Preprimitive streak and mesendoderm cells

• US 20070004038A1
• Filed: 06/23/2006
• Published: 01/04/2007
• Est. Priority Date: 07/09/2004
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Inventors: Kroon, Evert, D'Amour, Kevin, Eliazer, Susan, Baetge, Emmanuel, Agulnick, Alan
• Granted Patent: US 7,985,585 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• 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 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

Abstract

This disclosure relates to compositions comprising human preprimitive streak cells and/or human mesendoderm cells as well as methods for their production. Additionally, disclosed herein are methods of identifying factors useful in the further differentiation of preprimitive streak and mesendoderm cell types.

Claims

1. A cell culture comprising human cells wherein at least about 5% of said human cells are mesendoderm cells, said mesendoderm cells being multipotent cells that can differentiate into mesoderm cells or definitive endoderm cells.

2. The cell culture of claim 1, wherein at least about 10% of said human cells are mesendoderm cells.

3. The cell culture of claim 1, wherein at least about 20% of said human cells are mesendoderm cells.

4. The cell culture of claim 1, wherein at least about 30% of said human cells are mesendoderm cells.

5. The cell culture of claim 1, wherein at least about 40% of said human cells are preprimitive streak cells.

6. The cell culture of claim 1, wherein at least about 50% of said human cells are mesendoderm cells.

7. The cell culture of claim 1, wherein at least about 60% of said human cells are mesendoderm cells.

8. The cell culture of claim 1, wherein at least about 70% of said human cells are mesendoderm cells.

9. The cell culture of claim 1, wherein at least about 80% of said human cells are mesendoderm cells.

10. The cell culture of claim 1, wherein at least about 90% of said human cells are mesendoderm cells.

11. The cell culture of claim 1, wherein human feeder cells are present in said culture, and wherein at least about 5% of human cells other than said human feeder cells are mesendoderm cells.

12. The cell culture of claim 1, wherein human feeder cells are present in said culture, and wherein at least about 25% of human cells other than said human feeder cells are mesendoderm cells.

13. The cell culture of claim 1, wherein human feeder cells are present in said culture, and wherein at least about 50% of human cells other than said human feeder cells are mesendoderm cells.

14. The cell culture of claim 1, wherein human feeder cells are present in said culture, and wherein at least about 75% of human cells other than said human feeder cells are mesendoderm cells.

15. The cell culture of claim 1, wherein said mesendoderm cells express a marker selected from the group consisting of brachyury, FGF4 and SNAI1.

16. The cell culture of claim 15, wherein the expression of a marker selected from the group consisting of brachyury, FGF4 and SNAI1 is greater than the expression of a marker selected from the group consisting of OCT4, SOX17, CXCR4, FOXA2, SOX7 and SOX1 in said mesendoderm cells.

17. The cell culture of claim 15, wherein said mesendoderm cells do not substantially express a marker selected from the group consisting of OCT4, SOX17, CXCR4, FOXA2, SOX7 and SOX1.

18. The cell culture of claim 1, wherein said mesendoderm cells express brachyury, FGF4 and SNAI1.

19. The cell culture of claim 18, wherein the expression of brachyury, FGF4 and SNAI1 is greater than the expression of OCT4, SOX17, CXCR4, FOXA2, SOX7 and SOX1 in said mesendoderm cells.

20. The cell culture of claim 18, wherein said mesendoderm cells do not substantially express OCT4, SOX17, CXCR4, FOXA2, SOX7 and SOX1.

21. The cell culture of claim 1, wherein said cell culture is substantially free of cells selected from the group consisting of visceral endodermal cells, parietal endodermal cells, primitive endodermal cells, definitive endodermal cells, ectodermal cells and mesodermal cells.

22. The cell culture of claim 1 further comprising human embryonic stem cells (hESCs).

23. The cell culture of claim 22, wherein at least about 2 mesendoderm cells are present for about every 1 hESC in said cell culture.

24. The cell culture of claim 22, wherein at least about 10 mesendoderm cells are present for about every 1 hESC in said cell culture.

25. The cell culture of claim 22, wherein said hESC is derived from a tissue selected from the group consisting of the morula, the inner cell mass (ICM) of an embryo and the gonadal ridges of an embryo.

26. The cell culture of claim 1 further comprising a medium that comprises less than about 2% (v/v) serum.

27. The cell culture of claim 1 further comprising a medium that comprises less than about 1% (v/v) serum.

28. The cell culture of claim 1 further comprising a medium that comprises less than about 0.5% (v/v) serum.

29. The cell culture of claim 1 further comprising a medium that comprises less than about 0.2% (v/v) serum.

30. The cell culture of claim 1 further comprising a medium that lacks serum or lacks serum replacement.

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

superfamily.

32. The cell culture of claim 31, wherein said growth factor of the Nodal/Activin subgroup of the TGFβ

superfamily comprises activin A.

33. A cell population comprising cells wherein at least about 90% of said cells are human mesendoderm cells, said mesendoderm cells being multipotent cells that can differentiate into mesoderm cells or definitive endoderm cells.

34. The cell population of claim 33, wherein at least about 95% of said cells are human mesendoderm cells.

35. The cell population of claim 33, wherein at least about 98% of said cells are human mesendoderm cells.

36. The cell population of claim 33, wherein said human mesendoderm cells express a marker selected from the group consisting of brachyury, FGF4 and SNAI1.

37. The cell population of claim 36, wherein the expression of a marker selected from the group consisting of brachyury, FGF4 and SNAI1 is greater than the expression of a marker selected from the group consisting of OCT4, SOX17, CXCR4, FOXA2, SOX7 and SOX1 in said human mesendoderm cells.

38. The cell population of claim 36, wherein said human mesendoderm cells do not substantially express a marker selected from the group consisting of OCT4, SOX17, CXCR4, FOXA2, SOX7 and SOX1.

39. The cell population of claim 33, wherein said human mesendoderm cells express brachyury, FGF4 and SNAI1.

40. The cell population of claim 39, wherein the expression of brachyury, FGF4 and SNAI1 is greater than the expression of OCT4, SOX17, CXCR4, FOXA2, SOX7 and SOX1 in said human mesendoderm cells.

41. The cell population of claim 39, wherein said human mesendoderm cells do not substantially express OCT4, SOX17, CXCR4, FOXA2, SOX7 and SOX1.