PANCREATIC ENDOCRINE PROGENITOR CELLS DERIVED FROM PLURIPOTENT STEM CELLS

• US 20090280096A1
• Filed: 05/11/2009
• Published: 11/12/2009
• Est. Priority Date: 05/09/2008
• Status: Abandoned Application

• Current Assignee: VistaGen Therapeutics, Inc.
• Original Assignee: VistaGen Therapeutics, Inc.
• Inventors: KUBO, Atsushi, Snodgrass, H. Ralph, Bonham, Kristina, Stull, Robert
• Application Number:
  US12/464,005
• Publication Number:
  US 20090280096A1
• Time in Patent Office: Days
• US Class Current: 424/93.700
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C07K 14/4705 stimulating, promoting or activating activity
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/60 Transcription factors
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2830/003 tet inducible
• CPC Class Codes: C12N 2830/20 transcription of more than one cistron
• CPC Class Codes: C12N 2840/203 having an IRES
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: G01N 33/507 Pancreatic cells

Abstract

The invention provides pluripotent cells modified to overexpress Pdx1 and Ngn3. Pluripotent cells include embryonic stem cells and induced pluripotent stem cells. Methods of producing pancreatic endocrine progenitor cells from ES cells or from iPS cells by forced expression of Pdx1 and Ngn3 are provided. Pancreatic endocrine progenitor cells are useful for drug discovery and cell replacement therapy.

Claims

1. A pluripotent stem cell modified to overexpress Pdx1 and Ngn3.

2. A pluripotent stem cell of claim 1, wherein expression of Pdx1 and Ngn3 are under the control of one or more inducible promoters.

3. The pluripotent stem cell of claim 1, wherein the cell is an embryonic stem cell or an induced pluripotent stem (iPS) cell.

4. The cell of claim 1, wherein the overexpression of Pdx1 and Ngn3 is simultaneous.

5. The cell of claim 1, wherein the overexpression of Pdx1 and Ngn3 is sequential.

6. The cell of claim 1 further comprising a reporter molecule.

7. The cell of claim 6, wherein the reporter molecule is operably linked to a promoter expressed in pancreatic endocrine progenitor cells or derivatives thereof but not expressed in primitive endoderm.

8. The cell of claim 2 further comprising a reporter molecule.

9. The cell of claim 8, wherein the reporter molecule is operably linked to a promoter expressed in pancreatic endocrine progenitor cells or derivatives thereof but not expressed in primitive endoderm.

10. The cell of claim 1 further modified to overexpress MafA.

11. The cell of claim 2 further modified to overexpress MafA under the control of an inducible promoter.

12. The cell of claim 11 further comprising a reporter molecule.

13. The cell of claim 12, wherein the reporter molecule is operably linked to a promoter expressed in pancreatic endocrine progenitor cells or derivatives thereof but not expressed in primitive endoderm.

36. A method of producing pancreatic endocrine progenitor cells from pluripotent stem cells, the method comprising the steps of:

a) preparing embryonic bodies (EB) from the pluripotent stem cells of claim 2,

b) dissociating the cells and incubating the cells in the presence of activin A on about day 2,

c) dissociating the cells and inducing expression of Pdx1 and Ngn3 starting about day 4-about day 6,

d) plating the cells on low attachment plates about day 6-about day 9, and

e) culturing the cells for sufficient time to identify pancreatic endocrine progenitor cells.

37. A method of producing pancreatic endocrine progenitor cells from pluripotent stem cells, the method comprising the steps of:

a) culturing pluripotent stem cells of claim 2 as a monolayer,

b) dissociating the cells and incubating the cells in the presence of activin A on about day 2,

c) dissociating the cells and inducing expression of Pdx1 and Ngn3 starting about day 4-about day 6,

d) plating the cells on about day 6-about day 9, and

e) culturing the cells for sufficient time to identify pancreatic endocrine progenitor cells.

38. The method of claim 36 or 37, wherein the pluripotent stem cells are embryonic stem cells or iPS cells.

39. The method of claim 36 or 37, wherein the pancreatic endocrine progenitor cells are identified by expression of insulin.

40. The method of claim 36 or 37 wherein a nucleic acid encoding a reporter molecule is introduced to the cells prior to identifying pancreatic endocrine progenitor cells.

41. The method of claim 40, wherein the nucleic acid encoding a reporter molecule is operably linked to a promoter expressed in pancreatic endocrine progenitor cells or derivatives thereof but not expressed in primitive endoderm.

