DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS

• US 20070254359A1
• Filed: 04/18/2007
• Published: 11/01/2007
• Est. Priority Date: 04/28/2006
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Lifescan Incorporated (Duv Holding Corporation)
• Inventors: Xu, Jean, Rezania, Alireza
• Granted Patent: US 8,741,643 B2
• Time in Patent Office: Days
• US Class Current: 435/325
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• 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 2501/415 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 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 5/067 Hepatocytes
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

The present invention provides methods to promote the differentiation of pluripotent stem cells. In particular, the present invention provides an improved method for the formation of pancreatic endoderm, pancreatic hormone expressing cells and pancreatic hormone secreting cells. The present invention also provides methods to promote the differentiation of pluripotent stem cells without the use of a feeder cell layer.

Claims

1. A method for differentiating pluripotent stem cells into cells expressing markers characteristic of the pancreatic endocrine lineage, comprising the steps of:

a. Culturing the pluripotent stem cells,

b. Differentiating the pluripotent stem cells into cells expressing markers characteristics of the definitive endoderm lineage, by treating the pluripotent cells with activin A,

c. Differentiating the cells expressing markers characteristics of the definitive endoderm lineage into cells expressing markers characteristics of the pancreatic endoderm lineage, by treating the cells expressing markers characteristics of the definitive endoderm lineage with at least one fibroblast growth factor, or with retinoic acid and at least one fibroblast growth factor, and

d. Differentiating the cells expressing markers characteristics of the pancreatic endoderm lineage into cells expressing markers characteristics of the pancreatic endocrine lineage, by treating the cells expressing markers characteristics of the pancreatic endoderm lineage with a gamma secretase inhibitor.

2. The method of claim 1, wherein the step of differentiating the pluripotent stem cells into cells expressing markers characteristics of the definitive endoderm lineage is achieved by treating the pluripotent stem cells with activin A for up to about eight days.

3. The method of claim 1, wherein the pluripotent stem cells are treated with activin A at a concentration of about 1 pg/ml to about 100 μ

g/ml.

4. The method of claim 1, wherein the pluripotent stem cells are treated with activin A at a concentration of 100 ng/ml.

5. The method of claim 1, wherein the step of differentiating the pluripotent stem cells into cells expressing markers characteristics of the definitive endoderm lineage is achieved my a method comprising the steps of:

a. Plating the pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix, and

b. Culturing the pluripotent stem cells with activin A and a Wnt ligand.

6. The method of claim 5, wherein the pluripotent stem cells are cultured in culture medium having a serum concentration in the range of about 0% to about 10%.

7. The method of claim 5, wherein the pluripotent stem cells are cultured in culture medium having a serum concentration in the range of about 0% to about 5%.

8. The method of claim 5, wherein the pluripotent stem cells are cultured in culture medium having a serum concentration in the range of about 0% to about 2%.

9. The method of claim 5, wherein the pluripotent stem cells are cultured in culture medium having a serum concentration of about 2%.

10. The method of claim 5, wherein the time of culture with activin A and a Wnt ligand is in the range from about 1 day to about 7 days.

11. The method of claim 5, wherein the time of culture with activin A and a Wnt ligand is in the range from about 1 day to about 3 days.

12. The method of claim 5, wherein the time of culture with activin A and a Wnt ligand is about 3 days.

13. The method of claim 5, wherein the activin A is used at a concentration of about 1 pg/ml to about 100 μ

g/ml.

14. The method of claim 5, wherein the activin A is used at a concentration of about 1 pg/ml to about 1 μ

g/ml.

15. The method of claim 5, wherein the activin A is used at a concentration of about 1 pg/ml to about 100 ng/ml.

16. The method of claim 5, wherein the activin A is used at a concentration of about 50 ng/ml to about 100 ng/ml.

17. The method of claim 5, wherein the activin A is used at a concentration of about 100 ng/ml.

18. The method of claim 5, wherein the Wnt ligand is used at a concentration of about 1 ng/ml to about 1000 ng/ml.

19. The method of claim 5, wherein the Wnt ligand is used at a concentration of about 10 ng/ml to about 100 ng/ml.

20. The method of claim 5, wherein the Wnt ligand is selected from the group consisting of Wnt-1, Wnt-3a, Wnt-5a and Wnt-7a.

21. The method of claim 20, wherein the Wnt ligand is Wnt-1.

22. The method of claim 20, wherein the Wnt ligand is Wnt-3.

23. The method of claim 5, wherein the pluripotent stem cells are cultured with a GSK-3B inhibitor.

26. The method of claim 23, wherein the GSK-3B inhibitor is used at a concentration from about 1 nM to about 1000 nM.

27. The method of claim 23, wherein the GSK-3B inhibitor is used at a concentration from about 10 nM to about 100 nM.

28. The method of claim 5, wherein the extracellular matrix is selected from the group consisting of MATRIGEL, growth factor-reduced MATRIGEL, laminin, fibronectin, and human serum.

33. The method of claim 1, wherein the step of differentiating the pluripotent stem cells into cells expressing markers characteristics of the definitive endoderm lineage is achieved my a method comprising the steps of:

a. Plating the pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix,

b. Culturing the pluripotent stem cells with activin A and a Wnt ligand in a first culture medium, and

c. Culturing the pluripotent stem cells with activin A in a second culture medium.

