Methods of generating and isolating midbrain dopamine neurons
AssigneeMemorial Sloan Kettering Cancer Center
InventorsLorenz Studer, Taewan Kim, Stefan Irion, So Yeon Koo
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Abstract
The present disclosure provides methods for generating midbrain dopamine neurons (mDAs) and precursors thereof, mDAs and precursors thereof generated by such methods and compositions comprising such cells, and uses thereof for preventing and/or treating neurological disorders. The present disclosure further provides methods of isolating mDAs and precursors thereof from a cell population using novel surface markers.
Claims27
An in vitro method for inducing differentiation of stem cells, comprising:
- contacting the stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling, at least one activator of Sonic hedgehog (SHH) signaling, and at least one activator of wingless (Wnt) signaling;
- and contacting the cells with at least one activator of fibroblast growth factor (FGF) signaling to obtain a population of differentiated cells expressing at least one marker indicating a midbrain dopamine neuron (mDA) or a precursor thereof, wherein:
- (i) the at least one activator of FGF signaling is selected from the group consisting of FGF18, FGF17, FGF8a, and combination thereof, and/or
- (ii) the initial contact of the cells with the at least one activator of FGF signaling is at least about 5 days from the initial contact of the cells with the at least one inhibitor of SMAD signaling.
A cell population of in vitro differentiated cells, wherein at least about 50% of the cells express at least one marker indicating a midbrain dopamine neuron or a precursor thereof, and less than about 50% of the differentiated cells express at least one marker selected from the group consisting of PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, HOXA2, HOXB2, POU5F1, NANOG, and combinations thereof.
A method for isolating midbrain dopamine neurons and precursors thereof from a population of cells, comprising isolating cells that (a) do not express a detectable level or express a reduced level of at least one negative surface marker as compared to the mean expression level of the at least one negative surface marker in the population of cells;
- and (b) an increased level of at least one positive surface marker as compared to the mean expression level of the at least one positive marker in the population of cells.
A cell population of in vitro differentiated cells, wherein at least about 50% of the cells express an increased level of at least one positive surface marker as compared to the mean expression level of the at least one positive marker in the population of cells;
- and do not express a detectable level or express a reduced level of at least one negative surface marker as compared to the mean expression level of the at least one negative surface marker in the population of cells.
A kit for inducing differentiation of stem cells to midbrain dopamine neurons or precursors thereof, comprising:
- (a) at least one inhibitor of SMAD signaling;
- (b) at least one activator of SHH signaling;
- (c) at least one activator of Wnt signaling;
- and
- (d) at least one activator of FGF signaling.
Dependencies are inferred from the claim text.