Functional oligodendrocytes derived from pluripotent stem cells and methods of making and using the same
Described is the efficient and robust generation of oligodendrocyte progenitor cells (OPCs) and oligodendrocytes from pluripotent stem cells (PSCs). The protocols provided recapitulate the major steps of oligodendrocyte differentiation, from neural stem cells to OLIG2 + progenitors, and then to O4 + OPCs, in a significantly shorter time than the 120-150 days required by previous protocols. Furthermore, O4 + OPCs are able to differentiate into MBP + mature oligodendrocytes in vitro, and to myelinate axons in vivo when injected into immuno-compromised Shiverer mice, providing proof of concept that transplantation of PSC-derived cells for remyelination is technically feasible.
1. A method of generating O4+ oligodendrocyte progenitor cells (OPCs), the method comprising:
a) culturing three-dimensional cell aggregates of OLIG2+ OPCs in suspension in a medium comprising SAG and a low concentration of RA for about 8 days;
b) culturing the cell aggregates of (a) in suspension in a medium comprising platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), and neurotrophin 3 (NT3) for about 10 days;
c) re-plating the cell aggregates (b) at a density of 2 spheres/cm2; and
d) culturing the re-plated cell aggregates of (c) in an adherent culture in a medium comprising (i) ascorbic acid (AA) or (ii) PDGF, HGF, IGF-1, and NT3 until cells are O4+;
thereby generating O4+ OPCs.
2. The method of claim 1 , further comprising culturing the O4+ OPCs generated in (d) in the absence of PDGF, HGF, IGF-1, and NT3 for about three weeks; thereby generating oligodendrocytes, wherein the oligodendrocytes are myelin basic protein (MBP)+.
3. The method of claim 1 , wherein the concentration of RA is about 10 nM to 250 nM.
4. The method of claim 3 , wherein the concentration of RA is about 100 nM.
5. The method of claim 1 , wherein the medium comprising SAG and RA of (a) lacks SHH.
6. The method of claim 1 , wherein the re-plating of the cell aggregates of (c) is on a surface comprising an extracellular matrix protein and a positively charged poly-amino acid.
7. The method of claim 6 , wherein the extracellular matrix protein is laminin.
8. The method of claim 6 , wherein the poly-amino acid is poly-ornithine.
9. The method of claim 1 , wherein at least 25% of the re-plated cell aggregates cultured in (d) are O4+ after 25 days of culture in the medium comprising AA.
10. The method of claim 1 , wherein at least 30% of the re-plated cell aggregates cultured in (d) are O4+ after 25 days of culture in the medium comprising AA.
11. The method of claim 1 , wherein at least 40% of the re-plated cell aggregates cultured in (d) are O4+ after 45 days of culture in the medium comprising AA.
12. The method of claim 1 , wherein at least 50% of the re-plated cell aggregates cultured in (d) are O4+ after 45 days of culture in the medium comprising AA.
13. The method of claim 1 , wherein at least 60% of the re-plated cell aggregates cultured in (d) are O4+ after 45 days of culture in the medium comprising AA.
14. The method of claim 1 , wherein at least 40% of the re-plated cell aggregates cultured in (d) are O4+ after 45 days of culture in the medium comprising PDGF, HGF, IGF-1, and NT3.
15. The method of claim 1 , wherein at least 50% of the re-plated cell aggregates cultured in (d) are O4+ after 45 days of culture in the medium comprising PDGF, HGF, IGF-1, and NT3.
16. The method of claim 1 , wherein at least 60% of the re-plated cell aggregates cultured in (d) are O4+ after 45 days of culture in the medium comprising PDGF, HGF, IGF-1, and NT3.
17. The method of claim 1 , wherein at least 70% of the re-plated cell aggregates cultured in (d) are O4+ after 45 days of culture in the medium comprising PDGF, HGF, IGF-1, and NT3.