Definitive endoderm

• US 7,510,876 B2
• Filed: 12/23/2004
• Issued: 03/31/2009
• Est. Priority Date: 12/23/2003
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: CyThera, Inc. (Vertex Pharmaceuticals Incorporated)
• Inventors: Agulnick, Alan D., D'Amour, Kevin Allen, Baetge, Emmanuel E.
• Primary Examiner(s): Ton; Thaian N.
• Assistant Examiner(s): SHEN, WU CHENG WINSTON
• Application Number:
  US11/021,618
• Publication Number:
  US 20050158853A1
• Time in Patent Office: 1,559 Days
• Field of Search: 435/366, 435/325
• US Class Current: 435/366
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• 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: C12N 5/068 Stem cells; Progenitors

Abstract

Disclosed herein are cell cultures comprising definitive endoderm cells and methods of producing the same. Also disclosed herein are cell populations comprising substantially purified definitive endoderm cells as well as methods for enriching, isolating and purifying definitive endoderm cells from other cell types.

Claims

1. An in vitro cell culture comprising human cells wherein at least 15% of said human cells are definitive endoderm cells, said definitive endoderm cells being multipotent cells that can differentiate into cells of the gut tube or organs derived therefrom.

2. The cell culture of claim 1, wherein at least 50% of said human cells are definitive endoderm cells.

3. The cell culture of claim 1, wherein at least 80% of said human cells are definitive endoderm cells.

4. The cell culture of claim 1, wherein at least 2 definitive endoderm cells are present for every 1 pluripotent cell in said cell culture.

5. The cell culture of claim 4, wherein said pluripotent cell comprises an embryonic stem cell.

6. The cell culture of claim 4, wherein said pluripotent cell is an embryonic germ cell.

7. The cell culture of claim 5, wherein said embryonic stem cell is derived from a tissue selected from the group consisting of the morula and the inner cell mass (ICM) of an embryo.

8. The cell culture of claim 1 further comprising a medium which comprises less than 2% serum.

9. The cell culture of claim 1 further comprising a growth factor of the Nodal/Activin subgroup of the TGFβ

superfamily.

10. The cell culture of claim 1, further comprising a growth factor selected from the group consisting of Nodal, Activin A, Activin B and combinations thereof.

11. The cell culture of claim 1, wherein said definitive endoderm cells have a normal karyotype.

12. The cell culture of claim 11, wherein said definitive endoderm cells are non-recombinant.

13. The cell culture of claim 5, wherein said human embryonic stem cell has a normal karyotype.

14. The cell culture of claim 13, wherein said human embryonic stem cell is non-recombinant.

15. The cell culture of claim 1, wherein said definitive endoderm cells adhere to a surface of a culture vessel.

16. The cell culture of claim 15, wherein said definitive endoderm cells are present as a monolayer.

17. The cell population of claim 11, wherein said definitive endoderm cells adhere to a surface of a culture vessel.

18. The cell population of claim 17, wherein said definitive endoderm cells are present as a monolayer.

19. The cell culture of claim 1, wherein at least 20% of said human cells are definitive endoderm cells.

20. The cell culture of claim 1, wherein at least 30% of said human cells are definitive endoderm cells.

21. The cell culture of claim 1, wherein at least 40% of said human cells are definitive endoderm cells.

22. The cell culture of claim 1, wherein at least 60% of said human cells are definitive endoderm cells.

23. The cell culture of claim 1, wherein at least 70% of said human cells are definitive endoderm cells.

24. The cell culture of claim 12, wherein at least 20% of said human cells are definitive endoderm cells.

25. The cell culture of claim 12, wherein at least 30% of said human cells are definitive endoderm cells.

26. The cell culture of claim 12, wherein at least 40% of said human cells are definitive endoderm cells.

27. The cell culture of claim 12, wherein at least 60% of said human cells are definitive endoderm cells.

28. The cell culture of claim 12, wherein at least 70% of said human cells are definitive endoderm cells.

29. The cell culture of claim 12, wherein at least 80% of said human cells are definitive endoderm cells.

30. The cell culture of claim 15, wherein at least 20% of said human cells are definitive endoderm cells.

31. The cell culture of claim 15, wherein at least 30% of said human cells are definitive endoderm cells.

32. The cell culture of claim 15, wherein at least 40% of said human cells are definitive endoderm cells.

33. The cell culture of claim 15, wherein at least 60% of said human cells are definitive endoderm cells.

34. The cell culture of claim 15, wherein at least 70% of said human cells are definitive endoderm cells.

35. The cell culture of claim 15, wherein at least 80% of said human cells are definitive endoderm cells.

36. An in vitro cell population comprising cells wherein at least 90% of said cells are human definitive endoderm cells, said human definitive endoderm cells being multipotent cells that can differentiate into cells of the gut tube or organs derived therefrom.

37. The cell population of claim 36, wherein at least 95% of said cells are human definitive endoderm cells.

38. The cell population of claim 36, wherein at least 98% of said cells are human definitive endoderm cells.

39. The cell population of claim 36, wherein at least 2 human definitive endoderm cells are present for every 1 pluripotent cell in said cell population.

40. The cell population of claim 39, wherein said pluripotent cell is an embryonic germ cell.

41. The cell population of claim 39, wherein said pluripotent cell comprises an embryonic stem cell.

42. The cell population of claim 41, wherein said embryonic stem cell is derived from a tissue selected from the group consisting of the morula and the ICM of an embryo.

43. The cell population of claim 36, wherein said human definitive endoderm cells have a normal karyotype.

44. The cell population of claim 43, wherein said human definitive endoderm cells are non-recombinant.

45. The cell population of claim 41, wherein said embryonic stem cell has a normal karyotype.

46. The cell population of claim 45, wherein said embryonic stem cell is non-recombinant.

47. An in vitro cell culture comprising human cells wherein at least 25% of said human cells are definitive endoderm cells, said definitive endoderm cells being multipotent cells that can differentiate into cells of the gut tube or organs derived therefrom.

48. The cell culture of claim 47, wherein at least 45% of said human cells are definitive endoderm cells.

49. The cell culture of claim 48, wherein said definitive endoderm cells are non-recombinant.

50. The cell culture of claim 48, wherein said definitive endoderm cells adhere to a surface of a culture vessel.

51. An in vitro cell culture comprising human cells wherein at least 55% of said human cells are definitive endoderm cells, said definitive endoderm cells being multipotent cells that can differentiate into cells of the gut tube or organs derived therefrom.

52. The cell culture of claim 51, wherein at least 65% of said human cells are definitive endoderm cells.

53. The cell culture of claim 52, wherein said definitive endoderm cells are non-recombinant.

54. The cell culture of claim 52, wherein said definitive endoderm cells adhere to a surface of a culture vessel.

55. An in vitro cell culture comprising human cells wherein at least 75% of said human cells are definitive endoderm cells, said definitive endoderm cells being multipotent cells that can differentiate into cells of the gut tube or organs derived therefrom.

56. The cell culture of claim 55, wherein at least 85% of said human cells are definitive endoderm cells.

57. The cell culture of claim 56, wherein said definitive endoderm cells are non-recombinant.

58. The cell culture of claim 56, wherein said definitive endoderm cells adhere to a surface of a culture vessel.