34. The method of claim 33, wherein the first culture medium contains serum at a low concentration, and the second culture medium contains serum at a higher concentration than the first culture medium.

35. The method of claim 33, wherein the second culture medium does not contain a Wnt ligand.

36. The method of claim 33, wherein the second culture medium contains a Wnt ligand.

37. The method of claim 33, wherein the first culture medium contains serum at a concentration from about 0% to about 10%.

38. The method of claim 33, wherein the first culture medium contains serum at a concentration from about 0% to about 2%.

39. The method of claim 33, wherein the first culture medium contains serum at a concentration from about 0% to about 1%.

40. The method of claim 33, wherein the first culture medium contains serum at a concentration of about 0.5%.

41. The method of claim 33, wherein the second culture medium contains serum at a concentration from about 0.5% to about 10%.

42. The method of claim 33, wherein the second culture medium contains serum at a concentration from about 0.5% to about 5%.

43. The method of claim 33, wherein the second culture medium contains serum at a concentration from about 0.5% to about 2%.

44. The method of claim 33, wherein the second culture medium contains serum at a concentration of about 2%.

45. The method of claim 33, wherein the pluripotent stem cells are cultured in the first culture medium for about 1 day to about 3 days.

46. The method of claim 33, wherein the pluripotent stem cells are cultured in the second culture medium for about 1 day to about 4 days.

47. The method of claim 33, wherein the activin A is used at a concentration of about 1 pg/ml to about 100 μ

g/ml.

48. The method of claim 33, wherein the activin A is used at a concentration of about 1 pg/ml to about 1 μ

g/ml.

49. The method of claim 33, wherein the activin A is used at a concentration of about 1 pg/ml to about 100 ng/ml.

50. The method of claim 33, wherein the activin A is used at a concentration of about 50 ng/ml to about 100 ng/ml.

51. The method of claim 33, wherein the activin A is used at a concentration of about 100 ng/ml.

52. The method of claim 33, wherein the Wnt ligand is used at a concentration of about 1 ng/ml to about 100 ng/ml.

53. The method of claim 33, wherein the Wnt ligand is used at a concentration of about 10 ng/ml to about 100 ng/ml.

54. The method of claim 33, wherein the Wnt ligand is selected from the group consisting of Wnt-1, Wnt-3a, Wnt-5a and Wnt-7a.

55. The method of claim 54, wherein the Wnt ligand is Wnt-1.

56. The method of claim 54, wherein the Wnt ligand is Wnt-3.

57. The method of claim 33, wherein the pluripotent stem cells are cultured with a GSK-3B inhibitor.

58. The method of claim 57, wherein the GSK-3B inhibitor is added to the first culture medium.

59. The method of claim 57, wherein the GSK-3B inhibitor is added to the second culture medium.

60. The method of claim 57, wherein the GSK-3B inhibitor is added to both the first and second culture media.

61. The method of claim 57, wherein the GSK-3B inhibitor is selected from the group consisting of GSK-3B inhibitor IX and GSK-3B inhibitor XI.

63. The method of claim 57, wherein the GSK-3B inhibitor is used at a concentration from about 1 nM to about 1000 nM.

64. The method of claim 57, wherein the GSK-3B inhibitor is used at a concentration from about 10 nM to about 100 nM.

65. The method of claim 33, wherein the extracellular matrix is selected from the group consisting of MATRIGEL, growth factor-reduced MATRIGEL, laminin, fibronectin, and human serum.

66. The method of claim 65, wherein the extracellular matrix is MATRIGEL.

68. The method of claim 65, wherein the extracellular matrix is growth factor-reduced MATRIGEL.

69. The method of claim 68, wherein the growth factor-reduced MATRIGEL is used at a dilution from about 1:

60 to about 1;

10.

70. The method of claim 1, wherein the step of differentiating the cells expressing markers characteristics of the definitive endoderm lineage into cells expressing markers characteristics of the pancreatic endoderm lineage is achieved by treating the cells expressing markers characteristic of the definitive endoderm lineage with a factor selected from the group consisting of retinoic acid, FGF-2, FGF-4 and FGF-10.

71. The method of claim 70, wherein the cells expressing markers characteristics of the definitive endoderm lineage are treated with a factor selected from the group consisting of retinoic acid, FGF-2, FGF-4 and FGF-10 for about one to about six days.

72. The method of claim 70, wherein the cells expressing markers characteristics of the definitive endoderm lineage are treated with a factor selected from the group consisting of retinoic acid, FGF-2, FGF-4 and FGF-10 for about six days.

73. The method of claim 70, wherein the step of differentiating the cells expressing markers characteristics of the definitive endoderm lineage into cells expressing markers characteristics of the pancreatic endoderm lineage is achieved by treating the cells expressing markers characteristic of the definitive endoderm lineage with retinoic acid and a factor selected from the group consisting of FGF-2, FGF-4 and FGF-10.

74. The method of claim 73, wherein the cells expressing markers characteristics of the definitive endoderm lineage are treated with retinoic acid for about one to about three days.

75. The method of claim 73, wherein the cells expressing markers characteristics of the definitive endoderm lineage are treated with retinoic acid for about three days.

76. The method of claim 73, wherein the cells expressing markers characteristics of the definitive endoderm lineage are treated with a factor selected from the group consisting of FGF-2, FGF-4 and FGF-10 for about one to about three days.

