Differentiation of human embryonic stem cells

• US 20090170198A1
• Filed: 11/25/2008
• Published: 07/02/2009
• Est. Priority Date: 11/27/2007
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Lifescan Incorporated (Duv Holding Corporation)
• Inventors: Rezania, Alireza
• Granted Patent: US 9,062,290 B2
• Time in Patent Office: Days
• US Class Current: 435/377
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/117 Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
• CPC Class Codes: C12N 2501/12 Hepatocyte growth factor [HGF]
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• 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 2501/335 Glucagon; Glucagon-like peptide [GLP]; Exendin
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/40 Regulators of development
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/42 Notch; Delta; Jagged; Serrate
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0678 : Stem cells; Progenitor cells; Precursor cells

Abstract

The present invention provides methods to promote the differentiation of pluripotent stem cells and the products related to or resulting from such methods. In particular, the present invention provides an improved method for the formation of pancreatic hormone expressing cells and pancreatic hormone secreting cells. In addition, the present invention also provides methods to promote the differentiation of pluripotent stem cells without the use of a feeder cell layer and the products related to or resulting from such methods. The present invention also provides methods to promote glucose-stimulated insulin secretion in insulin-producing cells derived from pluripotent stem cells.

Claims

1. A method for producing cells capable of glucose-stimulated insulin secretion from pluripotent stem cells, comprising the steps of:

a. Culturing the pluripotent stem cells,

b. Differentiating the pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage,

c. Differentiating the cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage, and

d. Differentiating the cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine lineage.

2. The method of claim 1, wherein the step of differentiating the cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage is achieved by treating the cells expressing markers characteristic of the definitive endoderm lineage with at least one factor selected from the group consisting of retinoic acid, FGF-2, FGF-4, FGF-7, FGF-10, a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, and a netrin.

3. The method of claim 2, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with at least one factor selected from the group consisting of retinoic acid, FGF-2, FGF-4, FGF-7, FGF-10, a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, and a netrin for about one to about six days.

4. The method of claim 2, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with at least one factor selected from the group consisting of retinoic acid, FGF-2, FGF-4, FGF-7, FGF-10, a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, and a netrin for about six days.

5. The method of claim 2, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about three days, followed by treating the cells with a sonic hedgehog inhibitor, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about four days.

6. The method of claim 5, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about three days.

7. The method of claim 5, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about four days.

8. The method of claim 2, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about three days, followed by treating the cells with a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about four days.

9. The method of claim 8, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about three days.

10. The method of claim 8, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about four days.

11. The method of claim 2, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about three days, followed by treating the cells with a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, a netrin, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about four days.

12. The method of claim 11, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about three days.

13. The method of claim 11, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, a netrin, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about four days.

14. The method of claim 2, wherein the factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 is FGF-7.

15. The method of claim 14, wherein the cells expressing markers characteristic of the definitive endoderm are treated with FGF-7 at a concentration of about 50 pg/ml to about 50 μ

g/ml.

16. The method of claim 14, wherein the cells expressing markers characteristic of the definitive endoderm are treated with FGF-7 at a concentration of 20 ng/ml.

17. The method of claim 2, wherein the cells expressing markers characteristic of the definitive endoderm are treated with retinoic acid at a concentration from about 1 nM to about 11 mM.

18. The method of claim 2, wherein the cells expressing markers characteristic of the definitive endoderm are treated with retinoic acid at a concentration of 1 μ

M.

19. The method of claim 2, wherein the sonic hedgehog inhibitor is cyclopamine.

20. The method of claim 19, wherein cyclopamine is used at a concentration from about 0.1 μ

M to about 10 μ

M.

21. The method of claim 19, wherein cyclopamine is used at a concentration of 0.25 μ

M.

22. The method of claim 2, wherein the factor capable of inhibiting BMP is a BMP4 inhibitor.

23. The method of claim 22, wherein the BMP4 inhibitor is noggin.

24. The method of claim 22, wherein the cells expressing markers characteristic of the definitive endoderm are treated with noggin at a concentration from about 500 ng/ml to about 100 μ

g/ml.

25. The method of claim 22, wherein the cells expressing markers characteristic of the definitive endoderm are treated with noggin at a concentration of 100 ng/ml.

26. The method of claim 2, wherein the cells expressing markers characteristic of the definitive endoderm are treated with the netrin at a concentration from about 500 ng/ml to about 100 μ

g/ml.

27. The method of claim 2, wherein the cells expressing markers characteristic of the definitive endoderm are treated with the netrin at a concentration of 100 ng/ml.

