Differentiation of human embryonic germ cells

• US 6,562,619 B1
• Filed: 04/20/2000
• Issued: 05/13/2003
• 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): Crouch, Deborah
• Assistant Examiner(s): WOITACH, JOSEPH T
• Application Number:
  US09/553,640
• Time in Patent Office: 1,118 Days
• Field of Search: 435/325, 435/366, 435/440, 435/455, 800/8
• US Class Current: 435/366
• 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 of producing a cell population comprising differentiating in culture human embryonic germ (hEG) cells, wherein the hEG cells have the following culture characteristics during maintenance:

(i) dependence on an agent that binds to gp 130 or that binds to a receptor that can associate with gp 130; and

(ii) dependence on a growth factor.

2. The method of claim 1 wherein the primordial germ cells are from gonadal ridge tissue collected 3 to 13 weeks post-fertilization.

3. The method of claim 1 wherein the agent is selected from the group consisting of oncostatin-M, leukemia inhibitory factor, and an antibody to gp 130.

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

5. The method of claim 1 wherein the hEG cells have been obtained by culturing primordial germ cells in a medium that also contains a factor that elevates intracellular cAMP.

6. The method of claim 5 wherein the factor that elevates intracellular cAMP is selected from the group consisting of forskolin, cholera toxin, isobutylmethylxanthine and dibutyladenosine cyclic monophosphate.

7. The method of claim 1 wherein the hEG cells are differentiated by culturing for at least two weeks without passage onto a fresh layer of feeder cells.

8. The method of claim 1 wherein the hEG cells are differentiated by passaging them into a suspension culture.

9. The method of claim 8 wherein the cell population comprises embryoid bodies.

10. The method of claim 1 wherein the cell population comprises a member of the group consisting of neuroepithelial cells, hematopoietic progenitor cells, cardiomyocytes, and skeletal muscle cells.

11. The method of claim 1 wherein the hEG cells are differentiated by culturing in the presence of a member of the group consisting of retinoic acid, IL-3, IL-6, IL-11, GM-CSF, EPO, M-CSF, and G-CSF, or by withdrawing the growth factor with which the cells have been maintained.

12. A method of producing a population of restricted developmental lineage cells comprising:

(a) maintaining hEG cells by culturing in a medium comprising a growth factor and an agent that binds to a receptor that can associate with gp 130, wherein the hEG cells have the following culture characteristics during maintenance;

(i) dependence on an agent that binds to gp 130 or that binds to a receptor that can associate with gp 130; and

(ii) dependence on a growth factor; and

then

(b) differentiating the hEG cells.

13. The method of claim 12 wherein the hEG cells are differentiated by culturing the hEG cells for at least two weeks without passage onto a fresh layer of feeder cells, or by passaging the hEG cells into a suspension culture.

14. The method of claim 12 wherein the cell population comprises embryoid bodies.

15. The method of claim 12 wherein the cell population comprises a member of the group consisting of neuroepithelial cells, hematopoietic progenitor cells, cardiomyocytes, and skeletal muscle cells.

16. A method for producing human pluripotent embryonic germ (hEG) cells comprising:

(a) preparing a suspension of primordial germ cells;

(b) culturing cells of the suspension with feeder cells in a medium comprising a factor that elevates cellular cAMP levels; and

(c) harvesting pluripotent embryonic germ cells from the culture.

17. The method of claim 16 wherein the factor that elevates cellular cAMP levels is forskolin.

18. A human pluripotent embryogenic germ (hEG) cell prepared according to the method of claim 16.

19. The cells of claim 18, which are alkaline phosphatase positive.

20. The cells of claim 18, which express cell surface antigen SSEA-4, and cell surface antigens that bind with antibodies having the binding specificity of monoclonal antibodies TRA-1-60 and TRA-1-81.

21. The cells of claim 20, which express cell surface antigen SSEA-3.

22. The cells of claim 18, which express cell surface antigen SSEA-1.

23. The method of claim 16, wherein the medium further comprises

(d) dependence on an agent that binds to gp130 or that binds to a receptor that can associate with gp130; and

(e) a growth factor.

24. The method of claim 23, wherein the growth factor is basic fibroblast growth factor (bFGF).

25. The method of claim 23, wherein the ligand that binds to a receptor which can associate with gp130 is oncostatin-M or LIF.

26. The method of claim 23, wherein the agent that binds to and activates gp 130 is an antibody.

27. The method of claim 16, wherein the cells that are produced have the following culture characteristics during maintenance:

(i) dependence on an agent that binds to gp130 or that binds to a receptor that can associate with gp130; and

(ii) dependence on a growth factor.

28. A method for producing a population of restricted developmental lineage cells, comprising producing hEG cells according to the method of claim 16, and then causing them to differentiate.