77. The method of claim 73, wherein the cells expressing markers characteristics of the definitive endoderm lineage are treated with a factor selected from the group consisting of FGF-2, FGF-4 and FGF-10 for about three days.

78. The method of claim 70, wherein the cells expressing markers characteristics of the definitive endoderm are treated with retinoic acid at a concentration of about 1 nM to about 1 mM.

79. The method of claim 70, wherein the cells expressing markers characteristics of the definitive endoderm are treated with retinoic acid at a concentration of 1 μ

M.

80. The method of claim 70, wherein the cells expressing markers characteristics of the definitive endoderm are treated with FGF-2 at a concentration of about 50 pg/ml to about 50 μ

g/ml.

81. The method of claim 70, wherein the cells expressing markers characteristics of the definitive endoderm are treated with FGF-2 at a concentration of 50 ng/ml.

82. The method of claim 70, wherein the cells expressing markers characteristics of the definitive endoderm are treated with FGF-4 at a concentration of about 50 pg/ml to about 50 μ

g/ml.

83. The method of claim 70, wherein the cells expressing markers characteristics of the definitive endoderm are treated with FGF-4 at a concentration of 50 ng/ml.

84. The method of claim 70, wherein the cells expressing markers characteristics of the definitive endoderm are treated with FGF-10 at a concentration of about 50 pg/ml to about 50 μ

g/ml.

85. The method of claim 70, wherein the cells expressing markers characteristics of the definitive endoderm are treated with FGF-10 at a concentration of 50 ng/ml.

86. The method of claim 1, wherein the step of differentiating cells expressing markers characteristics of the pancreatic endoderm lineage into cells expressing markers characteristics of the pancreatic endocrine lineage is achieved by treating the cells expressing markers characteristics of the pancreatic endoderm lineage with the γ

-secretase inhibitor L-685,458.

87. The method of claim 86, wherein the cells expressing markers characteristics of the pancreatic endoderm lineage are treated with L-685,458 for about three to about five days.

88. The method of claim 86, wherein the cells expressing markers characteristics of the pancreatic endoderm lineage are treated with L-685,458 for about five days.

89. The method of claim 86, wherein L-685,458 is used at a concentration from about 0.1 M to about 100 μ

M.

90. The method of claim 86, wherein L-685,458 is used at a concentration of about 90 μ

M.

91. The method of claim 86, wherein L-685,458 is used at a concentration of about 80 μ

M.

92. The method of claim 86, wherein L-685,458 is used at a concentration of about 70 μ

M.

93. The method of claim 86, wherein L-685,458 is used at a concentration of about 60 μ

M.

94. The method of claim 86, wherein L-685,458 is used at a concentration of about 50 μ

M.

95. The method of claim 86, wherein L-685,458 is used at a concentration of about 40 μ

M.

96. The method of claim 86, wherein L-685,458 is used at a concentration of about 30 μ

M.

97. The method of claim 86, wherein L-685,458 is used at a concentration of about 20 μ

M.

98. The method of claim 86, wherein L-685,458 is used at a concentration of about 10 μ

M.

248. The method of claim 1, wherein the cells expressing markers characteristic of the definitive endoderm lineage are definitive endoderm cells.

249. The method of claim 1, wherein the cells expressing markers characteristic of the pancreatic endoderm lineage are pancreatic endoderm cells.

250. The method of claim 1, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are pancreatic endocrine cells.

251. The method of claim 1, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are pancreatic hormone expressing cells.

252. The method of claim 1, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are pancreatic secreting expressing cells.

253. The method of claim 252, wherein the pancreatic hormone secreting cells are cells expressing markers that are characteristic of the β

cell lineage.

254. The method of claim 253, wherein the cells expressing markers that are characteristic of the β

cell lineage are β

cells.

279. The method of claim 1, wherein the pluripotent stem cells are embryonic stem cells.

280. The method of claim 279, wherein the embryonic stem cells are human.

281. The method of claim 279, wherein the embryonic stem cells are obtained from an embryonic stem cell line.

282. The method of claim 279, wherein the embryonic stem cells are obtained from pre-embryonic tissue.

283. The method of claim 282, wherein the pre-embryonic tissue is blastocyst tissue.

284. The method of claim 279, wherein the embryonic stem cells are obtained from embryonic tissue.

24. The method of claim 24, wherein the GSK-3B inhibitor is selected from the group consisting of GSK-3B inhibitor IX and GSK-3B inhibitor XI.

25. The method of claim 24, wherein the GSK-3B inhibitor is GSK-3B inhibitor IX.

29. The method of claim 29, wherein the extracellular matrix is MATRIGEL.

31. The method of claim 29, wherein the extracellular matrix is growth factor-reduced MATRIGEL.

32. The method of claim 31, wherein the growth factor-reduced MATRIGEL is used at a dilution from about 1:

60 to about 1;

10.

30. The method of claim 30, wherein the MATRIGEL is used at a dilution from about 1:

60 to about 1;

10.

62. The method of claim 62, wherein the GSK-3B inhibitor is GSK-3B inhibitor IX.

67. The method of claim 67, wherein the MATRIGEL is used at a dilution from about 1:

60 to about 1;

10.

