EARLY MESODERM CELLS, A STABLE POPULATION OF MESENDODERM CELLS THAT HAS UTILITY FOR GENERATION OF ENDODERM AND MESODERM LINEAGES AND MULTIPOTENT MIGRATORY CELLS (MMC)

• US 20100166713A1
• Filed: 01/30/2008
• Published: 07/01/2010
• Est. Priority Date: 01/30/2007
• Status: Active Grant

• Current Assignee: The University of Georgia Research Foundation, Inc.
• Original Assignee: David Reynolds, Stephen Dalton
• Inventors: Reynolds, David, Dalton, Stephen
• Granted Patent: US 9,175,260 B2
• Time in Patent Office: Days
• US Class Current: 424/93.700
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2500/99 Serum-free medium
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/195 Heregulin, neu differentiation factor
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696

Abstract

The present invention relates to the production of multipotent migratory cell (MMCs) which can be differentiated into mesoderm or endoderm lineages. Multipotent Migratory Cells (MMC) are stable and robust and can be passaged at least 20 times (perhaps indefinitely), can be recovered after freezing, reamplified and differentiated into multiple lineages. They are therefore storage stable. The method of producing these cells points to a way to generate a multipotent cell type (mesendoderm) from blastocycts for the generation of therapeutically useful cell types without going through a classical hESC state. The production of multipotent migratory cells, mesendoderm cells and mesoderm cells (Isl1+) is also described.

Claims

1. A method of differentiating primate Pluripotent Stem Cells (pPSCs) into mesendoderm cells comprising (a) providing pPSCs and (b) contacting the pPSCs with an effective amount of a GSK inhibitor in a cell differentiation medium for a period of at least about 18 hours to produce mesendoderm cells, and (c) optionally, isolating said mesendoderm cells.

2. The method according to claim 1 wherein said pPSCs are human embryonic stem cells (hESCs).

3. The method according to claim 1 wherein said GSK inhibitor is selected from the group consisting of

BIO (2′

Z,3′

E)-6-Bromoindirubin-3′

-oxime (GSK3 Inhibitor IX),

BIO-Acetoxime (2′

Z,3′

E)-6-Bromoindirubin-3′

-acetoxime (GSK3 Inhibitor X),

(5-Methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine (GSK3-Inhibitor XIII),

Pyridocarbazole-cyclopenadienylruthenium complex (GSK3 Inhibitor XV),

TDZD-8 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (GSK3β

Inhibitor

     1),

2-Thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK3β

Inhibitor II),

OTDZT 2,4-Dibenzyl-5-oxothiadiazolidine-3-thione (GSK3β

Inhibitor III),

α

-4-Dibromoacetophenone (GSK3β

Inhibitor VII),

AR-A014418 N-(4-Methoxybenzyl)-N′

-(5-nitro-1,3-thiazol-2-yl)urea (GSK-3β

Inhibitor VIII),

3-(1-(3-Hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione (GSK-3β

Inhibitor XI),

TWS119 pyrrolopyrimidine compound (GSK3β

Inhibitor XII),

L803H-KEAPPAPPQSpP-NH₂ or its Myristoylated form (GSK3β

Inhibitor XIII),

2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone (GSK3β

Inhibitor VI), and mixtures thereof.

4. The method according to claim 1 wherein said GSK inhibitor is a Wnt protein.

5. The method according to claim 4 wherein said Wnt protein is selected from the group consisting of Wnt1, Wnt2, Wnt3, Wnt3a, Wnt4, Wnt10, Wnt 14, Wnt14b, Wnt15, Wnt16 and mixtures thereof.

6. The method according to claim 1 wherein said GSK inhibitor is BIO (2′

Z,3′

E)-6-Bromoindirubin-3′

-oxime (GSK3 Inhibitor IX).

7. The method according to any of claim 4

wherein said Wnt protein is Wnt 3a.

8. The method according to claim 1 wherein said

contacting step occurs for a period ranging from about 18 to 72 hours.

9. The method according to claim 1 wherein said cell differentiation medium is at least a minimum essential medium plus one or more optional components selected from the group consisting of growth factors, ascorbic acid, glucose, non-essential amino acids, salts (including trace elements), glutamine, insulin, Activin A, transferrin and beta mercaptoethanol.

