Co-culturing mammalian embryonic stem cells with human foreskin fibroblasts

• US 8,318,486 B2
• Filed: 01/28/2010
• Issued: 11/27/2012
• Est. Priority Date: 10/07/2002
• 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:
  US12/695,199
• Publication Number:
  US 20100129907A1
• Time in Patent Office: 1,034 Days
• Field of Search: None
• US Class Current: 435/373
• CPC Class Codes: C12N 2500/00 Specific components of cell culture medium
• CPC Class Codes: C12N 2500/99 Serum-free medium
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2502/1323 Adult fibroblasts
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2533/50 Proteins
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0031 Serum-free culture media
• CPC Class Codes: C12N 5/0043 Medium free of human- or animal-derived components
• CPC Class Codes: C12N 5/0056 Xeno-free medium
• 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 5/0611 Primordial germ cells, e.g. embryonic germ cells [EG]
• CPC Class Codes: C12N 5/0665 Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
• CPC Class Codes: C12N 5/0695 Stem cells; Progenitor cells; Precursor cells
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

A cell culture comprising human foreskin cells, the human foreskin cells being capable of maintaining stem cells in an undifferentiated state when co-cultured therewith.

Claims

1. A method of maintaining mammalian pluripotent stem cells in an undifferentiated state comprising co-culturing the mammalian pluripotent stem cells with a human foreskin fibroblast feeder cell line, wherein said mammalian pluripotent stem cells are capable of forming teratomas containing cells of the endoderm, mesoderm and ectoderm germ layers.

2. The method of claim 1, wherein said human foreskin feeder cell line is prepared by:

(a) isolating foreskin cells from foreskin tissue;

(b) culturing said foreskin cells in a culture medium including serum and/or serum replacement thereby preparing said human foreskin feeder cell line.

3. The method of claim 1, wherein said mammalian pluripotent stem cells are of embryonic origin.

4. The method of claim 1, wherein said mammalian pluripotent stem cells are of human origin.

5. The method of claim 2, wherein said foreskin tissue is obtained from an 8-14 day old male individual.

6. The method of claim 2, wherein step (a) is effected by:

(i) mincing said foreskin tissue;

(ii) dissociating said foreskin tissue resultant from step (i) into single cells.

7. The method of claim 6, wherein step (ii) is effected via treatment with trypsin.

8. The method of claim 2, wherein said serum and/or said serum replacement are provided at a concentration of at least 10%.

9. The method of claim 2, wherein said serum is human serum.

10. The method of claim 2, wherein said serum and/or said serum replacement are provided at a concentration of 15%.

11. A cell culture comprising:

(i) mammalian pluripotent stem cells, wherein said mammalian pluripotent stem cells are capable of forming teratomas containing cells of the endoderm, mesoderm and ectoderm germ layers; and

(ii) human foreskin fibroblast cells capable of maintaining said mammalian pluripotent stem cells in an undifferentiated state.

12. The cell culture of claim 11, wherein said mammalian pluripotent stem cells are of embryonic origin.

13. The cell culture of claim 11, wherein said mammalian pluripotent stem cells are of human origin.

14. The cell culture of claim 11, wherein said human foreskin cells are capable of forming a mono-layer in the cell culture.

15. The cell culture of claim 11, wherein said cell culture includes a culture medium including serum replacement and/or human serum.

16. The cell culture of claim 15, wherein said serum replacement and/or said human serum are provided at a concentration of at least 10%.

17. The cell culture of claim 15, wherein said serum replacement and/or said human serum are provided at a concentration of 15%.

18. The cell culture of claim 11, wherein said human foreskin cells of said cell culture maintain said mammalian pluripotent stem cells in an undifferentiated, proliferative state through at least 87 passages.