DEFINITIVE ENDODERM CELL POPULATIONS

• US 20120114613A1
• Filed: 07/14/2010
• Published: 05/10/2012
• Est. Priority Date: 05/17/2002
• Status: Active Grant

• Current Assignee: Icahn School of Medicine at Mount Sinai
• Original Assignee: Icahn School of Medicine at Mount Sinai
• Inventors: KELLER, GORDON M., KOUSKOFF, VALERIE, KUBO, ATSUSHI, FEHLING, HANS JOERG
• Granted Patent: US 8,748,171 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/04 Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: G01N 33/5073 Stem cells

Abstract

The present invention provides cell populations that are enriched for mesendoderm and mesoderm, and cell populations that are enriched for endoderm. The cell populations of the invention are useful for generating cells for cell replacement therapy.

Claims

1-10. -10. (canceled)

11. A cell population enriched for endoderm cells.

12. The cell population of claim 11 comprising at least about 50% endoderm cells.

13. The cell population of claim 11 comprising at least about 75% endoderm cells.

14. The cell population of claim 11 comprising at least about 90% endoderm cells.

15. The cell population of claim 11 obtained by culturing human embryonic stem cells in the presence of serum for about two to about ten days, isolating cells that express brachyury, and culturing said cells that express brachyury in the absence of serum for about one to about fifteen days.

16. A method of isolating the cell population of claim 11 comprising culturing human embryonic stem cells in the presence of serum for about two to about ten days, isolating cells that express brachyury, and culturing said cells that express brachyury in the absence of serum for about one to about fifteen days.

17. The method of claim 16 wherein a selectable marker gene is inserted into the brachyury locus of said embryonic stem cells, and cells that express brachyury are isolated by selecting for cells that express the selectable marker.

18. The method of claim 17 wherein the selectable marker is enhanced green fluorescence protein.

19. A method of isolating the cell population of claim 11 comprising culturing embryonic stem cells in the absence of serum and in the presence of a sufficient amount of activin to induce differentiation of embryonic stem cells to mesendoderm cells that express brachyury, isolating said mesendooderm cells that express brachyury, and culturing said isolated cells to obtain endoderm cells that do not express brachyury.

21. A method of identifying endodermal cells capable of differentiating into mature hepatocytes comprising isolating the cell population of claim 11, transplanting a subpopulation of said population into an animal exhibiting a disorder associated with hepatocyte destruction or dysfunction, and assaying for liver function, wherein an improvement in liver function in said animal is indicative of the identification of endodermal cells capable of differentiating into mature hepatocytes.

22. The method of claim 21 wherein said animal is a fumarylacetoacetate deficient mouse.

24. A method of identifying an agent that affects the proliferation, differentiation or survival of endoderm cells comprising culturing the cell population of claim 11 in the presence of an agent to be tested and comparing the proliferation, differentiation or survival of said cells or the progeny of said cells in the presence and absence of said agent, wherein a difference in the presence of said agent is indicative of the identification of an agent that affects the proliferation, differentiation or survival of said cells or the progeny of said cells.

29. A method for generating mammalian cells comprising culturing the cell population of claim 11 under conditions effective for the differentiation of endoderm into liver cells, lung cells or pancreatic cells.

47. A method of identifying an agent that affects the proliferation, differentiation or survival of cells derived from endoderm comprising culturing the cell population of claim 11 under conditions effective for differentiation to obtain a population of differentiated cells, culturing the population of differentiated cells in the presence of an agent to be tested, and comparing the proliferation, differentiation or survival of said differentiated cells in the presence and absence of said agent, wherein a difference in the presence of said agent is indicative of the identification of an agent that affects the proliferation, differentiation or survival of said differentiated cells.

48. The method of claim 47 wherein said differentiated cells are selected from the group consisting of hepatocytes, pancreatic cells and lung cells.

49. A method of cell replacement comprising transplanting endoderm-derived cells into a mammal in need of cell replacement wherein said endoderm-derived cells are obtained by culturing the cell population of claim 11 under conditions effective for the differentiation of endoderm.

50. The method of claim 49 wherein the endoderm-derived cells are selected from the group consisting of hepatocytes, pancreatic cells and lung cells.

20. (canceled)

23. (canceled)

25-28. -28. (canceled)

30. (canceled)

31. A cell population enriched for brachyury⁻

endoderm cells.

32. The cell population of claim 31 that generates hepatocytes under hepatocyte differentiation-inducing conditions.

33. The cell population of claim 31 that generates pancreatic cells under pancreatic cell differentiation-inducing conditions.

34. The cell population of claim 31 that generates lung cells under lung cell differentiation-inducing conditions.

35. A cell population enriched for endoderm cells produced by a method comprising culturing embryonic stem cells in the presence of a sufficient concentration of activin to induce differentiation of the embryonic stem cells to endoderm.

36. The cell population of claim 35 wherein said cells do not express brachyury after about nine days in culture.

37. The cell population of claim 35 wherein said endoderm cells express at least one gene selected from the group consisting of HNF3β

, Mixl-1, Sox17, Hex-2 and pdx-1.

38. The cell population of claim 35 wherein said endoderm cells do not express at least one of GATA-1 and PAX6.

39. A hepatocyte cell population made by obtaining brachyury⁻

endoderm cells and culturing said brachyury⁻

endoderm cells under conditions effective for differentiation of endoderm to hepatocytes.

40. A hepatocyte cell population made by culturing embryonic stem cells in the presence of a sufficient concentration of activin to induce differentiation of the embryonic stem cells to endoderm cells, and culturing said endoderm cells under conditions effective for differentiation of endoderm to hepatocytes.

41. A pancreatic cell population made by obtaining brachyury⁻

endoderm cells and culturing said brachyury⁻

endoderm cells under conditions effective for differentiation of endoderm to pancreatic cells.

43. The pancreatic cell population of claim 41 wherein the pancreatic cells are pancreatic islet cells.

42. A pancreatic cell population made by culturing embryonic stem cells in the presence of a sufficient concentration of activin to induce differentiation of the embryonic stem cells to endoderm cells, and culturing said endoderm cells under conditions effective for differentiation of endoderm to pancreatic cells.

44. The pancreatic cell population of claim 42 wherein the pancreatic cells are pancreatic islet cells.

45. A lung cell population made by obtaining brachyury⁻

endoderm cells and culturing said brachyury⁻

endoderm cells under conditions effective for differentiation of endoderm to lung cells.

46. A lung cell population made by culturing embryonic stem cells in the presence of a sufficient concentration of activin to induce differentiation of the embryonic stem cells to endoderm cells, and culturing said endoderm cells under conditions effective for differentiation of endoderm to lung cells.

51. A method for generating mammalian cells comprising culturing embryonic stem cells in the presence of a sufficient concentration of activin to induce differentiation of the embryonic stem cells to endoderm to obtain a cell population enriched for endoderm, and culturing the cell population enriched for endoderm cells under conditions effective for the differentiation of endoderm into liver cells, lung cells or pancreatic cells.