42. A method of producing pancreatic endocrine progenitor cells from pluripotent stem cells, the method comprising the steps of:

a) preparing embryonic bodies (EB) from the pluripotent stem cell of claim 9,

b) dissociating the cells and incubating the cells in the presence of activin A on about day 2,

c) dissociating the cells and inducing expression of Pdx1 and Ngn3 starting about day 4-about day 6,

d) plating the cells on low attachment plates about day 6-about day 9, and

e) culturing the cells for sufficient time to identify pancreatic endocrine progenitor cells by identifying cells expressing the reporter molecule.

43. A method of producing pancreatic endocrine progenitor cells from pluripotent stem cells, the method comprising the steps of:

a) incubating a population of cells of claim 9 to initiate differentiation,

b) dissociating the cells and incubating the cells in the presence of activin A on about day 2,

c) dissociating the cells and inducing expression of Pdx1 and Ngn3 starting about day 4-about day 6,

d) plating the cells on about day 6-about day 9,

e) culturing the cells for sufficient time to identify pancreatic endocrine progenitor cells by identifying cells expressing the reporter molecule.

44. The method of claim 42 or 43, wherein the pluripotent stem cells are embryonic stem cells or iPS cells.

45. A method of producing primitive beta-islet cells from pluripotent stem cells, the method comprising the steps of:

a) preparing embryonic bodies (EB) from the pluripotent stem cell of claim 11,

b) dissociating the cells and incubating the cells in the presence of activin A on about day 2,

c) dissociating the cells and inducing expression of Pdx1 and Ngn3 starting about day 4-about day 6,

d) inducing expression of MafA,

e) plating the cells on low attachment plates about day 6-about day 9, and

f) culturing the cells for sufficient time to identify primitive beta-islet cells.

46. A method of producing primitive beta-islet cells from pluripotent stem cells, the method comprising the steps of:

a) incubating a population of cells of claim 11 to initiate differentiation,

b) dissociating the cells and incubating the cells in the presence of activin A on about day 2,

c) dissociating the cells and inducing expression of Pdx1 and Ngn3 starting about day 4-about day 6,

d) inducing expression of MafA,

e) plating the cells on about day 6-about day 9, and

f) culturing the cells for sufficient time to identify pancreatic endocrine progenitor cells.

47. The method of claim 45 or 46, wherein the pluripotent stem cells are embryonic stem cells or iPS cells.

48. A method of producing primitive beta-islet cells from pluripotent stem cells, the method comprising the steps of:

a) preparing embryonic bodies (EB) from the pluripotent stem cell of claim 13,

b) dissociating the cells and incubating the cells in the presence of activin A on about day 2,

c) dissociating the cells and inducing expression of Pdx1 and Ngn3 starting about day 4-about day 6,

d) inducing expression of MafA,

e) plating the cells on low attachment plates about day 6-about day 9, and

f) culturing the cells for sufficient time to identify primitive beta-islet cells by identifying cells expressing the reporter molecule.

49. A method of producing primitive beta-islet cells from pluripotent stem cells, the method comprising the steps of:

a) incubating a population of cells of claim 13 to initiate differentiation,

b) dissociating the cells and incubating the cells in the presence of activin A on about day 2,

c) dissociating the cells and inducing expression of Pdx1 and Ngn3 starting about day 4-about day 6,

d) inducing expression of MafA,

e) plating the cells on about day 6-about day 9, and

f) culturing the cells for sufficient time to identify pancreatic endocrine progenitor cells by identifying cells expressing the reporter molecule.

50. The method of claim 48 or 49, wherein the pluripotent stem cells are embryonic stem cells or iPS cells.

51. A method of producing pancreatic endocrine progenitor cells from pluripotent stem cells, the method comprising the steps of:

a) culturing a population of cells of claim 2 to initiate differentiation on about day −

4,

b) passaging the cells on about day −

2,

c) preparing EBs from the pluripotent cells on about day 0,

d) dissociating the cells and incubating the cells in the presence of activin A on about day 2,

e) dissociating the cells, inducing expression of Pdx1 and Ngn3 starting about day 4-about day 6

f) plating the cells on about day 6-about day 9,

g) culturing the cells for sufficient time to identify pancreatic endocrine progenitor cells.

