Defined Culture Conditions of Human Embryonic Stem Cells

• US 20110076253A1
• Filed: 07/28/2006
• Published: 03/31/2011
• Est. Priority Date: 07/29/2005
• Status: Active Grant

• Current Assignee: Yale University
• Original Assignee: Yale University
• Inventors: Snyder, Michael P., Lu, Joyce J.
• Granted Patent: US 9,101,590 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• CPC Class Codes: A61K 38/1825 Fibroblast growth factor [FGF]
• CPC Class Codes: A61K 38/191 Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
• CPC Class Codes: A61K 38/28 Insulins
• CPC Class Codes: A61K 38/40 Transferrins, e.g. lactoferrins, ovotransferrins

Abstract

The present invention relates to compositions and methods for culturing stem cells, particularly embryonic stem cells. Specifically, the invention relates to a culture medium that supports proliferation of substantially undifferentiated stem cells, while maintaining potency of the cells. An an embodiment, the culture medium is defined and supports proliferation of substantially undifferentiated embryonic stem cells in essentially serum free and feeder cell free conditions. Compositions for making the medium and methods using the culture medium are also provided.

Claims

1. A culture medium for stem cells, wherein said culture medium comprises a basal medium, a protein comprising a member of the tumor necrosis factor (TNF) family, a protein comprising a member of the Wnt family, a protein comprising insulin, a protein comprising transferrin and a protein comprising fibroblast growth factor (FGF).

2. The culture medium of claim 1, wherein said protein comprising a TNF member is selected from the group consisting of April, an April substitute, BAFF, a BAFF substitute and combinations thereof.

3. The culture medium of claim 1, wherein said protein comprising a member of the Wnt family is Wnt3a or a Wnt3a substitute.

4. The culture medium of claim 1, wherein said basal medium is selected from the group consisting of DMEM and DMEM/F12.

5. The culture medium of claim 1, further comprising a protein comprising albumin.

6. The culture medium of claim 1, further comprising cholesterol.

7. The culture medium of claim 1, wherein said cholesterol is chemically-defined cholesterol.

8. The culture medium of claim 1, wherein said protein comprising FGF is basic fibroblast growth factor (bFGF).

10. The culture medium of claim 1, wherein any of said proteins are recombinantly synthesized and/or chemically synthesized.

11. The culture medium of claim 1, wherein said medium is essentially free of animal serum.

9. The culture medium of claim, wherein any of said proteins are recombinantly synthesized, chemically synthesized or isolated from a human biological sample.

12. A culture system for culturing stem cells, said culture system comprising a culture medium and a matrix, wherein said culture medium comprises a basal medium, a protein comprising a member of the tumor necrosis factor (TNF) family, a protein comprising a member of the Wnt family, a protein comprising insulin, a protein comprising transferrin and a protein comprising fibroblast growth factor (FGF).

13. The culture system of claim 12, wherein said matrix is selected from the group consisting of fibronectin, collagen, laminin and combinations thereof.

14. The culture system of claim 12, wherein said matrix is selected from the group consisting of fibronectin, collagen, and combinations thereof

15. The culture system of claim 12, wherein said matrix is recombinantly synthesized, chemically synthesized or isolated from a human biological sample.

16. The culture system of claim 12, wherein said matrix is recombinantly synthesized or chemically synthesized.

17. A composition for preparing a culture medium, said composition comprising a protein comprising a member of the tumor necrosis factor family and a protein comprising a member of the Wnt family.

18. The composition of claim 17, wherein said protein comprising a TNF member is selected from the group consisting of April, an April substitute, BAFF, a BAFF substitute and combinations thereof.

19. The composition of claim 17, wherein said protein comprising a WNT family member is Wnt3a or a Wnt3a substitute.

20. The composition of claim 17, further comprising a protein comprising insulin.

21. The composition of claim 17, further comprising a protein comprising transferrin.

22. The composition of claim 17, further comprising a protein comprising albumin.

23. The composition of claim 17, further comprising cholesterol.

24. The composition of claim 23, wherein said cholesterol is chemically-defined cholesterol.

25. The composition of claim 17, further comprising a protein comprising FGF.

26. The composition of claim 25, wherein said protein comprising FGF is basic fibroblast growth factor (bFGF).

27. The composition of claim 17, wherein any of said proteins are recombinantly synthesized or chemically synthesized.

28. A kit for preparing a culture medium, said kit comprising a composition comprising a protein comprising a member of the tumor necrosis factor family and a protein comprising a member of the Wnt family;

and instructional material for the use of the composition to prepare a culture medium.

29. A composition useful for preparing a culture medium, said composition comprising a protein comprising a member of the tumor necrosis factor family, a protein comprising a member of the Wnt family, a protein comprising insulin, a protein comprising transferrin, a protein comprising albumin, cholesterol and a basal medium.