99. A method for differentiating cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine lineage, comprising the steps of:

a. Treating the cells expressing markers characteristics of the definitive endoderm lineage into cells expressing markers characteristics of the pancreatic endoderm lineage with at least one fibroblast growth factor, or with retinoic acid and at least one fibroblast growth factor, and

b. Differentiating the cells expressing markers characteristics of the pancreatic endoderm lineage into cells expressing markers characteristics of the pancreatic endocrine lineage, by treating the cells expressing markers characteristics of the pancreatic endoderm lineage with a gamma secretase inhibitor.

100. The method of claim 99, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a factor selected from the group consisting of retinoic acid, FGF-2, FGF-4 and FGF-10.

101. The method of claim 100, wherein the cells expressing markers characteristics of the definitive endoderm lineage are treated with a factor selected from the group consisting of retinoic acid, FGF-2, FGF-4 and FGF-10 for about one to about six days.

102. The method of claim 100, wherein the cells expressing markers characteristics of the definitive endoderm lineage are treated with a factor selected from the group consisting of retinoic acid, FGF-2, FGF-4 and FGF-10 for about six days.

103. The method of claim 99, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with retinoic acid and a factor selected from the group consisting of FGF-2, FGF-4 and FGF-10.

104. The method of claim 103, wherein the cells expressing markers characteristics of the definitive endoderm lineage are treated with retinoic acid for about one to about three days.

105. The method of claim 103, wherein the cells expressing markers characteristics of the definitive endoderm lineage are treated with retinoic acid for about three days.

106. The method of claim 103, wherein the cells expressing markers characteristics of the definitive endoderm lineage are treated with a factor selected from the group consisting of FGF-2, FGF-4 and FGF-10 for about one to about three days.

107. The method of claim 103, wherein the cells expressing markers characteristics of the definitive endoderm lineage are treated with a factor selected from the group consisting of FGF-2, FGF-4 and FGF-10 for about three days.

108. The method of claim 99, wherein the cells expressing markers characteristics of the definitive endoderm are treated with retinoic acid at a concentration of about 1 nM to about 1 mM.

109. The method of claim 99, wherein the cells expressing markers characteristics of the definitive endoderm are treated with retinoic acid at a concentration of 1 μ

M.

110. The method of claim 99, wherein the cells expressing markers characteristics of the definitive endoderm are treated with FGF-2 at a concentration of about 50 pg/ml to about 50 μ

g/ml.

111. The method of claim 99, wherein the cells expressing markers characteristics of the definitive endoderm are treated with FGF-2 at a concentration of 50 ng/ml.

112. The method of claim 99, wherein the cells expressing markers characteristics of the definitive endoderm are treated with FGF-4 at a concentration of about 50 pg/ml to about 50 μ

g/ml.

113. The method of claim 99, wherein the cells expressing markers characteristics of the definitive endoderm are treated with FGF-4 at a concentration of 50 ng/ml.

114. The method of claim 99, wherein the cells expressing markers characteristics of the definitive endoderm are treated with FGF-10 at a concentration of about 50 pg/ml to about 50 μ

g/ml.

115. The method of claim 99, wherein the cells expressing markers characteristics of the definitive endoderm are treated with FGF-10 at a concentration of 50 ng/ml.

116. The method of claim 99, wherein the step of differentiating cells expressing markers characteristics of the pancreatic endoderm lineage into cells expressing markers characteristics of the pancreatic endocrine lineage is achieved by treating the cells expressing markers characteristics of the pancreatic endoderm lineage with the γ

-secretase inhibitor L-685,458.

117. The method of claim 116, wherein the cells expressing markers characteristics of the pancreatic endoderm lineage are treated with L-685,458 for about three to about five days.

118. The method of claim 116, wherein the cells expressing markers characteristics of the pancreatic endoderm lineage are treated with L-685,458 for about five days.

119. The method of claim 116, wherein L-685,458 is used at a concentration from about 0.1 μ

M to about 100 μ

M.

120. The method of claim 116, wherein L-685,458 is used at a concentration of about 90 μ

M.

121. The method of claim 116, wherein L-685,458 is used at a concentration of about 80 μ

M.

122. The method of claim 116, wherein L-685,458 is used at a concentration of about 70 μ

M.

123. The method of claim 116, wherein L-685,458 is used at a concentration of about 60 μ

M.

124. The method of claim 116, wherein L-685,458 is used at a concentration of about 50 μ

M.

125. The method of claim 116, wherein L-685,458 is used at a concentration of about 40 μ

M.

126. The method of claim 116, wherein L-685,458 is used at a concentration of about 30 μ

M.

127. The method of claim 116, wherein L-685,458 is used at a concentration of about 20 μ

M.

128. The method of claim 116, wherein L-685,458 is used at a concentration of about 10 μ

M.

255. The method of claim 99, wherein the cells expressing markers characteristic of the definitive endoderm lineage are definitive endoderm cells.

256. The method of claim 99, wherein the cells expressing markers characteristic of the pancreatic endoderm lineage are pancreatic endoderm cells.

257. The method of claim 99, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are pancreatic endocrine cells.

258. The method of claim 99, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are pancreatic hormone expressing cells.

259. The method of claim 99, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are pancreatic secreting expressing cells.

260. The method of claim 259, wherein the pancreatic hormone secreting cells are cells expressing markers that are characteristic of the β

cell lineage.

