Isolated primate embryonic cells derived from extended blastocysts

• US 8,354,277 B2
• Filed: 10/11/2005
• Issued: 01/15/2013
• Est. Priority Date: 10/12/2004
• Status: Active Grant

• Current Assignee: TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD.
• Original Assignee: TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD.
• Inventors: Amit, Michal, Itskovitz-Eldor, Joseph
• Primary Examiner(s): Crouch, Deborah
• Application Number:
  US11/665,248
• Publication Number:
  US 20090196860A1
• Time in Patent Office: 2,653 Days
• Field of Search: None
• US Class Current: 435/373
• 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 line that exhibits for at least 20 passages an undifferentiated proliferative state, the ability to differentiate to derivatives of each of an endoderm, mesoderm, and ectoderm tissue and a double staining expression of brachyury and Octamer binding transcription factor 4 (OCT-4), but not of SSEA-1.

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

3. The isolated primate embryonic cell line 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 line 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.

9. A pharmaceutical composition comprising as an active ingredient the isolated primate embryonic cell line of claim 1 and a pharmaceutically acceptable carrier.

10. The isolated primate embryonic cell line of claim 1, maintaining a stable normal karyotype for at least one year.

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

12. The isolated primate embryonic cell line 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.

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

14. The isolated primate embryonic cell line 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 brachyury using identical assay conditions.

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

16. The isolated primate embryonic cell line of claim 1, being genetically modified.

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

21. The isolated primate embryonic cell line of claim 1 being immortalized.

22. The isolated primate embryonic cell line of claim 1, wherein the primate is a human.

31. The isolated primate embryonic cell line of claim 1, wherein the cell line exhibits staining expression of SRY-related HMG-box-2 (SOX2) and Nanog.

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

(a) providing a pre-implantation stage blastocyst;

(b) ex vivo culturing said pre-implantation stage blastocyst on fibroblast feeder cells or on a synthetic extracellular matrix for at least nine days to fourteen days post fertilization to produce a pre-gastrulation, extended blastocyst;

(c) isolating cells from said extended blastocyst; and

(d) culturing said isolated extended blastocyst cells on fibroblast feeder cells, an extracellular matrix or media comprising basic fibroblast growth factor, thereby generating a primate embryonic cell culture comprising embryonic cells that express brachyury and Oct-4, are maintained in an undifferentiated proliferative states for at least 20 passages and have the capability to develop in to each of the three germ layers;

endoderm, mesoderm and ectoderm.

17. The method of claim 7, wherein said fibroblast feeder cells are mouse fibroblast feeder cells or human fibroblast feeder cells.

18. The method of claim 17, wherein said mouse fibroblast feeder cells are mitotically inactivated mouse embryonic fibroblasts or primary mouse embryonic fibroblasts.

19. The method of claim 17, wherein said human fibroblast feeder cells are selected from the group comprising embryonic fibroblast cells, adult fallopian epithelial cells and foreskin cells.

23. The method of claim 7, wherein said blastocyst is a human blastocyst.

25. An isolated primate embryonic cell line generated according to the method of claim 7 and comprising cells that exhibit for at least 20 passages an undifferentiated proliferative state, the ability to differentiate to derivatives of each of an endoderm, mesoderm, and ectoderm tissue and a double staining expression of brachyury and Octamer binding transcription factor 4 (OCT-4), but not of SSEA-1.

26. An isolated primate embryonic cell line generated according to the method of claim 7, and comprising cells that exhibit for at least 20 passages an undifferentiated proliferative state, the ability to differentiate to derivatives of each of an endoderm, mesoderm, and ectoderm tissue and a double staining expression of fibroblast growth factor 5 (FGF5) and Octamer binding transcription factor 4 (OCT-4), but not of SSEA-1.

32. The isolated primate embryonic cell line of claim 25, wherein the cell line exhibits staining expression of SRY-related HMG-box-2 (SOX2) and Nanog.

33. The isolated primate embryonic cell line of claim 26, wherein the cell line exhibits staining expression of SRY-related HMG-box-2 (SOX2) and Nanog.

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

(a) providing a pre-implantation stage blastocyst;

(b) ex vivo culturing said pre-implantation stage blastocyst on fibroblast feeder cells or on a synthetic extracellular matrix for at least nine days to fourteen days post fertilization to produce an extended blastocyst

(c) isolating cells from said extended blastocyst;

(d) culturing said isolated extended blastocyst cells on fibroblast feeder cells, an extracellular matrix or media comprising basic fibroblast growth factor; and

(d) cloning at least one of said isolated cells, thereby generating a primate embryonic cell line comprising embryonic cells that express brachyury and Oct-4, are maintained in an undifferentiated proliferative state for at least 20 passages and have the capability to develop in to each of the three germ layers;

endoderm, mesoderm and ectoderm.

24. The method of claim 8, wherein said blastocyst is a human blastocyst.

27. An isolated clonal primate embryonic cell line generated according to the method of claim 8, and comprising cells that exhibit for at least 20 passages an undifferentiated proliferative state, the ability to differentiate to derivatives of each of an endoderm, mesoderm, and ectoderm tissue and a double staining expression of brachyury and Octamer binding transcription factor 4 (OCT-4), but not of SSEA-1.

28. An isolated clonal primate embryonic cell line generated according to the method of claim 8, and comprising cells that exhibit for at least 20 passages an undifferentiated proliferative state, the ability to differentiate to derivatives of each of an endoderm, mesoderm, and ectoderm tissue and a double staining expression of fibroblast growth factor 5 (FGF5) and Octamer binding transcription factor 4 (OCT-4), but not of SSEA-1.

34. The isolated clonal primate embryonic cell line of claim 27, wherein the cell line exhibits staining expression of SRY-related HMG-box-2 (SOX2) and Nanog.

35. The isolated clonal primate embryonic cell line of claim 28, wherein the cell line exhibits staining expression of SRY-related HMG-box-2 (SOX2) and Nanog.

29. An isolated clonal primate embryonic cell line comprising cells that exhibit for at least 20 passages an undifferentiated proliferative state, the ability to differentiate to derivatives of each of an endoderm, mesoderm, and ectoderm tissue and a double staining expression of brachyury and Octamer binding transcription factor 4 (OCT-4), but not of SSEA-1.

36. The isolated clonal primate embryonic cell line of claim 29, wherein the cell line exhibits staining expression of SRY-related HMG-box-2 (SOX2) and Nanog.

30. An isolated primate embryonic cell line that exhibits for at least 20 passages an undifferentiated proliferative state, the ability to differentiate to derivatives of each of an endoderm, mesoderm, and ectoderm tissue and a double staining expression of brachyury and Octamer binding transcription factor 4 (OCT-4), but not of SSEA-1, wherein the cell line is not genetically modified.