studies (high green fluorescence). We were interested in evaluating if the sensitivity have documented downstream target genes of Oct4, however, only limited to differentiation inducers is cell cycle-phase dependent. Fucci-mESCs were information is available on the Oct4 protein complexes and their intrinsic FACS-sorted accordingly to their color and treated with retinoic acid, a well- protein-protein interactions that form the transcriptional machinery regulat- known powerful inducer of differentiation. Self-renewal was subsequently ing expression of the Oct4 target genes. We have employed an improved tested in a colony-forming assay associated with transient LIF deprivation. affinity purification approach combined with mass spectrometry to purify This aims to evaluate the capacity of mESCs to resist transient loss of self- the Oct4 protein complexes in mouse embryonic stem cells (ESCs), which renewal-inducing signals, therefore to remain pluripotent in sub-optimal has led us to the discovery of novel self renewal regulators and signaling culture conditions. We showed that mESCs are more likely to differentiate pathways important for stem cell pluripotency. We have identified 198 if they are in G1 phase, as compared to the other phases of the cell cycle. Oct4-interacting proteins constituting numerous transcription factors and Thus, G1 is a phase of increased susceptibility to differentiation inducers. We multiple chromatin modifying complexes with documented as well as newly next examined the role of cyclin E and cyclin E:CDK2 kinase in the control proved functional significance in stem cell maintenance. Using RNAi-based of self-renewal. Treatment of mESCs with roscovitin, an inhibitor of CDK2, functional assay we have confirmed several novel self renewal regulators resulted in extensive differentiation. The effect was more pronounced if that are important for ESC maintenance and lineage specification. Combined mESCs were in G1 phase at the time of treatment, which indicates that with available Chip-chip and Chip-seq datasets, we have constructed an CDK2 kinase activity prior to S-phase entry is critical for the maintenance integrated Oct4 network encompassing the protein-protein and protein- of pluripotency. Knockdown of cyclin E expression with interfering lentiviral DNA interactions of numerous pluripotency and reprogramming factors. Our vectors resulted in lowering the proliferation rate, lengthening the G1 phase, studies offer novel insights into the mechanisms controlling ESC self renewal and dramatically accelerating the kinetics of differentiation induced with and pluripotency, and provide a great resource for exploring alternative and/ embryoid bodies (EB). Similarly, mESCs harboring a double inactivation of or improved factor-based reprogramming strategies. cyclin E1 and E2 (E1-/-E2-/- null), display reduced self-renewal abilities in a colony-forming assay. The phenotype of E1-/-E2-/- null mESCs could be rescued by overexpressing human cyclin E. Surprisingly, overexpression of a kinase deficient mutant of human cyclin E also rescued the wild-type phe- notype. Together, these results suggest that cyclin E opposes differentiation

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Thursday Poster Abstracts

Poster Board Number: 3577 Poster Board Number: 3579 A PROTEOMIC APPROACH FOR HUNTING INSULIN IN EMBRYO CULTURE MEDIUM ELUSIVE STEMNESS PROTEINS FROM THE CLEAVAGE STAGE; A SUCCESSFUL Arauzo-Bravo, Marcos J., Tapia, Natalia, Graumann, Johannes, STRATEGY FOR IMPROVING THE Kim, Jeong Beom, Ko, Kinarm, Esch, Daniel, Cox, Jurgen, Mann, PLURIPOTENTIAL OF EMBRYOS Matthias, Scholer, Hans R. Campbell, Jared M., Vassiliev, Ivan, Nottle, Mark B., Lane, Michelle Dept Cell and Developmental Biology, Max Planck Institute for Molecular Research Centre for Reproductive Health, University of Adelaide, Adelaide, Biomedicine, Muenster, Germany, Proteomics Facility, Weill Cornell Australia Medical College, Doha, Qatar, School of Nano-Bioscience and Chemical Engineering, Hans Schoeler Stem Cell Research Center (HSSCRC), Ulsan, The efficiency with which hESCs can be derived is limited by embryo qual- Korea, Republic of, Dept of Neuroscience, Konkuk University, Konkuk, ity - low quality embryos give rise to hESCs at lower efficiencies. Human Korea, Republic of, Department of Proteomics and Signal Transduction, embryos donated for this purpose have often been cryopreserved during Max Planck Institute for Biochemistry, Martinsried, Germany the cleavage (4-8cell) stage up to ten years previously in relatively simple media. As hESC are derived from blastocyst stage embryos a window of Identification of stemness proteins is a cornerstone in stem cell biology. opportunity exists as thawed embryos are cultured, during which treatments Several trials using transcriptomics measurements were made in the past could be applied to improve embryo quality. The present study investigated for searching stemness genes, though the results were controversial. We whether culture with insulin from the 8-cell stage increased the number of applied a proteomic approach to identify stemness proteins based on a ESC progenitor cells in expanded blastocysts and the generation of primary label free quantitative mass spec approach on mouse embryo fibroblasts ESC colonies using a mouse model. In addition, we investigated molecular (MEF), embryonic stem cells (ESC), germ stem cells (GSC) and neural stem mechanisms which may underlie insulin’s effect. In vivo fertilised C57Bl6 cells (NSC). Our results span a bridge between the previous transcriptomic zygotes were cultured in G1 medium for 48h to the 8-cell stage, then approaches, showing the significant relevance of the DNA repair, and the cultured individually in G2 medium with insulin (0, 0.17, 1.7 and 1700pM) helicase activity. The synteny analysis found potential stemness-hot spots for 68h in 5% O2, 6% CO2, 89% N2 at 37oC. ESC progenitor number in the chromosomes 4 and 17. The mechanisms allowing the pluripotent was determined by immunohistochemical staining for Oct-4 and Nanog. cells to differentiate into multiple cells lineages are not totally understood. ESC progenitor cells are a subpopulation of the ICM (inner cell mass) which It is known that such mechanisms are driven by some transcription factors stain positive of Oct-4 and Nanog, while the rest of the ICM stains positive such as Oct4 that plays a pivotal role governing the pluripotent networks. for Oct-4 only. To investigate the mechanisms underlying insulin’s effect Another related mechanism, the transcription factor-induced reprogram- we examined the PI3K pathway, which is activated by insulin signalling ming of somatic cells into different types including induced pluripotent stem and has been shown to inactivate GSK3 and p53; two second messengers cells, is almost unapprehended. It is assumed that the induced reprogram- which inhibit Nanog transcription. To do this we studied the effect on ESC ming mechanism is based on the stochastic crosstalk between genetic and progenitor number of inhibition of PI3K, GSK3 and p53 by LY294002, epigenetic networks but the mechanism remains elusive. Thus, many basic CT99021 and pifithrin-α respectively as well as activation of GSK3 and questions about pluripotency and reprogramming still remain unanswered. p53 by H89 and Nicotinamide respectively, in the presence and absence of To what extent is stemness a universal mechanism that is ruled by the same insulin. Blastocysts, control and insulin treated, were plated on MEFs at 68h genes in all the cellular types? Does a stemness molecular circuitry exist? then outgrown for 48h. Outgrowths were trypsinized and replated. Cell Which are the additional or the substitutive genes that allow improving the colonies were then stained for Oct4 and Nanog to confirm ESC identity. reprogramming process? Transcriptomics approaches were applied in the Continuous data was analysed using Univariate ANOVA with between past to answer such questions but the results were controversial since they treatment differences assessed by the LSD method and replicate fitted as had very small overlap with each other. Here, we try to answer the afore- a covariate. Categorical data was assessed via chi square analysis. Culture mentioned questions using a proteomics approach that complements the in 1.7 M insulin increased ESC progenitor number and mean percentage high sensitivity of the transcriptomics measurements with the higher func- (as a function of ICM cell number) in blastocysts (4.1±0.5 vs 6.6±0.6, tionality of the quantitative proteomics data. Gene expression is much easier P<0.01 and 25.8±3.4% vs 38.9±3.7%, P<0.05, N≥20 respectively). This to measure than protein abundance. Methods to measure gene expression effect was shown to be dependent on PI3K signalling as inhibition of PI3K such as qRT-PCR require less material than the western-blot technique for reversed the increase in ESC progenitor cell number (4.5±0.3 cells reduced measuring protein expression. Such a requirement is even more demanding to 2.2±0.3 cells, P<0.001, N≥34). The inhibition of GSK3 and p53, increased on the omics scale where the gene expression microarrays are widely used ESC progenitor numbers (3.2±0.3 vs 5.1±0.3, P<0.001, N≥46, and 4.1±0.7 but the MS quantification of protein abundance is still of restricted use. This vs 7.1±0.8, P=0.001, N≥21, respectively). While GSK3 and p53 activation makes us to forget that the proteins and not the genes are the effectors of reversed the insulin mediated increase in ESC progenitor numbers (6.0±0.5 the cellular machinery and structure building blocks. Some of the concerns reduced to 4.3±0.5, P<0.05, N≥26, and 7.2±0.5 reduced to 4.7±0.5, of the previous works were raised by the heterogeneity of the used stem P<0.05, N≥38, respectively). The efficiency with which primary ESC colonies cells. In difference to such approaches we substituted the heterogeneous were obtained was also increased in the insulin treated group (13 vs 6% hematopoietic stems cells (HSCs) by the homogeneous GSCs and a NSC P<0.05, N=156). In conclusion, culture of 8-cell embryos in the presence of line. Also, to contrast the results we used a negative control consisting of insulin results in a higher percentage of ESC progenitor cells in the ICM and terminally differentiated MEFs. Finally, as in the transcriptomics studies we a higher percentage of embryos giving rise to primary ESC colonies. This use as the stemness gold standard embryonic stem cells (ESCs). It is captivat- suggests that insulin can shift cell fate determination towards pluripotency. ing that though we did not find exactly the very same stemness markers as The increase is mediated via the activation of PI3K which inactivates GSK3 in the transcriptomics approaches our dataset highlights similar functions and p53. This pathway exists in human embryos, suggesting that insulin and hot spot chromosome locations. In some sense we can see our stemness could be used to improve the efficiency of hESC derivation. proteins as a bridge connecting the stemness genes found by the previ- ous transcriptomics studies. Thus, our approach sheds a new light over the stemness searching problem, where the proteomics measurements provide a synergetic view to the transcriptomics approaches.

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Poster Board Number: 3581 of stem cell is maintained by high-level of expression of stem cell-specific transcription factors including Oct-3/4, Sox2, Klf4 and c-Myc (OSKM). SUMOYLATION AND AKT-MEDIATED hESC possess an unusual “open” chromatin structure that allows read- PHOSPHORYLATION OF OCT4 ily access of the human genome by transcription factors and transcription machinery thus resulting in elevated plasticity. Active chromatin domains REGULATE THE PRIMITIVE PLURIPOTENT STATE are marked by histones H3 and H4 lysines acetylation and di,tri-methylated Campbell, Pearl A., Rudnicki, Michael A. H3K4, and transcription factors and chromatin remodeling complexes are key determinants of hESCs identity. We sought to investigate the role of Sprott Centre for Stem Cell Research, Ottawa Hospital Research Institute, bromodomain (BrDs)-containing proteins in governing hESCs self-renewal Ottawa, ON, Canada and pluripotency. BRD-containing proteins regulate chromatin dynam- Pluripotent stem cells are characterized by the presence of simultaneous ics through modulating acetylation-mediated protein-protein interactions epigenetic modifications of gene activation and repression at the promot- in control of gene transcription. We discovered a small-molecule chemi- ers of developmentally regulated loci. Sustained pluripotency requires the cal compound that inhibits acetyl-lysine binding activities of BrDs in gene presence of Oct4, Polycomb, and Trithorax Group proteins. However, the transcription, and is capable of effectively reducing the expression of OSKM mechanisms by which they are recruited and retained to maintain this genes and the number of undifferentiated colonies. Instead the expression poised state are poorly understood. Coordinate Post-translational modifi- of neuroectodermal lineage, neural crest and melanoblast markers increased cation (PTM)-dependent modulation of transcription factor (TF) function following compound treatment, without any overt effect on cell viability. via the formation and recruitment of distinct co-activator or co-repressor RNA sequencing (Illumina) of compound-treated ESCs revealed differentially complexes has emerged as an important regulatory mechanism to influence expressed genes of the PDGF, TGFβ and WNT signaling pathways. Further, locus specific gene expression and cell fate. Moreover, the fine-tuning of we have observed that this BrD inhibitor (BrDi) affects RNA polymerase TF function by PTMs provides a precise mechanism whereby transcriptional recruitment to promoters of stem cell genes, with concomitant reduction of outcomes may be influenced by the signal transduction cascades active active histone marks and displacement of BrD-containing proteins. Using within a specific cellular state. The pluripotent state in particular requires this novel chemical probe, we are dissecting the bromodomain regulated- finely balanced levels of Oct4 protein to maintain appropriate activation and mechanisms that control differentiation and stem cell self-renewal. repression of its transcriptional targets. This is mediated in part by ubiquit- ination, which marks Oct4 for degradation via the 26S Proteasome and SU- Poster Board Number: 3585 MOylation, which enhances its stability and binding to chromatin. Although POST-TRANSCRIPTIONAL REGULATION OF SUMOylation has generally been implicated in transcriptional repression, the impact of this modification upon the formation and recruitment of Oct4 PLURIPOTENT CELL FATE transcriptional complexes to specific loci has not been characterized. Recent Darr, Henia, Lee, Dung-Fang, Schaniel, Christoph, Lemischka, Ihor proteomic studies have demonstrated functionally non-equivalent roles for R. Oct4 N- and C-terminal transcriptional regulatory domains (TRDs). While the N-terminal TRD interacts with transcriptional repressors, the C-terminal Gene & Cell Medicine, Mount Sinai School of Medicine, NYC, NY, USA TRD has been shown to interact with transcriptional activators, chromatin Pluripotent cells hold great promise for use in cell replacement therapy. Prior remodelers, and genes implicated in the response to DNA damage. We to clinical application it is vital to understand mechanisms orchestrating pluri- therefore hypothesized that the regulatory switch responsible for converting potent cell fate for the optimization of cell derivation, maintenance and dif- Oct4 from a repressor to an activator might be PTM dependent and involve ferentiation. Currently, most research in the field focuses on characterization coordinate regulation of Oct4 N- and C-terminal TRDs. In this study we of the pluripotent cell’s transcriptome and epigenome. RNA is not simply investigated the PTM-dependent role of Oct4 dimerization in maintaining an intermediate enabling flow of information from the genome to a protein the actively repressed, pluripotent state through the promotion of cell cycle product, but is in fact a regulatory core on which multiple proteins operate progression and the retention of bivalent domains. Here we show that Oct4 from transcription, through translation, until its ultimate degradation. This SUMOylation and phosphorylation act in concert to restrain the function of is a regulatory level of pluripotency for which we have little understanding the C-terminal transcriptional activation domain of Oct4. While SUMOyla- and knowledge. Indeed, work from our lab shows that upon differentiation, tion of Oct4 promotes stable interactions between the N- and C-terminal up to 50% of nuclear genes analyzed show discordance between changes in transcriptional regulatory domains, Akt-mediated phosphorylation dynami- mRNA and protein levels. We focus on pathways involved in post transcrip- cally promotes interaction with Hmgb2 to maintain the repressed state. tional regulation of RNA fate with an emphasis on RNA binding proteins In the absence of these restraints cell cycle checkpoint function, loss of (RBPs) and proteins involved in translation. In order to study the involve- bivalent domains, and the ectopic expression of lineage restricted genes are ment of post-transcriptional regulation in maintenance of pluripotency we observed. These findings suggest that Oct4 may direct the spatio-temporal have defined candidate genes as RBPs highly expressed in undifferentiated formation of activating and repressing complexes in a PTM-dependent man- cells. shRNA (short hairpin RNA) encoding viruses were employed to down- ner to maintain pluripotency. regulate these candidate genes, and identify RBPs whose down-regulation Poster Board Number: 3583 impairs mouse ES cells self-renewal and pluripotency. Such RBPs are poten- tial post-transcriptional regulators of pluripotency. Among these identified EPIGENETIC REGULATION OF STEM CELL SELF- potential regulators are genes affecting mRNA stability, translation initiation, termination and rRNA processing. Current work focuses on uncovering RENEWAL AND DIFFERENTIATION the modus operandi of these identified post-transcriptional regulators. This Di Micco, Raffaella, Zhang, Guangtao, Ohlmeyer, Michael, Zhang, includes analysis of consequences of gene knockdown temporally, analyz- Weijia, Walsh, Martin, Zhou, Ming-Ming, Hernando, Eva ing their RNA targets and their effects on translation and protein levels. This in turn, will elucidate the role of post-transcriptional regulation in the Pathology, New York University School of Medicine, NY, NY, USA, maintenance of pluripotency, and how it drives alterations in pluripotent cell Structural and Chemical Biology, Mount Sinai School of Medicine, NY, NY, fate. To conclude, the identified post-transcriptional regulators of pluripo- USA, Medicine, Mount Sinai School of Medicine, NY, NY, USA tency can be incorporated with existing knowledge on the transcriptional The full potential of hESCs in research and clinical applications requires a and epigenetic regulators of pluripotent cells to form a more comprehensive detailed understanding of the genetic and epigenetic mechanisms that gov- understanding of the pluripotent state. ern the unique properties of hESCs. Studies show that the pluripotent state