261. The method of claim 260, wherein the cells expressing markers that are characteristic of the β

cell lineage are β

cells.

129. A method for differentiating cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine lineage, comprising the step of treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway.

130. The method of claim 129, wherein the factor that inhibits the Notch signaling pathway is a γ

-secretase inhibitor.

131. The method of claim 130, wherein the γ

-secretase inhibitor L-685,458.

132. The method of claim 131, wherein the cells expressing markers characteristics of the pancreatic endoderm lineage are treated with L-685,458 for about three to about five days.

133. The method of claim 131, wherein the cells expressing markers characteristics of the pancreatic endoderm lineage are treated with L-685,458 for about five days.

134. The method of claim 131, wherein L-685,458 is used at a concentration from about 0.1 μ

M to about 100 μ

M.

135. The method of claim 131, wherein L-685,458 is used at a concentration of about 90 μ

M.

136. The method of claim 131, wherein L-685,458 is used at a concentration of about 80 μ

M.

137. The method of claim 131, wherein L-685,458 is used at a concentration of about 70 μ

M.

138. The method of claim 131, wherein L-685,458 is used at a concentration of about 60 μ

M.

139. The method of claim 131, wherein L-685,458 is used at a concentration of about 50 μ

M.

140. The method of claim 131, wherein L-685,458 is used at a concentration of about 40 μ

M.

141. The method of claim 131, wherein L-685,458 is used at a concentration of about 30 μ

M.

142. The method of claim 131, wherein L-685,458 is used at a concentration of about 20 μ

M.

143. The method of claim 131, wherein L-685,458 is used at a concentration of about 10 μ

M.

262. The method of claim 129, wherein the cells expressing markers characteristic of the pancreatic endoderm lineage are pancreatic endoderm cells.

263. The method of claim 129, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are pancreatic endocrine cells.

264. The method of claim 129, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are pancreatic hormone expressing cells.

265. The method of claim 129, wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are pancreatic secreting expressing cells.

266. The method of claim 265, wherein the pancreatic hormone secreting cells are cells expressing markers that are characteristic of the β

cell lineage.

267. The method of claim 266, wherein the cells expressing markers that are characteristic of the β

cell lineage are β

cells.

144. A method for differentiating pluripotent stem cells into 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 characteristics of the definitive endoderm lineage, by treating the pluripotent cells with activin A, and

c. Differentiating the cells expressing markers characteristics of the definitive endoderm lineage into cells expressing markers characteristics of the pancreatic endoderm lineage, by treating the cells expressing markers characteristics of the definitive endoderm lineage with at least one fibroblast growth factor, or with retinoic acid and at least one fibroblast growth factor.

145. The method of claim 144, wherein the step of differentiating the pluripotent stem cells into cells expressing markers characteristics of the definitive endoderm lineage is achieved by treating the pluripotent stem cells with activin A for up to about eight days.

146. The method of claim 144, wherein the pluripotent stem cells are treated with activin A at a concentration of about 1 pg/ml to about 100 μ

g/ml.

147. The method of claim 144, wherein the pluripotent stem cells are treated with activin A at a concentration of 100 ng/ml.

148. The method of claim 144, wherein the step of differentiating the cells expressing markers characteristics of the definitive endoderm lineage into cells expressing markers characteristics of the pancreatic endoderm lineage is achieved by treating the cells expressing markers characteristic of the definitive endoderm lineage with a factor selected from the group consisting of retinoic acid, FGF-2, FGF-4 and FGF-10.

149. The method of claim 148, wherein the cells expressing markers characteristics of the definitive endoderm lineage are treated with a factor selected from the group consisting of retinoic acid, FGF-2, FGF-4 and FGF-10 for about one to about six days.

150. The method of claim 148, wherein the cells expressing markers characteristics of the definitive endoderm lineage are treated with a factor selected from the group consisting of retinoic acid, FGF-2, FGF-4 and FGF-10 for about six days.

151. The method of claim 144, wherein the step of differentiating the cells expressing markers characteristics of the definitive endoderm lineage into cells expressing markers characteristics of the pancreatic endoderm lineage is achieved by treating the cells expressing markers characteristic of the definitive endoderm lineage with retinoic acid and a factor selected from the group consisting of FGF-2, FGF-4 and FGF-10.

152. The method of claim 151, wherein the cells expressing markers characteristics of the definitive endoderm lineage are treated with retinoic acid for about one to about three days.

153. The method of claim 151, wherein the cells expressing markers characteristics of the definitive endoderm lineage are treated with retinoic acid for about three days.

154. The method of claim 151, wherein the cells expressing markers characteristics of the definitive endoderm lineage are treated with a factor selected from the group consisting of FGF-2, FGF-4 and FGF-10 for about one to about three days.

155. The method of claim 151, wherein the cells expressing markers characteristics of the definitive endoderm lineage are treated with a factor selected from the group consisting of FGF-2, FGF-4 and FGF-10 for about three days.

156. The method of claim 144, wherein the cells expressing markers characteristics of the definitive endoderm are treated with retinoic acid at a concentration of about 1 nM to about 1 mM.

157. The method of claim 144, wherein the cells expressing markers characteristics of the definitive endoderm are treated with retinoic acid at a concentration of 1 μ

M.