28. The method of claim 2, wherein the netrin is selected from the group consisting of netrin1, netrin 2, and netrin 4.

29. The method of claim 1, wherein the step of differentiating the cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine lineage is achieved by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the TGF-β

R-1 pathway.

30. The method of claim 29, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are treated with a factor that inhibits the TGF-β

R-1 pathway for about one to about twelve days.

31. The method of claim 29, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are treated with a factor that inhibits the TGF-β

R-1 pathway for about five to about twelve days.

32. The method of claim 29, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are treated with a factor that inhibits the TGF-β

R-1 pathway for about twelve days.

33. The method of claim 29, wherein the factor that inhibits the TGF-β

R-1 pathway is an inhibitor of TGF-β

R-1 kinase.

34. The method of claim 33, wherein the factor that inhibits the TGF-β

R-1 signaling pathway is an inhibitor of TGF-β

R-1 kinase.

35. The method of claim 34, wherein the TGF-β

R-1 kinase is used at a concentration from about 0.1 μ

M to about 100M.

36. The method of claim 34, wherein the inhibitor of TGF-β

R-1 kinase is 2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine.

37. The method of claim 34, wherein the inhibitor of TGF-β

R-1 kinase is [3-(Pyridin-2-yl)-4-(4-quinonyl)]-1H-pyrazole.

38. The method of claim 29, wherein the cells expressing markers characteristic of the pancreatic endoderm lineage are further treated with at least one factor selected from the group consisting of a factor capable of inhibiting BMP and a netrin.

39. The method of claim 1, wherein the step of differentiating the cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine lineage is achieved by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway, and a factor that inhibits the TGF-β

R-1 pathway.

40. The method of claim 39, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are treated with a factor that inhibits the Notch signaling pathway, and a factor that inhibits the TGF-β

R-1 pathway for about one to about twelve days.

41. The method of claim 39, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are treated with a factor that inhibits the Notch signaling pathway, and a factor that inhibits the TGF-β

R-1 pathway for about five to about twelve days.

42. The method of claim 39, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are treated with a factor that inhibits the Notch signaling pathway, and a factor that inhibits the TGF-β

R-1 pathway for about twelve days.

43. The method of claim 39, wherein the factor that inhibits the Notch signaling pathway is a 7-secretase inhibitor.

44. The method of claim 43, wherein the 7-secretase inhibitor is L-685,458.

45. The method of claim 43, wherein L-685,458 is used at a concentration from about 0.1 μ

M to about 100 μ

M.

46. The method of claim 39, wherein the factor that inhibits the TGF-β

R-1 pathway is an inhibitor of TGF-β

R-1 kinase.

47. The method of claim 46, wherein the factor that inhibits the TGF-β

R-1 signaling pathway is an inhibitor of TGF-β

R-1 kinase.

48. The method of claim 47, wherein the TGF-β

R-1 kinase is used at a concentration from about 0.1 μ

M to about 100 μ

M.

49. The method of claim 47, wherein the inhibitor of TGF-β

R-1 kinase is 2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine.

50. The method of claim 47, wherein the inhibitor of TGF-β

R-1 kinase is [3-(Pyridin-2-yl)-4-(4-quinonyl)]-1H-pyrazole.

51. The method of claim 29, wherein the cells expressing markers characteristic of the pancreatic endoderm lineage are further treated with at least one factor selected from the group consisting of a factor capable of inhibiting BMP and a netrin.

52. A method for producing cells capable of glucose-stimulated insulin secretion from pluripotent stem cells, comprising the steps of:

a. Culturing the pluripotent stem cells,

b. Differentiating the pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage,

c. Differentiating the cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage, and

d. Differentiating the cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine lineage.

53. The method of claim 52, wherein the step of differentiating the cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage is achieved by treating the cells expressing markers characteristic of the definitive endoderm lineage with at least one factor selected from the group consisting of retinoic acid, FGF-2, FGF-4, FGF-7, FGF-10, a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, and a netrin.

54. The method of claim 53, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with at least one factor selected from the group consisting of retinoic acid, FGF-2, FGF-4, FGF-7, FGF-10, a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, and a netrin for about one to about six days.

55. The method of claim 53, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with at least one factor selected from the group consisting of retinoic acid, FGF-2, FGF-4, FGF-7, FGF-10, a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, and a netrin for about six days.

56. The method of claim 53, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about three days, followed by treating the cells with a sonic hedgehog inhibitor, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about four days.

