Compound screening using chondrocytes derived from primate pluripotent stem cells

• US 8,546,101 B2
• Filed: 02/04/2011
• Issued: 10/01/2013
• Est. Priority Date: 12/07/2001
• Status: Expired due to Fees

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Geron Corporation
• Inventors: Thies, R. Scott
• Primary Examiner(s): Crouch, Deborah
• Application Number:
  US13/021,497
• Publication Number:
  US 20110129867A1
• Time in Patent Office: 970 Days
• Field of Search: None
• US Class Current: 435/29
• 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: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: C12N 2500/44 Thiols, e.g. mercaptoethanol
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/395 Thyroid hormones
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0655 Chondrocytes; Cartilage

Abstract

This invention provides a system for obtaining cells of the chondrocyte lineage by differentiating primate pluripotent stem cells. The process involves culturing the cells as a micromass or other aggregate form in a cocktail of differentiation agents that facilitates outgrowth of the desired cell type. Progeny are capable of synthesizing Type II collagen or aggrecan, or other products that are characteristic of the chondrocyte lineage. Chondrocytes and chondrocyte precursor cells obtained according to this disclosure are suitable for use in both research and clinical therapy.

Claims

1. A method of screening a compound for an effect on cultured chondrocytes, comprising:

a) providing primate pluripotent stem cells which express SSEA3, SSEA4, Tra-1-60 and Tra-1-81;

b) culturing the primate pluripotent stem cells which express SSEA3, SSEA4, Tra-1-60 and Tra-1-81 in a medium comprising one or more of transforming growth factor (TGF), fibroblast growth factor (FGF), growth differentiation factor (GDF), bone morphogenic protein (BMP), hedgehog protein, L-ascorbic acid and para-thyroid hormone related protein to produce cultured chondrocytes;

c) contacting the cultured chondrocytes with the compound; and

d) observing a change in the cultured chondrocytes.

2. The method of claim 1, wherein the compound is a peptide.

3. The method of claim 1, wherein the compound is an oligonucleotide.

4. The method of claim 1, wherein the compound is a toxin.

5. The method of claim 1, wherein the compound is a small molecule.

6. The method of claim 1, wherein the compound is a solvent.

7. The method of claim 1, wherein the cultured chondrocytes have chromosomal DNA that is at least 99.6% identical to the chromosomal DNA of an established line of cells which express SSEA3, SSEA4, Tra-1-60 and Tra-1-81.

8. The method of claim 1, wherein the cultured chondrocytes have chromosomal DNA that is at least 99.7% identical to the chromosomal DNA of an established line of cells which express SSEA3, SSEA4, Tra-1-60 and Tra-1-81.

9. The method of claim 1, wherein the cultured chondrocytes have chromosomal DNA that is at least 99.8% identical to the chromosomal DNA of an established line of cells which express SSEA3, SSEA4, Tra-1-60 and Tra-1-81.

10. The method of claim 1, wherein the cultured chondrocytes have chromosomal DNA that is at least 99.9% identical to the chromosomal DNA of an established line of cells which express SSEA3, SSEA4, Tra-1-60 and Tra-1-81.

11. The method of claim 1, wherein the cultured chondrocytes have chromosomal DNA that is genetically identical to the chromosomal DNA of an established line of cells which express SSEA3, SSEA4, Tra-1-60 and Tra-1-81.

12. A method of screening a compound for an effect on cultured cells expressing collagen type II and/or aggrecan chondrocytes, comprising:

a) providing primate pluripotent stem cells which express SSEA3, SSEA4, Tra-1-60 and Tra-1-81;

b) culturing the primate pluripotent stem cells which express SSEA3, SSEA4,Tra-1-60 and Tra-1-81 in a medium comprising one or more of transforming growth factor (TGF), fibroblast growth factor (FGF), growth differentiation factor (GDF), bone morphogenic protein (BMP), hedgehog protein, L-ascorbic acid and para-thyroid hormone related protein to produce cultured cells expressing collagen type II and/or aggrecan;

c) contacting the cultured cells expressing collagen type II and/or aggrecan with the compound; and

d) observing a change in the cultured cells expressing collagen type II and/or aggrecan.

13. The method of claim 12, wherein the compound is a peptide.

14. The method of claim 12, wherein the compound is an oligonucleotide.

15. The method of claim 12, wherein the compound is a toxin.

16. The method of claim 12, wherein the compound is a small molecule.

17. The method of claim 12, wherein the compound is a solvent.

18. The method of claim 12, wherein the cultured collagen type II expressing chondrocytes have chromosomal DNA that is at least 99.6% identical to the chromosomal DNA of an established line of cells which express SSEA3, SSEA4, Tra-1-60 and Tra-1-81.

19. The method of claim 12, wherein the cultured collagen type II expressing chondrocytes have chromosomal DNA that is at least 99.7% identical to the chromosomal DNA of an established line of cells which express SSEA3, SSEA4, Tra-1-60 and Tra-1-81.

20. The method of claim 12, wherein the cultured collagen type II expressing chondrocytes have chromosomal DNA that is at least 99.8% identical to the chromosomal DNA of an established line of cells which express SSEA3, SSEA4, Tra-1-60 and Tra-1-81.

21. The method of claim 12, wherein the cultured collagen type II expressing chondrocytes have chromosomal DNA that is at least 99.9% identical to the chromosomal DNA of an established line of cells which express SSEA3, SSEA4, Tra-1-60 and Tra-1-81.

22. The method of claim 12, wherein the cultured collagen type II expressing chondrocytes have chromosomal DNA that is identical to the chromosomal DNA of an established line of cells which express SSEA3, SSEA4, Tra-1-60 and Tra-1-81.