Derivation of terminally differentiated dopaminergic neurons from human embryonic stem cells
First Claim
1. A method of generating a differentiated neural cell population from primate pluripotent stem cells comprising the following steps:
- (a) expanding a culture of primate pluripotent stem cells;
(b) culturing the pluripotent stem cells to select for neuroprogenitor cells that are positive for nestin;
(c) sorting the nestin-positive neuroprogenitor cells for enrichment of NCAM-positive cells;
(d) differentiating the nestin-positive, NCAM-positive cells to generate a differentiated neural cell population by culturing the cells in a differentiation media which comprises N-acetyl cysteine and TGF-β
3 or interleukin-1β
or both, wherein the differentiated neural cell population comprises at least about 60% dopaminergic neurons or at least about 30% serotonergic neurons.
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Abstract
The present disclosure is directed to improved methods for efficiently producing neuroprogenitor cells and differentiated neural cells such as dopaminergic neurons and serotonergic neurons from pluripotent stem cells, for example human embryonic stem cells. Using the disclosed methods, cell populations containing a high proportion of cells positive for tyrosine hydroxylase, a specific marker for dopaminergic neurons, have been isolated. The neuroprogenitor cells and terminally differentiated cells of the present disclosure can be generated in large quantities, and therefore may serve as an excellent source for cell replacement therapy in neurological disorders such as Parkinson'"'"'s disease.
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Citations
28 Claims
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1. A method of generating a differentiated neural cell population from primate pluripotent stem cells comprising the following steps:
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(a) expanding a culture of primate pluripotent stem cells; (b) culturing the pluripotent stem cells to select for neuroprogenitor cells that are positive for nestin; (c) sorting the nestin-positive neuroprogenitor cells for enrichment of NCAM-positive cells; (d) differentiating the nestin-positive, NCAM-positive cells to generate a differentiated neural cell population by culturing the cells in a differentiation media which comprises N-acetyl cysteine and TGF-β
3 or interleukin-1β
or both, wherein the differentiated neural cell population comprises at least about 60% dopaminergic neurons or at least about 30% serotonergic neurons. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)
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27. A method of generating dopaminergic neurons from neuroprogenitor cells, comprising enriching the neuroprogenitor cells for cells that are positive for nestin and positive for NCAM, and differentiating the nestin-positive, NCAM-positive cells to generate dopaminergic neurons by culturing the cells in the presence of N-acetyl cysteine and TGF-β
- 3 or interleukin-1β
or both, wherein at least about 60% of the nestin-positive, NCAM-positive cells differentiate into dopaminergic neurons.
- 3 or interleukin-1β
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28. A method of generating serotonergic neurons from neuroprogenitor cells, comprising enriching the neuroprogenitor cells for cells that are positive for nestin and NCAM, and differentiating the nestin-positive, NCAM-positive cells to generate serotonergic neurons by culturing the cells in the presence of N-acetyl cysteine and TGF-β
- 3 or interleukin-1β
or both, wherein at least about 30% of the nestin-positive, NCAM-positive cells differentiate into serotonergic neurons.
- 3 or interleukin-1β
Specification