Differentiation of stem cells to pancreatic endocrine cells

• US 20040121460A1
• Filed: 12/08/2003
• Published: 06/24/2004
• Est. Priority Date: 01/24/2001
• Status: Abandoned Application

• Current Assignee: United States Department Of Health And Human Services
• Original Assignee: United States Department Of Health And Human Services
• Inventors: Kim, Jong-Hoon, Blondel, Oliver, McKay, Ronald D, Lumelsky, Nadya L
• Application Number:
  US10/470,030
• Publication Number:
  US 20040121460A1
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2510/02 Cells for production
• CPC Class Codes: C12N 5/0677 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells

Abstract

A method is provided for differentiating embryonic stem cells to endocrine cells. The method includes generating embryoid bodies from a culture of undifferentiated embryonic stem cells, selecting endocrine precursor cells, expanding the endocrine precursor cells by culturing endocrine cells in an expansion medium that comprises a growth factor, and differentiating the expanded endocrine precursor cells in a differentiation media to differentiated endocrine cells produced by this method are also provided. Artificial islets are disclosed, as well as method for using the pancreatic endocrine cells and the artificial islets.

Claims

7. A method for differentiating embryonic stem cells to endocrine cells, comprising

selecting endocrine precursor cells from embryonic stem cells or from embryoid bodies differentiated from embryonic stem cells;

expanding the endocrine precursor cells by culturing endocrine cells in an expansion medium that comprises a growth factor; and

differentiating the expanded endocrine precursor cells in a differentiation medium to differentiated endocrine cells.

8. The method of claim 7, wherein the selection of endocrine precursor cells comprises selecting cells that express nestin.

9. The method of claim 7, wherein the expansion medium is N2 medium containing B27 media supplement.

10. The method of claim 7, wherein the growth factor is bFGF.

11. The method of claim 7, wherein the differentiation medium comprises N2 medium containing B27 media and nicotinamide in the absence of the growth factor.

12. The method of claim 7, wherein the endocrine cells secrete insulin, glucagon, somatostatin, pancreatic polypeptide, or a combination thereof.

13. The method of claim 7, wherein the embryonic stem cells comprise murine, procine, or human embryonic stem cells.

14. The method of claim 13, wherein the embryonic stem cells are human embryonic stem cells.

15. The method of claim 7, wherein the endocrine cells are pancreatic endocrine cells.

16. The method of claim 15, wherein the pancreatic endocrine cells comprises a β

-cell, an α

-cell, a δ

-cell or a PP cell, or a combination thereof.

17. The method of claim 7, wherein the endocrine precursor cells are selected from embryoid bodies.

18. The method of claim 7, wherein the generation of embryoid bodies comprises culturing expanded undifferentiated embryonic stem cells in suspension.

19. The method of claim 7, wherein the step of culturing the embryoid bodies to select endocrine precursor cells comprises culturing the embryoid bodies in a serum-free medium.

20. The method of claim 7, wherein the step of culturing the embryoid bodies to select for endocrine precursor cells comprises culturing the embryoid bodies on a fibronectin-coated surface.

21. The method of claim 7, wherein the step of culturing the embryoid bodies to select for endocrine precursor cells comprises culturing the embryoid bodies for about 6 to about 8 days.

22. The method of claim 7, further comprising aggregating the differentiated endocrine cells.

23. A differentiated endocrine cell produced by the method of claim 7.

24. The endocrine cell of claim 23, wherein the endocrine cell is a pancreatic endocrine cell.

25. The endocrine cell of claim 23, wherein the pancreatic endocrine cell secrete insulin, glucagon, somatostatin, pancreatic polypeptide, or a combination thereof.

26. An artificial islet of Langerhans comprising the pancreatic endocrine cell produced by the method of claim 23.

42. A method of enhancing insulin production in a subject, comprising:

administering to the subject a therapeutically effective amount of a pancreatic endocrine cell produced by the method of claim 7.

