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 creating a population of human ventral spinal progenitor cells, the method comprising the steps of:
- (a) obtaining a synchronous population of cells that are cultured from human embryonic stem cells, that are characterized by an early rosette morphology and that are Pax6+/Sox1−
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(b) culturing the cells of step (a) with a sufficient amount of retinoic acid in adherent culture, wherein the resulting cells are positive for Hoxb4 and are negative for at least one of Otx2 or Bf1;
(c) expanding the resulting cells of step (b) in a suspension culture with a sufficient amount of retinoic acid; and
(d) culturing the cells of step (c) in a suspension culture with a sufficient amount of retinoic acid and an agent selected from the group consisting of sonic hedgehog and purmorphamine, wherein at least 80% of the resulting cells are Olig2+, Nkx2.2+, Irx3+ and Pax7−
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Abstract
A method of differentiating embryonic stem cells into ventral spinal progenitor 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 retinoic acid, wherein the cells express Hoxb4, but not Otx2 or Bf1.
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Citations
10 Claims
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1. A method of creating a population of human ventral spinal progenitor cells, the method comprising the steps of:
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(a) obtaining a synchronous population of cells that are cultured from human embryonic stem cells, that are characterized by an early rosette morphology and that are Pax6+/Sox1−
;(b) culturing the cells of step (a) with a sufficient amount of retinoic acid in adherent culture, wherein the resulting cells are positive for Hoxb4 and are negative for at least one of Otx2 or Bf1; (c) expanding the resulting cells of step (b) in a suspension culture with a sufficient amount of retinoic acid; and (d) culturing the cells of step (c) in a suspension culture with a sufficient amount of retinoic acid and an agent selected from the group consisting of sonic hedgehog and purmorphamine, wherein at least 80% of the resulting cells are Olig2+, Nkx2.2+, Irx3+ and Pax7−
. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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Specification