248 www.isscr.org Thursday Poster Abstracts

Thursday Poster Abstracts

Poster Board Number: 3587 analysis (RIP-chip). RIP-chip analyses identified over 3500 mRNA transcripts selectively associated with MKRN1. Of these ~3500 MKRN1-target tran- THE PLURIPOTENT TRANSCRIPTION FACTOR scripts, 339 were differentially expressed upon the transient knockdown of ZIC3 DIRECTLY ACTIVATES NANOG IN MKRN1 in ESCs suggesting that MKRN1 post-transcriptionally regulates the gene expression of a subset of its targeted mRNAs. Taken together our data EMBRYONIC STEM CELLS indicates that MKRN1 is a novel component of a ribonucleoprotein complex Hong, Huimei, Lim, Shushan, Lawrence, Stanton in ESCs. Moreover, MKRN1 selectively associates with specific mRNAs to modulate stability and thus expression at the post-transcriptional level. Our NUS Graduate School / GIS, Singapore, Singapore, GIS, Singapore, data begins to unravel novel post-transcriptional gene regulatory mecha- Singapore nisms in ESCs that are likely important in shaping downstream stem cell fate The pluripotent state of embryonic stem (ES) cells is maintained by an decisions. intricate interplay of transcription factors within a complex transcriptional circuitry. Transcription factors Oct4, Sox2 and Nanog constitute the core Poster Board Number: 3593 regulators of this circuitry and they collaboratively maintain their own ALKBH1 IS A HISTONE H2A DEMETHYLASE expression via interconnected autoregulatory loops. This transcription fac- tor trio also co-occupy genes to concertedly maintain the transcriptional INVOLVED IN REGULATION OF PLURIPOTENCY program required for pluripotency. We previously reported that Zic3 is a AND SELF-RENEWAL IN EMBRYONIC STEM zinc-finger transcription factor that is co-occupied and directly regulated by Oct4, Sox2 and Nanog. In addition, we reported that depletion of Zic3 only CELLS modestly downregulated Oct4 and Sox2 but strikingly, Nanog expression Ougland, Rune, Nordstrand, Line, Klungland, Arne, Larsen, was significantly downregulated which suggests that Zic3 is a regulator of Elisabeth Nanog. Hence, we sought to investigate the underlying mechanisms of Rikshospitalet, Oslo University Hospital, Oslo, Norway the regulation of Nanog by Zic3. In this study, we show that Zic3 directly activates Nanog, and together, the duo form an independent autoregulatory Embryonic stem cells (ES cells) are characterized by the ability to divide loop in ES cells to maintain pluripotency. indefinitely, and still maintain the capacity to differentiate into function- ally distinct cell types. Since proliferation and differentiation occur without Poster Board Number: 3589 any detectable genetic changes, it is believed that the ES cell properties are MAKORIN-1 IS A NOVEL COMPONENT OF THE regulated by epigenetic marks e.g. methylated histones. Here, we show that a homozygous disruption of Alkbh1, a member of the 2-oxoglutarate- and POST-TRANSCRIPTIONAL GENE REGULATORY iron dependent dioxygenase family, leads to an increase in the levels of NETWORK IN EMBRYONIC STEM CELLS methylated K118 or K119 on histone H2A. Histone demethylases were dis- covered in 2004, and to our knowledge, this is the first time a histone H2A Cassar, Paul A., Samavarchi-Tehrani, Payman, Chang, Wing Y., demethylase that has been identified. Most of the Alkbh1 deficient mice Carpenedo, Richard L., Wrana, Jeffrey L., Stanford, William L. die during embryonic development due to defects in tissue originating from University of Toronto, Toronto, ON, Canada, Samuel Lunenfeld Research the ectodermal lineage i.e. bones, eyes and neural tissues. To gain further Institute, Toronto, ON, Canada insight into the role of ALKBH1 during embryonic development, we have characterized ALKBH1 in ES cells. Like NANOG, OCT4 and SOX2, which are Embryonic stem cell (ESC) behavior is regulated by a complex network of the key factors of pluripotency and self-renewal, ALKBH1 is expressed in the molecular events that ultimately lead to activation and repression of gene inner cell mass of mouse blastocysts. In ES cells, ALKBH1 is a nuclear protein expression. The dynamics of gene expression is often most attributed to regulated by the ES cell transcription factors NANOG and SOX2. Alkbh1 stoichiometric fluctuations at the level of gene transcription; however, it is deficient ES cells proliferate normally and retrain stem cell characteristics. becoming increasingly evident that post-transcriptional regulatory mecha- However, immunostaining show early signs of differentiation and these cells nisms also contribute to control both the abundance and kinetics of gene ex- differentiate more easily under stress. A whole genome microarray analysis pression within the ESC gene regulatory network (GRN). In a previous study, comparing the transcriptome of Alkbh1 knockout ES cells with the corre- we identified a cluster of genes that are preferentially expressed in undif- sponding wild type ES cell line identified 1497 transcripts to be differentially ferentiated ESCs, and are faithfully down-regulated in early committed ESCs. expressed. Of these 1497 genes, 840 were up regulated while 657 were We have demonstrated that this cluster of genes encode for components of down regulated indicating substantial dysregulation of gene expression upon the ESC GRN that individually function to regulate the expression of specific deletion of Alkbh1. Hierarchical clustering of the differentially expressed subsets of genes at multiple levels. Makorin-1 is a gene within this cluster genes revealed five discrete clusters of commonly regulated transcripts that encodes for the RING finger protein Makorin-1 (MKRN1). Although the including genes involved in stem cells renewal, pluripotency, differentia- molecular function of MKRN1 in ESCs is unknown, the selective expression tion, Bmp and Wnt signaling pathways. This result corresponds well with of MKRN1 in undifferentiated ESCs indicates its selective function within the the phenotype of the Alkbh1 deficient embryos. Thus, our work identifies a ESC. MKRN1 contains four C3H zinc finger RNA-binding motifs and an E3 histone demethylase and links its function to pluripotency and self-renewal ubiquitin ligase RING domain, which are common to other post-transcrip- in mouse ES cells. tional regulators of gene expression. To dissect the underlying mechanism for MKRN1 function in ESCs, we immunoprecipitated MKRN1 protein from transgenic FLAG:MKRN1 ESCs followed by mass spectrometry analysis. We found that MKRN1 is a novel component of a ribonucleoprotein complex in ESCs as it interacts with twenty-nine other known mRNA-binding proteins, including the poly(A)-binding protein (PABPC1) and eIF4G, that are associ- ated with translated mRNA. We also found that MKRN1 localizes to stress granules upon arsenite-induced oxidative stress in ESCs where it co-localizes with a number of its interacting partners - further corroborating MKRN1 as a novel component of a ribonucleoprotein complex. To identify the subset of mRNA associated with MKRN1, we subsequently performed ribonu- cleoprotein-immunoprecipitation of FLAG:MKRN1 coupled to microarray

249 ISSCR 9th Annual Meeting www.isscr.org

Thursday Poster Abstracts

Poster Board Number: 3595 Poster Board Number: 3599 DISSECTING THE REGULATION OF ESC NOVEL LIVE ALKALINE PHOSPHATASE PROPERTIES BY MEMBERS OF THE CATENIN SUBSTRATE FOR IDENTIFICATION OF SUPERFAMILY PLURIPOTENT STEM CELLS Kelly, Kevin F., Doble, Bradley W. Lakshmipathy, Uma, Singh, Upinder, Quintanilla, Rene, Grecian, Stem Cell and Cancer Research Institute, McMaster University, Hamilton, Scott, Gee, Kyle, Rao, Mahendra ON, Canada Life Technologies, Carlsbad, CA, USA, Life Technologies, Eugene, OR, USA Embryonic stem cell (ESC) properties are controlled by a multitude of inte- Pluripotent stem cells such as embryonic and induced pluripotent stem cells grated cellular signaling pathways, including the β-catenin/TCF pathway. are commonly identified and characterized based on biomarker expression. Stimulation of the β-catenin/TCF pathway through inhibition/ablation of Notably, the surface antigens SSEA4 and Tra antibodies facilitate a live meth- GSK-3 results in elevated cytosolic and nuclear β-catenin levels, increased od for labeling pluripotent stem cells. An additional measure of pluripotency expression of β-catenin/TCF target genes, and reinforced ESC pluripotency. is relative high levels of the enzyme alkaline phosphatase compared to feed- Surprisingly, we recently demonstrated that the effects of β-catenin on ers, somatic cells and differentiated progeny. Current methods for detecting pluripotency do not require the activation of β-catenin/TCF target genes. alkaline phosphatase expression utilizes chromogenic or fluorogenic com- Instead, β-catenin interacts with and enhances the activity of the core pounds. These methods require cell fixation and/or results in end products pluripotency regulator, Oct-4. Recent studies suggest that, in addition to that are toxic to cells. Because of these issues, alkaline phosphatase staining β-catenin, at least two other members of the catenin superfamily, p120 and is an end-point assay and the stained pluripotent stem cell colonies cannot γ-catenin/plakoglobin, are GSK-3 substrates. Indeed, we find that p120 and be further propagated and is therefore limiting. An ideal solution would be plakoglobin are stabilized upon treatment of mouse ESCs with GSK-3 inhibi- a live alkaline phosphatase method that would stain pluripotent stem cell tors, or in mouse ESCs lacking both isoforms of GSK-3. We are currently colonies without adverse effects and hence amenable to further isolation examining whether these catenins, like β-catenin, regulate the acquisition or and expansion. Such a method would be superior to antibody-based meth- maintenance of pluripotency. Collectively, these studies will reveal mecha- ods that are often variable due to antibody quality, expensive and hence nistic insight into how different members of the catenin superfamily regulate not ideal for large scale or high density usage. We have developed a novel the pluripotent state. fluorogenic substrate for alkaline phosphatase that shows robust labeling of Poster Board Number: 3597 ESC and iPSC colonies 20-30 minutes after addition to the basal culture me- dia. The level of background with nonpluripotent cells such as MEF feeders RETINOL/VITAMIN A SIGNALING AND and human fibroblasts are minimal. Fluorescent pluripotent colonies attained with this novel alkaline phosphatase substrate show normal morphology and REGULATION OF SELF RENEWAL OF STEM the fluorescence can be completely eliminated from the pluripotent colonies CELLS 60-90 minutes after removal of the dye from the media. These cells can be further propagated and the cells retain their pluripotency based on marker Khillan, Jaspal Singh, Bhatia, Himanshu, Yang, Minying, Johnson, expression and differentiation potential. Our results indicate that the novel Carla, Miki, Toshio live alkaline phosphatase substrate offers a simple and easy tool to not only Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, identify pluripotent colonies but further propagated the identified colony. PA, USA, Biochemistry and Molecular Genetics, University of Southern This tool will provide an easy live monitoring method to track cells during California, Los Angeles, CA, USA reprogramming or during routine culture of ESC and iPSC cells. Small molecules present a great promise for developing novel systems for maintaining the pluripotency of stem cells in vitro. Recently we have identi- fied a new function of retinol, the alcohol form of vitamin A in regulating the self renewal of mouse and human ES cells by elevating the expression of OCT4 and NANOG. Significantly, this function is independent of retinoic acid as ES cells do not contain enzymes such as alcohol dehydrogenase and retinaldehyde dehydrogenase required for metabolism of retinol into retinoic acid and Stra6, the receptor for retinol binding protein. Retinol regulates the self renewal of mouse ES cells by activating IGFII/IGF1 receptor axis by engaging PI3 Kinase signaling pathway. We have now established retinol based novel feeder free culture systems for large scale production of human and mouse pluripotent stem cells using simple culture conditions. Since retinol is a readily available physiologically relevant small molecule, our system represents a safe and inexpensive culture for large scale production of clinically applicable pluripotent stem cells.

250 www.isscr.org Thursday Poster Abstracts

Presenter Index

Aalto-SeTälä, Katriina...... 17, 19 Akbarian, Vahe...... 112 Andersen, MarnellE...... 84 Asuzu, David T...... 74 Abasi, Mozhgan...... 99 Akcali, K. Can...... 137 Anderson, Aileen J...... 50 Atalay, Arzu...... 77 Abdallah, Basem M...... 158 Akcali, Kamil C...... 137 Anderson, Eric C...... 68 Atashi, Amir...... 134 Abdelrahman, Sara...... 83 Akcora, Dilara -...... 29 Anderson, Erica...... 21 Atique, Rodrigo Ft...... 121 Abdou, Laila1...... 6 Åkerblom, Malin...... 59 Ando, Shunsuke...... 177 Atkinson, Kerry M...... 124 Abdul Jalil, Rufaihah...... 20 Åkerman, Karl E...... 225 Ando, Yuji...... 125 Atze, Kristin...... 83 Abdulrazzak, Hassan...... 153 Akhan, Ece...... 137 Andrade, Pedro Z...... 147 Atzet, Sarah...... 95, 145 Abe, Masumi...... 177 Akimoto, Keiko...... 140 Andrade, Silvia P...... 22 Audet, Julie...... 112 Abe, Tomoyuki...... 105 Akinci, Ersin...... 198 Andre, Charles...... 95 Aujia, Tijess P...... 5 Abecasis, Manuel M...... 147 Al Madhoun, Ashraf...... 52 André-Schmutz, Isabelle...... 107 Aumont, Anne....51, 51, 58, 191 Abhold, Eric...... 66 Alaie, Sara...... 44 Angel, Matthew...... 200 Auriat, Angela M...... 92 Aboody, Karen S...... 148 Alarcon, Vernadeth B...... 105 Angelini, Paola...... 67 Auvergne, Romane M...... 63 Abou El-Kheir, Wael...... 2 Alberto, Miryan L...... 120 Anguera, Monserrat C...... 43 Au-Young, Janice...... 128 Abou-Kheir, Wassim...... 67 Alcantara Llaguno, Sheila R....64 Ankrum, James A...... 153 Avrith, Nita...... 51 Abramyants, Yelena...... 148 Alexander, Jeffrey M...... 18, 163 Annala, Alexander J...... 148 Ayaloglu Butun, Fatma...... 137 Abroun, Saeed...... 61 Alexanian, Arshak R...... 126 Ansari, Saeed...... 25 Ayetey, Harold...... 17 Accili, Domenico...... 30 Alexeeva, Vera...... 170 Anselmo, Achille...... 123 Azabdaftari, Gissou...... 171 Achanta, Pragathi...... 63 Ali, Rouknuddin Q...... 210 Anstadt, Emily...... 15 Azarnier, Ronak Ghanbari...... 73 Adair, Henry...... 142 Alini, Mauro...... 135 Antonchuk, Jennifer...... 208, 209 Azem, Foad...... 242 Adams, TIm...... 83 Aliotta, Jason...... 75 Apáti, Ágota...... 240 Aznar-Benitah, Salvador...... 26 Adhami, Bahareh...... 209 Alitalo, Kari...... 186 Apgar, John R...... 214 Azzola, Lisa...... 221 Adjaye, James...... 222 Alizadeh, Shaban...... 116 Apostolou, Effie...... 161 Baba, Shiro...... 17 Aflatoonian, Abbass...... 76, 87 Allen, Randall...... 185 Aractingi, Selim...... 27 BabaeijandaghI, Farshad...... 145 Aflatoonian, Ali...... 87 Almada, Bruno Vinicius Pimenta. Arai, Takuma...... 22 Babaloo, Hamideh...... 99 Aflatoonian, Behrouz...... 133 Araki, Ryoko...... 177 Babona-Pilipos, Rob...... 191 ...... 26 , 76, 87 Alman, Benjamin...... 39, 73 ArauZo Bravo, Marcos...... 185 Bachelard-Cascales, Elodie....129 Aflatoonian, Nastaran...... 26 Almeida, Brígida G...... 22 Arauzo-Bravo, Marcos J...... 247 Bachoo, Robert...... 64 Aflatoonian, Reza...... 76 Almeida, Danilo C...... 8 Araya, Claudia...... 57 Baddour, Nahed...... 6 Afzal, Aqeela...... 25 Alonso, Nivaldo...... 121, 133 Arbel, Gil...... 13 Badowski, Michael...... 26 Aghdami, Nasser...... 133, 209 Alper, Michael...... 198, 198 Arindrarto, Wibowo...... 195 Bae, Jae-Sung...... 150 Aguena, Meire...... 133 Alt, Rüdiger...... 139 Ariza, Carolina B...... 53 Bae, Leon...... 116 Aguila, Héctor L...... 81, 106 Altamirano, José...... 9 Armstrong, Kelly...... 66 Bae, Weon...... 91 Aguila, Julio Cesar...... 55 Altarescu, Gheona...... 242 Arnold, Christopher P...... 73 Baek, Hyunjung...... 27 Aguilar-Gallardo, Cristobal...... 69 Altun, Gulsah...... 205 Arora, Natasha...... 109 Bagdonas, Edvardas...... 121 Ahmad, Faizzan S...... 164 Altuwaijri, Saleh A...... 66 Arslan, Y. Emre...... 130 Baghaban Eslaminejad, Ahmad, Ruhel...... 209 Amanpour, Saeid...... 134 Arthur, Agnes...... 156 Mohamadreza...... 93, 127 Ahmad, Sajjad...... 36 Ambrósio, Carlos E...... 121 Arthur, Agnieszka...... 141 Bagheban Islami Nejad, Mohamad Reza...... 133 Ahmadbeigi, Naser...... 134, 145 Ambruzs, Dana...... 29 Artus, Jerome...... 104, 104 Baharvand, Hossein.....7, 43, 44, Ahmed, Ishtiaq M...... 124 Amendola, Mario...... 79 Arufe Gonda, Maria...... 144 ...... 47, 133, 156, 162, 180, 234 Ahmed, Sharif...... 72 Amin, Caitlin...... 95 Arvanites, Anthony...... 41, 213 Bai, Qiang...... 232 Ahn, Ji Yeon...... 244 Amini-Nik, Saeid...... 39 Asfaw, Dawit...... 84 Baizabal, José-Manuel...... 55 Ahn, Woosung...... 195 Amit, Ami...... 242 Asgari, Samira...... 7 Baker, Wendy A...... 80 Aho, Joy...... 84 Amit, Michal...... 203 Ashley, Rebekah K...... 35 Balci, Deniz...... 147 Ahola, Antti...... 19 Amsellem, Sophie...... 107 Askari, Janet A...... 241 Ballios, Brian G...... 34, 35 Ährlund-Richter, Lars...... 210 An, Hwa...... 204 Assinck, Peggy...... 57 Ban, Hiroshi...... 105, 174 Ahuja, Tijess P...... 5 An, Jeung Hee...... 45 Assmann, Gerald...... 16 Bandi, Sriram...... 4, 4 Ailles, Laurie...... 73 An, Songzhu (Michael)...... 144 Astrauskiene, Danute Linute...... Banerjee, Ipsita...... 1 Ajeian, Jila N...... 241 An, Su Yeon...... 189 ...... 121

251 ISSCR 9th Annual Meeting www.isscr.org

Presenter Index

Banfi, Andrea...... 131 Behmanesh, Mehrdad...... 116 Biancotti, Juan C...... 237 Borges, Luciene...... 179 Banga, Anannya...... 198 Beldjord, Kheïra...... 107 Bien, Mauo-Ying...... 132 Borjesson, Dori L...... 151 Bao, Siqin...... 199 Belicchi, Marzia...... 160 Bigas, Anna...... 113 Bornhäuser, Martin...... 102 Barabé, Frédéric...... 63, 99 Bell, Gillian I...... 189 Bigildeev, Alexey...... 122 Borowsky, Mark...... 161 Baranov, Petr Y...... 42 Belloni, Elena...... 10 Bignone, Paola A...... 82 Borrego-Diaz, Emma...... 70, 71 Barbacid, Mariano...... 10 Beltrão-Braga, Patricia...... 160, Bilodeau, Melanie...... 11 Boscolo, Francesca...... 205 Barberi, Tiziano...... 215, 233 ...... 176, 176, 177 Bilodeau, Steve...... 161, 234 Bosen, Felicitas...... 224 Barboni, Barbara...... 31 Beltrão-Braga, Patricia C B....176 Binet, Christian...... 154 Bottinelli, Roberto...... 160 Barbosa Da Fonseca, Lea Mirian. Ben Azouna, Nesrine...... 141 Biran, Alva...... 161 Botvinick, Elliot...... 218 ...... 95 Ben Yosef, Dalit...... 242 Biswas, Anuradha...... 183 Bouab, Meriem...... 51 Barcelos, Lucíola S...... 22 Benderitter, Marc...... 143 Biswas, Arpita...... 123 Boucher, Shayne...... 128, 147 Bard, Frederic...... 237 BenneTt, Margaux...... 184 Bitirim, Verda C...... 137 Bouhout, Sara...... 98 Bardsley, Michael R...... 74 Bentzinger, Florian...... 40 Bjerkvig, Rolf...... 64 Boulland, Jean-Luc...... 122 Bardwell, Lee...... 215 Benvenisty, Nissim...... 165, 165, Björklund, Anders...... 232 Boulting, Gabriella L...... 166 ...... 168, 237 Bareiss, Petra M...... 70, 230 Björquist, Petter...... 7 Bourette, Roland P...... 88 Berardineli, Paolo...... 31 Barish, Michael E...... 148 Blair, Chris...... 124 Bowman, Teresa V...... 117 Berezikov, Eugene...... 195 Barnabe, Gabriela F...... 53 Blak, Alexandra...... 55, 209 Bowtell, David...... 70 Berger, André...... 108 Barnes, Brian...... 154 Blanco García, Fco...... 144 Boyer, Laurie A...... 18, 163 Bergeron, Anne...... 99 Bar-Nur, Ori...... 165, 165 Blanco, Carmino...... 4 Boza, Maria G...... 167 Berggren, Magnus...... 61 Baron, Margaret H...... 112 Blatt, Alexander...... 60 Bradley, Cara...... 86 Berks, Richard...... 109 Barriga, Francisco M...... 69 Blau, Helen M...... 200 Brady, Jennifer J...... 200 Berlin, Dorit S...... 185 Barrios Llerena, Martin...... 236 Blauwkamp, Tim...... 243 Brand, Michael...... 194 Bernabò, Nicola...... 31 Barron, Matthew R...... 13 Blelloch, Robert...... 166 Brandenberger, Ralph...... 81 Bernal, Carolina A...... 57 Barta, Tomas...... 236, 237 Bletsa, Ritsa...... 238 Brandt, Matthias...... 174 Bernal, Giovanna M...... 57 Bartek, Jiri...... 63 Bloda, Martin...... 75, 143 Brasch, Helen D...... 101 Bernard, Claude...... 30 Bartlett, Perry F...... 55 Blügermann, Carolina...... 155 Breault, David...... 29 Bernard, Geneviève...... 98 Baruchel, Sylvain...... 67 Blumenthal, Jacob...... 210 Bredesen, Dale E...... 241 Bernard, Marie-Christine...... 154 Barzin, Jalal...... 99, 100 Blystone, Scott...... 224 Breitbach, Martin...... 138 Bernardo, Andreia S...... 14, 215, Basak, Indranil...... 159 Böckenhoff, Annika...... 174 Brennand, Kristen J...... 187 ...... 215 Basu, Joydeep...... 193 Bodla, Ahmed Salman...... 162 Brenner, Sebastian...... 102 Bernemann, Christof...... 185 Basu, Sudipta...... 196 Bogomazova, Alexandra...... 164 Breuer, Nadine...... 83 Berninger, Benedikt...... 197 Batada, Nizar N...... 186 Boimer, Corey...... 39 Brick, David J...... 207 Bernotiene, Eiva...... 121 Bataller, Ramon...... 9 Boisvert, Erin M...... 211 Brickman, Joshua M...... 236 Berry, Suzanne E...... 19, 194 Batchelor, Alex...... 83 Bokermann, Gudrun...... 124 Briggs, Sharon...... 202 Berthiaume, Sara...... 99 Batchu, Chandana...... 94 Bolduc, Stéphane...... 98 Brill, Laurence...... 86 Bertin, Enrica...... 89 Batlle, Eduard...... 69 Boles, Nathan...... 211 Broccoli, Vania...... 79 Bertolotti, Roger...... 165 Battistella, Valeria...... 95 Bolli, Roberto...... 14 Brodarac A, Nitschke M...... 31 Bertram, Tim...... 193 Baudet, Aurelie...... 107 Bolontrade, Marcela F...... 125 Brolén, Gabriella...... 7 Betel, Doron...... 241 Bazett-Jones, David P....177, 186 Bonaguidi, Michael...... 96 Bronfman, Miguel...... 57 Bevilacqua, Estela...... 177 Beanlands, Robert S...... 82 Bongaarts, Rico...... 91 Brooke, Gary P...... 124 Bhadriraju, Kiran...... 217 Beaudoin, Stéfanny...... 191 Bonham, Kristina...... 4 Broughton, Heather...... 189 Bharathan, Sumitha P....166, 178 Becker, Fabienne...... 232 Bonhoeffer, Sebastian...... 120 Broxmeyer, Hal E...... 239 Bhatia, Himanshu...... 250 Becker, Sandy...... 207 Boquest, Andrew C...... 122 Bruck, Tal...... 168 Bhatia, Mickie...... 222 Beckstead, Robert B...... 183 Borbely, Alexandre...... 177 Bruestle, Oliver...... 105 Bhatnagar, Aruni...... 14 Bedoya, Francisco J...... 243 Borchardt, Erin R...... 157 Bruneau, Benoit G...... 18, 163 Bhise, Nupura S...... 86 Beeretz, Michael...... 230 Borchin, Bianca E...... 215 Bruneau, Julie...... 107 Bhutani, Nidhi...... 200 Begum, Aynun N...... 204 Bordbar, Sima...... 93 Bruscia, Emanuela...... 140 Bi, Kun...... 60 Behie, Leo A...... 144 Bordini, Daniela...... 176 Brüstle, Oliver...... 169, 174, 209