30. A composition useful for preparing a culture medium, said composition comprising a protein comprising a member of the tumor necrosis factor family, a protein comprising insulin, a protein comprising transferrin, a protein comprising albumin, cholesterol and a basal medium.

31. A composition of matter comprising stem cells and a culture medium, wherein said culture medium comprises a basal medium, a protein comprising a member of the tumor necrosis factor (TNF) family, a protein comprising a member of the Wnt family, a protein comprising insulin, a protein comprising transferrin and a protein comprising fibroblast growth factor (FGF).

32. The composition of claim 31 wherein said protein comprising a TNF member is selected from the group consisting of April, an April substitute, BAFF, a BAFF substitute and combinations thereof.

33. The composition of claim 31, wherein said protein comprising a WNT family member is Wnt3a or a Wnt3a substitute.

34. The composition of claim 31, wherein said basal medium is DMEM or DMEM/F12.

35. The composition of claim 31, further comprising a protein comprising albumin, and cholesterol.

36. The composition of claim 31, wherein said cholesterol is chemically-defined cholesterol.

37. The composition of claim 31, wherein said protein comprising FGF is basic fibroblast growth factor (bFGF).

38. The composition of claim 31, wherein any of said proteins are recombinantly synthesized or chemically synthesized.

39. The composition of claim 31, wherein said stem cells are embryonic stem cells.

40. The composition of claim 31, wherein said stem cells are human embryonic stem cells.

41. A method for culturing a stem cell, said method comprising:

a) providing a culture system,

b) adding a stem cell to said culture system, and

c) culturing said stem cell,

wherein said culture system comprises a culture medium and a matrix, wherein said culture medium comprises a basal medium, a protein comprising a member of the tumor necrosis factor (TNF) family, a protein comprising a member of the Wnt family, a protein comprising insulin, a protein comprising transferrin and a protein comprising fibroblast growth factor (FGF).

42. The method of claim 41 wherein said stem cell is an embryonic stem cell.

43. The method of claim 41, wherein said stem cell is a human embryonic stem cell.

44. The method of claim 41, wherein said culture medium further comprises a protein comprising albumin, and cholesterol.

45. The method of claim 41, wherein said matrix in said culture system is selected the group consisting of fibronectin, collagen, laminin and combinations thereof.

46. The method of claim 42, wherein said embryonic stem cell is cultured for at least about 4 passages and wherein said embryonic stem cell proliferates in a substantially undifferentiated state while maintaining the potential to differentiate into derivatives of endoderm, mesoderm and ectoderm tissues and while maintaining the karyotype of said embryonic stem cell.

47. A kit for maintaining at least one stem cell in a culture system, said kit comprising:

a) a culture system comprising a culture medium and a matrix, wherein said culture medium comprises a basal medium, a protein comprising a member of the Wnt family, a protein comprising insulin, a protein comprising transferrin and a protein comprising a member of the TNF family and; and

b) instructional material;

wherein said instructional material comprises instructions for the use of said kit to maintain at least one stem cell in said culture system.

48. The kit of claim 47, wherein said culture medium further comprises a protein comprising FGF.

51. The kit of claim 47, wherein said culture medium is a defined culture medium and is essentially free of animal serum and feeder cells.

52. The kit of claim 47, wherein any of said proteins are recombinantly synthesized or chemically synthesized.

49. A kit for proliferating at least one stem cell in a culture system, said kit comprising:

a) a culture system comprising a culture medium and a matrix, wherein said culture medium comprises a basal medium, a protein comprising a member of the Wnt family, a protein comprising insulin, a protein comprising transferrin, a protein comprising a member of the tumor necrosis factor (TNF) family, a protein comprising albumin and cholesterol; and

b) instructional material comprising instructions for the use of said kit to proliferate at least one stem cell in said culture system.

50. The kit of claim 49, wherein said culture medium further comprises a protein comprising FGF.

53. A method of cell therapy, said method comprising:

culturing stem cells in a defined culture medium to produce a population of isolated stem cells, and

administering a therapeutically effective amount of said population of stem cells to a recipient in need of such cells,

wherein said defined culture medium comprises a basal medium, a protein comprising a member of the Wnt family, a protein comprising FGF, a protein comprising insulin, a protein comprising transferrin, a protein comprising a member of the tumor necrosis factor (TNF) family, a protein comprising albumin and cholesterol, and wherein any of said proteins are recombinantly synthesized or chemically synthesized, and further wherein said culturing is essentially free of animal serum and feeder cells.

54. The method of claim 53, wherein said stem cells are human embryonic stem cells.

55. The method of claim 53, wherein said method further comprises inducing differentiation of said stem cells prior to administering them to said recipient.