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Stem Cell Biology HESI Workshop on Alternative Assays for Developmental Toxicology Stem Cell Biology Embryonic Stem Cells as Model System to Study Embryotoxic Effects Cary, N.C. February 27- 28, 2007 Anna M. Wobus ([email protected]) Leibniz Institute (IPK) Gatersleben, Germany UniqueUnique PropertiesProperties ofof EmbryonicEmbryonic StemStem (ES)(ES) CellsCells in vivo Oocyte Sperm Zygote in vitro Inner cell mass Cultivation (ICM) ES cells Proliferation Blastocyst Cultivation Differentiation Primordial germ cells EG cells (PGC) "embryoid body" (EB) Endoderm Ectoderm Liver cells Neuronal cells Pancreas Mesoderm cells Cardiac cells Skeletal muscle cells Blood cells Fat cells ESES cellscells differentiatedifferentiate inin vitrovitro intointo cellcell typestypes ofof allall threethree primaryprimary germgerm layerslayers C D E Mesoderm Cardiomyocytes Skeletal muscle Smooth muscle A B F G H Ectoderm Neurons Glial Epithelial Embryonic stem cells Embryoid body I K L Endoderm Pancreatic cells Hepatocytes Hepatocytes ESES cellcell differentiationdifferentiation inin vitrovitro recapitulatesrecapitulates processesprocesses ofof earlyearly embryonicembryonic developmentdevelopment DifferentiationDifferentiation ofof ESES cellscells inin vitrovitro 1) Embryoid body formation (3 germ layers!) • ‘Mass culture‘ • ‘Hanging drop culture‘ • Methylcellulose • ‘Spinner culture‘ 2) Differentiation in monolayer culture (neural cells) 3) Co-culture systems (PA-6, END-2) DifferentiationDifferentiation ofof ESES cellscells inin vitrovitro 1) Embryoid body formation (3 germ layers!) • ‘Mass culture‘ • ‘Hanging drop culture‘ • Methylcellulose • ‘Spinner culture‘ 2) Differentiation in monolayer culture (neural cells) 3) Co-culture systems (PA-6, END-2) ParametersParameters thatthat affectaffect ESES CellCell DifferentiationDifferentiation Culture media, supplements Concentration and quality of FCS ES cell line, passage number Number of cells in the EBs Time of EB plating ⇒ Threshold concentrations of ECM and growths factors in the EB regulate ES cell differentiation CCardiacardiac differentiationdifferentiation DifferentiationDifferentiation ofof beatingbeating cardiomyocytescardiomyocytes Titin (Z-disk)-positive cardiac cells CardiacCardiac differentiationdifferentiation ES cells EBs Multilineage Cardiac clusters progenitors Stage 123 4 nestin/desmin titin Culture (d) 055+65+24 Differentiation induction Media Additives L-Glut, NEAA, MTG Basal medium DMEM + 15%FCS IMDM + 20% FCS Substrate MEF Gelatine or Laminin ExpressionExpression ofof mesodermmesoderm-- andand cardiaccardiac--specificspecific genesgenes duringduring ESES--derivedderived cardiogenesiscardiogenesis 1 2 3 4 5 6 7 8 9101214day T (Brachyury) BMP-4 plating ES 3 5 + 2 + 5 + 7 + 12 + 18 day Nkx2.5 α−MHC β−MHC ANF MLC-2V CardiacCardiac--specificspecific actionaction potentialspotentials andand ionion currentscurrents Early Intermediate Terminal stage +60 0 mV Action potentials -80 Atrial-like Ventricle- Sinusnodal- 1 sec like like ICa + ++ ++ ++ ++ Ito + + + + + IK,ATP + + + + + IK - + + + + INa - + ++ ++ +- I - - + + - Ion currents K1 IK,Ach - - + - + - If - + - ++ ESES--derivedderived