Generation of a lineage specific cell from a primate extended blastocyst cell line

• US 9,206,392 B2
• Filed: 12/27/2012
• Issued: 12/08/2015
• Est. Priority Date: 10/12/2004
• Status: Expired due to Fees

• Current Assignee: TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD. (Technion-Israel Institute Of Technology)
• Original Assignee: TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD. (Technion-Israel Institute Of Technology)
• Inventors: Amit, Michal, Itskovitz-Eldor, Joseph
• Primary Examiner(s): Crouch, Deborah
• Application Number:
  US13/727,693
• Publication Number:
  US 20130108597A1
• Time in Patent Office: 1,076 Days
• Field of Search: 435/373, 435/377, 435/384, 435/395
• US Class Current: 1/1
• 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. A method of generating a lineage specific cell comprising culturing an isolated extended blastocyst cell line under conditions:

(i) that allow spontaneous differentiation;

(ii) that allow formation of teratoma;

or

(iii) that direct differentiation into cells of the mesoderm lineage, the ectoderm lineage or the endoderm lineage, wherein said isolated extended blastocyst cell line 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, thereby generating the lineage specific cell.

2. The method of claim 1, wherein said isolated extended blastocyst cell line further expresses at least one cartilage marker.

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

4. The method of claim 1, wherein said isolated extended blastocyst cell line maintains a stable normal karyotype for at least one year.

5. The method of claim 1, wherein said isolated extended blastocyst cell line expresses SSEA4 and TRA-1-60 markers.

6. The method of claim 1, wherein said isolated extended blastocyst cell line expresses less TRA-1-81 marker than an embryonic stem cell of the same primate species not expressing brachyury using identical assay conditions.

7. The method of claim 1, wherein said isolated extended blastocyst cell line is capable of colony organization of columnar epithelium with villi throughout the upper side of said colony.

8. The method of claim 1, wherein said isolated extended blastocyst cell line exhibits 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.

9. The method of claim 1, wherein said isolated extended blastocyst cell line expresses more mesodermal differentiating markers than an embryonic stem cell of an identical primate not expressing brachyury using identical assay conditions.

10. The method of claim 1, wherein said isolated extended blastocyst cell line being genetically modified.

11. The method of claim 1, wherein said inducing is effected in vitro.

12. The method of claim 1, wherein said inducing is effected ex-vivo.

13. The method of claim 1, wherein said lineage-specific cell comprises cardiomyocytes.

14. The method of claim 1, wherein said lineage-specific cell comprises neural or glial lineages.

15. The method of claim 1, wherein said lineage-specific cell comprises hematopoietic cells.

16. The method of claim 1, wherein said lineage-specific cell comprises insulin-secreting beta cells.

17. The method of claim 1, wherein said differentiation is directed by genetic modification.

18. The method of claim 1, wherein said differentiation comprises formation of embryoid bodies.

19. The method of claim 1, wherein said primate is a human.