252 www.isscr.org Thursday Poster Abstracts

Presenter Index

Brzustowicz, Linda M...... 172 Cañamero, Marta1...... 88 Chapellier, Marion...... 129 Cherman, Natasha...... 135 Bshara, Wiam...... 65 Cancino, Gonzalo...... 49, 58 Charter, Suellen J...... 185 Cherry, Simon R...... 151 Buard, Valérie...... 143 Canela, Andres...... 195 Chase, Lucas...... 147 Chettri, Pranav...... 178 Buchstaller, Johanna...... 74 Cano, Agustina...... 55 Chaudhary, Kunal...... 8 Cheuh, Hee Won...... 146, 148 Buckley, Shannon...... 116 Cantz, Tobias...... 198 Chaudhry, Aneeka...... 146 Cheung, Aaron Yl...... 168 Buehler, Nicole...... 237 Cao, Tong...... 222 Chaudhry, Faisal...... 143 Chi, Guang Fan...... 195 Bueno, Daniela F...... 133 Caprariello, Andrew V...... 227 Chaudhry, G. Rasul...... Chia, Na-Yu...... 237 Bueren, Juan...... 118 Carbone, Catherine...... 35 ...... 75, 143, 231 Chiamvimonvat, Nipavan...... 16 Bufalino, Mary Rose...... 59 Carelli, Stephana...... 191 Chavez, Ferman...... 231 Chiang, Yung-Hsiao...... 64 Bugdayci, Emre...... 137 Carlin, Stephen M...... 106 Chayosumrit, Methichit...... 75 Chiao, Eric T...... 21 Bühring, Hans-Jörg...... 70, 133 Carlone, Diana...... 29 Che, Jeong Hwan...... 151 Chicha, Laurie...... 234 Bulgin, Dmitry...... 101 Carman, Christopher V...... 153 Chellapan, Malathi...... 15 Chien, Chiao-Yun...... 3 Burch, Jaclyn...... 63 Carpenedo, Richard L...... 249 Chen, Aaron...... 218 Chien, Chung-Liang...... 11 Burden, Steven...... 123 Carrade, Danielle M...... 151 Chen, Alice...... 198 Chien, Ming-Hsien...... 132 Burgold, Thomas...... 51 Carrel, Laura...... 168 Chen, Antony...... 217 Chilton, Jamie M...... 45 Burgon, Patrick G...... 82 Carson, Christian T...... 211, 214 Chen, Caifu...... 73, 128, 204 Chin, Alan...... 82 Burkhardt, Matthew...... 44 Carter, Richard L...... 92 Chen, Chang-Zheng...... 73 Ching, Reagan W...... 186 Burks, Scott R...... 146 Carvalho, Ana Flávia...... 160 Chen, Chi-Long...... 132 Chiou, Shih-Hwa...... 36 Burns, Dennis...... 64 Carvalho, Isabel...... 15 Chen, Huei-Sheng Vincent.....16 Chiu, Wen-Ta...... 64 Burns, Sarah...... 53, 56 Caspi, Inbal...... 165 Chen, Huei-Wen...... 11 Chmelar, Renee...... 88 Bursac, Nenad...... 15 Cassar, Paul A...... 249 Chen, Hung-Kuan...... 11 Cho, Candy H-H...... 216 Burton, Doug...... 66 Castro, Tiago B. R...... 22 Chen, Jiekai...... 201 Cho, Hyun-Jai...... 12, 199 Bushman, Jared...... 93 Cavanaugh, Christopher...... 243 Chen, Kai-Yun...... 64 Cho, Jaejin...... 88 Buszczak, Michael...... 78 Cavazzana Calvo, Marina.....107 Chen, Kevin G...... 81, 135 Cho, Ka Hee...... 151 Buttigieg, Josef...... 54 Cebers, Gvido...... 49 Chen, Kui...... 72 Cho, Mee-Young...... 88 Byers, Blake Hampton...... 171 Ceh, Katerina...... 125 Chen, Pei-Min...... 153 Cho, Myung Soo...... 33, 204 Byers, Peter H...... 201 Cerletti, Massimiliano...... 41 Chen, Qing...... 141 Cho, Wheemoon...... 103 Byron, Adam...... 241 Cernat, Laura...... 124 Chen, Ruey-Jien...... 136 Choe, Leila...... 229 Caballero, Montserrat...... 135 Céspedes, María Virtudes...... 69 Chen, Seng Mei...... 213 Choi, Cheonggab...... 182 Cacace, Angela...... 47 Cezar, Gabriela G...... 48, 49 Chen, Shuibing...... 213 Choi, Chulhee...... 21 Caccavelli, Laure...... 107 Chae, Jung Il...... 205 Chen, Ting...... 27 Choi, Hong Seo....239, 239, 239 Cady, Nathaniel...... 95 Chakraborty, Syandan...... 15 Chen, Vincent C...... 237 Choi, Hyeongwon...... 195 Cahuana, Gladys M...... 243 Chamberlain, Jeffrey S...... 179 Chen, Wannhsin...... 84 Choi, Hyung Jun...... 151 Cai, Chenleng...... 117 Chambers, Stuart M.....213, 219 Chen, Weihsu Claire...... 62 Choi, Jee In...... 159 Cai, Qing...... 216 Chan, Anthony W.S...... 92 Chen, Weika...... 120 Choi, Jeong Woo...... 45 Caisander, Gunilla...... 171 Chan, David N...... 214 Chen, Wen Li Kelly...... 139 Choi, Kang Yell...... 91 Cakouros, Dimitrios...... 141 Chan, Jerry...... 20 Chen, Xiaodong...... 141 Choi, Michael...... 243 Calder, Ashley...... 218 Chan, Shing Fai...... 55 Chen, Yao-Chang...... 189 Choi, Soo Jin...... 128 Callamaras, Nick...... 92 Chanda, Bidisha...... 114 Chen, Yen-Shun...... 136 Choi, Soon Won...... 159 Callery, Elizabeth M...... 215 Chandler-Militello, Devin...... 63 Chen, Ying...... 40 Choi, Yong Jin...... 184 Calon, Frédéric...... 51 Chandran, Jayanth S...... 58 Chen, Yi-Shiou...... 69 Choi, Young Min....33, 204, 208 Campbell, Andrew...... 147 Chandran, Yogeswari...... 184 Chen, Zhong...... 117 Choi, Young Wook...... 27 Campbell, Jared M...... 247 Chang, Da-Jeong...... 182 Cheng, Chialin...... 45 Choi, Yu Suk...... 132 Campbell, Pearl A...... 248 Chang, Fang-Pei ...... 200 Cheng, Chieh-Feng...... 132 Chong, Fenny...... 87 Campos De Carvalho, Antonio Chang, Howard Y...... 163 Cheng, Linzhao...... 20 Chong, Lynn...... 70 C...... 150 Chang, Wing Y...... 249 Cheng, Yenfu...... 210 Chong, Mark...... 20 Can, Alp...... 147 Chang, Young-Tae...... 184 Chen-Konak, Limor...... 210 Choolani, Mahesh A...... 20 Canaday, Pamela S...... 2 Chang, Yun-Chuang.....132, 132 Chenoweth, Josh...... 135

253 ISSCR 9th Annual Meeting www.isscr.org

Presenter Index

Chowdhury, Mohammad Cook, Karen...... 92 Dahl, John P...... 135 Deng, David...... 204 Mahfuz...... 186 Cook, Matthew...... 124 Dailey, William...... 143 Denhardt, David T...... 141 Christalla, Peter...... 91 Cooke, John...... 20, 188 Dal Cortivo, Liliane...... 107 Derynck, Rik...... 166 Christoforou, Nicolas...... 15 Cooke, Michael...... 100 Daley, George Q...... 109, 113, Desmarais, Bryan...... 238 Chu, Vi...... 199 Cooper, Lachlan...... 141 ...... 117, 182, 230 Detamore, Michael...... 125 Chua, Shawn...... 101 Cooper-White, Justin J...... 79 Daliri, Morteza...... 116 Devaraju, Karthikeyan...... 54 Chuang, Ching-Yu...... 76 Cord, Branden J...... 171 Daniunaite, Kristina...... 121 Deveale, Brian...... 52, 59 Chugh, Atul...... 14 Corneo, Barbara...... 217 Darabi, Radbod...... 96, 179 Devito, Loren...... 49 Chun, Ju Lan...... 19, 194 Coronado, Diana...... 246 Darabie, Audrey...... 61 DevrekeR, Fabienne...... 219 Chung, Dai-Jung...... 151 Corso, Andrew...... 203 Darr, Henia...... 248 Deyle, David R...... 201 Chung, Eunkyung...... 195 Costa, Yael...... 203 Das, Shreyasi...... 82, 169 Dhar, Madhu...... 142 Chung, Henry C.Y...... 167 Cotgreave, Ian...... 49 Dasilva, Jean N...... 82 Dhara, Sujoy K...... 46 Chung, Hyung-Min...... 30, 205 Couture, Larry A...... 237 Datta, Krishnalekha...... 94 Di Fiore, Pier Paolo...... 10 Chung, Meng-Hong...... 90, 154 Couture, Sylvana...... 237 Daughters, Randy...... 40 Di Giulio, Anna Maria...... 191 Chung, Tung-Liang...... 72 Covarrubias, David...... 54 Davern, Kathleen...... 2 Di Maggio, Nunzia...... 131 Chy, Hun...... 83 Covarrubias, Luis...... 55 Davey, Rhonda...... 78 Di Micco, Raffaella...... 248 Cianci, Amelia...... 198 Covas, Dimas T. David, Robert...... 16 Di Nardo, Paolo...... 15 Ciavattone, Nicholas...... 75, 143, ...... 8, 120, 121, 123 Davidow, Lance S...... 41 Di Stefano, Bruno...... 79 ...... 231 Covas, Dimas Tadeu...... 176 Davidson, Alan J...... 230 Diaz, Irene...... 243 Cibella, Javier...... 123 Cowley, Sally...... 196 Davidson, Lindsay...... 171 Dick, John E...... 62, 62, 62, 63 Cibelli, Jose...... 230 Cox, Andrew G...... 9 Davies, Paige S...... 28 Didangelos, Athanasios...... 91 Cifelli, Carlo...... 18 Cox, Brian J...... 104 Davila-Kruger, Diana...... 241 Didié, Michael...... 91 Ciolfi, Federico...... 125 Cox, Jurgen...... 247 Davis, Jeffrey M...... 193 Diienno, AmanDa R...... 223 Civin, Curt I...... 109 Craig, Susan E...... 241 Dawes, Glenn N...... 14 Dileo, Anthony...... 85 Clapcote, Steven J...... 58 Crain, Andrew...... 86 Day, Darren John...... 101 Dilworth, F. Jeffrey...... 177 Clark, Amander...... 237 Crispino, John...... 164 De Arriba, María Del Carmen..... Dimmeler, Stefanie...... 97 Clarke, Ian D...... 64 Cromwell, Evan F...... 92 ...... 88 Dimos, John...... 96 Clegg, Dennis O.1...... 35 Cross, Michael...... 139 De Coppi, Paolo...... 40, 89, 153 Dinarvand, Peyman...... 100 Clementson, Christine Ekdahl.54 Cross, Rosie...... 84 De Freitas, GaBriel R...... 95 Dincer, Zehra...... 211 Clevers, Hans...... 69 Crovace, Antonio...... 142 De La Fuente González, Dinda, Sumi...... 75, 143, 231 Coelho-Aguiar, Juliana M...... 59 Crowe, Suzanne...... 82 Alexandre...... 144 Ding, Junjun...... 246 Coetzee, William...... 224 Crum, Christopher P...... 10 De Mulder, Katrien...... 195 Ding, Lei...... 110 Cogswell, Cathy...... 123 Csaszar, Elizabeth...... 108 De Toro, Fco...... 144 Dirks, Peter B...... 64 Coles, Mark...... 109 Cui, Hao...... 146 De Verneuil, Hubert...... 118 Ditadi, Andrea...... 220 Coles-Takabe, Brenda...... 35 Cunha, Celia...... 204 De Vos, John...... 232 Divoky, Vladimir...... 63 Collas, Philippe...... 122 Cuniberti, Luis...... 137 Dean, Dana...... 146 Divsalar, Adeleh...... 228 Collins, Tony...... 218 Curini, Valentina...... 31 Deftos, Leonard J...... 66 Djabrayan, Nareg J...... 202 Colman, Alan...... 235 Curran, Matthew...... 113 Degoul, Olivier...... 127 Djuric, Ugljesa...... 177 Colombini, Amanda M...... 123 D’amour, Kevin...... 3 Dekemp, Rob A...... 82 Doble, Bradley W...... 250 Colovai, Adriana I...... 108 D’antona, Giuseppe...... 160 Del Alamo, Juan Carlos...... 142 Dobrowolska, Hanna M...... 108 Colton, Clark K...... 223, 224 D’ecclessis, Michael...... 196 Del Tatto, Michael...... 75 Dobson, Stephanie M...... 63 Côme, Julien...... 167 D’escamard, Valentina...... 238 Delay, Emmanuel...... 129 Docherty, Hilary M...... 2 Conard, Kevin...... 48, 49 D’souza, Sunita...... 170, 217 Deleu, Sandrine...... 219 Docherty, Kevin...... 2, 227 Conget, Paulette...... 7 Da Silva-Diz, Victoria...... 69 Delgado-Olguin, Paul...... 18 Dodla, Mahesh...... 80 Conklin, Bruce R...... 17 Dadi, Srividya...... 128 Demircan, Turan...... 195 Doerr, Jonas...... 169 Connolly, E Sander...... 25 Dadwal, Ushashi...... 196 Deneault, Eric...... 99 Doi, Daisuke...... 205, 206 Connor, Bronwen...... 192 Daghers, Rania...... 10 Denecke, Bernd...... 124 Dolezalova, Dasa...... 236, 237 Conradi, Lenard...... 97 Daheron, Laurence...... 46 Deneubourg, Laurence...... 219

