USOO9029146B2

(12) United States Patent (10) Patent No.: US 9,029,146 B2 Lim et al. (45) Date of Patent: May 12, 2015

(54) MESENCHYMAL STEM CELL 2500/99 (2013.01); C12N 2501/115 (2013.01); CONDITIONED MEDUM CI2N 2501/135 (2013.01); C12N 2506/02 (2013.01): CI2N5/0662 (2013.01) (75) Inventors: Sai Kiang Lim, Singapore (SG); Elias USPC ...... 435/377; 435/404: 435/405; 435/406 Lye, Singapore (SG) (58) Field of Classification Search CPC ...... C12N5/0606; C12N 2501/115; C12N (73) Assignee: Agency for Science, Technology and 2506/02; C12N 2500/99: C12N5/0663; Research, Singapore (SG) C12N5/0611; C12N 2500/90; C12N 2509/00; C12N5/0018; C12N 5/0668: A01N 1/0221; (*) Notice: Subject to any disclaimer, the term of this CO7K 147475 patent is extended or adjusted under 35 See application file for complete search history. U.S.C. 154(b) by 1544 days. (56) References Cited (21) Appl. No.: 12/377,398 U.S. PATENT DOCUMENTS (22) PCT Filed: Aug. 15, 2007 7,592, 176 B2 * 9/2009 Pike et al...... 435/373 2003/0036.194 A1 2/2003 Xu et al...... 435,366 (86). PCT No.: PCT/SG2007/000257 2006/005.1334 A1 3/2006 Kornowski et al...... 424.93.21 S371 (c)(1), (2), (4) Date: Jun. 15, 2010 FOREIGN PATENT DOCUMENTS WO O1/32189 A1 5, 2001 (87) PCT Pub. No.: WO2O08/020815 WO O3,OO4605 A2 1, 2003 PCT Pub. Date: Feb. 21, 2008 WO 2005, O76845 8, 2005 OTHER PUBLICATIONS (65) Prior Publication Data Hausman et al. (2001) Prevention of fracture healing in rats by an US 2010/0323O27 A1 Dec. 23, 2010 inhibitor of angiogenesis. Bone 29(6): 560-564.* Schuldiner et al., PNAS, 97(21): 11307-11312 (2000). “Effects of Related U.S. Application Data E. growth factors O the differentiation of cells derived from uman embryonic stem cells. (63) Continuation of application No. PCT/SG2006/000232, filed on Aug. 15, 2006. * cited by examiner (60) Provisional application No. 60/878,222, filed on Jan. Primary Examiner — Deborah Crouch 3, 2007, provisional application No. 60/713.992, filed (74) Attorney, Agent, or Firm Nixon Peabody LLP; Mark on Sep. 2, 2005. J. FitzGerald (51) Int. Cl. (57) ABSTRACT EN Og 3. We disclose a method of preparing a conditioned cell culture ( .01) medium, the method comprising the steps of: (a) culturing a A6 IK35/28 (2006.01) mesenchymal stem cell (MSC), a descendent thereofor a cell C1 2N 5/0775 (2010.01) line derived therefrom in a cell culture medium; and (b) CI 2N 5/0735 (2010.01) optionally isolating the cell culture medium; in which the (52) U.S. Cl. mesenchymal stem cell (MSC) is obtained by propagating a CPC ...... A61K 35/28 (2013.01); C12N 5/0668 cell obtained by dispersing a embryonic stem (ES) cell (2013.01); C12N5/0663 (2013.01); C12N colony, or a descendent thereof, in the absence of co-culture 5/0018 (2013.01); C12N2509/00 (2013.01); in a serum free medium comprising FGF2. CI2N5/0606 (2013.01); C12N 2500/90 (2013.01); C12N 5/0611 (2013.01); C12N 16 Claims, 49 Drawing Sheets U.S. Patent May 12, 2015 Sheet 1 of 49 US 9,029,146 B2

FIGURE 1A

U.S. Patent May 12, 2015 Sheet 2 of 49 US 9,029,146 B2

FIGURE 1B

E. 5

-4C U.S. Patent May 12, 2015 Sheet 3 of 49 US 9,029,146 B2

FIGURE 1C

i. U.S. Patent May 12, 2015 Sheet 4 of 49 US 9,029,146 B2

FIGURE 1D

U.S. Patent May 12, 2015 Sheet 5 of 49 US 9,029,146 B2

FIGURE 1E

U.S. Patent May 12, 2015 Sheet 6 of 49 US 9,029,146 B2

FIGURE1F

Oil DN,

E

U.S. Patent May 12, 2015 Sheet 7 of 49 US 9,029,146 B2

FIGURE 1G

Dip

U.S. Patent May 12, 2015 Sheet 10 of 49 US 9,029,146 B2

FIGURE 3A

U.S. Patent May 12, 2015 Sheet 11 of 49 US 9,029,146 B2

FIGURE 3A (CONTINUED)

U.S. Patent May 12, 2015 Sheet 12 of 49 US 9,029,146 B2

FIGURE 3A (CONTINUED)

I s: se

iaido

ris E. 3. U.S. Patent May 12, 2015 Sheet 13 of 49 US 9,029,146 B2

FIGURE 3B

U.S. Patent May 12, 2015 Sheet 14 of 49 US 9,029,146 B2

FIGURE3B (CONTINUED) ii

2. I.

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FIGURE 3B (CONTINUED)

Collagen ||

HESS.E Day 14 Day2 --- CIOrldrogenesis U.S. Patent May 12, 2015 Sheet 16 of 49 US 9,029,146 B2

FIGURE 3C

U.S. Patent May 12, 2015 Sheet 17 of 49 US 9,029,146 B2

FIGURE 3C (CONTINUED)

s i 2. &

HLESS.E Day 14 Day 21 Osteocytic differentiation U.S. Patent May 12, 2015 Sheet 18 of 49 US 9,029,146 B2

FIGURE 3C (CONTINUED)

HESS.E. Day 4 sy 2.

Osteocytic differentiation U.S. Patent May 12, 2015 Sheet 19 Of 49 US 9,029,146 B2

FIGURE 4A

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FIGURE 4B

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FIGURE 4B (CONTINUED)

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U.S. Patent May 12, 2015 Sheet 22 of 49 US 9,029,146 B2

FIGURE 4C

i

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is girl T-shi- -Friuli

ill -i. ESC-MSC U.S. Patent May 12, 2015 Sheet 23 of 49 US 9,029,146 B2

FIGURE 4D

Metabolic processes

Cell roliferation

Differentiation

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U.S. Patent May 12, 2015 Sheet 25 Of 49 US 9,029,146 B2

FIGURE 5A

HESS.E.

F.

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MEF

H.E. U.S. Patent May 12, 2015 Sheet 26 of 49 US 9,029,146 B2

FIGURE SB

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FIGURESB (CONTINUED)

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FIGURE 5C

10s

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FIGURE SD

102 103 104 105 hESC-MSC U.S. Patent May 12, 2015 Sheet 30 of 49 US 9,029,146 B2

FIGURE SE

R. R. in East of Eart aaa. was alia as a rise are late Cl 8 U.S. Patent May 12, 2015 Sheet 31 of 49 US 9,029,146 B2

FIGURE 6

CO (N St N

U.S. Patent May 12, 2015 Sheet 32 of 49 US 9,029,146 B2

FIGURE 7 Cytokine Antibody Array W

Angiogenesis Antibody Array

NCM C

Metalloproteinase

Antibody Array

a t c NCW o U.S. Patent May 12, 2015 Sheet 33 of 49 US 9,029,146 B2

FIGURE 8

Metabolism

OO-02 --

1.OOE-00 *,glucosemetabolism š?cellularcatabolism No.?alcoholC?tabo?ism ¿|catabolism …|phosphorusmetabolism hexoseçatabolismi ____,|cellularcarbohydratecatabolism A?carbohydratemetabolism mainpathwaysof exocytosis secretion carbohydrateca?abolisin {cellularTlacTUITIO?eculecatabolism |monosaccharidecatabo?ism glucosecatabolism? OxidationofOrganiccompounds energyderivationby hexoseraetabolism cellularcarbohydratemetabolism ?monosaccharidemetabolism metabo?itesandenergy generationofprecursor carbohydratemetabolism macromp??CÙÎemetabolism phosphatemetabolism secretorypathway U.S. Patent May 12, 2015 Sheet 34 of 49 US 9,029,146 B2

FIGURE 8 (CONTINUED

Defense Response

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OOECO TT||taxis __,_,_,__…………]responsetostimulus heatgeneration |defenseresponse responsetoabioticstimulus responsetobioticstimulus responsetostress responsetowounding inflammatoryresponse immuneresponse thermoregulation chemotaxis responsetochemicalstimulus responsetoexternalstimulus responsetoexternalbioticstimulus responsetopest,pathogenorparasite U.S. Patent May 12, 2015 Sheet 35 of 49 US 9,029,146 B2

FIGURE 8 (CONTINUED

Tissue differentiation Non-specific 1.OOE-09

Vascularization OOE-07 Hematopoiesis 1.OOE-05

1.OOE-03 Bone development

1OOE-01 .…!Organdevelopment 10OE-01 º:?ossification !]development hemopoiesis organdevelopment boneTermodeling organmorphogenesis tissuedevelopment angiogenesis bloodvesseldevelopment vasculaturedevelopment bloodvesselmorphogenesis vasculogenesis hemopoieticorlymphoid skeletaldevelopment biomineralformation U.S. Patent May 12, 2015 Sheet 36 of 49 US 9,029,146 B2

FIGURE 9

junctionGap junctionTight adhesionFocal Apoptosis interactionECM-receptor signalingpathwayJak-STAT pathwaysignalingMAPK pathwaysignalingreceptorToll-like signalingpathwayTGF-beta pathwaysigna?ingmTOR pathwaysignalingepsilonRIFc cytoskeletonRegulationofactin presentationandAntigenprocessing pathwaysignalingreceptorTcell lineageHematopoieticcell interactionCytokine-cytokinereceptor infectionHelicobacterpykorisignalinginEpithelialcell migrationtransendothelialLeukocyte cascadescoagulationComplementand US 9,029,146 B2

metabolismMethane metabolismInositol cycle(TCAcycle)Citrate carboxylate(COReductivecycle U.S. Patent May 12, 2015 Sheet 38 of 49 US 9,029,146 B2

FIGURE 10

U.S. Patent May 12, 2015 Sheet 39 Of 49 US 9,029,146 B2

FIGURE 11A U.S. Patent May 12, 2015 Sheet 40 of 49 US 9,029,146 B2

FIGURE 11B

US 9,029,146 B2

Over-represented pathway

Callcycle

?eñawopurinebitisynthesis Cytoskeletatregula?ionbyRhoGTPase U.S. Patent May 12, 2015 Sheet 42 of 49 US 9,029,146 B2

FIGURE 13 Over-represented processes

U.S. Patent May 12, 2015 Sheet 43 of 49 US 9,029,146 B2

FIGURE 14

Over-represented functions

Growthfactor Cytokine Inter?eukin Othercytokir?e Chaperoric Isomerase Otherisomeras? ATPsynthase Hydratase Otheriyase Histone Signalingmolecule Chemokine Oxidoreductase Dehydrogenase Peroxidase Transamimase Otherenzymeregu?a?or Selectregulatorymo??cule Ribonuc?eoprotein Ribosoma?pºç?ein Transtationfactor OtherRNA-bändingproteín Calmodufinrelatedprotein Vesiclecoatprotein

Under-represented functions 100-01 100.06 100.11 100s-16 100-21 ,00E-26 100E-31 1.OOS-35 Transcriptionfactor Zincfingertranscriptionfactor KRABboxtranscriptionfactor Othertranscrip?jonfactor Chromatim/chromatiri-bindingprotein G-proteincoup?edreceptor U.S. Patent May 12, 2015 Sheet 44 of 49 US 9,029,146 B2

FIGURE 15

CM NCM Saline U.S. Patent May 12, 2015 Sheet 45 of 49 US 9,029,146 B2

FIGURE 16 minessms360---

-- e H .m.T.L.E.III. CM 3 NCM Crow CM rol 15% ge: s 1 OX 50X 100X FCS U.S. Patent May 12, 2015 Sheet 46 of 49 US 9,029,146 B2

FIGURE 17

ONCM X CM U.S. Patent May 12, 2015 Sheet 47 of 49 US 9,029,146 B2

FIGURE 18

U.S. Patent May 12, 2015 Sheet 48 of 49 US 9,029,146 B2

FIGURE 19 D. baseline ischemia d4 hours reperfusion 80.00 dobutamine 2.5 70.00 - dobutamine 5.0

60.00 -

50.00 -

40.00 -

30.00 -

20.00 -

10.00

0.00

-10.00

-20.00 U.S. Patent May 12, 2015 Sheet 49 of 49 US 9,029,146 B2

FIGURE 20 baseline ischemia 60.00 - 14 hours reperfusio dobutamine 2.5 50.00 - dobutamine 5.0

