Isolated Primate Embryonic Cells and Methods of Generating and Using Same

• US 20090196860A1
• Filed: 10/11/2005
• Published: 08/06/2009
• Est. Priority Date: 10/12/2004
• Status: Active Grant

• Current Assignee: TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD.
• Original Assignee: UAB Research Foundation (University of Alabama System)
• Inventors: Itskovitz-Eldor, Joseph, Amit, Michal
• Granted Patent: US 8,354,277 B2
• Time in Patent Office: Days
• US Class Current: 424/93.21
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• 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
• CPC Class Codes: C12N 5/0623 Stem cells
• CPC Class Codes: C12N 5/0654 Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
• CPC Class Codes: C12N 5/0663 Bone marrow mesenchymal stem cells (BM-MSC)
• CPC Class Codes: C12N 5/0672 Stem cells; Progenitor cells; Precursor cells; Oval cells
• CPC Class Codes: C12N 5/068 Stem cells; Progenitors

Abstract

An isolated primate embryonic cell is provided as well as cell cultures and cell lines derived therefrom. Also provided are methods of generating and using such cells.

Claims

1. An isolated primate embryonic cell characterized by expression of brachyury and ability to differentiate to derivatives of each of an endoderm, mesoderm, and ectoderm tissue.

2. The isolated primate embryonic cell of claim 1 further expressing at least one cartilage marker.

3. The isolated primate embryonic cell of claim 2 wherein said at least one cartilage marker is selected from the group consisting of COMP, aggrecan and collagen type II.

4. A cell culture comprising the isolated primate embryonic cell of claim 1.

5. The cell culture of claim 4, further comprising feeder cells.

6. The cell culture of claim 4, further comprising growth medium.

14. A pharmaceutical composition comprising as an active ingredient the stem cell of claim 1 and a pharmaceutically acceptable carrier.

15. The isolated primate embryonic cell of claim 1, maintaining a stable normal karotype for at least one year.

16. The isolated primate embryonic cell of claim 1, expressing SSEA4 and TRA-1-60 markers.

17. The isolated primate embryonic cell of claim 1, not expressing SSEA1 marker.

18. The isolated primate embryonic cell of claim 1, expressing less TRA-1-81 marker than an embryonic stem cell of the same primate species not expressing brachyury using identical assay conditions.

19. The isolated primate embryonic cell of claim 1, capable of colony organization of columnar epithelium with villi throughout the upper side of said colony.

20. The isolated primate embryonic cell of claim 1, having an OCT4 protein level lower than the OCT4 protein level in an embryonic stem cell of the same primate species not expressing brachycury using identical assay conditions.

21. The isolated primate embryonic cell of claim 1, expressing more mesodermal differentiating markers than an embryonic stem cell of an identical primate not expressing brachyury using identical assay conditions.

22. The isolated primate stem cell of claim 1 being genetically modified.

26. The isolated primate embryonic stem cell of claim 1 being in an undifferentiated proliferative state for at least 100 passages.

27. The isolated primate embryonic cell of claim 1 being immortalized.

28. The isolated primate embryonic cell, cell culture and methods of claim 1, wherein the primate is a human.

29. A method of treating or preventing a disease in which cell transplantation is therapeutically beneficial comprising;

(a) inducing cell differentiation in the isolated human embryonic cell of claim 28; and

(b) transplanting a therapeutically effective amount of the differentiated cells into a subject in need thereof,

thereby preventing or treating a disease in which cell transplantation is therapeutically beneficial.

30. The method of claim 29, further comprising genetically modifying the differentiated cell to express a therapeutic agent.

31. The method of claim 29, wherein said inducing cell differentiation comprises genetically modifying a plurality of said cells.

32. A method of generating a lineage specific cell comprising inducing a lineage-specific cell differentiation in the isolated primate embryonic cell of claim 1, thereby generating the lineage specific cell.

7. A method of generating a primate embryonic cell culture comprising;

(a) providing a blastocyst of at least nine days post fertilization;

(b) isolating cells from said blastocyst; and

(c) culturing said isolated cells, thereby generating the primate embryonic cell culture.

9. The methods of claim 7, wherein said providing said blastocyst is effected by ex-vivo culturing said blastocyst.

10. The method of claim 9, wherein said ex-vivo culturing said blastocyst is effected on feeder cells.

11. The method of claim 9, wherein said ex-vivo culturing said blastocyst is effected on a synthetic surface.

12. The methods of claim 7, wherein said step (c) is effected on feeder cells.

13. The methods of claim 7, wherein said step (c) is effected on a synthetic surface.

23. The methods of claims 10, wherein said feeder cells are mouse feeder cells or human feeder cells.

24. The method of claims 23, wherein said mouse feeder cells are mitotically inactivated mouse embryonic fibroblasts or primary mouse embryonic fibroblasts.

25. The method of claims 23, wherein said human feeder cells are selected from the group comprising embryonic fibroblast cells, adult fallopian epithelial cells and foreskin cells.

8. A method of generating a primate embryonic cell line comprising;

(a) providing a blastocyst of at least nine days post fertilization;

(b) isolating cells from said blastocyst;

(c) culturing said isolated cells; and

(d) cloning at least one of said isolated cells, thereby generating the primate embryonic cell line.