254 www.isscr.org Thursday Poster Abstracts

Presenter Index

Dolley-Sonneville, Paula...... Efremov, Ivan...... 42 Espinosa, Lluis...... 113 Finger, Jared M...... 157, 173 80, 81, 84 Eftekarpour, Eftekhar...... 102 Ethell, Doug W...... 204 Fiondella, Chris...... 46 Domenech, Jorge...... 154 Egert, Angela...... 224 Ezquer, Fernando...... 7 Firestein, Bonnie L...... 172 Domingo, Santiago...... 69 Eggan, Kevin...... 164, 166, Ezquer, Marcelo...... 7 Fischer, Anna...... 52 Donaldson, Laura...... 35 ...... 182, 198, 201 Faial, Tiago...... 215, 215 Fischer, Julia...... 174 Donley, Elizabeth L.R...... 49 Egli, Dieter...... 198 Fairey, Mark...... 208 Fischer, Stephen E...... 90 Donovan, David M...... 182 Eiges, Rachel...... 242 Faltus, Timo...... 39 Fisk, Nicholas M...... 136, 153 Dooner, Mark S...... 75 Eijken, Marco...... 134 Fan, Anran...... 181 Fleischmann, Bernd K...138, 178 Dorrell, Craig...... 2 Eijkholt, Marleen...... 38 Fan, Guoping...... 183 Flory, Egbert...... 108 Dorscheid, Delbert...... 10 Elcin, A. Eser...... 129, 130 Fan, Heng...... 152 Foley, Lucy A...... 36 Dos Santos, Francisco...... 147 Elcin, Y. Murat...... 129, 130 Fang, Li...... 52 Fonoudi, Hananeh...... 209 Doty, Nathaniel J...... 95 Eldar-Geva, Talia...... 242 Fanganiello, Roberto Dalto...133 Fontaine, Burr...... 49 Dowen, Jill...... 161 Elefanty, Andrew...... Farassati, Faris...... 70, 71 Fontes, Andrew D...... 60 Draper, Jon...... 218 ...... 14, 208, 221 Farber, Debora...... 36 Fontes, Aparecida M..8, 120, 121 Drize, Nina...... 122 Elio, Vanin...... 164 Farhadian, Shirin...... 100 Formstecher, Pierre...... 72 Droujinine, Ilia...... 146, 191 Elizabeth, Sato...... 67 Farini, Andrea...... 160 Forozandeh Moghadam, Mehdi. Drowley, Lauren...... 129 Elkabetz, Yechiel...... 241 Fasano, Christopher A...... 211 ...... 61 Drury-Stewart, Danielle...... 218 Elkaffash, Dalal...... 6 Fattahi, Faranak...... 7, 209 Forraz, Nico...... 127, 141 Duan, Yuyou...... 5, 5 Ellerström, Catharina C...... 171 Faust, Nicole...... 83 Forster, Andrew...... 244 Dubner, Ronald...... 150 Elliott, David A...... 14 Faustman, Denise...... 157 Forte, Giancarlo...... 15 Dubois, Nicole...... 18 Elliott, John...... 217 Favaedi, Raha...... 162 Foster, Barbara A...... 65 Dubois-Dauphin, Michel...... 231 Ellis, James....160, 168, 177, 202 Fazzari, Jennifer...... 218 Fowlkes, Charless C...... 218 Duchesneau, Pascal...... 11 Ellis, Rebecca...... 136 Fecenko-Tacka, Karen...... 185 Fradette, Julie...... 98 Dugas, CHristian...... 191 Elsalanty, Mohammed...... 138 Fehlings, Michael...... 54, 56, Fraidenraich, Diego...... 180 Duncan, Andrew W...... 6 Emre, Nil...... 211, 214 ...... 102, 126 Frampton, Garrett M...... 234 Duncan, Greg J...... 57 Endo, Morinobu...... 155 Fehm, Tanja...... 70 Francesco, Faiola...... 246 Dupin, Elisabeth...... 59 Engle, Sandra J...... 42, 211 Felkner, Daniel J...... 206 Franciolli, André Luis...... 160 Durán, Rocío...... 54 Engler, Adam...... 132, 142, 226 Fend, Falko...... 70 Francioso, Edda...... 142 Durnall, Jennifer...... 221 Englert, Yvon...... 219 Feneley, Michael P...... 124 Francke, Uta...... 21 Duryagina, Regina...... 102 Englund, Mikael C.O...... 171 Feng, JiAn...... 171 Franco, Diogo...... 133 Dustin, Michael...... 123 Enzmann, Volker...... 111 Fennell, Myles...... 47 Frank, Joseph A...... 146, 148 Dutton, James...... 198 Eppert, Kolja...... 62, 62, 62 Ferguson, Gabriel B...... 118 Franke, Steffi...... 83 Dvash, Tamar...... 80 Epsztejn-Litman, Silvina...... 242 Ferguson, Linda...... 80 Frankel, Victor...... 146 Dvorak, Petr...... 236, 237, 240 Era, Takumi...... 174 Fernandes, Andrielle C.....8, 120, Frankland, Paul...... 49 Dworski, Shaalee...... 57, 158 Erdei, Zsuzsa...... 240 ...... 121 Franz, Wolfgang M...... 16 Dwyer, Benjamin...... 2 Erdemli, Esra...... 77 Fernandes, Isabella...... 160, 176, Franzin, Chiara...... 40, 89 ...... 176, 177 Dynan, William...... 117 Eriksson, Gustav...... 7 Fraser, Stuart...... 112 Fernandes, Karl J.L.....51, 51, 58, Eaker, Shannon...... 142 Erker, Laura...... 2 Freire, Ana...... 15 ...... 191 Eaves, Allen C...... 10, 208, 209 Erneux, Christophe...... 219 Freitas, Marcela C...... 120 Fernandez Espinosa, Darío D..155 Eaves, Connie J...... 220 ERoglu, Elif...... 243 French, Deborah L...... 188 Fernandez, Francis...... 102 Eberle, Irina...... 198 Ervasti, James M...... 179 Freschauf, Lauren...... 218 Ferrari, Roberto...... 23 Ebihara, Yasuhiro...... 170 Erwes, Kim...... 174 Freudenberg, Johannes...... 245 Fesahat, Farzaneh...... 26, 76, 87 Ebrahimi, Marzieh...... 133 Erwin, William M...... 102 Freudenreich, Dorian...... 194 ffrench-Constant, Charles...... 58 Eckardt, Sigrid...... 209 Esch, Daniel...... 247 Freundt, Eric C...... 227 Fibbe, Willem E...... 110 Edenhofer, Frank...... 224 Eschenhagen, Thomas...... 97 Frey, Peter...... 89 Filareto, Antonio...... 179 Eder, Alexandra...... 97 Esfandyari, Tuba...... 70, 71 Frias, Ana M...... 131 Filipovic, Radmila...... 46 Edward, Christina...... 71 Eskandar Afshari, Andrea...... 10 Friedman, Aaron R...... 54 Filippo, Thais R. M...... 53 Efrat, Shimon...... 165 Esmaeilian, Yashar...... 77 Friedman, Ann...... 114

255 ISSCR 9th Annual Meeting www.isscr.org

Presenter Index

Friedrich Ben-Nun, Inbar...... 185 Gasparetto, Emerson L...... 95 Goldhan, Jörg...... 135 Grompe, Markus...... 2, 6 Friedrich, Amy M...... 42 Gauthier, Morgane...... 167 Goldman, Orit Rachel...... 217 Gronthos, Stan...... 141, 156 Friedrichs, Stephanie...... 178 Gauvin, Robert...... 98 Goldman, Steven A.1...... 63 Grosveld, Frank...... 163 Frisén, Jonas...... 54 Ge, Xiaohu...... 174 Goldstein, Lawrence S.B...... 167 Grskovic, Marica...... 44, 96, 140 Frumkin, Tsvia...... 242 Gee, Kyle...... 250 Golebiewska, Anna...... 64 Gruber Mica, Yvonne...... 219 Frye, Janie...... 194 Geis, Christian...... 209 Gomes, Manuela E...... 131 Grumet, Martin...... 196 Fuchs, Elaine...... 27 Geiser, Dennis...... 142 Gómez, Ricardo...... 155 Gu, Ming...... 150 Fuegemann, Christopher J....138 Genheimer, Christopher...... 193 Gonzales, Kevin Andrew Uy...... Gu, Zhen...... 183 Fuentes Boquete, Isaac...... 144 Gennery, Andrew R...... 79 ...... 186 Guenechea, Guillermo...... 118 Fugère, Claudia...... 27 Gentner, Bernhard...... 62 Gonzalez-Murillo, Africa...... 118 Gueven, Sinan...... 131 Fuhrmann, Alexander...... 226 George, Joshy...... 70 Goodwin, Richard L...... 193 Guillot, Pascale V...... 153 Fujii, Teruo...... 179 Georget, Marie-Therese...... 154 GoosenS, Ki A...... 54 Gumei, Maha...... 6 Fujio, Masato...... 125 Gepstein, Amira...... 13 Gopinathan, Ajay...... 218 Guo, Wei...... 150 Fujisaki, Joji...... 111 Gepstein, Lior...... 13 Gordon, Renee J...... 192 Gupta, Bhavik...... 196 Fujiyoshi, Kanehiro...... 195 Gerald, William...... 65 Gorges, Laura...... 202 Gupta, Sanjeev...... 4, 4 Fukasawa, Hiroko...... 158 Gerbal-Chaloin, Sabine...... 232 Gorio, Alfredo...... 191 Gur Dedeoglu, Bala...... 77 Fukusumi, Hayato...... 173 Gerbino, Elvira...... 10 Gottlieb, David I...... 53 Gustafsson, Karin...... 113 Fulzele, Sadanand...... 138 Gerecht, Sharon...... 20 Götz, Magdalena...... 197 Gutfilen, Bianca...... 95 Funabashi, Hisakage...... 225 Gerlach, Debora...... 174 Gouon-Evans, Valerie...... 217 Guthrie, Kelly...... 193 Funk, Walter...... 82, 169 Germain, Lucie...... 27 Gourabi, Hamid...... 44 Guthrie, Siobhan...... 101 Furue, Miho K...... 175, 222 Gerst, Jennifer...... 70 Goyal, Deepika...... 117 Gutova, Margarita...... 148 Fusaki, Noemi...... 174 Gerstein, Mark B...... 232 Goyer, Benjamin...... 27 Guzman, Raphael...... 92 Fusar, Fulvia...... 10 Ghaedi, Kamran...... 156 Grabel, Laura...... 207 Ha, Hyung-Ho...... 184 Fuseler, John W...... 193 Ghaedi, Mahboobe...... 171 Grabole, Nils...... 199 Ha, Jeong Won...... 242 Fussner, Eden...... 177 Ghaffari, Saghi...... 238 Grace, Andrew...... 17 Hadjantonakis, Kat...... 104, 104 Gabr, Hala...... 2 Ghanekar, Anand...... 72 Grad, Sibylle...... 135 Hafeez, Sana...... 202 Gadue, Paul...... 236 Ghazizadeh, Zaniar...... 209 Grafodatskaya, Daria...... 168 Hafner, Markus...... 233, 241 Gafni, Yosef...... 60 Gheisari, Yousof...... 134, 145 Graham, Monica...... 222 Haft, Annelise...... 2 Gage, Fred H...... 187 Ghoochani, Ali...... 156 Gramolini, Anthony...... 104 Hagerman, Randi J...... 207 Gai, Hui...... 44 Gibson, Jason...... 81 Grauer, Matthias...... 70, 230 Haggarty, Stephen...... 43, 44, Galadari, Helia...... 116 Gieselmann, Volkmar...... 174 Graumann, JohaNnes...... 247 ...... 45, 46, 169 Galat, Vasil V...... 164 Gillich, Astrid...... 199 Grawé, Jan...... 113 Haldipurkar, Suhas...... 32 Galat, Yekaterina...... 164 Gimble, Jeffrey...... 147 Gray, Peter P...... 87 Hall, Christine A...... 237 Galindo, Layla T...... 53 Gioia, Jason...... 204 Greally, John...... 4 Hallowell, Shawn E...... 211 Gallagher, Denis...... 49 Girard, Mathilde...... 167 Greber, Boris...... 185 Hämäläinen, Riikka...... 43, 186 Gallagher, Patrick G...... 112 Glancy, Dylan...... 39 Grecian, Scott...... 250 Hamamah, Sammir...... 232 Gallegos Cardenas, Amalia...... Glazewski, Lisa...... 229 Greder, Lucas...... 198 Haman, André...... 113 ...... 181, 183 Glover, Joel C...... 122 Green, Jordan J...... 86 Hamdorf, Matthias...... 108 Gallo, Marco...... 64 Go, Kathryn...... 198 Greenberg, Norman M...... 88 Hamilton, Amy...... 94 Gamm, David M...... 34 Goessling, Wolfram...... 9 Grégoire, Catherine-Alexandra... Hamilton, Brad...... 198 Ganat, Yosif...... 212 Goh, Hwee Ngee...... 225 ...... 58 Hamilton, Laura...... 51, 191 Gangavarapu, Kalyan J...... 65 Goland, Robin...... 198 Gregory, Cynthia...... 119 Hamilton, Rebecca...... 81, 135 Gao, Xiaolin...... 163 Gold, Eric...... 146 Gregory, Kenton...... 119 Hammerick, Kyle E...... 134 Garcia Ii, Bradley H...... 206 Goldberg, Laura R...... 75 Gregory, Philip D...... 79 Hammond, Scott M...... 234 Garcia, Mariana...... 125 Goldbrunner, Roland...... 197 Griffin, Tagan...... 172 Hammoud, Lamis...... 22 Gardner, Lucy...... 215 Goldenberg, David M...... 67 Grimm, Sabrina...... 70, 133 Hampl, Ales...... 236, 237, 240 Garitaonandia, Ibon...... 205 Goldenberg, Regina C. S...... 95 Grinshtein, Natalie...... 67 HamRick, Mark W...... 138 Garner, Jodi...... 104 Goldhamer, David J...... 123 Griswold-Prenner, Irene...... 44 Han, Dong Wook...... 185

256 www.isscr.org Thursday Poster Abstracts

Presenter Index

Han, Ho Jae...... 242, 245, 245 Heckmann, Manfred...... 209 Higa, Kazunari...... 101 Horiguchi, Ikki...... 186 Han, Ilkyu...... 73 Hector, Ralph...... 159 High, Katherine...... 188 Hornecker, Jacey L...... 182 Han, Ji You...... 189 Heer, Rakesh...... 66 Higuchi, Akon...... 69 Hornsby, Peter J...... 182 Han, Yong-Mahn....21, 136, 221 Heffner, Garrett....109, 113, 117 Higuchi, Yuichiro...... 175 Horvath, Lindsay M...... 168 Hanada, Sachiyo...... 170 Hei, Derek J...... 206 Hilcove, Simon...... 208 Hosseini Salekdeh, Ghasem...... Hanazono, Yutaka...... 105 Heider, Andreas...... 139 Hilgaertner, Jianhua...... 26 ...... 7, 209 Hannan, Nicholas...... 216 Heider, Fanie Barnabé...... 54 Hill, David...... 237 Hotta, Akitsu...... 168, 177 Hannon, Gregory J...... 184, 195 Hein, Marco Y...... 169 Hill, Jonathan...... 78 Hou, Junjie...... 86 Hans, Stefan...... 194 Heiser, Diane...... 109 Hill, William D...... 138 Hou, Sheng-Mou...... 136 Hansen, Arne...... 97 Helps, Stephen...... 156 Hinman, Cassidy R...... 35 Houck, Marlys L...... 185 Hansford, Loen M...... 67 HembergeR, Myriam...... 215 Hiramatsu, Hidefumi...... 62 Hovatta, Outi...... 76, 190 Hara, Kazuo...... 155 Hemmesi, Katayoon...... 44 Hiramoto, Takafumi...... 170 Hoxha, Eneda...... 18 Harfouche, Rania...... 196 Hemmesi, Katayoun...... 47 Hirata, Mitsuhi...... 175, 222 Hsieh, Matthew...... 115 Hari, Aarya...... 32 Heng, Dominic Jian-Chien....186 Hirata, Shuji...... 158 Hsieh, Sing-Ying...... 84 Harichandan, Abhishek...... 133 Henschler, Reinhard...... 124, 198 Hiroyuki, Fukushima...... 18 Hsu, David...... 237 Harkness, Linda...... 158 Henshall, Tanya...... 156 Hirst, Claire...... 221 Hsu, Hsiang-Chun...... 84 Harness, Julie V...... 219 Heo, June Seok...... 126 Hirt, Marc...... 97 Hsu, Jenny...... 46 Harnprasopwat, Ratanakanit...... Herault, Olivier...... 154 Hitos, Ana B...... 243 Hsu, Matthew...... 155 ...... 114 Herber, Renee L...... 45 Hitoshi, Seiji...... 50 Hsu, Pei-Ju...... 153 Harris, David...... 26 Herberg, Samuel...... 138 Ho, Jaclyn...... 184 Hu, Gangzheng...... 141 Harris, James M...... 9 Herbert, Kirsten...... 37 Ho, Jennifer H...... 132, 132 Hu, Guang...... 245 Hart, Ronald P...... 172, 184, 196 Herbert, Mary...... 80 Ho, Jenny...... 64 Hu, Yuanyu...... 10 Hartman, Nathaniel W...... 207 Herblot, Sabine...... 113 Hoang, Trang...... 113 Hu, Zhixing...... 171 Hasegawa, Kouichi...... 214 Herland, Anna...... 49, 61, 68 Hochedlinger, Konrad...... 161 Hua, Haiqing...... 164 Hasegawa, Mamoru.....105, 174 Herman, Amanda M...... 13 Hockenbery, David...... 243 Huang, Anderson H.C...... 92 Hashimoto, Haruo...... 175 Hermans, Karin G...... 62 Hodges, Kathryn L...... 45 Huang, George T...... 150 Haskins, Jeffrey R...... 130 Hermanson, Spencer...... 60 Hodzic, Enes...... 101 Huang, Jian...... 90 Hassane, Abbad...... 233 Hernandez, Kristina...... 172 Hoh, Brain...... 25 Huang, Jun-Jae...... 84 Hasskamp, Joanne H...... 130 Hernando, Eva...... 248 Hohenstein Elliott, Kristi A...... Huang, Kevin...... 50 Hatami, Maryam...... 44, 234 Hernando-Momblona, Xavier...... 197, 197 Huang, Wei...... 67 Hatou, Shin...... 101 ...... 69 Hoke, Heather...... 234 Huang, Yen-Chih...... 96 Haviv, Izahk...... 70 Herr, Greg...... 84 Hoki-Fujimori, Yuko...... 177 Huang, Yen-Hua...... 77 Hayashi, Katsuhiko...... 199 Herrid, Muren...... 78 Holland, Michael...... 78 Huang, Yin...... 141 Hayashi, Kei...... 151 Herrmann, Bethany R...... 106 Holle, Andrew W...... 142 Hubbell, Jeffrey A...... 89 Hayashi, Satoshi...... 105 Herrmann, Kelsey...... 148 Holloway, Alisha K...... 18, 163 Huber, Irit...... 13 Hayashi, TaKuya...... 206 Herszfeld, Daniella...... 30 Holmbeck, Kenn...... 115 Hudson, James...... 79 Hayashi, Yujiro...... 74 Hesley, Jayne...... 92 Holmes, Michael C...... 79 Hughes, Christopher...... 87 Hayashida, Midori...... 175 Hess, David A...... 189 Holubcova, Zuzana...... 236 Hughes-Large, Jennifer...... 189 Hayhurst, Monica...... 41, 41 Hesse, Michael...... 138 Holzenberger, Martin...... 27 Huh, Yong Jun...... 174 Haylock, David...... 221 Hessl, David R...... 207 Hong, Huimei...... 249 Hulkower, Keren I...... 45 Hazelbauer, Stephanie...... 173 Hessman, Crystal J...... 68 Hong, Hyun Sook...... 195 Humphries, Jonathan D...... 241 He, Fan...... 98 Hesson, Jennifer...... 243 Hong, Kwan Soo...... 182 Humphries, Martin J...... 241 He, Jia-Qiang...... 14 Heximer, Scott...... 18 Hong, Seok-Ho...... 222 Hunt, Greg...... 14 He, Lin...... 184 Hibi, Hideharu...... 152 Hoofd, Catherine...... 219 Hunter, Arwen...... 20, 188 He, Xiaoming...... 179 Hidalgo, Juan...... 69 Hooshmand, Mitra J...... 50 Huss, Wendy J...... 65, 66 He, Xingyue...... 184 Hidalgo-Gonzalez, Alejandro...... Hope, Kristin...... 62 Hussein, Khaled...... 138 He, Zhiyong...... 112 ...... 79 Hope, Shelly...... 78 Hussein, Samer M...... 186 Head, Renee...... 64 Hidari, Kentaro...... 93 Horie, Kyoji...... 244 Hyatt, Elzbieta...... 73

