Control of ES Cell Self Renewal and Lineage Specification, and Medium Therefor

• US 20080145936A1
• Filed: 01/07/2008
• Published: 06/19/2008
• Est. Priority Date: 05/08/2002
• Status: Abandoned Application

• Current Assignee: University of Edinburgh
• Original Assignee: University of Edinburgh
• Inventors: Ying, Qi-Long, Smith, Austin Gerard
• Application Number:
  US11/970,377
• Publication Number:
  US 20080145936A1
• Time in Patent Office: Days
• US Class Current: 435/455
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• 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

Self renewal of pluripotent cells in culture is promoted using a combination of an activator of a signalling pathway downstream of a receptor of the TGF-β superfamily and an activator of a gp130 downstream signalling pathway.

Claims

1. A method of self-renewing pluripotent cells in culture, comprising maintaining the cells in medium containing:

(a) a bone morphogenetic protein (BMP); and

(b) an agonist of a leukemia inhibitory factor (LIF) receptor selected from (i) LIF, and (ii) a combination of IL-6 and soluble IL-6 receptor, whereby the cells proliferate without differentiating.

4. The method of claim 1, wherein the BMP is BMP4.

6. The method of claim 1, wherein the agonist is LIF.

7. The method of claim 1, wherein the agonist is soluble IL-6 in combination with soluble IL-6 receptor.

9. The method of claim 1, wherein the cells are cultured in the presence of a FGF receptor inhibitor.

10. The method of claim 9 wherein the FGF receptor inhibitor is SU5402 or PD173074.

12. The method of claim 1, wherein the cells are pluripotent cells selected from mammalian and avian pluripotent cells.

13. The method of claim 1 wherein the cells are ES cells.

14. The method of claim 1, wherein the cells are selected from rodent, bovine, porcine, ovine and primate pluripotent stem cells.

16. The method of claim 1, wherein the cells are selected from rat cells and mouse cells.

17. The method of claim 1, wherein the cells are human cells.

18. The method of claim 1, comprising maintaining the cells in medium that is free of serum, free of serum extract, free of feeder cells and free of feeder cell extract.

19. The method of claim 1, wherein the medium is a fully defined medium.

2-3. -3. (canceled)

5. (canceled)

8. (canceled)

11. (canceled)

15. (canceled)

20. A method of culture of ES cells, comprising maintaining the ES cells in medium containing:

(a) a BMP; and

(b) an agonist of a LIF receptor selected from (i) LIF, and (ii) a combination of IL-6 and soluble IL-6 receptor.

24. The method of claim 20, wherein the agonist is LIF.

25. The method of claim 20, wherein the medium further comprises an inhibitor of a FGF receptor.

21-23. -23. (canceled)

26. A method of culture of ES cells, comprising:

maintaining the ES cells in a pluripotent state in culture, in the presence of an agonist of a LIF receptor selected from (i) LIF, and (ii) a combination of IL-6 and soluble IL-6 receptor, and serum or an extract of serum, optionally on feeders;

passaging the ES cells at least once;

withdrawing the serum or the serum extract from the medium and withdrawing the feeders if present, so that the medium is free of feeders, serum and serum extract; and

subsequently maintaining ES cells in a pluripotent state in the presence of

(a) a BMP; and

(b) an agonist of a LIF receptor selected from (i) LIF and (ii) a combination of IL-6 and soluble IL-6 receptor.

27. The method of claim 26, comprising culturing the ES cells in the presence of a FGF receptor inhibitor.

28. The method of claim 27 wherein the inhibitor is added to culture medium at the time that serum or serum extract is withdrawn.

29. The method of claim 28 wherein at the same time as or subsequent to maintenance of the cells in the presence of a BMP, the inhibitor is withdrawn.

30. A method of obtaining a transfected, pluripotent population of ES cells, comprising:

transfecting ES cells with a construct encoding a selectable marker;

plating the ES cells;

culturing the ES cells in the presence of

(a) a BMP;

(b) an agonist of a LIF receptor selected from (i) LIF, and (ii) a combination of IL-6 and soluble IL-6 receptor; and

selecting for ES cells that express the selectable marker thereby obtaining transfected, pluripotent ES cells.

31. The method of claim 30 wherein the selectable marker encodes antibiotic resistance or a cell surface marker.

32-51. -51. (canceled)