DIFFERENTIATION OF PRIMATE PLURIPOTENT CELLS TO HEPATOCYTE-LINEAGE CELLS

• US 20100086999A1
• Filed: 06/01/2007
• Published: 04/08/2010
• Est. Priority Date: 06/02/2006
• Status: Active Grant

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.), University of Edinburgh
• Original Assignee: University of Edinburgh
• Inventors: Cui, Wei, Zhao, Debiao, Hay, David C., Majumdar, Anish Sen
• Granted Patent: US 8,148,151 B2
• Time in Patent Office: Days
• US Class Current: 435/370
• CPC Class Codes: C12N 2500/30 Organic components metal chelators C12N2500/10; calcitonin C12N2500/14; transferrin C12N2500/24
• CPC Class Codes: C12N 2500/36 Lipids
• CPC Class Codes: C12N 2500/62 DMSO
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/12 Hepatocyte growth factor [HGF]
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/23 Interleukins [IL]
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/067 Hepatocytes

Abstract

Methods for differentiating primate pluripotent stem cells into hepatocyte-lineage cells are provided. In certain embodiments, the methods utilize sequential culturing of the primate pluripotent stem cells in certain growth factors to produce hepatocyte-lineage cells. In certain embodiments, the population of cells produced by the methods is further enriched for hepatocyte-lineage cells.

Claims

1. A method of obtaining hepatocyte-lineage cells comprising

sequentially culturing primate pluripotent stem cells in the following order:

culturing in a first culture medium comprising an Activin;

culturing in a second culture medium comprising at least one of an HGF polypeptide, an EGF polypeptide, and an OSM polypeptide.

2. The method of claim 1, wherein the primate pluripotent stem cells are human embryonic stem cells.

3. The method of claim 1, wherein the Activin comprises an Activin peptide.

4. The method of claim 3, wherein the Activin peptide comprises an active fragment of at least one of Activin A, Activin B, and Activin C.

6. The method of claim 1, wherein the Activin comprises at least one of Activin A, Activin B, and Activin C.

7. The method of claim 6, wherein the Activin is Activin A.

8. The method of claim 1, wherein the HGF polypeptide is an active fragment of an HGF.

10. The method of claim 1, wherein the HGF polypeptide is an HGF.

11. The method of claim 1, wherein the EGF polypeptide is an active fragment of an EGF.

13. The method of claim 1, wherein the EGF polypeptide is an EGF.

14. The method of claim 1, wherein the OSM polypeptide is an active fragment of an OSM.

16. The method of claim 1, wherein the OSM polypeptide is an OSM.

17. The method of claim 1, wherein the first culture medium further comprises sodium butyrate.

18. The method of claim 1, further comprising a step after step (a) and before step (b), comprising culturing in a third culture medium comprising DMSO.

19. The method of claim 1, wherein the second culture medium comprises an HGF polypeptide and an EGF polypeptide.

20. The method of claim 19, wherein the second culture medium further comprises dexamethasone.

21. The method of claim 19, further comprising a step after step (b), comprising culturing in a fourth culture medium comprising an HGF polypeptide, an EGF polypeptide, and an OSM polypeptide.

5. (canceled)

9. (canceled)

12. (canceled)

15. (canceled)

22. A method of obtaining hepatocyte-lineage cells comprising sequentially culturing primate pluripotent stem cells in the following order:

culturing in a first culture medium comprising an Activin and sodium butyrate;

culturing in a second culture medium comprising DMSO;

culturing in a third culture medium comprising an HGF polypeptide, an EGF polypeptide, and dexamethasone;

culturing in a fourth culture medium comprising an HGF polypeptide, an EGF polypeptide, and an OSM polypeptide.

23. The method of claim 22, further comprising a step after step (a) and before step (b) comprising culturing in a fifth culture medium comprising Activin and sodium butyrate, wherein the concentration of at least one of Activin and sodium butyrate is different than the concentration of the same factor in step (a).

24-55. -55. (canceled)