257 ISSCR 9th Annual Meeting www.isscr.org

Presenter Index

Hyllner, Johan...... 171 Itinteang, Tinte...... 101 Jeong, Sang Young...... 128 Kamath, Anant...... 19, 194 Hyman, Anthony A...... 169 Ito, Masataka...... 32 Jeong, Sang-Hee...... 226 Kamiyoshi, Akiko...... 22 Hynes, Kim...... 141 Itoh, Isamu...... 155 Jiang, Fang-Xu...... 2 Kamme, Fredrik...... 197, 197 Hynes, Paul...... 67 Itskovitz-ELdor, Joseph...... 203 Jiang, Houbo...... 171 Kamp, Timothy J...... 13, 17 Hynes, Richard F...... 95 Ivanova, Natalia...... 232 Jiang, Man...... 207 Kan, Natalia...... 197 Hyon, Min Kyong...... 151 Ivanovic, Zoran...... 118 Jiang, Mei Hua...... 195 Kanda, Seiji...... 157 Hyttinen, Jari...... 19 Iwaniuk, Katharina...... 233 Jiang, Xin...... 92 Kandoi, Sangeetha...... 85 Ibrahimova, Vusala...... 137 Iwase, Akira...... 125 Jiang, Yuan...... 57 Kanegae, Yumi...... 114 Ibsirlioglu, Tulin...... 129 Iyer, Vivek...... 13 Jin, Hee Kyung...... 150 Kanematsu, Daisuke...... 173 Ichida, Justin K...... 182, 201 Izawa, Kiyoko...... 114 Jin, Sha...... 175 Kanemura, Yonehiro.....173, 175 Ichikawa-Shindo, Yuka...... 22 Jacoby-Alner, Tamara...... 2 Jin, Ying...... 109, 230 Kang Sk, Stamm C...... 31 Ichinose, Akihiro...... 148 Jadhaw, Neera...... 50 Jin, Yiran...... 152 Kang, Byeong Cheol...... 151 Ichisaka, Tomoko...... 231 Jager, Edwin W.H...... 61 Jincho, Yuko...... 177 Kang, Eun-Ju...... 180 Iemura, Masashi...... 175 Jain, Deepak...... 193 Jing, Donghui...... 98 Kang, Hoon-Chul...... 48, 174 Igarashi, Hiroshi...... 183 Jakobsson, Johan...... 59, 232 Jing, Naihe...... 207 Kang, Hwan-Goo...... 226 Iglesias, Mar...... 69 James, William...... 162 Jinno, Hiroyuki...... 158 Kang, Kyung Sun...... Ignatchenko, Vladimir...... 104 Jamieson, Emma...... 2 Johannisson, Jenny...... 171 ...... 158, 159, 159 Ihn, Hironobu...... 174 Janahmadi, Mahyar...... 47 John, Annie...... 102, 103 Kang, Minjung...... 104 Iida, Akihiro...... 174 Janan, Arghavan...... 76 Johnson, Carla...... 250 Kang, Nam-Young...... 184 Ikenaka, Kazuhiro...... 50 Jang, Il-Ho...... 109 Johnson, Julie A...... 157, 173 Kang, Soo Kyung...... 158, 159 Ikeno, Masayuki...... 125 Jang, Jiho...... 174 Johnson, Julie C...... 206 Kang, Tae Wook...... 159 Ilagan, Roger...... 193 Jang, Jin Hwa...... 158, 159 Johnson, Mahlon...... 63 Kanz, Lothar...... 70 Illes, Judy...... 38 Jang, Min Woo...... 245 Jokubaitis, Vanta J...... 221 Kaplan, David...... Im, Young BIn...... 158 Jang, Moon Ju...... 30 Jones, Andrew...... 226 ...... 49, 53, 56, 58, 67 Inaba, Mayu...... 78 Jang, Young Joo...... 239 Jones, Gemma N...... 153 Karamali, Fereshteh...... 156 Incorvaia, Elisabetta...... 51 Jang, Yu Jin...... 189 Jones, Michael...... 80 Karbalaei, Khadijeh...... 156 Ingrungruanglert, Praewphan..... Janich, Peggy...... 26 Jones, Morgan...... 114 Kardel, Melanie D...... 220 ...... 199 Janowska-Wieczorek, Anna...... Jönsson, Marie...... 232 Kareta, Michael...... 163, 202 Inlay, Matthew A...... 23 ...... 190 Jordan, Brian J...... 183 Karimi, Negar...... 127 Inman, Robert D...... 102 Jansson, Linda C...... 225 Jordan, Erin T...... 183 Karlsson, Christine...... 107 Inoue, Haruhisa...... 172 January, Craig T...... 17 Jordan, Kay...... 146 Karmacharya, Rakesh...... 45 Inoue, Kazutomo...... 221 Jaramillo, Maria...... 1 Joseph, Brigid...... 4 Karp, Jeffrey M...... 146, 153 Inoue, Makoto...... 105 Jarmalaite, Sonata...... 121 Josselyn, Sheena A...... 58 Kasama, Yasuji...... 177 Inoue, Shin-Ich...... 222 Jaros, Josef...... 237 Jothi, Raja...... 245 Kasar, Siddha...... 180 Iohara, Koichiro...... 151 Jasmin, Fnu...... 150 Joussen, Sylvia...... 124 Kaslin, Jan...... 194 Irion, Stefan...... 140 Javaherian, Ashkan...... 44 Judson, Robert...... 166 Kasper, F. Kurtis...... 134 Irion, Stephan...... 96 Javan, Mohammad...... 43 Juliano, Luiz...... 53 Kass, Robert S...... 12, 13 Irwin, Meredith S...... 67 Jazedje, Tatiana...... 133 Juliano, Maria A...... 53 Kassem, Moustapha...... 158 Isailovic, Lisa...... 90 Jeannet, Robin...... 114 Jun, Yamashita K...... 18 Kassmer, Susannah H...... 140 Isasi, Rosario...... 38 Jee, Min Ki...... 159 Jung, Hye Lim...... 146, 148 Kataoka, Masateru...... 3 Isenmann, Sandra...... 141 Jelicks, Linda A...... 150 Jung, Jin Sun...... 158, 159 Katayama, Shiori...... 229 Isern, Joan...... 112 Jenkin, Graham...... 30 Jung, Ji-Won...... 159 Katsman, Diana...... 36 Ishino, Yugo...... 50 Jensen, Janne...... 7 Jung, Peter...... 69 Katsuda, Takeshi...... 145 Islam, Diana...... 102 Jeon, Hong Bae...... 128 Jung, Sunghoon...... 144 Kattman, Steve...... 12, 18 Isobe, Eri...... 41 Jeon, Iksoo...... 182 Junqueira-Reis, Luiza C...... 176 Kaufer, Daniela...... 54 Israel, Mason A...... 167 Jeon, Ji Hoon...... 245, 245 Jurga, Marcin...... 127 Kaur, Pritinder...... 70 Israsena, Nipan...... 199 Jeon, Jongwook...... 21 Ka Yan, Ng...... 1 Kaviani, Saeed...... 61 Issaragrisil, Surapol...... 24, 75 Jeon, Young Joo...... 205 Kahler, David J...... 164 Kaviani, Saeid...... 119

258 www.isscr.org Thursday Poster Abstracts

Presenter Index

Kawabata, Kenji...... 175 Khurana, Satish...... 116 Kim, Seok Hyun...... 208 Kohara, Arihiro...... 175 Kawada, Jiro...... 179 Kiang, Alan...... 66 KiM, Sung Joon...... 208 Kohmoto, TakuShi...... 15 Kawai, Shinji...... 175 Kiani, Abbas...... 156 Kim, Vanuytsel...... 216 Kohn, Joachim...... 93 Kawakita, Tetsuya...... 32 Kiani, Sahar...... 43, 44, 47 Kim, Won-Tae...... 239 Kokaia, Zaal...... 54 Kawamura, Suguru...... 222 Kido, Tsuneo...... 85 Kim, Yeji...... 221 Kokorsch, Philipp...... 70 Kawasaki, Toshiyuki...... 206 Kiessling, Ann A...... 238 Kim, Yoon Young...... 208 Kokubu, Chikara...... 244 Kawashima, Motoko...... 32 Kikkawa, Fumitaka...... 125, 152 Kim, Young-June...... 239 Kollar, Katarina...... 124, 124 Kawate, Hisaka...... 22 Kikuchi, Tetsuhiro.172, 205, 206 Kimber, Susan...... 241 Komljenovic-Blitva, Danijela...... Kazanis, Ilias...... 58 Kilmare, Eva...... 171 Kimmel, Lida H...... 42 ...... 101 Kazim, A L...... 65 Kilpatrick, Lauren A...... 50 Kimura, Hiroshi...... 179 Konantz, Martina...... 230 Keating, Armand...... 71, 137 Kim, Bong Sun...... 158, 159 Kimura, Kenichi...... 140 Kondrikova, Galina...... 138 Keegan, Catherine E...... 114 Kim, Boyon...... 244 Kinehara, Masaki...... 222 Koo, Hong Hoe...... 146, 148 Kehoe, Daniel E...... 85 Kim, Cheoljin...... 94 Kingham, Paul J.1...... 192 Koole, Wouter...... 243 Keirstead, Hans S...... 190, 219 Kim, Cheorl-Ho....239, 239, 239 Kinoshita, HaruYuki...... 179 Kopparam, Jawahar...... 196 Keller, Charles...... 39 Kim, Dae Seong...... 146, 148 Kirby, Elizabeth D...... 54 Korzh, Vladimir...... 235 Keller, Gordon...... 12, 12, 13, Kim, Dae Wook...... 195 Kirkeby, Agnete...... 232 Kositzke, Beth...... 173 ...... 18, 18, 49, 220, 234 Kim, Dae-Sung...... 48, 212 Kirouac, Daniel C...... 108 Kostyszyn, Beata...... 210 Keller, Jonathan...... 24 Kim, Dohoon...... 221 Kiselev, Sergey...... 164 Kotton, Darrell...... 12, 188 Keller, Margaret A...... 185 Kim, Dong-Wook...48, 174, 212 Kish, Vincent L...... 98 Kouhkan Kahangi, Fatemeh.. 116 Kelley, Jessica J...... 81 Kim, Eu-Joo...... 226 Kishore, Raj...... 18 Koyama, Teruhide...... 22 Kelley, Rusty...... 193 Kim, Eunkyoung...... 126 Kiskinis, Evangelos...... 166 Kozhich, Olga...... 81 Kellner, C P...... 25 Kim, Han-Soo...... 126 Kislinger, Thomas...... 104 Kozlov, Sergei...... 164 Kelly, Kathy...... 67 Kim, Hye Eun...... 205 Kleiman, Robin J...... 42 Kraus, Petra...... 89 Kelly, Kevin F...... 250 Kim, Hye Jin...... 146, 148 Klein, Peter...... 90 Krause, Diane S...... 140 Kelly, Michael...... 92 Kim, Hye Ryung...... 146, 148 Kletzel, Morris...... 67, 127 Krause, Karl-Heinz...... 231 Kelsey, Peter...... 9 Kim, Hye Sun...... 30 Klindworth, Amber...... 198 Kremer, Karlea...... 156 Kempf, Volkhard A.J...... 97 Kim, Hyo-Jin...... 189 Klungland, Arne...... 249 Krieg, Lisa...... 16 Kennedy, James...... 63 Kim, Hyo-Soo...... 12, 199 Knopf, Anne...... 223 Kriks, Sonja...... 212 Kennedy, Marion...... 220 Kim, Hyun Ok...... 126 Knoppers, Bartha Maria....38, 38 Krishnamurthy, Prasanna...... 18 Kerboeuf, Dominique...... 154 Kim, Hyung-Sik...... 159 Ko, Kinarm...... 247 Krishnan-Kutty, Viknish...... 47 Kerkelä, Erja...... 19 Kim, Jaemin...... 212 Ko, Minoru S.H...... 242 Kroehne, Volker...... 194 Kerr, Douglas...... 229 Kim, Jeong Beom...... 247 Ko, Si-Hwan...... 126 Krueger, Winfried H...... 187 Khalili, Saeed...... 157 Kim, Ji Young...... 174, 212 Koba, Wade...... 150 Krug, Edward L...... 50 Khamisipour, Gholamreza...... 61 Kim, Jiyoung...... 195 Kobayashi, Gerson S...... 133 Krupa, Rachel A...... 82 Khan, Iram F...... 201 Kim, Jong Min...... 151 Kobayashi, Mami...... 41 Ku, Chia-Chi...... 132 Khankaldyyan, Vazgen...... 148 Kim, Jong-Hoon...... 189 Kobayashi, Seiichiro...... 114 Ku, Seung-Yup...... 33, 204, 208 Khanna, Anu...... 94 Kim, Jong-Hyun...... 189 Kobayashi, Yoshiomi...... 195 Kubaczka, Caroline...... 105 Khattak, Shahryar...... 202 Kim, Jumi...... 205 Koblar, Simon A...... 156 Kuckenberg, Peter...... 105 Kheolamai, Pakpoom...... 24 Kim, Jun Young...... 184 Koch, Carmen M...... 124 Kuehn, Mathias...... 83 Khillan, Jaspal Singh...... 250 Kim, Ju-Young...... 12 Koch, Philipp...... 169, 209 Kujala, Kirsi...... 19 Khine, Michelle...... 218 Kim, Kun-Yong...... 168 Koch, Steffen...... 223 Kulea, Tony...... 93 Kho, Jordan...... 201 Kim, Kyoung Soo...... 239 Kockx, Christel E.M...... 163 Kuligowski, Sandra...... 147 Khoja, Suhail...... 196 Kim, Kyung Sul...... 30 Kodama, Shohta...... 157 Kumar, Ajit...... 85 Khoo, Melissa L.M...... 106 Kim, Mi Ok...... 242, 245 Koedam, Marijke...... 134 Kumar, Namit...... 146 Khoobyari, Shiva...... 5 Kim, Minjung...... 109 Koekkoek, Karin...... 110 Kumar, Pooja Ashok...... 10 Khorad-Mehr, Arezoo...... 76, 87 Kim, Natalie...... 123 Koga, Takeshi...... 151 Kume, Kazuhiko...... 3 Khoshchehreh, Reyhaneh.....133 Kim, Nury...... 221 Koh, Gou Young...... 21 Kume, Shoen...... 3 Khosrotehrani, Kiarash...... 27 Kim, Sang Yong...... 184 Koh, Young Jun...... 21 Kunova, Michaela...... 240

259 ISSCR 9th Annual Meeting www.isscr.org

Presenter Index

Kuo, Hung-Chih...... 76 Larsson, Jonas...... 107 Lee, Man Ryul...... 239 Li, Shuyi...... 117 Kuo, Min-Liang...... 136 Laslett, Andrew L...... 83 Lee, Min Ji...... 205 Li, Steven Sl...... 240 Kuo, Selena...... 66 Lassiter, Chelsea...... 207 Lee, Myoung Woo...... 146, 148 Li, Ting...... 25 Kuraitis, Drew...... 82 Lau, Singting...... 68 Lee, Nayeon...... 182 Li, Wan-Ju...... 97 Kuranda, Klaudia...... 72 Lauber, Kirsten...... 97 Lee, Sahmin...... 12 Li, Wenzhao...... 141 Kurdistani, Siavash K...... 23 Laurent, Louise...... 60, 83, Lee, Seung Tae...... 89 Li, Xiao Bing...... 222 Kurek, Dorota...... 243 ...... 169, 185 205 Lee, Seunghee...... 159 Li, Xiaojie...... 63 Kusuma, Sravanti...... 20 Lavin, Martin F...... 164 Lee, Soo Hyun...... 146, 148 Li, Xinmin...... 23 Kuznetsov, Sergei A...... 135 Lavoie, Amélie...... 27 Lee, Soo Youn...... 45 Li, Xue-Jun...... 211 Kwek, Joly...... 215 Lavon, Neta...... 237 Lee, Sung Chan...... 184 Li, Yingchun...... 30 Kwok, Showming...... 43, 169 Lawrence, Stanton...... 249 Lee, Wenqing Jean...... 89 Li, Yuanyuan...... 207 Kwon, Brian...... 57 Lazzaro Jr, John T...... 42 Lee, Won I...... 178 Li, Yukui...... 152 Kwon, Euna...... 151 Le Bihan, Thierry...... 236 Lee, Yu Jin...... 242, 245, 245 Li, Yun...... 78 Kwon, Heechung...... 27 Le Douarin, Nicole M...... 59 Leeder, Rachel...... 52, 77 Li, Zhe...... 117 Kwon, Jihye...... 182 Le Roy, Helene...... 141 Lehnert, Sigrid...... 78 Li, ZhenshenG...... 80 Kwon, Yoo-Wook...... 12, 199 Le, Sheila...... 211 Lei, Ienglam...... 163 Li, Zhentian...... 117 Kyba, Michael...... 96, 179 Lebkowski, Jane S...... 14 Leibel, Rudolph...... 198 Li, Zhi-Hua...... 235 Laabi, Yacine...... 167 Leblanc, Sarah...... 146 Lemischka, Ihor R...... 248 Li, Ziyi...... 181 Lacitignola, Luca...... 142 Lecaudé, Stéphanie...... 111 Lengerke, Claudia...... 70, 230 Lian, Xiaojun...... 13 Lackford, Brad...... 245 Lechman, Eric R...... 62 Leonard, Brian...... 21 Liao, Chih-Ching...... 84 Lacombe, Julie...... 113 Lecuyer, Camille...... 167 Leonardo, Trevor...... 205 Liau, Brian...... 15 Lacorazza, Daniel...... 116 Lee, Dongjin R...... 212 Leong, Cheryl...... 184 Lichtner, Björn...... 222 Lacroix, Steve...... 191 Lee, Dong-Sup...... 88 Leong, Kam W...... 15 Lie, D. Chichung...... 197 Ladurner, Peter...... 195 Lee, Dung-Fang...... 248 Leong, Wai K...... 156 Lie, Khun H...... 167 Laevsky, Ilana...... 203 Lee, Eunah...... 103 Lesende Rodriguez, Iván Aarón.. Liedtke, Christian...... 8 Lagarkova, Maria...... 164 Lee, Gabsang...... 213, 219 ...... 144 Lightowler, Sally...... 29 Lahesmaa, Riitta...... 186 Lee, Gene...... 88 Lessard, Martine...... 115 Lilja, Anna M...... 190 Lai, Hsing-Hua...... 84 Lee, Haksup...... 204 Levadoux-Martin, Marilyne...... Lily, Ngotran...... 207 Lajoie, Gilles...... 87 Lee, Heekyung...... 27 ...... 218 Lim, Bo Young...... 48, 212 Lakshmipathy, Uma...... Lee, Heon-Jin...... 115 Levenberg, Shulamit...... 210 Lim, Boyoung...... 174 ...... 60, 80, 250 Lee, Hey Jin...... 30 Levern, Yves...... 154 Lim, Daniel A...... 51 Lam, Alexander...... 5 Lee, Hoong-Chien...... 69 Levin, Trevor...... 28 Lim, Jeong Mook...... 244 Lam, Clarrie K...... 57 Lee, Hsuan-Shu...... 3 Levine, Stuart S...... 163 Lim, Ji Eun...... 195 Lam, Kelvin...... 213 Lee, Hyojin...... 55 Levison, Steven W...... 52 Lim, Shushan...... 249 Lambers, Erin...... 18 Lee, Hyo-Sang...... 180 Levy Lahad, Ephrat...... 242 Lim, Siew Joo...... 10 Lambshead, Jack...... 83 Lee, Hyun...... 150 Lew, Valerie...... 218 Lim, Sunki...... 103 Lamouille, Samy...... 166 Lee, Hyun-Seung...... 182 Lewis, Martin D...... 156 Lima, Maria J...... 2 Lampron, Antoine...... 115 Lee, Jeannie T...... 43 Lezama, LindsEy...... 173 Lin, Chao-Po...... 184 Lamrissi-Garcia, Isabelle...... 118 Lee, Jeong-Ah...... 174 Li, Fugen...... 163 Lin, Charles P...... 111 Lane, Michelle...... 247 Lee, Jieun...... 188 Li, Guangnan...... 52 Lin, Charles Y...... 161 Lang, Hainan...... 50 Lee, Jin-Sung...... 45 Li, Gui-Rong...... 156 Lin, I-Ying...... 76 Langston, William...... 171 Lee, Jong-Hee...... 222 Li, Jingting...... 128 Lin, Jia-Wei...... 64 Lanner, Fredrik...... 104, 177 Lee, Joo-Eun...... 12 Li, Kelly...... 204 Lin, Kuo-I...... 76 Lanz, Thomas A...... 42 Lee, Jung Bok...... 222 Li, Ming...... 199 Lin, Meng-Hsueh...... 84 Larocca, David...... 82, 169 Lee, Kelvin...... 229 Li, Mingming...... 222 Lin, Qiong...... 124, 228 Larouche, Danielle...... 27 Lee, Kibum...... 94 Li, Mira...... 101 Lin, Ruby C.Y...... 167 Larsen, Elisabeth...... 249 Lee, Kyung Il...... 205 Li, Pulin...... 117 Lin, Su-Yo...... 84 Larsson, Hans Mattias...... 89 Lee, Lilian...... 64 Li, Qing...... 108 Linay, Fabien...... 27