10. The method according to claim 1 wherein said cell differentiation medium is selected from the group consisting of Dulbecco'"'"'s modified Eagle'"'"'s medium (DMEM), Knockout Dulbecco'"'"'s modified Eagle'"'"'s medium (KO DMEM) and Ham'"'"'s F12/DMEM basal medium (50:

50) further including albumin, antibiotics, trace minerals, ascorbic acid, β

-mercaptoethanol, fibroblast growth factor (FGF) and insulin-like growth factor analog (LR-IGF).

11. The method according to claim 1 wherein said cell differentiation medium is DMEM/F12 (50:

50) containing effective amounts of proalbumin, pen/strep antibiotic, non-essential amino acids, trace minerals, ascorbic Acid, β

-Mercaptoethanol, fibroblast growth factor (FGF), insulin-like growth factor analogue (LR-IGF), Activin A and Heregulin.

12. A method of differentiating primate Pluripotent Stem Cells (pPSCs) into mesoderm (Isl1+) cells comprising (a) providing pPSCs;

(b) contacting the pPSCs with an effective amount of a GSK inhibitor in a cell differentiation medium for a period of at least about 18 hours to produce mesendoderm cells; and

(c) subsequently contacting the cells obtained from step (b) with an effective amount of a bone morphogenic protein (BMP) and optionally, a GSK inhibitor for a period of at least about 2 days to produce said mesoderm cells; and

(d) optionally, isolating said mesoderm cells.

13. The method according to claim 12 wherein said pPSCs are human embryonic stem cells (hESCs).

14. The method according to claim 12 wherein said GSK inhibitor is selected from the group consisting of

BIO (2′

Z,3′

E)-6-Bromoindirubin-3′

-oxime (GSK3 Inhibitor IX),

BIO-Acetoxime (2′

Z,3′

E)-6-Bromoindirubin-3′

-acetoxime (GSK3 Inhibitor X),

(5-Methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine (GSK3-Inhibitor XIII),

Pyridocarbazole-cyclopenadienylruthenium complex (GSK3 Inhibitor XV),

TDZD-8 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (GSK3β

Inhibitor I),

2-Thio(3-iodobenzyl)-5-(1-pyridyl)[1,3,4]-oxadiazole (GSK3β

Inhibitor II),

OTDZT 2,4-Dibenzyl-5-oxothiadiazolidine-3-thione (GSK3(3β

Inhibitor III),

α

-4-Dibromoacetophenone (GSK3β

Inhibitor VII),

AR-A014418 N-(4-Methoxybenzyl)-N′

-(5-nitro-1,3-thiazol-2-yl)urea (GSK-3β

Inhibitor VIII),

3-(1-(3-Hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione (GSK-3β

Inhibitor XI),

TWS119 pyrrolopyrimidine compound (GSK3β

Inhibitor XII),

L803H-KEAPPAPPQSpP-NH₂ or its Myristoylated form (GSK3β

Inhibitor XIII),

2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone (GSK3β

Inhibitor VI), and mixtures thereof.

15. The method according to claim 12 wherein said GSK inhibitor is a Wnt protein.

16. The method according to claim 15 wherein said Wnt protein is selected from the group consisting of Wnt1, Wnt2, Wnt3, Wnt3a, Wnt4, Wnt10, Wnt 14, Wnt14b, Wnt15, Wnt16 and mixtures thereof.

17. The method according to claim 12 wherein said GSK inhibitor is BIO (2′

Z,3′

E)-6-Bromoindirubin-3′

-oxime (GSK3 Inhibitor IX).

18. The method according to claim 15 wherein said Wnt protein is Wnt 3a.

19. The method according to claim 12 wherein said contacting step (b) occurs for a period ranging from about 18 to 72 hours.

20. The method according to claim 12 wherein said bone morphogenic protein is selected from the group consisting of BMP-2, BMP-4, BMP-6, BMP-7 and mixtures thereof.

21. The method according to claim 12 wherein said contacting step (c) occurs for a period ranging from about 3 days to about 9 days.