cardiomyocytescardiomyocytes respondrespond pharmacologicallypharmacologically similarsimilar toto cardiaccardiac cellscells developeddeveloped inin vivovivo Isradipin Nisoldipin Bay K Gallopamil CarbacholAtropin Forskolin Isoproterenol 8644 Diltiazem Dihydropyridine Ca2+ senstitive GTPγS GTPγS channel (L-type) Cholinoceptor Adenylyl cyclase β-adrenoceptor 2+ N N Ca GGi s Gs Phoshat- P ases C N ATP CCcAMP N cAMP-dependent PDEs protein kinase A C ATP Ca2+ PDEs inhibitors 8-bromo PKA PKA ATPγS okadaic IBMX, EHNA, cAMP inhibitor catalytic acid milrinone, rolipram peptide subunit ESES--derivedderived cardiomyocytescardiomyocytes forfor drugdrug screeningscreening Embryoid body (EB) Beating clusters in EB outgrowths Estimation of basal level of beating frequency by the LUCIA 'Heart' Pharmacological analysis imaging system of cardioactive drugs BayK 8644 Diltiazem 100 Diltiazem BayK 8644 Dose-response-effects 80 60 40 20 0 -20 Schlagfrequenz (Schläge/min) -40 -60 -8 -7 -6 -5 log Koncentration (M) NNeuronaleuronal differentiationdifferentiation NeuronalNeuronal differentiationdifferentiation ES cells EBs Neural Neuronal Dopaminergic progenitors cells neurons Stage 12 3 4 5 nestin ß III-tubulin TH Culture (d) 0 4 4+7 4+14 C 4+30 Selection Expansion Differentiation induction Media Additives B1 (-FCS: 4+1) B2 + EGF;bFGF Neurobasal + B27 (2%) + SPFs Basal medium IMDM+20%FCS DMEM/F12 DMEM/F12 +10%FCS Substrate Gelatine poly-L-Ornithine/Laminin 100 ES EG C 80 60 cells (%) ESES cellscells differentiatedifferentiate 40 20 nestin-positive intointo neuralneural precursorprecursor Percentage cell-derived of ES 0 4+ (ES) 2681012144 cellscells andand TH+TH+ (EG)5+ dopaminedopamine--producingproducing EB plating Replating 100 -SPF E neuronsneurons 80 + SPF 60 Immunofluorescence analysis of nestin-positive neuronal precurso cells (A,B,C), tyrosine hydroxylase (TH)- (D,E,) and dopamine transporter (DAT)-positive dopaminergic neurons (F,G) * * 40 labelled cells TH+- (%)Syn- of cells 20 0 4+ 16 23 30 100 -SPF + SPF G 80 60 * 40 * labelled cells DAT+-Syn- (%) of cells 20 0 4+ 16 23 30 Time of differentiation (d) Increase of Nurr1, TH and bcl-2 mRNA levels by SPFs Embryoid bodies plated at day 4 Genes 4+ 4 7 12 16 16 23 23 30 30 35 35 40 40 44 44 En-1 A D2R-L TH Nurr1 bcl-2 β-tubulin 100 80 En-1 B 60 40 20 0 100 80 D2R-L C 60 40 20 R-L, TH, Nurr1 and 2 0 100 TH D 80 * 60 40 20 0 100 Nurr1 80 * ** ** E 60 40 20 0 100 bcl-2 F 80 ** * 60 ** 40 20 Relative mRNA levels of En-1, D bcl-2 (%) 0 4+ 4 7 12 16 23 30 35 40 44 Time of Differentiation -SPF (4+14-4+44d) +SPF (4+14-4+44d) Intensity (mv) 1200 600 0 135678 012345 Dopamine synthesis of TH+ neurons neurons TH+ of synthesis Dopamine Dopamine synthesis of TH+ neurons neurons TH+ of synthesis Dopamine External standard External Retention time (min) time Retention ---- 5.79 (Dopamine) analyzed by HPLC by analyzed analyzed by HPLC by analyzed 12345 678 12345 678 Cultures wi Cultures Retention time (min) time Retention ---------- 5.69 (Dopamine) SPF thout Retention time (min) time Retention Cultures with SPF with Cultures ---------- 5.73 (Dopamine) NeurotoxicNeurotoxic effectseffects ofof 100100 μμMM MPTPMPTP onon TH+TH+ neuronsneurons A B C D Control MPTP treatment 48h PancreaticPancreatic