Method of in vitro differentiation of neural stem cells, motor neurons and dopamine neurons from primate embryonic stem cells
A method of differentiating embryonic stem cells into neural and motor cells is disclosed. In one embodiment, the invention comprises culturing a population of cells comprising a majority of cells that are characterized by an early rosette morphology and are Sox1 − /Pax6 + in the presence of FGF2, FGF4, FGF8, FGF 9, or RA wherein the cells are characterized by an neural tube-like rosette morphology and are Pax6 + /Sox1 + .
1. A method of generating a population of human spinal motor neurons comprising:
(a) culturing human Sox 1 − , Pax 6 + cells characterized by an early rosette morphology for 4-6 days in the presence of retinoic acid (RA), wherein a population of Hox + cells are created;
(b) culturing the cells resulting from step (a) in the presence of RA and sonic hedgehog (SHH); and
(c) culturing the cells resulting from step (b) in neuronal differentiation medium and in the presence of SHH, wherein the resulting cells express HB9, HoxB1, HoxB6, HoxC5, HoxC8, choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) and produce acetylcholine.
2. The method of claim 1 wherein the cells are exposed to SHH between 6-7 days in step (b).
3. A population of human spinal motor neurons generated by the method of claim 1 , wherein the spinal motor neurons express HB9, HoxB1, HoxB6, HoxC5, HoxC8, ChAT and VAChT and produce acetylcholine.
4. The method of claim 1 wherein the cells are exposed to SHH between 6-10 days in step (b).
5. The method of claim 1 wherein the cells are exposed to neuronal differentiation medium between 7-21 days in step (c).