Methods of generating tendon tissue in vitro from connective tissue progenitor cells

• US 8,541,234 B2
• Filed: 06/19/2012
• Issued: 09/24/2013
• Est. Priority Date: 01/11/2006
• 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: Itskovitz-Eldor, Joseph, Cohen, Shahar
• Primary Examiner(s): Crouch, Deborah
• Application Number:
  US13/526,633
• Publication Number:
  US 20120253463A1
• Time in Patent Office: 462 Days
• Field of Search: None
• US Class Current: 435/377
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2533/40 Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
• CPC Class Codes: C12N 5/0652 Cells of skeletal and connective tissues; Mesenchyme
• CPC Class Codes: C12N 5/0662 Stem cells

Abstract

Methods of generating and expanding proliferative, multipotent connective tissue progenitor cells from adult stem cells are provided. Also provided are methods of generating functional tendon grafts in vitro and bone, cartilage and connective tissues in vivo using the isolated cell preparation of connective tissue progenitor cells.

Claims

1. A method of generating a tendon in vitro, comprising:

(a) culturing connective tissue progenitor cells in the absence of a scaffold or a carrier to thereby obtain a sheet-like tissue, and

(b) rolling and/or folding said sheet-like tissue to form a rounded cylinder,

thereby generating the tendon tissue in vitro.

2. The method of claim 1, wherein said culturing is performed in a culture medium which comprises ascorbic acid and/or dexamethasone.

3. The method of claim 1, wherein said culturing is effected without culture splitting.

4. The method of claim 3, wherein said culturing without said culture splitting is effected for about 4-5 weeks.

5. The method of claim 1, further comprising removing said sheet-like tissue from said culturing vessel prior to said rolling and/or to said folding of said sheet-like tissue.

6. The method of claim 5, wherein said removing is effected using a scraper.

7. The method of claim 1, wherein said connective tissue progenitor cells are capable of differentiating into at least two cell lineages of a connective tissue.

8. The method of claim 7, wherein said at least two cell lineages of said connective tissue are selected from the group consisting of an osteogenic lineage, a chondrogenic lineage, an adipocytic lineage and a tendon and ligament lineage.

9. The method of claim 1, wherein said culturing is effected at a cell density of about 5-10×

10⁵ cells/cm².

10. The method of claim 2, wherein said culture medium further comprises serum or serum replacement.

11. The method of claim 1, wherein said connective tissue progenitor cells express CD105, CD166, CD44, CD29 and HLA-ABC.

12. The method of claim 1, wherein said connective tissue progenitor cells not expressing CD45 and HLA-DR.

13. The method of claim 1, wherein said connective tissue progenitor cells express core binding factor alpha 1 (CBFA1).

14. The method of claim 1, wherein said connective tissue progenitor cells are devoid of feeder cells.

15. The method of claim 1, wherein said connective tissue progenitor cells are xeno-free.