40.00 -

30.00 –

20.00 -

10.00 -

O.OO - US 9,029, 146 B2 1. 2 MESENCHYMAL STEM CELL The therapeutic capacity of MSCs to treat a wide spectrum CONDITIONED MEDIUM of diseases in clinical and preclinical applications to treat a wide range of diseases A1-A2) e.g. GVHDA1 in muscu CROSS-REFERENCE TO RELATED loskeletal tissue bioengineering A3, A4 and heart disease APPLICATIONS A5,A6 has been attributed to their potential to differentiate into many different reparative cell types. This application is a S371 National Stage of International However, the efficiency of transplanted MSCs to differen application PCT/SG2007/000257, filed Aug. 15, 2007, which tiate into functional reparative cells in the injured tissues or designated the U.S. PCT/SG2007/000257 claims priority organs, and in therapeutically relevant numbers have never under 35 U.S.C. S 119(e) of U.S. Provisional application Ser. 10 been adequately documented or demonstrated. No. 60/878,222, filed Jan. 3, 2007, and is a continuation-in This invention seeks to solve this and other problems with part of International application PCT/SG2006/000232, filed methods in the art. Aug. 15, 2006, which designates the U.S. and which claimed the priority under 35 U.S.C. S 119(e) of U.S. Provisional SUMMARY application Ser. No. 60/713,992, filed Sep. 2, 2005. The entire 15 contents of each of the priority applications is incorporated According to the invention, instead of using stem cells, herein by reference. The foregoing application, and each document cited or injured or lost tissues may be regenerated or repaired through referenced in each of the present and foregoing applications, enhancement of endogenous tissue repair by applying secre including during the prosecution of each of the foregoing tions from MSCs instead of, or in addition to, MSCs them application (“application and article cited documents'), and selves. any manufacturers instructions or catalogues for any prod Specifically, we provide for the use of conditioned media in ucts cited or mentioned in each of the foregoing application which the MSCs derived from human embryonic stem cells and articles and in any of the application and article cited are cultured in the treatment of disease. Thus, conditioned documents, are hereby incorporated herein by reference. Fur 25 media may be used to treat any disease for which ES cells, thermore, all documents cited in this text, and all documents specifically MSCs, are prescribed as being suitable for treat cited or reference in documents cited in this text, and any 1ng. manufacturers instructions or catalogues for any products We disclose the identity of polypeptides secreted by MSCs, cited or mentioned in this text or in any document hereby and which form the components of the conditioned media. It incorporated into this text, are hereby incorporated herein by 30 will be evident that compositions comprising one or more reference. Documents incorporated by reference into this text specific biologically active compounds in the secretions of or any teachings therein may be used in the practice of this MSCs, in particular one or more of the 794 polypeptides, may invention. Documents incorporated by reference into this text be used instead of, or in addition to, the conditioned media in are not admitted to be prior art. Such treatment. 35 With this approach, the present confounding issues asso FIELD ciated with cell based therapy i.e. immune compatibility, The present invention relates to the fields of development, tumorigenicity, Xenozootic infections, costs, and waiting time cell biology, molecular biology and genetics. More particu if autologous cell preparations are used will be eliminated. larly, the invention relates to a method of deriving mesenchy 40 Such an approach could potentially provide for the devel mal stem cells from embryonic stem cells. opment of “off-the-shelf MSC-based therapeutics at afford able costs and with better quality control and consistency. BACKGROUND The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, Stem cells, unlike differentiated cells have the capacity to 45 molecular biology, microbiology, recombinant DNA and divide and either self-renew or differentiate into phenotypi immunology, which are within the capabilities of a person of cally and functionally different daughter cells (Keller, Genes ordinary skill in the art. Such techniques are explained in the Dev. 2005; 19:1129-1155: Wobus and Boheler, Physiol Rev. literature. See, for example, J. Sambrook, E. F. Fritsch, and T. 2005: 85:635-678; Wiles, Methods in Enzymology. 1993: Maniatis, 1989, Molecular Cloning: A Laboratory Manual, 225:900-918; Choi et al, Methods Mol Med. 2005: 105:359 50 Second Edition, Books 1-3, Cold Spring Harbor Laboratory 368). Press: Ausubel, F. M. et al. (1995 and periodic supplements: Mesenchymal stein cells (MSCs) are multipotent stein Current Protocols in Molecular Biology, ch. 9, 13, and 16, cells that have documented evidence oftherapeutic efficacy in John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and treating musculoskeletal injuries, improving cardiac function A. Kahn, 1996, DNA Isolation and Sequencing. Essential in cardiovascular disease and ameliorating the severity of 55 Techniques, John Wiley & Sons; J. M. Polak and James O'D. GVHD (Le Blanc and Pittenger, 2005). Being lineage McGee, 1990, Oligonucleotide Synthesis: A Practical restricted, they have limited but robust potential to differen Approach, Irl Press; D. M. J. Lilley and J. E. Dahlberg, 1992, tiate into mesenchymal cell types, e.g. adipocytes, chondro Methods of Enzymology: DNA Structure Part A. Synthesis cytes and osteocytes, and have negligible risk of teratoma and Physical Analysis of DNA Methods in Enzymology, Aca formation. Host immune rejection of transplanted MSCs is 60 demic Press; Using Antibodies: A Laboratory Manual: Por routinely circumvented through autologous or allogeneic table Protocol NO. I by Edward Harlow, David Lane, Ed transplantation. MSCs can be isolated from several adult Harlow (1999, Cold Spring Harbor Laboratory Press, ISBN tissues including bone marrow (BM), adipose tissues (ad), 0-87969-544-7); Antibodies: A Laboratory Manual by Ed cord blood and expanded ex vivo. Harlow (Editor), David Lane (Editor) (1988, Cold Spring However, availability of tissues for their isolation remains 65 Harbor Laboratory Press, ISBN 0-87969-314-2), 1855; and limiting and requires risky invasive procedures, and ex vivo Lab Ref: A Handbook of Recipes, Reagents, and Other Ref expansion of MSCs while significant, is nonetheless finite. erence Tools for Use at the Bench, Edited Jane Roskams and US 9,029, 146 B2 3 4 Linda Rodgers, 2002, Cold Spring Harbor Laboratory, ISBN measured by Taqman quantitative RT-PCR. All values are 0-87969-630-3. Each of these general texts is herein incor normalized to that of HuBS9.E.1 porated by reference. FIGS. 4A-4E. Gene expression analysis. FIG. 4A. Hierarchical clustering of expressed genes in BRIEF DESCRIPTION OF THE DRAWINGS three hESC-MSC cultures consisting of HuES9.E.1, HuES9.E3, and H1. E2, three BMMSC samples, three ad FIGS. 1A-1G. Characterisation of hESC-MSC cultures. MSC samples and three hESC lines consisting of HuES9, H1 FIG. 1A. Cellular morphology under phase contrast. and Hes3. FIG. 1B. Expression of pluripotency-associated genes in FIG. 4B. Pairwise comparison of gene expression between hESC-MSC. Transcript levels are measured by Taqman 10 hESC-MSCs and BMMSCs (left) and between hESC-MSCs based quantitative RT-PCR and normalized to that of hESC. and hESCs (right). The transcript level in hESC is derived from the average of FIG. 4C. Analysis of commonly expressed genes (<2 fold HuES9 and H1 hESC lines. difference) in hESC-MSCs and BM-MSCs. The genes are FIG.1C. Western blot analysis for pluripotency-associated classified into biological processes using the Panther classi 15 fication system. Each biological process is determined if it is genes in HuES9 and H1 hESC lines, HuES9.E1 HuES9.E3 significantly over- or under-represented (p<0.01) by compar and H1. E2 hESC-MSC cultures and E14 mouse ESC line. ing the observed frequency of genes to the expected fre FIG. 1D. Renal subcapsular transplantation of HuES9 and quency of genes in the NCBI. H. sapiens gene database of HuES9.E.1. Paraffin-embedded, H&E stained cross sections 23481 genes for each biological process. Significantly over of kidney four months after transplantation with either or under-represented processes are grouped and graphically HuES9.E.1 (top) or HuES9 (bottom). presented. FIG. 1E. Alkaline phosphatase activity in human HuES9 FIG. 4D. Analysis of differentially expressed genes (>2 ESC line, mouse E14 ESC line, mouse embryonic fibroblast fold difference) in hESC-MSCs and BM-MSCs. Biological (MEF) feeder and HuES9.E1. processes that are significantly over- or under-represented FIG.1F. Genomic DNA analysis by PCR for the presence 25 (p<0.01) by genes highly expressed in hESC-MSCs or BM of human Alu and mouse c-mos repeat sequences. MSCs are grouped and graphically presented. FIG.1G. Chromosomal analysis of HuES9.E1 by G-band FIG. 4E. Gene expression analysis. ing (top panel) and Spectral Karyotyping (SKY) (middle FIGS. 5A-5E. Positive and negative sorting for generation panel). Inversion of chromosome 9 shown in bottom panel. of hESC-MSC. FIGS. 2A-2B. Surface antigen profiling by FACS analysis. 30 FIG.5A. FACS analysis HuBS9.E1 HuES9.E3 and H1.E2 FIG. 2A. HuES9.E.1 HuES9.E3 and H1.E.2 hESC-MSCs, hESC-MSCs, hESCs, and murine embryonic fibroblast HuES9 hESCs, and murine embryonic fibroblastfeeder cells feeder cells are stained and analyzed for the presence of CD24 are stained and analyzed on a Cyan LX (Dako North America, on a Cyan LX (Dako North America, Inc., Carpinteria, Calif.) Inc., Carpinteria, Calif.) instrument using WinMDI software. instrument using WinMDI software. Nonspecific fluores Nonspecific fluorescence is determined by incubation of 35 cence is determined by incubation of similar cell aliquots similar cell aliquots with isotype-matched mouse mono with isotype-matched mouse monoclonal antibodies. clonal antibodies. FIG. 5B. Sorting for CD105+, CD24-cells from HuES9 FIG.2B. HuBS9.E.1 and HuES9.E3 hESC-MSCs are pas cells that have been trypsinized and propagated without saged twice in serum-containing BM-MSC media before feeder in media supplemented with PDGF and FGF2 for one being analyzed in parallel with BM-MSCs by FACS analysis. 40 week. CD105+, CD24-cells represented in Q4 are selected FIGS 3A-3C. Differentiation of HuBS9.E.1. HuBS9.E.1 for culture. cells are induced to undergo adipogenesis, chondrogenesis FIG.5C. Pairwise comparison of gene expression between and osteogenesis using standard protocols. Q4.1 and each of the other Q4 cultures, namely Q4.2 to Q4.5. FIG.3A. Adipogenesis. i.) Day 14 after inducing adipogen FIG.5D. Pairwise comparison of gene expression between esis, cells are stained for oil droplets by oil red; ii) PPARY 45 all Q4 cultures and hESC-MSCs consisting of HuES9.E.1, mRNA at day 7 and day 14 are measured by Taqman quanti HuES9.E3, and H1.E.2, and between all Q4 cultures and BM tative RT-PCR. All values are normalized to that of MSCs; e) SKY analysis of Q4.3. HuES9.E1; iii) Relative PPARY mRNA levels in HuES9 and FIG. 5E. Positive and negative sorting for generation of HI hESCs, their derivative MSC cell cultures (HuES9.E.1, hESC-MSC. HuES9.E3 and H1.E2) and adult tissue-derived MSCs (BM 50 FIG. 6. Protein analysis of media conditioned by MSC and ad-MSC) as measured by Taqman quantitative RT HuES9.E1 MSC culture. 80% confluent HuBS9.E1 cell cul PCR. All values are normalized to that of HuBS9.E1. tures are washed 3 times with PBS, cultured overnight in a FIG. 3B. Chondrogenesis. i.) Day 21 after inducing chon chemically defined media consisting of DMEM media with drogenesis, cells are stained for proteoglycans by alcian blue out phenol red and supplemented with ITS, 5 ng/ml FGF2, 5 (left) and immunoreactivity to collagen type II using a HRP 55 ng/ml PDGF AB glutamine-penicillin-streptomycin and based visualization assay; ii) Aggrecan and PPARY mRNA at B-mercaptoethanol. The cultures are then rinsed three times day 7 and day 14 are measured by Taqman quantitative RT with PBS and then replaced with fresh defined media. An PCR. All values are normalized to that of HuES9.E1; iii) aliquot of media are removed at 0, 24, 48 and 72 hours, Relative PPARY mRNA levels in HuES9 and HI hESCs, their centrifuged at 500xg and the supernatant is 0.2L, filtered. 10 derivative MSC cell cultures (HuES9.E1, HuBS9.E3 and 60 ul of the media is separated on a 4-12% SDS-PAGE and H1.E2) and adult tissue-derived MSCs (BM-MSC and ad silver-stained. MSC) as measured by Taqman quantitative RTPCR. All val FIG. 7. Antibody array. One ml of conditioned (CM) or ues are normalized to that of HuES9.E1. non-conditioned media (NCM) is incubated with a RayBio(R) FIG. 3C. Osteogenesis i) Day 21 after inducing chondro Cytokine Antibody Arrays according to manufacturers genesis, cells are stained for mineralization by Von Kossa 65 instruction (RayBio Norcross, Ga.). The antibody map for stain, ii., iii) Bone-specific alkaline phosphatase (ALP) and each array is listed in the Examples. Binding of ligands to bone sialoprotein (BSP) mRNA at day 7 and day 14 are specific antibody is visualized using HRP-based chemilumi US 9,029, 146 B2 5 6 nescence assay. Different exposures of each membrane is FIG. 17 shows 6 CEM cells incubated with either 50 or 500 analysed. An antigen is scored as presentifa signal is present uM H2O2 in the presence of 1xCM or 10xCM. The relative on the membrane hybridized with CM but absent on that number of viable cells was determined at 12 and 24 hours by hybridized with NCM. Data from analysis of 4 independent trypan blue staining. batches of CM and NCM is summarized in the Examples. 5 FIG. 18. Relative infarct size after acute ischemia-reperfu FIG. 8. Distribution of 201 gene products into biological sion in pigs treated with CM, NCM or saline. Acute ischemia processes. The 201 genes are classified into different biologi was induced by ligation of LCX for 75 minutes followed by cal processes in the GO classification system. 58 biological release of ligation for reperfusion. Five minutes before the processes that are over-represented by the frequency of genes onset of reperfusion, the pigs were treated intravenously with in the secretory proteome relative to the frequency of the CM, NCM or saline. Immediately after reperfusion, CM genes in a database collated from Unigene, Entrez and Gen NCM or saline was infused locally into the LCx coronary Bank with a p-value of <0.05 are grouped into three major artery. Four hours after reperfusion, relative infarct size in the groups: metabolism, defense response and tissue differentia area at risk was assessed. tion. FIGS. 19 and 20. Echocardiography. FIG. 19: Systolic wall 15 thickening. FIG. 20. Fractional area shortening. Cardiac FIG. 9. Distribution of 201 gene products into pathways. Measurements were performed before ischemia, during The 201 genes are classified into different pathways in the GO ischemia and 4 hours after ischemia. Four hours after classification system. 30 biological processes that are over ischemia, additional measurements were performed under represented by the frequency of genes in the secretory pro pharmacologically induced stress by intravenous infusion of teome relative to the frequency of the genes in a database the B1-adrenergic receptor agonist dobutamine (2.5 and 5.0 collated from Unigene, Entrez and GenBank with a p-value of microg/kg/min) to challenge stunned myocardium. Short <0.05 are categorised into several major categories: receptor axis epicardial ultrasound images (Prosound SSD-5000, binding, signal transduction, cell-cell interaction, cell migra 5-MHz probe UST-5280-5, Aloka Holding Europe AG, Zug, tion, immune response and metabolism. Switzerland) were obtained at the midpapillary level. Wall FIG. 10. SDS gel electrophoresis showing presence of 25 thickness (WT) of the infarct area and LV internal area (LVia) MMP gelatinase activity in ES cell conditioned media (CM) were measured at end diastole (ED) and end systole (ES). but not a non-conditioned media (NCM) control Systolic wall thickening (SWT) was calculated as (WT FIG. 11A. Graph showing mortality in a mouse model of (ES)-WT(ED))/WT(ED)*100% and fractional area short acute myocardial infarction (AMI). CM: mice treated with ES ening (FAS) as (LVia(ED)-LVia(ES))/. cell conditioned media; NCM: mice treated with non-condi 30 tioned media control. DETAILED DESCRIPTION FIG. 11B. Graph showing left ventricular ejection fraction Conditioned Media in a mouse model of acute myocardial infarction (AMI). CM: The Examples describe experiments which analyse the mice treated with ES cell conditioned media; NCM: mice 35 proteome of human ESC-derived MSCs (hESC-MSCs). treated with non-conditioned media control. As shown in Examples 7 to 15, the hESC-MSCs have an FIG. 12. Predicted pathways driven by the secreted pro expression profile which is similar to that of adult bone mar teins. Frequency of genes in each pathway was significantly row derived MSCs (BM-MSCs). These results demonstrate higher in the secretome than that in the NCBI homo sapiens that the MSCs derived by the methods described herein have database (p<0.01). 40 significant biological similarities to their bone marrow FIG. 13. Predicted processes driven by the secreted pro derived counterparts, e.g., in their ability to secrete paracrine teins. Frequency of genes in each pathway was significantly factors. Accordingly, the hESC-MSCs may be used for any higher or lower in the secretome than that in the NCBI homo purpose for which BM-MSCs are suitable. sapiens database (p<0.01). We further provide a medium which is conditioned by FIG. 14. Predicted molecular driven by the secreted pro 45 culture of hESC-MSCs. Such a conditioned medium com teins. Frequency of genes in each pathway was significantly prises molecules secreted by the hESC-MSC, including higher in the secretome than that in the NCBI homo sapiens unique gene products. Such a conditioned medium, and com database (p<0.01). binations of any of the molecules comprised therein, includ FIG. 15 shows THP-1 cells that are exposed to 100 ug CM ing in particular proteins or polypeptides, may be used in the proteins or NCM proteins or PBS for 30 mins. Cell lysate is 50 treatment of disease. They may be used to Supplement the then prepared and analyzed by standard western blot hybrid activity of, or in place of the hESC-MSCs, for the purpose of ization using anti-phosphorylated Smad3 antibody and a for example treating or preventing a disease. chemiluminescence-based detection system. The signals Conditioned medium may be made by culturing MSCs in a were quantitated by densitometry and the relative intensity of medium, Such as a cell culture medium, for a predetermined the signals was estimated. 55 length of time. The MSCs may in particular comprise those FIG.16 shows 1x105 HUVECs that are placed in the upper produced by any of the methods described in this document. chamber of Transwell(R)plates with membrane pore size of 5 The conditioned medium will comprise polypeptides uM. The lower chamber is filled with RPMI media with 10x. secreted by the MSCs, as described in the Examples. 50x100x dilution of a 25x concentrated CM or NCM Stock The conditioned medium may be used in therapy as is, or solution. The positive and negative controls were RPMI with 60 after one or more treatment steps. For example, the condi 15% and 0% fetal calf serum (FCS), respectively. After 24 tioned medium may be UV treated, filter sterilised, etc. One hours at 37° C. in CO2 incubator, the number of cells that or more purification steps may be employed. migrated to the lower chamber was counted. The number of In particular, the conditioned media may be concentrated, cells in the chamber with 10x dilution of the concentrated CM for example by dialysis or ultrafiltration. For example, the was normalized to 100. The number of cells that have 65 medium may be concentrated using membrane ultrafiltration migrated in the other chambers was expressed relative to that with a nominal molecular weight limit (NMWL) of for in the 10x dilution of the concentrated CM. example 3K. US 9,029, 146 B2 7 8 We also provide for a composition comprising one or more, nucleoprotein, ribosomal protein, translation factor, other preferably all, of the polypeptides described in the Examples, RNA-binding protein, histone, calmodulin related protein, in lieu of or to Supplement Such a conditioned medium. vesicle coat protein. Diseases Treatable by MSC Conditioned Medium Furthermore, the hESC-MSCs may be used to treat dis Analysis of the proteome of the hESC-MSCs shows that eases which these functions may have a role in, or whose the proteins expressed are involved in a number of biological repair or treatment involves any one or more of these biologi processes including: metabolism, defense response, and tis cal processes. Similarly, the proteins expressed by the hESC Sue differentiation including vascularization, hematopoiesis MSCs, singly or in combination, preferably in the form of a and skeletal development. Wetherefore provide generally for conditioned medium, may be used to Supplement the activity 10 of, or in place of the hESC-MSCs, for the purpose of for the use of hESC-MSCs or medium conditioned by hESC example treating or preventing Such diseases. MSCs in the regulation of any of these biological processeses, The gene products expressed by the hESC-MSCs are as detailed below. shown to activate important signalling pathways in cardio We provide for the use of hESC-MSCs or medium condi vascular biology, bone development and hematopoiesis Such tioned by hESC-MSCs in the regulation of pathways includ 15 as Jak-STAT, MAPK, Toll-like receptor, TGF-beta signalling ing any one or more of the following: cytoskeletal regulation and mTOR signaling pathways. Accordingly, the hESC by Rho GTPase, cell cycle, integrin signalling pathway, MSCs, proteins expressed by them, etc., may be used to pre Inflammation mediated by chemokine & cytokine signaling vent or treat a disease in which any of these signalling path pathway, FGF signaling pathway, EGF receptor signaling ways is involved, or whose aetiology involves one or more pathway, angiogenesis, plasminogen activating cascade, defects in any one or more of these signalling pathways. blood coagulation, glycolysis, ubiquitin proteasome path Accordingly, Such a conditioned medium may be used to way, de novo purine biosynthesis, TCA cycle, phenylalanine treat cardiac failure, bone marrow disease, skin disease, burns biosynthesis, heme biosynthesis. and degenerative diseases such as diabetes, Alzheimer's dis We provide also for the use of hESC-MSCs or medium ease, Parkinson's disease and cancer. conditioned by hESC-MSCs in the regulation of processes 25 Such a conditioned medium may also be used to treat including any one or more of the following: cell structure and myocardial infarction, a cutaneous wound, a dermatologic motility, cell structure, cell communication, cell motility, cell disorder, a dermatological lesion, dermatitis, psoriasis, adhesion, endocytosis, mitosis, exocytosis, cytokinesis, cell condyloma, Verruca, hemangioma, keloid, skin cancer, atopic cycle, immunity and defense, cytokine/chemokine mediated dermatitis, Behcet disease, chronic granulomatous disease, immunity, macrophage-mediated immunity, granulocyte 30 cutaneous T cell lymphoma, ulceration, a pathological con mediated immunity, ligand-mediated signaling, cytokine and dition characterised by initial injury inducing inflammation chemokine mediated signaling pathway, signal transduction, and immune dysregulation leading to chronic tissue remod extracellular matrix protein-mediated signaling, growth fac eling including fibrosis and loss of function, renal ischemic tor homeostasis, receptor protein tyrosine kinase signaling injury, cystic fibrosis, sinusitis and rhinitis or an orthopaedic pathway, cell adhesion-mediated signaling, cell Surface 35 disease. receptor mediated signal transduction, JAK-STAT cascade, The conditioned medium may be used to aid wound heal antioxidation and free radical removal, homeostasis, stress ing, scar reduction, bone formation, a bone graft or bone response, blood clotting, developmental processes, meso marrow transplantation in an individual. derm development, skeletal development, angiogenesis, In particular, the conditioned medium may be used to regu muscle development, muscle contraction, protein metabo 40 late the processes involved in vascularisation, hematology lism and modification, proteolysis, protein folding, protein (specifically immune processes) or musculoskeletal develop complex assembly, amino acid activation, intracellular pro ment, etc. tein traffic, other protein targeting and localization, amino Furthermore, any one or more proteins secreted from the acid metabolism, protein biosynthesis, protein disulfide MSCs described here, including in the form of conditioned isomerase reaction, carbohydrate metabolism, glycolysis, 45 media, may be used for the same purposes as the BM-MSCs pentose-phosphate shunt, other polysaccharide metabolism, described herein. We therefore provide a composition com purine metabolism, regulation of phosphate metabolism, prising one or more, preferably Substantially all, the polypep Vitamin metabolism, amino acid biosynthesis, pre-mRNA tides which are present in the conditioned medium. Specifi processing, translational regulation, mRNA splicing. cally, we provide a composition comprising one or more, We further provide for the use of hESC-MSCs or medium 50 preferably substantially all, the polypeptides set out in the conditioned by hESC-MSCs in the supply of functions Examples, including Example 9. Example 13, Example 14 or including any one or more of the following: signaling mol Example 14A. ecule, chemokine, growth factor, cytokine, interleukin, other Such a composition may be used for any purpose the con cytokine, extracellular matrix, extracellular matrix structural ditioned medium may be used. Unless the context dictates protein, other extracellular matrix, extracellular matrix gly 55 otherwise, the term “conditioned medium’ should be taken to coprotein, protease, metalloprotease, other proteases, pro include not only cell culture medium exposed to MSCs as tease inhibitor, metalloprotease inhibitor, serine protease well as Such a composition comprising one or more, prefer inhibitor, oxidoreductase, dehydrogenase, peroxidase, chap ably substantially all, the polypeptides which are present in erone, chaperonin, Hsp70 family chaperone, other chaper the conditioned medium. ones, synthetase, synthase and synthetase, select calcium 60 The hESC-MSCs may also be used as sources for any of the binding protein, aminoacyl-tRNA synthetase, lyase, proteins secreted or expressed by them, as listed in the isomerase, other isomerase, ATP synthase, hydratase, tran Examples including Examples 10 to 14, particularly the saminase, other lyase, other enzyme regulator, select regula tables therein. In particular, the hESC-MSCs may be used as tory molecule, actin binding cytoskeletal protein, cytoskel a source of conditioned media, We therefore provide for a etal protein, non-motor actin binding protein, actin and actin 65 method of producing a polypeptide as shown in any of the related protein, annexin, tubulin, cell adhesion molecule, Examples including Examples 10 to 14, the method compris actin binding motor protein, intermediate filament, ribo ing obtaining a mesenchymal Stein cell as described, cultur US 9,029, 146 B2 10 ing the mesenchymal stem cell and isolating the polypeptide in clinical or therapeutic applications; replacement of adult from the mesenchymal stem cell, preferably from a medium tissue-derived MSCs as feeders for propagation of other cell in which the mesenchymal stem cell is growing. types such as human ESCs, expansion of cord blood or bone Obtaining Mesenchymal StemCells (MSC) marrow stem cell populations; and preparation of MSC-con MSCs suitable for use in the production of conditioned 5 ditioned media for treatment of cardiovascular disease. media may be made by any method known in the art. In Disaggregating Embryonic StemCell Colonies particular, MSCs may be made by propagating a cell obtained Our methods of producing mesenchymal stem cells com by dispersing a embryonic stem (ES) cell colony, or a descen prise dispersing or disaggregating an embryonic stem cell dent thereof, in the absence of co-culture in a serum free colony into cells. medium comprising FGF2. This is described in detail in the 10 The embryonic stein cell colony may comprise a huES9 sections below. colony (Cowan Calif., Klimanskaya I, McMahon J. Atienza J. The prior art methods of obtaining mesenchymal stem cells Witmyer J, et al. (2004) Derivation of embryonic stem-cell (MSC) or MSC-like cells from hESCs involve either trans lines from human blastocysts. NEnglJ Med 350: 1353-1356) fection of a human telomerase reverse transcriptase (hTERT) or a H1 ESC colony (Thomson JA, Itskovitz-Eldor J, Shapiro gene into differentiating hESCs (Xu et al., 2004) or coculture 15 SS, Waknitz MA, Swiergiel JJ, et al. (1998) Embryonic Stem with mouse OP9 cell line (Barberi et al., 2005). The use of Cell Lines Derived from Human Blastocysts. Science 282: exogenous genetic material and mouse cells in these deriva 1145-1147). tion protocols introduces unacceptable risks of tumorigenic Preferably, the cells in the colony are disaggregated or ity or infection of Xenozootic infectious agents. dispersed to a substantial extent, i.e., at least into clumps. In contrast, our method provides for a clinically relevant More preferably, the colony is disaggregated or dispersed to and reproducible protocol for isolating similar or identical the extent that all the cells in the colony are single, i.e., the (preferably homogenous) MSC populations from differenti colony is completely disaggregated. ating hESCs. In general, our method comprises dispersing a The disaggregation may be achieved with a dispersing embryonic stem (ES) cell colony into cells. The cells are then agent. plated out and propagated. The cells are propagated in the 25 The dispersing agent may be anything that is capable of absence of co-culture in a serum free medium comprising detaching at least Some embryonic stem cells in a colony from fibroblast growth factor 2 (FGF2), in order to obtain mesen each other. The dispersing agent may preferably comprise a chymal stem cells (MSCs). reagent which disrupts the adhesion between cells in a colony, Thus, our protocol does not require serum, use of mouse or between cells and a substrate, or both. Preferably, the cells or genetic manipulations and requires less manipula 30 dispersing agent may comprise a protease. tions and time, and is therefore highly scalable. The Examples In preferred embodiments, the dispersing agent comprises describe the isolation of MSCs from two different hESC trypsin. The treatment with trypsin may last for example for 3 lines, HuES9 and H-1 and also a third one, Hes-3', and minutes orthereabouts at 37 degrees C. The cells may then be demonstrates the robustness of the protocol. Human ES cell neutralised, centrifuged and resuspended in medium before derived MSCs (hESC-MSCs) obtained by the methods and 35 plating out. compositions described here are remarkably similar to bone In preferred embodiments, the method comprises dispers marrow derived MSCs (BM-MSCs). ing a confluent plate of human embryonic stem cells with In preferred embodiments, the embryonic stem cell culture trypsin and plating the cells out. comprises a human embryonic stem cell (hESC) culture. The disaggregation may comprise at least Some of the In a one embodiment, a method of generating mesenchy 40 following sequence of steps: aspiration, rinsing, trypsiniza mal stem cells (MSC) comprises trypsinizing and propagat tion, incubation, dislodging, quenching, re-seeding and ali ing hESCs without feeder support in media supplemented quoting. The following protocol is adapted from the Hedrick with FGF2 and optionally PDGF AB before sorting for Lab, UC San Diego (http://hedricklab.ucsa.edu/Protocol/ CD105+CD24-cells. COSCell.html). In a preferred embodiment, the method comprises sorting 45 In the aspiration step, the media is aspirated or generally for CD 105+, CD24-cells from trypsinized hESCs one week removed from the vessel. Such as a flask. In the rinsing step, after feeder-free propagation in a media Supplemented with the cells are rinsed with a volume, for example 5-10 mls, of a FGF2 and optionally PDGF AB will generate to generate a buffered medium, which is preferably free from Ca" and hESC-MSC cell culture in which at least some, preferably Mg". For example, the cells may be rinsed with calcium and substantially all, more preferably all cells are similar or iden 50 magnesium free PBS. In the trypsinization step, an amount of tical (preferably homogenous) to each other. dispersing agent in buffer is added to the vessel, and the vessel The methods described here for generating clinically rel rolled to coat the growing Surface with the dispersing agent evant hESCMSC cultures that are physically, biologically solution. For example, 1 ml of trypsin in Hank’s BSS may be and functionally similar to BM-MSCs could potentially alle added to a flask. viate the limiting supply of BM for isolation of BM-MSCs 55 In the incubation step, the cells are left for some time at a that have demonstrated therapeutic efficacy in many clinical maintained temperature. For example, the cells may be left at and preclinical animal studies''. This will also remove the 37° C. for a few minutes (e.g., 2 to 5 minutes). In the dislodg need for risky invasive BM aspiration procedure, reduce the ing step, the cells may be dislodged by mechanical action, for waiting time and cost of preparing BM-MSCs on aper-patient example by Scraping or by whacking the side of the vessel basis, and reduce batch to batch variations. Furthermore, the 60 with a hand. The cells should come off in sheets and slide robust derivation of MSCs from a defined cell type such as down the surface. hESC provides a useful model to study and better understand In the quenching step, a Volume of medium is added to the the derivation and biology of MSC that has remain an enigma flask. The medium preferably contains a neutralising agent to despite its present widespread clinical and preclinical appli stop the action of the dispersing agent. For example, if the cations. 65 dispersing agent is a protease such as trypsin, the medium Further uses of the mesenchymal stem cells generated by may contain a protein, such as a serum protein, which will the process include replacement of adult tissue-derived MSCs mop up the activity of the protease. In a particular example, 3 US 9,029, 146 B2 11 12 ml of serum containing cell culture medium is added to the Serum Free Media flask to make up a total of 4 mls. The cells may be pipetted to The dissociated or disaggregated embryonic stem cells are dislodge or disperse the cells. cultured in a medium which is preferably a serum-free In the re-seeding step, the cells are re-seeded into fresh medium. 5 The term "serum-free media” may comprise cell culture culture vessels and fresh medium added. A number of re media which is free of serum proteins, e.g., fetal calf serum. seedings may be made at different split ratios. For example, Serum-free media are known in the art, and are described for the cells may be reseeded at 1/15 dilution and 1/5 dilution. In example in U.S. Pat. Nos. 5,631,159 and 5,661,034. Serum a particular example, the cells may be re-seeded by adding 1 free media are commercially available from, for example, drop of cells into a 25 cm flask and 3 drops into another to Gibco-BRL (Invitrogen). re-seed the culture, and 7-8 mls media is then added to each to 10 The serum-free media may be protein free, in that it may provide for 1/15 dilution and 1/5 dilution from for example a lack proteins, hydrolysates, and components of unknown 75 cm flask. In the aliquoting step, the cells may be aliquoted composition. The serum-free media may comprise chemi into new dishes or whatever split ratio is desired, and media cally-defined media in which all components have a known added. chemical structure. Chemically-defined serum-free media is 15 advantageous as it provides a completely defined system In a specific embodiment, the method includes the follow which eliminates variability allows for improved reproduc ing steps: human ES cells are first grown Suspended in non ibility and more consistent performance, and decreases pos adherent manner to form embryoid bodies (EBs). 5-10 day sibility of contamination by adventitious agents. old EBs are then trypsinized before plating as adherent cells In a preferred embodiment, the serum-free media com on gelatine coated tissue culture plates. prises Knockout DMEM media (Invitrogen-Gibco, Grand Maintenance as Cell Culture Island, N.Y.). The disaggregated cells are plated and maintained as a cell The serum-free media may be supplemented with one or culture. more components, such as serum replacement media, at a The cells may be plated onto a culture vessel or substrate concentration of for example, 5%, 10%, 15%, etc. The serum Such as a gelatinized plate. Crucially, the cells are grown and 25 free media is preferably supplemented with 10% serum propagated without the presence of co-culture, e.g., in the replacement media from Invitrogen-Gibco (Grand Island, absence offeeder cells. N.Y.). The cells in the cell culture are grown in a serum-free Growth Factor medium which is Supplemented by one or more growth fac The serum-free medium in which the dissociated or disag tors such as fibroblast growth factor 2 (FGF2) and optionally 30 gregated embryonic stem cells are cultured preferably com platelet-derived growth factor AB (PDGFAB), at for example prises one or more growth factors. A number of growth fac 5 ng/ml. The cells in the cell culture are preferably split or tors are known in the art, including PDGF, EGF, TGF-a, FGF, subcultured 1:4 when confluent, by treatment with trypsin, NGF, Erythropoietin, TGF-b, IGF-I and IGF-II. washing and replating. The growth factor may comprise fibroblast growth factor 2 Absence of Co-Culture 35 (FGF2). The medium may also contain other growth factors In highly preferred embodiments, our methods involve such as platelet-derived growth factor AB (PDGFAB). Both culturing cells in the absence of co-culture. The term “co of these growth factors are known in the art. In a highly culture” refers to a mixture of two or more different kinds of preferred embodiment, the method comprises culturing cells cells that are grown together, for example, stromal feeder in a medium comprising both FGF2 and PDGF AB. cells. 40 Alternatively, or in addition, the medium may comprise or Thus, in typical ES cell culture, the inner surface of the further comprise epidermal growth factor (EGF). Use of EGF culture dish is usually coated with a feeder layer of mouse may enhance growth of MSCs. EGF may be used at any embryonic skin cells that have been treated so they will not suitable concentration, for example 5-10 ng/ml EGF. EGF divide. The feeder layer provides an adherent surface to may be used in place of PDGF. EGF is a protein well known enable the ES cells to attach and grow. In addition, the feeder 45 in the art, and is referred to as symbol EGF. Alt. Symbols cells release nutrients into the culture medium which are URG, Entrez 1950, HUGO 3229, OMIM 131530, RefSeq, required for ES cell growth. In the methods and compositions NM 001963, UniProt PO1133. described here, the ES and MSC cells are cultured in the Thus, we disclose the use of media comprising (i) FGF2, absence of Such co-culture. (ii) FGF2 and PDGF and (iii) FGF2 and EGF and other Preferably, the cells are cultured as a monolayer or in the 50 combinations. absence offeeder cells. According to preferred embodiments FGF2 is a wide-spectrum mitogenic, angiogenic, and neu of the methods described here, the embryonic stem cells are rotrophic factor that is expressed at low levels in many tissues cultured in the absence offeeder cells to establish mesenchy and cell types and reaches high concentrations in brain and mal stem cells (MSC). pituitary. FGF2 has been implicated in a multitude of physi In preferred embodiments, the dissociated or disaggre 55 ologic and pathologic processes, including limb develop gated embryonic stem cells are plated directly onto a culture ment, angiogenesis, wound healing, and tumor growth. FGF2 Substrate. The culture Substrate may comprise a tissue culture may be obtained commercially, for example from Invitrogen vessel, such as a Petri dish. The vessel may be pre-treated. In Gibco (Grand Island, N.Y.). preferred embodiments, the cells are plated onto, and grow Platelet Derived Growth Factor (PDGF) is a potent mito on, a gelatinised tissue culture plate. 60 gen for a wide range of cell types including fibroblasts, An example protocol for the gelatin coating of dishes fol smooth muscle and connective tissue. PDGF, which is com lows. A solution of 0.1% gelatin in distilled water is made and posed of a dimer of two chains termed the A chain and B autoclaved. This may be stored at room temp. The bottom of chain, can be present as AA or BB homodimers or as an AB a tissue culture dish is covered with the gelatin Solution and heterodimer. Human PDGF-AB is a 25.5 kDa homodimer incubated for 5-15 min. Remove gelatin and plates are ready 65 protein consisting of 13.3 kDa A chain and 12.2 B chain. to use. Medium should be added before adding cells to pre PDGFAB may be obtained commercially, for example from vent hypotonic lysis. Peprotech (Rocky Hill, N.J.). US 9,029, 146 B2 13 14 The growth factor(s), preferably FGF2 and optionally ments, the selection or sorting may specifically make use of PDGF AB, are preferably present in the medium at concen any of the antigens shown in Table E1A and E1 B below. trations of about 100 pg/ml, preferably about 500 pg/ml, In preferred embodiments, the selection or sorting step of preferably about 1 ng/ml, preferably about 2 ng/ml, prefer our method may positively select formesenchymal stem cells ably about 3 ng/ml, preferably about 4 ng/ml, most preferably based on the expression of antigens which are identified as about 5 ng/ml. In preferred embodiments, the medium con expressed on MSCs, but not expressed on ES cells such as tains FGF2 at about 5 ng/ml. The medium may also contain hESCS. PDGF AB, preferably at about 5 ng/ml. Thus, as shown in the Examples, we demonstrate that Splitting Cells CD73 is highly expressed on MSCs, while being not highly 10 expressed on hESCs (FIG. 4A). Furthermore, as the Cells in culture will generally continue growing until con Examples demonstrate that both CD73 and CD105 are highly fluence, when contact inhibition causes cessation of cell divi expressed surface antigens in MSCs and are among the top 20 sion and growth. Such cells may then be dissociated from the highly expressed surface antigens in hESC-MSCs relative to Substrate or flask, and 'split. Subcultured or passaged, by hESC (FIG. 3, table), the use of either CD73 or CD105 (or dilution into tissue culture medium and replating. 15 both) as selectable marker for putative MSCs will be equally The methods and compositions described here may there effective in sorting for putative MSCs generated by differen fore comprise passaging, or splitting during culture. Prefer tiating hESCs. ably, the cells in the cell culture are split at a ratio of 1:2 or Alternatively, or in addition, the selection or sorting step more, preferably 1:3, more preferably 1:4, 1:5 or more. The may negatively select againstantigens based on Surface anti term "passage’ designates the process consisting in taking an gens that are highly expressed as Surface antigen on embry aliquot of a confluent culture of a cell line, in inoculating into onic stem cells (ES cells) such as hESCs, and not mesenchy fresh medium, and in culturing the line until confluence or mal stem cells e.g., hESC-MSC. Selection or sorting may be saturation is obtained. based on known or previously identified hESC-specific sur Selection, Screening or Sorting Step face antigens such as MIBP, ITGB1BP3 and PODXL’, and In highly preferred embodiments, the method further com 25 CD24. prises a selection or sorting step, to further isolate or select for The Examples show that FACS analysis confirms the mesenchymal stem cells. expression of CD24 on hESC but not hESC-MSCs. There The selection or sorting step may comprise selecting mes fore, CD24 may be used as a negative selection or sorting enchymal stem cells (MSC) from the cell culture by means of marker either on its own, or in conjunction with CD105 as a one or more surface antigenmarkers. The use of a selection or 30 positive selectable marker for isolating putative MSCs from sorting step further enhances the stringency of sorting and differentiating hESC cultures. selection specificity for MSCs and furthermore potentially Importantly, the mesenchymal stem cells are able to main reduces possible contamination from embryonic stem cells tain self-renewal without the requirement for transformation. such as hESCs and other hESC-derivatives from the starting Thus, for example, known transformation treatments such as material. This would then further reduce the risk of teratoma 35 fusion with immortalised cells such as tumour cells or tumour formation and further increase the clinical relevance of the cell lines, viral infection of a cell line with transforming protocol we describe. viruses such as SV40, EBV, HBV or HTLV-1, transfection A number of methods are known for selection or sorting with specially adapted vectors, such as the SV40 vector com based on antigen expression, and any of these may be used in prising a sequence of the large T antigen (R. D. Berry et al., the selection or sorting step described here. In particularly 40 Br. J. Cancer, 57, 287-289, 1988), telomerase (Bodnar-A-G. preferred embodiments, the selection or sorting is achieved et. al., Science (1998) 279: p. 349-52) or a vector comprising by means of fluorescence activated cell sorting (FACS). Thus, DNA sequences of the human papillomavirus (U.S. Pat. No. as known in the art, FACS involves exposing cells to a 5,376,542), introduction of a dominant oncogene, or by muta reporter, such as a labelled antibody, which binds to and labels tion are therefore not required in the methods described here antigens expressed by the cell. Methods of production of 45 for making mesenchymal stem cells. antibodies and labelling thereof to form reporters are known In preferred embodiments, the mesenchymal stein cells in the art, and described for example in Harlow and Lane. The and cell lines (or the differentiated cells derived from them) cells are then passed through a FACS machine, which sorts do not display one or more characteristics of embryonic stem the cells from each other based on the labelling. Alternatively cells. Preferred such characteristics include expression of the or in addition, magnetic cell sorting (MACS) may be 50 OCT4 gene and alkaline phosphatase activity. Preferably, the employed to sort the cells. mesenchymal stem cell exhibits reduced expression of one or We have realised that while a number of candidate surface more characteristic markers of pluripotency. Such pluripo antigens known to be associated with MSCs e.g. CD105, tency markers are described in further detail below, but CD73, ANPEP ITGA4 (CD49d), PDGFRA, some of the include Nanog, BMP4, FGF5, Octa, Sox-2 and Utf1. MSC associated surface antigens e.g. CD29 and CD49e are 55 Mesenchymal stem cells made by the methods described also highly expressed in ES cells such as hESCs and their here are preferably non-tumorigenic. Preferably, the mesen expression are verified by FACS analysis. The association of chymal Stein cells when implanted into an immune compro a surface antigen with MSCs may not be sufficient to qualify mised or immunodeficient host animal do not result in the antigen as a selectable marker for isolating MSCs from ES tumours, compared to implantation of parental embryonic cells such as hESC. Accordingly, the selection or sorting step 60 stem cells which results in tumour formation. Preferably, the preferably employs antigens which are differentially immune compromised or immunodeficient host animal is a expressed between MSCs and ES cells. SCID mouse or a Rag1-/- mouse. Preferably, the mesenchy The selection or sorting step of our method may positively mal stem cells do not form tumours after prolonged periods of select formesenchymal stem cells based on the expression of implantation, preferably greater than 2 weeks, more prefer antigens. Such antigens may be identified by, for example, 65 ably greater than 2 months, most preferably greater than 9 comparing the gene expression profiles of hESCs and hESC months. Detailed protocols for tumourigenicity testing are set MSCs as described in the Examples. In particular embodi out in the Examples. US 9,029, 146 B2 15 16 Mesenchymal stem cells made by the methods described obtaining a mesenchymal stem cell (MSC) by providing a cell here are also preferably display one or more of the following obtained by dispersing a embryonic stem (ES) cell colony, or characteristics. They have a substantially stable karyotype as a descendent thereof, deriving one or more progenitor cells or assessed by chromosome number, preferably when main progenitor cell lines from the embryonic stem cell and propa tained in cell culture for at least 10 generations. They also gating the cell in the absence of co-culture in a serum free preferably display a Substantially stable gene expression pat medium comprising FGF2. tern from generation to generation. By this we mean that the Preferably, the progenitor cell line is selected based on its expression levels one or more, preferably substantially all, of ability to self-renew, or the method may select against a chosen set of genes does not vary significantly between a somatic cells based on their inability to self-renew, or both. mesenchymal stem cells in one generation and mesenchymal 10 stem cells in the next generation. In a preferred embodiment, the progenitor cell line is Preferably, the set of genes comprises one or more, a Sub derived or established in the absence of co-culture, preferably set, or all of the following: cerberus (GenBank Accession in the absence of feeder cells. Preferably, the absence of nos: NM 009887, AF031896, AF035579), FABP (GenBank co-culture selects against embryonic stem cells. Accession nos: NM 007980, M65034, AY523818, 15 In preferred embodiments, the progenitor cell line is estab AY523819), Foxa2 (GenBank Accession nos: NM 010446, lished without transformation. The progenitor cell line may X74937, L10409), Gata-1 (GenBank Accession nos: be established by exposing embryonic stem cells or their NM 008089, X15763, BC052653), Gata-4 (GenBank descendants to conditions which promote self-renewal of Accession nos: NM 008092, AF 179424, U85046, M98339, putative progenitor cells. Preferably, the self-renewal-pro AB075549), HesX1 (GenBank Accession nos: NM 010420, moting conditions discourage the propagation of embryonic X80040, U40720, AK082831), HNF4a (GenBank Accession Stein cells. nos: NM 008261, D29015, BC039220), c-kit (GenBank The self-renewal-promoting conditions may comprise Accession nos: NM 021099, Y00864, AY536430, growth in rich media. More preferably, the self-renewal-pro BC075716, AK047010, BC026713, BC052457, AK046795), moting conditions comprise growing cells in the absence of PDGFRa. (NM 011058, M57683, M84607, BC053036), 25 LIF. Preferably, the self-renewal-promoting conditions com Octá (GenBank Accession nos: NM 013633, X52437, prise serial passages. Preferably, the self-renewal promoting M34381, BC068268), Runx1 (GenBank Accession nos: conditions comprise at least 12 serial passages. NM 009821, D26532, BC069929, AK051758), Sox17 In preferred embodiments, the progenitor cell line has (GenBank Accession nos: NM 011441, D49474, L29085, reduced potential compared to the embryonic stem cell. Pref AK004781), Sox2 (GenBank Accession nos: NM 011443, 30 erably, the progenitor cell line is lineage restricted, preferably U31967, AB108673), Brachyury (NM 009309, X51683), non-pluripotent. Preferably, the progenitor cell line is non TDGF1 (GenBank Accession nos: NM 011562, M87321) tumorigenic. and Tie-2 (GenBank Accession nos: NM 013690, X67553, Preferably, the step of deriving the progenitor cell line X71426, D13738, BC050824). comprises a step of exposing the embryonic stem cell to The methods described here enable the production of mes 35 conditions that enhance differentiation to a specific lineage. enchymal stem cells as well as differentiated cells, which Preferably, the differentiation enhancing-conditions com comprise clonal descendants of mesenchymal stem cells. The prises generating an embryoid body from the embryonic stem term “clonal descendant’ of a cell refers to descendants of the cell. Preferably, the cells are removed from differentiation cells which have not undergone Substantially any transform enhancing-conditions after pluripotency is lost. ing treatment or genetic alteration. Such clonal descendants 40 Preferably, the removing of the cells from lineage restric have not undergone substantial genomic changes are Substan tion-promoting conditions comprises disaggregating an tially genetically identical to the parent cell, or an ancestor, embryoid body. Preferably, the method comprises disaggre preferably, the embryonic stem cell (save with reduced gating embryoid bodies which have been grown from potency). The term “mesenchymal stem cells' should also between about 3 to 6 days. preferably be taken to include cell lines derived from mesen 45 In preferred embodiments, the progenitor cell line displays chymal stem cells, i.e., mesenchymal stem cell lines, and vice reduced expression of or does not substantially express either WSa. or both of OCT4 and alkaline phosphatase activity. Derivation of Progenitor Cells Preferably, the progenitor cell line displays reduced In preferred embodiments, the methods described here expression of a pluripotency marker compared to an embry may employ further steps to selector Screen formesenchymal 50 onic stem cell from which it is derived, the pluripotency stem cells. marker preferably selected from the group consisting of Such further steps may take place prior to the steps Nanog, BMP4, FGF5, Octá, Sox-2 and Utfl. described above, in between such steps, or after these steps. In preferred embodiments, the progenitor cell lines display The further steps may be in fact be conducted independently, one or more of the following characteristics: (a) are maintain and may specifically comprise deriving one or more progeni 55 able in cell culture for greater than 40 generations; (b) have a tor cells or cell lines from the ES cells. Substantially stable karyotype or chromosome number when We therefore disclose an alternative method of deriving maintained in cell culture for at least 10 generations; (c) have mesenchymal stem cells. The method comprises: (a) provid a Substantially stable gene expression pattern from generation ing an embryonic stem (ES) cell; and (b) establishing a pro to generation. genitor cell line from the embryonic stein cell; in which the 60 Preferably, the progenitor cell line does not substantially progenitor cell line is selected based on its ability to self induce formation of teratoma when transplanted to a recipient CW. animal, preferably an immune compromised recipient ani However, preferably, the methods are conducted together. mal, preferably after 3 weeks, more preferably after 2 to 9 Thus, for example, our methods may comprise deriving pro months. genitor cells or progenitor cell lines from the embryonic stem 65 Preferably, the embryonic stein cell or progenitor cell line cell prior to, during, or after the dispersal step or the propa is a mammalian, preferably mouse or human, embryonic Stein gation step. Thus, the method may for example comprise cell or progenitor cell line. US 9,029, 146 B2 17 18 Preferably, the progenitor cell line comprises an endothe endodermal differentiation. Examples of such growth factors lial progenitor cell line, preferably a E-RoSH cell line. Alter and drugs include activin A, FGF4, dexamethasone and ret natively, or in addition, the progenitor cell line may comprise inoic acid. a mesenchymal progenitor cell line, preferably a huES9.E.1 Hematopoietic and Endothelial Progenitor Cells cell line. On the other hand, where it is desired to bias differentiation In some embodiments, the method further comprises the of embryonic stein cells towards hematopoietic or endothelial step of (d) deriving a differentiated cell from the progenitor lineages, the disaggregated embryoid bodies may be exposed cell line. to growth factors or drugs or combinations thereofthat induce Preferably, the progenitor cell line is propagated for at least hematopoietic or endothelial differentiation. Examples of 5 generations prior to differentiation. 10 such growth factors and drugs include GM-CSF, G-CSF, We provide a method of generating a differentiated cell SCF, PDGF, IL-3, erythropoietin, thrombopoeittin, TNFC. from an embryonic stem (ES) cell, the method comprising: and rapamycin. (a) deriving a progenitor cell line from the embryonic stem Cardiac Mesodermand Skeletal Myoblast Progenitor Cells cell; (b) propagating the progenitor cell line; and (c) deriving On the other hand, where it is desired to bias differentiation a differentiated cell from the progenitor cell line. 15 of embryonic stem cells towards cardiac mesoderm or skel There is provided a method comprising: (a) providing an etal myoblast lineages, the disaggregated embryoid bodies embryonic stem (ES) cell; (b) deriving a progenitor cell from may be exposed to growth factors or drugs or combinations the embryonic stem cell; and (c) establishing a progenitor cell thereof that induce cardiac mesoderm or skeletal myoblast line from the progenitor cell, in which progenitor cells are differentiation. Examples of Such growth factors and drugs selected based on their ability to self-renew. include dexamethasone, inhibitors of PPARY and testosterone The method may specifically be used for generating a or its analogs. differentiated cell from an embryonic stem (ES) cell. Prefer The second step may comprise plating the differentiating ably, the differentiated cell is an endothelial cell or a mesen cells in a rich media. In such embodiments, continued propa chymal cell. More preferably, the differentiated cell is an gation will selectively enrich for progenitor cells which can adipocyte or an osteocyte. 25 then be cloned. We provide a progenitor cell line produced by a method Formation of Embryoid Bodies according to any preceding aspect of the invention. In some embodiments, the differentiation-enhancing step The methods and compositions described here may also comprises formation of embryoid bodies from embryonic further comprise further steps which employ other factors or stem cells. Embryoid bodies, and methods for making them, characteristics of such MSCs for selection or screening or 30 are known in the art. The term "embryoid body” refers to both. spheroid colonies seen in culture produced by the growth of Biasing Differentiation embryonic stein cells in suspension. Embryoid bodies are of In preferred embodiments, the method for generating mixed cell types, and the distribution and timing of the embryonic stem cell-derived progenitor cell lines of specific appearance of specific cell types corresponds to that observed lineages preferably further comprises a first step of biasing 35 within the embryo. Preferably, the embryoid bodies are gen differentiation of embryonic stem cells towards a specific erated by plating out embryonic stem cells onto semi-solid desired lineage or lineage of interest. Our methods may also media, preferably methylcellulose media as described in Lim comprise a second step of encouraging self-renewal of puta et al, Blood. 1997: 90:1291-1299. Preferably, the embryoid tive progenitor cells and discouraging the propagation of bodies are between 3 to 6 days old. embryonic stem cells. 40 In Such embodiments, the embryoid body is disaggregated, The first step may comprise promoting the growth or i.e., separating the component cells from each other, e.g., by propagation of a specific lineage of interest. Different pro collagenase or trypsin treatment, in order to remove the cells genitor cell lines of specific lineages of interest may be made from lineage restriction-promoting conditions. by exposing the cells to conditions that promote the differen The method in preferred embodiments comprises a step of tiation of those lineages of interest. For example, the embry 45 choosing a putative progenitor cell for the desired specific onic stem cells may be exposed to growth factors or Small lineage. The choosing may be conducted based on morphol molecules Such as ligands that promote or enable differentia ogy of the cell, or by expression or markers, etc. Gene expres tion. sion profiling or antigen profiling may also be used to choose Thus, the methods described here for establishing embry specific progenitor cells which are of a desired lineage. The onic stem cell-derived cell lines of specific lineages prefer 50 chosen putative progenitor cell for the desired specific lineage ably include a step of enhancing differentiation of embryonic may then be cultured, or further choosing steps conducted stem cells towards that specific lineage. Preferably, the dif thereon. ferentiation-enhancing step is carried out for a predetermined In preferred embodiments, the differentiation-enhancing period of time. Thus, preferably, the embryonic stem cells or step is followed by exposing differentiating cells to condi their descendants are transiently exposed to differentiation 55 tions which encourage self-renewal of putative progenitor enhancing environment. cells and discourage the propagation of embryonic stem cells. The choice of the method of enhancing or biasing differ Such conditions may preferably comprise culture in the entiation will depend on the specific cell lineage of interest for absence of co-culture or feeder cells (see above). which it is desired to produce progenitor cells. The person Rich Media skilled in the art will be aware of the various methods which 60 Alternatively, or in addition, Such conditions comprise may be used for different cells. plating in rich media. The term "rich media” as used in this Endodermal Progenitor Cells document is intended to refer to media which is nutrient rich. Where it is desired to bias differentiation of embryonic Preferably, such media comprises essential nutrients required stem cells towards endodermal types of tissues, for example, for growth of the relevant cell. Preferably, the rich media embryoid bodies may be formed and disaggregated (see 65 contain serum. More preferably, it comprises substantially all later). The disaggregated embryoid bodies may be exposed to the nutrients required for such growth. Most preferably, the growth factors or drugs or combinations thereof that induce rich medium Supports, promotes and encourages growth of US 9,029, 146 B2 19 20 the relevant cells. in highly preferred embodiments, the rel cells. In particular, they are not capable of giving rise to all evant cell is a progenitor cell or a putative progenitor cell of three germ layers. In highly preferred embodiments, the pro interest. An example of a rich medium is DMEM with 4500 genitor cell lines are preferably non-pluripotent. mg/l D-glucose, supplemented with 20% fetal calf serum, Characteristics of Progenitor Cells non essential amino acids, L-glutamine and B-mercaptoetha In preferred embodiments, the progenitor cells and cell nol. lines (or the differentiated cells derived from them) do not In preferred embodiments, such rich media does not com display one or more characteristics of embryonic stem cells. prise additional growth regulators or hormones that allow, Preferred such characteristics include expression of the promote or encourage growth of embryonic stem cells. Such OCT4 gene and alkaline phosphatase activity. Preferably, the as Leukemia Inhibitory Factor (LIF). 10 progenitor cell line exhibits reduced expression of one or According to Such embodiments, continued propagation more characteristic markers of pluripotency. Such pluripo will selectively enrich for progenitor cells which can then be tency markers are described in further detail below, but cloned. include Nanog, BMP4, FGF5, Octa, Sox-2 and Utf1. Long-Term Maintenance in Culture Progenitor cells made by the methods described here are Preferably, the methods described here involve culturing 15 preferably non-tumorigenic. Preferably, the progenitor cells the embryonic stem cells or their descendants for more than when implanted into an immune compromised or immuno one generation. Preferably, the cells are cultured for more deficient host animal do not result in tumours, compared to than 5, more than 10, more than 15, more than 20, more than implantation of parental embryonic stem cells which results 25, more than 50, more than 40, more than 45, more than 50, in tumour formation. Preferably, the immune compromised more than 100, more than 200, more than 500 or more than or immunodeficient host animal is a SCID mouse or a 800 generations. In particular, the cell lines may be main Rag1-/- mouse. Preferably, the progenitor cells do not form tained for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, tumours after prolonged periods of implantation, preferably 30, 35, 40, 45, 50, 75, 100, 200, 500 or more generations. greater than 2 weeks, more preferably greater than 2 months, Cells in culture will generally continue growing until con most preferably greater than 9 months. Detailed protocols for fluence, when contact inhibition causes cessation of cell divi 25 tumourigenicity testing are set out in the Examples. sion and growth. Such cells may then be dissociated from the Progenitor cells made by the methods described here are Substrate or flask, and 'split' or passaged, by dilution into also preferably display one or more of the following charac tissue culture medium and replating. The progenitor cells teristics. They have a substantially stable karyotype as may therefore be passaged, or split during culture; preferably assessed by chromosome number, preferably when main they are split at a ratio of 1:2 or more, preferably 1:3, more 30 tained in cell culture for at least 10 generations. They also preferably 1:4, 1:5 or more. The term “passage' designates preferably display a Substantially stable gene expression pat the process consisting in taking an aliquot of a confluent tern from generation to generation. By this we mean that the culture of a cell line, in inoculating into fresh medium, and in expression levels one or more, preferably substantially all, of culturing the line until confluence or saturation is obtained. a chosen set of genes does not vary significantly between a The progenitor cells derived according to the methods 35 progenitor cell in one generation and a progenitor cell in the described here may however be maintained for a large num next generation. ber of generations, based on their capacity to self-renew. On Preferably, the set of genes comprises one or more, a Sub the other hand, it has been established that “normal' (i.e., set, or all of the following: cerberus (GenBank Accession untransformed Somatic) cells derived directly from an organ nos: NM 009887, AF031896, AF035579), FABP (GenBank ism are not immortal. In other words, such somatic cells have 40 Accession nos: NM 007980, M65034, AY523818, a limited life span in culture (they are mortal). They will not AY523819), Foxa2 (GenBank Accession nos: NM 010446, continue growing indefinitely, but will ultimately lose the X74937, L10409), Gata-1 (GenBank Accession nos: ability to proliferate or divide after a certain number of gen NM 008089, X15763, BC052653), Gata-4 (GenBank erations. On reaching a “crisis phase' such cells die after Accession nos: NM 008092, AF179424, U85046, M98339, about 50 generations. Thus, such somatic cells may only be 45 AB075549), HesX1 (GenBank Accession nos: NM 010420, passaged a limited number of times. X80040, U40720, AK082831), HNF4a (GenBank Accession Importantly, the progenitor cells are able to maintain self nos: NM 008261, D29015, BC039220), c-kit (GenBank renewal without the requirement for transformation. Thus, for Accession nos: NM 021099, Y00864, AY536430, example, known transformation treatments such as fusion BC075716, AK047010, BC026713, BC052457, AK046795), with immortalised cells such as tumour cells or tumour cell 50 PDGFRa. (NM 011058, M57683, M84607, BC053036), lines, viral infection of a cell line with transforming viruses Octá (GenBank Accession nos: NM 013633, X52437, such as SV40, EBV, HBV or HTLV-1, transfection with spe M34381, BC068268), Runx1 (GenBank Accession nos: cially adapted vectors, such as the SV40 vector comprising a NM 009821, D26532, BC069929, AK051758), Sox17 sequence of the large T antigen (R. D. Berry et al., Br. J. (GenBank Accession nos: NM 011441, D49474, L29085, Cancer, 57,287-289, 1988), telomerase (Bodnar-A-G. et. al., 55 AK004781), Sox2 (GenBank Accession nos: NM 011443, Science (1998) 279: p. 349-52) or a vector comprising DNA U31967, AB108673), Brachyury (NM 009309, X51683), sequences of the human papillomavirus (U.S. Pat. No. 5,376, TDGF1 (GenBank Accession nos: NM 011562, M87321) 542), introduction of a dominant oncogene, or by mutation and Tie-2 (GenBank Accession nos: NM 013690, X67553, are therefore not required in the methods described here for X71426, D13738, BC050824). making progenitor cell lines. 