Methods of deriving mammalian pluripotent stem cell lines

• US 9,255,247 B2
• Filed: 08/19/2014
• Issued: 02/09/2016
• 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:
  US14/462,589
• Publication Number:
  US 20140356950A1
• Time in Patent Office: 539 Days
• US Class Current: 1/1
• 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 deriving a mammalian pluripotent stem cell line, comprising:

(a) obtaining a mammalian pluripotent stem cell which is capable of forming teratomas; and

(b) culturing said mammalian pluripotent stem cell on a foreskin fibroblast feeder cell line under culturing conditions which allow expansion of said mammalian pluripotent stem cell in an undifferentiated state,

thereby deriving the mammalian pluripotent stem cell line.

2. The method of claim 1, wherein said human foreskin fibroblast 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 cell is a human embryonic stem cell.

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

5. 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.

6. The method of claim 5, wherein step (ii) is effected via treatment with trypsin.

7. The method of claim 2, wherein said serum or said serum replacement is provided at a concentration of at least 10%.

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

9. The method of claim 2, wherein said serum or said serum replacement is provided at a concentration of 15%.

10. The method of claim 2, wherein said human foreskin cells form a mono-layer in the cell culture.

11. The method of claim 1, wherein said conditions include a culture medium including serum replacement and/or human serum.

12. The method of claim 1, wherein said serum replacement or said human serum is provided at a concentration of at least 10%.

13. The method of claim 1, wherein said human foreskin cells maintain said mammalian pluripotent stem cells in an undifferentiated, proliferative state through at least 11 passages.

14. The method of claim 1, wherein said mammalian pluripotent stem cell is an embryonic stem cell from an embryonic tissue.

15. The method of claim 14, wherein said embryonic cell is obtained from a pre-implantation stage blastocyst, a genital tissue of a fetus or an expanded embryonic cell.

16. The method of claim 1, further comprising passaging said mammalian pluripotent stem cell every four to six days.

17. The method of claim 14, wherein said embryonic stem cell is comprised in an inner cell mass (ICM) of said pre-implantation stage blastocyst.

18. The method of claim 17, wherein said ICM is isolated by removing the zona pellucida of said pre-implantation stage blastocyst.

19. The method of claim 1, wherein said embryonic stem cell is comprised in a genital ridge of an 8-11 weeks human fetus.

20. The method of claim 16, wherein said passaging begins following 9-15 days of initial growth of said mammalian pluripotent stem cell on said foreskin fibroblast feeder cell line.