158. The method of claim 144, wherein the cells expressing markers characteristics of the definitive endoderm are treated with FGF-2 at a concentration of about 50 pg/ml to about 50 μ

g/ml.

159. The method of claim 144, wherein the cells expressing markers characteristics of the definitive endoderm are treated with FGF-2 at a concentration of 50 ng/ml.

160. The method of claim 144, wherein the cells expressing markers characteristics of the definitive endoderm are treated with FGF-4 at a concentration of about 50 pg/ml to about 50 μ

g/ml.

161. The method of claim 144, wherein the cells expressing markers characteristics of the definitive endoderm are treated with FGF-4 at a concentration of 50 ng/ml.

162. The method of claim 144, wherein the cells expressing markers characteristics of the definitive endoderm are treated with FGF-10 at a concentration of about 50 pg/ml to about 50 pg/ml.

163. The method of claim 144, wherein the cells expressing markers characteristics of the definitive endoderm are treated with FGF-10 at a concentration of 50 ng/ml.

235. The method of claim 144, wherein the cells expressing markers characteristics of the definitive endoderm lineage are treated with retinoic acid and a factor selected from the group consisting of FGF-2, FGF-4 and FGF-10.

236. The method of claim 235, wherein the cells expressing markers characteristics of the definitive endoderm lineage are treated with retinoic acid for about one to about three days.

237. The method of claim 235, wherein the cells expressing markers characteristics of the definitive endoderm lineage are treated with retinoic acid for about three days.

238. The method of claim 235, wherein the cells expressing markers characteristics of the definitive endoderm lineage are treated with a factor selected from the group consisting of FGF-2, FGF-4 and FGF-10 for about one to about three days.

239. The method of claim 235, wherein the cells expressing markers characteristics of the definitive endoderm lineage are treated with a factor selected from the group consisting of FGF-2, FGF-4 and FGF-10 for about three days.

268. The method of claim 144, wherein the cells expressing markers characteristic of the definitive endoderm lineage are definitive endoderm cells.

269. The method of claim 144, wherein the cells expressing markers characteristic of the pancreatic endoderm lineage are pancreatic endoderm cells.

285. The method of claim 144, wherein the pluripotent stem cells are embryonic stem cells.

286. The method of claim 285, wherein the embryonic stem cells are human.

287. The method of claim 285, wherein the embryonic stem cells are obtained from an embryonic stem cell line.

288. The method of claim 285, wherein the embryonic stem cells are obtained from pre-embryonic tissue.

289. The method of claim 288, wherein the pre-embryonic tissue is blastocyst tissue.

290. The method of claim 285, wherein the embryonic stem cells are obtained from embryonic tissue.

164. A method for differentiating pluripotent stem cells into cells expressing markers that are characteristic of the definitive endoderm lineage, comprising the steps of:

a. Plating the pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix, and

b. Culturing the pluripotent stem cells with activin A and a Wnt ligand.

165. The method of claim 164, wherein the pluripotent stem cells are cultured in culture medium having a serum concentration in the range of about 0% to about 10%.

166. The method of claim 164, wherein the pluripotent stem cells are cultured in culture medium having a serum concentration in the range of about 0% to about 5%.

167. The method of claim 164, wherein the pluripotent stem cells are cultured in culture medium having a serum concentration in the range of about 0% to about 2%.

168. The method of claim 164, wherein the pluripotent stem cells are cultured in culture medium having a serum concentration of about 2%.

169. The method of claim 164, wherein the time of culture with activin A and a Wnt ligand is in the range from about 1 day to about 7 days.

170. The method of claim 164, wherein the time of culture with activin A and a Wnt ligand is in the range from about 1 day to about 3 days.

171. The method of claim 164, wherein the time of culture with activin A and a Wnt ligand is about 3 days.

172. The method of claim 164, wherein the activin A is used at a concentration of about 1 pg/ml to about 100 g/ml.

173. The method of claim 164, wherein the activin A is used at a concentration of about 1 pg/ml to about 1 μ

g/ml.

174. The method of claim 164, wherein the activin A is used at a concentration of about 1 pg/ml to about 100 ng/ml.

175. The method of claim 164, wherein the activin A is used at a concentration of about 50 ng/ml to about 100 ng/ml.

176. The method of claim 164, wherein the activin A is used at a concentration of about 100 ng/ml.

177. The method of claim 164, wherein the Wnt ligand is used at a concentration of about 1 ng/ml to about 1000 ng/ml.

178. The method of claim 164, wherein the Wnt ligand is used at a concentration of about 10 ng/ml to about 100 ng/ml.

179. The method of claim 164, wherein the Wnt ligand is selected from the group consisting of Wnt-1, Wnt-3a, Wnt-5a and Wnt-7a.

180. The method of claim 179, wherein the Wnt ligand is Wnt-1.

181. The method of claim 179, wherein the Wnt ligand is Wnt-3.

182. The method of claim 164, wherein the pluripotent stem cells are cultured with a GSK-3B inhibitor.

183. The method of claim 182, wherein the GSK-3B inhibitor is selected from the group consisting of GSK-3B inhibitor IX and GSK-3B inhibitor XI.