60 The methods described here enable the production of pro According to preferred embodiments of the methods genitor cells and progenitor cell lines as well as differentiated described here, progenitor cells may be propagated without cells, which comprise clonal descendants of progenitor cells. transformation for more than 50 generations. In preferred The term “clonal descendant’ of a cell refers to descendants embodiments, the progenitor cells may be propagated indefi of the cells which have not undergone substantially any trans nitely and without transformation as progenitor cell lines. The 65 forming treatment or genetic alteration. Such clonal descen progenitor cells and progenitor cell lines are preferably lin dants have not undergone Substantial genomic changes are eage restricted compared to their parental embryonic stem Substantially genetically identical to the parent cell, or an US 9,029, 146 B2 21 22 ancestor, preferably, the embryonic stem cell (save with Parkinson's disease, etc. Stem cells, for example may be used reduced potency). The term “progenitor cell' should also as sources of progenitors for NK or dendritic cells for immu preferably be taken to include cell lines derived from progeni notherapy for cancer, which progenitors may be made by the tor cells, i.e., progenitor cell lines, and Vice versa. methods and compositions described here. Regulators of Self-Renewal and Differentiation It will be evident that the methods and compositions Our methods may also be used to identify putative regula described here enable the production of progenitor cells, tors of self-renewal or differentiation. The methods involve which may of course be made to differentiate using methods conducting the methods described for production of progeni known in the art. Thus, any uses of differentiated cells will tor cell lines or differentiated cells in the presence and equally attach to those progenitor cells for which they are absence of a candidate molecule, and identifying if the pres 10 SOUCS. ence of the molecule has any effect on the process. For Progenitor cells produced by the methods and composi example, a molecule which accelerates the production of tions described here may be used for, or for the preparation of progenitor cells or differentiated cells may be used as a posi a pharmaceutical composition for, the treatment of a disease. tive regulator of differentiation (or alternatively as an inhibi Such disease may comprise a disease treatable by regenera tor of self-renewal). Conversely, a molecule which retards the 15 tive therapy, including cardiac failure, bone marrow disease, process can be considered an inhibitor of differentiation or a skin disease, burns, degenerative disease Such as diabetes, promoter of self-renewal. Alzheimer's disease, Parkinson's disease, etc and cancer. In preferred embodiments, we also provide a cell, prefer We therefore describe a method of treatment of a disease ably a progenitor, of a selected lineage, obtainable according comprising: (a) providing an embryonic Stein (ES) cell; (b) to the method. Hitherto, preparations of progenitors were too establishing a progenitor cell line from the embryonic Stein impure for certainty as to whether any chosen cell was a cell in which the progenitor cell line is selected based on its progenitor cell. With culture according to the invention that ability to self-renew; (d) optionally deriving a differentiated can give rise to substantially 100% pure preparations of pro cell from the progenitor cell line; and (e) administering the genitors, isolation of a single progenitor is achieved. progenitor cell line or the differentiated cell into a patient. We further provide in preferred embodiments a composi 25 Characteristics of Obtained MSCs tion comprising a plurality of cells, wherein a majority of the The MSCs obtained by the methods and compositions cells are progenitor cells of a selected lineage. Preferably, at described here preferably satisfy the morphologic, pheno least 60% of the cells are progenitor cells of the selected typic and functional criteria commonly used to identify lineage. More preferably, at least 60% of the cells are pro MSCs, as known in the art. For ease of reference, the mes genitor cells. In addition, the invention provides an isolated 30 enchymal stem cells obtained by the methods and composi progenitor cell. The term cell line preferably refers to cells tions described here, particularly as derived from human that can be maintained and grown in culture and display an embroynic stem cells, may be referred to as “hESC-MSC's. immortal or indefinite life span. Thus, the MSCs obtained by the methods and composi The methods described here may be combined with tions described here may preferably exhibit one or more decreasing the activity of mTOR to promote differentiation, 35 morphological characteristics of mesenchymal stem cells. as described in U.S. 60/609.216, herein incorporated by ref For example, the MSCs obtained may form an adherent CCC. monolayer with a fibroblastic phenotype. Uses of Progenitor Cells Furthermore, the MSCs obtained may preferably display a Our methods are capable of producing of progenitor cells Surface antigen profile which is similar or identical to mes and cell lines of various types. 40 enchymal stem cells. Thus, the Surface antigen profile of the For example, we disclose a method of making peripheral MSCs obtained may include one or more, preferably all, of blood progenitor cells (PBPC), neuronal progenitor cells, CD29--, CD44+, CD49a and e--, CD105+, CD166+ and haematopoeitic progenitor cells, myeloid progenitor cells, CD34-, CD45-''. epithelial progenitor cells, bone marrow Stromal cells, skel The MSCs obtained may be differentiated into any mesen etal muscle progenitor cells, pancreatic islet progenitor cells, 45 chymal lineage, using methods known in the art and mesenchymal progenitor cells, cardiac mesodermal stem described below. Thus, the MSCs obtained by the methods cells, lung epithelial progenitor cells, liver progenitors, and and compositions described here may display a differentia endodermal progenitor cells. tion potential that include adipogenesis, chondrogenesis and Progenitor cells made according to the methods described osteogenesis. here can be used for a variety of commercially important 50 The mesenchymal stem cells obtained as described, e.g., research, diagnostic, and therapeutic purposes. These uses are hESC-MSCs, can have a substantial proliferative capacity in generally well known in the art, but will be described briefly vitro. In some embodiments, the mesenchymal stem cells here. obtained may undergo at least 10 population doublings while For example, stem cells may be used to generate progenitor maintaining a normal diploid karyotype. Preferably, however, cell populations for regenerative therapy. Progenitor cells 55 the MSCs are capable of undergoing at least 20-30 population may be made by ex vivo expansion or directly administered doublings while maintaining a normal diploid karyotype. In into a patient. They may also be used for the re-population of preferred embodiments, the MSCs display a stable gene damaged tissue following trauma. expression and Surface antigen profile throughout this time. Thus, hematopoietic progenitor cells may be used for bone Preferably, the MSCs obtained do not display any defects, marrow replacement, while cardiac progenitor cells may be 60 Such as chromosomal aberrations and/or alterations in gene used for cardiac failure patients. Skin progenitor cells may be expression. In preferred embodiments, such defects are not employed for growing skin grafts for patients and endothelial evident until after 10 passages, preferably after 13 passages, progenitor cells for endothelization of artificial prosthetics more preferably after 15 passages. Such as stents or artificial hearts. Homogeneity Embryonic stem cells and their tissue stem cell derivatives 65 In a preferred embodiment, the mesenchymal stem cells may be used as Sources of progenitor cells for the treatment of produced by the method described here are similar or identi degenerative diseases such as diabetes, Alzheimer's disease, cal (preferably homogenous) in nature. That is to say, mes US 9,029, 146 B2 23 24 enchymal stem cell (MSC) clones isolated by the protocol In some embodiments, the method described here gener show a high degree of similarity or identity with each other, ates mesenchymal stem cells whose gene expression correla whether phenotypically or otherwise. tion coefficient between any two or more isolates of mesen Similarity or identity may be gauged by a number of ways chymal stem cells so obtained is in the same order as, or and measured by one or more characteristics. In a preferred 5 slightly less than, the correlation coefficient between techni embodiment, the clones are similar or identical in gene cal replicates of the same RNA sample, performed a period of expression. Preferably, the method is such that any two or time apart such as 1 month apart. In other embodiments, the more mesenchymal stein cells selected by the method exhibit gene expression correlation coefficient between any two or Substantially identical or similar gene expression profiles, more isolates of mesenchymal stem cells is greater than 0.90, 10 preferably greater than 0.95. that is to say, a combination of the identity of genes expressed Preferably, the gene expression correlation coefficients are and the level to which they are expressed. Preferably, sub in Such ranges for cells which have undergone the selection or stantially all of the mesenchymal stem cells isolated exhibit sorting procedure described above. Preferably, the gene Substantially identical or similar gene expression profiles. expression correlation coefficient between the majority of Homogeneity of gene expression may be measured by a 15 isolates, preferably all isolates, is in Such ranges. number of methods. Preferably, genome-wide gene profiling Thus, as shown in the Examples, the correlation coefficient is conducted using, for example, array hybridisation of shows a high degree of similarity between five mesenchymal extracted RNA as described in the Examples. Total RNA may stem cell cultures obtained, with a correlation coefficient be extracted and converted into cDNA, which is hybridised to between four of the lines of 0.96 and the one with 0.90; in an array chip comprising a plurality of gene sequences from contrast, the correlation coefficient between technical repli a relevant genome. Preferably, the array comprises NCBI cates of a RNA sample analysed one month apart is between Reference Sequence (RefSeq) genes, which are well charac 0.97 and 0.98. terised genes validated, annotated and curated on an ongoing Accordingly, we provide for a method of generating mes basis by National Center for Biotechnology Information enchymal stem cells which are Substantially similar or iden (NCBI) staff and collaborators. 25 tical (preferably homogenous) with each other. The isolates Gene expression between samples is then compared using preferably display a near identical gene expression profile. analysis software. In a preferred embodiment, the similarity As well as the “internal’ homogeneity described above or identity of gene expression expressed as a "correlation (i.e., homogeneity between the isolates of MSCs from the coefficient'. In Such measures, a high correlation coefficient method), homogeneity may also be assessed between Such between two samples indicates a high degree of similarity 30 isolates and other cells or cell types. In particular, compari between the pattern of gene expression in the two samples. Sons may be made with mesenchymal stem cells derived by Conversely, a low correlation coefficient between two other methods, such as bone-marrow derived mesenchymal samples indicates a low degree of similarity between the stein cells (BM-MSCs). In preferred embodiments, the MSCs pattern of gene expression in the two samples. Normalisation obtained by the methods and compositions described here may be conducted to remove systematic variations or bias 35 display a gene expression profile which is similar to, homog (including intensity bias, spatial bias, plate bias and back enous with, or identical with a BM-MSC. Thus, the MSCs ground bias) prior to data analysis. obtained may show a correlation coefficient of gene expres Correlation tests are known in the art and include a T-test sion of greater than 0.5, preferably greater than 0.6, prefer and Pearson's test, as described in for example Hill, T. & ably greater than 0.7, with BM-MSCs. Lewicki, P. (2006). Statistics. Methods and Applications. 40 Thus, as shown in the Examples, pairwise comparison of StatSoft, Tulsa, Okla., ISBN: 1884233597 (also StatSoft, Inc. gene expression between three independently derived hESC (2006). Electronic Statistics Textbook. Tulsa, Okla.: StatSoft. MSC populations and three individual BM-MSC samples are WEB: http://www.statsoft.com/textbook/stathome.html). found to be similar with a correlation coefficient of 0.72. Reference is made to Khojasteh et al., 2005. A stepwise Regulators of Mesenchymal Stem Cell Formation framework for the normalization of array CGH data, BMC 45 Our methods may also be used to identify putative regula Bioinformatics 2005, 6:274. An Intra-class correlation coef tors of mesenchymal stem cell formation from embryonic ficient (ICC) may also be conducted, as described in Khojas stem cells. teh et al. Supra. The methods involve conducting the methods described for In preferred embodiments, a Pearson's test is conducted to production of mesenchymal stem cells in the presence and generate a Pearson’s correlation coefficient. A correlation 50 absence of a candidate molecule, and identifying if the pres coefficient of 1.0 indicates an identical gene expression pat ence of the molecule has any effect on the process. For tern. example, a molecule which accelerates the production of In a preferred embodiment, the cDNA is hybridised to a mesenchymal stem cells may be used as a positive regulator Sentrix Human Ref-8 Expression BeadChip and scanned of mesenchymal stem cell formation. Conversely, a molecule using a Illumina BeadStation 500x. Preferably, the data is 55 which retards the process can be considered an inhibitor of extracted, normalised and analysed using Illumina BeadStu mesenchymal stem cell formation. dio (Illumina, Inc, San Diego, Calif., USA). It will be clear to In preferred embodiments, we also provide a cell, prefer the reader however that any Suitable chip and Scanning hard ably a mesenchymal stem cell, obtainable according to the ware and Software (which outputs a correlation measure method. Hitherto, preparations of mesenchymal stem cells ment) may be used to assay similarity of gene expression 60 were either too impure, or not substantially phenotypically profile. similar or identical (e.g., with respect to gene expression), or Preferably, the gene expression correlation coefficient were not suitable for clinical purposes as they are produced by between any two isolates as preferably measured by the above methods involving co-culture or presence of serum. With means is greater than 0.65, preferably greater than 0.70, more culture according to the invention, this can give rise to Sub preferably greater than 0.80, more preferably greater than 65 stantially 100% pure preparations of mesenchymal stein cells 0.85, more preferably greater than 0.90, most preferably more which are similar or identical (preferably homogenous) to than 0.95. each other. US 9,029, 146 B2 25 26 In addition, we describe a process for producing differen We further provide for the use of such a delivery system in tiated cells, the method comprising obtaining a mesenchymal a method of delivering a conditioned medium to a target. stem cell by a method as described herein, and differentiating Delivery systems for delivering fluid into the body are the mesenchymal stem cell. For example, we provide for known in the art, and include injection, Surgical drips, cathe methods of differentiating to adipocytes, chondrocytes and thers (including perfusion cathethers) such as those described osteocytes, etc. We further provide differentiated cells obtain in U.S. Pat. No. 6,139.524, for example, drug delivery cath able by such methods. Cell lines made from such mesenchy eters such as those described in U.S. Pat. No. 7,122,019. mal stein cells and differentiated cells are also provided. The Delivery to the lungs or nasal passages, including intrana term cell line preferably refers to cells that can be maintained sal delivery, may beachieved using for example a nasal spray, 10 puffer, inhaler, etc as known in the art (for example as shown and grown in culture and display an immortal or indefinite life in U.S. Design Pat. No. D544,957. Span. Delivery to the kidneys may be achieved using an intra The methods described here may be combined with aortic renal delivery catheter, such as that described in U.S. decreasing the activity of mTOR to promote differentiation, Pat. No. 7,241,273. as described in U.S. 60/609.216, herein incorporated by ref 15 It will be evident that the particular delivery should be CCC. configurable to deliver the required amount of conditioned Uses of Conditioned Media from Mesenchymal StemCells medium at the appropriate interval, in order to achieve opti It will be evident that the methods and compositions mal treatment. described here enable the production of conditioned media The conditioned medium may for example be used for the from mesenchymal stem cells. Thus, any uses of mesenchy treatment or prevention of atherosclerosis. Here, perfusion of mal stem cells will equally attach to conditioned media from conditioned media may be done intravenously to stabilize mesenchymal stem cells. atherosclerotic plaques or reduce inflammation in the Mesenchymal stem cells and differentiated cells produced plaques. The conditioned medium may be used for the treat by the methods and compositions described here may be used ment or prevention of Septic shock by intravenous perfusion. for, or for the preparation of a pharmaceutical composition 25 The conditioned medium may be used for the treatment or for, the treatment of a disease. Such disease may comprise a prevention of heart failure. This may be achieved by chronic disease treatable by regenerative therapy, including cardiac intracoronary or intramyocardially perfusion of conditioned failure, bone marrow disease, skin disease, burns, degenera media to retard remodeling or retard heart failure. The con tive disease such as diabetes, Alzheimer's disease, Parkin ditioned medium may be used for the treatment or prevention son's disease, etc and cancer. Accordingly, conditioned 30 of lung inflammation by intranasal delivery. media from MSCs may be used to treat such diseases. The conditioned medium may be used for the treatment or Conditioned media from mesenchymal stem cells such as prevention of dermatological conditions e.g. psoriasis. Long those made according to the methods and compositions term delivery of conditioned media may be employed using described here may be used for a variety of commercially transdermal microinjection needles until the condition is important research, diagnostic, and therapeutic purposes. 35 resolved. The conditioned media from mesenchymal stem cells may It will be evident that the delivery method will depend on in particular be used for the preparation of a pharmaceutical the particular organ to which the conditioned medium is to be composition for the treatment of disease. Such disease may delivered, and the skilled person will be able to determine comprise a disease treatable by regenerative therapy, includ which means to employ accordingly. ing cardiac failure, bone marrow disease, skin disease, burns, 40 As an example, in the treatment of cardiac inflammation, degenerative disease such as diabetes, Alzheimer's disease, the conditioned medium may be delivered for example to the Parkinson's disease, etc and cancer. cardiac tissue (i.e., myocardium, pericardium, or endocar As shown in the Examples, the mesenchymal stem cells dium) by direct intracoronary injection through the chest wall made by the methods and compositions described here have or using standard percutaneous catheter based methods under similar or identical properties to bone marrow derived mes 45 fluoroscopic guidance for direct injection into tissue such as enchymal stem cells (BM-MSCs). Therefore, the mesenchy the myocardium or infusion of an inhibitor from a stent or mal stem cells, and any differentiated cells made from these, catheter which is inserted into a bodily lumen. as well as conditioned media derived therefrom, may be used Any variety of coronary catheter, or a perfusion catheter, in any of the applications for which BM-MSCs are known to may be used to administer the compound. Alternatively the be used, or in which it is possible for them to be used. 50 conditioned medium may be coated or impregnated on a stent Delivery of Conditioned Media that is placed in a coronary vessel. The conditioned medium as described in this document Tissue Regeneration may be delivered to the human or animal body by any suitable Mesenchymal stem cells and differentiated cells made CaS. according to the methods and compositions described here, We therefore describe a delivery system for delivering a 55 and conditioned media derived therefrom, may also be used conditioned medium as described in this document to a target for tissue reconstitution or regeneration in a human patient in cell, tissue, organ, animal body or human body, and methods need thereof. The cells are administered in a manner that for using the delivery system to deliver conditioned medium permits them to graft to the intended tissue site and reconsti to a target. tute or regenerate the functionally deficient area. The delivery system may comprise a source of conditioned 60 For example, the methods and compositions described here medium, Such as a container containing the conditioned may be used to modulate the differentiation of stem cells. medium. The delivery system may comprise a dispenser for Mesenchymal stem cells and differentiated cells and condi dispensing the conditioned medium to a target. tioned media derived therefrom may be used for tissue engi Accordingly, we provide a delivery system for delivering a neering, such as for the growing of skin grafts. Modulation of conditioned medium, comprising a source of conditioned 65 stem cell differentiation may be used for the bioengineering medium as described in this document together with a dis of artificial organs or tissues, or for prosthetics, such as Stents. penser operable to deliver the conditioned medium to a target. Cancer US 9,029, 146 B2 27 28 Mesenchymal stem cells and differentiated cells made by (such as diethylstilbestrol (DES), Tamoxifen, LHRH antago the methods and compositions described here and condi nizing drugs, progestins, anti-progestins etc). tioned media derived therefrom may be used for the treatment Stem Cells of cancer. As used in this document, the term “stem cell refers to a The terms “cancer and “cancerous” refer to or describe the 5 cell that on division faces two developmental options: the physiological condition in mammals that is typically charac daughter cells can be identical to the original cell (self-re terized by unregulated cell growth. Examples of cancer newal) or they may be the progenitors of more specialised cell include but are not limited to, carcinoma, lymphoma, blas types (differentiation). The stem cell is therefore capable of toma, sarcoma, and leukemia. adopting one or other pathway (a further pathway exists in 10 which one of each cell type can be formed). Stem cells are More particular examples of Such cancers include squa therefore cells which are not terminally differentiated and are mous cell cancer, Small-cell lung cancer, non-Small cell lung able to produce cells of other types. cancer, gastric cancer, pancreatic cancer, glial cell tumors Stem cells as referred to in this document may include Such as glioblastoma and neurofibromatosis, cervical cancer, totipotent stem cells, pluripotent stem cells, and multipotent ovarian cancer, liver cancer, bladder cancer, hepatoma, breast 15 stem cells. cancer, colon cancer, colorectal cancer, endometrial carci Totipotent StemCells noma, salivary gland carcinoma, kidney cancer, renal cancer, The term “totipotent cell refers to a cell which has the prostate cancer, Vulval cancer, thyroid cancer, hepatic carci potential to become any cell type in the adult body, or any cell noma and various types of head and neck cancer. Further of the extraembryonic membranes (e.g., placenta). Thus, the examples are solid tumor cancer including colon cancer, 20 only totipotent cells are the fertilized egg and the first 4 or so breast cancer, lung cancer and prostrate cancer, hematopoi cells produced by its cleavage. etic malignancies including leukemias and lymphomas, Pluripotent StemCells Hodgkin’s disease, aplastic anemia, skin cancer and familiar "Pluripotent stem cells' are true stem cells, with the poten adenomatous polyposis. Further examples include brain neo tial to make any differentiated cell in the body. However, they plasms, colorectal neoplasms, breast neoplasms, cervix neo- 25 cannot contribute to making the extraembryonic membranes plasms, eye neoplasms, liver neoplasms, lung neoplasms, which are derived from the trophoblast. Several types of pancreatic neoplasms, ovarian neoplasms, prostatic neo pluripotent stem cells have been found. plasms, skin neoplasms, testicular neoplasms, neoplasms, Embryonic Stem Cells bone neoplasms, trophoblastic neoplasms, fallopian tube Embryonic Stem (ES) cells may be isolated from the inner neoplasms, rectal neoplasms, colonic neoplasms, kidney neo- 30 cell mass (ICM) of the blastocyst, which is the stage of plasms, stomach neoplasms, and parathyroid neoplasms. embryonic development when implantation occurs. Breast cancer, prostate cancer, pancreatic cancer, colorectal Embryonic Germ Cells cancer, lung cancer, malignant melanoma, leukaemia, lympy Embryonic Germ (EG) cells may be isolated from the homa, ovarian cancer, cervical cancer and biliary tract carci precursor to the gonads in aborted fetuses. noma are also included. 35 Embryonic Carcinoma Cells The mesenchymal stem cells and differentiated cells made Embryonic Carcinoma (EC) cells may be isolated from according to the methods and compositions described here teratocarcinomas, a tumor that occasionally occurs in agonad may also be used in combination with anticancer agents such of a fetus. Unlike the first two, they are usually aneuploid. All as endostatin and angiostatin or cytotoxic agents or chemo three of these types of pluripotent stem cells can only be therapeutic agent. For example, drugs such as Such as adria- 40 isolated from embryonic or fetal tissue and can be grown in mycin, daunomycin, cis-platinum, etoposide, taxol, taxotere culture. Methods are known in the art which prevent these and alkaloids, such as Vincristine, and antimetabolites such as pluripotent cells from differentiating. methotrexate. The term “cytotoxic agent” as used herein Adult Stem Cells refers to a substance that inhibits or prevents the function of Adult stem cells comprise a wide variety of types including cells and/or causes destruction of cells. The term is intended 45 neuronal, skin and the blood forming stem cells which are the to include radioactive isotopes (e.g. I.Y. Pr), chemotherapeu active component in bone marrow transplantation. These lat tic agents, and toxins such as enzymatically active toxins of ter stem cell types are also the principal feature of umbilical bacterial, fungal, plant or animal origin, or fragments thereof. cord-derived stem cells. Adult stem cells can mature both in Also, the term includes oncogene product/tyrosine kinase the laboratory and in the body into functional, more special inhibitors, such as the bicyclic ansamycins disclosed in WO 50 ised cell types although the exact number of cell types is 94/22867; 1,2-bis(arylamino) benzoic acid derivatives dis limited by the type of stem cell chosen. closed in EP 600832: 6,7-diamino-phthalazin-1-one deriva Multipotent Stem Cells tives disclosed in EP 600831; 4.5-bis(arylamino)-phthalim Multipotent stem cells are true stem cells but can only ide derivatives as disclosed in EP 516598; or peptides which differentiate into a limited number of types. For example, the inhibit binding of a tyrosine kinase to a SH2-containing sub- 55 bone marrow contains multipotent stem cells that give rise to strate protein (see WO94/07913, for example). A “chemo all the cells of the blood but not to other types of cells. therapeutic agent' is a chemical compound useful in the Multipotent stem cells are found in adult animals. It is thought treatment of cancer. Examples of chemotherapeutic agents that every organ in the body (brain, liver) contains them where include Adriamycin, Doxorubicin, 5-Fluorouracil (5-FU), they can replace dead or damaged cells. Cytosine arabinoside (Ara-C), Cyclophosphamide. Thiotepa, 60 Methods of characterising stem cells are known in the art, Busulfan, Cytoxin, Taxol. Methotrexate, Cisplatin, Mel and include the use of standard assay methods such as clonal phalan, Vinblastine, Bleomycin, Etoposide. Ifosfamide, assay, flow cytometry, long-term culture and molecular bio Mitomycin C. Mitoxantrone, Vincristine, VP-16, Vinorel logical techniques e.g. PCR, RT-PCR and Southern blotting. bine, Carboplatin, Teniposide, Daunomycin, Carminomycin, In addition to morphological differences, human and Aminopterin, Dactinomycin, Mitomycins, Nicotinamide, 65 murine pluripotent stem cells differ in their expression of a Esperamicins (see U.S. Pat. No. 4.675, 187), Melphalan and number of cell Surface antigens (stem cell markers). Antibod other related nitrogen mustards, and endocrine therapies ies for the identification of stem cell markers including the US 9,029, 146 B2 29 30 Stage-Specific Embryonic Antigens 1 and 4 (SSEA-1 and pocytes and red cells. These cells reportedly can be expanded SSEA-4) and Tumor Rejection Antigen 1-60 and 1-81 (TRA and cultured to produce hormones and conditioned culture 1-60, TRA-1-81) may be obtained commercially, for example media. from Chemicon International, Inc (Temecula, Calif., USA). Stem cells of any vertebrate species can be used. Included The immunological detection of these antigens using mono are Stein cells from humans; as well as non-human primates, clonal antibodies has been widely used to characterize pluri domestic animals, livestock, and other non-human mammals potent stem cells (Shamblott M. J. et al. (1998) PNAS 95: Such as rodents, mice, rats, etc. 13726-13731; Schuldiner M. et al. (2000). PNAS97: 11307 Amongst the stem cells Suitable for use in this invention are 11312: Thomson J. A. et al. (1998). Science 282: 1145-1147: primate pluripotent stem (pPS) cells derived from tissue Reubinoff B. E. et al. (2000). Nature Biotechnology 18: 10 formed after gestation, Such as a blastocyst, or fetal or embry 399-404; Henderson J. K. et al. (2002). Stem Cells 20:329 onic tissue taken any time during gestation. Non-limiting 337; Pera M. et al. (2000). J. Cell Science 113: 5-10). examples are primary cultures or established lines of embry Sources of Stem Cells onic stem cells. Stem cells of various types, which may include the follow Media and Feeder Cells ing non-limiting examples, may be used in the methods and 15 Media for isolating and propagating pluripotent stem cells compositions described here for producing mesenchymal can have any of several different formulas, as long as the cells stem cells and differentiated cells. obtained have the desired characteristics, and can be propa U.S. Pat. No. 5,851,832 reports multipotent neural stein gated further. cells obtained from brain tissue. U.S. Pat. No. 5,766,948 Suitable sources are as follows: Dulbecco's modified reports producing neuroblasts from newborn cerebral hemi Eagles medium (DMEM), Gibcoil 11965-092: Knockout spheres. U.S. Pat. Nos. 5,654,183 and 5,849,553 report the Dulbecco's modified Eagles medium (KO DMEM), use of mammalian neural crest stem cells. U.S. Pat. No. Gibco#10829-018; 200 mM L-glutamine, Gibcoil 15039 6,040,180 reports in vitro generation of differentiated neu 027; non-essential amino acid solution, Gibco 11140-050; rons from cultures of mammalian multipotential CNS stem beta-mercaptoethanol, Sigmail M7522; human recombinant cells. WO98/50526 and WO99/01159 report generation and 25 basic fibroblast growth factor (bFGF), Gibcoil 13256-029. isolation of neuroepithelial Stein cells, oligodendrocyte-as Exemplary serum-containing embryonic Stein (ES) medium trocyte precursors, and lineage-restricted neuronal precur is made with 80% DMEM (typically KO DMEM), 20% sors. U.S. Pat. No. 5,968,829 reports neural stem cells defined fetal bovine serum (FBS) not heat inactivated, 0.1 obtained from embryonic forebrain and cultured with a mM non-essential amino acids, 1 mM L-glutamine, and 0.1 medium comprising glucose, transferrin, insulin, selenium, 30 mM beta-mercaptoethanol. The medium is filtered and stored progesterone, and several other growth factors. at 4 degrees C. for no longer than 2 weeks. Serum-free embry Primary liver cell cultures can be obtained from human onic stem (ES) medium is made with 80% KODMEM, 20% biopsy or Surgically excised tissue by perfusion with an serum replacement, 0.1 mM non-essential amino acids, 1 mM appropriate combination of collagenase and hyaluronidase. L-glutamine, and 0.1 mM beta-mercaptoethanol. An effective Alternatively, EP 0953 633 A1 reports isolating liver cells by 35 serum replacement is Gibcoil 10828-028. The medium is fil preparing minced human liver tissue, resuspending concen tered and stored at 4 degrees C. for no longer than 2 weeks. trated tissue cells in a growth medium and expanding the cells Just before use, human bFGF is added to a final concentration in culture. The growth medium comprises glucose, insulin, of 4 ng/mL (Bodnar et al., Geron Corp, International Patent transferrin, T, FCS, and various tissue extracts that allow the Publication WO99/20741). hepatocytes to grow without malignant transformation. The 40 In a preferred embodiment, the media comprises Knockout cells in the liver are thought to contain specialized cells DMEM media (Invitrogen-Gibco, Grand Island, N.Y.), including liver parenchymal cells, Kupffer cells, sinusoidal supplemented with 10% serum replacement media (Invitro endothelium, and bile duct epithelium, and also precursor gen-Gibco, Grand Island, N.Y.), 5 ng/ml FGF2 (Invitrogen cells (referred to as “hepatoblasts' or "oval cells') that have Gibco, Grand Island, N.Y.) and 5 ng/ml PDGF AB (Pepro the capacity to differentiate into both mature hepatocytes or 45 tech, Rocky Hill, N.J.). biliary epithelial cells (L. E. Rogler, Am. J. Pathol. 150:591, Feeder cells (where used) are propagated in mEF medium, 1997: M. Alison, Current Opin. Cell Biol. 10:710, 1998: containing 90% DMEM (Gibcoil 11965-092), 10% FBS (Hy Lazaro et al., Cancer Res. 58:514, 1998). clone#3007 1-03), and 2 mM glutamine. mEFs are propagated U.S. Pat. No. 5,192,553 reports methods for isolating in T150 flasks (Coming#430825), splitting the cells 1:2 every human neonatal or fetal hematopoietic stem or progenitor 50 other day with trypsin, keeping the cells subconfluent. To cells. U.S. Pat. No. 5,716,827 reports human hematopoietic prepare the feeder cell layer, cells are irradiated at a dose to cells that are Thy-1 positive progenitors, and appropriate inhibit proliferation but permit synthesis of important factors growth media to regenerate them in vitro. U.S. Pat. No. 5,635, that support human embryonic stem cells (about.4000 rads 387 reports a method and device for culturing human hemato gamma irradiation). Six-well culture plates (such as Fal poietic cells and their precursors. U.S. Pat. No. 6,015.554 55 conii.304) are coated by incubation at 37 degrees C. with 1 mL describes a method of reconstituting human lymphoid and 0.5% gelatin per well overnight, and plated with 375,000 dendritic cells. irradiated mEF's per well. Feeder cell layers are typically used U.S. Pat. No. 5,486.359 reports homogeneous populations 5h to 4 days after plating. The medium is replaced with fresh of human mesenchymal Stein cells that can differentiate into human embryonic stem (hES) medium just before seeding cells of more than one connective tissue type. Such as bone, 60 pPS cells. cartilage, tendon, ligament, and dermis. They are obtained Conditions for culturing other stem cells are known, and from bone marrow or periosteum. Also reported are culture can be optimized appropriately according to the cell type. conditions used to expand mesenchymal stem cells. WO Media and culture techniques for particular cell types referred 99/01145 reports human mesenchymal stem cells isolated to in the previous section are provided in the references cited. from peripheral blood of individuals treated with growth fac 65 Embryonic Stem Cells tors such as G-CSF or GM-CSF. WO 00/53795 reports adi Embryonic stem cells can be isolated from blastocysts of pose-derived stem cells and lattices, substantially free of adi members of the primate species (Thomson et al., Proc. Natl. US 9,029, 146 B2 31 32 Acad. Sci. USA 92:7844, 1995). Human embryonic stem hyaluronidase type V, 0.002% DNAse I, 0.1% collagenase (hES) cells can be prepared from human blastocyst cells using type IV, all from Sigma prepared in EG growth medium). the techniques described by Thomson et al. (U.S. Pat. No. Tissue is minced and incubated 1 h or overnight at 37 degrees 5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol. C., resuspended in 1-3 mL of EG growth medium, and plated 38:133 ff., 1998) and Reubinoffetal, Nature Biotech. 18:399, onto a feeder layer. 2OOO. Ninety-six well tissue culture plates are prepared with a Briefly, human blastocysts are obtained from human in sub-confluent layer of feeder cells cultured for 3 days in vivo preimplantation embryos. Alternatively, in vitro fertil modified EG growth medium free of LIF, bFGF or forskolin, ized (IVF) embryos can be used, or one cell human embryos inactivated with 5000 rady-irradiation. Suitable feeders are can be expanded to the blastocyst stage (BongSo et al., Hum 10 STO cells (ATCC Accession No. CRL 1503). 0.2 mL of Reprod 4: 706, 1989). Human embryos are cultured to the primary germ cell (PGC) suspension is added to each of the blastocyst stage in G1.2 and G2.2 medium (Gardner et al., wells. The first passage is conducted after 7-10 days in EG Fertil. Steril. 69:84, 1998). Blastocysts that develop are growth medium, transferring each well to one well of a selected for embryonic stem cell isolation. The Zona pellucida 24-well culture dish previously prepared with irradiated STO is removed from blastocysts by brief exposure to pronase 15 mouse fibroblasts. The cells are cultured with daily replace (Sigma). The inner cell masses are isolated by immunosur ment of medium until cell morphology consistent with EG gery, in which blastocysts are exposed to a 1:50 dilution of cells are observed, typically after 7-30 days or 1-4 passages. rabbit anti-human spleen cell antiserum for 30 minutes, then Self-Renewal and Differentiation washed for 5 minutes three times in DMEM, and exposed to Self-Renewal a 1:5 dilution of Guinea pig complement (Gibco) for 3 min Stem cells which are self-renewing may be identified by utes (see Solter et al., Proc. Natl. Acad. Sci. USA 72:5099, various means known in the art, for example, morphology, 1975). After two further washes in DMEM, lysed trophecto immunohistochemistry, molecular biology, etc. derm cells are removed from the intact inner cell mass (ICM) Such stem cells preferably display increased expression of by gentle pipetting, and the ICM plated on mEF feeder layers. Octá and/or SSEA-1. Preferably, expression of any one or After 9 to 15 days, inner cell mass-derived outgrowths are 25 more of Flk-1, Tie-2 and c-kit is decreased. Stem cells which dissociated into clumps either by exposure to calcium and are self-renewing preferably display a shortened cell cycle magnesium-free phosphate-buffered saline (PBS) with 1 mM compared to stem cells which are not self-renewing. EDTA, by exposure to dispase or trypsin, or by mechanical For example, in the two dimensions of a standard micro dissociation with a micropipette; and then replated on mEF in scopic image, human embryonic Stein cells display high fresh medium. Dissociated cells are replated on mEF feeder 30 nuclear/cytoplasmic ratios in the plane of the image, promi layers in fresh embryonic stem (ES) medium, and observed nent nucleoli, and compact colony formation with poorly for colony formation. Colonies demonstrating undifferenti discernable cell junctions. Cell lines can be karyotyped using ated morphology are individually selected by micropipette, a standard G-banding technique (available at many clinical mechanically dissociated into clumps, and replated embry diagnostics labs that provides routine karyotyping services, onic stem cell-like morphology is characterized as compact 35 Such as the Cytogenetics Lab at Oakland Calif.) and com colonies with apparently high nucleus to cytoplasm ratio and pared to published human karyotypes. prominent nucleoli. Resulting embryonic stem cells are then Human embryonic stem and human embryonic germ cells routinely split every 1-2 weeks by brief trypsinization, expo may also be characterized by expressed cell markers. In gen sure to Dulbecco's PBS (without calcium or magnesium and eral, the tissue-specific markers discussed in this disclosure with 2 mM EDTA), exposure to type IV collagenase 40 can be detected using a suitable immunological technique— (..about.200U/mL. Gibco) or by selection of individual colo Such as flow cytometry for membrane-bound markers, immu nies by micropipette. Clump sizes of about 50 to 100 cells are nohistochemistry for intracellular markers, and enzyme optimal. linked immunoassay, for markers secreted into the medium. Embryonic Germ Cells The expression of protein markers can also be detected at the Human Embryonic Germ (hEG) cells can be prepared from 45 mRNA level by reverse transcriptase-PCR using marker-spe primordial germ cells present in human fetal material taken cific primers. See U.S. Pat. No. 5,843,780 for further details. about 8-11 weeks after the last menstrual period. Suitable Stage-specific embryonic antigens (SSEA) are character preparation methods are described in Shamblott et al., Proc. istic of certain embryonic cell types. Antibodies for SSEA Natl. Acad. Sci. USA 95:13726, 1998 and U.S. Pat. No. markers are available from the Developmental Studies Hybri 6,090,622. 50 doma Bank (Bethesda Md.). Other useful markers are detect Briefly, genital ridges are rinsed with isotonic buffer, then able using antibodies designated Tra-1-60 and Tra-1-81 (An placed into 0.1 mL 0.05% trypsin/0.53 mM sodium EDTA drews et al., Cell Lines from Human Germ. Cell Tumors, in E. solution (BRL)andcut into <1 mm chunks. The tissue is then J. Robertson, 1987, supra). Human embryonic stem cells are pipetted through a 100/LL tip to further disaggregate the cells. typically SSEA-1 negative and SSEA-4 positive. hEG cells It is incubated at 37 degrees C. for about 5 min, then about 3.5 55 are typically SSEA-1 positive. Differentiation of pPS cells in mL EG growth medium is added. EG growth medium is vitro results in the loss of SSEA-4, Tra-1-60, and Tra-1-81 DMEM, 4500 mg/L D-glucose, 2200 mg/L mM sodium expression and increased expression of SSEA-1. pPS cells bicarbonate; 15% embryonic stem (ES) qualified fetal calf can also be characterized by the presence of alkaline phos serum (BRL); 2 mM glutamine (BRL); 1 mM sodium pyru phatase activity, which can be detected by fixing the cells with vate (BRL); 1000-2000 U/mL human recombinant leukemia 60 4% paraformaldehyde, and then developing with Vector Red inhibitory factor (LIF, Genzyme): 1-2 ng/ml human recom as a substrate, as described by the manufacturer (Vector Labo binant basic fibroblast growth factor (bFGF, Genzyme); and ratories, Burlingame Calif.). 10 uM forskolin (in 10% DMSO). In an alternative approach, Embryonic stem cells are also typically telomerase positive EG cells are isolated using hyaluronidase/collagenase/ and OCT-4 positive. Telomerase activity can be determined DNAse. Gonadalanlagen or genital ridges with mesenteries 65 using TRAP activity assay (Kim et al., Science 266:2011, are dissected from fetal material, the genital ridges are rinsed 1997), using a commercially available kit (TRAPeze(R) XK in PBS, then placed in 0.1 ml HCD digestion solution (0.01% Telomerase Detection Kit, Cat. s.7707; Intergen Co., Purchase US 9,029, 146 B2 33 34 N.Y.; or TeloTAGGGTM Telomerase PCR ELISA plus, Cat. and myf5. For endothelial cells: PECAM (platelet endothe 2.013,89; Roche Diagnostics, Indianapolis). htERT expres lial cell adhesion molecule), Flk-1, tie-i, tie-2, vascular endot sion can also be evaluated at the mRNA level by RT-PCR. The helial (VE) cadherin, MECA-32, and MEC-14.7. For smooth LightCycler TeloTAGGGTM hTERT quantification kit (Cat. muscle cells: Specific myosin heavy chain. For cardiomyo 3.012,344; Roche Diagnostics) is available commercially for cytes: GATA-4, NkX2.5, cardiac troponin I, alpha-myosin research purposes. heavy chain, and ANF. For pancreatic cells, pdx and insulin Differentiation secretion. For hematopoietic cells and their progenitors: Differentiating cells, including mesenchymal stem cells GATA-1, CD34, AC133, B-major globulin, and B-major and differentiated cells derived from these, preferably display globulin like gene PH1. enhanced dephosphorylation of 4E-BP1 and/or S6K1. They 10 preferably display decreased expression of Oct4 and/or Certain tissue-specific markers listed in this disclosure or SSEA-1. Preferably, expression of any one or more of Flk-1, known in the art can be detected by immunological tech Tie-2 and c-kit is increased. Preferably, expression of any one niques—such as flow immunocytochemistry for cell-surface or more of Brachyury, AFP, nestin and nurr1 expression markers, immunohistochemistry (for example, offixed cells increased. Stem cells which are self-renewing preferably dis 15 or tissue sections) for intracellular or cell-surface markers, play a lenghtened cell cycle compared to stem cells which are Western blot analysis of cellular extracts, and enzyme-linked not self-renewing. immunoassay, for cellular extracts or products secreted into Differentiating stem cells, i.e., cells which have started to, the medium. The expression of tissue-specific gene products or are committed to a pathway of differentiation can be char can also be detected at the mRNA level by Northern blot acterized according to a number of phenotypic criteria, analysis, dot-blot hybridization analysis, or by reverse tran including in particular transcript changes. The criteria include scriptase initiated polymerase chain reaction (RT-PCR) using but are not limited to characterization of morphological fea sequence-specific primers in standard amplification methods. tures, detection or quantitation of expressed cell markers and Sequence data for the particular markers listed in this disclo enzymatic activity, gene expression and determination of the sure can be obtained from public databases such as GenBank functional properties of the cells in vivo. In general, differen 25 (URL www.ncbi.nlm.nih.gov:80/entrez). tiating stem cells will have one or more features of the cell type which is the final product of the differentiation process, EXAMPLES i.e., the differentiated cell. For example, if the target cell type is a muscle cell, a stem cell which is in the process of differ Example 1 entiating to such a cell will have for example a feature of 30 myosin expression. Methods In many respects, therefore, the criteria will depend on the fate of the differentiating stem cell, and a general description Derivation of hESC-MSC (Mesenchymal StemCells) of various cell types is provided below. Hues9 and H1 hESCs are grown as previously Markers of interest for differentiated or differentiating neu 35 described 7. ral cells include beta-tubulin EIII or neurofilament, charac To differentiate hESCs, a confluent 6 cm plate of hESCs is teristic of neurons; glial fibrillary acidic protein (GFAP), trypsinized for 3 mins, 37° C., neutralized, centrifuged and present in astrocytes; galactocerebroside (GalC) or myelin resuspended in Knockout DMEM media (Invitrogen-Gibco, basic protein (MBP); characteristic of oligodendrocytes; Grand Island, N.Y.), supplemented with 10% serum replace OCT-4, characteristic of undifferentiated human embryonic 40 ment media (Invitrogen-Gibco, Grand Island, N.Y.), 5 ng/ml stem cells; nestin, characteristic of neural precursors and FGF2 (Invitrogen-Gibco, Grand Island, N.Y.), and 5 ng/ml other cells. A2B5 and NCAM are characteristic of glial pro PDGFAB (Peprotech, Rocky Hill, N.J.) on a gelatinized 10 genitors and neural progenitors, respectively. Cells can also cm plate. The cells are trypsinized when confluent and split be tested for secretion of characteristic biologically active 1:4. Substances. For example, GABA-secreting neurons can be 45 Sorting for CD105+ and CD24- is performed one week identified by production of glutamic acid decarboxylase or after hESCs have been trypsinized. The differentiating hESCs GABA. Dopaminergic neurons can be identified by produc are trypsinized for 3 mins, neutralized, centrifuged, resus tion of dopa decarboxylase, dopamine, or tyrosine hydroxy pended in the culture media and then plated on bacterial lase. culture dish. After 2 hours at 37°C. in CO incubator, the cells Markers of interest for differentiated or differentiating liver 50 are harvested, washed with PBS and incubated with CD24 cells include alpha-fetoprotein (liver progenitors); albumin, PE and CD105-FITC (PharMingen, San Diego, Calif.) for 90 C-antitrypsin, glucose-6-phosphatase, cytochrome p450 mins at room temperature. activity, transferrin, asialoglycoprotein receptor, and glyco The cells are then washed with PBS and sorted on a FACS gen storage (hepatocytes); CK7, CK19, and gamma-glutamyl Aria using FACS Diva software (BD Biosciences Pharmin transferase (bile epithelium). It has been reported that hepa 55 gen, San Diego, Calif.). BMMSCs are prepared as previously tocyte differentiation requires the transcription factor BNF-4 described. The cells are cultured in DMEM supplemented alpha (Liet al., Genes Dev. 14:464, 2000). Markers indepen with penicillin-streptomycin-glutamine, non-essential amino dent of HNF-4 alpha expression include alpha-antitrypsin, acids and 10% fetal calf serum (Invitrogen-Gibco, Grand alpha-fetoprotein, apoE, glucokinase, insulin growth factors Island, N.Y.) 1 and 2, IGF-1 receptor, insulin receptor, and leptin. Markers 60 Differentiation into adipocytes, chondrocytes and osteo dependent on HNF-4 alpha expression include albumin, cytes is performed as previously described. Oil red, alcian apoAI, apoAII, apoB, apoCIII, apoCII, aldolase B, phenyla blue and Von Kossa staining is performed using standard lanine hydroxylase, L-type fatty acid binding protein, trans techniques. Immunoreactivity for collagen type II is per ferrin, retinol binding protein, and erythropoietin (EPO). formed on paraformaldehyde fixed, paraffin-embedded sec Cell types in mixed cell populations derived from pPS cells 65 tions using a goat anti-collagenal Type II and donkey anti can be recognized by characteristic morphology and the goat IgG antibody conjugated with HRP (Santa Cruz, Santa markers they express. For skeletal muscle: myoD, myogenin, Cruz, Calif.). US 9,029, 146 B2 35 36 Karyotyping Illumina Gene Chip Analysis Cells received at about 80% confluence in Petri dish. Cells Total RNA (2 ug) from 3 samples each of primary BM and are treated with colcemid for mitotic arrest and harvested by adipose-derived MSCs, from two biological replicates of standard hypotonic treatment and methanol: acetic acid (3:1) HuES9.E.1, HuES9.E3, HuES9.E.1 and three undifferentiated fixation. Slides are prepared by standard air drying method 5 hESC lines, H1, Hes3 and HuES9 are converted to biotiny and hybridized with SKY paint probe (ASI). Post hybridiza lated crNA using the Illumina RNA Amplification Kit (Am tion washes are performed according to the protocols pro bion, Inc., Austin, Tex.) according to the manufacturers vided by the manufacturer and established in our laboratory. directions. 20-30 metaphase cells per culture were are. The karyotype of Samples are purified using the RNeasy kit (Qiagen, Valen each culture is representative of >80% metaphase cells. 10 cia, Calif.). Hybridization to the Sentrix Human Ref-8 Transplantation Studies Expression BeadChip (Illumina, Inc., San Diego, Calif.), 2x10 cells are resuspended in 30 ul of saline and trans washing and Scanning are performed according to the Illu ferred into the renal Subcapsular space as previously mina BeadStation 500x manual. The data are extracted, nor described. After four months, the mice are sacrificed and the 15 malized and analyzed using Illumina BeadStudio provided by kidneys are removed, fixed in 4% paraformaldehyde, paraf the manufacturer. Transcript signals that are below the limit of fin-embedded, sectioned at 4 LM and stained with H&E. detection (LOD) at 99% confidence are eliminated as genes Western Blot Analysis not expressed. Standard procedures are used. Briefly, cells are lysed in RIPA buffer and centrifuged at 14,000 rpm for 15 minutes at Example 2 4°C. 20 g supernatant is denatured, separated on 10 SDS polyacrylamide gel, electro-blotted onto a nitrocellulose Generating MSC Cultures from Human ES Cell membrane and membrane is incubated sequentially with a Lines primary antibody, then either a HRP conjugated-secondary antibody or a biotinylated secondary antibody followed by 25 When hESC colonies are dispersed by trypsin and then neutroavidin-HRP, and finally, a HRP enhanced chemilumi passaged on gelatinized tissue culture plates in the absence of nescent substrate, ECS (Pierce, Rockford, Ill.). feeder, and in serum-free media that is Supplemented with Primary antibodies used are 1:200 dilution of anti-OCT4, serum replacement media, FGF2 and optionally PDGFAB, a anti-SOX-2 and B-actin (Santa Cruz, Biotechnology, CA), culture of fibroblast-like cells which is similar or identical Secondary antibodies are HRP-conjugated goat anti-rabbit, 30 (preferably homogenous) to each other is generated within rabbit anti-goat and rabbit antimouse. two weeks. PCR The cultures have a fibroblastic cellular morphology that Genomic PCR for mouse- and human-specific repeat resembled BM-MSCs (FIG. 1A). Dispersing hESC colonies sequences are performed as previously described. Genomic by collagenase is not efficient in generating these fibroblast PCR for mouse- and human-specific repeat sequences are 35 performed as previously described. Real time RT-PCR is like cells. performed by reverse transcribing 1 g of total RNA using a Two polyclonal cultures, huFS9.E.1 and huES9.E3, are High Capacity cDNA Archive Kit (Applied Biosystems, Fos independently generated from huES9 ESC line, while the ter City, Calif.). The cDNA is diluted 10x in water and ampli third, H1.E2 is generated from H1 ESC line. Expression of fied by Taqman primers (Applied Biosystems, Foster City, 40 several pluripotency-associated genes is generally reduced. Calif.) for 40 cycles. For example, transcript levels of HESX1, POUFL5, SOX-2, Surface Antigen Analysis UTF-1 and ZFP42 are >10' fold below that in the hESCs Cell surface antigens on hESC-MSCs, and hESCs are ana (FIG. 1B). Protein levels of OCT4, NANOG and SOX2 are lyzed using FACS. The cells are tryspinized for 5 minutes, also reduced (FIG. 1C). As typified by huES9.E.1, these cells centrifuged, resuspended in culture media and incubated in a 45 did not have detectable alkaline phosphatase activity (FIG. bacterial culture dish for 2-3 hours in a 37° C., 5% CO 1D). incubator. Cell surface antigens on hESC-MSCs and hESCs Unlike its parental HuES9 cells, renal subcapsular trans are analyzed by FACS. plantation of 1x10'HuES9.E1 cells in immune compromised The cells are trypsinized for 1 minute, centrifuged, washed SCID mice did not induce the formation of a teratoma during with PBS, fixed in 4% paraformaldehyde for 0.5 hour at room 50 a four-month observation period (FIG. 1E). To assess the temperature, washed and blocked in 2% FCS for 0.5 hour at possibility that these cells are contaminated or fused with room temperature with agitation. 1.5x10 cells are then incu mouse feeder cells, these cultures are tested and shown to be bated with each of the following conjugated monoclonal anti negative for mouse-specific c-mos repeat sequences but posi bodies: CD24-PE, CD29-PE, CD44-FITC, CD49a-PE, tive for human specific alu repeat sequences (FIG.1F). CD49e-PE, CD105-FITC, CD166-PE, CD34-FITC, CD45 55 The average population doubling time of HuES9.E.1, FITC (PharMingen, San Diego, Calif.) for 90 mins at room HuES9.E3 and H1. E2 are 72, 72 and 120 hours respectively. temperature. Population doubling time is highly dependent on cell density After incubation, cells are washed and resuspended in and is most optimal at between 30-80% confluency. After PBS. Nonspecific fluorescence is determined by incubation about 80 population doublings, HnES9.E1 have a 46 XX of similar cell aliquots with isotype-matched mouse mono 60 inv(9) (p13q12 karyotype. The chromosome 9 inversion origi clonal antibodies (PharMingen, San Diego, Calif.). Data are nated from the parental huES9 hESC line. HuES9.E3 also analyzed by collecting 20,000 events on a Cyan LX (Dako have 46 XX inv(9)(p13q12 karyotype while H1.E1 has a 46, North America, Inc., Carpinteria, Calif.) instrument using XY karyotype after 36 and 32 population doubling time. We WinMDI software. Nonspecific fluorescence is determined observed that the cells began to manifest abnormal, nonclonal by incubation of similar cell aliquots with isotype-matched 65 chromosomal aberrations in ~10-30% of cells after about 35 mouse monoclonal antibodies (PharMingen, San Diego, population doublings (data not shown) and cells are not used Calif.), or with secondary antibody alone. after 35 population doublings. US 9,029, 146 B2 37 38 Example 3 Example 5 Surface Antigen Profile Gene Expression Profile Surface antigen profiling of HuES9.E1, HuES9.E3 and 5 Gene expression profiling of the hESC-MSCs are per H1. E2 by FACS analysis revealed a surface antigen profile formed to 1) assess the relatedness of hESC-MSC cultures that is qualitatively similar to that defined for BMMSCs i.e. with adult tissue-derived MSCs using BM-MSCs and adipose CD29--, CD44+, CD49a and e--, CD105+, CD166+ and derived (ad)-MSCs from three different individuals, and three CD34-, CD45-' (FIG. 2A) The intensity of fluorescent human ESC lines; 2) assess the relatedness between each of 10 the three hESC-MSC cultures; 3) compare the similarity and labelling and distribution of labelled cells varied with each of differences between MSCs derived from hESC and those the hESC-MSC cultures (FIG. 2A). derived from BM. To compare the surface antigen profile of these cells to that Labeled cDNA prepared from total RNA RNA are hybrid of BM-MSCs, HuES9.E.1, HuES9.E3 and H1.E.1 are grownin ized to Illumina BeadArray containing about 24,000 unique the same BM-MSC culture media supplemented with 10% 15 features. Hierarchical clustering of expressed genes in three fetal calf serum for two passages. Despite the change in hESC-MSC cultures i.e. HuES9.E.1, HuES9.E3 and H1.E.2, culture condition, HuES9.E.1, HuES9.E3 and H1. E1 contin three BM-MSC samples and three adipose derived (ad)-MSC ued to be CD29+, CD44+, CD49a+, CD105+, CD166+ and samples revealed that the gene expression profile of hESC CD34-, CD45- (FIG.2B: data not shown for H1.E1) and are MSCs is more closely related to that of adult tissue-derived largely similar to that of BM-MSCs. An exception is CD49a MSCs, namely BM-MSC and ad-MSC than to their parent which had a much lower expression in BM-MSCs. These data hESCs (FIG. 4A). indicated that the hESC-MSCs exhibited characteristic BM Interestingly, MSCs clustered according to their tissue of MSC Surface antigen profiles that are stable and are not sig origin, and this can be further demarcated into adult versus nificantly influenced by changes in their microenvironment. embryonic tissue as Suggested by the clustering of ad-MSCs 25 and BMMSCs as a distinct group from hESC-MSCs. Pair Example 4 wise comparison of gene expression between hESC-MSCs and BM-MSCs revealed a correlation coefficient of 0.72 Sug Differentiation Potential of hESC-MSC: gesting that while there is significant conservation of gene Adipogenesis, Chondrogenesis and Osteogenesis expression in both hESC-MSCs and BM-MSCs, there are 30 also significant differences (FIG. 4B). Pairwise comparison As all of the surface antigens associated with MSCs are between hESC-MSCs and hESCs confirmed the distinction also expressed on many other cell types and the expression of of hESC-MSCs from hESCs with a low correlation coeffi these surface antigens are variable, identification of presump cient of 0.65 (FIG. 4C). tive MSCs have traditionally relies on functional To assess the relatedness between each of the three hESC 35 MSC cultures, HuES9.E.1, H1. E2 and HuES9.E3 are each parameters . It is reported that the default differentiation compared to the same reference consisting of HuES9.E.1, pathway of MSCs in culture is osteogenesis with varying H1. E2 and HuBS9.E3. The correlation coefficients of degrees of adipogenesis and chondrogenesis. HuES9.E.1, H1. E2 and HuES9.E3 to the same reference are Differentiation potential of HuES9.E1 cells is therefore virtually identical i.e. 0.93, 0.95 and 0.93, respectively sug tested using standard differentiation conditions for adipogen 40 gesting that HuES9.E.1, H1. E2 and HuES9.E3 are highly esis, chondrogenesis and osteogenesis using published pro similar (FIG. 4C). tocols. Of 8699 and 8505 genes that are expressed above the limit Adipocytic differentiation is highly efficient with oil drop of detection at 99% confidence level in hESC-MSC and BM lets observed in >99% of the cells (FIG.3A). Consistent with MSC, respectively, 6376 genes are expressed in both hESC its role as important transcription factor in adipogenesis', 45 MSCs and BM-MSCs at <2.0 fold difference. As these genes PPARY mRNA in the hESC derived MSCs which is about are likely to provide insights into the fundamental biology of 10-100 fold higher than that in their respective parental ESC MSCs, we examine the biological processes that are driven by lines increased by a further 2 fold (FIG. 3A). these genes. Of the 6376 commonly expressed genes, 4.064 Chondrogeneiss or the formation of cartilage is also effi are found in the Panther classified gene list (March 2006; cient with >90% of cells producing proteoglycan in extracel 50 http://www.pantherdb.org/). lular matrix as detected by alcian blue staining (FIG.3B) and Classification of these genes into different biological pro ~20% of the cells being immunoreactive for collagen II. cesses revealed that the frequency of genes in Some of the Transcript level of aggrecan, an cartilage-specific extracellu biological processes are significantly over or under repre lar matrix protein is also increased' (FIG. 3B). However, sented (p<0.01) when compared to the reference list consist 55 ing of 23481 genes in NCBI. H. sapiens gene database. For transcript level of collagen II, another cartilage-specific example, there are an overrepresentation of genes in meta extracellular matrix protein is decreased despite the presence bolic processes that are likely to be important for growth and of collagen II immunoreactivity in the matrix. The reason is self-renewal of putative stein cells. These processes include not known but some mRNAS particularly those with AU-rich elements are known to be destabilized when translated'''. basal metabolic processes for catabolic and anabolic activi 60 ties, biosynthesis of secretory products that require extensive When HuES9.E1 cells are induced to undergo osteogenesis post-translational modifications e.g. glycosylation, and cel or the formation of bone, expression of bone-specific alkaline lular proliferation (FIG. 4C). phosphatase (ALP) and bonesialoprotein (BSP) is upregu Consistent with their mesenchymal potential, there is also lated by 2-3 fold (FIG. 3C). However, mineralization, a more an under-representation of genes involved in ectoderm differ advanced stage of bone formation'7 as determined by von 65 entiation particularly neural development. The gene expres Kossa staining is poor (FIG. 3C). There is <1% positive sion analysis also suggested that MAPKKK signalling is staining in the differentiated culture. prominent in both BM-MSC and hESC-MSCs. MAPKKK US 9,029, 146 B2 39 40 signalling which consists of at least three Subfamilies, namely cesses within each category are differently represented in the classical MAPK (also known as ERK), stress-activated hESC-MSC and BM-MSC. For example, differentiation pro protein kinase/c-Jun N-terminal kinase (JNK) and p38 cesses that are associated with early embryonic development kinase, are associated with proliferation, differentiation, Such as embryogenesis and segmentation are over-repre sented in hESC-MSC while those associated with late embry development, regulation of responses to cellular stresses, cell onic development e.g. skeletal development and muscle cycle, death and survival''. development are overrepresented in BM-MSC. Similarly, Further analysis of the gene expression profiles of hESC extracellular matrix protein-mediated signalling and MAP MSC and BM-MSC revealed that 1142 and 1134 genes are KKK cascade are overrepresented in BM-MSC. Together, expressed at >2.0 fold in hESC-MSC and BM-MSC, respec 10 these observations suggest that differentiation potential and tively. Of these, 738 and 880 genes respectively, are located in signalling pathway utilisation in hESC-MSC and BM-MSC Panther classified gene list (March 2006: http://www.pan may not be identical. therdb.org/) and classified into biological processes. Biologi cal processes that are significantly over- or under-represented Example 5 (p<0.01) when compared to the reference list of 23481 genes in NCBI. H. sapiens gene database are selected. 15 Distinguishing Surface Markers for hESCs and Genes that are preferentially expressed in BM-MSCs are hESC-Derived MSCs for Isolating of Single clustered in biological processes that are involved in meta Cell-Derived MSC Population bolic processes, cell structure, differentiation and signalling while preferentially expressed hESC-MSC genes are clus The genome-wide gene expression is queried for highly tered in those processes involved in proliferation, differentia expressed genes in either hESC-MSC or hESC that encode tion, immunity and signal transduction (FIG. 3C). The over for membrane proteins to facilitate the isolation of MSCs representation of genes in biological processes associated from differentiating hESCs. From a list of top 20 highly with proliferation is consistent with the higher proliferative expressed genes encoding for putative membrane proteins in capacity of hESC-MSC over BM-MSC. either hESC-MSCs or hESCs, candidate genes are selected Although highly expressed genes in either hESC-MSC or 25 for which antibodies against their gene product is commer BM-MSC are over-represented in the general categories of cially available (Table E1A and E1 B below). Among those differentiation and signalling, the specific biological pro candidate genes that are highly expressed in hESC derived TABLE E1A Highly expressed membrane proteins in hESC-derived MSC over hESC

