Differentiation of human pluripotent stem cells to multipotent neural crest cells
AssigneeUniversity of Georgia Research Foundation, Inc.
InventorsStephen Dalton, Laura M. Menendez
Life of this patent
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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.
Claims16
1-50. (canceled)
A method of producing p75+Hnk1+Ap2+ multipotent neural crest-like cells from human pluripotent stem cells comprising differentiating said pluripotent stem cells in a differentiation medium consisting essentially of an effective amount of at least one glycogen synthase kinase (GSK) inhibitor, at least one Wnt protein or a mixture of at least one GSK inhibitor and at least one Wnt protein, wherein said GSK inhibitor is selected from the group consisting of Acetoxime (2′Z,3′E)-6-Bromoindirubin-3′-acetoxime (BIO, GSK3 Inhibitor X);
- (5-Methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine (GSK3 Inhibitor XIII);
- Pyridocarbazole-cyclopenadienylruthenium complex (GSK3 Inhibitor XV);
- 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8, GSK3β Inhibitor I);
- 2-Thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK3β Inhibitor II);
- 2,4-Dibenzyl-5-oxothiadiazolidine-3-thione (OTDZT, GSK3β Inhibitor III);
- α-4-Dibromoacetophenone (GSK3β Inhibitor VII);
- N-(4-Methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418, GSK-3β Inhibitor VIII);
- 3-(1-(3-Hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione (GSK-3β Inhibitor XI);
- pyrrolopyrimidine compound TWS119 (GSK3β Inhibitor XII);
- H-KEAPPAPPQSpP-NH2 or its Myristoylated form (L803, GSK3β Inhibitor XIII);
- 2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone (GSK3β Inhibitor VI) and mixtures thereof, and said Wnt protein is selected from the group consisting of Wnt1, Wnt2, Wnt3, Wnt3a, Wnt4, Wnt10, Wnt 14, Wnt14b, Wnt15, Wnt16 and mixtures thereof, said GSK inhibitor(s), said Wnt protein(s) or said mixture of said GSK inhibitor(s) and said Wnt protein(s) being combined with an effective amount of at least one Activin A inhibitor selected from the group consisting of SB-431542, follistatin, follistatin gene related protein (FGRP), bone morphogenic protein (BMP) and Activin Membrane Bound Inhibitor (BAMBI), anti-BAMBI (monoclonal antibody), Smad7 (Mothers Against Decapentaplegic Homolog 7) and TGF RI inhibitor, said differentiation medium optionally containing an effective amount of at least one bone morphogenic protein (BMP) inhibitor selected from the group consisting of noggin, chordin, sclerostin, follistatin, gremlin, dorsomorphorin, and connective tissue growth factor (CTGF) to produce a population of cells comprising neural crest-like stem cells and PAX6+ neural progenitor cells, wherein said neural progenitor cells comprise no more than about 15% of the population of neural crest-like stem cells and neural progenitor cells;
- and optionally isolating said neural crest-like stem cells from said population of cells.
A method of producing p75+Hnk1+Ap2+ multipotent neural crest-like cells from human pluripotent stem cells comprising differentiating said pluripotent stem cells in a differentiation medium consisting essentially of an effective amount of at least one GSK inhibitor, at least one Wnt protein or a mixture of at least one GSK inhibitor and at least one Wnt protein further in combination with an effective amount of at least one Activin A inhibitor, and optionally, an effective amount of at least one bone morphogenic protein inhibitor to produce a population of cells comprising neural crest-like cells and PAX6+ neural progenitor cells, wherein said PAX6+ neural progenitor cells comprise no more than about 15% of the population of neural crest-like cells and PAX6+ neural progenitor cells, and optionally isolating said neural crest-like cells from said population of cells.
Dependencies are inferred from the claim text.