Methods of differentiating stem cells into chondrocytes

• US 10,724,005 B2
• Filed: 09/27/2013
• Issued: 07/28/2020
• Est. Priority Date: 09/28/2012
• Status: Active Grant

• Current Assignee: Scripps Health
• Original Assignee: Scripps Health
• Inventors: D'Lima, Darryl D., Olee, Tsaiwei, Colwell, Clifford W.
• Primary Examiner(s): Lankford, Blaine
• Application Number:
  US14/431,893
• Publication Number:
  US 20150259648A1
• Time in Patent Office: 2,496 Days
• Field of Search: None
• CPC Class Codes: A61K 35/32 Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 2537/10 Cross-linking
• CPC Class Codes: C12N 5/0655 Chondrocytes; Cartilage

Abstract

Disclosed herein are methods of producing chondrocytes from pluripotent stem cells. The invention further provides methods of regenerating cartilaginous tissue.

Claims

1. A method of differentiating pluripotent cells to a population of chondrogenic precursors, comprising:

a) passaging a population of mechanically-dissected pluripotent cell aggregates (PCA) directly on untreated tissue culture-grade plastic until confluency, at least three times to generate mesenchymal stem cell (MSC)-like cells; and

b) culturing the MSC-like cells in a three-dimensional culture, to generate the population of chondrogenic precursors;

wherein the MSC-like cells are positive for CD90, CD73, and CD105.

2. The method of claim 1, wherein cells of the population of mechanically-dissected pluripotent cell aggregates are:

a) less than 20% positive for CD73;

b) positive for expression of Oct3/4; and

c) lacking immunoreactivity to vimentin.

3. The method of claim 1, wherein culturing the MSC-like cells comprises culturing the MSC-like cells in serum-free chondrogenic media.

4. The method of claim 1, wherein culturing the MSC-like cells comprises culturing in the presence of insulin-like growth factor (IGF-1) or basic fibroblast factor (bFGF).

5. The method of claim 1, wherein the population of mechanically-dissected PCA are passaged at least five times.

6. The method of claim 1, wherein the pluripotent cell aggregates are induced pluripotent cell aggregates derived from fibroblasts.

7. The method of claim 1, wherein the pluripotent cell aggregates are embryonic stem cell aggregates.

8. The method of claim 1, wherein the population of chondrogenic precursors express one or more markers selected from:

aggrecan, annexin A6, CD44, CD151, Collagen II, collagen type 2A1, Collagen IV, CRTAC1, DSPG3, FoxC1, FoxC2, Sialoprotein II, ITM2A, Matrilin-1, Matrilin-3, Matrilin-4, MIA, Otoraplin, SOX5, SOX6, SOX9 and URB.

9. The method of claim 8, wherein the population of chondrogenic precursors is at least 85% positive for CD73 and CD105.

10. The method of claim 9, wherein the population of chondrogenic precursors is at least 95% positive for CD73.

11. The method of claim 1, wherein the method does not comprise enzymatic digestion of the population of mechanically-dissected pluripotent cell aggregates.

12. The method of claim 1, wherein the three-dimensional culture comprises a protein selected from laminin, entactin, tenascin, fibrillin or fibronectin, osteocalcin, and osteonectin.

13. The method of claim 1, wherein passaging the population of mechanically-dissected PCA comprises plating cells from the population of mechanically-dissected PCA at a density of from 1×

10² cells/cm² to 10⁴ cells/cm².

14. The method of claim 1, wherein the MSC-like cells express one or more markers selected from:

vimentin, fibronectin, receptors for Bone Morphogenetic Proteins (BMPs), CD44, CD45, nucleostemin, integrin alpha 1, integrin alpha V, integrin beta 1, NCAM-1, PDGF-R alpha, Scal/Ly6, SCF-4/c-kit, SSEA-4, STRO-1, VCAM-1/CD106, ALCAM, CD45RO, CDCP1, CD166, ANPEP, ALK-3, ALK-6, N-Cadherin, HLA Class I, CD54, CD49a, CD49e, CD51, CD29, CD56, TNFRSF16, Nucleostemin, STRO-1, Transferrin R, CD 106, Endolin, and Stem Cell Factor Receptor.

15. The method of claim 14, wherein the MSC-like cells are at least 75% positive for CD44.

16. The method of claim 1, wherein the MSC-like cells are less than 10% positive for CD11b, CD19, CD34, CD34, CD45, HLA-DR, or a combination thereof.

17. The method of claim 14, wherein the MSC-like cells are at least 85% positive for CD90.

18. The method of claim 1, wherein the three-dimensional culture comprises a cell pellet.

19. The method of claim 1, wherein the three-dimensional culture comprises a matrix gel material.

20. The method of claim 1, wherein culturing the MSC-like cells comprises culturing the MSC-like cells in the presence of a chondrogenic growth factor, to generate the population of chondrogenic precursors.

21. The method of claim 1, wherein passaging the population of mechanically-dissected PCA comprises culturing in a medium comprising DMEM/F12 supplemented with non-essential amino acids and 10% fetal bovine serum.