Fold Symbol change Accession Synonyms ANPEP 715.50 NM 001150.1 CD13; LAP1; PEPN: gp150 ENG 479. SO NM 000118.1 END, ORW: HHT1: ORW1; CD105 SCN9A 251.20 NM OO2977.1 PN1; NE-NA TRPV2 187.90 NM 016113.3 VRL: VRL1; VRL-1; MGC12549 RAMP1 182.30 NM OO5855.1 F2RL2 1S2O3 NM 004101.2 PAR3 NTSR1 141.15 NM OO2531.1 NTR GABRA2 122.OS NM 000807.1 SLC16A4 106.60 NM 004696.1 MCT4 TGA4 103.60 NM OOO885.2 NCAM2 93.31 NM 004540.2 NCAM21; MGC51008 L1R1 86.8O NM OOO877.2 P80; IL1R; IL1RA, CD121A: D2S1473; IL-1R alpha PDGFRA 80.25 NM OO62O6.2 CD140A; PDGFR2 WCAM1 71.30 NM 080682.1 INCAM-1OO SSFA2 69.74 NM OO6751.3 CS1; CS-1; KRAP: SPAG13; KIAA1927 TRHDE 58.63 NM 013381.1 PAP-II EDG2 55.82 NM OO1401.3 LPA1; edg-2; Vzg-1; Gpcr26; Mrec1.3; rec.1.3 NTSE 48.15 NM OO2526.1 eN; NTS; NTE: eNT, CD73; E5NT FLRT2 46.51 NM 013231.2 KIAA0405 FAP 44.43 NM 00446.0.2 FAPA: DPPIV: SEPRASE