22. The method according to claim 12 wherein said cell differentiation medium is at least a minimum essential medium plus one or more optional components selected from the group consisting of growth factors, ascorbic acid, glucose, non-essential amino acids, salts (including trace elements), glutamine, insulin, Activin A, transferring and beta mercaptoethanol.

23. The method according to claim 12 wherein said cell differentiation medium is selected from the group consisting of Dulbecco'"'"'s modified Eagle'"'"'s medium (DMEM), Knockout Dulbecco'"'"'s modified Eagle'"'"'s medium (KO DMEM) and Ham'"'"'s F12/DMEM basal medium (50:

50) further including albumin, antibiotics, trace minerals, ascorbic acid, β

-mercaptoethanol, fibroblast growth factor (FGF) and insulin-like growth factor analog (LR-IGF).

24. The method according to claim 12 wherein said cell differentiation medium is DMEM/F12 (50:

50) containing effective amounts of proalbumin, pen/strep antibiotic, non-essential amino acids, trace minerals, ascorbic Acid, β

-Mercaptoethanol, fibroblast growth factor (FGF), insulin-like growth factor analogue (LR-IGF), Activin A and Heregulin.

65. A population of isolated mesoderm (Isl+) cells obtained by the method of any of claims 12-24 and 39-51 having the following characteristics:

a) express Isl1, Tbx20, Nkx2.5, Fgf10, GATA4, KDR (Flk1), FoxF1, PDGFRα

b) karyotypically normal;

c) do not express Oct4, Nanog, T, eomesodermin;

d) can differentiate into cardiomyocytes, smooth muscle cells and endothelial cells.

66. A population of cryopreserved mesoderm (Isl+) cells obtained by the method of claim 12.

67. A population of cardiomyocytes obtained by differentiating a mesoderm (Isl+) cell obtained by the method of claim 12.

25. A method of producing multipotent migratory cells (MMCs) comprising (a) providing pPSCs;

(b) contacting the pPSCs with an effective amount of a GSK inhibitor in a cell differentiation medium in combination with at least one additional agent selected from the group consisting of an Activin A signaling inhibitor, an inhibitor of bone morphogenic protein and mixtures thereof for a period of at least about 3 days to produce multipotent migratory cells (MMCs); and

(d) optionally, isolating said multipotent migratory cells.

26. The method according to claim 25 wherein said pPSCs are human embryonic stem cells (hESCs).

27. The method according to claim 25 wherein said GSK inhibitor is selected from the group consisting of

BIO (2′

Z,3′

E)-6-Bromoindirubin-3′

-oxime (GSK3 Inhibitor IX),

BIO-Acetoxime (2′

Z,3′

E)-6-Bromoindirubin-3′

-acetoxime (GSK3 Inhibitor X),

(5-Methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine (GSK3-Inhibitor XIII),

Pyridocarbazole-cyclopenadienylruthenium complex (GSK3 Inhibitor XV),

TDZD-8 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (GSK3β

Inhibitor I),

2-Thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK3β

Inhibitor II),

OTDZT 2,4-Dibenzyl-5-oxothiadiazolidine-3-thione (GSK3β

Inhibitor III),

α

-4-Dibromoacetophenone (GSK3β

Inhibitor VII),

AR-A014418 N-(4-Methoxybenzyl)-N′

-(5-nitro-1,3-thiazol-2-yl)urea (GSK-3β

Inhibitor VIII),

3-(1-(3-Hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione (GSK-3β

Inhibitor XI),

TWS119 pyrrolopyrimidine compound (GSK3β

Inhibitor XII),

L803H-KEAPPAPPQSpP-NH₂ or its Myristoylated form (GSK3β

Inhibitor XIII),

2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone (GSK3β

Inhibitor VI), and mixtures thereof.

28. The method according to claim 25 wherein said GSK inhibitor is a Wnt protein.

29. The method according to claim 28 wherein said Wnt protein is selected from the group consisting of Wnt1, Wnt2, Wnt3, Wnt3a, Wnt4, Wnt10, Wnt 14, Wnt14b, Wnt15, Wnt16 and mixtures thereof.

