Differentiation of Human Embryonic and Induced Pluripotent Stem Cells

• US 20110229440A1
• Filed: 11/24/2010
• Published: 09/22/2011
• 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: Kosher, Robert, Dealy, Caroline
• Granted Patent: US 8,349,609 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• 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;

wherein said hESCs directly differentiate into a substantially uniform population of 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 Matrigel or other polymeric or gelatinous substrates or scaffolds.

4. The method of claim 1, further comprising administering transforming growth factor beta to said hESCs.

5. The method of claim 1, wherein said BMP comprises BMP-2 and wherein said BMP-2 is administered about 24 hours after establishing said high-density micromass culture.

6. 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.

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

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

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

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

11. The method of claim 1, wherein at least about 85% of said hESCs differentiate into cells of the chondrogenic lineage.

12. The method of claim 1, wherein at least about 95% of said hESCs differentiate into cells of the chondrogenic lineage.

13. The method of claim 1, wherein said hESCs are dissociated with trypsin, TrypLE Select, or Accutase.

14. The method of claim 1, further comprising the administration of Rho-associated kinase (ROCK) inhibitor.

16. The method of claim 1 or 15, wherein said hESCs are in the form of a micromass suspension culture or a pellet culture.

17. A population of chondrogenic cells, wherein said population is substantially free of non-chondrogenic cells, said population having been differentiated from ESCs by the method of claim 1.

21. A method of repairing or restoring cartilage, comprising contacting damaged or diseased cartilage with the population of claim 17.

22. A method of preventing or treating arthritis, comprising administering to an articulating joint the population of claims 17.

23. A method of treating fracture repair comprising contacting a bone fracture site with the population of claim 17.

24. A method of treating or repairing torn or ruptured ligaments or menisci comprising administering to articulating joints the population of claim 17.

25. A method of regenerating fingers or limbs with the population of claim 17.

15. 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 thereby directing the differentiation of iPSCs into the chondrogenic lineage.

18. A population of chondrogenic cells, wherein said population is substantially free of non-chondrogenic cells, said population having been differentiated from iPSCs by the method of claim 15.

26. A method of identifying a gene involved in the development of a cartilage disorder, comprising providing the population of claim 18 and detecting an increase or decrease in gene expression compared to normal control cells, wherein said increase or decrease indicates that said gene is involved in the development of said cartilage disorder.

27. The method of claim 26, wherein said cartilage disorder comprises chondrodysplasia or achondroplasia.

28. A method of identifying a cartilage promoting agent, comprising contacting the population of claim 18 with a candidate compound and detecting chondrogenesis, wherein an increase in chondrogenesis in the presence of the said compound compared to in the absence of said compound indicates that said compound promotes the formation of cartilage.

29. The method of claim 15, wherein said substrate is a feeder layer comprising mouse embryonic fibroblasts (MEF).

30. The method of claim 15, wherein said substrate is Matrigel or other polymeric or gelatinous substrates or scaffolds.

31. The method of claim 15, further comprising administering transforming growth factor beta to said hESCs.

32. The method of claim 15, wherein said BMP-2 is administered about 24 hours after establishing iPSC high-density micromass culture.

33. The method of claim 15, wherein said BMP-2 is administered 48 hours after initiation of said iPSC high-density micromass culture.

34. The method of claim 15, wherein said iPSCs differentiate into full differentiated chondrocytes within about 14 days.

35. The method of claim 15, wherein said iPSCs differentiate into fully differentiated chondrocytes within 7 days.

36. The method of claim 15, wherein said iPSCs differentiate into chondroprogenitor cells within about 2-3 days.

37. The method of claim 15, wherein said iPSCs differentiate into early chondrocytes within about 4 days.

38. The method of claim 15, wherein at least about 85% of said iPSCs differentiate into cells of the chondrogenic lineage.

39. The method of claim 15, wherein at least about 95% of said iPSCs differentiate into cells of the chondrogenic lineage.

40. The method of claim 15, wherein said iPSCs are dissociated with trypsin, TrypLE Select, or Accutase.

41. The method of claim 15, further comprising the administration of Rho-associated kinase (ROCK) inhibitor.

19. A uniformly differentiated population of chondrogenic cells, at least 95% of said cells being at a single defined stage of differentiation, wherein said population is substantially free of fully differentiated chondrocytes.

20. The population of claim 19, wherein said stage is selected from the group consisting of day 2 or 3 chondrogenic cells (chondroprogenitors), or day 4 chondrogenic cells (early chondrocytes).