TABLE E1B Highly expressed membrane proteins in hESC overhESC-derived MSC

Fold Symbol change Accession Synonyms 2642.33 NM O14446.1 PTPRZ1 2126.50 NM 002851.1 CNTN1 430.00 NM 175038.1 PCDH1 342.08 NM OO2587.3 PODXL 303.06 NM OO5397.2 GPR64 217.67 NM O05756.1 US 9,029, 146 B2 41 42 TABLE E 1 B-continued Highly expressed membrane proteins in hESC overhESC-derived MSC

Fold Symbol change Accession Synonyms PROM1 209.04 NM OO6017.1 AC133: CD133; PROML.1 GPRC5C 205.00 NM 022036.2 RAIG3: RAIG-3 CD24 166.98 NM 01323.0.1 CD24A CLDN3 166.42 N.M OO1306.2 RVP1; HRVP1; CPE-R2; CPETR2 TACSTD1 163.81 NM 002354.1 EGP; KSA; M4S1; MK 1: EGP40; MIC18; TROP1; Ep-CAM; hEGP 2: CO17-1A; GA733-2 HTR3A 140.00 NM OOO869.1 HTR3 FGFR4 139.96 NM 022963.1 TKF; JTK2 ADCY1 127.44 NM 021116.1 FGFR3 123.27 NM 022965.1 ACH; CEK2: JTK4; HSFGFR3EX IL17RB 91.70 NM 018725.2 CRL4; EVI27: IL17BR: IL17RH1, MGC5245 SORL1 66.79 NM 003105.3 LR11; LRP9; SORLA; gp250; SorLA-1 GPM6B 60.70 NM OO5278.2 M6B KCNS3 35.26 NM 002252.3 KV9.3; MGC9481

Tables E1A and E1B above show highly expressed surface formed on randomly selected Q4.3 culture. Q4.3 has a normal antigen encoding genes in hESC-derived MSCs (Table E1A) karyotype with a chromosome 9 inversion that originated or their parental hESCs (Table E1B). Based on gene expres- as from its parental HuES9 hESC line (FIG. 5E). Together sion analysis by microarray hybridization, the top twenty these observations show that highly homogenous MSC cul genes that were highly expressed in hESC-derived MSCs tures can be generated by sorting for CD105+ and CD24 (HuES9.E.1, HuES9.E3 and H1.E2) vs. hESCs (HuES9, H1 cells from trypsinized hESC culture after propagation in and Hes3 hESCs) (Table E1A) and vice versa (Table E1B). MSCs are ENG (CD105), ITGA4 (CD49d), PDGFRA, media supplemented with bFGF2 and optionally PDGF BB NT5E (CD73) that are characteristic surface markers of 30 for one week. MSCs derived from adult tissues '' and among those can The incorporation of positive and negative selectable didate genes that are highly expressed in hESC are previously markers into the derivation protocol resulted in the derivation identified as highly expressed hESC-specific genes, of five monoclonal isolates with a genome-wide expression profile that is almost identical to each other and confirmed the ITGB1BP3 and PODXL and CD 24 whose expression has specificity of the selection or sorting criteria. Global pairwise not been associated with hESCs. We confirmed that CD24 is gene expression comparison between the five isolates reveal highly expressed in hESC vs. hESC-MSC (FIG. 5A). a near identical gene expression profile that is comparable to that observed for technical replicates using the same RNA Example 6 samples. Deriving a Homogenous hESC-MSC Population 40 Examples 7 to 15 describe experiments to analyse the proteome of human ESC-derived MSCs (hESC-MSCs). In We next tested the utility of these markers to enhance the these experiments, a chemically defined serum-free culture homogeneity of hESC-MSCs. One week after trypsinization media is conditioned by human ESC-derived MSCs (hESC and culture in media Supplemented with serum replacement MSCs) is analysed using a clinically compliant protocol. The media, FGF2 and optionally PDGFAB, the cells in the culture conditioned media is analyzed by multidimensional protein is sorted by FACS for CD105 and against CD24. identification technology (MuldPIT) and cytokine antibody CD105+ and CD24-cells constituted ~5% of the culture array analysis, and reveals the presence of 201 unique gene (FIG.5B). Sorted cells are plated onto 10x96 well plates at 1 products. cell/well, 1x24 well plate at 100 cells/well and 3x6 well 86-88% of these gene products have detectable transcript plates at 1000 cells/well. Of these, only five of the eighteen 50 levels by microarray or qRT-PCR assays. Computational 1000 cells/wells generated MSC-like cultures suggesting that analysis predicts that these gene products will significantly these cultures are likely to be generated from a single cell. drive three major groups of biological processes: metabolism, defense response, and tissue differentiation including vascu Genome-wide gene expression profiling of these five cul larization, hematopoiesis and skeletal development. It also tures, Q4.1 to Q4.5 using the Illumina BeadArray containing predicts that the 201 gene products activate important signal about 24,000 unique features revealed a high degree of simi 55 ling pathways in cardiovascular biology, bone development larity among the five cultures with four of the lines having a and hematopoiesis such as Jak-STAT, MAPK, Toll-like correlation coefficient of 0.96 and the remaining one with receptor, TGF-beta signalling and mTOR signaling path 0.90 (FIG. 5C). In our hands, the correlation coefficient ways. between technical replicates performed at least one month apart using the same RNA sample is routinely in the range of 60 Example 7 0.97 to 0.98. Q4.1 to Q4.5 are also highly similar to the hESC-MSCs consisting of huES9.E.1, H1.E2 and huES9.E3, Analysis of Proteome of Human ESC-Derived MSCs and BM-MSCs with a correlation coefficient of 0.87 and 0.81, (hESC-MSCs): Materials and Methods respectively (FIG.5D). In contrast, the correlation coefficient of Q4.1 to Q4.5 to 65 Preparation of Conditioned Media their parental HuES9.E1 hESC line is a low 0.55 (FIG. 5D). HuES9.E1 cells are cultured as described above. 80% con Chromosomal analysis using G banding and SKY is per fluent HuES9.E1 cell cultures are washed 3 times with PBS, US 9,029, 146 B2 43 44 cultured overnight in a chemically defined media consisting Da for peptide precursor and fragmentions respectively. Pro of DMEM media without phenol red (Catalog 31053: Invit tein identifications are accepted as true positive if two differ rogen-Gibco, Grand Island, N.Y.) and supplemented with ITS ent peptides are found with score 50 or above. Since many (Invitrogen-Gibco, Grand Island, N.Y.), 5 ng/ml FGF2 (In growth factors and cytokines are Small proteins/peptides and vitrogen-Gibco, Grand Island, N.Y.), 5 ng/ml PDGF AB secreted in small amount, the corresponding MS/MS spectra (Peprotech, Rocky Hill, N.J.) glutamine-penicillin-strepto will be weak and only one peptide per protein will be identi mycin and B-mercaptoethanol. The cultures are then rinsed fied. For those peptides with Mascot score of between 20 and three times with PBS and then replaced with fresh defined 50, manual validation of the MS/MS spectra is performed. media. After three days, the media are collected, centrifuged Bioinformatics at 500xg and the supernatant is 0.2L filtered. For LCMS/MS 10 The validated proteins are collated by removing the back analysis, the conditioned media is placed in dialysis cassettes ground proteins identified in the non-conditioned medium. with MW cutoff of 3500 (Pierce Biotechnology, Rockford, The IPI identifier of each protein is then converted to gene Ill.), dialyzed against 3 changes of 10 vol. 0.9% NaCl, then symbol by using the protein cross-reference table. Gene prod concentrated 20 times using Slide-A-Lyzer Concentrating ucts are classified into the different biological processes or Solution, then dialysing against 10 changes of 100 vol. 0.9% 15 pathways of the GO classification system Gene Ontology NaCl before filtering with a 0.2L filter. A same volume of (GO) classification on GeneSpring GX7.3 Expression Analy non-conditioned media is dialyzed and concentrated in par sis (Agilent Technologies, Palo Alto, Calif.). and then com allel with the conditioned media. paring the frequency of genes in each process or pathway to Instead of concentrating the medium by dialysis, the that in the Genbank human genome database, those processes medium may be concentrated using membrane ultrafiltration of pathways with significantly higher gene frequency with a nominal molecular weight limit (NMWL) of 3K. The (p<0.05) are assumed to be significantly modulated by the resulting concentrated medium retains most of the proteins Secretion of MSC. and biological activities observed in the medium concen qRT-PCR trated by dialysis. Total RNA is extracted from HuBS9.E1 cells with Trizol Cytokine Antibody Blot Assays 25 Reagent (Gibco-BRL) and purified over a spin column (Nu One ml of conditioned or non-conditioned media is cleospin RNA II System, Macherey-Nagel GmbH & Co., assayed for the presence of cytokines and other proteins using Diren, Germany) according to the manufacturers’ protocol. 1 RayBio R. Cytokine Antibody Arrays according to manufac ug total RNA is converted to cDNA with random primers in a turer's instruction (RayBio Norcross, Ga.). 50 ul reaction volume using a High Capacity cDNA Archive LCMS/MS Analysis 30 Kit (Applied Biosystems, Foster City, Calif., USA). The Proteins in two ml of dialyzed conditioned (CM) or non cDNA is diluted with distilled water to a volume of 100 ul. 1 conditioned media (NCM) are reduced, alkylated, and tryptic ul is used for each primer set in a pathway-specific RT digested as described Washburn, 2001 #2997. The samples ProfilerPCR Arrays (SuperArray, Frederick, USA) according are then desalted by passing the digested mixture through a to the manufacturer's protocol. The plates used for the analy conditioned Sep-Pak C-18 SPE cartridge (Waters, Milford, 35 sis are: Chemokines & Receptors PCR Array (cat. no. APH Mass., USA), washed twice with a 3% acetonitrile (ACN) (JT 022), NFkB Signaling Pathway PCR Array (cat. no. APH Baker, Phillipsburg, N.J.) and 0.1% formic acid (FA) buffer, 025), Inflammatory Cytokines & Receptors PCR Array (cat. and eluted with a 70% ACN and 0.1% FA buffer. The eluted no. APH-011), Common Cytokine PCR Array (cat. no. APH samples are then dehydrated by speedvac to about 10% of 021), JAK/STAT Signaling Pathway PCR Array (cat. no. their initial volumes and adjusted to 50 ul with 0.1% formic 40 APH-039). acid. The samples are kept at 4° C. prior to LC-MS/MS analysis. Example 8 The desalted peptide mixture is analyzed by MudPIT with a LC-MS/MS system (LTO, ThermoFinnign, San Jose, Calif., Analysis of Proteome of Human ESC-Derived MSCs USA). The sample is loaded into a strong cation exchange 45 (hESC-MSCs): Preparation of Conditioned Media (SCX) column (Biobasic SCX, 5um, Thermo Electron, San (CM) and Non-Conditioned Media (NCM) Jose, USA) and fractioned by 6 salt steps with 50 ul of buffers (0.2, 5, 10, 100, and 1000 mMofammonium chloride in a 5% To ensure that there is minimal contamination of condi ACN and 0.1% FA) in first dimension. The peptides eluted tioned media by media Supplements such as serum replace from SCX column are concentrated and desalted in a Zorbax 50 ment media, HuES9.E1 MSCs are grown to about 80% con peptide trap (Agilent, Pola Alto, Calif., USA). The second fluency, washed three times with PBS, incubated overnight in dimensional chromatographic separation is carried out with a a chemically defined media consisting of DMEM supple home-packed nanobored C18 column (75 um i.dx10 cm, 5 mented with ITS (insulin, transferrin and selenoprotein), 5 um particles) directly into a pico-frit nanospray tip (New ng/ml FGF2, 5 ng/ml PDGFAB, glutamine-penicillin-strep Objective, Wubrun, Mass., USA), operating at a flow rate of 55 tomycin and B-mercaptoethanol. HuES9.E1 MSCs can be 200 mL/min with a 120 min gradient. propagated in this minimal media for at least a week. The next The LTQ is operated in a data-dependent mode by perform day, the cell culture is againwashed three times with PBS, and ing MS/MS scans for the 3 of the most intense peaks from incubated with the fresh defined media. each MS Scan. For each experiment, ms/ms (dta) spectra of The media is collected after three days of conditioning. The the 6 salt steps are combined into a single mascot generic file 60 conditioned media (CM) is always analyzed or processed in by a home-written program. Protein identification is achieved paralled with an equivalent Volume of non-conditioned media by searching the combined data against the IPI human protein (NCM). For LCMS/MS analysis, the media is concentrated database via an in-house Mascot server. The search param ~10x before extensive dialysis against 0.9% saline as eters are: a maximum of 2 missed cleavages using trypsin; described in the materials and methods. The average protein fixed modification is carbaminomethylation and variable 65 concentration of concentrated CM and NCM are 98.017.9 modifications are oxidation of methionine and protein N-ter ug/ml and 41.6-1.2 ug/ml (n-3), respectively. The condition minal acetylation. The mass tolerances are set to 2.0 and 0.8 ing of media by MSCs is monitored by running aliquots of the US 9,029, 146 B2 45 46 media on protein gels. Protein composition of the media of Collagen alpha-3(VI) chain precursor; 57. IPIO0072917 increased in complexity with time (FIG. 6). The CM had a alpha 3 type VI collagen isoform 3 precursor; 58. more complex protein composition than NCM. IPIO0021828 Cystatin B: 59. IPI00007778 Di-N-acetylchito biase precursor; 60. IPI00295741 Cathepsin B precursor: Example 9 61. IPI002992.19 Protein CYR61 precursor; 62. IPIO0514900 42 kDa protein; 63. IPI00333770 Similar to Analysis of Proteome of Human ESC-Derived MSCs Dedicator of cytokinesis protein 10; 64. IPI00478332 Similar (hESC-MSCs): Analysis of MSC Conditioned Media to Dedicator of cytokinesis protein 9: 65. IPIO0000875 Elon by LCMS/MS and Antibody Array gation factor 1-gamma; 66. IPIOO465248 Alpha-enolase; 67. 10 IPIO0013769 Alpha-enolase, lung specific; 68. IPIO0216171 LCMS/MS analysis identified 250 proteins that are present Gamma-enolase; 69. IPI00218803 Splice Isoform B of Fibu in two independently prepared batches of CM, but not in a lin-1 precursor: 70. IPIO0296537 Splice Isoform C of Fibu similarly processed NCM. These are: lin-1 precursor; 71. IPI00328113 Fibrillin-1 precursor; 72. 1. IPIO0021428 Actin, alpha skeletal muscle; 2. IPIO0019439 fibrillin 2 precursor; 73. IPI00385645 Splice IPIO0414057 Actin alpha 1 skeletal muscle protein; 3. 15 Isoform 2 of Fibroblast growth factor 17 precursor; 74. IPIO0008603 Actin, aortic smooth muscle; 4. IPI00021439 IPIO0216602 Splice Isoform 5 of Fibroblast growth factor Actin, cytoplasmic 1: 5. IPIO0023006 Actin, alpha cardiac; 6. receptor 2 precursor; 75. IPIO0216604 Splice Isoform 8 of IPIO0021440 Actin, cytoplasmic 2: 7. IPI00025416 Actin, Fibroblast growth factor receptor 2 precursor; 76. gamma-enteric smooth muscle; 8. IPIOO479925 agrin: 9. IPIO0034099 Hypothetical protein FLJ21918; 77. IPIO0015102 CD166 antigen precursor; 10. IPI00007423 IPIO0333541 Filamin-A: 78. IPI00302592 FilaminA, alpha; Acidic leucine-rich nuclear phosphoprotein 32 family mem 79. IPI00339227 Hypothetical protein DKFZp686O1166; ber B: 11. IPI0041333136 kDa protein; 12. IPI0041257734 80. IPI00414283 Fibronectin precursor (FN) (Cold-insoluble kDa protein: 13. IPIOO413506 33 kDa protein; 14. globulin) (CIG). Splice isoform 3: 81. IPI00339225 Splice IPIO0418169 Hypothetical protein DKFZp686P03159; 15. Isoform 5 of Fibronectin precursor; 82. IPI00339319 Splice IPIO0003815 Rho GDP-dissociation inhibitor 1; 16. 25 Isoform 11 of Fibronectin precursor; 83. IPI00556632 Splice IPIO0004656 Beta-2-microglobulin precursor; 17. Isoform 12 of Fibronectin precursor; 84. IPIO041 1462 Hypo IPIO0218042 Splice Isoform BMP1-5 of Bone morphoge thetical protein DKFZp686B18150; 85. IPI00029723 Fol netic protein 1 precursor; 18. IPI00009054 Splice Isoform listatin-related protein 1 precursor; 86. IPIO0005401 BMP1-3 of Bone morphogenetic protein 1 precursor; 19. Polypeptide N-acetylgalactosaminyltransferase 5: 87. IPIO0014021 Splice Isoform BMP1-1 of Bone morphoge 30 IPIO0219025 Glutaredoxin-1; 88. IPI00171411 Golgi phos netic protein 1 precursor; 20. IPI00218040 Splice Isoform phoprotein 2: 89. IPI00026314 Gelsolin precursor: BMP1-4 of Bone morphogenetic protein 1 precursor; 21. 90. IPI002 19757 Glutathione S-transferase P: 91. IPIO0006980 Protein C14orf166; 22. IPI00296.165 Comple IPIO0027569 Heterogeneous nuclear ribonucleoprotein ment C1r subcomponent precursor; 23. IPI00152540 C-like 1; 92. IPI00003881 HNRPF protein;93. IPI00442294 OTTHUMP00000016748; 24. IPI00305064 Splice Isoform 35 Splice Isoform 1 of Neurotrimin precursor; 94. IPI00003865 CD44 of CD44 antigen precursor; 25. IPIO0297160 Hypo Splice Isoform 1 of Heat shock cognate 71 kDa protein; 95. thetical protein DKFZp451K1918; 26. IPI00293539 Splice IPIO0037070 Splice Isoform 2 of Heat shock cognate 71 kDa Isoform 2 of Cadherin-11 precursor; 27. IPIO0304227 Splice protein: 96. IPIO0220362 10 kDa heat shock protein, mito Isoform 1 of Cadherin-11 precursor; 28. IPIO0386476 Cad chondrial: 97. IPIO0024284 Basement membrane-specific herin 11, type 2, isoform 1 preproprotein; 29. IPI00024046 40 heparan Sulfate proteoglycan core protein precursor; 98. Cadherin-13 precursor: IPIO0297284 Insulin-like growth factor binding protein 2 30. IPIO0290085 Neural-cadherin precursor; 31. precursor; 99. IPI00297284 Insulin-like growth factor bind IPIO0029739 Splice Isoform 1 of Complement factor H pre ing protein 2 precursor; 100. IPI00029236 Insulin-like cursor; 32. IPIO0012011 Cofilin, non-muscle isoform; 33. growth factor binding protein 5 precursor; 101. IPIO0029236 IPIO0007257 callsyntenin 1 isoform 2: 34. IPI00218539 45 Insulin-like growth factor binding protein 5 precursor; 102. Splice Isoform B of Collagen alpha-1(XI) chain precursor: IPIO0029235 Insulin-like growth factor binding protein 6 35. IPI00477350 Collagen, type XI, alpha 1:36. IPI00329573 precursor; 103. IPI00029235 Insulin-like growth factor bind Splice Isoform Long of Collagen alpha-1(XII) chain precur ing protein 6 precursor; 104. IPI00016915 Insulin-like sor;37. IPI00221384 Splice Isoform Short of Collagen alpha growth factor binding protein 7 precursor; 105. IPIO0016915 1(XII) chain precursor; 38. IPIO0400935 Collagen alpha-1 50 Insulin-like growth factor binding protein 7 precursor; 106. (XVI) chain precursor; 39. IPI00297646 Collagen alpha-1 (I) IPIO0328.163 K-ALPHA-1 protein; 107. IPI00021396 Vascu chain precursor; 40. IPIO0164755 Prepro-alpha2(I) collagen lar endothelial growth factor receptor 2 precursor; 108. precursor; 41. IPI00304962 Collagen alpha-2(I) chain pre IPIO0298281 Laminin gamma-1 chain precursor; 109. cursor; 42. IPI00021033 Collagen alpha-1 (III) chain precur IPIO0219219 Galectin-1; 110. IPIO0023673 Galectin-3 bind sor; 43. IPI00167087 COL3A1 protein; 44. IPI00021034 55 ing protein precursor; 111. IPI00021405 Splice Isoform A of Collagen alpha-1 (IV) chain precursor; 45. IPIOO479324 Lamin-A/C: 112. IPIO0216953 Splice Isoform ADelta10 of alpha 2 type IV collagen preproprotein; 46. IPIO0306322 Lamin-A/C: 113. IPIOO180173 PREDICTED: similar to tro Collagen alpha-2(IV) chain precursor; 47. IPI00303313 Col pomyosin 4; 114. IPI004.01614 PREDICTED: similar to lagen alpha-1 (V) chain precursor; 48. IPI00477611 184 kDa FKSG30; 115. IPI00374397 PREDICTED: similar to tro protein;49. IPI00293881 COL5A2 protein; 50. IPIO0018279 60 pomyosin 4: 116. IPI00374732 PREDICTED: similar to Collagen alpha-3(V) chain precursor; 51. IPI00291 136 Col PPIA protein: 117. IPI00402104 PREDICTED: similar to lagen alpha-1 (VI) chain precursor; 52. IPIO0304840 Splice peptidylprolyl isomerase A isoform 1: cyclophilin A; pepti Isoform 2C2 of Collagen alpha-2(VI) chain precursor; 53. dyl-pro: 118. IPI00455415 PREDICTED: similar to Hetero IPIO0220613 Splice Isoform 2C2A of Collagen alpha-2(VI) geneous nuclear ribonucleoprotein C-like d845O24.4; 119. chain precursor; 54. IPI00022200 alpha 3 type VI collagen 65 IPIO0454722 PREDICTED: similar to Phosphatidylethano isoform 1 precursor; 55. IPI00072918 alpha 3 type VI col lamine-binding protein; 120. IPI00454852 PREDICTED: lagen isoform 4 precursor; 56. IPIO0220701 Splice Isoform 2 similar to Teratocarcinoma-derived growth factor 1; US 9,029, 146 B2 47 48 121. IPIO0002802 Protein-lysine 6-oxidase precursor: alpha 1 precursor; 197. IPI00654685 Similar to SPARC pre 122. IPI004 10152 latent transforming growth factor beta cursor; 198. IPI00641961 Collagen, type XII, alpha 1: 199. binding protein 1 isoform LTBP-1L: 123. IPI00220249 IPIO064.5849 Extracellular matrix protein 1: 200. Latent transforming growth factor beta-binding protein, iso IPIO0554786 Thioredoxin reductase 1: 201. IPIOO645.018 form 1L precursor; 124. IPIO0220249 Latent transforming Plasminogen activator, urokinase; 202. IPIO0552339 Tissue growth factor beta-binding protein, isoform 1L precursor"; inhibitor of metalloproteinase 1: 203. IPI0064.2997 Actin, 125. IPI004 10152 latent transforming growth factor beta cytoplasmic 2: 204. IPI007 19778 Similar to Annexin A2; 205. IPI00647915 Transgelin 2: 206. IPI00552815 Collagen, binding protein 1 isoform LTBP-1L: 126. IPI00020986 type V, alpha 1: 207. IPIO0552981 CDNA PSEC0266 fis, Lumican precursor; 127. IPIO0291006 Malate dehydroge clone NT2RP3003649, highly similar to Homo sapiens fibu nase, mitochondrial precursor; 128. IPIO0005707 Macroph 10 lin-1D mRNA; 208. IPI00180776 29 kDa protein: 209. age mannose receptor 2 precursor; 129. IPIO0020501 Myo IPIO0552416 FilaminA, alpha; sin-11: 130. IPI00019502 Myosin-9; 131. IPI00604620 210. IPIO064.0698 Actin, gamma-enteric smooth muscle; Nucleolin; 132. IPI00220740 Splice Isoform 2 of Nucleophosmin: 133. IPI00219446 Phosphatidylethanola 211. IPIO0514530 Actin, alpha 1, skeletal muscle; 212. mine-binding protein: 134. IPI00299738 Procollagen C-en IPIO0556442 Insulin-like growth factorbinding protein 2 dopeptidase enhancer 1 precursor; 135. IPI00015902 Beta 15 variant (Fragment): 213. IPI00513782 Gelsolin; 214. platelet-derived growth factor receptor precursor; 136. IPIOO478731 29 kDa protein; 215. IPI00396479 24 kDa pro IPIO0216691 Profilin-1; 137. IPI00169383 Phosphoglycer tein: 216. IPI00334627 39 kDa protein; 217. IPI00555762 ate kinase 1: 138. IPI00219568 Phosphoglycerate kinase, PTK7 protein tyrosine kinase 7 isoform a variant (Fragment); testis specific; 139. IPIO0296.180 Urokinase-type plasmino 218. IPI0065820297 kDa protein; 219. IPIO0006273 CYR61 gen activator precursor; 140. IPI00215943 Splice Isoform 3 protein; 220. IPI00719405 TMSL6 protein: 221. of Plectin 1: 141. IPI00215942 Splice Isoform 2 of Plectin 1: IPIO0658096 Thymosin beta-4: 222. IPI003761635 kDa pro 142. IPIO0014898 Splice Isoform 1 of Plectin 1: 143. tein; 223. IPIO0556217 Fibrillin 1 variant (Fragment): 224. IPIO0398777 plectin1 isoform 8: 144. IPI00398776 plectin 1 IPIO0514817. Similar to Lamin A/C; 225. IPIO0644087 Pro isoform 7: 145. IPIO0186711 plectin 1 isoform 6: 146. gerin; 226. IPI00655812 Rhabdomyosarcoma antigen MU IPIO0420096 plectin1 isoform 3: 147. IPI00398779 plectin 1 isoform 11: 148. IPIO0398778 plectin 1 isoform 10: 149. 25 RMS-40.12: 227. IPIO0604517 Similar to Nucleolin; 228. IPIO0398.002 plectin 1 isoform 1: 150. IPI00419585 Pepti IPIOO444262 CDNA FLJ45706 fis, clone FEBRA2028457, dyl-prolyl cis-trans isomerase A: highly similar to Nucleolin; 229. IPI00412473 Protein: 230. 151. IPIO0472718 peptidylprolyl isomerase A isoform 2: IPIOO414489 Protein: 231. IPIOO411463 Protein; 232. 152. IPIO0000874 Peroxiredoxin-1: 153. IPIO0024.915 Per IPIO0556415 Transgelin variant (Fragment): 233. oxiredoxin-5, mitochondrial precursor; 154. IPIO0375306 30 IPIO0718825 Calmodulin; 234. IPI0047815617 kDa protein; peroxiredoxin 5 precursor, isoform b: 155. IPI000 12503 235. IPI00386621 CALM3 protein: 236. IPIO0647001 Splice Isoform Sap-mu-0 of Proactivator polypeptide precur Acidic;237. IPIO0642650 Similar to Stanniocalcin 2 precur sor; 156. IPI00374179 proteasome activator subunit 1 iso sor; 238. IPIO0641471 Collagen-like protein: 239. form 2: 157. IPI 100030154 Proteasome activator complex IPIO0514669 SH3 domain binding glutamic acid-rich protein subunit 1; 158. IPIO0168812 PTK7 protein tyrosine kinase 7 35 like 3: 240. IPIO0719422 Triosephosphate isomerase (Frag isoform d precursor; 159. IPIO04.19941 PTK7 protein tyrosine kinase 7 isoform a precursor; 160. IPI00003590 ment); 241. IPIO0003734 Putative S100 calcium-binding pro Quiescin Q6, isoform a: 161. IPI00015916 Bone-derived tein H NH0456N16.1; 242. IPI00029574 11 kDa protein; growth factor (Fragment); 162. IPI00015916 Bone-derived 243. IPIO0641047 Gelsolin: 244. IPIO0647556 Gelsolin; 245. growth factor; 163. IPI00298289 Splice Isoform 2 of Reticu IPIO0654821 hypothetical protein L0054845 isoform 1: 246. lon-4; 164. IPI00021766 Splice Isoform 1 of Reticulon-4: 40 IPIO0647572 Dickkopf related protein-3 precursor; 247. 165. IPIO0013895 Calgizzarin; 166. IPI00010402 Hypotheti IPIO0639879 Similar to Cytokinesis protein sepa; 248. cal protein; 167. IPI002 18733 Superoxide dismutase; 168. IPIO0657746 Similar to Dedicator of cytokinesis protein 8: IPIO001.4572 SPARC precursor; 169. IPI00005614 Splice 249. IPI00555993 Vascular endothelial growth factor recep Isoform Long of Spectrin beta chain, brain 1: 170. tor 3 variant: 250. IPIO0552545 Dedicator of cytokinesis IPIO0008780 Stanniocalcin-2 precursor; 171. IPI003.01288 45 protein 8. SEL-OB protein; 172. IPI00216138 Transgelin; 173. Together, these 250 proteins are encoded by 132 unique IPIO0018219 Transforming growth factor-beta-induced pro tein ig-h3 precursor; 174. IPI00304865 transforming growth known genes: ACTA1: COL5A2; HSPA8; PSAP; ACTA2; factor, beta receptor III"; 175. IPIO0296099 Thrombospon COL5A3; HSPE1; PSME1; ACTB; COL6A1; HSPG2: din-1 precursor; 176. IPIO0032292 Metalloproteinase inhibi PTK7; ACTC: COL6A2; IGFBP2: QSCN6; ACTG1; tor 1 precursor; 177. IPI00027166 Metalloproteinase inhibi 50 COL6A3; IGFBP5; RTN4; ACTG2: CSTB; IGFBP6; tor 2 precursor; 178. IPI00220828 Thymosin beta-4; 179. S100A11; AGRN, CTBS; IGFBP7; SH3BGRL3: ALCAM; IPIO0180240 thymosin-like 3: CTSB; K-ALPHA-1; SOD1: ANP32B: CYR61; KDR; 180. IPI00299633 OTTHUMP00000031270 (Fragment); SPARC: ANXA2: DOCK10; LAMC1; SPTBN1; ARHG 181. IPI00465028 Triosephosphate isomerase 1 variant DIA: DOCK8; LGALS1; STC2: B2M; ECM1; LGALS3BP: (Fragment); 182. IPI00451401 Splice Isoform 2 of Triose 55 SVEP1, BMP1; EEF1G: LMNA; TAGLN; C14orf166; phosphate isomerase: 183. IPI00010779 Tropomyosin 4; ENO1; LOX; TAGLN2; C1R: ENO1B: LTBP1: TGFBI; 184. IPI00216975 Splice Isoform 2 of Tropomyosin alpha-4 CALM1; ENO2: LUM: TGFBR3: CD109; FBLN1; MDH2: chain; 185. IPIO0180675 Tubulin alpha-3 chain; 186. THBS1: CD44; FBN1; MRC2: TIMP1; CDH11; FBN2: IPIO0218343 Tubulin alpha-6 chain; 187. IPI00216298 Thioredoxin; 188. IPIOO472175 CDNA FLJ46672 fis, clone MYH11: TIMP2. CDH13; FGF17; MYH9; TMSB4X; TRACH3009008, highly similar to Thioredoxin reductase: 60 CDH2; FGFR2: NCL; TMSL3; CFH/HF 1: FLJ21918; 189. IPI00450472 Ubiquitin-conjugating enzyme E2I; 190. NPM1; TMSL6; CFL1; FLNA; PBP: TPI1; CLSTN1; FN1; IPIO0018352 Ubiquitin carboxyl-terminal hydrolase PCOLCE; TPM4: COL11A1: FSTL1, PDGFRB, TUBA3; isozyme L1; 191. IPI00010207 Ubiquitin-fold modifier 1 COL12A1; GALNT5; PFN1; TUBA6: COL16A1 GLRX; precursor; 192. IPI00260630 URB; 193. IPI00021263 14-3-3 PGK1; TXN; COL1A1: GOLPH2: PGK2: TXNRD1; protein zeta/delta; 194. IPIO0642991 Hypothetical protein 65 COL1A2: GSN; PLAU, UBE2I; COL3A1; GSTP1; PLEC1; DKFZp686F10164; 195. IPI00470919 Hypothetical protein UCHL1; COL4A1; HNRPCL1: PPIA; UFM1; COL4A2; DKFZp686K08164; 196. IPI00719088 collagen, type VI, HNRPF; PRDX1; URB: COL5A1; HNT PRDX5; YWHAZ. US 9,029, 146 B2 49 50 There are 32 unknown proteins: IPIO0642991 Hypotheti H NH0456N16.1; IPI00029574 11 kDa protein; cal protein DKFZp686F10164; IPI00470919 Hypothetical IPIO0654821 hypothetical protein L0054845 isoform 1: protein DKFZp686K08164; IPI00654685 Similar to SPARC IPIO0647572 Dickkopf related protein-3 precursor: precursor; IPI007 19778 Similar to Annexin A2; IPIO0552.981 IPIO0639879 Similar to Cytokinesis protein sepa: IPIO0657746 Similar to Dedicator of cytokinesis protein 8: CDNA PSEC0266 fis, clone NT2RP3003649, highly similar IPIO0555993 Vascular endothelial growth factor receptor 3 to Homo sapiens fibulin-1D mRNA; IPIO0180776 29 kDa variant. protein; IPI00478731 29 kDa protein; IPI00396479 24 kDa The MSCs described in this document can therefore be protein; IPI00334627 39 kDa protein; IPI0065820297 kDa used as sources of any or all of these proteins, or any proteins protein; IPI003761635 kDa protein; IPI00514817 Similar to or other molecules which are secreted or expressed by them. Lamin A/C; IPIO0644087 Progerin;- . IPI00655812 Rhab- 10 MSCs have been shown to secrete a broad spectrum of domyosarcoma antigen MU-RMS-40.12; IPIO0604517 cytokines and growth factors that affect cells on their vicin Similar to Nucleolin; IPIOO444262 CDNA FLJ45706 fis, ity. Many of these factors are small molecules that are not clone FEBRA2028457, highly similar to Nucleolin; easily detectable during shot-gun LC MS/MS analysis. IPIO0412473 Protein; IPI00414489 Protein; IPI00411463 Therefore, the CM and NCM are also analyzed by hybridiza Protein; IPI0047815617 kDa protein; IPI00386621 CALMS tion to 5 different antibody arrays that together carried anti protein; IPI00647001 Acidic; IPI00642650 Similar to Stan- bodies against 101 cytokines/growth factors (See also FIG. niocalcin 2 precursor; IPI00641471 Collagen-like protein; 7). IPIO0514669 SH3 domain binding glutamic acid-rich protein Layout of Antibody Arrays like 3: IPIO0003734 Putative S100 calcium-binding protein RayBio R. Angiogenesis Array I

