Human embryonic germ cell line and methods of use

• US 20030148514A1
• Filed: 02/07/2003
• Published: 08/07/2003
• Est. Priority Date: 03/31/1997
• Status: Abandoned Application

• Current Assignee: Johns Hopkins University
• Original Assignee: Johns Hopkins University
• Inventors: Shamblott, Michael Joseph, Gearhart, John D.
• Application Number:
  US10/359,917
• Publication Number:
  US 20030148514A1
• Time in Patent Office: Days
• US Class Current: 435/368
• 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 cell having the characteristics of a human pluripotent embryonic germ (EG) cell.

2. The cell of claim 1, wherein the cell is capable of differentiating spontaneously into a cell from each of the mesoderm, endoderm, or ectoderm germ layers.

3. The cell of claim 1, wherein the cell is alkaline phosphatase positive.

4. The cell of claim 1, wherein the cell is telomerase positive.

5. The cell of claim 1, wherein the cell expresses cell surface antigens SSEA-1 and SSEA-4, and cell surface antigens which bind with antibodies having the binding specificity of monoclonal antibodies TRA-1-60 (ATCC HB-4783) and TRA-1-81 (ATCC HB4784).

6. The cell of claim 5, wherein the cell expresses cell surface antigen SSEA-3.

7. The cell of claim 1, wherein the cell is in fluid contact with an EG cell growth supportive cell and culture media containing an effective amount of:

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

     130) or an antibody that binds to and activates gp130; and

(b) a growth factor.

8. The cell of claim 7, wherein the ligand is oncostatin-M.

9. The cell of claim 7, wherein the ligand is leukemia inhibitory factor (LIF).

10. The cell of claim 7, wherein the growth factor is basic Fibroblast Growth Factor (bFGF).

11. The cell of claim 7, wherein the growth supportive cells is selected from the group consisting of a STO cell, a human embryonic lung fibroblast.

12. A method for producing EG, comprising culturing human primordial germ cells (PCGs) in the presence of STO cells in a culture medium containing an effective amount of:

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

     130) or an antibody that binds to and activates gp130; and

(b) a growth factor.

13. The method of claim 12, wherein the PCG are from gonadal ridge tissue.

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

15. The method of claim 13, wherein the gonadal ridge tissue is collected 8 to 11 weeks post last menstrual period.

16. The method of claim 12, wherein the ligand is oncostatin-M.

17. The method of claim 12, wherein the ligand is leukemia inhibitory factor (LIF).

18. The method of claim 12, wherein the growth factor is basic fibroblast growth factor (bFGF).

19. The method of claim 12, wherein the culture medium further comprises a factor which elevates intracellular cAMP.

20. The method of claim 19, wherein the factor is selected from the group consisting of forskolin, cholera toxin, isobutylmethylxanthine and dibutyladenosine cyclic monophosphate.

21. A human EG cell obtained using the method of claim 12.

22. A method for identifying a compound which modulates EG cell function comprising:

(a) incubating components comprising the compound and at least one EG cell under conditions sufficient to allow the components to interact; and

(b) determining the effect of the compound on an EG cell function before and after incubating in the presence of the compound.

23. The method of claim 22, wherein the modulating is inhibition of a cell function.

24. The method of claim 22, wherein the modulating is stimulation of a cell function.

25. The method of claim 22, wherein the EG cell function is selected from the group consisting of differentiation, gene expression, production of growth factors, response to growth factors and modulation of cell membrane permeability.

26. A method of using an EG cell to produce restricted developmental lineage cells, comprising:

contacting the EG cell with a differentiation agent, thereby producing restricted developmental lineage cells from the EG cell.

27. The method of claim 26, wherein the agent is retinoic acid.

28. The method of claim 26, further comprising:

(a) expressing a selectable marker in a restricted developmental lineage cell, wherein the restricted developmental lineage cell contains a recombinant polynucleotide, and wherein the recombinant polynucleotide encodes the selectable marker in operable linkage with a restricted developmental lineage cell specific promoter; and

(b) selecting the developmental lineage cells that express the selectable marker.

29. The method of claim 26, wherein the restricted developmental lineage cells are neuroepithelial cells.

30. The method of claim 29, wherein the restricted developmental lineage cells are epithelial cells.

31. The method of claim 26, wherein the restricted developmental lineage cells are cardiomyocytes.

32. A method of using an EG cell to produce an immortalized human restricted developmental lineage cell, comprising:

(a) generating a differentiation signal in an EG cell thereby producing restricted developmental lineage cells from the EG cell; and

(b) expressing recombinant telomerase in the restricted developmental lineage cell, thereby producing immortalized human cells having the characteristics of a restricted developmental lineage cell.

33. A method for identifying a compound which can differentiate EG cells, comprising:

(a) incubating components comprising the compound and an EG cell under conditions sufficient to allow the components to interact; and

(b) determining the effect of the compound on the EG cell wherein the appearance in culture of a restricted developmental lineage cells indicates differentiation of the EG cell.