184. The method of claim 183, wherein the GSK-3B inhibitor is GSK-3B inhibitor IX.

185. The method of claim 182, wherein the GSK-3B inhibitor is used at a concentration from about 1 nM to about 1000 nM.

186. The method of claim 182, wherein the GSK-3B inhibitor is used at a concentration from about 10 nM to about 100 nM.

187. The method of claim 164, wherein the extracellular matrix is selected from the group consisting of MATRIGEL, growth factor-reduced MATRIGEL, laminin, fibronectin, and human serum.

188. The method of claim 187, wherein the extracellular matrix is MATRIGEL.

189. The method of claim 188, wherein the MATRIGEL is used at a dilution from about 1:

60 to about 1;

10.

190. The method of claim 187, wherein the extracellular matrix is growth factor-reduced MATRIGEL.

191. The method of claim 190, wherein the growth factor-reduced MATRIGEL is used at a dilution from about 1:

60 to about 1;

10.

270. The method of claim 164, wherein the cells expressing markers characteristic of the definitive endoderm lineage are definitive endoderm cells.

274. The method of claim 164, wherein the ells expressing markers that are characteristic of the definitive endoderm lineage are purified from the pluripotent stem cells by exposing the cell culture to an agent that specifically recognizes a protein marker expressed by the cells expressing markers that are characteristic of the definitive endoderm lineage.

275. The method of claim 274, wherein the protein marker is CXCR4.

291. The method of claim 164, wherein the pluripotent stem cells are embryonic stem cells.

292. The method of claim 291, wherein the embryonic stem cells are human.

293. The method of claim 291, wherein the embryonic stem cells are obtained from an embryonic stem cell line.

294. The method of claim 291, wherein the embryonic stem cells are obtained from pre-embryonic tissue.

295. The method of claim 294, wherein the pre-embryonic tissue is blastocyst tissue.

296. The method of claim 291, wherein the embryonic stem cells are obtained from embryonic tissue.

192. A method for differentiating pluripotent stem cells into cells expressing markers that are characteristic of the definitive endoderm lineage, comprising the steps of:

a. Plating the pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix,

b. Culturing the pluripotent stem cells with activin A and a Wnt ligand in a first culture medium, and

c. Culturing the pluripotent stem cells with activin A in a second culture medium.

193. The method of claim 192, wherein the first culture medium contains serum at a low concentration, and the second culture medium contains serum at a higher concentration than the first culture medium.

194. The method of claim 192, wherein the second culture medium does not contain a Wnt ligand.

195. The method of claim 192, wherein the second culture medium contains a Wnt ligand.

196. The method of claim 192, wherein the cells that express markers characteristic of the definitive endoderm lineage may be definitive endoderm cells.

197. The method of claim 192, wherein the pluripotent stem cells are embryonic stem cells.

198. The method of claim 192, wherein the first culture medium contains serum at a concentration from about 0% to about 10%.

199. The method of claim 192, wherein the first culture medium contains serum at a concentration from about 0% to about 2%.

200. The method of claim 192, wherein the first culture medium contains serum at a concentration from about 0% to about 1%.

201. The method of claim 192, wherein the first culture medium contains serum at a concentration of about 0.5%.

202. The method of claim 192, wherein the second culture medium contains serum at a concentration from about 0.5% to about 10%.

203. The method of claim 192, wherein the second culture medium contains serum at a concentration from about 0.5% to about 5%.

204. The method of claim 192, wherein the second culture medium contains serum at a concentration from about 0.5% to about 2%.

205. The method of claim 192, wherein the second culture medium contains serum at a concentration of about 2%.

206. The method of claim 192, wherein the pluripotent stem cells are cultured in the first culture medium for about 1 day to about 3 days.

207. The method of claim 192, wherein the pluripotent stem cells are cultured in the second culture medium for about 1 day to about 4 days.

208. The method of claim 192, wherein the activin A is used at a concentration of about 1 pg/ml to about 100 μ

g/ml.

209. The method of claim 192, wherein the activin A is used at a concentration of about 1 pg/ml to about 1 μ

g/ml.

210. The method of claim 192, wherein the activin A is used at a concentration of about 1 pg/ml to about 100 ng/ml.

211. The method of claim 192, wherein the activin A is used at a concentration of about 50 ng/ml to about 100 ng/ml.

212. The method of claim 192, wherein the activin A is used at a concentration of about 100 ng/ml.

213. The method of claim 192, wherein the Wnt ligand is used at a concentration of about 1 ng/ml to about 1000 ng/ml.

214. The method of claim 192, wherein the Wnt ligand is used at a concentration of about 10 ng/ml to about 100 ng/ml.

215. The method of claim 192, wherein the Wnt ligand is selected from the group consisting of Wnt-1, Wnt-3a, Wnt-5a and Wnt-7a.

216. The method of claim 215, wherein the Wnt ligand is Wnt-1.

217. The method of claim 215, wherein the Wnt ligand is Wnt-3.

218. The method of claim 192, wherein the pluripotent stem cells are cultured with a GSK-3B inhibitor.

219. The method of claim 218, wherein the GSK-3B inhibitor is added to the first culture medium.

220. The method of claim 218, wherein the GSK-3B inhibitor is added to the second culture medium.

221. The method of claim 218, wherein the GSK-3B inhibitor is added to both the first and second culture media.

222. The method of claim 218, wherein the GSK-3B inhibitor is selected from the group consisting of GSK-3B inhibitor IX and GSK-3B inhibitor XI.

