Differentiation of human pluripotent stem cells to multipotent neural crest cells
First Claim
1. A method of producing p75+ Hnk1+ Ap2+ multipotent neural crest-like cells from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) comprising differentiating said stem cells in a differentiation medium in the absence of feeder cells consisting essentially of an effective amount of a glycogen synthase kinase (GSK) inhibitor in combination with an effective amount of an Activin A inhibitor to produce a population of cells comprising p75+ Hnk1+ Ap2+ multipotent neural crest-like stem cells and PAX6+ neural progenitor cells, wherein said GSK inhibitor is (2′
- Z,3′
E)-6-Bromoindimbin-3′
-oxime (BIO) and said Activin A inhibitor is SB 431542 and wherein said PAX6+ neural progenitor cells produced comprise no more than 10% of the total population of p75+ Hnk1+ Ap2+ multipotent neural crest-like cells and PAX6+ neural progenitor cells; and
optionally isolating said neural p75+ Hnk1+ Ap2+ multipotent neural crest-like cells from said population of cells.
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Abstract
The present invention relates to the differentiation of human pluripotent cells, including human pluripotent stems cells to produce a self-renewing multipotent neural crest cell population in a single step method without the requirement of isolation of intermediate cells and without appreciable contamination (in certain preferred instances, virtually none) with Pax6+ neural progenitor cells in the population of p75+ Hnk1+ Ap2+ multipotent neural crest-like cells. The multipotent neural crest cell population obtained can be clonally amplified and maintained for >25 passages (>100 days) while retaining the capacity to differentiate into peripheral neurons, smooth muscle cells and mesenchymal precursor cells.
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Citations
24 Claims
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1. A method of producing p75+ Hnk1+ Ap2+ multipotent neural crest-like cells from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) comprising differentiating said stem cells in a differentiation medium in the absence of feeder cells consisting essentially of an effective amount of a glycogen synthase kinase (GSK) inhibitor in combination with an effective amount of an Activin A inhibitor to produce a population of cells comprising p75+ Hnk1+ Ap2+ multipotent neural crest-like stem cells and PAX6+ neural progenitor cells, wherein said GSK inhibitor is (2′
- Z,3′
E)-6-Bromoindimbin-3′
-oxime (BIO) and said Activin A inhibitor is SB 431542 and wherein said PAX6+ neural progenitor cells produced comprise no more than 10% of the total population of p75+ Hnk1+ Ap2+ multipotent neural crest-like cells and PAX6+ neural progenitor cells; and
optionally isolating said neural p75+ Hnk1+ Ap2+ multipotent neural crest-like cells from said population of cells. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
- Z,3′
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13. A method of producing p75+ Hnk1+ Ap2+ multipotent neural crest-like cells from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) comprising differentiating said stem cells in a differentiation medium in the absence of feeder cells consisting essentially of an effective amount of a Wnt protein in combination with an effective amount of an Activin A inhibitor to produce a population of cells comprising p75+ Hnk1+ Ap2+ multipotent neural crest-like stem cells and PAX6+ neural progenitor cells, wherein said Wnt protein is Wnt3a and said Activin A inhibitor is SB 431542 and wherein said PAX6+ neural progenitor cells produced comprise no more than 10% of the total population of p75+ Hnk1+ Ap2+ multipotent neural crest-like cells and PAX6+ neural progenitor cells;
- and optionally isolating said neural p75+ Hnk1+ Ap2+ multipotent neural crest-like cells from said population of cells.
- View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)
Specification