Human embryonic germ cell line and methods of use

• US 6,245,566 B1
• Filed: 03/31/1998
• Issued: 06/12/2001
• Est. Priority Date: 03/31/1997
• Status: Expired due to Fees

• Current Assignee: Johns Hopkins University
• Original Assignee: Johns Hopkins University
• Inventors: Gearhart, John D., Shamblott, Michael Joseph
• Primary Examiner(s): Weber, Jon P.
• Application Number:
  US09/052,772
• Time in Patent Office: 1,169 Days
• Field of Search: 435/383, 435/384, 435/366, 435/372
• US Class Current: 435/384
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2502/1323 Adult fibroblasts
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• CPC Class Codes: C12N 5/0611 Primordial germ cells, e.g. embryonic germ cells [EG]
• CPC Class Codes: G01N 33/56966 Animal cells
• CPC Class Codes: G01N 33/689 related to pregnancy or the gonads

Abstract

Primordial germ cells isolated from human embryonic tissue, such as from the gonadal ridges of human embryo, are disclosed. The primordial germ cells are cultured resulting in cells that resemble embryonic stem cells or embryonic germ cells in morphology and pluripotency. The cells are maintained several months in culture and can be genetically manipulated using transgenic technology to insert heterologous genetic material.

Claims

1. A method for producing human pluripotent embryonic germ (hEG) cells, comprising culturing human primordial germ cells (PGCs) in a culture medium comprising:

(a) a ligand which binds to a receptor which can associate with glycoprotein 130 (gp

     130); and

(b) a growth factor;

until cells with the morphology of human pluripotent embryonic germ cells are observed, and wherein the cells exhibit the following culture characteristics during maintenance;

(i) dependence on a ligand that binds to a receptor, which can associate with gp 130; and

(ii) dependence on a growth factor.

2. The method of claim 1, wherein the PGCs are from gonadal ridge tissue.

3. The method of claim 2, wherein the gonadal ridge tissue is collected 3 to 13 weeks post-fertilization.

4. The method of claim 2, wherein the gonadal ridge tissue is collected 8 to 11 weeks post last menstrual period.

5. The method of claim 1, wherein the ligand is selected from the group consisting of oncostatin-M and leukemia inhibitory factor.

6. The method of claim 1, wherein the growth factor is basic fibroblast growth factor.

7. The method of claim 6, wherein the basic fibroblast growth factor is human basic fibroblast growth factor.

8. The method of claim 1 wherein the culture medium further comprises a factor which elevates intracellular cAMP.

9. The method of claim 8, wherein the factor is selected from the group consisting of forskolin, cholera toxin, isobutylmethylxanthine and dibutyladenosine cyclic monophosphate.

10. The method of claim 8, wherein the factor is forskolin.

11. The method of claim 1, wherein the culture medium further comprises conditioned media obtained by culturing feeder cells.

12. The method of claim 11, wherein the feeder cells are murine cells.

13. The method of claim 12, wherein the murine cells are STO cells.

14. The method of claim 13, wherein the STO cells have ATCC accession number CRL 1503.

15. The method of claim 12, wherein the murine cells are murine embryonic cells.

16. The method of claim 1, comprising culturing the PGCs in the presence of feeder cells that support the growth of human embryonic germ cells.

17. The method of claim 16, wherein the feeder cells are STO cells.

18. The method of claim 17, wherein the STO cells have ATCC accession number CRL 1503.

19. The method of claim 1, comprising culturing the PGCs in the presence of an extracellular matrix derived from feeder cells.

20. The method of claim 1, wherein the hEG cells are SSEA-1 positive.

21. The method of claim 1, further comprising freezing the hEG cells.

22. A method of maintaining a culture of human pluripotent embryonic germ (hEG) cells in a substantially undifferentiated state, wherein the cells are characterized as requiring a ligand which binds to a receptor which can heterodimerize with glycoprotein 130 (gp130) and a growth factor, the method comprising passaging the hEG cells in a culture medium comprising:

(a) a ligand which binds to a receptor which can associate with gp130; and

(b) a growth factor.

23. The method of claim 22, wherein the ligand is selected from the group consisting of oncostatin-M and leukemia inhibitory factor.

24. The method of claim 22, wherein the growth factor is basic fibroblast growth factor.

25. The method of claim 22, wherein the culture medium further comprises a factor which elevates intracellular cAMP.

26. The method of claim 22, wherein the hEG cells are SSEA-1 positive.

27. A method for producing and maintaining human pluripotent embryonic germ (hEG) cells, comprising culturing human primordial germ cells (PGCs) in a culture medium comprising:

(a) a ligand which binds to a receptor which can associate with glycoprotein 130 (gp

     130); and

(b) a growth factor;

until cells with the morphology of human pluripotent embryonic germ cells are observed, and wherein the cells exhibit the following culture characteristics during maintenance;

(i) dependence on a ligand that binds to a receptor which can associate with gp 130; and

(ii) dependence on a growth factor;

and maintaining the hEG cells in a substantially undifferentiated state by culturing the cells in a medium containing a factor that elevates cellular cAMP levels, a growth factor, and a factor that stimulates gp 130.

28. A method for producing human pluripotent embryonic germ (hEG) cells, comprising culturing human primordial germ cells (PGCs) in a culture medium comprising:

(a) an agent that binds to and activates gp130; and

(b) a growth factor;

until cells with the morphology of human pluripotent embryonic germ cells are observed, and wherein the cells exhibit the following culture characteristics during maintenance;

(i) dependence on an agent that binds to and activates gp130;

(ii) dependence on a growth factor.

29. The method of claim 28, wherein the agent that binds to and activates gp130 is an antibody.

30. The method of claim 28, wherein the PGCs are from gonadal ridge tissue.

31. The method of claim 30, wherein the gonadal ridge tissue is collected 8 to 11 weeks post last menstrual period.

32. The method of claim 28, wherein the growth factor is basic fibroblast growth factor.

33. The method of claim 28, wherein the culture medium further comprises a factor which elevates intracellular cAMP.

34. The method of claim 33, wherein the factor is selected from the group consisting of forskolin, cholera toxin, isobutylmethylxanthine and dibutyladenosine cyclic monophosphate.

35. A method of maintaining a culture of human pluripotent embryonic germ (hEG) cells in a substantially undifferentiated state, wherein the cells are characterized as requiring an agent that binds to and activates gp130 and a growth factor, the method comprising passaging the hEG cells in a culture medium comprising:

(a) an agent that binds to and activates gp130; and

(b) a growth factor.

36. The method of claim 35, wherein the agent that bind to and activates gp130 is an antibody.