Methods of generating oligodendrocytes
A method of generating human mature oligodendrocytes is disclosed. The method comprises contacting a cell population which comprises human pre-oligodendrocytes with an inhibitor of the MAPK/ERK pathway under conditions that allow the pre-oligodendrocytes to differentiate into mature oligodendrocytes. Use of the MAPK/ERK pathway inhibitor for treating diseases is also disclosed.
1 . A method of generating human mature oligodendrocytes comprising:
(a) culturing human pluripotent stem cells under conditions that produce a cell population comprising pre-oligodendrocytes which express O4, wherein no more than 70% of the cells of said cell population are oligodendrocyte progenitor cells (OPCs); and
(b) contacting said cell population which comprises said pre-oligodendrocytes with an inhibitor of the MAPK/ERK pathway under conditions that allow the pre-oligodendrocytes to differentiate into mature oligodendrocytes.
2 . The method of claim 1 , the number of human pre-oligodendrocytes in the cell population is greater than the number of oligodendrocyte progenitor cells in the cell population.
3 . The method of claim 1 , wherein said pre-oligodendrocytes express at least a 20% decrease in nkx2.2 as compared to oligodendrocyte progenitor cells, as measured by RT-PCR.
4 . The method of claim 1 , wherein said pre-oligodendrocytes do not express nkx2.2, as measured by immunofluorescence.
5 . The method of claim 1 , wherein said inhibitor of the MAPK/ERK pathway comprises a MEK1/2 inhibitor.
6 . The method of claim 5 , wherein said MEK1/2 inhibitor is PD0325901 or trametinib.
7 . The method of claim 1 , further comprising contacting the population of pre-oligodendrocytes with a tankyrase inhibitor.
8 . The method of claim 1 , further comprising contacting the population of pre-oligodendrocytes with a BMP antagonist and/or a γ-secretase inhibitor.
9 . The method of claim 8 , wherein said BMP antagonist comprises noggin.
10 . The method of claim 8 , wherein said γ-secretase inhibitor comprises (N—N-(3,5-difluorphenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT).
11 . The method claim 1 , wherein said contacting comprises culturing said pre-oligodendrocytes in a culture medium comprising said inhibitor of said MAPK/ERK pathway.
12 . The method of claim 11 , wherein said culture medium further comprises at least one agent selected from the group consisting of a tankyrase inhibitor, a γ-secretase inhibitor and a BMP antagonist.
13 . The method of claim 1 , wherein said human pre-oligodendrocytes were exposed to hypoxic conditions for at least 6 hours prior to said contacting.