Differentiation of human embryonic and induced pluripotent stem cells

• US 8,349,609 B2
• Filed: 11/24/2010
• Issued: 01/08/2013
• Est. Priority Date: 11/24/2009
• Status: Active Grant

• Current Assignee: University of Connecticut (State of Connecticut)
• Original Assignee: University of Connecticut (State of Connecticut)
• Inventors: Dealy, Caroline, Kosher, Robert
• Primary Examiner(s): Hama, Joanne
• Application Number:
  US12/954,088
• Publication Number:
  US 20110229440A1
• Time in Patent Office: 776 Days
• Field of Search: None
• US Class Current: 435/377
• 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: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 5/0655 Chondrocytes; Cartilage

Abstract

The invention relates to culture systems, methods, and conditions that allow pluripotent undifferentiated hESCs or iPSCs to progressively and uniformly differentiate into cells of the chondrogenic lineage.

Claims

1. A method for direct differentiation of human embryonic stem cells (hESCs) into chondrogenic cells comprising:

providing a population of hESCs on a substrate, said population being substantially free of embryoid bodies (EBs);

detaching said hESCs from said substrate and dissociating said hESCs;

establishing a high-density micromass culture of said hESCs;

contacting said population with a bone morphogenetic protein (BMP) no sooner than 24 hours

after establishing the high-density micromass culture;

wherein said hESCs directly differentiate without EB formation into a population comprising at least 85% chondrogenic cells.

2. The method of claim 1, wherein said substrate is a feeder layer comprising mouse embryonic fibroblasts (MEF).

3. The method of claim 1, wherein said substrate is a polymeric or gelatinous substrate or scaffold.

4. The method of claim 1, further comprising administering contacting said hESCs with transforming growth factor beta no sooner than 24 hours after establishing the high-density micromass culture.

5. The method of claim 1, wherein said BMP comprises BMP-2 and wherein said BMP-2 is administered about 48 hours after establishing said high-density micromass culture.

6. The method of claim 1, wherein said hESCs differentiate into fully differentiated chondrocytes within about 14 days.

7. The method of claim 1, wherein said hESCs differentiate into fully differentiated chondrocytes within about 7 days.

8. The method of claim 1, wherein said hESCs differentiate into chondroprogenitor cells within about 2-3 days.

9. The method of claim 1, wherein said hESCs differentiate into early chondrocytes within about 4 days.

10. The method of claim 1, wherein the population comprises at least about 95% chondrogenic cells.

11. The method of claim 1, wherein said hESCs are dissociated with trypsin, TrypLE Select™

, or Accutase™

.

12. The method of claim 1, further comprising contacting said hESCs with Rho-associated kinase (ROCK) inhibitor.

26. The method of claim 1, wherein the BMP is BMP-2, and wherein the BMP-2 is added at a concentration of between 25 ng/ml to 200 ng/ml.

27. The method of claim 26, further comprising administering contacting said hESCs with transforming growth factor beta (TGF-β

) no sooner than 24 hours after establishing the high-density micromass culture.

28. The method of claim 27, wherein the ratio of BMP-2 to TGF-β

is between 5;

1 to 20;

1.

13. A method for direct differentiation of induced pluripotent stem cells (iPSCs) into chondrogenic cells comprising:

providing a population of iPSCs on a substrate, said population being substantially free of EBs;

detaching said iPSCs from said substrate and dissociating said iPSCs;

establishing a high-density culture of said iPSCs;

contacting said iPSCs with a bone morphogenetic protein (BMP) no sooner than 24 hours after establishing the high-density micromass culture;

wherein said iPSCs directly differentiate without EB formation into a population comprising at least 85% chondrogenic cells.

14. The method of claim 13, wherein said substrate is a feeder layer comprising mouse embryonic fibroblasts (MEF).

15. The method of claim 13, wherein said substrate is a polymeric or gelatinous substrate or scaffold.

16. The method of claim 13, further comprising contacting said iPSCs with transforming growth factor beta no sooner than 24 hours after establishing the high-density micromass culture.

17. The method of claim 13, wherein said BMP is BMP-2.

18. The method of claim 17, wherein said BMP-2 is administered 48 hours after initiation of said iPSC high-density micromass culture.

19. The method of claim 13, wherein said iPSCs differentiate into full differentiated chondrocytes within about 14 days.

20. The method of claim 13, wherein said iPSCs differentiate into fully differentiated chondrocytes within 7 days.

21. The method of claim 13, wherein said iPSCs differentiate into chondroprogenitor cells within about 2-3 days.

22. The method of claim 13, wherein said iPSCs differentiate into early chondrocytes within about 4 days.

23. The method of claim 13, wherein at least about 95% of said iPSCs differentiate into cells of the chondrogenic lineage.

24. The method of claim 13, wherein said iPSCs are dissociated with trypsin, TrypLE Select™

, or Accutase™

.

25. The method of claim 13, further comprising contacting said iPSCs with Rho-associated kinase (ROCK) inhibitor.