Preprimitive streak and mesendoderm cells

• US 7,985,585 B2
• Filed: 06/23/2006
• Issued: 07/26/2011
• Est. Priority Date: 07/09/2004
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Inventors: D'Amour, Kevin Allen, Agulnick, Alan D., Baetge, Emmanuel E.
• Primary Examiner(s): SHEN, WU CHENG WINSTON
• Application Number:
  US11/474,211
• Publication Number:
  US 20070004038A1
• Time in Patent Office: 1,859 Days
• Field of Search: 435/366, 435/325
• 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. An in vitro cell culture comprising a medium and human cells wherein at least 10% 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 20% of said human cells are mesendoderm cells.

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

4. The cell culture of claim 1, wherein at least 40% of said human cells are mesendoderm cells.

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

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

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

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

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

10. The cell culture of claim 1, wherein human feeder cells are present in said culture, and wherein at least 10% of human cells other than said human feeder cells are mesendoderm cells.

11. The cell culture of claim 1, wherein human feeder cells are present in said culture, and wherein at least 25% 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 50% 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 75% of human cells other than said human feeder cells are mesendoderm cells.

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

15. The cell culture of claim 14, 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, wherein expression is determined by quantitative PCR.

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

17. The cell culture of claim 16, 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, wherein expression is determined by quantitative PCR.

18. The cell culture of claim 1, wherein said cell culture includes less than 5% cells selected from the group consisting of visceral endodermal cells, parietal endodermal cells, primitive endodermal cells, definitive endodermal cells, ectodermal cells and mesodermal cells.

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

20. The cell culture of claim 19, wherein at least 2 mesendoderm cells are present for about every 1 hESC in said cell culture.

21. The cell culture of claim 19, wherein at least 10 mesendoderm cells are present for about every 1 hESC in said cell culture.

22. The cell culture of claim 19, 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.

23. The cell culture of claim 1, wherein said medium comprises less than 2% (v/v) serum.

24. The cell culture of claim 1, wherein said medium comprises less than 1% (v/v) serum.

25. The cell culture of claim 1, wherein said medium comprises less than 0.5% (v/v) serum.

26. The cell culture of claim 1, wherein said medium comprises less than 0.2% (v/v) serum.

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

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

superfamily.

29. The cell culture of claim 28, wherein said growth factor of the Nodal/Activin subgroup of the TGFβ

superfamily comprises activin A.

30. An in vitro cell population comprising a medium and human cells, wherein at least 90% of said human cells are human mesendoderm cells, said mesendoderm cells being multipotent cells that can differentiate into mesoderm cells or definitive endoderm cells.

31. The cell population of claim 30, wherein at least 95% of said human cells are mesendoderm cells.

32. The cell population of claim 30, wherein at least 98% of said human cells are mesendoderm cells.

33. The cell population of claim 30, wherein said human mesendoderm cells express a marker selected from the group consisting of brachyury, FGF4 and SNAI1.

34. The cell population of claim 33, 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, wherein expression is determined by quantitative PCR.

35. The cell population of claim 30, wherein said human mesendoderm cells express brachyury, FGF4 and SNAI1.

36. The cell population of claim 35, 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, wherein expression is determined by quantitative PCR.

37. An in vitro cell culture comprising a medium and human cells, wherein at least 10% of said human cells are mesendoderm cells, said mesendoderm cells being multipotent cells that can differentiate into mesoderm cells or definitive endoderm cells, and wherein less than 5% of the human cells are mesoderm cells.

38. The cell culture of claim 37 free of human mesoderm cells.

39. The cell culture of claim 37, wherein said medium comprises serum at a concentration of not more than 2%.

40. The cell culture of claim 37, wherein said medium lacks B27.

41. The cell culture of claim 37, wherein at least 40% of the human cells are mesendoderm cells.

42. The cell culture of claim 37, wherein at least 60% of the human cells are mesendoderm cells.