30. The method according to claim 25 wherein said GSK inhibitor is BIO (2′

Z,3′

E)-6-Bromoindirubin-3′

-oxime (GSK3 Inhibitor IX).

31. The method according to claim 28 wherein said Wnt protein is Wnt 3a.

32. The method according to claim 25 wherein said contacting step (b) occurs for a period ranging from about 4 to 12 days.

33. The method according to claim 25 wherein said Activin A signaling inhibitor is selected from the group consisting of SB-431542, follistatin, follistatin gene related protein (FGRP), Bone Morphogenic Protein and Activin Membrane Bound Inhibitor (BAMBI), anti-BAMBI monoclonal antibody, Smad7 (Mothers Against Decapentaplegic Homolog 7), TGF RI inhibitor and mixtures thereof.

34. The method according to claim 25 wherein said bone morphogenic protein inhibitor is selected from the group consisting of noggin, sclerostin, gremlin (Drm/Gremlin), USAG-1, and mixtures thereof.

35. The method according to claim 25 wherein said contacting step (b) occurs for a period ranging from about 4 days to about 9 days.

36. The method according to claim 25 wherein said cell differentiation medium is at least a minimum essential medium plus one or more optional components selected from the group consisting of growth factors, ascorbic acid, glucose, non-essential amino acids, salts (including trace elements), glutamine, insulin, Activin A, transferrin and beta mercaptoethanol.

37. The method according to claim 25 wherein said cell differentiation medium is selected from the group consisting of Dulbecco'"'"'s modified Eagle'"'"'s medium (DMEM), Knockout Dulbecco'"'"'s modified Eagle'"'"'s medium (KO DMEM) and Ham'"'"'s F12/DMEM basal medium (50:

50) further including albumin, antibiotics, trace minerals, ascorbic acid, β

-mercaptoethanol, fibroblast growth factor (FGF) and insulin-like growth factor analog (LR-IGF).

38. The method according to claim 25 wherein said cell differentiation medium is DMEM/F12 (50:

50) containing effective amounts of proalbumin, pen/strep antibiotic, non-essential amino acids, trace minerals, ascorbic Acid, β

-Mercaptoethanol, fibroblast growth factor (FGF), insulin-like growth factor analogue (LR-IGF), Activin A and Heregulin.

61. A multipotent migratory cell population produced by a method according to claim 25 having the following characteristics:

a) The cells are multipotent and self renewing;

a. The cells can be differentiated into multiple cell types including endoderm and mesoderm;

c) The cells are dynamic cells which can alternate between MMCs (E-cad−

Oct4−

Nanog−

SSEA3−

CXCR4+) and an alternative cell type which his E-cad+Oct4+Nanog+SSEA3−

CXCR4+ (high density (epithelial sheet))−

have significant developmental plasticity;

d) The cells are not hESC'"'"'s based upon marker profiling.

63. A cryopreserved population of MMCs produced according to the method of claim 25.

39. A method of producing mesoderm (Isl1+) cells from multipotent migratory cells comprising a) providing a population of multipotent migratory cells;

b) contacting said multipotent migratory cells with an effective amount of a bone morphogenic factor in a cell differentiation medium, optionally in combination with a GSK inhibitor for a period of at least about 2 days to produce mesoderm (Isl1+) cells;

+ and c) optionally isolating said mesoderm (Isl1+) cells.

40. The method according to claim 39 wherein said cell differentiation medium contains an absence of an Activin A inhibitor and an absence of an inhibitor of bone morphogenic protein.

41. The method according to claim 39 wherein said MMCs are obtained from human embryonic stem cells contacted with a cell differentiation medium comprising an effective amount of a GSK inhibitor in combination with an Activin A inhibitor and/or a bone morphogenic protein inhibitor.