260 www.isscr.org Thursday Poster Abstracts

Presenter Index

Lindsey, Benjamin W...... 61 Lundin, Vanessa...... 61, 68 Mandefro, Berhan...... 237 Masterson, Keith...... 180 Lindvall, Olle...... 54, 182 Lünskens, Raphaela...... 174 Mandegar, Mohammad A....196 Masuda, Shigeo...... 105 Ling, Qing-Dong...... 69 Luo, Chenmei...... 198 Mändle, Tanja...... 97 Mathieu, Chantal...... 216 Ling, Thai-Yen...... 11, 77, 189 Luo, Jiesi...... 230 Mangues, Ramón...... 69 Matsubara, Hiroki...... 125 Lipchina, Inna...... 241 Luo, Wylie...... 114 Manian, Kannan V...... 166, 178 Matsumura, Hiroko...... 175, 222 Lipman, Tatiana...... 67 Lushaj, Entela...... 15 Mann, Matthias...... 169, 247 Matsuoka, Hideaki...... 225 Lipton, Stuart A...... 55 Lutz, Dominique...... 169 Manninen, Tuula...... 43 Matsushita, Shonosuke...... 140 Liu, Er...... 93 Luzna, Pavla...... 63 Manochantr, Sirikul...... 24, 75 Matsuyama, Akifumi...... Liu, Jie...... 57 Ly, Irene...... 151 Manz, Markus G...... 120 ...... 39, 149, 149 Liu, Jing...... 183 Lynch, Candace...... 60, 83, Marceau Fortier, Guillaume.....98 Matsuzaka, Emiko...... 170 Liu, Ting...... 152 ...... 169, 205 Marchand, Melanie...... 21 Mattioli, Mauro...... 31 Liu, Yen-Nien...... 67 Lyons, Ian...... 80 Marcin, Jurga...... 141 Mattout, Anna...... 161 Liu, Ying...... 60, 183 M.S. Cabral, Joaquim...... 147 Marfia, Giovanni...... 191 Matulka, Kamil...... 240 Liu, Younan...... 157 Ma, David D.F...... 106 Margineantu, Daciana...... 243 Matushita, Hamilton...... 121 Liu, Yubing...... 181 Ma, Feng...... 170 Maric, Dragan...... 115 Matusyama, Akifumi...... 148 Liu, Zhengian...... 70 Ma, Hong...... 180 Marinka-Kalmani, Keren...... 60 Matzner, Ulrich...... 174 Livak, Kenneth J...... 94 Ma, Jian...... 42 Marino, Elena...... 10 Maucksch, Christof...... 192 Lo Riso, Pietro...... 79 Ma, Lijun...... 152 Marques, Alexandra P...... 131 Maulion, Christopher...... 224 Lo, Mandy Y...... 160 Ma, Xiaocui...... 5, 5 Marques, Fernanda F...... 22 Maurino, Christopher...... 75 Lobato Da Silva, Cláudia...... 147 Ma, Xiaona...... 152 Marquez-Curtis, Leah A...... 190 Mauritz, Christina...... 228 Lomax, Michael A...... 227 Ma’ayan, Avi...... 246 Marrano, Marco A...... 67 Maury, Yves...... 167 Lombardo, Angelo...... 79 Maass, Karen...... 224 Marrano, Paula...... 67 Mayer, Balazs...... 115 Lomberk, Gwen A...... 74 Mackiewicz, Zygmunt...... 121 Martin, Franz...... 243 Mayr, Manuel...... 91 Long, Xiaoxia...... 127 Macmillan, Annette...... 185 Martin, Ivan...... 131 Mazurier, Frédéric...... 118 Longaker, Michael T...... 21 Madaschi, Laura...... 191 Martin, Jody...... 211 Mazzarol, Giovanni...... 10 Lopez Rodriguez, Yelica V.....125 Madden, David T...... 241 Martin, Philip...... 67 Mazzolini, Guillermo...... 125 López, Javier E...... 16 Madison, Jon...... 42, 46 Martin, Ulrich...... 228 Mazzon, Cristina...... 123 López-Barneo, José...... 54 Magalhães, Danielle A...... 120 Martinat, Cecile...... 167 Mccann, Anna...... 60 Loring, Jeanne...... 60, 83, Magnúsdóttir, Erna...... 199 Martinez, Fernando J...... 44 Mccarrey, John R...... 182 ...... 169, 185, 205 Maguer-Satta, Veronique...... 129 Martinez, Joaquin...... 71 Mcclendon, Britney N...... 135 Losordo, Douglas...... 18 Mahon, Kerry...... 198 Martinez, Rita...... 44 Mccollough, Amanda...... 145 Loturco, Joseph...... 46 Maillard, Ivan...... 114 Martinez, Yannick...... 231 Mcconnell, Michael J...... 187 Lotz, Steven...... 211 Maiman, Dennis J...... 126 Martinez-Agosto, Julian...... 118 Mccreedy, Dylan A...... 53 Louis, Sharon A...... 10, 208, 209 Maines, Jean...... 78 Martinez-Arroyo, Ana...... 69 Mcdonald, Courtney...... 30 Loutradis, Dimitris...... 238 Maiolino, Angelo...... 95 Martins, Daniele S...... 120, 121 Mcgehee, Cordelia...... 74 Lu, Bin...... 23 Majdic, Gregor...... 125 Martins, Marilia T...... 133 Mcgill, Sean...... 207 Lu, Chi-Wei...... 83, 216 Majumder, Anirban...... 46 Marty, Andrea L...... 206 Mcguckin, Colin...... 127, 141 Lu, Jonathan...... 13 Makieski, Jonathan C...... 17 Maruotti, Julien...... 34 Mcivor, Scott R...... 179 Lu, Lin...... 188 Malan, Daniela...... 178 Marusciac, Laura...... 124 Mckay, Ronald...... 81, 135 Lu, Yangqing...... 183 Malaterre, Jordane...... 29 Marutle, Amelia...... 190 Mckearin, Dennis...... 78 Luce, Sonia...... 107 Malavarca, Richard...... 47 Mason, Clark...... 90 Mckee, Christina.....75, 143, 231 Lucero, Jovanny...... 50 Malchenko, Sergey...... 164 Mason, Mike...... 140 Mckeever, Paul...... 74 Ludlow, John W...... 193 Malcov, Mira...... 242 Mason, Robert W...... 229 Mckeon, Frank...... 10 Ludwig, Tenneille E...... 206 Mali, Prashant...... 20 Masoudi, Najmeh...... 44 Mckercher, Scott R...... 55 Lufkin, Thomas...... 89 Mallon, Barbara...... 81, 135 Masselot, Bernadette...... 72 Mckinney-Freeman, Shannon..... Lugo-Martinez, Veronica- Malmegrim, Kelen C.R...... 123 Masserdotti, GiacomO...... 197 ...... 109, 113, 117 Haydée...... 143 Malone, Cindy S...... 235 Massumi, Mohammad...... Mclaughlin, K. John...... 209 Lujan, Ernesto...... 56 Manavis, Jim...... 156 ...... 99, 100, 228 Mcleod, Jessica...... 62

261 ISSCR 9th Annual Meeting www.isscr.org

Presenter Index

Mcrae, Scott...... 80 Milhavet, Ollivier...... 232 Moon, Byoung San...... 91 Nagano, Masumi...... 140 Meani, Natalia...... 10 Miller, Freda D.....49, 53, 56, 57, Moon, Jung-Il...... 221 NaGao, Yoshikazu...... 105 Medin, Jeffrey A...... 137 ...... 58, 158 Moon, Shin Yong...... Nagy, Andras...... 186, 203 Mehrabi, Nasim...... 192 Miller, Robert H...... 227 ...... 33, 204, 208 Naik, Pooja...... 72 Mehrkens, Arne...... 131 Millman, Jeffrey R...... 223, 224 Moore, Jennifer C...... Najbauer, Joseph...... 148 Mei Raz, Nave...... 242 Mills, Jason A...... 188 ...... 172, 184, 196 Najm, Fadi J...... 227 Meiler, Steffen E...... 117 Minami, Itsunari...... 17, 17 Moore, John J...... 106 Nakagawa, Masato...... 231 Meis, Judy E...... 94 Minden, Mark...... 62, 62 Morahan, Grant...... 2 Nakamura, Kenta...... 17 Meisner, Lorraine F...... 157, 173 Ming, Guo-Li...... 96 Moralli, Daniela...... 196 Nakamura, Masato...... 173, 175 Melkoumian, Zara...... Mir, Alain...... 90, 94 Moreira, Caroline M...... 53 Nakamura, Masaya...... 195, 232 ...... 20, 32, 80, 81, 84 Miremadi, A...... 28 Moretto Zita, Matteo...... 30 Nakamura, Michiko...... 76 Melkounian, Zara...... 175 Miriuka, Santiago G...... 155 Morey, Robert...... 169 Nakamura, Miki...... 177 Mello, Fernanda U...... 120 Mirnajafi-Zadeh, Seyad-Javad..... Morizane, Asuka...... Nakamura, Tomonori...... 231 Mello, Luiz E...... 53 ...... 47 ...... 172, 205, 206 Nakano, Austin...... 23 Mellott, Aj...... 125 Miropolsky, Yael...... 203 Morley, Gregory...... 224 Nakano, Haruko...... 23 Melo, Fernanda U F...... 8 Mirza, Asma...... 143 Morozova, Olena...... 67 Nakashima, Misako...... 151 Melton, Douglas...... 198, 213 Mishima, Hajime...... 140 Morrison, Sean...... 74, 110 Nakaso, Kazuhiro...... 171 Mendez-Otero, Rosalia...... Mishima, Kenji...... 32 Morshead, Cindi...... 52, 77, Naldini, Luigi...... 62, 79 ...... 95, 150 Miskinyte, Giedre...... 121 ...... 100, 191 Nall, Dara...... 73 Meneghello, Giulia...... 61 Mital, Seema...... 202 Mortazavi, Yousef...... 145 Nam, Seungwoo...... 103 Menezes, Gustavo G...... 22 Mitalipov, Shoukhrat...... 180 Mortens, Daniel...... 75 Narwani, Kavita...... 237 Mengarelli, Isabella...... 233 Mitchell, Amanda...... 62 Mortier, Laurent...... 72 Nascimento, Diana...... 15 Menke, Sandra...... 228 Miyagawa, Shigeru...... 148 Moschidou, Dafni...... 153 Nasello, Cara Marie...... 83 Mercadante, Marcos...... 176 Miyagawa, Shinichi...... 22 Moslem, Mohsen...... 180 Nasr-Esfahani, Mohammad Mercola, Mark...... 55, 197 Miyagoe-Suzuki, Yuko...... 41 Mosley, Adriane...... 23 Hossein...... 156 Meregalli, Mirella...... 160 Miyashita, Hideyuki...... 101 Mossahebi Mohammadi, Majid.. Natarajan, Anuradha...... 194 Merli, Devide...... 191 Mizuguchi, Hiroyuki...... 175 ...... 61, 116 Natesan, Sridaran3...... 63 Merlos-Suárez, Anna...... 69 Moats, Rex...... 148 Mostoslavsky, Gustavo...... Navara, Christopher S...... 182 Meschino, Michael...... 189 Mocco, J...... 25 ...... 42, 188 Navarro, Claire...... 160 Meshorer, Eran...... 161 Mochizuki, Shinji...... 170 Moussa, Leon...... 30 Nayak, Shreya...... 227 Messaggio, Fanuel...... 191 Moffett, Ashley...... 215 Mraz, Marek...... 236 Nayler, Samuel P...... 164 Metz, Marianne...... 148 Moghe, Prabhas...... 93 Mueller-Hoecker, Josef...... 16 Nazar, Ely...... 196 Mewe, Marco...... 97 Mohamad, Osama...... 218 Mukherjee, Sach...... 187 Neilsen, Nancy...... 142 Meyer, Jason S...... 32 Mohammadi, Shahin...... 116 Müller, Albrecht M...... 209 Nelander, Jenny...... 232 Meyer, Mona...... 64 Mohammed, Noor...... 112 Müller, Hans-Werner...... 233 Nelson, Angel...... 243 Meyn Iii, Malcolm A...... 229 Mohan, Beena...... 103 Mumaw, Jennifer L...... 183 Nelson, Craig E...... 81 Mezey, Eva...... 115 Molina, Javier...... 118 Muñoz, Purificación...... 69 Nemati, Shiva...... 44 Mica, Yvonne...... 213 Mollamohammadi, Sepideh...... Munro, Trent P...... 87 Nemeth, Krisztian...... 115 Michael, Young J...... 34 ...... 162 Munsie, Megan...... 37, 37, 37 Nemeth, Michael...... 115 Miglino, Maria A...... 120, 121 Momen Dizghandi, Hassan...... Muotri, Alysson...... 176 Neo, Boon Hoe...... 10 Miglino, Maria Angélica...... 228 Murakami, Rumi...... 41 Nethercott, Hubert E...... 207 ...... 160, 176, 176, 177 Monaco, Zoia L...... 196 Murphy, Christopher J...... 151 Neto, Mário S A...... 8 Mihaila, Silvia M...... 131 Monge, Matthieu...... 110 Murphy, William...... 95 Ng, Darice J...... 20 Mihailovic, Aleksandra...... 241 Monsma, Scott A...... 206 Musheer Aalam, Syed Ng, Elizabeth...... 208, 221 Mohammed...... 166, 178 Mi-Jin, Jang...... 136 Montague, Susanne C...... 185 Ng, Huck-Hui...... 186, 237 Muttini, Aurelio...... 31 Miki, Toshio...... 250 Montel-Hagen, Amelie...... 23 Ng, Siemon...... 186 Myrglot, Katelin S...... 84 Mikkola, Hanna K.A...... 23 Montgomery, Karen D...... 206 Ngan, Elly...... 68 Nagai, Mika...... 155 Mikos, Antonios G...... 134 Montgomery, Robert...... 29 Nguyen, Diep...... 218

262 www.isscr.org Thursday Poster Abstracts

Presenter Index

Nguyen, Ha Nam...... 21, 171 Okamura, Ross M...... 14 Panckeri, Kimberly...... 185 Paula, Ana M...... 22 Nguyen, Leana...... 140 Okano, Hideyuki...... 195, 232 Panella, James...... 173 Paulson, Robert F...... 110 Nguyen, Ngoc Tue...... 5, 5 Okazaki, Haruka...... 157 Panigrahy, Dipak...... 117 Pawlak, Piotr...... 231 Nguyen, Thi Mai Uyen...... 219 Okimura, Ayano...... 22 Panula, Sarita P...... 76 Payne, Natalie...... 30 Nguyen, Thu Minh...... 38 Okita, Keisuke...... 172 Pao, Hsiang-Yin...... 132, 132 Pease, Jim...... 94 Ni, Jessie H.T...... 84 Okura, Hanayuki...... Papa, Elaine...... 75 Pedersen, Roger A...... Niakan, Kathy K...... 215 ...... 148, 149, 149 Pappa, Andrew K...... 135 ...... 14, 215, 215 Nichol, Helen...... 92 Oliveira, Gislane L. V...... 123 Parada, Luis F...... 64 Pei, Duanqing...... 201 Niclou, Simone P...... 64 Oliveira, Maria E...... 120, 121 Paramban, Rosanto I...... 214 Pei, Ming...... 98, 128 Nilsson, Susie...... 221 Olson, Carl...... 162 Paramchuk, Wendy J...... 144 Peitz, Michael...... 105, 174 Ninagawa, Nana...... 41 Olson, Eric N...... 234 Parast, Mana...... 30 Pekkanen-Mattila, Mari...... 19 Nishi, Rebecca...... 50 Olsson, Cia...... 186 Pardal, Ricardo...... 54 Pelekanos, Rebecca...... 136 Nishihama, Natsumi...... 170 Olszewski, Marie...... 67 Pare, Jean-Francois...... 1 Pelicci, Pier Giuseppe...... 10 Nishimoto, Kevin P...... 14 Omidkhoda, Azadeh...... 134 Parikh, Abhirath...... 98 Pellegrini, Matteo...... 23 Nishiyama, Toshimasa...... 157 Ongkeko, Weg M...... 66 Park, Ae Kyung...... 244 Peng, Chi-Hsien...... 36 Nisler, Benjamin S...... 206 Oni, Eileen...... 184 Park, Chun Shik...... 116 Peng, Fu-Chou...... 189 Nistor, Gabriel...... 190, 219 Ono, Michele Y...... 207 Park, Dae Hwi...... 51 Peng, Hsiao-Wen...... 136 Nitta, Suguru...... 105 Ono, Yusuke...... 41 Park, Han-Soo...... 221 Peng, Xiao-Rong...... 129 Niu, Lei...... 213 Onoe, Hirotaka...... 206 Park, In-Hyun...... 182, 244 Pepin, Andrea...... 111 Noer, Agate...... 122 Ooka, Hisashi...... 157 Park, Jae Hong...... 245 Pera, Martin F...... 214 Noggle, Scott A...... 164 Orbán, Tamás I...... 240 Park, Jihwan...... 199 Pereira, Mandy...... 75 Nordberg, Agneta...... 190 Ordog, Tamas...... 74 Park, Jong Heum...... 244 Perez, Harvey...... 50 Nordstrand, Line...... 249 Ordovas, Laura...... 216 Park, Jung Hyun...... 33 Perez-Iratxeta, Carolina...... 177 Norén, Helena...... 171 Org, Tonis...... 23 Park, Kye-Yoon...... 81, 135 Peri, Meital...... 203 Nori, Satoshi...... 195 Orkin, Stuart H...... 23 Park, Myoung Hee...... 12 Perlingeiro, Rita C.R...... 96, 179 North, Trista E...... 9 Orlando, David A...... 161, 234 Park, Sang Young...... 151 Perlis, Roy...... 42 Nottle, Mark B...... 247 Oron, Efrat...... 232 Park, Sang-Wook...... 21, 221 Perreault, Marie-Claude...... 122 Novikov, Lev N...... 192 Ortega, SandrA...... 170 Park, Shin Ae...... 151 Perruisseau-Carrier, Claire.....127 Nowrouzi, Ali...... 118 Ortiz, Mariaestela...... 14, 24 Park, Su Shin...... 245, 245 Persson, Kristin...... 61 Nusse, Roel...... 243 Ortmann, Bodo...... 83 Park, Sung-Jin...... 184 Peschanski, Marc...... 167 O’brien, Carmel...... 83 Ortmann, Daniel...... 14, 215 Park, Un Chul...... 33 Pessolato, Alicia Greyce G...... O’brien, Robert...... 19 Osawa, Gail A...... 114 Park, Woong Yang...... 244 ...... 120, 121 O’brien, Vincent...... 159 Osteen, Jeremiah D...... 13 Park, Yonsil...... 216 Petersen, Alan...... 37 O’rourke, Fiona...... 97 Otonkoski, Timo...... 43, 186 Park, Young-Bae...... 12, 199 Petersen, Bruce...... 117 O’rourke, Matthew...... 185 Ougland, Rune...... 249 Parker, Sarah...... 179 Peterson, Alan...... 157 O’shea, Patrick...... 129 Ovchinnikov, Dmitry...... 72 Parmar, Malin...... 232 Peterson, Alexander...... 217 Ochiya, Takahiro...... 145 Ozawa, Yutaka...... 175 Parmelee, Alissa...... 115 Peterson, Cory...... 197, 197 Ochs, Matthias...... 228 Ozdag, Hilal...... 129 Parolini, Daniele...... 160 Peterson, Daniel A...... 57 Ogawa, Shinichiro...... 22 Pacienza, Natalia...... 137 Parsons, Stephanie...... 82, 169 Petravicz, Jeremy C...... 43 Oh, Byung-Hee...... 12 Pai, Sadashiva K...... 80 Paruzynski, Anna...... 118 Petrighi Polidori, Gioia...... 227 Oh, Sun Kyung...... 204, 208 Pakpoom, Kheolamai...... 75 Pasceri, Peter...... 168 Petzold, Petzold N...... 80 Oh, Wonil...... 128 Palecek, Sean P...... 13 Pascual, Gloria...... 26 Peymani, Maryam...... 156 Ohlmeyer, Michael...... 248 Paliouras, Grigorios N...... 51 Passos-Bueno, Maria Rita...... Phetfong, Jitrada...... 75 Ohneda, Osamu...... 140 Palmer, Jessica A...... 48, 49 ...... 121, 133 Phillips, Matthew...... 135 Ohsumi, Toshiro K...... 161 Palmer, Theo...... 171 Pasut, Alessandra...... 40 Phoenix, Timothy N...... 211 Okada, Aki...... 201 Palumbo, Sunmi...... 97 Patel, Neel...... 145 Piaggio, Eduardo...... 125 Okada, Naoko...... 32 Panaviene, Violeta...... 121 Patel, Nisha A...... 3 Piao, Jinghua...... 212 Okada, Yohei...... 232 Panchalingam, Krishna M.....144 Paul, Gadue...... 188 Piao, Yulan...... 242