57. The method of claim 56, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about three days.

58. The method of claim 56, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about four days.

59. The method of claim 53, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about three days, followed by treating the cells with a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about four days.

60. The method of claim 59, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about three days.

61. The method of claim 59, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about four days.

62. The method of claim 53, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about three days, followed by treating the cells with a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, a netrin, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about four days.

63. The method of claim 62, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about three days.

64. The method of claim 62, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, a netrin, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about four days.

65. The method of claim 53, wherein the factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 is FGF-7.

66. The method of claim 65, wherein the cells expressing markers characteristic of the definitive endoderm are treated with FGF-7 at a concentration of about 50 pg/ml to about 50 μ

g/ml.

67. The method of claim 65, wherein the cells expressing markers characteristic of the definitive endoderm are treated with FGF-7 at a concentration of 20 ng/ml.

68. The method of claim 53, wherein the cells expressing markers characteristic of the definitive endoderm are treated with retinoic acid at a concentration from about 1 nM to about 1 mM.

69. The method of claim 53, wherein the cells expressing markers characteristic of the definitive endoderm are treated with retinoic acid at a concentration of 1 μ

M.

70. The method of claim 53, wherein the sonic hedgehog inhibitor is cyclopamine.

71. The method of claim 70, wherein cyclopamine is used at a concentration from about 0.1 μ

M to about 10 μ

M.

72. The method of claim 70, wherein cyclopamine is used at a concentration of 0.25 μ

M.

73. The method of claim 53, wherein the factor capable of inhibiting BMP is a BMP4 inhibitor.

74. The method of claim 73, wherein the BMP4 inhibitor is noggin.

75. The method of claim 73, wherein the cells expressing markers characteristic of the definitive endoderm are treated with noggin at a concentration from about 500 ng/ml to about 100 μ

g/ml.

76. The method of claim 73, wherein the cells expressing markers characteristic of the definitive endoderm are treated with noggin at a concentration of 100 ng/ml.

77. The method of claim 53, wherein the cells expressing markers characteristic of the definitive endoderm are treated with the netrin at a concentration from about 500 ng/ml to about 100 μ

g/ml.

78. The method of claim 53, wherein the cells expressing markers characteristic of the definitive endoderm are treated with the netrin at a concentration of 100 ng/ml.

79. The method of claim 53, wherein the netrin is selected from the group consisting of netrin1, netrin 2, and netrin 4.

80. The method of claim 52, wherein the step of differentiating the cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine lineage is achieved by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the TGF-β

R-1 pathway.

81. The method of claim 80, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are treated with a factor that inhibits the TGF-β

R-1 pathway for about one to about twelve days.

82. The method of claim 80, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are treated with a factor that inhibits the TGF-β

R-1 pathway for about five to about twelve days.

83. The method of claim 80, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are treated with a factor that inhibits the TGF-β

R-1 pathway for about twelve days.

84. The method of claim 80, wherein the factor that inhibits the TGF-β

R-1 pathway is an inhibitor of TGF-β

R-1 kinase.

85. The method of claim 84, wherein the factor that inhibits the TGF-β

R-1 signaling pathway is an inhibitor of TGF-β

R-1 kinase.

86. The method of claim 85, wherein the TGF-β

R-1 kinase is used at a concentration from about 0.1 μ

M to about 100 μ

M.

87. The method of claim 85, wherein the inhibitor of TGF-β

R-1 kinase is 2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine.

88. The method of claim 85, wherein the inhibitor of TGF-β

R-1 kinase is [3-(Pyridin-2-yl)-4-(4-quinonyl)]-1H-pyrazole.

89. The method of claim 80, wherein the cells expressing markers characteristic of the pancreatic endoderm lineage are further treated with at least one factor selected from the group consisting of a factor capable of inhibiting BMP and a netrin.

90. The method of claim 52, wherein the step of differentiating the cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine lineage is achieved by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway, and a factor that inhibits the TGF-β

R-1 pathway.

91. The method of claim 90, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are treated with a factor that inhibits the Notch signaling pathway, and a factor that inhibits the TGF-β

R-1 pathway for about one to about twelve days.

92. The method of claim 90, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are treated with a factor that inhibits the Notch signaling pathway, and a factor that inhibits the TGF-β

R-1 pathway for about five to about twelve days.

93. The method of claim 90, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are treated with a factor that inhibits the Notch signaling pathway, and a factor that inhibits the TGF-β

R-1 pathway for about twelve days.