42. The method according to claim 39 wherein said GSK inhibitor is selected from the group consisting of

BIO (2′

Z,3′

E)-6-Bromoindirubin-3′

-oxime (GSK3 Inhibitor IX),

BIO-Acetoxime (2′

Z,3′

E)-6-Bromoindirubin-3′

-acetoxime (GSK3 Inhibitor X),

(5-Methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine (GSK3-Inhibitor XIII),

Pyridocarbazole-cyclopenadienylruthenium complex (GSK3 Inhibitor XV),

TDZD-8 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (GSK3β

Inhibitor I),

2-Thio(3-iodobenzyl)-5-(1-pyridyl)[1,3,4]-oxadiazole (GSK3β

Inhibitor II),

OTDZT 2,4-Dibenzyl-5-oxothiadiazolidine-3-thione (GSK3β

Inhibitor III),

α

-4-Dibromoacetophenone (GSK3β

Inhibitor VII),

AR-A014418 N-(4-Methoxybenzyl)-N′

-(5-nitro-1,3-thiazol-2-yl)urea (GSK-3β

Inhibitor VIII),

3-(1-(3-Hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione (GSK-3β

Inhibitor XI),

TWS119 pyrrolopyrimidine compound (GSK3β

Inhibitor XII),

L803H-KEAPPAPPQSpP-NH₂ or its Myristoylated form (GSK3β

Inhibitor XIII),

2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone (GSK3β

Inhibitor VI), and mixtures thereof.

43. The method according to claim 39 wherein said GSK inhibitor is a Wnt protein.

44. The method according to claim 43 wherein said Wnt protein is selected from the group consisting of Wnt1, Wnt2, Wnt3, Wnt3a, Wnt4, Wnt10, Wnt 14, Wnt14b, Wnt15, Wnt16 and mixtures thereof.

45. The method according to claim 39 wherein said GSK inhibitor is BIO (2′

Z,3′

E)-6-Bromoindirubin-3′

-oxime (GSK3 Inhibitor IX).

46. The method according to claim 43 wherein said Wnt protein is Wnt3a.

47. The method according to claim 39 wherein said contacting step occurs for a period ranging from about 3 to 9 days.

48. The method according to claim 39 wherein said contacting step occurs for a period ranging from about 4 days to about 7 days.

49. The method according to claim 39 wherein said cell differentiation medium is at least a minimum essential medium plus one or more optional components selected from the group consisting of growth factors, ascorbic acid, glucose, non-essential amino acids, salts (including trace elements), glutamine, insulin, Activin A, transferrin and beta mercaptoethanol.

50. The method according to claim 39 wherein said cell differentiation medium is selected from the group consisting of Dulbecco'"'"'s modified Eagle'"'"'s medium (DMEM), Knockout Dulbecco'"'"'s modified Eagle'"'"'s medium (KO DMEM) and Ham'"'"'s F12/DMEM basal medium (50:

50) further including albumin, antibiotics, trace minerals, ascorbic acid, β

-mercaptoethanol, fibroblast growth factor (FGF) and insulin-like growth factor analog (LR-IGF).

51. The method according to claim 39 wherein said cell differentiation medium is DMEM/F12 (50:

50) containing effective amounts of proalbumin, pen/strep antibiotic, non-essential amino acids, trace minerals, ascorbic Acid, 13-Mercaptoethanol, fibroblast growth factor (FGF), insulin-like growth factor analogue (LR-IGF), Activin A and Heregulin.

52. A method of producing definitive endoderm cells from multipotent migratory cells (MMCs) comprising a) providing a population of multipotent migratory cells;

b) contacting said multipotent migratory cells with an effective amount of Activin A and optionally an inhibitor of PI3 kinase signaling for a period of at least about 2 days to produce definitive endoderm cells; and

c) optionally isolating said definitive endoderm cells.

53. The method according to claim 52 wherein said contact step occurs over a period of about 4-5 days.

54. The method according to claim 52 wherein said PI3 kinase signaling inhibitor is selected from the group consisting of Rapamycin, LY 294002, wortmannin, lithium chloride, Akt inhibitor I, Akt inhibitor II (SH-5), Akt inhibitor III (SH-6), NL-71-101, and mixtures thereof.

55. A method of producing pancreatic endoderm cells from multipotent migratory cells (MMCs) comprising a) providing a population of multipotent migratory cells;

b) contacting said multipotent migratory cells with an effective amount of Activin A and optionally an inhibitor of PI3 kinase signaling in a cell differentiation medium for a period of at least about 2 days to produce definitive endoderm cells;

c) optionally isolating said definitive endoderm cells;

d) contacting said definitive endoderm cells to an effective amount of retinoic acid and FGF10 in a cell differentiation medium for a period of at least about 24 hours to produce pancreatic endoderm cells; and

e) optionally isolating said pancreatic endoderm cells.