263 ISSCR 9th Annual Meeting www.isscr.org

Presenter Index

Piazzolla, Daniela2...... 88 Presnell, Sharon...... 193 Rathjen, Peter D...... 225, 229 Robey, Pamela G...... 135 Picanço-Castro, Virgínia...... 176 Prestwich, Glenn D...... 145 Rauen, Katherine A...... 187 Robins, Allan...... 3 Piccoli, Martina...... 40, 89 Prewitz, Marina...... 102 Raval, Kunil K...... 13 Robinson, Anibal...... 125 Pignatari, Graciela...... Preynat-Seauve, Olivier...... 231 Raveche, Elizabeth Scott...... 180 Robson, Craig...... 66 ...... 160, 176, 176, 177 Price, Feodor...... 226 Ravindran, Geeta...... 143, 152 Roccio, Marta...... 89 Pilcher, Webster...... 63 Prior, Faith...... 42 Reddy, Anita...... 14 Rodova, Mariana...... 73 Piliszek, Anna...... 104 Prowse, Andrew B...... 87 Regatieri, Caio...... 42 Rodrigo-Torres, Daniel...... 9 Pineau, Isabelle...... 191 Pugach, Emily K...... 117 Reggatt, Liza...... 136 Roeder, Olga...... 124 Pinheiro, Irina...... 68 Pulak, Rock...... 91 Regoes, Roland R...... 120 Rogers, Ian...... 101 Pinho, Vanessa...... 22 Qi, Yuchen...... 213 Reichen, Marcel...... 95 Roh, Tae-Young...... 199 Pinto-Do-Ó, Perpétua...... 15 Qian, Yun...... 207 Reijo Pera, Renee...... 188 Rohrschneider, Larry R...... 88 Piper, Julia...... 187 Qin, Gangjian...... 18 Reijo, Renee...... 21, 76, 171 Rojas-Sutterlin, Shanti...... 113 Pirraco, Rogério P...... 131 Qiu, Yuanyuan...... 190 Reijo-Pera, Rene...... 174 Rolong, Andrea...... 96 Pitaru, Sandu...... 60 Qu, Kun...... 163 Reilly, Christopher M...... 151 Romorini, Leonardo...... 155 Plant, Anne...... 217 Quandel, Tamara...... 174 Reimann, Christian...... 107, 107 Rooney, Gemma E...... 187 Platero-Luengo, Aida...... 54 Quesenberry, Peter J...... 75 Reinhardt, Jens...... 108 Roos, Eric...... 147 Pleasure, Samuel...... 52 Quesnel, Bruno...... 72 Reis, Rui L...... 131 Root, Sierra H...... 81, 106 Plummer, Mark...... 196 Questa, María...... 155 Reis, Surya...... 43, 46 Rorne, Rachel...... 136 Po Sing, Leung...... 1 Quinones-Hinojosa, Alfredo...63 Ren, Gaoying...... 201 Rosa, Nathália G...... 120 Pochon, Gaetan...... 129 Quintanilla, Rene...... 60, 250 Ren, Ke...... 150 Rosado De Castro, Paulo Podhajcer, Osvaldo...... 125 Quist, Sven R...... 28 Ren, Yong...... 171 Henrique...... 95 Poentitzsch, Ashley...... 48 Qunitanilla, Rene H...... 80 Renbaum, Pinhas...... 242 Rose, Keeley...... 77 Poirier, Valerie...... 78 Ra, Jeong Chan...... 151 Renin, Gil...... 90 Rose, Keeley L...... 52 Polakowska, Renata...... 72 Rabelink, Ton J...... 110 Renteria, Roberto...... 155 Rosen, Barry...... 14 Pollard, Katherine S...... 18 Rachima, Heled...... 60 Renwick, Neil...... 233 Rosenberg, Lawrence...... 144 Polo, Jose M...... 161 Rada, Tommaso...... 131 Repele, Andrea...... 40 Rosenfeld, Michael G...... 3 Poo, Maria...... 69 Radzhisheuskaya, Aliaksandra..... Reuckert, Erroll...... 46 Rossant, Janet...... 11, 22, 104 Poon, Kar Lai...... 235 ...... 203 Reyna, Kristen...... 84 Rossell, David...... 69 Popescu, Andreea...... 62 Raghunathan, Srividhya...... 85 Reynolds, Susan...... 90 Rossi, Fabio M. V...... 194 Popovic, Milos R...... 191 Rahimian, Ali...... 119 Rezvani, Hamid Reza...... 118 Rossi, Giacomo...... 142 Porcionatto, Marimelia A...... 53 Rahimy, Elham...... 66 Rhee, Dong-Yoon Rhee...... 221 Rostami, Shahrbanoo...... 145 Porteous, David...... 58 Rajasekar, Reena...... 85 Rho, Kyoung-Hwan...... 159 Roth, Monica...... 83 Porteus, Matthew...... 117 Rajasekhar, Vinagolu K...... 65 Riazi, Gholamhossein...... 99 Rothman, Joel H...... 202 Portier, Nate...... 91 Ralston, Amy...... 244 Riboldi, Marcia...... 69 Rouault-Pierre, Kevin...... 118 Poser, Ina...... 169 Ramirez, Jean-Marie...... 232 Richmond, Camilla...... 29 Rouiller, Julien...... 107, 107 Pospisilova, Sarka...... 236 Ramos, Carla...... 44 Ricupero, Christopher L...... 196 Rousseau, Alexandre...... 98 Postovit, Lynne...... 87 Ramsamooj, Rajendra...... 5 Riddle, Misty R...... 202 Roy, Edwige...... 27 Potts, Jay D...... 193 Ramsay, Robert G...... 29, 29 Riedel, Michael J...... 10, 208 Roy, Rajika...... 31 Pourfatolah, Ali Akbar...... 61 Ramsey, Cathy...... 180 Rigas, Anastasia...... 66 Ruban, Ludmila...... 95 Pouya, Alireza...... 43 Ranade, Aarati...... 4 Riggs, Arthur D...... 237 Rubin, Lee L...... 41, 41, 213 Powe, Allan C...... 45, 92 Rao, Mahendra...... 60, 250 Riley, Elizabeth B...... 117 Rudnicki, Michael...... Powell, Anne M...... 182 Raposo-Amaral, Cassio Eduardo. Rimmele, PaulIne...... 238 ...... 40, 163, 248, 226 ...... 133 Powers, Mark...... 80 Ripple, Kyle...... 206 Ruel, Marc...... 82 Rasmussen, Theodore P...... 187 Pozzobon, Michela...... 40, 89 Ritz, Anita...... 164 Ruff, Crystal...... 56 Raspa, Andrea...... 191 Prado, Karen...... 177 Rival-Gervier, Sylvie...... 160 Rugg-Gunn, Peter...... 104 Rastegar, Mojgan...... 162 Prasain, Nutan...... 239 Rivest, Serge...... 115 Ruiz-Herguido, Cristina...... 113 Rath, Sasmita...... 96 Prathalingam, Nilendran...... 80 Rizk, Pamela...... 47 Ruohola-Baker, Hannele...... 243 Rathjen, Joy...... 225, 229 Pratt, Scott L...... 181 Roach, Marsha...... 47 Russ, Holger A...... 165

264 www.isscr.org Thursday Poster Abstracts

Presenter Index

Russell, Daniela Fischer...... 222 Sanchez, Eva...... 69 Scherberich, Arnaud...... 131 Senner, Claire E...... 215 Russell, David W...... 201 Sánchez, Rodrigo...... 197 Schiavo, Andrea...... 107 Senzel, Stanislava...... 101 Russell, Jennifer...... 218 Sanchez-Pernaute, Rosario.....55 Schiavo, Andrea Alex...... 107 Seo, Bit Na...... 245 Russell, Paul...... 151 Sancho Bru, Pau...... 9 Schichor, Christian...... 197 Serban, Geo...... 108 Russo, Fabiele...... Sancho, Elena...... 69 Schira, Jessica...... 233 Sermon, Karen...... 219 ...... 160, 176, 176, 177 Sander, Chris...... 241 Schlaeger, Thorsten M...... 117 Serrano, Manuel2...... 88 Russo, Valentina...... 31 Sander, Maike...... 3 Schluter, Holger...... 70 Serwold, Thomas...... 23 Russo-Carbolante, Elisa Maria Sanderson, AimEe...... 37 Schmeckebier, SabriNa...... 228 Sevillano, Marta...... 69 De Sousa...... 176 Sandri, Silvana...... 177 Schmidlen, Tara J...... 185 Sevlever, Gustavo E...... 155 Ryan, Jennifer...... 136 Sangha, Namrata...... 193 Schmidt, Anne G...... 230 Seyedjafari, Ehsan...... 134 Ryan, Tammy...... 52 Santa-Olalla, Jesús...... 55 Schmidt, Felipe R...... 95 Sganga, Leonardo...... 125 Ryder, Oliver A...... 185 Santhosh Kumar, Saranya...... 46 Schmidt, Johannes...... 169 Shah, Khooshbu K...... 214 Ryu, Chun Jeih.....239, 239, 239 Santos, Mark...... 155 Schmidt, Manfred...... 118 Shah, Sanjay...... 32 Ryu, Jung Min...... 242, 245 Sanzey, Morgane...... 64 Schmidt, Uli...... 86 Shah, Shreyas...... 94 Säämänen, Anna-Marja...... 158 Saphier, Genevieve...... 198 Schmiedl, Andreas...... 228 Shah, Smitesh...... 32 Sachan, Shailendra...... 32 Sapolsky, Robert M...... 54 Schmoelzl, Sabine...... 78 Shahbazi, Ebrahim...... 47 Sachdev, PerminDer S...... 167 Saramipoor Bahbahan, Iman.....5 Schneider, Joel Solomon...... 180 Shahhoseini, Maryam...162, 234 Sachdeva, Rohit...... 59, 232 Saramipoor Behbahan, Iman.....5 Schnitzler, Aletta C...... 85 Shahosseini, Maryam...... 127 Sachewsky, Nadia...... 52, 77 Sarang, Shabari...... 143 Scholer, Hans R...... 247 Sham, Mai Har...... 163 Sadeghian, Fatemeh...... Saraswati, Sarika...... 193 Schöler, Hans R...... 185 Shankar, Sharmila...... 73 ...... 26, 76, 87 Sarkadi, Balázs...... 240 Schoordijk, Wenda...... 134 Shariki, Kohava...... 203 Safi, Mojtaba...... 100, 228 Sarkar, Debanjan...... 146 Schorle, Hubert...... 105, 224 Sharma, Himanshu...... 218 Saga, Ayami...... 148, 149, 149 Sarukhan, Adelaida...... 123 Schouteden, Sarah...... 116 Sharma, Parveen...... 104 Sagar, Vinay...... 146 Sasai, Yoshiki...... 173, 206 Schreiner, A...... 28 Sharov, Alexei...... 242 Sage, Julien...... 202 Sasaki, Erika...... 183, 232 Schüle, Birgitt...... 171 Sharova, Lioudmila...... 242 Saito, Ichiro...... 32 Sasaki, Katsunori...... 155 Schüle, Silke...... 108 Shaw, Sheng-Wen Steven...... 89 Saito, Izumu...... 114 Sasidharan, Rajkumar...... 23 Schüler, Herdit...... 124 She, Bin-Ru...... 84 Saito, Masato...... 93 Sasse, Philipp...... 178 Schultheiz, Heather...... 205 Sheldon, Michael...... 172 Saito, Mikako...... 225 Satarian, Leila...... 43 Schulz, Julia C...... 141 Shen, Chia-Ning...... 3 Saito, Naoto...... 155 Satkunendrarajah, Kajana...... 56 Schulz, Vincent...... 112 Shen, Chia-Ning ...... 200 Sakai, Kiyoshi...... 125 Sato, Shingo...... 73 Schulze, Wiebke...... 228 Sheng, Nengyin...... 207 Sakai, Yasuyuki...... 145, 186 Sats, Natalya...... 122 Schwartz, Joseph...... 108 Shenoy, Sachin...... 102, 103 Sakano, Daisuke...... 3 Sauvageau, Guy...... 99 Schwartz, Michael P...... 95 Sheridan, Robert...... 241 Sakiyama-Elbert, Shelly E...... 53 Sawa, Yoshiki...... 148 Schwartz, Philip H...... 207 Sheridan, Steven...... Sakthivel, Ramasamy...... 90, 154 Sayed, Nazish...... 188 Schwarze, Ulrike...... 201 ...... 43, 44, 45, 46, 169 Sakurai, Takayuki...... 22 Scadden, David T...... 111 Scott, Edward...... 25 Sherley, James L...... 1 Salazar, Georgina...... 140 Scassa, María E...... 155 Scott, Heather...... 86 Sherlock, Jon...... 128 Salcedo, Rosalba...... 24 Schaaf, Gerben...... 134 Sears, Marie A...... 17 Shetty, Prathibha...... 152 Saltanatpour, Manijeh...... 134 Schaaf, Sebastian...... 97 Sebastiano, Vittorio...... 174 Shi, Songhai...... 213 Salykin, Anton...... 240 Schaaf, Tory...... 179 Sebe, Attila...... 240 Shi, Xingming...... 138 Samani, Fazel...... 133 Schaller, Martin...... 97 Sedlackova, Miroslava...... 237 Shi, Yufang...... 141 Samant, Mugdha D...... 66 Schaniel, Christoph...... 248 Seear, Kate...... 37 Shibamiya, Aya...... 97 Samavarchi-TehrAni, Payman..... Schärer, Lukas...... 195 Segard, Pascaline...... 72 Shibata, Shinsuke...... 195 ...... 249 Schätti, Oliver...... 135 Seifinejad, Ali...... 7 Shigeto, Hajime...... 225 Sampson, Kevin J...... 13 Scheinman, Melvin...... 17 Seifried, Erhard...... 124 Shim, Jae-Won...... 212 Sams, Alexandria...... 80 Schellenberg, Anne...... 124 Seita, Yasunari...... 83, 216 Shimada, Hiroko...... 232 Samuvel, Devadoss J...... 50 Schenke-Layland, Katja...... 223 Seng, Victoria...... 67 Shimmura, Shigeto...... 32, 101 Sances, Sam A...... 55 Scher, Howard I...... 65 Sengutpa, Shiladitya...... 196 Shin, Tae-Hoon...... 159

265 ISSCR 9th Annual Meeting www.isscr.org

Presenter Index

Shindo, Takayuki...... 22 Smith, Jim C...... 215 Stambrook, Peter J...... 243 Surani, Azim...... 199 Shinkai, Yoichi...... 229 Smith, Kristen M...... 67 Stanford, William L...... 249 SuryapRakash, Shruthi...... 146 Shipounova, Irina...... 122 Smith, Susan...... 48 Stanley, Edouard G...... 14, 221 Suuronen, Erik J...... 82 Shiraki, Nobuaki...... 3 Smithgall, Thomas E...... 229 Stendardo, Massimo...... 10 Suzuki, Hiroko...... 157 Shirasawa, Sakiko...... 155 Snodgrass, Ralph...... 4 Steward, Melissa M...... 32 Swartz, James...... 188 Shofuda, Tomoko...... 173, 175 Snyder, Evan...... 82, 86, 169 Stice, Steven L...... Swift, Brenna...... 71 Shohara, Ryutaro...... 125, 152 Snyder, Michael...... 232 ...... 45, 46, 92, 181, 183 Syed, Fraz...... 160 Shoichet, Molly...... 34, 35, 100 So, Ah-Young...... 159 Stockwell, Sally...... 78 Sykes, Megan...... 111 Shou, Peishun...... 141 So, Leslie...... 194 Stoddart, Martin J...... 135 Synnergren, Jane...... 7 Shoukat-Mumtaz, Uzma...... 44 SoareS, Bento M...... 164 Stoehr, Andrea...... 97 Sytwu, Huey-Kang ...... 200 Shu, Yousheng...... 207 Socci, Nicholas D...... 65 Stover, Alexander E...... 207 Szita, Nicolas...... 95 Shudo, Yasuhiro...... 148 Sodha, Anirudhasingh...... 90 Stowe, Heather...... 181 Taapken, Seth M...... 206 Shutova, Maria...... 164 Soeda, Mayumi....148, 149, 149 Stranecky, Viktor...... 63 Tabar, Viviane...... 212 Siatskas, Christopher...... 30 Solagna, Francesca...... 40, 89 Streeter, Philip R...... 2 Tachibana, Makoto...... 229 Sidhu, Kuldip S...... 167 Solanki, Aniruddh...... 94 Streetz, Konrad...... 8 Tachibana, Masahito...... 180 Siebelt, Michiel...... 110 Soldani, Cristiana...... 123 Strehl, Raimund...... 171 Taghizadeh, Zeinab...... 234 Sii-Felice, Karine...... 143 Soleimani, Masoud...... Strom, Stephen C...... 6 Tahara, Shinya...... 148 Silva, José C.R...... 203 ...... 61, 100, 116, 119, 134, 145 Strom, Terry B...... 111 Taher, Yousry...... 6 Silva, Rafaella M...... 95 Soleimani, Vahab...... 40, 163 Strulovici, Berta...... 44, 140 Taipaleenmaki, Hanna...... 158 Silverman, Cara R...... 53 Soleimani-Salehabadi, Studer, Lorenz...... Takacova, Sylvia...... 63 Sim, Fraser...... 63 Mohammad...... 26 ...... 65, 211, 212, 213, 219, 241 Takahashi, Jun...... 172, 205, 206 Simanov, Daniil...... 195 Son, Esther Y...... 201 Sturgeon, Chris...... 220 Takahashi, Kazutoshi...... 76, 201 Simler, Jancie L...... 85 Son, Youngsook...... 103, 195 Sturgeon, Christopher M...... 234 Takahashi, Keisuke...... 114 Simmons, Craig A...... 139 Song, Guangqi...... 181 Su, Hong-Lin...... 213 Takahashi, Yuichiro...... 195 Simon, Carlos...... 69 Song, Hongjun...... 96 Su, I-Chang...... 69 Takano, Morito...... 195 SiMon, Valeska...... 7 Song, Jihwan...... 182 Su, Jing...... 216 Takashima, Yasuhiro...... 17 Simonsson, Stina...... 246 Song, Mingke...... 218 Su, Juanjuan...... 141 Takeda, Junji...... 244 Singec, Ilyas...... 86 Sonntag, Kai C...... 55 Subramanyam, Deepa...... 166 Takeda, Shin’ichi...... 41 Singh, Karun...... 42 Soria, Bernat...... 243 Suciu-Foca, Nicole...... 108 Takenaka, Katsuto...... 62 Singh, Upinder...... 250 Sorkin, Adam M...... 154 Sudchada, Sakchai...... 24 Takeshita, Fumitaka...... 145 Sirén, Anna-Leena...... 209 Sorrentino, Miguel Angel...... 125 Suemizu, Hiroshi...... 173, 175 Takikawa, Sachiko...... 125, 152 Sirenko, Oksana...... 92 Sosunov, S A...... 25 Suemori, Hirofumi...... 206 Takizawa, Hitoshi...... 120 Sirish, Padmini...... 16 Sourour, Michel...... 186 Suh, Han Na...... 242, 245, 245 Talchai, Chutima...... 30 Sirokmany, G...... 28 Sousa, Amaia...... 55 Sullivan, Mike...... 129 Tam, Paul...... 68 Sitzia, Clementina...... 160 Souza, Lucas E B...... 8 Sullivan, Stephen...... 30 Tamarat, Radia...... 143 Sivasubramaniyan, Kavitha...... Souza, Sergio A. L...... 95 Sumi, Shoichro...... 221 Tamashiro, Dana Ann A...... 105 ...... 133 Sparks, Wendy O...... 182 Sun, Guizhi...... 30 Tamiya, Eiichi...... 93 Skerjanc, Ilona S...... 52 Sparling, Joseph...... 57 Sun, Nawei...... 172 Tan, Boon Siang Nicholas...... 229 Skinner, Rebecca...... 37, 37, 37 Spencer, C. Ian...... 17 Sun, Shawn...... 51 Tan, Cherise E...... 101 Sklar, Pamela...... 46 Spencer, Thomas...... 193 Sun, Wayne G...... 54 Tan, Jit Hin...... 224 Slack, Jonathan...... 40, 198 Spray, David C...... 150 Sun, Yan...... 10 Tan, Kar Tong...... 235 Slany, Robert...... 63 Squiban, Claire...... 143 Sun, Zhuo...... 24 Tan, Siah Hong...... 20 Slavin, Ileana...... 169, 205 Sridhar, Akshayalakshmi...... 32 Sunayama, Misato...... 177 Tan, Swee T...... 101 Slot, Edith M...... 110 Srivastava, Alok...... 166, 178 Sung, Ki Woong...... 146, 148 Tan, Yu-Zhen...... 25, 25, 235 Smet, Francis...... 91 Srivastava, Rakesh K...... 73 Suomalainen, Anu...... 43 Tanabe, Koji...... 201 Smith, Alan M...... 49 Stachelscheid H, Tschoepe C...... Supokawej, Aungkura...... 75 Tanaka, Yujiro...... 105 Smith, Austin...... 17 ...... 31 Supokawej, Aungura...... 24 Tanasijevic, Borko...... 187 Smith, James C...... 215 Staebler, Annette...... 70 Sur, Mriganka...... 43, 169 Tandeski, Terry...... 145

