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 forebrain dopamine neurons, comprising the steps of:
(a) providing a synchronous population of human Sox1−, Pax6+ cells characterized by an early rosette morphology;
(b) culturing the human Sox1−, Pax6+ cells of step (a) in the presence of fibroblast growth factor 2 (FGF2) for 4-6 days, wherein a population of cells expressing brain factor 1 (Bf1) and orthodenticle homeobox2 (Otx2) is generated;
(c) culturing the Bf1+ cells resulting from step (b) in the presence of sonic hedgehog (SHH) and FGF8 for 6-7 days; and
(d) culturing the cells resulting from step (c) in a neuronal differentiation medium to obtain a cell population in which about one third of the differentiated cells are human forebrain dopamine-producing neurons that express tyrosine hydroxylase (TH), aromatic acid decarboxylase (AADC), Bf1, Otx2, but do not express dopamine beta-hydroxylase (DβH) and phenylethanolamine N-methyltransferase (PNMT).
2. The method of claim 1 wherein FGF2 is present at a concentration of 10-20 ng/ml in step (b).
3. The method of claim 1 wherein SHH is present at a concentration of 50-250 ng/ml in step (c).
4. The method of claim 1 wherein the cells resulting from step (c) are exposed to the neuronal differentiation medium between 7-21 days.