Differentiation method for production of glial cell populations
The present invention provides methods for generating oligodendrocyte progenitor cells from pluripotent cells, as well as methods for sustaining these oligodendrocyte progenitor cells in relatively pure cultures for long periods of time. The present invention also provides methods for further differentiating these oligodendrocyte progenitor cells into various glial cells.
1. A method for generating mammalian oligodendrocyte progenitor cells comprising: providing a pluripotent cell; inducing development of the pluripotent cell into neuroectoderm by culturing the pluripotent cell in the presence of one or more inhibitors of an activin-nodal pathway and one or more inhibitors of a bone morphogenetic protein pathway; inducing development of patterned neuroectoderm by culturing the neuroectoderm in the presence of two or more of sonic hedgehog, retinoic acid and noggin; and inducing development of oligodendrocyte progenitor cells by culturing the patterned neuroectoderm cells in the presence of one or more of a fibroblast growth factor, a platelet-derived growth factor and sonic hedgehog.
2. The method of claim 1 , wherein two or more of a fibroblast growth factor, a platelet-derived growth factor and sonic hedgehog are used in the culture of the patterned neuroectoderm cells.
3. The method of claim 2 , wherein a fibroblast growth factor, a platelet-derived growth factor and sonic hedgehog are used in the culture of the patterned neuroectodermal cells.
4. The method of claim 1 , wherein the fibroblast growth factor is FGF2 and the platelet-derived growth factor is PDGF-AA.
5. The method of claim 1 , wherein retinoic acid, sonic hedgehog and noggin are used in the culture of the neuroectodermal cells.
6. The method of claim 1 , wherein at least 75% or more of cells resulting from the culture of the patterned neuroectodermal cells are oligodendrocyte progenitor cells.
7. The method of claim 6 , wherein at least 90% or more of cells resulting from the culture of the patterned neuroectodermal cells are oligodendrocyte progenitor cells.
8. The method of claim 7 , wherein at least 95% or more of cells resulting from the culture of the patterned neuroectodermal cells are oligodendrocyte progenitor cells.
9. The method of claim 8 , wherein at least 99% or more of cells resulting from the culture of the patterned neuroectodermal cells are oligodendrocyte progenitor cells.
10. The method of claim 1 , wherein the one or more inhibitors of an activin-nodal pathway is SB431542.
11. The method of claim 1 , wherein the one or more inhibitors of a bone morphogenic protein pathway is selected from dorsomorphin, LDN-193189 or noggin.
12. A method for generating mammalian oligodendrocyte progenitor cells and maintaining the mammalian oligdendrocyte progenitor cells in culture comprising: providing pluripotent cells; inducing development of the pluripotent cells into neuroectoderm by culturing the pluripotent cells in the presence of one or more inhibitors of an activin-nodal pathway and one or more inhibitors of a bone morphogenetic protein pathway; inducing development of patterned neuroectoderm by culturing the neuroectoderm in the presence of sonic hedgehog, retinoic acid and noggin; inducing development of oligodendrocyte progenitor cells by culturing the patterned neuroectoderm cells in the presence of a fibroblast growth factor, a platelet-derived growth factor and sonic hedgehog; and maintaining the oligodendrocyte progenitor cells by culturing the oligodendrocyte progenitor cells in the presence of an activator of Wnt-β-catenin signaling.
13. The method of claim 12 , wherein the one or more inhibitors of an activinnodal pathway is SB431542 and the one or more inhibitors of a bone morphogenetic protein pathway is selected from dorsomorphin, LDN-193189 or noggin.
14. The method of claim 12 , wherein the fibroblast growth factor is FGF2 and the platelet-derived growth factor is PDGF-AA.
15. The method of claim 12 , wherein at least 95% of cells in culture are oligodendrocyte progenitor cells.
16. A method for generating oligodendrocyte progenitor cells from patterned neuroectoderm comprising culturing the patterned neuroectoderm cells in the presence of a fibroblast growth factor, a platelet-derived growth factor and sonic hedgehog.