A. B C D E F G H 1. POS POS NEG NEG Angiogenin EGF ENA-78 b FGF 2 POS POS NEG NEG Angiogenin EGF ENA-78 b FGF 3 GRO IFN- IGF-I IL-6 IL-8 LEPTIN MCP-1 PDGF-BB 4 GRO IFN- IGF-I IL-6 IL-8 LEPTIN MCP-1 PDGF-BB 5 PIGF RANTES TGF-D, TIMP-1 TIMP-2 Thrombopoietin VEGF VEGF-D 6 PIGF RANTES TGF-D, TIMP-1 TIMP-2 Thrombopoietin VEGF VEGF-D 7 BLANK BLANK BLANK BLANK BLANK BLANK Neg POS 8 BLANK BLANK BLANK BLANK BLANK BLANK Neg POS

RayBio(R. Human Chemokine Antibody Array I

A. B C D E F G H I J K L 1. POS POS NEG NEG BLC CCL28 CkB8-1 CTACK CXCL16 ENA-78 Eotaxin Eotaxin-2 2 POS POS NEG NEG BLC CCL28 CkB8-1 CTACK CXCL16 ENA-78 Eotaxin Eotaxin-2 3 Eotaxin-3 Fractalkine GCP-2 GRO GROC. HCC-4 I-309 I-TAC IL-8 IP-10 Lymphotactin MCP-1 4 Eotaxin-3 Fractalkine GCP-2 GRO GROC. HCC-4 I-309 I-TAC IL-8 IP-10 Lymphotactin MCP-1 S MCP-2 MCP-3 MCP-4 MDC MIG MIP-1C. MIP-1B MIP-18 MIP-3ol. MIP-3| MPIF-1 NAP2 6 MCP-2 MCP-3 MCP-4 MDC MIG MIP-1C. MIP-1B MIP-18 MIP-3ol. MIP-3| MPIF-1 NAP2 7 PARC RANTES SDF-1 C. SDF-1 B TARC TECK BLANK BLANK BLANK BLAND BLANK POS 8 PARC RANTES SDF-1 C. SDF-1 B TARC TECK BLANK BLANK BLANK BLAND BLANK POS

RayBio(R) Matrix Metalloproteinases Antibody Array I

A. B C D E F G H

1 POS POS NEG NEG MMP-1. MMP-2 MMP-3 MMP-8 2 POS POS NEG NEG MMP-1. MMP-2 MMP-3 MMP-8 3 MMP-9 MMP-10 MMP-13 TIMP-1. TIMP-2 TIMP-3 TIMP-4 POS 4 MMP-9 MMP-10 MMP-13 TIMP-1. TIMP-2 TIMP-3 TIMP-4 POS

RayBio(R. Human Cytokine Antibody Array I

A. B C D E F G H

1 POS POS NEG NEG GCSF GM-CSF GRO GROC. 2 POS POS NEG NEG GCSF GM-CSF GRO GROC. 3 IL-1 C. IL-2 IL-3 IL-5 IL-6 IL-7 IL-8 IL-10 4 IL-1 C. IL-2 IL-3 IL-5 IL-6 IL-7 IL-8 IL-10 5 IL-13 IL-15 IFN-y MCP-1 MCP-2 MCP-3 MIG RANTES 6 IL-13 IL-15 IFN-y MCP-1 MCP-2 MCP-3 MIG RANTES 7 TGF-B1 TNF-ct, TNF-B BLANK BLANK BLANK BLANK POS 8 TGF-B1 TNF-ct, TNF-B BLANK BLANK BLANK BLANK POS

US 9,029, 146 B2 55 56 TABLE E2-continued observed for gene products identified by LCMS/MS and whose gene transcripts are detectable by Illumina BeadArray. Alphabetical list of 201 unique gene products In addition, all 15 that have detectable transcript levels by identified by LC-MS/MS and antibody array. Illumina BeadArray, also have detectable transcript levels by TGFB1 qRT-PCR (Table E3). 21 of 27 (78%) gene products that did TGFB2* not have detectable transcript levels by Illumina BeadArray TGFBI* have transcript level detectable by qRT-PCR (Table E3). TGFBR3 THEBS1 THPO TABLE E3 TIMP1 10 TIMP2 Quantitative RT-PCR assay for the presence of transcripts. 42 of the 72 TIMP3 gene products identified by antibody array are randomly selected for TIMP4 QRT-PCR analysis. TMSB4X TMSL3 Symbol Illumina BeadArray Normalized C, TMSL6 15 1. BDNF >LOD 3.38 TNFs 2. CCL2 >LOD 1.28 TNFRSF11B: 3. CCL7 >LOD 7.35 TPI1 4. CCL8 >LOD 30.28 TPM4 5. CXCL1 >LOD 4.09 TUBA3 6. CXCL12 >LOD 6.83 TUBA6 7. CXCLS >LOD 9.93 TXN 8. IL1A >LOD 6.6 TXNRD1 9. IL1B >LOD 1.44 UBE2I 10 IL6 >LOD 892 UCHL1 11 IL8 >LOD 8.53 UFM1 12 MIF >LOD 6.23 URB 25 13 MMP3 >LOD S.96 VEGFt 14 TGFB2 >LOD S.16 XCL1 15 TNFRSF11B >LOD 2.28 YWHAZ 16 CCL1 35 45 38 CCL16 35 Expression Analysis 39 CXCL13 35 40 IFNG 35 Comparison of the 201 gene products to a genome-wide 41 VEGF 35 gene expression profile of the hESC-MSCs generated by 42 XCL1 35 hybridizing total RNA to a Illumina BeadArray revealed that 12 gene products have transcript levels that are above the limit of detection (LOD) at 99% 134 or 67% of the gene products had gene transcript levels 50 confidence on the Illumina BeadArray, a high throughput genome-wide gene expression assay, that are present at above the limit of detection (LOD) with a The C, value for each gene is normalized against B-actin, 99% confidence (Table E2). While 115 or 88% of the 132 gene products identified by LCMS/MS had detectable tran script levels (Table E2), 27 or 38% of the 72 gene products Example 11 identified by antibody array had detectable transcript levels and 45 or 62% had no detectable transcript level (Table E2). 55 Analysis of Proteome of Human ESC-Derived MSCs Probes for two of the gene products, ENO1B and SVEP1 are (hESC-MSCs): Biological Processes that are not present on the Illumina BeadArray. It is possible that Modulated by the Secreted Proteins transcript levels for most of the 72 gene may be too low in abundance for detection by Illumina BeadArray as mRNAs To investigate if the secreted products have the potential to 60 repair the injured tissues or organs, gene products are first encoding for cytokines/chemokines are known to contain classified according to their biological processes and path AU-rich elements that caused rapid degradation of the mRNA ways according to the Gene Ontology (GO). The frequency of during translation (22.23). unique genes in the secreted MSC proteome associated with More sensitive qRT-PCR assays are therefore performed. each process or pathway is then compared to the gene-fre 42 of the 72 gene products are randomly selected and tested. quency for the respective pathway or process in a database 36 or 86% of the 42 gene products have detectable transcript 65 collated from Unigene, Entrez and GenBank. Significantly levels as defined as having a normalized C, value of <35 higher frequencies of genes (p<0.05) are associated with 58 (Table E3 below). This frequency is similar to 88% frequency biological processes and 30 pathways. US 9,029, 146 B2 57 58 Biological processes classified by GO modulated by the 201 unique gene products:

# genes # genes GO Biological Processes (ref) 96 (ref) (expt) % (expt) p-value 1. GO:42221: response to chemical stimulus 130 O.S3 23 11.56 2.5OE-24 2. GO: 42330: taxis 130 O.S3 23 11.56 2.5OE-24 3. GO: 6935: chemotaxis 130 O.S3 23 11.56 2.5OE-24 4. GO:9605: response to external stimulus 157 O.65 24 12.06 945E-24 5. GO: 50896: response to stimulus 158 O.6SO 24 12.06 111E-23 6. GO: 9628: response to abiotic stimulus 150 O.62 23 11.56 7.83E-23 7. GO:485.13: organ development 116 O.48 11 5.53 3.06E-09 8. GO: 7275: development 827 3.40 23 11.56 3.27E-07 9. GO: 16052: carbohydrate catabolism 51 O.21 6 3.02 3.66E-O6 10. GO: 19320:hexose catabolism 51 O.21 6 3.02 3.66E-O6 11. GO: 44248: cellular catabolism 51 O.21 6 3.02 3.66E-O6 12. GO: 44265: cellular macromolecule catabolism 51 O.21 6 3.02 3.66E-O6 13. GO: 44275: cellular carbohydrate catabolism 51 O.21 6 3.02 3.66E-O6 14. GO: 46164: alcohol catabolism 51 O.21 6 3.02 3.66E-O6 15. GO: 46365: monosaccharide catabolism 51 O.21 6 3.02 3.66E-O6 16. GO: 6007: glucose catabolism 51 O.21 6 3.02 3.66E-O6 17. GO: 6096: glycolysis 51 O.21 6 3.02 3.66E-O6 18. GO:9057: macromolecule catabolism 51 O.21 6 3.02 3.66E-O6 19. GO:9056: catabolism 53 O.22 6 3.02 4.6OE-06 20. GO: 15980: energy derivation by oxidation of organic 65 0.27 6 3.02 1.53E-OS compounds 21. GO: 19318: hexose metabolism 65 0.27 6 3.02 1.53E-OS 22. GO: 44262: cellular carbohydrate metabolism 65 0.27 6 3.02 1.53E-OS 23. GO: 5975: carbohydrate metabolism 65 0.27 6 3.02 1.53E-OS 24. GO: 5996: monosaccharide metabolism 65 0.27 6 3.02 1.53E-OS 25. GO: 6006: glucose metabolism 65 0.27 6 3.02 1.53E-OS 26. GO: 6066: alcohol metabolism 65 0.27 6 3.02 1.53E-OS 27. GO: 6091: generation of precursor metabolites and energ 65 0.27 6 3.02 1.53E-OS 28. GO: 6092: main pathways of carbohydrate metabolism 65 0.27 6 3.02 1.53E-OS 29. GO: 43170: macromolecule metabolism 571 2.35 16 8.04 2.03E-OS 30. GO: 1525: angiogenesis 44 O.18 5 2.5 2.90E-OS 31. GO: 1568: blood vessel development 47 O.19 5 2.5 4.02E-05 32. GO: 1944: vasculature development 47 O.19 5 2.5 4.02E-05 33. GO:48514: blood vessel morphogenesis 47 O.19 5 2.5 4.02E-05 34. GO: 6950: response to stress 10 O.04 3 .5 6.18E-05 35. GO: 9611: response to wounding 10 O.04 3 .5 6.18E-05 36. GO: 1660: fewer 2 O.O1 2 O 6.64E-05 37. GO: 31649: heat generation 2 O.O1 2 O 6.64E-05 38. GO:43207: response to external biotic stimulus 2 O.O1 2 O 6.64E-05 39. GO: 6952: defense response 2 O.O1 2 O 6.64E-05 40. GO: 6954: inflammatory response 2 O.O1 2 O 6.64E-05 41. GO: 6955: immune response 2 O.O1 2 O 6.64E-05 42. GO:9607: response to biotic stimulus 2 O.O1 2 O 6.64E-05 43. GO: 96.13: response to pest, pathogen or parasite 2 O.O1 2 O 6.64E-05 44. GO: 9887: organ morphogenesis 53 O.22 5 2.5 7.23E-OS 45. GO: 1659: thermoregulation 3 O.O1 2 O 198E-04 46. GO: 300.97: hemopoiesis 26 O.11 3 .5 1.22E-O3 47. GO:48534: hemopoietic or lymphoid organ development 26 O.11 3 .5 1.22E-O3 48. GO: 1501: skeletal development 38 O16 3 .5 3.67E-03 49. GO: 1503: ossification 38 O16 3 .5 3.67E-03 50. GO: 31214: biomineral formation 38 O16 3 .5 3.67E-03 51.. GO:46849: bone remodeling 38 O16 3 .5 3.67E-03 52. GO: 6793: phosphorus metabolism 13 O.OS 2 O 4.88E-03 53. GO: 6796: phosphate metabolism 13 O.OS 2 O 4.88E-03 54. GO: 9888: tissue development S4 O.22 3 .5 9.82E-O3 55. GO: 45045: secretory pathway 34 O.14 2 O 3.14E-O2 56. GO: 6887: exocytosis 34 O.14 2 O 3.14E-O2 57. GO: 46903: Secretion 36 O.15 2 O 3.49E-O2 58. GO: 1570: vasculogenesis 5 O.O2 1 O.SO 4.02E-O2

55 -continued GO Pathways p-value GO Pathways p-value 1. Cytokine-cytokine receptor interaction 4.97E-47 2. ECM-receptor interaction 3.21E-17 60 10. Cell Communication 1.17E-17 3. Jak-STAT signaling pathway 1.19E-10 11. Gap junction 0.00077 4. MAPK signaling pathway 1.19E-08 12. Tightjunction O.O459 5. Toll-like receptor signaling pathway 1.34E-OS 13. Focal adhesion 1.54E-21 6. TGF-beta signaling pathway O.OOO292 14. Regulation of actin cytoskeleton 2.65E-13 7. mTOR signaling pathway O.0143 15 . Leukocyte transendothelial migration 3.88E-OS 8. Fe epsilon RI signaling pathway O.OO267 65 16 . Complement and coagulation cascades O.O488 9. Epithelial cell signaling in Helicobacter pylori infection O.O183 17 . Antigen processing and presentation O.O1O2 US 9,029, 146 B2 59 60 -continued MSCs (8). Here we describe the composition of the secreted proteome ofhESC-MSCs through a combination of two tech GO Pathways p-value niques, LC-LC-MS and antibody arrays. 18. Apoptosis O.OO648 Although shot-gun proteomic analysis by LC-LC-MS is a 19. T cell receptor signaling pathway O.OOO866 sensitive technique and has high throughput capability, it is 20. Hematopoietic cell lineage 3.32E-14 difficult to detect small proteins/peptides that include most of 21. Type I diabetes mellitus 4.6OE-06 22. Carbon fixation O.OOO2S7 the cytokines, chemokines and growth factors. This is par 23. Glycolysis or Gluconeogenesis O.OO106 tially mitigated by the use of antibody arrays. The qualitative 24. Stilbene, coumarine and lignin biosynthesis O.OO348 proteomic profile of the MSC secretion using the two tech 25. Phenylalanine metabolism O.OO488 10 26. Phenylalanine, tyrosine and tryptophan biosynthesis O.OOS67 niques is highly reproducible. Proteins identified by LC 27. Methane metabolism O.OO739 MS/MS are present in two independently prepared batches of 28. Reductive carboxylate cycle (CO2 fixation) O.OO739 CM while those identified by antibody array are present in at 29. Inositol metabolism O.O163 30. Citrate cycle (TCA cycle) O.O404 least three of four independently prepared batches of CM. 15 The resulting proteomic profile of secretion by hESC-MSCs included almost all the factors that are previously reportedly The 58 biological processes could be approximated into secreted by adult tissue-derived MSCs (13,16, 19-21) as well three major groups: metabolism, defense response and tissue as many others that have not been described. The robustness differentiation while the 30 pathways could be broadly cat of the proteomic profiling is further substantiated by the egorized into: receptor binding, signal transduction, cell-cell detection of transcripts for 86-88% of gene products in the interaction, cell migration, immune response and metabolism proteomic profile using a high throughput microarray-based (FIG. 8, FIG. 9). The postulated biological processes and gene expression analysis. pathways both suggest that the secreted proteins have a major To evaluate and assess the potential functions of the MSC impact on the cellular metabolism that will modulate energy secretion on a global scale, we utilized the more readily production, breakdown, biosynthesis and secretion of mac 25 available computational tools for gene expression analysis, romolecules, processes essential for the removal of damaged based on gene products rather than on post-translationally tissues and regeneration of new tissues (FIG.8. FIG. 9). modified proteins. Consistent with the predominance of Consistent with the predominant presence of cytokines and cytokines and chemokines in the secretion, computational chemokines in the MSC-conditioned media, the analysis also analysis predicted many processes and pathways that are predicted that the secreted factors could elicit many cellular 30 generally associated with the functions of cytokines and responses that are dependent on external stimuli e.g. chemo chemokines such as chemotaxis, taxis, cellular response to taxis, taxis and many immune responses (FIG. 8). Notably, external stimuli, breakdown, biosynthesis and secretion of the conditioned media could also induce biological processes macromolecules, cytokine-cytokine receptor interactions, that are important in tissue differentiation particularly pro cell-cell communication, and basal metabolism e.g. glucose cesses that promote vascularization, hematopoiesis and bone 35 and amino acid metabolism. The MSCs described here can be development (FIG. 8). used to treat diseases which these functions may have a role In those pathways predicted to be modulated by the 1. secreted proteome, receptor-mediated binding of cytokine Although these processes and pathways are not specific to and ECM pathways are consistent with the predominance of the process of injury, repair and regeneration in any particular cytokines and ECM components in the secreted proteome 40 cell or tissue type, their facilitation of immune cell migration (FIG.9). The main signal transduction pathways that could be to the site of injury, ECM remodelling and increase in the activated by the secreted proteome include Jak-STAT signal cellular metabolism will have reparative effects on most ing pathway, MAPK signaling pathway, Toll-like receptor injured or diseased tissues. signaling pathway, TGF-beta signaling pathway, mTOR sig Aside from these generic pathways associated with cytok naling pathway, Fc epsilon RI signaling pathway and Epithe 45 ines and chemokines, computational analysis also predicted lial cell signaling in Helicobacter pylori infection. The com that the Secreted proteins regulate many processes involved in putational analysis of the secreted proteome also suggested vascularization, hematopoiesis and skeletal development. that MSC secretion could enhance cell-cell interaction, Coincidentally, most reported MSC-mediated tissue repair or migration and immune responses. regeneration are associated with cardiovascular, hematopoi 50 etic and musculoskeletal tissues (3-5,24-27). Pathway analy Example 12 sis further uncovered candidate pathways that may be involved in mediating some of the paracrine effects of MSCs. Analysis of Proteome of Human ESC-Derived MSCs In fact, many of these candidate pathways have already been (hESC-MSCs): Discussion implicated in many aspects of cardiovascular, hematopoietic 55 and musculoskeletal biology. For example, Jak-STAT signal MSCs have been used in pre-clinical and clinical trials to ing is associated with cardioprotection (28), hematopoiesis treat a myriad of diseases (3-5,24-27). However the underly (29.30), and skeletal repair and remodelling (31.32): MAPF ing mechanism has remained imprecisely understood. signaling plays a crucial role in many aspects of cardiovas Although MSCs have to potential to differentiate into numer cular responses (33.34), skeletal repair and remodelling (32. ous cell type e.g. endothelial cells, cardiomyctes, chondro 60 35), and hematopoiesis (36); Toll-like receptor signalling has cytes that can potentially repair or regenerate damaged tis been implicated in the initiation and progression of cardio sues, the therapeutic effects of MSCs cannot be solely vascular pathologies (37), and modulation of innate and adap mediated by generation of MSC-derived reparative cell types tive immunity (38); TGF-beta signalling is critical in correct as differentiation of MSCs is generally too inefficient to heart development cardiac remodeling, progression to heart mediate tissue repair or restore tissue function. It has been 65 failure and vascularization (39-41) hematopoiesis (42), for increasingly proposed that some of the therapeutic effects of mation and remodelling of bone and cartilage (27.43) as well MSCs may be mediated by paracrine factors secreted by as general wound healing (44); and mTOR as an important US 9,029, 146 B2 61 62 regulator of cell growth and proliferation plays a non-specific thermore, any one or more proteins secreted from the MSCs yet critical role in both normal physiology and diseases (45 described here, including in the form of conditioned media, 47). may be used for the same purposes as the MSCs described In conclusion, our analysis of the secreted proteome in herein. hESC-derived MSCs, however, have several advantages hESC-derived MSCs which includes many of the cytokines over adult tissue-derived MSCs. The use ofhESC cell lines as reportedly secreted by adult tissue-derived MSCs suggests a tissue source of MSC constitutes an infinitely renewable and that the Secreted proteome could potentially exert modulating expansible tissue source, and enhances the reproducible and effects on tissue repair and regeneration particularly in the consistent batch to batch preparation of MSCs and therefore cardiovascular, hematopoietic and musculoskeletal tissues, CM in a clinically compliant manner. It also boosts the Scal and therefore provide molecular support for a MSC-mediated 10 ability of preparing CM and the potential of developing low paracrine effect on tissue repair and regeneration in MSC cost off-the-shelf therapeutics. In addition, the development transplantation studies. This secreted proteome also uncov of a serum-free chemically defined medium for the prepara ered many highly testable hypotheses for the molecular tion of hESC-derived MSC CM reduces confounding and mechanisms in MSC-mediated tissue repair and also poten variable contaminants associated with complex media tial "druggable' targets to modulate tissue repair and regen 15 Supplements such as serum or serum replacement media. Therefore, our elucidation of the CM is very relevant for the eration. The significant similarity between hESC-derived translation of MSC-based biologics towards clinical applica MSCs and adult tissue-derived derived MSCs suggest that tions. conditioned media of either MSC cultures are likely to have similar biological activities. Accordingly, this demonstrates that the MSCs derived by our methods have significant biological similarities to their Example 13 bone marrow derived counterparts. These findings demon Strate the resemblance of the hESC-MSCs to adult BM-de Listing of 201 Genes in Each of the 58 Biological rived MSCs in their ability to secrete paracrine factors. Fur Processes

Alcohol Metabolism 16507966-SENO2 enolase 2 (gamma, neuronal) 16507965-S ENO1 enolase 1, (alpha) 22095338-SPGK1 phosphoglycerate kinase 1 31543396-SPGK2 phosphoglycerate kinase 2 21735620-S MDH2 malate dehydrogenase 2, NAD (mitochondrial) 26024330-STPI1 triosephosphate isomerase 1 Angiogenesis 11321596-S KDR kinase insert domain receptor (a type III receptor tyrosine kinase) 30172563-S VEGF vascular endothelial growth factor 2861.0153-SIL8 interleukin 8 42716312-SANG angiogenin, ribonuclease, RNase A family, 5 10337586-S FGF6 fibroblast growth factor 6 Biomineral Formation 27262662-A CDH 11 cadherin 11, type 2, OB-cadherin (osteoblast) 5902810-A BMP1 bone morphogenetic protein 1 4507170-SS PARC Secreted protein, acidic, cysteine-rich (osteonectin) Blood Vessel Devolopment

11321596-SKDR kinase insert domain receptor (a type III receptor tyrosine kinase) 30172563-S VEGF vascular endothelial growth factor 2861.0153-SIL8 interleukin 8 42716312-SANG angiogenin, ribonuclease, RNase A family, 5 10337586-S FGF6 fibroblast growth factor 6 Blood Vessel Morphogenesis

11321596-SKDR kinase insert domain receptor (a type III receptor tyrosine kinase) 30172563-S VEGF vascular endothelial growth factor 2861.0153-SIL8 interleukin 8 42716312-SANG angiogenin, ribonuclease, RNase A family, 5 10337586-S FGF6 fibroblast growth factor 6 Bone Remodeling 27262662-A CDH 11 cadherin 11, type 2, OB-cadherin (osteoblast) 5902810-A BMP1 bone morphogenetic protein 1 MP1 : 4507170-S SPARC Secreted protein, acidic, cysteine-rich (osteonectin) Carbohydrate Metabolism 16507966-SENO2 enolase 2 (gamma, neuronal) 16507965-S ENO1 enolase 1, (alpha) 22095338-SPGK1 phosphoglycerate kinase 1 31543396-SPGK2 phosphoglycerate kinase 2 21735620-S MDH2 malate dehydrogenase 2, NAD (mitochondrial) 26024330-STPI1 triosephosphate isomerase 1 US 9,029, 146 B2 63 64 -continued

Catabolism 16507966-SENO2 enolase 2 (gamma, neuronal) 16507965-S ENO1 enolase 1, (alpha) 22095338-SPGK1 phosphoglycerate kinase 31543396-SPGK2 phosphoglycerate kinase 2 21735620-S MDH2 malate dehydrogenase 2, NAD (mitochondrial) 26024330-STPI1 triosephosphate isomerase 1 ellular Carbohydra e Catabolism 16507966-SENO2 enolase 2 (gamma, neuronal) 16507965-S ENO1 enolase 1, (alpha) 22095338-SPGK1 phosphoglycerate kinase 31543396-SPGK2 phosphoglycerate kinase 2 21735620-S MDH2 malate dehydrogenase 2, NAD (mitochondrial) 26024330-STPI1 triosephosphate isomerase 1 Cellular Carbohydra e Metabolism 16507966-SENO2 enolase 2 (gamma, neuronal) 16507965-S ENO1 enolase 1, (alpha) 22095338-SPGK1 phosphoglycerate kinase 31543396-SPGK2 phosphoglycerate kinase 2 21735620-S MDH2 malate dehydrogenase 2, NAD (mitochondrial) 26024330-STPI1 triosephosphate isomerase 1 Cellular Catabolism

16507966-SENO2 enolase 2 (gamma, neuronal) 16507965-S ENO1 enolase 1, (alpha) 22095338-SPGK1 phosphoglycerate kinase 31543396-SPGK2 phosphoglycerate kinase 2 21735620-SMDH2 malate dehydrogenase 2, NAD (mitochondrial) 26024330-STPI1 triosephosphate isomerase 1 Cellular Macromolecule Catabolism

16507966-SENO2 enolase 2 (gamma, neuronal) 16507965-S ENO1 enolase 1, (alpha) 22095338-SPGK1 phosphoglycerate kinase 1 31543396-SPGK2 phosphoglycerate kinase 2 21735620-SMDH2 malate dehydrogenase 2, NAD (mitochondrial) 26024330-STPI triosep hosphate isomerase 1 Chemotaxis 2726.2654-AIL16 interleukin 16 (lymp hocyte chemoattractant factor) 22538807-ACCL23 CO kine (C-C motif) ligan 23 34335180-ACCL15 CO kine (C-C motif) ligan 15 40316922-I CXCL12 CO kine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) 41872613-SCXCL5 CO kine (C-X-C motif) ligand 5 22538812-S CCL2 CO kine (C-C motif) ligan 2 4504098-S CXCR3 CO kine (C-X-C motif) receptor 3 22538399-S CCL11 CO kine (C-C motif) ligan 11 5453.576-S CXCL13 CO kine (C-X-C motif) ligand 13 (B-cell chemoattractant) 22538815-S CCL8 kine (C-C motif) ligan 8 34222286-S CYR61 ysteine-rich, angiogenic inducer, 61 4506832-S CCL kine (C-C motif) ligan 1 2861O153-SIL8 interleukin 8 22538800-S CCL16 CO kine (C-C motif) ligan 16 14790145-S CXCL11 CO kine (C-X-C motif) ligand 11 22165426-SCCL24 CO kine (C-C motif) ligan 24 22538813-SCCL5 CO kine (C-C motif) ligan 5 13435401-SCCL7 CO kine (C-C motif) ligan 7 4505862-S PLAU asminogen activator, urokinase 4504152-S CXCL1 CO kine (C-X-C motif) ligand 1 (melanoma growth stimulating ctivity, alpha) 27894329-SIL1A interleukin 1, alpha 22547151-SCCL26 ClelO kine (C-C motif) ligand 26 4506852-S XCL1 ClelO kine (C motif) ligand 1 Defense Response 278943 05-SIL1B interleukin 1, beta 27894329-SIL1A interleukin 1, alpha Development

42716312-SANG angiogenin, ribonuclease, RNase A family, 5 5902810-A BMP1 bone morphogenetic protein 1 27262662-A CDH11 cadherin 11, type 2, OB-cadherin (osteoblast) 27262662-A CSF1 colony stimulating factor 1 (macrophage) 27437029-S CSF2 colony stimulating factor 2 (granulocyte-macrophage) 27437048- A CSF3 colony stimulating factor 3 (granulocyte) 34222286-S CYR61 cysteine-rich, angiogenic inducer, 61 US 9,029, 146 B2 65 66 -continued

24430140-S FBN1 fibril in 1 (Marfan syndrome) 4755135-S FBN2 fibril in 2 (congenital contractural arachnodactyly) 10337586-S FGF6 fibroblast growth factor 6 24430216-SIL10 inter eukin 10 2861O153-SIL8 inter eukin 8 11321596-SKDR kinas e insert domain receptor (a type III receptor tyrosine kinase) 4580419-A KITLG KIT igand 600601 8-S LIF leukemia inhibitory factor (cholinergic differentiation factor) 7262388-SPCOLCE procollagen C-endopeptidase enhancer 4507170-S SPARC secreted protein, acidic, cysteine-rich (osteonectin) 1086.3872-S TGFB1 trans orming growth factor, beta 1 (Camurati-Engelmann disease) 4507470-STOFBR3 trans orming growth factor, beta receptor III (betaglycan, 300 kDa) 40317625-S THEBS1 thrombospondin 1 40805871-S THPO thrombopoietin (myeloproliferative leukemia virus oncogene ligand, megakaryocyte growth and development factor) 4507508-S TIMP1 TIMP metallopeptidase inhibitor 1 30172563-S VEGF vascular endothelial growth factor Energy Derivation by Oxidation of Organic Compounds 16507966-SENO2 enolase 2 (gamma, neuronal) 16507965-S ENO1 enolase 1, (alpha) 22095338-SPGK1 phosphoglycerate kinase 1 31543396-SPGK2 phosphoglycerate kinase 2 21735620-SMDH2 malate dehydrogenase 2, NAD (mitochondrial) 26024330-STPI1 triosephosphate isomerase 1 Exocytosis 22538815-S CCL8 chemokine (C-C motif) ligand 8 22538813-SCCL5 chemokine (C-C motif) ligand 5 Fewer 278943 05-SIL1B interl eukin 1, beta 27894329-SIL1A interl eukin 1, alpha Generation of Precursor Metabolites and Energy 16507966-SENO2 enolase 2 (gamma, neuronal) 16507965-S ENO1 enolase 1, (alpha) 22095338-SPGK1 phosphoglycerate kinase 1 31543396-SPGK2 phosphoglycerate kinase 2 21735620-SMDH2 malate dehydrogenase 2, NAD (mitochondrial) 26024330-STPI1 triosephosphate isomerase 1 Glucose Catabolism