52. A method of producing pancreatic endocrine progenitor cells from embryonic stem cells, the method comprising the steps of:

a) culturing a population of cells of claim 2 to initiate differentiation on about day −

4,

b) passaging the cells on about day −

2,

c) passaging the cells maintained as monolayer on about day 0,

d) dissociating the cells and incubating the cells in the presence of activin A on about day 2,

e) dissociating the cells, inducing expression of Pdx1 and Ngn3 starting about day 4-about day 6

f) plating the cells on about day 6-about day 9,

g) culturing the cells for sufficient time to identify pancreatic endocrine progenitor cells.

53. The method of claim 51 or 52, wherein the pluripotent stem cells are embryonic stem cells or iPS cells.

54. A method of producing pancreatic endocrine progenitor cells from pluripotent stem cells, the method comprising the steps of:

a) culturing a population of cells of claim 9 to initiate differentiation on about day −

4,

b) passaging the cells on about day −

2,

c) preparing EBs from the pluripotent stem cells on about day 0,

d) dissociating the cells and incubating the cells in the presence of activin A on about day 2,

e) dissociating the cells, inducing expression of Pdx1 and Ngn3 in the cells starting about day 4-about day 6

f) plating the cells on about day 6-about day 9,

g) culturing the cells for sufficient time to identify pancreatic endocrine progenitor cells by identifying cells expressing the reporter molecule.

55. A method of producing pancreatic endocrine progenitor cells from pluripotent stem cells, the method comprising the steps of:

a) culturing a population of cells of claim 9 to initiate differentiation on about day −

4,

b) passaging the cells on about day −

2,

c) passaging the cells maintained as monolayer on about day 0,

d) dissociating the cells and incubating the cells in the presence of activin A on about day 2,

e) dissociating the cells, inducing expression of Pdx1 and Ngn3 in the cells starting about day 4-about day 6

f) plating the cells on about day 6-about day 9,

g) culturing the cells for sufficient time to identify pancreatic endocrine progenitor cells by identifying cells expressing the reporter molecule.

56. The method of claim 54 or 55, wherein the pluripotent stem cells are embryonic stem cells or iPS cells.

57. A method of producing primitive beta-islet cells from embryonic stem cells, the method comprising the steps of:

a) culturing a population of cells of claim 11 to initiate differentiation on about day −

4,

b) passaging the cells on about day −

2,

c) preparing EBs from pluripotent stem cells on about day 0,

d) dissociating the cells and incubating the cells in the presence of activin A on about day 2,

e) dissociating the cells and inducing expression of Pdx1, Ngn3 and MafA in the cells starting about day 4-about day 6,

f) plating the cells on about day 6-about day 9,

g) culturing the cells for sufficient time to identify pancreatic endocrine progenitor cells.

58. A method of producing primitive beta-islet cells from pluripotent stem cells, the method comprising the steps of:

a) culturing a population of cells of claim 11 to initiate differentiation on about day −

4,

b) passaging the cells on about day −

2,

c) passaging the cells maintained as monolayer on about day 0,

d) dissociating the cells and incubating the cells in the presence of activin A on about day 2,

e) dissociating the cells, inducing expression of Pdx1, Ngn3 and MafA in the cells starting about day 4-about day 6

f) plating the cells on about day 6-about day 9,

g) culturing the cells for sufficient time to identify pancreatic endocrine progenitor cells.

59. The method of claim 57 or 58 wherein the pluripotent stem cells are embryonic stem cells or iPS cells.

60. A method of producing primitive beta-islet cells from embryonic stem cells, the method comprising the steps of:

a) culturing a population of cells of claim 13 to initiate differentiation on about day −

4,

b) passaging the cells on about day −

2,

c) preparing EBs from pluripotent stem cells on about day 0,

d) dissociating the cells and incubating the cells in the presence of activin A on about day 2,

e) dissociating the cells and inducing expression of Pdx1, Ngn3 and MafA in the cells starting about day 4-about day 6,

f) plating the cells on about day 6-about day 9,

g) culturing the cells for sufficient time to identify pancreatic endocrine progenitor cells by identifying cells expressing the reporter molecule.

61. A method of producing primitive beta-islet cells from pluripotent stem cells, the method comprising the steps of:

a) culturing a population of cells of claim 11 to initiate differentiation on about day −

4,

b) passaging the cells on about day −

2,

c) passaging the cells maintained as monolayer on about day 0,

d) dissociating the cells and incubating the cells in the presence of activin A on about day 2,

e) dissociating the cells, inducing expression of Pdx1, Ngn3 and MafA in the cells starting about day 4-about day 6

f) plating the cells on about day 6-about day 9,

g) culturing the cells for sufficient time to identify pancreatic endocrine progenitor cells by identifying cells expressing the reporter molecule.

