Chondrogenic progenitor cells, protocol for derivation of cells and uses thereof

• US 9,029,145 B2
• Filed: 04/08/2011
• Issued: 05/12/2015
• Est. Priority Date: 04/08/2010
• Status: Active Grant

• Current Assignee: University of Edinburgh
• Original Assignee: University of Edinburgh
• Inventors: Noble, Brendon Stewart, Pier, David Matthew
• Primary Examiner(s): BERTOGLIO, VALARIE E
• Application Number:
  US13/082,727
• Publication Number:
  US 20110256109A1
• Time in Patent Office: 1,495 Days
• Field of Search: None
• US Class Current: 435/375
• CPC Class Codes: A61K 35/32 Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 19/04 for non-specific disorders of the connective tissue
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/84 from mammals
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/03 from non-embryonic pluripotent stem cells
• CPC Class Codes: C12N 5/0655 Chondrocytes; Cartilage
• CPC Class Codes: C12N 5/0662 Stem cells
• CPC Class Codes: G01N 33/5044 involving specific cell types

Abstract

The present invention provides an isolated population of chondrocyte precursor cells wherein 1% or less of the cells express Oct4, Nanog and/or TRA-1-60, 7% or less of the cells express no collagen II, collagen X, CD105 or Stro-1 and 85% or more of the cells express CBFA1, methods for preparing such cells and uses of chondrocyte cells derived from said precursor cells.

Claims

1. A method for preparing chondrocyte precursor cells, comprising the steps of:

(a) providing a culture of primate pluripotent stem (pPS) cells in chondrogenic media, wherein the pPS cells are greater than 75% confluent;

(b) differentiating the cells of (a) by further culturing in chondrogenic media until the cells form colonies; and

(c) culturing the cells obtained in (b) in de-differentiation medium that lacks factors necessary for chondrogenic differentiation, until cells de-differentiate and migrate out of the colonies, to provide a population of de-differentiated chondrocyte precursor cells, wherein the de-differentiated chondrocyte precursor cells are characterized by the loss of transdifferentiation potential to form osteoblasts when cultured in osteogenic medium.

2. The method of claim 1, wherein the population of de-differentiated chondrocyte precursor cells obtained in (c) are characterized in that 1% or less of the cells express Oct4, Nanog and tra-1-60, and 85% or more of the cells express CBFA 1.

3. The method of claim 1, wherein the pPS cells are human embryonic stem cells.

4. The method of claim 2, wherein the pPS cells are human embryonic stem cells.

5. The method of claim 1, wherein at least 25% of the population of de-differentiated chondrocyte precursor cells generated in step (c) synthesize either Type II collagen or aggrecan.

6. The method of claim 1, wherein 7% or less of the population of de-differentiated chondrocyte precursor cells generated in step (c) express no collagen II.

7. The method of claim 1, wherein 7% or less of the population of de-differentiated chondrocyte precursor cells generated in step (c) express collagen X.

8. The method of claim 1, wherein 7% or less of the population of de-differentiated chondrocyte precursor cells generated in step (c) express CD105.

9. The method of claim 1, wherein 7% or less of the population of de-differentiated chondrocyte precursor cells generated in step (c) express Stro-1.