Methods of differentiating stem cells into chondrocytes
Disclosed herein are methods of producing chondrocytes from pluripotent stem cells. The invention further provides methods of regenerating cartilaginous tissue.
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 1×10 2 cells/cm 2 to 10 4 cells/cm 2 .
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.