62. The method of claim 60 or 61 wherein the pluripotent stem cells are embryonic stem cells or iPS cells.

14. A method of producing a pluripotent stem cell to overexpress Pdx1 and Ngn3, the method comprising the step of introducing nucleic acid encoding Pdx1 and Ngn3 into the cell.

15. The method of claim 14, wherein the pluripotent stem cell is an embryonic stem cell or an iPS cell.

16. The method of claim 14, wherein the nucleic acid encoding Pdx1 and the nucleic acid encoding Ngn3 are operably linked to one or more inducible promoters.

17. The method of claim 14, wherein the method further comprises the step of introducing a reporter molecule to the cell.

18. The method of claim 17, wherein the reporter molecule is operably linked to a promoter expressed in pancreatic endocrine progenitor cells or derivatives thereof but not expressed in primitive endoderm.

19. A method of producing a pluripotent stem cell to overexpress Pdx1, Ngn3 and MafA;

the method comprising the steps of;

a) introducing nucleic acid encoding Pdx1 and Ngn3 into the cells, and

b) introducing nucleic acid encoding MafA into the cells.

20. The method of claim 19, wherein the pluripotent stem cell is an embryonic stem cell or an iPS cell.

21. The method of claim 19, wherein the nucleic acid encoding Pdx1 and the nucleic acid encoding Ngn3 are operably linked to one or more inducible promoters.

22. The method of claim 19, wherein the nucleic acid encoding MafA is operably linked to an inducible promoter.

23. The method of claim 19, wherein the method further comprises the step of introducing a reporter molecule to the cell.

24. The method of claim 23, wherein the reporter molecule is operably linked to a promoter expressed in pancreatic endocrine progenitor cells or derivatives thereof but not expressed in primitive endoderm.

25. A method of producing pancreatic endocrine progenitor cells from pluripotent stem cells, the method comprising the steps of

a) producing definitive endoderm cells from the pluripotent stem cells,

b) expressing Pdx1 and Ngn3 in the definitive endoderm cells, and

c) culturing the cells for sufficient time to identify pancreatic endocrine progenitor cells.

26. The method of claim 25, wherein the pluripotent stem cells are embryonic stem cells or iPS cells.

27. The method of claim 25, wherein the pancreatic endocrine progenitor cells are identified by expression of insulin.

28. The method of claim 25, wherein the method includes an additional step of culturing the pancreatic endocrine progenitor cells in a monolayer.

63. A method of screening a compound for its ability to modulate pancreatic endocrine cell function, comprising combining the compound with an pancreatic endocrine progenitor cell according to claim 25, determining any phenotypic or metabolic changes in the cell that result from being combined with the compound, and correlating the change with an ability of the compound to modulate secretion of insulin, glucagon, gherlin, or somatostatin or proliferation of insulin secreting cells.

65. A method of screening a compound for its ability to modulate pancreatic endocrine cell function, comprising combining the compound with a pancreatic endocrine progenitor cell according to claim 25, culturing the cells for varying amounts of time, determining any phenotypic or metabolic changes in the cell that result from being combined with the compound, and correlating the phenotypic or metabolic change with the time of culturing the cells.

66. A method of screening a compound for its ability to modulate pancreatic endocrine cell function, comprising isolating pancreatic endocrine progenitor cells that express Pdx1 and Ngn3 according to claim 25 at fixed time points following induction of differentiation, combining the compound and the isolated cells, and determining any phenotypic or metabolic changes in the cell that result from being combined with the compound.

67. A method of screening a compound for its ability to modulate pancreatic endocrine cell function, comprising combining the compound with an pancreatic endocrine progenitor cell according to claim 25, determining any phenotypic or metabolic changes in the cell that result from being combined with the compound, and correlating the change with an ability of the compound to modulate secretion of insulin.

69. A method of screening a compound for its ability to modulate pancreatic endocrine cell function, comprising combining the compound with a pancreatic endocrine progenitor cell according to claim 25;

wherein the pancreatic endocrine progenitor cell further comprises a reporter molecule operably linked to a promoter expressed in pancreatic endocrine progenitor cells or derivatives thereof but not expressed in primitive endoderm; and

determining changes in expression of the reporter molecule.

70. A method of pancreatic cell therapy comprising administering to a subject in need of such treatment a composition comprising pancreatic endocrine progenitor cells produced by the method of claim 25.

