Method of in vitro differentiation of neural stem cells, motor neurons and dopamine neurons from primate embryonic stem cells
First Claim
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1. A method of obtaining a population of midbrain dopamine neurons, comprising:
- (a) obtaining a synchronous population of cells that are cultured from embryonic stem cells, and which are characterized by an early rosette morphology and are Sox1−
, Pax6+;
(b) culturing the cells obtained in (a) in a neural basal medium with fibroblast growth factor-8 (FGF8), wherein resulting cells are Pax6+, Sox1+ and engrailed 1 positive (EN-1+) neuroepithelial cells;
(c) expanding the cells from step (b) in a neural basal medium with sonic hedgehog (SHH) and FGF8, wherein resulting cells are dopaminergic neural precursors; and
(d) culturing the cells from step (c) in a neural basal differentiation medium without SHH and FGF8 until resulting cells express tyrosine hydroxylase (TH), aromatic acid decarboxylase (AADC), EN-1, vesicular monoamine transporter 2 (VMAT2) and dopamine transporter (DAT) but do not express dopamine beta hydroxylase (DbH) and phenylethanolamine N-methyltransferase (PNMT), and produce dopamine.
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Abstract
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+.
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Citations
2 Claims
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1. A method of obtaining a population of midbrain dopamine neurons, comprising:
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(a) obtaining a synchronous population of cells that are cultured from embryonic stem cells, and which are characterized by an early rosette morphology and are Sox1−
, Pax6+;(b) culturing the cells obtained in (a) in a neural basal medium with fibroblast growth factor-8 (FGF8), wherein resulting cells are Pax6+, Sox1+ and engrailed 1 positive (EN-1+) neuroepithelial cells; (c) expanding the cells from step (b) in a neural basal medium with sonic hedgehog (SHH) and FGF8, wherein resulting cells are dopaminergic neural precursors; and (d) culturing the cells from step (c) in a neural basal differentiation medium without SHH and FGF8 until resulting cells express tyrosine hydroxylase (TH), aromatic acid decarboxylase (AADC), EN-1, vesicular monoamine transporter 2 (VMAT2) and dopamine transporter (DAT) but do not express dopamine beta hydroxylase (DbH) and phenylethanolamine N-methyltransferase (PNMT), and produce dopamine. - View Dependent Claims (2)
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Specification