CO-CULTURING MAMMALIAN EMBRYONIC STEM CELLS WITH HUMAN FORESKIN FIBROBLASTS

• US 20130084563A1
• Filed: 11/26/2012
• Published: 04/04/2013
• 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
• Granted Patent: US 8,846,400 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• 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 culturing the mammalian pluripotent stem cells with a human foreskin fibroblast conditioned medium.

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

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

4. The method of claim 1, wherein foreskin cells for said human foreskin fibroblast conditioned medium are obtained from an 8-14 day old male individual.

5. The method of claim 1, wherein said culturing is effected under culturing conditions including a growth medium which comprises serum and/or serum replacement.

6. The method of claim 5, wherein said serum and/or said serum replacement are provided at a concentration of at least 10%.

7. The method of claim 5, wherein said serum and/or said serum replacement are provided at a concentration of 15%.

8. The method of claim 5, wherein said serum is human serum.

9. The method of claim 1, wherein said human foreskin fibroblast conditioned medium being capable of maintaining said mammalian pluripotent stem cells in an undifferentiated, proliferative state through at least 87 passages.

10. The method of claim 1, wherein said culturing is effected under culturing conditions including a growth medium which comprises a factor selected from the group consisting of a growth factor, an anti oxidant and an amino acid.

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 conditioned medium 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 human pluripotent stem cells.

14. The cell culture of claim 11, wherein human foreskin cells for said human foreskin fibroblast conditioned medium are obtained from an 8-14 day old male individual.

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.

19. A method of producing a conditioned medium suitable for maintaining mammalian pluripotent stem cells in an undifferentiated state, the method comprising:

(a) culturing human foreskin fibroblast cells in a growth medium under conditions suitable for producing the conditioned medium; and

(b) collecting said growth medium to thereby produce the conditioned medium, and

(c) validating the ability of the conditioned medium to maintain said mammalian pluripotent stem cells in an undifferentiated state following step (b),

thereby producing the conditioned medium suitable for maintaining the mammalian pluripotent stem cells in an undifferentiated state.

20. The method of claim 19, wherein said validating is effected by a differentiation assay selected from the group consisting of morphology analysis, karyotype analysis and surface marker analysis.