Oligodendrocytes derived from human embryonic stem cells for remyelination and treatment of spinal cord injury
View Patent ↗This invention provides populations of neural cells bearing markers of glial cells, such as oligodendrocytes and their precursors. The populations are generated by differentiating pluripotent stem cells such as human embryonic stem cells under conditions that promote enrichment of cells with the desired phenotype or functional capability. Various combinations of differentiation factors and mitogens can be used to produce cell populations bearing markers of oligodendrocyte precursor cells. Upon further differentiation form complex processes characteristic of mature oligodendrocytes. The cells are capable of forming myelin sheaths, and can be used therapeutically improve function of the central nervous system.
1. A method for producing glial cells from human embryonic stem (hES) cells, comprising
culturing hES cells in suspension in a culture medium comprising basic fibroblast growth factor (bFGF), thyroid hormone T3, and retinoic acid (RA).
2. The method of claim 1 , wherein the concentration of thyroid hormone T3 is 20 ng/ml and the concentration of RA is 10 μM.
3. The method of claim 1 , wherein prior to said culturing, the hES cells are cultured in suspension in a culture medium comprising bFGF and thyroid hormone T3.
4. The method of claim 1 , wherein prior to said culturing, the hES cells are cultured in suspension in a culture medium comprising bFGF at a concentration of 4 ng/ml and thyroid hormone T3 at a concentration of 20 ng/ml.
5. The method of claim 1 , wherein the cells are cultured in the presence of RA for 8 days.
6. The method of claim 5 , wherein RA is present at a concentration of 10 μM.
7. The method of claim 1 , comprising
following said culturing, replacing the culture medium with a replacement culture medium comprising thyroid hormone T3 and retinoic acid (RA), wherein the replacement culture medium does not contain bFGF; and
culturing cells in the replacement culture medium.
8. The method of claim 7 , wherein thyroid hormone T3 is present in the replacement culture medium in a concentration of 40 ng/ml thyroid hormone T3 and RA is present in the replacement culture medium at a concentration of 10 μM.
9. The method of claim 7 , wherein the cells are cultured in the culture medium comprising bFGF, thyroid hormone T3, and RA for one day prior to said replacing.
10. The method of claim 7 , wherein following said culturing in the replacement culture medium, the method comprises:
plating cells on an adherent surface; and
harvesting cells that adhere to the surface.
11. The method of claim 10 , wherein following plating, the plated cells are cultured in a medium comprising epidermal growth factor (EGF) and thyroid hormone T3.
12. The method of claim 11 , wherein EGF in the medium following plating is present at a concentration of 20 ng/ml and thyroid hormone T3 is present in the medium following plating at a concentration of 40 ng/ml.
13. The method of claim 10 , further comprising separating glial cells from non-glial cells.
14. The method of claim 13 , wherein the separating is performed by plating cells from the culture onto a solid surface, and harvesting cells that adhere to the surface.
15. A method for producing glial cells from human embryonic stem (hES) cells, comprising
a) culturing the hES cells in suspension in a culture medium comprising basic fibroblast growth factor (bFGF), and thyroid hormone T3;
b) adding retinoic acid (RA) to the culture medium and continuing said culturing;
c) replacing the culture medium with a replacement culture medium comprising thyroid hormone T3 and RA, wherein the replacement culture medium does not contain bFGF and culturing cells in the replacement culture medium;
d) plating cells on an adherent surface; and
e) culturing the plated cells in a culture medium comprising epidermal growth factor (EGF) and thyroid hormone T3; and
f) harvesting cells that adhere to the surface.
16. The method of claim 15 , wherein in step a) bFGF is present in the culture medium at a concentration of 4 ng/ml and thyroid hormone T3 is present in the culture medium at a concentration of 20 ng/ml.
17. The method of claim 15 , wherein in step b), RA is added at a concentration of RA is 10 μM.
18. The method of claim 15 , wherein the cells are cultured in the presence of RA for one day prior to said replacing.
19. The method of claim 15 , wherein EGF in the culture medium following said plating is present at a concentration of 20 ng/ml and thyroid hormone T3 is present at a concentration of 40 ng/ml.
20. A method for producing glial cells from human embryonic stem (hES) cells, comprising
a) culturing the hES cells in suspension in a culture medium comprising basic fibroblast growth factor (bFGF), thyroid hormone T3, and retinoic acid (RA); and
b) replacing the culture medium with a replacement culture medium comprising thyroid hormone T3 and RA, wherein the replacement culture medium does not contain bFGF and culturing cells in the replacement culture medium;
c) plating cells on an adherent surface; and
d) culturing the plated cells in a culture medium comprising epidermal growth factor (EGF) and thyroid hormone T3; and
e) harvesting cells that adhere to the surface;
wherein the cells are cultured in the presence of RA for 8 days.
21. The method of claim 20 , wherein at step b) RA is present at a concentration of 10 μM.
22. The method of claim 20 , wherein prior to step a), the hES cells are cultured in suspension in a culture medium comprising bFGF and thyroid hormone T3.