16507966-SENO2 enolase 2 (gamma, neuronal) 16507965-S ENO1 enolase 1, (alpha) 22095338-SPGK1 phosphoglycerate kinase 1 31543396-SPGK2 phosphoglycerate kinase 2 21735620-SMDH2 malate dehydrogenase 2, NAD (mitochondrial) 26024330-STPI1 triosephosphate isomerase 1 Glucose Metabolism

16507966-SENO2 enolase 2 (gamma, neuronal) 16507965-S ENO1 enolase 1, (alpha) 22095338-SPGK1 phosphoglycerate kinase 1 31543396-SPGK2 phosphoglycerate kinase 2 21735620-SMDH2 malate dehydrogenase 2, NAD (mitochondrial) 26024330-STPI1 triosephosphate isomerase 1 Glycolysis 16507966-SENO2 enolase 2 (gamma, neuronal) 16507965-S ENO1 enolase 1, (alpha) 22095338-SPGK1 phosphoglycerate kinase 1 31543396-SPGK2 phosphoglycerate kinase 2 21735620-SMDH2 mala e dehydrogenase 2, NAD (mitochondrial) 26024330-STPI triosephosphate isomerase 1 Heat Generation

278943 05-S IL1B inter eukin 1, beta 27894329-SIL1A inter eukin 1, alpha Hemopoiesis

27262662-A CSF1 colony stimulating factor 1 (macrophage) 4580419-AKITLG KIT igand 24430216-SIL10 inter eukin 10 Hemo poietic or Lymphoid Organ Development

27262662-A CSF1 colony stimulating factor 1 (macrophage) 4580419-AKITLG KIT igand 24430216-SIL10 inter eukin 10 US 9,029, 146 B2 67 68 -continued

Hexose Catabolism 16507966-SENO2 enolase 2 (gamma, neuronal) 16507965-S ENO1 enolase 1, (alpha) 22095338-SPGK1 phosphoglycerate kinase 31543396-SPGK2 phosphoglycerate kinase 2 21735620-S MDH2 malate dehydrogenase 2, NAD (mitochondrial) 26024330-STPI1 triosephosphate isomerase 1 Hexose Metabolism 16507966-SENO2 enolase 2 (gamma, neuronal) 16507965-S ENO1 enolase 1, (alpha) 22095338-SPGK1 phosphoglycerate kinase 31543396-SPGK2 phosphoglycerate kinase 2 21735620-S MDH2 malate dehydrogenase 2, NAD (mitochondrial) 26024330-STPI1 triosephosphate isomerase 1 Immune Response

278943 05-S IL1B interleukin 1, beta 27894329-SIL1A interleukin 1, alpha Inflammatory Response 278943 05-S IL1B interleukin 1, beta 27894329-SIL1A interleukin 1, alpha Macromolecule Metabolism

5902810-A BMP1 bone morphogenetic protein 1 31542249-SC1R complement component 1, r Subcomponent 2253.8429-A CTSB cathepsin B 16507965-S ENO1 enolase 1, (alpha) 16507966-SENO2 enolase 2 (gamma, neuronal) 33.859834-S HGF hepatocyte growth factor (hepapoietin A; Scatter factor) 21735620-SMDH2 malate dehydrogenase 2, NAD (mitochondrial) 13.027798-S MMP1 matrix metallopeptidase 1 (interstitial collagenase) 4505204-S MMP10 matrix metallopeptidase 10 (stromelysin 2) 13.027796-SMMP13 matrix metallopeptidase 13 (collagenase 3) 130278O3-SMMP3 matrix metallopeptidase 3 (stromelysin 1, progelatinase) 4826835-S MMP9 matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase) 22095338-SPGK1 phosphoglycerate kinase 1 31543396-SPGK2 phosphoglycerate kinase 2 4505862-S PLAU plasminogen activator, urokinase 26024330-STPI1 triosephosphate isomerase 1 Macromolecule Catabolism 16507966-SENO2 enolase 2 (gamma, neuronal) 16507965-S ENO1 enolase 1, (alpha) 22095338-SPGK1 phosphoglycerate kinase 1 31543396-SPGK2 phosphoglycerate kinase 2 21735620-S MDH2 malate dehydrogenase 2, NAD (mitochondrial) 26024330-STPI1 triosephosphate isomerase 1 Main Pathways of Carbohydrate Metabolism 16507966-SENO2 enolase 2 (gamma, neuronal) 16507965-S ENO1 enolase 1, (alpha) 22095338-SPGK1 phosphoglycerate kinase 1 31543396-SPGK2 phosphoglycerate kinase 2 21735620-SMDH2 malate dehydrogenase 2, NAD (mitochondrial) 26024330-STPI1 triosephosphate isomerase 1 Monosaccharide Catabolism

16507966-SENO2 enolase 2 (gamma, neuronal) 16507965-S ENO1 enolase 1, (alpha) 22095338-SPGK1 phosphoglycerate kinase 1 31543396-SPGK2 phosphoglycerate kinase 2 21735620-SMDH2 malate dehydrogenase 2, NAD (mitochondrial) 26024330-STPI1 triosephosphate isomerase 1 Monosaccharide Metabolism

16507966-SENO2 enolase 2 (gamma, neuronal) 16507965-S ENO1 enolase 1, (alpha) 22095338-SPGK1 phosphoglycerate kinase 1 31543396-SPGK2 phosphoglycerate kinase 2 21735620-S MDH2 malate dehydrogenase 2, NAD (mitochondrial) 26024330-STPI1 triosephosphate isomerase 1 Organ Development

27262662-A CSF1 colony stimulating factor 1 (macrophage) 27262662-A CDH11 cadherin 11, type 2, OB-cadherin (osteoblast) US 9,029, 146 B2 69 70 -continued

4580419-AKITLG KIT ligand 5902810-A BMP1 bone morphogenetic protein 1 11321596-S KDR kinase insert domain receptor (a type III receptor tyrosine kinase) 30172563-S VEGF vascular endothelial growth factor 2861O153-SIL8 interleukin 8 4507170-S SPARC secreted protein, acidic, cysteine-rich (osteonectin) 42716312-SANG angiogenin, ribonuclease, RNase A family, 5 24430216-SIL10 interleukin 10 10337586-SFGF6 fibroblast growth factor 6 Organ Morphogenesis 11321596-SKDR kinase insert domain receptor (a type III receptor tyrosine kinase) 30172563-S VEGF vascular endothelial growth factor 2861O153-SIL8 interleukin 8 42716312-SANG angiogenin, ribonuclease, RNase A family, 5 10337586-SFGF6 fibroblast growth factor 6 Ossification 27262662-A CDH11 cadherin 11, type 2, OB-cadherin (osteoblast) 5902810-A BMP1 bone morphogenetic protein 1 4507170-S SPARC secreted protein, acidic, cysteine-rich (osteonectin) Phosphate Metabolism 22095338-SPGK1 hosphoglycerate kinase 1 31543396-SPGK2 hosphoglycerate kinase 2 Phosphorus Metabolism 22095338-SPGK1 hosphoglycerate kinase 1 31543396-SPGK2 hosphoglycerate kinase 2 Response to Abiotic Stimulus 2726.2654-AIL16 interleukin 16 (lymphocyte chemoattractant factor) 22538807-ACCL23 hemokine (C-C motif) ligand 23 34335180-ACCL15 hemokine (C-C motif) ligand 15 40316922-I CXCL12 hemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) 41872613-SCXCL5 hemokine (C-X-C motif) ligand 5 225.38812-S CCL2 hemokine (C-C motif) ligand 2 4504098-S CXCR3 hemokine (C-X-C motif) receptor 3 22538399-S CCL11 hemokine (C-C motif) ligand 11 5453.576-S CXCL13 hemokine (C-X-C motif) ligand 13 (B-cell chemoattractant) 22538815-S CCL8 hemokine (C-C motif) ligand 8 34222286-S CYR61 ysteine-rich, angiogenic inducer, 61 4506832-S CCL1 hemokine (C-C motif) ligand 1 2861O153-SIL8 interleukin 8 22538800-S CCL16 hemokine (C-C motif) ligand 16 14790145-S CXCL11 hemokine (C-X-C motif) ligand 11 22165426-SCCL24 hemokine (C-C motif) ligand 24 22538813-SCCL5 hemokine (C-C motif) ligand 5 13435401-SCCL7 hemokine (C-C motif) ligand 7 4505862-S PLAU asminogen activator, urokinase 4504152-S CXCL1 hemokine (C-X-C motif) ligand 1 (melanoma growth stimulating ctivity, alpha) 27894329-SIL1A interleukin 1, alpha 22547151-SCCL26 hemokine (C-C motif) ligand 26 4506852-S XCL1 hemokine (C motif) ligand 1 Response to Biotic Stimulus 278943 05-SIL1B interleukin 1, beta 27894329-SIL1A interleukin 1, alpha Response to Chemical Stimulus 2726.2654-AIL16 interleukin 16 (lymphocyte chemoattractant factor) 22538807-ACCL23 chemokine (C-C motif) ligand 23 34335180-ACCL15 hemokine (C-C motif) ligand 15 40316922-I CXCL12 hemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) 41872613-SCXCL5 hemokine (C-X-C motif) ligand 5 22538812-S CCL2 hemokine (C-C motif) ligand 2 4504098-S CXCR3 hemokine (C-X-C motif) receptor 3 22538399-S CCL11 hemokine (C-C motif) ligand 11 5453.576-S CXCL13 hemokine (C-X-C motif) ligand 13 (B-cell chemoattractant) 22538815-S CCL8 hemokine (C-C motif) ligand 8 34222286-S CYR61 ysteine-rich, angiogenic inducer, 6 4506832-S CCL1 hemokine (C-C motif) ligand 1 2861O153-SIL8 interleukin 8 22538800-S CCL16 hemokine (C-C motif) ligand 16 14790145-S CXCL11 hemokine (C-X-C motif) ligand 11 22165426-S CCL24 hemokine (C-C motif) ligand 24 22538813-SCCL5 hemokine (C-C motif) ligand 5 13435401-SCCL7 hemokine (C-C motif) ligand 7 US 9,029, 146 B2 71 72 -continued

4505862-S PLAU plasminogen activator, urokinase 4504152-S CXCL1 chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha) 27894329-SIL1A interleukin 1, alpha 22547151-SCCL26 chemokine (C-C motif) ligand 26 4506852-S XCL1 chemokine (C motif) ligand 1 Response to External Biotic Stimulus 278943 05-SIL1B interleukin 1, beta 27894329-SIL1A interleukin 1, alpha Response to External Stimulus 2726.2654-AIL16 interleukin 16 (lymp hocyte chemoattractant factor) 22538807-ACCL23 CO kine (C-C motif) ligand 23 34335180-ACCL15 CO kine (C-C motif) ligand 15 40316922-I CXCL12 CO kine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) 41872613-SCXCL5 CO kine (C-X-C motif) ligand 5 22538812-S CCL2 CO kine (C-C motif) ligand 2 4504098-S CXCR3 CO kine (C-X-C motif) receptor 3 22538399-S CCL11 CO kine (C-C motif) ligand 11 5453.576-S CXCL13 CO kine (C-X-C motif) ligand 13 (B-cell chemoattractant) 22538815-S CCL8 lel kine (C-C motif) ligand 8 34222286-S CYR61 e-rich, angiogenic inducer, 61 4506832-S CCL1 CO kine (C-C motif) ligand 1 2861O153-SIL8 interleukin 8 22538800-S CCL16 C CO kine (C-C motif) ligand 16 14790145-S CXCL11 C CO kine (C-X-C motif) ligand 11 22165426-SCCL24 C CO kine (C-C motif) ligand 24 22538813-SCCL5 C CO kine (C-C motif) ligand 5 278943 05-S IL1B interleukin 1, beta 13435401-SCCL7 CO kine (C-C motif) ligand 7 4505862-S PLAU asminogen activator, urokinase 4504152-S CXCL1 CO kine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha) 27894329-SIL1A interleukin 1, alpha 22547151-SCCL26 C CO kine (C-C motif) ligand 26 4506852-S XCL1 C O kine (C motif) ligand 1 Res ponse to Pest, Pathogen or Parasite 278943 05-SIL1B interleukin 1, beta 27894329-SIL1A interleukin 1, alpha Response to Stimulus 2726.2654-AIL16 interleukin 16 (lymp hocyte chemoattractant factor) 22538807-ACCL23 CO kine (C-C motif) ligand 23 34335180-ACCL15 CO kine (C-C motif) ligand 15 40316922-I CXCL12 CO kine (C-X-C motif) ligan 12 (stromal cell-derived factor 1) 41872613-SCXCL5 CO kine (C-X-C motif) ligan 5 22538812-S CCL2 CO kine (C-C motif) ligand 2 4504098-S CXCR3 CO kine (C-X-C motif) recep or 3 22538399-S CCL11 CO kine (C-C motif) ligand 11 5453.576-S CXCL13 CO kine (C-X-C motif) ligan 13 (B-cell chemoattractant) 22538815-S CCL8 CO kine (C-C motif) ligand 8 34222286-S CYR61 ysteine-rich, angiog enic inducer, 6 4506832-S CCL1 CO kine (C-C motif) ligand 1 2861O153-SIL8 interleukin 8 22538800-S CCL16 C CO kine (C-C motif) ligand 16 14790145-S CXCL11 C CO kine (C-X-C motif) ligan 11 22165426-SCCL24 C CO kine (C-C motif) ligand 24 22538813-SCCL5 C CO kine (C-C motif) ligand 5 278943 05-S IL1B interleukin 1, beta 13435401-SCCL7 CO kine (C-C motif) ligand 7 4505862-S PLAU asminogen activator, urokinase 4504152-S CXCL1 CO kine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha) 27894329-SIL1A interleukin 1, alpha 22547151-SCCL26 C CO kine (C-C motif) ligand 26 4506852-S XCL1 C CO kine (C motif) ligand 1 Response to Stress 278943 05-SIL1B interleukin 1, beta 4505862-S PLAU asminogen activator, urokinase 27894329-SIL1A interleukin 1, alpha Response to Wounding

278943 05-S IL1B interleukin 1, beta 4505862-S PLAU plasminogen activator, urokinase 27894329-SIL1A interleukin 1, alpha US 9,029, 146 B2 73 74 -continued

Secretion GI 22538815-S CCL8 chemokine (C-C motif) ligand 8 GI 22538813-SCCL5 chemokine (C-C motif) ligand 5 Secretory Pathway 22538815-S CCL8 chemokine (C-C motif) ligand 8 22538813-SCCL5 chemokine (C-C motif) ligand 5 Skeletal Development 27262662-A CDH11 cadherin 11, type 2, OB-cadherin (osteoblast) 5902810-A BMP1 bone morphogenetic protein 1 MP1 : 4507170-S SPARC secreted protein, acidic, cysteine-rich (osteonectin) 8XIS

2726.2654-AIL16 interleukin 16 (lymphocyte chemoattractant factor) 22538807-ACCL23 hemokine (C-C motif) ligand 23 34335180-ACCL15 hemokine (C-C motif) ligand 15 40316922-I CXCL12 hemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) 41872613-SCXCL5 hemokine (C-X-C motif) ligand 5 22538812-S CCL2 hemokine (C-C motif) ligand 2 4504098-S CXCR3 hemokine (C-X-C motif) receptor 3 22538399-S CCL11 hemokine (C-C motif) ligand 11 5453.576-S CXCL13 hemokine (C-X-C motif) ligand 13 (B-cell chemoattractant) 22538815-S CCL8 hemokine (C-C motif) ligand 8 34222286-S CYR61 ysteine-rich, angiogenic inducer, 61 4506832-S CCL1 hemokine (C-C motif) ligand 1 2861O153-SIL8 interleukin 8 22538800-S CCL16 hemokine (C-C motif) ligand 16 14790145-S CXCL11 hemokine (C-X-C motif) ligand 11 22165426-SCCL24 hemokine (C-C motif) ligand 24 22538813-SCCL5 hemokine (C-C motif) ligand 5 13435401-SCCL7 hemokine (C-C motif) ligand 7 4505862-S PLAU asminogen activator, urokinase 4504152-S CXCL1 hemokine (C-X-C motif) ligand 1 (melanoma growth stimulating ctivity, alpha) 27894329-SIL1A interleukin 1, alpha 22547151-SCCL26 chemokine (C-C motif) ligand 26 4506852-S XCL1 chemokine (C motif) ligand 1 Thermoregulation GI 278943 05-SIL1B interleukin 1, beta GI 27894329-SIL1A interleukin 1, alpha Tissue Development GI 27262662-A CDH11 cadherin 11, type 2, OB-cadherin (osteoblast) GI 5902810-A BMP1 bone morphogenetic protein 1 BMP1 GI 4507170-S SPARC secreted protein, acidic, cysteine-rich (osteonectin) Vasulogenesis GI 30172563-S VEGF vascular endothelial growth factor Vasculature Development GI 11321596-SKDR kinase insert domain receptor (a type III receptor tyrosine kinase) GI 30172563-S VEGF vascular endothelial growth factor GI 2861 O153-SIL8 interleukin 8 GI 42716312-SANG angiogenin, ribonuclease, RNase A family, 5 GI 10337586-S FGF6 fibroblast growth factor 6

Example 14 Listing of 201 Genes in Each of the 30 Pathways 55

Antigen processing and presentation - Homo sapiens (human) GI 2253.8429-A CTSB cathepsin B GI 37704380-S B2M beta-2-microglobulin GI 24234685-A HSPA8 heat shock 70 kDa protein 8 GI 30581139-APSME1 proteasome (prosome, macropain) activator Subunit 1 (PA28 alpha) Apoptosis - Homo sapiens (human)

GI 41281560-S CLSTN1 calsyntenin 1 GI 28416914-SIL3 interleukin 3 (colony-stimulating factor, multiple) US 9,029, 146 B2 75 -continued 27894329-SIL1A interleukin 1, alpha 278943 05-SIL1B interleukin 1, beta 2595211O-STNF tumor necrosis factor (TNF Superfamily, member 2) Carbon fixation - Homo sapiens (human) 21735620-SMDH2 malate dehydrogenase 2, NAD (mitochondrial) 26024330-STPI1 triosephosphate isomerase 1 22095338-SPGK1 phosphoglycerate kinase 1 31543396-SPGK2 phosphoglycerate kinase 2 Cell Communication - Homo sapiens (human) 27436944-A LMNA lamin AC 5O16088-S ACTB actin, beta 10938011-SACTC actin, alpha, cardiac muscle 11038618-SACTG1 actin, gamma 1 14719826-S COL1A1 collagen, type I, alpha 1 2153.6289-S COL1A2 collagen, type I, alpha 2 151494.80-S COL3A1 collagen, type III, alpha 1 (Ehlers-Danlos Syndrome type IV, autosomal dominant) 45580690-S COLAA1 collagen, type IV, alpha 1 17986276-S COLA-A2 collagen, type IV, alpha 2 16554578-S COL5A1 collagen, type V, alpha 1 16554580-S COLSA2 collagen, type V, alpha 2 1501 1912-S COL6A1 collagen, type VI, alpha 1 17402876-A COL6A2 collagen, type VI, alpha 2 17149810-A COL6A3 collagen, type VI, alpha 3 18375521-A COL11A1 collagen, type XI, alpha 1 16933.543-AFN fibronectin 9845497-SLAMC1 laminin, gamma 1 (formerly LAMB2) 16554581-S COLSA3 collagen, type V, alpha 3 40317625-S THEBS1 thrombospondin 1 Citrate cycle (TCA cycle) - Homo sapiens (human) 4504374-S CFH1/HF1 complement factor H 21735620-SMDH2 malate dehydrogenase 2, NAD (mitochondrial) Complement and coagulation cascades - Homo Sapiens (human) 4505862-S PLAU plasminogen activator, urokinase 4504374-S CFH1. HF1 complement factor H 31542249-SC1R complement component 1, r subcomponent Cytokine-cytokine receptor interaction - Homo sapiens (human) 4506832-S CCL1 hemokine (C-C motif) ligand 1 22538399-S CCL11 hemokine (C-C motif) ligand 11 34335180-ACCL15 hemokine (C-C motif) ligand 15 22538800-S CCL16 hemokine (C-C motif) ligand 16 22538812-S CCL2 hemokine (C-C motif) ligand 2 22538807-ACCL23 hemokine (C-C motif) ligand 23 22165426-SCCL24 hemokine (C-C motif) ligand 24 22547151-SCCL26 hemokine (C-C motif) ligand 26 22538813-SCCL5 hemokine (C-C motif) ligand 5 13435401-SCCL7 hemokine (C-C motif) ligand 7 22538815-S CCL8 hemokine (C-C motif) ligand 8 27262662-A CSF1 olony stimulating factor 1 (macrophage) 27437029-SCSF2 olony stimulating factor 2 (granulocyte-macrophage) 27437048- A CSF3 olony stimulating factor 3 (granulocyte) 4506856-S CX3CL1 hemokine (C-X3-C motif) ligand 1 4504152-S CXCL1 hemokine (C-X-C motif) ligand 1 (melanoma growth stimulating ctivity, alpha) 14790145-S CXCL11 hemokine (C-X-C motif) ligand 11 40316922-I CXCL12 hemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) 5453.576-S CXCL13 hemokine (C-X-C motif) ligand 13 (B-cell chemoattractant) 41872613-SCXCL5 hemokine (C-X-C motif) ligand 5 4504098-S CXCR3 hemokine (C-X-C motif) receptor 3 6031163-SEGF pidermal growth factor (beta-urogastrone) 384.55415-S FLT3LG fms-related tyrosine kinase 3 ligand 33.859834-S HGF hepatocyte growth factor (hepapoietin A; Scatter factor) 10835170-SIFNG interferon, gamma 24430216-SIL10 interleukin 10 24497437-SIL12B interleukin 12B (natural killer cell stimulatory factor 2, cytotoxic lymphocyte maturation factor 2, p.40) 26787977-SIL13 interleukin 13 27894329-SIL1A interleukin 1, alpha 278943 05-S IL1B interleukin 1, beta 28178860-SIL2 interleukin 2 284.16914-SIL3 interleukin 3 (colony-stimulating factor, multiple) 10834983-SIL6 interleukin 6 (interferon, beta 2) 2861O152-SIL7 interleukin 7 2861O153-SIL8 interleukin 8 US 9,029, 146 B2 77 78 -continued

11321596-SKDR kinase insert domain receptor (a type III receptor tyrosine kinase) 4580419-AKITLG KIT ligand 4557714-S LEP leptin (obesity homolog, mouse) 600601 8-S LIF leukemia inhibitory factor (cholinergic differentiation factor) 1545.1785-APDGFB platelet-derived growth factor beta polypeptide (simian sarcoma viral (v- sis) oncogene homolog) 15451788-SPDGFRB platelet-derived growth factor receptor, beta polypeptide 1086.3872-STGFB1 transforming growth factor, beta 1 (Camurati-Engelmann disease) 4507462-STGFB2 transforming growth factor, beta 2 40805871-S THPO thrombopoietin (myeloproliferative leukemia virus oncogene ligand, megakaryocyte growth and development factor) 4507508-S TIMP1 TIMP metallopeptidase inhibitor 1 25952110-STNF tumor necrosis factor (TNF Superfamily, member 2) 22547122-STNFRSF11B tumor necrosis factor receptor Superfamily, member 11b (osteoprotegerin) 30172563-S VEGF vascular endothelial growth factor 4506852-S XCL1 chemokine (C motif) ligand 1 ECM-receptor interaction - Homo sapiens (human) 9845497-SLAMC1 laminin, gamma 1 (formerly LAMB2) 14719826-S COL1A1 collagen, type I, alpha 1 2153.6289-S COL1A2 collagen, type I, alpha 2 151494.80-S COL3A1 collagen, type III, alpha 1 (Ehlers-Danlos Syndrome type IV, autosomal dominant) 45580690-S COLAA1 collagen, type IV, alpha 1 17986276-S COLA-A2 collagen, type IV, alpha 2 16554578-S COL5A1 collagen, type V, alpha 1 16554580-S COLSA2 collagen, type V, alpha 2 1501 1912-S COL6A1 collagen, type VI, alpha 1 17402876-A COL6A2 collagen, type VI, alpha 2 17149810-A COL6A3 collagen, type VI, alpha 3 1837.5521-A COL11A1 collagen, type XI, alpha 1 16554581-S COL5A3 collagen, type V, alpha 3 16933.543-AFN1 fibronectin 40317625-S THEBS1 thrombospondin 1 7427516-S HSPG2 heparan Sulfate proteoglycan 2 (perlecan) 21361192-S CD44 CD44 antigen (homing function and Indian blood group System) Epithelial cell signaling in Helicobacter pylori infection - Homo sapiens (human) 22538813-SCCL5 chemokine (C-C motif) ligand 5 4504152-S CXCL1 chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha) 2861O153-SIL8 interleukin 8 Fc epsilon RI signaling pathway - Homo sapiens (human) 2595211O-STNF tumor necrosis factor (TNF Superfamily, member 2) 26787977-SIL13 interleukin 13 27437029-SCSF2 colony stimulating factor 2 (granulocyte-macrophage) 41281560-S CLSTN1 calsyntenin 1 28416914-SIL3 interleukin 3 (colony-stimulating factor, multiple) Focal adhesion - Homo sapiens (human) 5O16088-S ACTB actin, beta O938O11-S ACTC actin, alpha, cardiac muscle 11038618-SACTG1 actin, gamma 1 41281560-S CLSTN1 calsyntenin 1 837SS21-A COL11A1 collagen, type XI, alpha 1 4719826-S COL1A1 collagen, type I, alpha 1 2153.6289-S COL1A2 collagen, type I, alpha 2 5149.480-S COL3A1 collagen, type III, alpha 1 (Ehlers-Danlos Syndrome type IV, autosomal dominant) 45580690-S COLAA1 collagen, type IV, alpha 1 7986276-S COL4A2 collagen, type IV, alpha 2 6SS4578-S COLSA1 collagen, type V, alpha 1 6SS4580-S COLSA2 collagen, type V, alpha 2 6SS4581-S COLSA3 collagen, type V, alpha 3 SO11912-S COL6A1 collagen, type VI, alpha 1 7402876-A COL6A2 collagen, type VI, alpha 2 7149810-A COL6A3 collagen, type VI, alpha 3 6031163-SEGF epidermal growth factor (beta-urogastrone) 4503744-S FLNA filamin A, alpha (actin binding protein 280) 6933S43-AFN1 fibronectin 33.859834-S HGF hepatocyte growth factor (hepapoietin A; Scatter factor) 99.231.11-SIGF1 insulin-like growth factor 1 (somatomedin C) O834983-SIL6 interleukin 6 (interferon, beta 2) 1321596-SKDR kinase insert domain receptor (a type III receptor tyrosine kinase) 9845497-SLAMC1 laminin, gamma 1 (formerly LAMB2) S4S1785-APDGFB platelet-derived growth factor beta polypeptide (simian sarcoma viral (v- sis) oncogene homolog) US 9,029, 146 B2

-continued 15451788-SPDGFRB platelet-derived growth factor receptor, beta polypeptide 40317625-S THEBS1 thrombospondin 1 30172563-S VEGF vascular endothelial growth factor Gap junction - Homo sapiens (human) 5174476-S K-ALPHA-1 tubulin, alpha, ubiquitous 1798.6282-STUBA3 tubulin, alpha 3 31880337-STUBA6 tubulin, alpha 6 15451788-SPDGFRB platelet-derived growth factor receptor, beta polypeptide 6031163-SEGF epidermal growth factor (beta-urogastrone) 15451785-APDGFB platelet-derived growth factor beta polypeptide (simian sarcoma viral (v- sis) oncogene homolog) Glycolysis or Gluconeogenesis - Homo sapiens (human) 16507965-S ENO1 enolase 1, (alpha) 16507966-SENO2 enolase 2 (gamma, neuronal) 26024330-STPI1 triosephosphate isomerase 1 22095338-SPGK1 phosphoglycerate kinase 1 31543396-SPGK2 phosphoglycerate kinase 2 Hematopoietic cell lineage - Homo sapiens (human) 21361192-S CD44 CD44 antigen (homing function and Indian blood group system) 4503744-S FLNA filamin A, alpha (actin binding protein 280) 40805871-S THPO thrombopoietin (myeloproliferative leukemia virus oncogene ligand, megakaryocyte growth and development factor) 4507508-S TIMP1 TIMP metallopeptidase inhibitor 1 25952110-STNF tumor necrosis factor (TNF Superfamily, member 2) 27262662-A CSF1 colony stimulating factor 1 (macrophage) 27437048- A CSF3 colony stimulating factor 3 (granulocyte) 384.55415-S FLT3LG fms-related tyrosine kinase 3 ligand 10834983-SIL6 interleukin 6 (interferon, beta 2) 27437029-SCSF2 colony stimulating factor 2 (granulocyte-macrophage) 27894329-SIL1A interleukin 1, alpha 278943 05-S IL1B interleukin 1, beta 284.16914-SIL3 interleukin 3 (colony-stimulating factor, multiple) 2861O152-SIL7 interleukin 7 4580419-A KITLG KIT ligand Inositol metabolism - Homo sapiens (human) 26024330-STPI1 triosephosphate isomerase 1 Insulin signaling pathway - Homo sapiens (human) 31377794-S CALM1 calmodulin 1 (phosphorylase kinase, delta) 41281560-S CLSTN1 calsyntenin 1 Jak-STAT signaling pathway - Homo sapiens (human) 41281560-S CLSTN1 calsyntenin 1 27437029-SCSF2 colony stimulating factor 2 (granulocyte-macrophage) 27437048- A CSF3 colony stimulating factor 3 (granulocyte) 10835170-SIFNG interferon, gamma 24430216-SIL10 interleukin 10 24497437-SIL12B interleukin 12B (natural killer cell stimulatory factor 2, cytotoxic lymphocyte maturation factor 2, p.40) 26787977-SIL13 interleukin 13 28178860-SIL2 interleukin 2 284.16914-SIL3 interleukin 3 (colony-stimulating factor, multiple) 10834983-SIL6 interleukin 6 (interferon, beta 2) 2861O152-SIL7 interleukin 7 4557714-SLEP leptin (obesity homolog, mouse) 600601 8-S LIF leukemia inhibitory factor (cholinergic differentiation factor) 40805871-S THPO thrombopoietin (myeloproliferative leukemia virus oncogene ligand, megakaryocyte growth and development factor) 4507508-S TIMP1 TIMP metallopeptidase inhibitor 1 Leukocyte transendothelial migration - Homo sapiens (human) 4826835-S MMP9 matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase) 5453.576-S CXCL13 chemokine (C-X-C motif) ligand 13 (B-cell chemoattractant) 2861O153-SIL8 interleukin 8 40316922-I CXCL12 chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) 41281560-S CLSTN1 calsyntenin 1 5O16088-S ACTB actin, beta 10938011-SACTC actin, alpha, cardiac muscle 11038618-SACTG1 actin, gamma 1 MAPK signaling pathway - Homo sapiens (human) 34106709-A BDNF brain-derived neurotrophic factor 6031163-SEGF epidermal growth factor (beta-urogastrone) 4503692-S FGF17 fibroblast growth factor 17 US 9,029, 146 B2 81 82 -continued

4503700-S FGF4 fibroblast growth factor 4 (heparin Secretory transforming protein 1, Kaposi sarcoma oncogene) 10337586-S FGF6 broblast growth factor 6 15147344-S FGF7 broblast growth factor 7 (keratinocyte growth factor) 4503706-S FGF9 broblast growth factor 9 (glia-activating factor) 13186266-AFGFR2 broblast growth factor receptor 2 (bacteria-expressed kinase, eratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon yndrome, Pfeiffer syndrome, Jackson-Weiss syndrome) 4503744-S FLNA amin A, alpha (actin binding protein 280) 405494-01-A. GDNF ial cell derived neurotrophic factor 24234685-A HSPA8 heat shock 70 kDa protein 8 27894329-SIL1A interleukin 1, alpha 278943 05-SIL1B interleukin 1, beta 1545.1785-APDGFB platelet-derived growth factor beta polypeptide (simian sarcoma viral (v- sis) oncogene homolog) 15451788-SPDGFRB platelet-derived growth factor receptor, beta polypeptide 1086.3872-STGFB1 ransforming growth factor, beta 1 (Camurati-Engelmann disease) 4507462-STGFB2 ransforming growth factor, beta 2 2595211O-STNF umor necrosis factor (TNF Superfamily, member 2) Methane metabolism - Homo sapiens (human) 40805871-S THPO hrombopoietin (myeloproliferative leukemia virus oncogene ligand, megakaryocyte growth and development factor) 324.55261-A PRDX5 peroxiredoxin 5 mTOR signaling pathway - Homo sapiens (human) 30172563-S VEGF vascular endothelial growth factor 41281560-S CLSTN1 calsyntenin 1 1992.3111-SIGF1 insulin-like growth factor 1 (somatomedin C) Phenylalanine, tyrosine and tryptophan biosynthesis - Homo sapiens (human) 16507965-S ENO1 enolase 1, (alpha) 16507966-SENO2 enolase 2 (gamma, neuronal) Phenylalanine metabolism - Homo Sapiens (human) 4505184-S MIF macrophage migration inhibitory factor (glycosylation-inhibiting factor) 40805871-S THPO thrombopoietin (myeloproliferative leukemia virus oncogene ligand, megakaryocyte growth and development factor) G 324.55261-A PRDX5 peroxiredoxin 5 Regulation of actin cytoskeleton - Homo sapiens (human) 5O16088-S ACTB actin, beta 10938011-SACTC actin, alpha, cardiac muscle 11038618-SACTG1 actin, gamma 1 5031634-S CFL1 cofilin 1 (non-muscle) 41281560-S CLSTN1 calsyntenin 1 6031163-SEGF epidermal growth factor (beta-urogastrone) 4503692-S FGF17 fibroblast growth factor 17 4503700-S FGF4 fibroblast growth factor 4 (heparin Secretory transforming protein 1, Kaposi sarcoma oncogene) 10337586-SFGF6 broblast growth factor 6 15147344-S FGF7 broblast growth factor 7 (keratinocyte growth factor) 4503706-S FGF9 broblast growth factor 9 (glia-activating factor) 13186266-AFGFR2 broblast growth factor receptor 2 (bacteria-expressed kinase, eratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon yndrome, Pfeiffer syndrome, Jackson-Weiss syndrome) 16933.543-AFN1 bronectin 1 38.044287-AGSN 9. elsolin (amyloidosis, Finnish type) 22507396-SMYH9 myosin, heavy polypeptide 9, non-muscle : 1545.1785-APDGFB platelet-derived growth factor beta polypeptide (simian sarcoma viral (v- sis) oncogene homolog) 15451788-SPDGFRB platelet-derived growth factor receptor, beta polypeptide 16753213-SPFN1 profilin 1 34.328943-STMSB4X hymosin, beta 4, X-linked 34O13529-STMSL3 hymosin-like 3 Stilbene, coumarine and lignin biosynthesis - Homo sapiens (human)