72. A method of pancreatic cell therapy comprising administering to a subject in need of such treatment a composition comprising pancreatic endocrine progenitor cells produced by the method of claim 25;

wherein the cells are autologous to the subject.

74. A method of pancreatic cell therapy comprising administering to a subject in need of such treatment a composition comprising pancreatic endocrine progenitor cells produced by the method of claim 25;

wherein the cells are allogeneic to the subject.

76. A composition comprising pancreatic endocrine progenitor cells produced by the method of claim 25.

78. Use of pancreatic endocrine progenitor cells produced by the method of claim 25 in the manufacture of a medicament for treatment of an individual in need of pancreatic cell therapy.

79. Use of pancreatic endocrine progenitor cells produced by the method of claim 25 in the manufacture of a medicament for the treatment of a condition associated with deficiency of a pancreatic endocrine hormone.

80. The use of claim 79, wherein the pancreatic endocrine hormone is selected from the group consisting of insulin, glucagon, somatostatin, gherlin and pancreatic polypeptide.

81. The use of claim 80, wherein the pancreatic endocrine hormone is insulin.

82. The use of claim 81, wherein the condition associated with deficiency of a pancreatic endocrine hormone is diabetes.

29. A method of producing pancreatic endocrine progenitor cells from pluripotent stem cells, the method comprising the steps of

a) producing definitive endoderm cells from the pluripotent stem cells,

b) initiating expression of Pdx1 in the definitive endoderm cells,

c) analyzing the Pdx1-expressing cells for the expression of insulin mRNA,

d) initiating expression of Ngn3 in the Pdx1-expressing cells, and

e) culturing the Pdx1/Ngn3-expressing cells for sufficient time to identify pancreatic endocrine progenitor cells.

30. The method of claim 29, wherein the pluripotent stem cells are embryonic stem cells or iPS cells.

31. The method of claim 29, wherein the pancreatic endocrine progenitor cells are identified by expression of insulin.

32. The method of claim 29, wherein the method includes an additional step of culturing the pancreatic endocrine progenitor cells in a monolayer.

33. A method of producing primitive beta-islet cells from pluripotent stem cells, the method comprising the steps of

a) producing definitive endoderm cells from the pluripotent stem cells,

b) expressing Pdx1 and Ngn3 in the definitive endoderm cells,

c) culturing the Pdx1/Ngn3-expressing cells for sufficient time to identify pancreatic endocrine progenitor cells by measuring expression of insulin,

d) expressing MafA in the pancreatic endocrine progenitor cells, and

e) culturing the cells for sufficient time to identify primitive beta-islet cells by measuring secretion of insulin.

34. The method of claim 33, wherein the pluripotent stem cells are embryonic stem cells or iPS cells.

35. The method of claim 33, wherein the method includes an additional step of culturing the pancreatic endocrine progenitor cells in a monolayer.

64. A method of screening a compound for its ability to modulate beta-islet cell function, comprising combining the compound with an pancreatic endocrine progenitor cell according to claim 33, determining any phenotypic or metabolic changes in the cell that result from being combined with the compound, and correlating the change with an ability of the compound to modulate secretion of insulin or proliferation of insulin secreting cells.

68. A method of screening a compound for its ability to modulate primitive beta-islet cell function, comprising combining the compound with a primitive beta-islet cell according to claim 33, determining any phenotypic or metabolic changes in the cell that result from being combined with the compound, and correlating the change with an ability of the compound to modulate secretion of insulin.

71. A method of pancreatic cell therapy comprising administering to a subject in need of such treatment a composition comprising primitive beta-islet cells produced by the method of claim 33.

73. A method of pancreatic cell therapy comprising administering to a subject in need of such treatment a composition comprising primitive beta-islet cells produced by the method of claim 33;

wherein the cells are autologous to the subject.

75. A method of pancreatic cell therapy comprising administering to a subject in need of such treatment a composition comprising primitive beta-islet cells produced by the method of claim 33;

wherein the cells are allogeneic to the subject.

77. A composition comprising primitive beta-islet cells produced by the method of claim 33.

83. Use of primitive beta-islet cells produced by the method of claim 33 in the manufacture of a medicament for treatment of an individual in need of pancreatic cell therapy.

84. Use of primitive beta-islet cells produced by the method of claim 33 in the manufacture of a medicament for the treatment of a condition associated with a deficiency of beta-islet cell function.

85. The use of claim 84, wherein the condition is diabetes.