In vitro propagation of embryonic stem cells

• US 20030064512A1
• Filed: 04/12/2002
• Published: 04/03/2003
• Est. Priority Date: 08/04/1988
• Status: Active Grant

• Current Assignee: Zenyth Operations Pty. Ltd.
• Original Assignee: Zenyth Operations Pty. Ltd.
• Inventors: Gough, Nicholas Martin, Hilton, Douglas James, Williams, Robert Lindsay
• Granted Patent: US 7,186,883 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• 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 the use of leukemia inhibitory factor (LIF) in the isolation and propagation of embryonic stem cells in vitro.

Claims

1. A method for the isolation of embryonic stem (ES) cells from animal embryos in vitro which method comprises deriving and maintaining said embryos in culture medium containing an effective amount of leukaemia inhibitory factor (LIF) for a time and under conditions sufficient for the development of said ES cells.

2. The method according to claim 1 wherein the culture medium is free of feeder cells.

3. The method according to claims 1 or 2 wherein the animal embryos are derived from humans, mice, birds, sheep, pigs, cattle, goats or fish.

4. The method according to claim 3 wherein the animal embryos are derived from mice.

5. The method according to claims 1 or 2 wherein the culture medium is Eagle'"'"'s medium or modifications thereof or equivalents thereto.

6. The method according to any one of the preceding claims wherein the LIF is recombinant LIF.

7. The method according to claim 6 wherein the LIF is recombinant human or murine LIF.

8. The method according to claim 7 wherein LIF is added to the culture medium at a concentration of from 10 to 1,000,000 units/ml.

9. The method according to claim 8 wherein the LIF is added to the culture medium at a concentration of from 100 to 100,000 units/ml.

10. The method according to claim 9 wherein the LIF is added to the culture medium at a concentration of from 500 to 10,000 units/ml.

11. A method according to any one of the preceding claims wherein the effective time is from 1 day to 20 weeks.

12. The method according to claim 11 wherein the effective time is from 1 to 8 weeks.

27. A chimaeric animal or transgenic progeny thereof generated using ES cells which have been isolated from animal embryos according to claim 1.

29. The chimaeric animal or transgenic progeny thereof according to claim 27 or 28 wherein said animal is a mouse.

30. The chimaeric animal or transgenic progeny thereof according to claims 27 or 28 or 29 wherein the ES cells contain additional genetic material inserted therein.

13. A method for maintaining animal embryonic stem (ES) cells in vitro while retaining their pluripotential phenotype which process comprises culturing said cells in a culture medium containing an effective amount of leukaemia inhibitory factor (LIF) under conditions sufficient to maintain said cells.

14. The method according to claim 13 wherein the culture medium is free of feeder cells.

15. The method according to claim 13 or 14 wherein the animal ES cells are derived from humans, mice, birds, sheep, pigs, cattle, goats or fish.

16. The method according to claim 15 wherein the animal ES cells are derived from mice.

17. The method according to claim 13 or 14 wherein the culture medium comprises Eagle'"'"'s medium or modifications thereof or equivalents thereto.

18. The method according to any one of claims 13 to 17 wherein the LIF is recombinant LIF.

19. The method according to claim 18 wherein the LIF is recombinant murine or human LIF.

20. The method according to claim 19 wherein the recombinant LIF is added to the culture medium at a concentration of from 10 to 1,000,000 units/ml.

21. The method according to claim 20 wherein the recombinant LIF is added to the culture medium at a concentration of from 100 to 100,000 units/ml.

22. The method according to claim 21 wherein LIF is added to the culture medium at a concentration of from 500 to 10,000 units/ml.

28. A chimaeric animal or transgenic progeny thereof generated using animal ES cells which have been maintained in vitro according to the method of claim 13.

23. Embryonic stem (ES) cells derived from animal embryos in vitro isolated by deriving and maintaining said embryos in culture medium, said culture medium containing an effective amount of leukaemia inhibitory factor (LIF) for a time and under conditions sufficient for the development of said ES cells.

24. The ES cells according to claim 23 wherein the culture medium is free of feeder cells.

25. The ES cells according to claim 23 or 24 derived from human, mouse, bird, sheep, pig, cattle, goat or fish embryos.

26. The ES cells according to claim 25 derived from mouse embryos.