94. The method of claim 90, wherein the factor that inhibits the Notch signaling pathway is a 7-secretase inhibitor.

95. The method of claim 94, wherein the 7-secretase inhibitor is L-685,458.

96. The method of claim 94, wherein L-685,458 is used at a concentration from about 0.1 μ

M to about 100 μ

M.

97. The method of claim 90, wherein the factor that inhibits the TGF-β

R-1 pathway is an inhibitor of TGF-β

R-1 kinase.

98. The method of claim 97, wherein the factor that inhibits the TGF-β

R-1 signaling pathway is an inhibitor of TGF-β

R-1 kinase.

99. The method of claim 98, wherein the TGF-β

R-1 kinase is used at a concentration from about 0.1 μ

M to about 100 μ

M.

100. The method of claim 98, wherein the inhibitor of TGF-β

R-1 kinase is 2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine.

101. The method of claim 98, wherein the inhibitor of TGF-β

R-1 kinase is [3-(Pyridin-2-yl)-4-(4-quinonyl)]-1H-pyrazole.

102. The method of claim 90, wherein the cells expressing markers characteristic of the pancreatic endoderm lineage are further treated with at least one factor selected from the group consisting of a factor capable of inhibiting BMP and a netrin.

103. A method for producing cells expressing markers characteristic of the pancreatic endoderm lineage, comprising the steps of:

a. Culturing the pluripotent stem cells,

b. Differentiating the pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage, and

c. Differentiating the cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage.

104. The method of claim 103, wherein the step of differentiating the cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage is achieved by treating the cells expressing markers characteristic of the definitive endoderm lineage with at least one factor selected from the group consisting of retinoic acid, FGF-2, FGF-4, FGF-7, FGF-10, a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, and a netrin.

105. The method of claim 104, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with at least one factor selected from the group consisting of retinoic acid, FGF-2, FGF-4, FGF-7, FGF-10, a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, and a netrin for about one to about six days.

106. The method of claim 104, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with at least one factor selected from the group consisting of retinoic acid, FGF-2, FGF-4, FGF-7, FGF-10, a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, and a netrin for about six days.

107. The method of claim 104, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about three days, followed by treating the cells with a sonic hedgehog inhibitor, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about four days.

108. The method of claim 107, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about three days.

109. The method of claim 107, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about four days.

110. The method of claim 104, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about three days, followed by treating the cells with a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about four days.

111. The method of claim 110, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about three days.

112. The method of claim 110, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about four days.

113. The method of claim 104, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about three days, followed by treating the cells with a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, a netrin, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about four days.

114. The method of claim 113, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about three days.

115. The method of claim 113, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, a netrin, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about four days.

116. The method of claim 104, wherein the factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 is FGF-7.

117. The method of claim 116, wherein the cells expressing markers characteristic of the definitive endoderm are treated with FGF-7 at a concentration of about 50 pg/ml to about 50 μ

g/ml.

118. The method of claim 116, wherein the cells expressing markers characteristic of the definitive endoderm are treated with FGF-7 at a concentration of 20 ng/ml.

119. The method of claim 104, wherein the cells expressing markers characteristic of the definitive endoderm are treated with retinoic acid at a concentration from about 1 nM to about 1 mM.

120. The method of claim 104, wherein the cells expressing markers characteristic of the definitive endoderm are treated with retinoic acid at a concentration of 1 μ

M.

121. The method of claim 104, wherein the sonic hedgehog inhibitor is cyclopamine.

122. The method of claim 121, wherein cyclopamine is used at a concentration from about 0.1 μ

M to about 10 μ

M.

123. The method of claim 121, wherein cyclopamine is used at a concentration of 0.25 μ

M.

124. The method of claim 104, wherein the factor capable of inhibiting BMP is a BMP4 inhibitor.

125. The method of claim 124, wherein the BMP4 inhibitor is noggin.

126. The method of claim 124, wherein the cells expressing markers characteristic of the definitive endoderm are treated with noggin at a concentration from about 500 ng/ml to about 100 μ

g/ml.

127. The method of claim 124, wherein the cells expressing markers characteristic of the definitive endoderm are treated with noggin at a concentration of 100 ng/ml.

128. The method of claim 104, wherein the cells expressing markers characteristic of the definitive endoderm are treated with the netrin at a concentration from about 500 ng/ml to about 100 μ

g/ml.

129. The method of claim 104, wherein the cells expressing markers characteristic of the definitive endoderm are treated with the netrin at a concentration of 100 ng/ml.

130. The method of claim 104, wherein the netrin is selected from the group consisting of netrin1, netrin 2, and netrin 4.