56. The method of claim 55 wherein said pancreatic endoderm cells are further differentiated to pancreatic β

cells by exposing said endoderm cells to an effective amount of retinoic acid and FGF10 in a cell differentiation medium for a period of about 10 to 24 days to produce pancreatic β

cells and optionally, isolating said pancreatic β

cells.

57. The method of claim 56 wherein said pancreatic endoderm cells are further differentiated to liver endoderm cells by exposing said endoderm cells to an effective amount of FGF10 in a cell differentiation medium in the absence of retinoic acid for a period of at least about 2 days to produce liver endoderm cells and optionally, isolating said liver endoderm cells.

58. The method according to claim 57 wherein said exposing step occurs for a period ranging from about 10 to about 24 days.

59. The method according to claim 57 wherein said PI3 kinase signaling inhibitor is selected from the group consisting of Rapamycin, LY 294002, wortmannin, lithium chloride, Akt inhibitor I, Akt inhibitor II (SH-5), Akt inhibitor 111 (SH-6), NL-71-101, and mixtures thereof.

60. A population of multipotent migratory cells having at least four of the following characteristics:

a) the cells can be cultured for at least 10 passages as a stable cell population;

b) the cells appear mesenchymal when plated at low density and grow into a sheet at high density;

c) the cells can be produced from a range of hESC lines including BG01, BG02, WA09;

d) the cells can be frozen and cryogenically preserved by standard methods;

e) the cells can be recovered after cryogenic storage, recovered and differentiated;

f) the cells can be passaged with high plating efficiency;

g) the cells do not exhibit the SSEA3 and SSEA4 antigens on their cell surface;

h) the cells do not express hESC markers Oct4 or Nanog;

i) the cells can express CXCR4 on their surface;

j) the cells express the following transcripts at high levels;

Zic1, Sox1, Sox2, HoxA9, HoxD4, HoxA5, HoxC10, HoxD3, Pax6, N-CAM, CXCR4

k) the cells are not mesendoderm cells but do T/brachyury or eomesodermin;

l) the cells are E-cadherin negative;

m) the cells do not express Sox17, Isl1, musashi, nestin at appreciable levels as measured by Q-PCR;

n) the cells retain a normal karyotype during passaging

o) the cells exhibit a migratory, mesenchymal phenotype

p) the cells have multipotent differentiation capacity (including mesoderm, endoderm)

q) the cells do not form teratomas when injected into SCID mice.

62. A cryopreserved population of MMCs.

64. A population of cells consisting essentially of isolated mesoderm (Isl1+) cells (mesoderm-derived Isl1+ multipotent progenitor cell) having the following characteristics:

a) express Isl1, Tbx20, Nkx2.5, Fgf10, GATA4, KDR (Flk1), FoxF1, PDGFRα

b) karyotypically normal;

c) do not express Oct4, Nanog, T, eomesodermin;

d) can differentiate into cardiomyocytes, smooth muscle cells and endothelial cells.

68. A method of method of treating myocardial infarction in a patient in need thereof comprising administering to said patient an effective number of mesoderm (Isl+) cells or cardiomyocytes.

69. The method according to claim 68 wherein said cells are administered directly into the patient'"'"'s heart.

70. A method of treating damaged or ischemic vascular tissue in a patient in need thereof comprising administering to the blood vessels to be repaired said patient an effective amount of mesoderm (Isl1+) cells.

71. A method of treating damaged or ischemic vascular tissue in a patient in need thereof comprising differentiating mesoderm (Isl1+) cells to smooth muscle cells by contacting said mesoderm (Isl1+) cells in a cell differentiation medium to an effective amount of a GSK inhibitor in combination with BMP for a period of at least about 4 days to produce smooth muscle cells;

isolating said smooth muscle cells and administering said smooth muscle cells to said damaged or ischemic vascular tissue in said patient.

72. The method according to claim 71 wherein said contacting step occurs over a period of about 5-6 days.