43. The method of claim 42, wherein the subject is a diabetic.

44. A pharmacological composition comprising

a pancreatic endocrine cell produced by the method of claim 7;

and

a pharmacologically acceptable carrier.

45. The method of claim 7, wherein selecting endocrine precursor cells from embryonic stem cells or from embryoid bodies differentiated from embryonic stem cells is performed in the absence of exogenously added LIF.

27. A method of producing an artificial islet of Langerhans, comprising

generating embryoid bodies from a culture of undifferentiated embryonic stem cells;

selecting pancreatic endocrine precursor cells;

expanding the pancreatic endocrine precursor cells by culturing pancreatic endocrine cells in an expansion medium that comprises a growth factor; and

differentiating the expanded pancreatic endocrine precursor cells in a differentiation mediun to form pancreatic endocrine cells, and wherein the differentiation produces the artificial islet.

1. An isolated pancreatic endocrine cell, wherein said cell is differentiated from an embryonic stem cell in vitro, and wherein said cell secretes a pancreatic hormone.

2. The isolated pancreatic endocrine cell of claim 1, wherein the pancreatic endocrine cell comprises a β

-cell, an α

-cell, a δ

-cell, or a PP cell, or combinations thereof.

3. The isolated pancreatic endocrine cell of claim 1, wherein the pancreatic endocrine cell is a β

-cell.

4. The isolated pancreatic endocrine cell of claim 1, wherein the pancreatic endocrine cell is a murine cell.

5. The isolated pancreatic endocrine cell of claim 1, wherein the pancreatic endocrine cell is a human cell.

6. The isolated pancreatic endocrine cell of claim 1, wherein the pancreatic hormone is insulin, glucagon, somatostatin, or pancreatic polypeptide.

28. The method of claim 27, wherein the selection of endocrine precursor cells comprises selecting cells that express nestin.

29. The method of claim 27, wherein the expansion expansion medium is N2 medium containing B27 media supplement.

30. The method of claim 27, wherein the growth factor is bFGF.

31. The method of claim 27, wherein the differentiation medium comprises N2 medium containing B27 medium in the absence of the growth factor.

32. The method of claim 27, wherein the endocrine cells secrete insulin.

33. The method of claim 27, wherein the embryonic stem cells are murine, procine, or human embryonic stem cells.

34. The method of claim 27, wherein the embryonic stem cells are human embryonic stem cells.

35. The method of claim 27, wherein the generation of embryoid bodies comprises culturing expanded embryonic stem cells for about 4 to about 7 days.

36. The method of claim 27, wherein the generation of embryoid bodies comprises culturing expanded undifferentiated embryonic stem cells in suspension.

37. The method of claim 27, wherein the step of culturing the embryoid bodies to select endocrine precursor cells comprises culturing the embryoid bodies in a serum-free medium.

38. The method of claim 27, wherein the step of culturing the embryoid bodies to select for endocrine precursor cells comprises culturing the embryoid bodies on a fibronectin-coated surface.

39. The method of claim 27, wherein the step of culturing the embryoid bodies to select for endocrine precursor cells comprises culturing the embryoid bodies for about 6 to about 8 days.

46. The method of claim 27, wherein selecting pancreatic endocrine precursor cells includes culturing embryoid bodies in the absence of exogenously added LIF.

32-1. The method of claim 27, wherein the endocrine cells secrete glucagon, somatostatin, pancreatic polypeptide, or a combination thereof.

40. A method for testing an agent to determine the effect of the agent on secretion or expression of a pancreatic hormone, comprising:

contacting pancreatic endocrine cells with the agent, wherein the pancreatic endocrine cells are differentiated from embryonic stem cells; and

assaying a parameter of the pancreatic endocrine cell to determine the effect of the agent on the secretion or expression of the pancreatic hormone.

41. The method of claim 40, wherein the pancreatic endocrine hormone is insulin.