266 www.isscr.org Thursday Poster Abstracts

Presenter Index

Tandon, Radhika...... 32 Thorn, Stephanie...... 82 Tsuji, Kohichiro...... 170 Van Zon, Patrick...... 195 Tang, Bo...... 181 Thorner, Paul S...... 67 Tsuji, Osahiko...... 195 Van Zonneveld, Anton Jan....110 Tang, Lina...... 181 Thu, Mia S...... 148 Tuck, David...... 112 Vanderweide, Stephanie...... 244 Tang, Roger...... 92 Tichy, Elisia D...... 243 Tucker, Budd...... 42 Várady, György...... 240 Tang, Su-Ni...... 73 Tillu, Parnita...... 143 Tucker, Budd A...... 34 Varma, Hari Krishna...... 102, 103 Tang, Yi...... 183 Timofeyev, Valeriy...... 16 Tuncel, Donus...... 137 Varshaver, Irit...... 242 Tang, Yixin...... 84 Tischfield, Jay A...... 172 Turner, Jennifer...... 79 Vasania, Viraf...... 32 Tang, Zhenya...... 185 Tisdale, John...... 115 Tuschl, Thomas...... 233, 241 Vasei, Mohammad...... 134, 145 Tani, Junko...... 148 Titmarsh, Drew...... 79 Tzanakakis, Emmanuel S...... 98 Vasquez, Louella J...... 187 Tanikawa, Daniela...... 133 Tizzano, Eduardo...... 167 Uchida, Nobuko...... 50 Vassiliev, Ivan...... 247 Tanowitz, Herbert B...... 150 Tobe, Brian...... 86 Uda, Masahiro...... 177 Velayudhan, Shaji R...... 166, 178 Tantrawatpan, Chairat...... 24, 75 Toepke, Michael W...... 95 Udolph, Gerald...... 47 Velluto, Diana...... 89 Tao, Rong...... 156 Tojo, Arinobu...... 114 Ueda, Minoru...... 125, 152 Vemuri, Mohan...... 147 Tapia Limonchi, Rafael A...... 243 Toma, Ildiko...... 174 Uetake, Ryuichi...... 22 Vendrell, Marc...... 184 Tapia, Natalia...... 185, 247 Tomioka, Ikuo...... 183, 232 Uhl, Elizabeth W...... 181 Veraitch, Farlan S...... 95 Tarraf, Pania...... 228 Tomishima, Mark J...... 219 Ulfendahl, Mats...... 210 Vered, Zvi...... 60 Tassone, Flora...... 207 Tomotsune, Daihachiro...... 155 Ullian, Erik M...... 187 Verfaillie, Catherine...... 116, 216 Tateyama, Daiki...... 175, 222 Tompkins, Joshua D...... 237 Underbayev, Chingiz...... 180 Verma, Suresh...... 18 Tay, Seok WEi...... 10 Tonge, Peter...... 203 Unguryte, Ausra...... 121 Vermeesch, Joris...... 187 Taylor, Sarah...... 71 Torihashi, Shigeko...... 41 Uosaki, Hideki...... 18 Veronesi, Giulia...... 10 Taylor-Weiner, Hermes...... 226 Toro-Ramos, Alana J...... 196 Uphill, Grant...... 78 Vessey, John P...... 56 Teitell, Michael A...... 235 Torrente, Yvan...... 160 U-Pratya, Yaowalak...... 24, 75 Vicente, Wilter R...... 120, 121 Teixeira, Ana...... 49, 61, 68 Touil, Yasmine...... 72 Urbani, Luca...... 40 Vidal, Jason G...... 211, 214 Teixeira, Mauro M...... 22 Tousey, Susan...... 84 Urbich, Carmen...... 97 ViDela Richardson, Guillermo A.. Tejedo, Juan R...... 243 Toyama, Yoshiaki...... 195 Urrutia, Raul A...... 74 ...... 155 Tellier, Helena...... 87 Tran, Ha...... 205 Usui, Yuki...... 155 Vieira, Natassaia...... 133 Ten Berge, Derk...... 243 Tran, Simon...... 157 Valarmathi, Mani T...... 193 Vierbuchen, Thomas...... 163 Teo, Grace Sock Leng...... 153 Tran, Tung N...... 136 Valdivia, Carmen R...... 17 Vignon, Christine...... 154 Teo, Weisuong...... 146 Trautwien, Christian...... 8 Valencia, Concepción...... 55 Vijayraghavan, Deepthi...... 142 Termglinchan, Vittavat...... 199 Treff, Nathan...... 216 Valente, Mariana...... 15 Villegas, S. Nahuel...... 236 Terraf, Panieh...... 100 Tremml, Gabi...... 199 Valentin, Silke...... 83 Vinarsky, Vladimir...... 236, 237 Terrenoire, Cecile...... 12, 13 Trevino, Elizabeth...... 125 Vallar, Laurent...... 64 Vincent, Ludovic...... 132 Tesar, Paul J...... 227 Trokovic, Ras...... 43 Vallaster, Markus...... 16 Vincent, Pasque...... 199 Testa, Giuseppe...... 51 Trompeter, Hans-Ingo...... 233 Vallier, Ludovic...... 216 Vink, Robert...... 156 Tetzlaff, Wolfram...... 57 Tropepe, Vincent...... 61 Valojerdi, Mojtaba Rezazadeh..... Viola, Antonella...... 123 Thakar, Nilay Y...... 72 Trotter, Matthew...... 180 Viswanathan, Chandra...... Thakur, Anirban...... 152 ...... 199, 215, 215 Van Aalst, John A...... 135 ...... 32, 143, 152 Thal, Melissa A...... 18 Tsai, Li-Huei...... 42 Van Arenbergen, Joris...... 55 Visweswaran, Malini...... 85 Theiss, Hans D...... 16 Tsai, Miao-Chih...... 163 Van De Peppel, Jeroen...... 134 Vizoso, Dita B...... 195 Theriault, Kraig...... 44, 45, 46 Tsai, Schickwann...... 24 Van Der Kooy, Derek...... Vlahos, Katerina...... 221 Theunissen, Thorold W...... 203 Tsai, Zong Yun...... 240 ...... 34, 35, 35, 52, 59, 77 Vo, Tien...... 148 Thieme, Sebastian...... 102 Tschopp, Markus...... 111 Van Der Ven, Karlijn...... 46 Voet, Thierry...... 187 Thiriot, Aude...... 146 Tse, Kai-Hei...... 192 Van Galen, Peter...... 62, 63 Volfson, Dmitri...... 44 Thomas, Terry E...... 10, 208, 209 Tseng, Pei-Chi...... 136 Van Handel, Ben...... 23 Voltarelli, Julio C...... 123 Thompson, Julia...... 91 Tskhovrebova, Leyla...... 164 Van Ijcken, Wilfred F.J...... 163 Von Andrian, Ulrich H...... 146 Thompson, Tadeo...... 202 Tsoi, Liz...... 68 Van Leeuwen, Hans...... 134 Von Kalle, Christof...... 118 Thomson, James A...... 13 Tsubota, Kazuo...... 32, 101 Van Pel, Melissa...... 110 Von Tigerstrom, Barbara...... 38 Thoppe, Karthiklal...... 85 Tsui, David...... 49, 53 Van Solingen, Coen...... 110 Voronova, Anastassia...... 52

267 ISSCR 9th Annual Meeting www.isscr.org

Presenter Index

Vu, Duc M...... 14 Weber, Mark...... 42 Wolf, Sebastian...... 111 Yamamoto, Atsuyo...... 173 Vulesevic, Branka...... 82 Wei, Chung-Fan...... 153 Wolfe, John H...... 172 Yamamoto, Yusuke...... 10 Vuoristo, Sanna...... 186 Wei, Feng...... 150 Wolff, Erin...... 180 Yamanaka, Shinya...... Waddell, Thomas K...... 11 Wei, Jun...... 152 Wolowczuk, Isabelle...... 72 ...... 17, 76, 201, 231 Wade, Marlene...... 117 Wei, Ling...... 218 Woltjen, Knut...... 203 Yamasaki, Mami...... 173 Wade-Gueye, Marième...... 143 Wei, Qingxia...... 73 Wolvetage, Ernst...... 136 Yamashita, Jun K...... 229 Wadsworth, Samuel...... 10 Weir-Hauptman, April M...... 49 Wolvetang, Ernst...... 72, 79, 164 Yamashita, Toshiharu...... 140 Wagers, Amy J...... 41, 41 Weiss, Mark...... 125 Won, Chunkil...... 221 Yamashita, Yukiko...... 78 Wagner, Amanda K...... 41, 41 Weiss, Mitchell J...... 188 Wong, Amy P...... 11 Yamauchi, Akihiro...... 22 Wagner, Wolfgang...... 124 Weissman, Irving L...... 23 Wong, Andrew...... 16 Yan, Xiurui...... 152 Wahlin, Karl J...... 34 Weksberg, Rosanna...... 168 Wong, Melissa H...... 28, 68 Yan, Zhen...... 171 Wainger, Brian J...... 201 Weldon, Don...... 155 Woo, Dong-Hun...... 189 Yañéz, Rafael...... 167 Wallace, Kyle...... 34 Wells, William...... 173 Wood, Joshua A...... 151 Yang, Fengchun...... 117 Wallwiener, Diethelm...... 70 Welsh, Michael...... 113 Wood, Teresa L...... 52 Yang, Han-Mo...... 12 Walsh, Martin...... 248 Wenzel, Pamela...... 109, 119 Woolf, Clifford J...... 201 Yang, Jae Won...... 45 Walter, Kevin...... 63 Werbowetski-Ogilvie, Tamra E.... Wörsdörfer, Philipp...... 224 Yang, Jin Mo...... 146, 148 Waltz, Shannon...... 205 ...... 222 Wosczyna, Michael N...... 123 Yang, Jingsheng...... 64 Wambua, Gerald K...... 205 Werner, Carsten...... 102 Wrana, Jeffrey L...... 249 Yang, Jiwei...... 81 Wamstad, Joseph A...... 18, 163 Wernet, Peter...... 233 Wu, Cheng-Yi...... 84 Yang, Minying...... 250 Wan, Yudi...... 54 Wernig, Marius...... 56, 163, 202 Wu, Chia-Yen...... 229 Yang, Nianlan...... 138 Wang, Chang Ye Yale...... 73 West, Franklin D...... 181, 183 Wu, Chung-Che...... 64 Yang, Pan-Chyr...... 11 Wang, De Yun...... 10 West, Michael D...... 82, 169 Wu, Dai-Chen...... 110 Yang, Phillip...... 174 Wang, George Kai...... 12 West, Paul...... 48, 49 Wu, Dai-Tze...... 83 Yang, Ruifeng...... 235 Wang, Hai-Jie...... 25, 25, 235 Whalley, Jason...... 155 Wu, Di L...... 57 Yang, Ting...... 69 Wang, Hu...... 150 Whetstone, Heather...... 39 Wu, Guangming...... 185 Yang, William...... 188 Wang, Ing-Kae...... 84 Whiteley, Jennifer...... 101 Wu, Jiaqian...... 232 Yang, Yoon Sun...... 128 Wang, I-Ning E...... 174 Whye, Dosh...... 229 Wu, Jin-Hong...... 235 Yanik, Mehmet F...... 200 Wang, Jean...... 62 Whyte, Warren A...... 234 Wu, Ping-Cheng...... 119 Yannarelli, Gustavo...... 137 Wang, Jianlong...... 246 Wiberg, Mikael2...... 192 Wu, Ruei-Ren...... 3, 200 Yao, Huantong...... 175 Wang, Jiapeng...... 117 Wicklund, Linn...... 190 Wunder, Jay S...... 73 Yao, Jing...... 42 Wang, Jing...... 49, 232 Wienholds, Erno...... 63 Wyatt, Tanya J...... 190 Yap, Charlotte...... 225 Wang, Kai...... 13 Wilcox, Jared T...... 56 Xenocostas, Anargyros...... 189 Yap, Sook Peng...... 89 Wang, Lei...... 19, 194 Wilhelm, Brian T...... 99 Xian, Hai-Qing...... 4 Yasuda, Akimasa...... 195 Wang, Libin...... 152 Willecke, Klaus...... 224 Xian, Wa...... 10 Yates, Frank...... 167 Wang, Min...... 164 Williams, Brenda...... 221 Xie, Yang...... 64 Ye, Hui...... 54 Wang, Peirong...... 201 Williams, Daniel A...... 167 Xu, Chunliang...... 141 Ye, Kaiming...... 175 Wang, Su...... 63 Williamson, Stuart C...... 66 Xu, Huilei...... 246 Yeh, Erika...... 121 Wang, Weijia...... 112 Wilson, Gisela F...... 13 Xu, Mingjiang...... 117 Yehia, Ghassan...... 180 Wang, Wenlan...... 229 Wingert, Rebecca A...... 230 Xu, Wenjun...... 77 Yen, B Linju...... 136, 153 Wang, Yu-Chieh...... 60, 83, 205 Winquist, Alicia M...... 197, 197 Xu, Xiu Qin...... 235 Yen, Men-Luh...... 136, 153 Wang, Zhong...... 163 Winterstern, Aaron...... 13 Xue, Haipeng...... 60 Yeoh, George...... 2 Wanisch, Klaus...... 167 Wise, Amanda...... 71 Yalcin, Safak...... 238 Yeon, Sooin...... 27 Wapinski, Orly L...... 163 Wisidagama, Dona R...... 235 Yam, Gary Hin-Fai...... 33 Yeung, Emily...... 18 Ward, Ryan J...... 64 Witkowski, Marybeth A...... 206 Yamada, Takeshi...... 116 Yi, David...... 66 Ware, Carol...... 243 Witty, Alec...... 12 Yamagata, Mari...... 125 Yi, Kevin...... 198 Watt, F. M...... 28 Wobus, Manja...... 102 Yamaguchi, Yoshinori...... 93 Yin, Hang...... 40, 163 Weber, Christian...... 118 Wolber, Wanja...... 209 Yamamizu, Kohei...... 229 Yin, Ivy...... 67 Weber, Jennifer...... 81 Wolf, Pavlina...... 46 Yamamoto, Akihito...... 125, 152 Ying, Carl...... 54

268 www.isscr.org Thursday Poster Abstracts

Presenter Index

Ying, Lei...... 236 Zhang, Guangtao...... 248 Zorko, Bojan...... 125 Yiu, Adelaide P...... 58 Zhang, Guoli...... 5 Zou, Gang-Ming...... 120 Yoder, Mervin C...... 239 Zhang, Jianhua...... 13, 17 Zou, Shiping...... 150 Yokoyama, Tadayuki...... 155 Zhang, Jin...... 235 Zuliani, Thomas...... 72 Yoo, Dae-Hoon...... 204 Zhang, Liang...... 146 Zupanc, Matej...... 125 Yoo, Jeong-Eun...... 48, 174 Zhang, Liying...... 141 Zwi-Dantsis, Limor...... 13 Yoo, Keon Hee...... 146, 148 Zhang, Min...... 207 Zwingenberger, Allison...... 151 Yoshida, Eileen...... 209 Zhang, Peng...... 181 Yoshida, Junko...... 244 Zhang, Ping-Xia...... 140 Yoshida, Satoru...... 101 Zhang, Regan-Heng...... 194 Yoshikawa, Tatsuya...... 206 Zhang, Weijia...... 248 Yoshimizu, Takao...... 42 Zhang, Xia...... 83 Yoshizawa, Takahiro...... 22 Zhang, Xin...... 238 You, Jungmok...... 126 Zhang, Xiong...... 229 Young, Jessica E...... 167 Zhang, Yan (Mary)...... 82 Young, Michael...... 42 Zhang, Yang...... 181 Young, Nilas J...... 16 Zhang, Ying-Ying...... 156 Young, Pampee P...... 193 Zhang, Zhiying...... 126 Young, Richard A...... 161, 234 Zhao, Li...... 184 Young, W Scott...... 115 Zhao, Weian...... 146 Yu, Hongyao...... 109 Zhao, Yukang...... 90 Yu, Hyeong Gon...... 33 Zheng, Bo...... 119 Yu, Junying...... 13 Zheng, Feifei...... 73 Yu, Mei...... 108 Zheng, Weili...... 92 Yu, Ping...... 181 Zheng, Xiaofeng...... 245 Yu, Qing C...... 221 Zhiromkina, Oxana...... 122 Yu, Shan Ping...... 218 Zhong, Bonan...... 80 Yuan, Tze-An...... 3 Zhong, Mei...... 232 Yue, Fengming...... 155 Zhong, Ping...... 171 Yue, Si-Biao...... 73 Zhong, Yingchao...... 184 Yue, Wei...... 207 Zhou, Baiyu...... 73 Yuen, Eunice Y...... 171 Zhou, Fen...... 46 Yum, Kyung Eun...... 244 Zhou, Jamie...... 23 Yun, Seong-Wook...... 184 Zhou, Ming-Ming...... 248 Yun, Seung Pil...... 245, 245 Zhou, Qi...... 83 Zachariah, Robby M...... 162 Zhou, Wenyu...... 243 Zack, Donald J...... 34 Zhu, John...... 199 Zahabi, Azadeh...... 33 Zhu, Juhong...... 50 Zandstra, Peter W...... 108 Zhu, Lili...... 230 Zannettino, Andrew C.W...... 141 Zhu, Yanan...... 144 Zaremba, Anita...... 227 Zhu, Yongzhao...... 152 Zarembinski, Thomas...... 95, 145 Ziadloo, Ali...... 146 Zeldin, Darryl C...... 117 Ziegler, Amber N...... 52 Zenke, Martin...... 124, 228 Zielonka, Elisabeth...... 10 Zern, Mark A...... 5, 5 Zimmermann, Heiko...... 141 Zhai, Shuya...... 198 Zimmermann, Wolfram Hubertus Zhang, Dan...... 25 ...... 91, 97 Zhang, Dawei...... 49 Zon, Leonard I...... 117

269 ISSCR 9th Annual Meeting www.isscr.org

Notes

270