Maintenance of embryonic stem cells by the GSK-3 inhibitor 6-bromoindirubin-3'-oxime

• US 20060030042A1
• Filed: 12/20/2004
• Published: 02/09/2006
• Est. Priority Date: 12/19/2003
• Status: Abandoned Application

• Current Assignee: Rockefeller University
• Original Assignee: Rockefeller University
• Inventors: Meijer, Laurent, Brivanlou, Ali, Sato, Noboru
• Application Number:
  US11/018,784
• Publication Number:
  US 20060030042A1
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/70 Enzymes
• 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

The present invention relates to methods for maintaining the undifferentiated state of embryonic stem cells without the use of a feeder layer by activating the Wnt signal transduction pathway or by inhibiting glycogen synthase kinase-3 activity by contacting the cell with, inter alia, 6-bromoindirubin-3′-oxime. The present invention also relates to embryonic stem cell lines and cells derived therefrom that have been isolated and cultured in the absence of a feeder layer.

Claims

1. A method of maintaining the undifferentiated state of an embryonic stem cell, said method comprising contacting the stem cell in vitro with a molecule that antagonizes GSK-3 activity such that the cell divides but does not differentiate, wherein said contacting is in the absence of a feeder layer.

2. The method according to claim 1, wherein the molecule is LiCl.

3. The method according to claim 1, wherein the molecule is 6-bromoindirubin-3′

-oxime.

4. The method according to claim 1 further comprising the step of removing the molecule from contact with the stem cell.

5. The method according to claim 1, wherein the stem cell is a human stem cell.

6. A method of maintaining the undifferentiated state of an embryonic stem cell, said method comprising contacting said stem cell with 6-bromoindirubin-3′

-oxime such that the cells divides but does not differentiate.

7. The method according to claim 6, wherein said contacting is in the absence of a feeder layer.

8. The method according to claim 6, wherein said contacting is in vitro.

9. The method according to claim 6, wherein the stem cell is a human stem cell.

10. An isolated embryonic stem cell in contact with 6-bromoindirubin-3′

-oxime.

11. An embryonic stem cell that is the progeny of a second embryonic stem cell that was previously contacted with 6-bromoindirubin-3′

-oxime.

12. The embryonic stem cell of claim 11 which is isolated.

13. An embryonic stem cell line produced by the process comprising isolating embryonic stem cells from an embryo and culturing the isolated embryonic stem cells in the presence of a molecule that antagonizes GSK-3 activity such that the isolated embryonic stem cells divide but do not differentiate, wherein said isolating and culturing is in the absence of a feeder layer.

14. The embryonic cell line according to claim 13, wherein said molecule is 6-bromoindirubin-3′

-oxime.

15. The embryonic cell line according to claim 13, wherein said molecule is LiCl.

16. The embryonic cell line according to claim 13, wherein the embryonic stem cells are human embryonic stem cells.

29. The embryonic stem cell line according to claim 13, wherein the embryonic stem cells are isolated and cultured in the absence of exogenous cell extract or serum.

30. The embryonic stem cell line according to claim 13, wherein the embryonic stem cells are recombinant embryonic stem cells.

31. The recombinant embryonic stem cells according to claim 30, which express a prophylactic or therapeutic protein.

17. An embryonic stem cell line produced by the process comprising isolating embryonic stem cells from an embryo and culturing the isolated cells in the presence of 6-bromoindirubin-3′

-oxime such that the isolated embryonic stem cells divide but do not differentiate.

18. The embryonic cell line of claim 17, wherein said culturing is in the absence of a feeder layer.

19. The embryonic cell line of claim 17, wherein the embryonic stem cells are human embryonic stem cells.

20. A method of obtaining an embryonic stem cell line comprising isolating embryonic stem cells from an embryo and culturing the isolated embryonic stem cells in the presence of a molecule that antagonizes GSK-3 activity such that the isolated embryonic stem cells divide but do not differentiate, wherein said isolating and culturing is in the absence of a feeder layer.

21. The method according to claim 20, wherein the molecule is LiCl.

22. The method according to claim 20, wherein the molecule is 6-bromoindirubin-3′

-oxime.

23. The method according to claim 20, wherein the embryo is a human embryo.

24. A method of obtaining an embryonic stem cell line comprising isolating embryonic stem cells from an embryo and culturing the isolated embryonic stem cells in the presence of 6-bromoindirubin-3′

-oxime such that the embryonic stem cells divide but do not differentiate.

25. The method according to claim 24, wherein said isolating and culturing is in the absence of a feeder layer.

26. The method according to claim 24, wherein the embryo is a human embryo.

27. A human embryonic cell line, which cell line has not been in contact with a feeder layer and has not been in contact with an exogenous cell extract.

28. A human embryonic cell line, which cell line has not been in contact with a feeder layer and has not been in contact with a recombinant human protein.