Hepatocyte lineage cells

• US 7,989,204 B2
• Filed: 04/27/2007
• Issued: 08/02/2011
• Est. Priority Date: 04/28/2006
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Inventors: D'Amour, Kevin, Baetge, Emmanuel E.
• Primary Examiner(s): Paras, Jr.; Peter
• Assistant Examiner(s): Montanari; David A
• Application Number:
  US12/298,898
• Publication Number:
  US 20090298170A1
• Time in Patent Office: 1,558 Days
• Field of Search: 435/70.1, 435/377
• US Class Current: 435/377
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/067 Hepatocytes
• CPC Class Codes: C12N 5/0672 Stem cells; Progenitor cells; Precursor cells; Oval cells

Abstract

Disclosed herein are methods for producing liver precursor cells as well as hepatocyte cells form pluripotent and/or multipotent cells. Also disclosed herein are methods of enriching isolating and/or purifying liver precursor cells and/or hepatocyte cells. Further disclosed are compositions comprising cell cultures and cell populations that are enriched for liver precursor cells or hepatocyte cells.

Claims

1. An in vitro method for producing human liver precursor cells, said method comprising contacting a cell population comprising human definitive endoderm cells with BMP4 and FGF10 for a time sufficient to allow the differentiation of human liver precursor cells from said human definitive endoderm cells.

2. The method of claim 1, wherein said BMP4 is provided to said cell population for about 1 day to about 8 days.

3. The method of claim 1, wherein said BMP4 is provided to said cell population for about 1 day to about 5 days.

4. The method of claim 1, wherein said BMP4 is provided to said cell population for about 1 day to about 3 days.

5. The method of claim 1, wherein said FGF10 is provided to said cell population for about 1 day to about 8 days.

6. The method of claim 1, wherein said FGF10 is provided to said cell population for about 1 day to about 5 days.

7. The method of claim 1, wherein said FGF10 is provided to said cell population for about 1 day to about 3 days.

8. The method of claim 1, wherein said FGF10 is provided to said cell population for about 1 day to about 2 days.

9. The method of claim 1, wherein BMP4 is provided to said cell population at a concentration ranging from about 0.1 ng/ml to about 100 ng/ml.

10. The method of claim 1, wherein BMP4 is provided to said cell population at a concentration ranging from about 1 ng/ml to about 5 ng/ml.

11. The method of claim 1, wherein BMP4 is provided to said cell population at a concentration of about 3 ng/ml.

12. The method of claim 1, wherein FGF10 is provided to said cell population at a concentration ranging from about 1 ng/ml to about 1000 ng/ml.

13. The method of claim 1, wherein FGF10 is provided to said cell population at a concentration ranging from about 5 ng/ml to about 500 ng/ml.

14. The method of claim 1, wherein said FGF10 is provided to said cell population at a concentration ranging from about 20 ng/ml to about 200 ng/ml.

15. The method of claim 1, wherein FGF10 is provided to said cell population at a concentration of about 50 ng/ml.

16. The method of claim 1 further comprising providing B27 to said cell population.

17. The method of claim 16, wherein said B27 is provided to said cell population at a concentration ranging from about 0.1% to about 20% of the total medium.

18. The method of claim 16, wherein said B27 is provided to said cell population at a concentration ranging from about 0.2% to about 2% of the total medium.

19. The method of claim 16, wherein said B27 is provided to said cell population at a concentration of about 0.5% of the total medium.

20. The method of claim 16, wherein said B27 is provided to said cell population for about 1 day to about 8 days.

21. The method of claim 16, wherein said B27 is provided to said cell population for about 2 days to about 6 days.

22. The method of claim 16, wherein said B27 is provided to said cell population for about 5 days.

23. The method of claim 1, wherein activin A is present in said cell population.

24. The method of claim 1, wherein activin A is not substantially present in said cell population.

25. The method of claim 1, wherein said human definitive endoderm cells are differentiated in vitro from human embryonic stem cells (hESCs).

26. The method of claim 25, wherein the differentiation of said hESCs to definitive endoderm cells comprises providing said hESCs with activin A.

27. The method of claim 26, wherein said hESCs are provided with activin A for about 1 day to about 8 days.

28. The method of claim 26, wherein said hESCs are provided with activin A for about 1 day to about 6 days.

29. The method of claim 26, wherein said hESCs are provided with activin A for about 1 day to about 5 days.

30. The method of claim 26, wherein said hESCs are provided with activin A for about 3 days.

31. The method of claim 26, wherein activin A is provided to said hESCs at a concentration ranging from about 0.1 ng/ml to about 1000 ng/ml.

32. The method of claim 26, wherein activin A is provided to said hESCs at a concentration ranging from about 1 ng/ml to about 500 ng/ml.

33. The method of claim 26, wherein activin A is provided to said hESCs at a concentration ranging from about 20 ng/ml to about 200 ng/ml.

34. The method of claim 26, wherein activin A is provided to said hESCs at a concentration of about 100 ng/ml.

35. The method of claim 1, wherein said cell population comprises at least about 10% human definitive endoderm cells.

36. The method of claim 1, wherein said cell population comprises at least about 25% human definitive endoderm cells.

37. The method of claim 1, wherein said cell population comprises at least about 50% human definitive endoderm cells.

38. The method of claim 1, wherein said cell population comprises at least about 75% human definitive endoderm cells.

39. The method of claim 1, wherein said cell population comprises at least about 90% human definitive endoderm cells.