Human embryonic pluripotent germ cells

• US 6,090,622 A
• Filed: 03/31/1997
• Issued: 07/18/2000
• Est. Priority Date: 03/31/1997
• Status: Expired due to Fees

• Current Assignee: Johns Hopkins School of Medicine
• Original Assignee: Johns Hopkins School of Medicine
• Inventors: Gearhart, John D., Shamblott, Michael Joseph
• Primary Examiner(s): Weber, Jon P.
• Application Number:
  US08/829,372
• Time in Patent Office: 1,205 Days
• Field of Search: 424/93.1, 424/9.1, 424/93.21, 435/172.3, 435/325, 435/352, 435/353, 435/366
• US Class Current: 435/366
• 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 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic 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 extracted from post blastocyst human embryos, such as from the gonadal ridges of a 8-11 week LMP human embryo, are disclosed. The primordial germ cells are cultured in long term culture (more than 30 days) resulting in cells that resemble embryonic stem 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. Human pluripotential embryonic germ cells, wherein the cells exhibit the following culture characteristics during maintenance:

(a) dependence on a ligand which binds to a receptor which can heterodimerize with glycoprotein 130 (gp

     130); and

(b) dependence on a growth factor.

2. The cells of claim 1, wherein the growth factor is basic fibroblast growth factor (bFGF).

3. The cells of claim 1, wherein the cells are alkaline phosphatase positive.

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

5. The cells of claim 4, wherein the cells express cell surface antigen SSEA-3.

6. The cells of claim 1, wherein the ligand is oncostatin-M.

7. The cells of claim 1, wherein the ligand is leukemia inhibitory factor (LIF).

8. The cells of claim 1, wherein the cells are derived from gonadal ridge tissue.

9. The cells of claim 8, wherein the gonadal ridge tissue is 3 to 13 weeks post-fertilization.

10. The cells of claim 8, wherein the gonadal ridge tissue is 8 to 11 weeks after the last menstrual period.

11. The cells of claim 1, wherein the cells are SSEA-1 positive.

12. The cells of claim 1, wherein the culture characteristics further comprise a factor which elevates intracellular cAMP.

13. The cells of claim 12, wherein the factor which elevates intracellular cAMP is selected from the group consisting of forskolin, cholera toxin, isobutylmethylxanthine and dibutyladenosine cyclic monophosphate.

14. The cells of claim 12, wherein the factor which elevates intracellular cAMP is forskolin.

15. The cells of claim 1, wherein the cells retain the ability to proliferate after being cryopreserved.