223. The method of claim 222, wherein the GSK-3B inhibitor is GSK-3B inhibitor IX.

224. The method of claim 218, wherein the GSK-3B inhibitor is used at a concentration from about 1 nM to about 1000 nM.

225. The method of claim 218, wherein the GSK-3B inhibitor is used at a concentration from about 10 nM to about 100 nM.

226. The method of claim 192, wherein the extracellular matrix is selected from the group consisting of MATRIGEL, growth factor-reduced MATRIGEL, laminin, fibronectin, and human serum.

227. The method of claim 226, wherein the extracellular matrix is MATRIGEL.

228. The method of claim 227, wherein the MATRIGEL is used at a dilution from about 1:

60 to about 1;

10.

229. The method of claim 226, wherein the extracellular matrix is growth factor-reduced MATRIGEL.

230. The method of claim 229, wherein the growth factor-reduced MATRIGEL is used at a dilution from about 1:

60 to about 1;

10.

271. The method of claim 192, wherein the cells expressing markers characteristic of the definitive endoderm lineage are definitive endoderm cells.

276. The method of claim 192, wherein the ells expressing markers that are characteristic of the definitive endoderm lineage are purified from the pluripotent stem cells by exposing the cell culture to an agent that specifically recognizes a protein marker expressed by the cells expressing markers that are characteristic of the definitive endoderm lineage.

277. The method of claim 276, wherein the protein marker is CXCR4.

297. The method of claim 192, wherein the pluripotent stem cells are embryonic stem cells.

298. The method of claim 297, wherein the embryonic stem cells are human.

299. The method of claim 297, wherein the embryonic stem cells are obtained from an embryonic stem cell line.

300. The method of claim 297, wherein the embryonic stem cells are obtained from pre-embryonic tissue.

301. The method of claim 300, wherein the pre-embryonic tissue is blastocyst tissue.

302. The method of claim 297, wherein the embryonic stem cells are obtained from embryonic tissue.

231. A method for differentiating cells expressing markers characteristic of the definitive endoderm into cells expressing markers characteristic of the pancreatic endoderm lineage, comprising the steps of treating the cells expressing markers characteristic of the definitive endoderm with at least one fibroblast growth factor, or with retinoic acid and at least one fibroblast growth factor.

232. The method of claim 231, wherein the cells expressing markers characteristics of the definitive endoderm lineage are treated with a factor selected from the group consisting of retinoic acid, FGF-2, FGF-4 and FGF-10.

233. The method of claim 232, wherein the cells expressing markers characteristics of the definitive endoderm lineage are treated with a factor selected from the group consisting of retinoic acid, FGF-2, FGF-4 and FGF-10 for about one to about six days.

234. The method of claim 232, wherein the cells expressing markers characteristics of the definitive endoderm lineage are treated with a factor selected from the group consisting of retinoic acid, FGF-2, FGF-4 and FGF-10 for about six days.

240. The method of claim 231, wherein the cells expressing markers characteristics of the definitive endoderm are treated with retinoic acid at a concentration of about 1 nM to about 1 mM.

241. The method of claim 231, wherein the cells expressing markers characteristics of the definitive endoderm are treated with retinoic acid at a concentration of 1 μ

M.

242. The method of claim 231, wherein the cells expressing markers characteristics of the definitive endoderm are treated with FGF-2 at a concentration of about 50 pg/ml to about 50 μ

g/ml.

243. The method of claim 231, wherein the cells expressing markers characteristics of the definitive endoderm are treated with FGF-2 at a concentration of 50 ng/ml.

244. The method of claim 231, wherein the cells expressing markers characteristics of the definitive endoderm are treated with FGF-4 at a concentration of about 50 pg/ml to about 50 μ

g/ml.

245. The method of claim 231, wherein the cells expressing markers characteristics of the definitive endoderm are treated with FGF-4 at a concentration of 50 ng/ml.

246. The method of claim 231, wherein the cells expressing markers characteristics of the definitive endoderm are treated with FGF-10 at a concentration of about 50 pg/ml to about 50 μ

g/ml.

247. The method of claim 231, wherein the cells expressing markers characteristics of the definitive endoderm are treated with FGF-10 at a concentration of 50 ng/ml.

272. The method of claim 231, wherein the cells expressing markers characteristic of the definitive endoderm lineage are definitive endoderm cells.

273. The method of claim 231, wherein the cells expressing markers characteristic of the pancreatic endoderm lineage are pancreatic endoderm cells.

278. A method for treating a patient suffering from diabetes, comprising the steps of:

a. Culturing pluripotent stem cells,

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

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

d. Differentiating the cells expressing markers characteristics of the pancreatic endoderm lineage into cells of a β

-cell lineage, and

e. Implanting the cells of a β

-cell lineage into the patient.

303. The method of claim 278, wherein the pluripotent stem cells are embryonic stem cells.

304. The method of claim 303, wherein the embryonic stem cells are human.

305. The method of claim 303, wherein the embryonic stem cells are obtained from an embryonic stem cell line.

306. The method of claim 303, wherein the embryonic stem cells are obtained from pre-embryonic tissue.

307. The method of claim 306, wherein the pre-embryonic tissue is blastocyst tissue.

308. The method of claim 303, wherein the embryonic stem cells are obtained from embryonic tissue.