40805871-S THPO hrombopoietin (myeloproliferative leukemia virus oncogene ligand, megakaryocyte growth and development factor) 324.55261-A PRDX5 peroxiredoxin 5 T cell receptor signaling pathway - Homo sapiens (human) 2595211O-STNF umor necrosis factor (TNF Superfamily, member 2) 27437029-SCSF2 colony stimulating factor 2 (granulocyte-macrophage) 10835170-SIFNG interferon, gamma 24430216-SIL10 interleukin 10 28178860-SIL2 interleukin 2 41281560-S CLSTN1 calsyntenin 1 US 9,029, 146 B2 83 84 -continued TGF-beta signaling pathway - Homo sapiens (human) 4557730-S LTBP1 latent transforming growth factor beta binding protein 1 40317625-S THEBS1 thrombospondin 1 1086.3872-STGFB1 transforming growth factor, beta 1 (Camurati-Engelmann disease) 4507462-STGFB2 transforming growth factor, beta 2 2595211O-STNF tumor necrosis factor (TNF Superfamily, member 2) 10835170-SIFNG interferon, gamma Tightjunction - Homo sapiens (human) 5O16088-S ACTB actin, beta 10938011-SACTC actin, alpha, cardiac muscle 11038618-SACTG1 actin, gamma 1 22507396-SMYH9 myosin, heavy polypeptide 9, non-muscle Toll-like receptor signaling pathway - Homo sapiens (human) 14790145-S CXCL11 chemokine (C-X-C motif) ligand 11 2861O153-SIL8 interleukin 8 24497437-SIL12B interleukin 12B (natural killer cell stimulatory factor 2, cytotoxic lymphocyte maturation factor 2, p.40) 278943 05-SIL1B interleukin 1, beta 41281560-S CLSTN1 calsyntenin 1 10834983-SIL6 interleukin 6 (interferon, beta 2) 22538813-SCCL5 chemokine (C-C motif) ligand 5 25952110-STNF tumor necrosis factor (TNF Superfamily, member 2) Type I diabetes mellitus - Homo sapiens (human) 24497437-SIL12B interleukin 12B (natural killer cell stimulatory factor 2, cytotoxic lymphocyte maturation factor 2, p.40) 28178860-SIL2 interleukin 2 10835170-SIFNG interferon, gamma 27894329-SIL1A interleukin 1, alpha 278943 05-SIL1B interleukin 1, beta 2595211O-STNF tumor necrosis factor (TNF Superfamily, member 2)

Example 14A -continued

593 Additional Proteins 35 Total 593 unique gene products: set 1-4(3of4 &4of 4) without common genes in 201 list Further analysis of the conditioned media shows that the media contains the further 593 proteins shown below, for a total of 794 unique gene products: 40

Total 593 unique gene products: set 1-4(3of4 &4of 4) without common genes in 201 list 02-Sep 07-Sep 45 AARS PI5 ACAA2 POA1BP ACAT2 POE ACO1 PP ACTN1 PRT ACTN2 50 RCN1 ACTN3 HGAP1 ACTN4 PC1A ACTR1A PC1B ACTR1B PC2 ACTR2 PC3 ACTR3 55 PC4 ACTR3B TS-1 ADAM9 TIC ADSL ATPSB ADSS ATP6AP1 AEBP1 RO1C AGA TL1 AHCY 60 P2 AK1 AK2 ASP1 AKR1A1 A.T1 BS1 ALDH2 CAT1 ALDHAA1 65 N ALDOA LVRA

US 9,029, 146 B2 91 92 -continued Example 14B Total 593 unique gene products: set 1-4(3of4 Analysis of 593 Further Proteins &4of 4) without common genes in 201 list SSB 5 The biological processes and pathways modulated by the ST13 secretome are further analysed as follows. ST6GAL2 STAT1 We performed the analysis using a free web-based com STC1 puter program (http://www.pantherdb.org/tools/geneXAnaly STIP1 sis.jsp). The frequency of genes in each functional categories SULF1 10 SYNCRIP was compared between the total list of 794 genes and that in TALDO1 the public database (NCBI: Homo sapiens). TARS We compared the frequency of genes in the different GO TCN2 (gene ontology) pathways, GO biological processes and GO TCP1 TFPI 15 molecular functions. Only those GO pathways, GO biologi THEBS2 cal processes and GO molecular functions where frequency THOP1 of genes is significantly higher or lower than the frequency of THY1 genes in the NCBI homo sapiens database (p<0.01) were used TKT TLN1 in our analysis. TMOD2 The results are shown in FIGS. 12, 13 and 14. TMOD3 FIG. 12 shows the predicted pathways driven by the TNC secreted proteins. Frequency of genes in each pathway was TNPO1 TP53I3 significantly higher in the secretome than that in the NCBI TPM1 homo sapiens database (p<0.01). TPM2 25 FIG. 13 shows the predicted processes driven by the TPM3 secreted proteins. Frequency of genes in each pathway was TRAP1 TRHDE significantly higher or lower in the secretome than that in the TROVE2 NCBI homo sapiens database (p<0.01). TSKU FIG. 14 shows the predicted molecular driven by the TUBA1A 30 secreted proteins. Frequency of genes in each pathway was TUBA8 TUBB significantly higher in the secretome than that in the NCBI TUBB2C homo sapiens database (p<0.01). TUBB3 TUBB4 Example 15 35 TUBB6 TUBB8 F 1 References for Examples 7 to 14 NL5 1. Le Blanc, K., and Pittenger, M. (2005) Cytotherapy 7. E2L3 40 36-45 E2N 2. Reiser, J., Zhang, X.Y., Hemenway, C. S., Mondal, D., E2V1 E3B Pradhan, L., and La Russa, V. F. (2005) Expert Opin Biol Ther HL3 5, 1571-1584 DH 3. Hui, J. H., Ouyang, H. W. Hutmacher, D.W., Goh, J. C., P2 45 and Lee, E. H. (2005) Ann Acad Med Singapore 34, 206-212 UROD USP14 4. Caplan, A. I. (2005) Tissue Eng 11, 1198-1211 USPS 5. Menasche, P. (2005) Curr Opin Mol Ther 7, 293–299 VARS 6. Laflamme, M.A., and Murry, C. E. (2005) Nat Biotech WASN nol 23, 845-856 WAT1 50 7. Kinnaird, T., Stabile, E., Burnett, M. S., Shou, M., Lee, VCL. VCP C. W., Barr, S., Fuchs, S., and Epstein, S. E. (2004) Circula VIL2 tion 109, 1543-1549 VIM 8. Caplan, A.I., and Dennis, J. E. (2006) J Cell Biochem VPS26A 9. Leedham, S. J., Brittan, M., McDonald, S. A., and VPS35 55 Wright, N.A. (2005) J Cell Mol Med 9, 11-24 VTN 10. Togel, F., Hu, Z. Weiss, K., Isaac, J., Lange, C., and WARS WDR1 Westenfelder, C. (2005) Am J Physiol Renal Physiol 289, WNTSA F31-42 WNTSB 11. Patschan, D., Plotkin, M., and Goligorsky, M.S. (2006) XPO1 60 Curr Opin Pharmacol 6, 176-183 YKT6 12. Miyahara, Y., Nagaya, N., Kataoka, M., Yanagawa, B., YWHAB Tanaka, K., Hao, H., Ishino, K., Ishida, H., Shimizu, T., YWHAE YWHAG Kangawa, K., Sano, S., Okano, T., Kitamura, S., and Mori, H. YWHAH (2006) Nat Med 12, 459-465 YWHAQ 65 13. Gnecchi, M., He, H., Noiseux, N., Liang, O. D., Zhang, L., Morello, F. Mu, H., Melo, L.G., Pratt, R.E., Ingwall, J.S., and Dzau, V.J. (2006) FasebJ 20, 661-669 US 9,029, 146 B2 93 94 14. Gnecchi, M., He, H., Liang, O. D., Melo, L. G., 46. Proud, C. G. (2004) Cardiovasc Res 63, 403-413 Morello, F. Mu, H., Noiseux, N., Zhang, L., Pratt, R. E., 47. Tee, A. R., and Blenis, J. (2005) Semin Cell Dev Biol Ingwall, J. S., and Dzau, V.J. (2005) Nat Med 11,367–368 16, 29-37 15. Mayer, H., Bertram, H., Lindenmaier, W., Korff, T., Weber, H., and Weich, H. (2005) J Cell Biochem 95,827-839 5 Example 16 16. Nakagami, H., Maeda, K., Morishita, R., Iguchi, S., Nishikawa, T., Takami, Y., Kikuchi, Y., Saito, Y., Tamai, K., Biological Activity in Conditioned Media (CM) of Ogihara, T., and Kaneda, Y. (2005) Arterioscler Thromb Vasc ES Cells Biol 25, 2542-2547 17. Van Overstraeten-Schlogel, N. Beguin, Y., and Gothot, 10 A major consideration is the consistent and reproducible production of biologically active secretory factors from the A. (2006) Eur J. Haematol 76, 488-493 MSCs. Additionally, the spectrum of secreted gene products 18. Cheng, L., Qasba, P., Vanguri, P., and Thiede, M. A. must provide a molecular basis for the reported paracrine (2000) J Cell Physiol 184,58-69 effects of MSCs on different diverse cellular systems and 19. Liu, C. H., and Hwang, S. M. (2005) Cytokine 32, 15 diseases A7-18. 270-279 By performing multidimensional protein identification 20. Haynesworth, S. E., Baber, M. A., and Caplan, A. I. technology (Mul)PIT) A23 and cytokine antibody array (1996) J Cell Physiol 166,585-592 analysis on a chemically defined culture media conditioned 21. Kinnaird, T., Stabile, E., Burnett, M.S., and Epstein, S. by the HuES9.E1 MSC and a control media consisting of only E. (2004) Circ Res 95, 354-363 chemically defined culture media, we have identified 201 22. Barreau, C., Paillard, L., and Osborne, H. B. (2005) unique gene products (see above Examples). As an estimate Nucleic Acids Res 33, 7138-7150 of the biological activity in the conditioned media (CM) we 23. Espel, E. (2005) Semin Cell Dev Biol 16, 59-67 tested the CM for MMP gelatinase activity that was predicted 24. Bhatia, R., and Hare, J. M. (2005) Congest Heart Fail by proteome analysis 11, 87-91; quiz 92-83 25 FIG. 10 shows MMP gelatinase activity in ES cell condi 25. Mauney, J. R., Volloch, V., and Kaplan, D. L. (2005) tioned medium. A Zymography using 0.1 ug protein con Tissue Eng 11, 787-802 firmed that MMP gelatinase activity, possibly MMP2 or 26. Zimmet, J. M., and Hare, J. M. (2005) Basic Res Car MMP9, is present in the CM but not the non-conditioned diol 100, 471-481 media (NCM) control (A24). 27. Lin, Z. Willers, C., Xu, J., and Zheng, M. H. (2006) 30 Tissue Eng Example 1.6A 28. Negoro, S. Kunisada, K., Tone, E., Funamoto, M. Oh, H. Kishimoto, T., and Yamauchi-Takihara, K. (2000) Cardio Biological Activity in Conditioned Media (CM) of vasc Res 47, 797-805 ES Cells 29. Ward, A. C., Touw, I., and Yoshimura, A. (2000) Blood 35 95, 19-29 A. We demonstrate that the TGF-beta protein present in the 30. Ungureanu, D., and Silvennoinen, O. (2005) Sci STKE secretome is biologically active. Exposure of THP-1 cells to 2005, pe49 the secretome induced increased phosphorylation of Smad3. 31. Blair, H. C., Robinson, L. J., and Zaidi, M. (2005) a downstream target of TGF-beta signalling (Euler-Taimor, Biochem Biophys Res Commun 328,728-738 40 G., and J. Heger. 2006. The complex pattern of SMAD sig 32. Malemud, C.J. (2004) Clin Orthop Relat Res, S145 naling in the cardiovascular system. Cardiovascular Research 151 69:15-25). This is shown in FIG. 15. 33. Nigro, J. Osman, N., Dart, A. M., and Little, P. J. FIG. 15 shows THP-1 cells that are exposed to 100 ug CM (2006) Endocr Rev 27, 242-259 proteins or NCM proteins or PBS for 30 mins. Cell lysate is 34. Pandya, N., Santani, D., and Jain, S. (2005) Cardiovasc 45 then prepared and analyzed by standard western blot hybrid Drug Rev 23, 247-254 ization using anti-phosphorylated Smad3 antibody and a 35. Berenbaum, F. (2004) Curr Opin Rheumatol 16, 616 chemiluminescence-based detection system. The signals 622 were quantitated by densitometry and the relative intensity of 36. Platanias, L. C. (2003) Blood 101, 4667-4679 the signals was estimated. 37. de Kleijn, D., and Pasterkamp, G. (2003) Cardiovasc 50 B. We have also demonstrated that the conditioned media Res 60, 58-67 can induce cell chemotaxis, a process that is predicted by 38. Ozato, K., Tsujimura, H., and Tamura, T. (2002) Bio computer analysis to be influenced by the CM (Example 11 techniques Suppl. 66-68, 70, 72 passim and Example 13). 39. Euler-Taimor, G., and Heger, J. (2006) Cardiovascular CM induces migration of human umbilical cord endothe Research 69, 15-25 55 lial cells (HUVECs) through membrane with 5uM pore size. 40. Bertolino, P., Deckers, M., Lebrin, F., and ten Dijke, P. This is shown in FIG. 16. The CM also induced similar (2005) Chest 128,585S-590S migration of THP1, a monocytic cell line and primary human 41. Bobik, A. (2006) Arterioscler Thromb Vasc Biol macrophages. 42. Ruscetti, F. W., Akel, S., and Bartelmez, S. H. (2005) FIG.16 shows 1x105 HUVECs that are placed in the upper Oncogene 24, 5751-5763 60 chamber of Transwell(R)plates with membrane pore size of 5 43. Janssens, K., ten Dijke, P.Janssens, S., and Van Hul, W. uM. The lower chamber is filled with RPMI media with 10x. (2005) Endocr Rev 26,743-774 50x, 100x dilution of a 25x concentrated CM or NCM stock 44. Faler, B. J., Macsata, R. A., Plummer, D., Mishra, L., solution. The positive and negative controls were RPMI with and Sidawy, A. N. (2006) Perspect Vasc Surg Endovasc Ther 15% and 0% fetal calf serum (FCS), respectively. After 24 18, 55-62 65 hours at 37° C. in CO2 incubator, the number of cells that 45. Wullschleger, S., Loewith, R., and Hall, M. N. (2006) migrated to the lower chamber was counted. The number of Cell 124, 471-484 cells in the chamber with 10x dilution of the concentrated CM US 9,029, 146 B2 95 96 was normalized to 100. The number of cells that have Prior to induction of the infarct, echocardiography is per migrated in the other chambers was expressed relative to that formed. In the ischemia reperfusion model, sutures will first in the 10x dilution of the concentrated CM. be tightened around the proximal left circumflex coronary C. The prediction that the CM has biological effects on artery (LCx) to induce an occlusion for 45 minutes before apoptosis (above) is tested by incubating CEM cells with removing for reperfusion to take place. Two groups of 10 hydrogen peroxide (H2O) to induce oxidative stress-induced animals are used. The test group will get the paracrine secre apoptosis in the presence of CM or NCM and determining the tion i.e. media conditioned by hESC-MSCs and the control number of remaining viable cells after 12 or 24 hours. CM group will get the non-conditioned control media. In each pig, significantly reduced oxidative stress-induced apoptosis. the medium is delivered directly into the three major coronary This is shown in FIG. 17. 10 arteries. First a bolus of 1 ml is given into the LCX after FIG. 17 shows 6 CEM cells incubated with either 50 or 500 removing of the sutures around the LCX after 90 minutes and uM H2O2 in the presence of 1xCM or 10xCM. The relative a bolus of 1 ml each in the RCA and LAD. Three osmotic number of viable cells was determined at 12 and 24 hours by trypan blue staining. pumps (Alzec) each with 2 ml of medium is implanted in 15 chest and a cathether is shuttled from the pump directly to Example 17 each of the arteries (LCX, RCA and LAD). The pumps will deliver the medium (10 ul/h) for 7 days after implantation into Cardioprotective Effect in Mouse Model of Acute the coronary arteries. Each pig will therefore receive locally 9 Myocardial Infarction (AMI) ml of medium. Survival is three weeks. Other routes of administration are: 1) each pig will receive a bolus of CM or AMI is induced in mice by permanent ligation of the left NCM (3 ml) intramyocardially in the peri-infarct area imme anterior descending coronary artery as previously described diately after ischemia-repeferfusion followed by IV admin A57. istration of 6 ml for the next 72 hours; 2) intracoronary admin 100 ul of 10x concentrated CM or NCM (control) made as istration of 3 ml CM or NCM by catheter immediately after described above is then administered to the mice via an 25 ischemia-repeferfusion followed by IV administration of 6 osmotic pump placed at the jugular vein over the next 72 ml for the next 72 hours. Internal defibrillation with 50 Joules hours. Heart function in these mice is assessed by MRI three is used if VF occurs. At implantation, measurement of cardiac weeks later. Mice treated with CM have a significantly output, mean arterial pressure, left ventricle pressure and reduced mortality rate (FIG.11A) and improved left ventricu echocardiography is performed before and after myocardial lar ejection fraction (FIG. 11B). 30 infarction. At termination this is extended with PV loops and measurement of infarct size. After stabilization of hemody Example 18 namics and heart rhythm, the thorax is closed and the animals are allowed to recover in the stable. Cardioprotective Effect in Porcine Model of Evaluation Ischemia-Repferfusion 35 Three weeks after induction of the myocardial infarction, the animals are anesthetized again and their sternums re The conditioned media will have a reparative effect on the opened. Echocardiography and conductance catheter based heart of a clinically more relevant porcine model of ischemia pressure-volume recordings are measured to assess cardiac repferfusion and improve heart function. This model and time function and geometry. After functional measurements, the is comparable with the myocardial infarction patient that 40 heart is explanted for laboratory analysis. arrives on average at 90 minutes after infarction in the hospi Hemodynamics tal where the occluded artery will be opened and a stent The electrocardiogram, arterial pressure, cardiac output placed. and left ventricular pressure (LVP) is digitized at a minimal Anesthesia sampling rate of 300 Hz, and stored for off-line analysis After an overnight fast, the pigs are sedated with ketamine 45 (Sonometrics Corporation, Ontario, Canada). (10 mg/kg i.m.) and anesthetized with thiopental (4 mg/kg, Echocardiography i.v.) before they are intubated and connected to a respirator for Short axis epicardial ultrasound (Prosound SSD-5000, 5 intermittent positive pressure ventilation with a mixture of MHz probe UST-5280-5, Aloka Holding Europe AG, Zug, oxygen and air (1:1 V/v). A venous catheter is placed in an ear Switzerland) is obtained at the mid-papillary level. Wall vein for continuous administration of saline and anesthetic 50 thickness of the infarct area (WT) and left ventricular internal drugs. Anesthesia is maintained by continuous infusion of areas (LVia) is measured at end-diastole (ED) and end-systole midazolam (0.3 mg/kg/h, i.v.), while analgesia is obtained by (ES). Systolic wall thickening (SWT) is calculated as (WT continuous infusion of Sufentanylcitrate (1 lug/kg/h, i.v.) and (ED)-WT(ES))/WT(ED)*100(%) and fractional area short muscle relaxation by infusion of pancuronium bromide (0.1 ening FAS as (LVia(ED)-LVia(ES))/LVia(ED)*100(%). mg/kg/h, i.v.). Prior to the Surgery 160 mg Sotalol is also 55 Conductance Catheter Protocol infused intravenously in 30 minutes to prevent cardiac The conductance catheter method provides a continuous arrhythmias. online measurement of LV volume and LV pressure and has Myocardial Infarction and Operational Procedure been performed as described previously A58, A59. Con During the entire operation, electrocardiogram, arterial tinuous LV pressure and Volume signals derived from the pressure and capnogram is continuously monitored. After 60 conductance catheter is displayed and acquired at a 250 HZ median sternotomy, a pacing lead is introduced into the right sampling rate using a Leycom CFL-512 (CD Leycom, atrium through a small hole in the right auricle to enable Zoetermeer, the Netherlands). Data is acquired during steady measurements at fixed heart rates. Left ventricular pressure state and during temporal caval vein occlusion, all with the (LVP) is measured using a pressure tipped Millar catheter that ventilator turned off at end-expiration. Acquisition is per is inserted through the apex into the left ventricle. A transonic 65 formed at a fixed atrial pacing rate of 80 beats/min. Analysis flow probe (Transonic Systems Inc, Ithaca, N.Y., USA) is of the pressure Volume loops is performed using custom placed around the proximal aorta to measure cardiac output. software. End-diastole is defined as the onset of rapid US 9,029, 146 B2 97 98 increase in left ventricular pressure and end-systole at the diac function under pharmacologically induced stress by maximal rate of decrease in left ventricular pressure (dP/ intravenous infusion of the B1-adrenergic receptor agonist dtmin). dobutamine (2.5 and 5.0 microg/kg/min) to challenge Infarct Size stunned myocardium. After explantation of the heart, the LV is isolated and cut Results into 5 slices from apex to base. The slices are incubated in 1% The results of the abbreviated experiment are shown in triphenyltetrazolium chloride (TTC, Sigma-Aldrich Chemi FIGS. 18, 19 and 20. cals, Zwijndrecht, Netherlands) in 37° C. Sörensen buffer FIG. 18. Relative infarct size after acute ischemia-reperfu (13.6 g/l KHPO+17.8 g/l NaH PO2H.O, pH 7.4) for 15 sion in pigs treated with CM, NCM or saline. Acute ischemia minutes to discriminate infarct tissue from viable myocar 10 was induced by ligation of LCX for 75 minutes followed by dium. All slices were scanned from both sides and in each release of ligation for reperfusion. Five minutes before the slide the infarct area was compared to total area using digital onset of reperfusion, the pigs were treated intravenously with planimetry software. After correction for the weight of the CM, NCM or saline. Immediately after reperfusion, CM slices, infarct size was calculated as a percentage of the LV. NCM or saline was infused locally into the LCx coronary At implantation, measurement of cardiac output, mean 15 artery. Four hours after reperfusion, relative infarct size in the arterial pressure, left ventricle pressure and echocardio area at risk was assessed. graphy is performed before and after myocardial infarction. FIGS. 19 and 20. Echocardiography At termination this is extended with PV loops and measure FIG. 19: Systolic wall thickening. FIG. 20. Fractional area ment of infarct size. shortening. Cardiac Measurements were performed before ischemia, Example 18B during ischemia and 4 hours after ischemia. Four hours after ischemia, additional measurements were performed under Cardioprotective Effect in Porcine Model of pharmacologically induced stress by intravenous infusion of Ischemia-Repferfusion the B1-adrenergic receptor agonist dobutamine (2.5 and 5.0 25 microg/kg/min) to challenge stunned myocardium. Short An “abbreviated' version of Example 18 is performed. The axis epicardial ultrasound images (Prosound SSD-5000, experiment is essentially similar with the major difference 5-MHz probe UST-5280-5, Aloka Holding Europe AG, Zug, being that the endpoint was reduced from three weeks to a Switzerland) were obtained at the midpapillary level. Wall shorter 4 hours. The procedure is identical apart from the thickness (WT) of the infarct area and LV internal area (LVia) following modification: 30 were measured at end diastole (ED) and end systole (ES). Myocardial Infarction and Operational Procedure Systolic wall thickening (SWT) was calculated as (WT During the entire operation, electrocardiogram, arterial (ES)-WT(ED))/WT(ED)*100% and fractional area short pressure and capnogram is continuously monitored. After ening (FAS) as (LVia(ED)-LVia(ES))/. median sternotomy, a pacing lead is introduced into the right Conductance Catheter Protocol to Measure PV Loop atrium through a small hole in the right auricle to enable 35 The conductance catheter method provides a continuous measurements at fixed heart rates. Left ventricular pressure online measurement of LV volume and LV pressure and was (LVP) is measured using a pressure tipped Millar catheter that performed as described previously (Timmers, L., J. P. G. is inserted through the apex into the left ventricle. A transonic Sluijter, C. W. J. Verlaan, P. Steendijk, M. J. Cramer, M. flow probe (Transonic Systems Inc, Ithaca, N.Y., USA) is Emons, C. Strijder, P. F. Grundeman, S. K. Sze, L. Hua, J. J. placed around the proximal aorta to measure cardiac output. 40 Piek, C. Borst, G. Pasterkamp, and D. P. V. de Kleijn. 2007. Prior to induction of the infarct, echocardiography is per Cyclooxygenase-2 Inhibition Increases Mortality, Enhances formed. In the ischemia reperfusion model, sutures will first Left Ventricular Remodeling, and Impairs Systolic Function be tightened around the proximal left circumflex coronary After Myocardial Infarction in the Pig. Circulation 115:326 artery (LCx) to induce an occlusion for 75 minutes before 332). removing for reperfusion to take place. Three groups of ani 45 LV pressure and Volume signals derived from the conduc mals were used. The test group were given the CM i.e. media tance catheter were displayed and acquired at a 250-Hz Sam conditioned by hESC-MSCs and the control groups were pling rate with a Leycom CFL-512 (CDLeycom, Zoetermeer, given either non-conditioned control media or saline. Five the Netherlands). Data were acquired during steady state and minutes before the onset of reperfusion, the pigs were treated during temporal caval vein occlusion, all with the ventilator intravenously with MSC-CM (2.0 mg protein, 1 ml), NCM or 50 turned off at end expiration. Analysis of the pressure-volume saline. Immediately after reperfusion, MSC-CM (4 ml, 8.0 loops was performed with custom Software. mg protein, N=7), NCM (N=5) or saline (N=7) was infused locally into the LCx coronary artery. The pigs were sacrificed Example 19 4 hours after reperfusion. Relative infarct size in the area at risk was assessed using Evans Blue and TTC staining. It was 55 Therapeutic Effect of Conditioned Media in markedly reduced in the pigs treated with CM relative to those Dermatologic Disorders treated with NCM (p=0.024) and treated with saline (p=0.007) (FIG. 7). Functional cardiac functions such as Sys The molecular composition of the CMby hESC-MSCs and tolic Wall Thickening (SWT) and Fractional Area Shortening the computational predictions of the collective functions of (FAS) were measured by Echocardiography (FIG. 8) while 60 the CM suggest that the CM induce reparative effects on continuous online measurement of LV Volume and LV pres cutaneous wounds or dermatological lesions disorders such Sure will be measured using a conductance catheter method. as dermatitis or psoriasis. Many of the molecular components At baseline and during ischemia, SWT and FAS in the three and the biological processes that are driven by the CM have experimental groups of pigs were not significantly different. been either used or targeted in the treatment of cutaneous However at 4 hours after reperfusion, SWT and FAS in CM 65 wound healing and dermatological disorders. treated pigs were significantly better than NCM and saline Specifically, secreted cyokines such as interferon-gamma treated animals. CM-treated pigs also exhibited Stronger car and interleukins-2 and -10 have been used in the in the treat US 9,029, 146 B2 99 100 ment of condyloma and Verruca, hemangiomas, keloids, skin hours. Following bronchoalveolar lavage collection, lungs cancers, atopic dermatitis, psoriasis, Behcet disease, chronic are fixed with 10% neutralized formalin. granulomatous disease, wound healing, and cutaneous T cell To investigate the effects of CM and the NCM on acute lymphoma A60. Other secreted proteins such as ECM pro airway inflammation and airway tein including the collagens, modulators of ECM e.g. MMPs A Blott 5 specific Th2 cell line which secretes high level of and their inhibitors. TIMPs, angiogenic factors e.g. VEGF, IL-4, IL-5, IL-13 and with undetectable level of IFN-Y, is used PIGF, growth factors e.g. TGF-3, EGF have also been impli to establish a mouse allergy model. Briefly, sensitization of cated in wound healing scar reduction ulceration A61-67. naive mice is done by adoptive transfer of 2.5x10 Blot 5 Several of the signaling pathways that may be modulated by specific Th2 cells intravenously in each mouse. These mice the CM of hESC-MSC have been implicated in many derma 10 are anesthetized and intranasal (IN) challenged with 50 ug of tological disorders e.g. MAPF signaling pathway is impli Blot 5 for three consecutive days. Twenty-four hours after the cated in psoriasis A68, A69 modulation of IGF axis through last IN challenge, airway hyperresponsivensess (AHR) is IGF and IGFBPs in psoriasis A70), Toll signaling in derma tologic disease A71. measured using invasive BUXCO A79. The mice are then Therefore the topical application of CM on cutaneous 15 anesthetized and given CM or NCM intranasally for three wounds or dermatological lesions or disorders such as der consecutive days. BAL fluid is collected at forty-eight hours matitis or psoriasis improves healing and reduce Scarring. It after the last Blot 5 challenge. Following bronchoalveolar should also maintain homeostasis. The CM may be delivered lavage collection, lungs are fixed with 10% neutralized for in liposome-based emulsion, gel or cream formulations and malin for histopathological analysis. part of standard wound dressing. The CM may also be used as Inclinical practice, the use of CM to treat lung disease may a Supplement in cosmetic skincare product to promote skin be administered effectively using standard aerosol therapy repair and healing. A80-86. A suitable animal model to test the efficacy of the CM on cutaneous disorders is a mouse model of dermatitis. Epicuta Example 21 neous sensitization of mice is performed as described earlier 25 A72, A73). Briefly, 50g of Blot 5 in 100 ul of PBS or PBS Therapeutic Effect of Conditioned Media in Other alone are applied to 1 cm gauze and patched to the skin with Diseases a transparent dressing (Smith Nephew). This procedure is repeated twice over a period of 50 days. CM and NCM are In general, we predict that the CMofhESC-MSCs is useful then applied to 1 cm2 gauze and patched to the skin as 30 in restoring homeotstasis and promoting tissue repair in described above for varying period of time. pathological conditions where the initial injury induced For histological examination of skin inflammation, speci inflammation and immune dysregulation leads to chronic mens are obtained from the patched skins and fixed in 10% tissue remodeling that includes fibrosis and loss of function. buffered neutral formalin immediately. 35 Other diseases include renal ischemic injury, cystic fibrosis, Example 20 sinusitis and rhinitis. Example 22 Therapeutic Effect of Conditioned Media in Asthma and Allergy 40 Therapeutic Effect of Conditioned Media in Asthma is a complex disease with an equally complex Orthopedics etiology caused by a poorly characterized set of genetic and environmental factors. The resulting pathology is immune Current therapeutic strategies for repair of musculoskeletal dysregulation leading to chronic inflammation of the airways tissue often include the use of a biomaterial (ceramics or and subepithelial fibrosis characterized by increase in smooth 45 polymers) not only to provide mechanical Support but also as muscle mass and increased deposition of extracellular matrix a scaffold to promote cell migration, cell adhesion, prolifera proteins and Subsequently, reduced lung function. tion and differentiation to initiate vascularization and ulti Current therapies include modulating several factors or mately new bone formation A87-90. Based on the compu signaling pathways e.g. the ECM, integrins, and mesenchy tation analysis of CM by hESC-MSC, incorporation of CM mal cell function A74, toll-like receptors A75, growth 50 into the scaffold design may enhance cell migration, prolif factors such as TGF-B and EGF LAT6, A77 and the IL6 eration, adhesion, skeletal differentiation and vascularization pathway A78). The CM by hESC-MSCs has been predicted of the scaffold. to have biological effects on these targeted area and we pre To test the effect of CMonbone regeneration indefects that dict that the CM helps restore immune regulation in asthmatic would otherwise have led to atrophic nonunions, New lungs and promote tissue repair and minimize scarring of lung 55 Zealand white rabbits receive a 15-mm critical size defect on tissues. one radius A91, which is filled with a suitable matrix such as To investigate the effects of CM and the NCM on chronic a collagen sponge or hydrogel coated with either CM or airway inflammation and airway, epicutaneous sensitization NCM. Radiographs are obtained every 3 weeks. After 6 or 12 of mice are performed as described above A72, A73. After weeks, animals are killed. New bone is measured by microCT 50 days, the patched mice are anesthetized and receive intra 60 scans and vascularity is measured using anti-CD31 staining nasal challenge with 50 lug of Blot 5 for three consecutive of endothelial cells in the implant. There should be increased days. Twenty-four hours after the last dose, airway hyperre vascularity at the least initially and also increased new bone sponsivensess (AHR) is measured using invasive BUXCO formation. A79. The mice are anesthetized and given CM or NCM To test the effects of CM on cartilage repair, a rabbit model intranasally for three consecutive days. Twenty-four hours 65 of osteochondral injury A92 is used. CM is coated on a after the last dose, airway hyperresponsivensess (AHR) is Suitable scaffold such as collagen or gydrogel and implanted measured. BAL fluid is collected after another twenty-four into 3-mm osteochondral knee defects A93. For clinical