differentiationdifferentiation PancreaticPancreatic DifferentiationDifferentiation ofof wtwt andand Pax4+Pax4+ ESES CellsCells ES cells EBs Multilineage Committed Islet-like progenitors progenitors clusters Stage 12345 Nestin/CK19 C-peptide/Nestin Insulin/C-peptide Culture (d) 0 5 5+9 5+16 5+28 Differentiation induction Media Additives L-Glut, NEAA, MTG NA + Laminin Basal medium DMEM + 15%FCS IMDM + 20% FCS N2 + B27 (- FCS: 5 + 10d to 5 + 28d) Substrate MEF Gelatine poly-Ornithine/Laminin UpregulationUpregulation ofof pancreaspancreas--specificspecific genesgenes andand differentiationdifferentiation ofof isletislet--likelike clustersclusters ES 5+9d 5+16d 5+28d panc brain Insulin/ proinsuli n InsulinInsulin II I Glucagon Amylase Somatostatin ß- tubulin C-peptide/insulin Pdx1 Pax4 IAPP ß-tubulin 5+95+16 5+28 5+28d wt Time of differentiation (d) ESES cellscells (Pax4+)(Pax4+) differentiatedifferentiate intointo functionalfunctional insulininsulin--producingproducing cellscells Insulin/C-peptide Voltage-activated Na+ channels ATP-sensitive K+ channels (Pax4+ cells) Wt 5+28d 30 Transplantation Insulin/C-peptide 25 20 15 10 Blood glucose [mM] wt (n=6) Sham control (n=6) + 5 Voltage-activated K channels + + STZ Pax4 (n=5) Pax4 , graft removal (n=2) 0 + 0481216202428323640 Pax4 5+28d Time (days) Bar = 20 µm Blyszczuk et al., PNAS 2003; 2004; Kania et al., IJDB 2004; Schroeder et al., Nature Prot. 2006 ESES cellscells recapitulaterecapitulate thethe programprogram ofof mesodermalmesodermal,, ectodermalectodermal andand endodermalendodermal lineagelineage developmentdevelopment Undifferentiated ESC Inner cell mass of blastocyst Proliferation and differentiation Cardiac cells Neuronal cells Pancreatic cells Wobus et al. (1991) Rolletschek et al. (2001) Blyszczuk et al. (2003) Wobus & Boheler, 2005 etc…. Blyszczuk et al. (2004) etc…. ESES CellCell TechnologyTechnology inin vitrovitro Embryotoxicity/Teratogenicity Cell Therapy Selective Differentiation Blastocyst Analysis of Embryotoxic Factors in vitro and Isolation 'G a in -o f- Injection Cultivation func In vitro t ++ io ES cells (A /A ) n Endoderm ' Ectoderm Pharmacology Differentiation Drug Screening embryoid body Mesoderm n' io Homologous ct fun Recombination f- -o A- oss 'L A-/A- Genomics/ Proteomics - cDNA Arrays/Microchips - SAGE CriticalCritical parametersparameters ofof embryotoxicityembryotoxicity teststests usingusing ESES cellscells • Treatment time (development-specific effects) • Concentration-dependent effects (threshold concentration) • Activity of chemicals under in vitro conditions (metabolic activity) • Genetic and epigenetic factors (long-term cultivation) • Several endpoints and lineages should be included into the test system. EffectsEffects ofof RARA onon ESES CellCell DDifferentiationifferentiation • Biologically active form of Vitamin A • Highly teratogenic in animals and human • Malformations of craniofacial structures and CNS • Cardiac (conotruncal heart, aortic arch, ventricular septum) defects (RAR-/- and RXR-/- mice!) StageStage-- andand concentrationconcentration--dependentdependent effectseffects ofof RARA onon ESES cellcell differentiationdifferentiation
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