| CPC A61L 27/3878 (2013.01) [A61K 35/545 (2013.01); A61L 27/383 (2013.01); A61L 27/3834 (2013.01); A61L 27/3895 (2013.01); C12N 5/0621 (2013.01); A61K 35/30 (2013.01); A61L 2400/06 (2013.01); A61L 2430/16 (2013.01); C12N 2501/10 (2013.01); C12N 2501/11 (2013.01); C12N 2501/115 (2013.01); C12N 2501/155 (2013.01); C12N 2501/385 (2013.01); C12N 2501/41 (2013.01); C12N 2501/415 (2013.01); C12N 2501/727 (2013.01); C12N 2501/999 (2013.01); C12N 2506/02 (2013.01)] | 11 Claims |
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1. A method for producing a ciliary marginal zone stem cell, comprising:
performing steps (A) and (B):
(A) subjecting pluripotent stem cells to floating culture in a serum-free medium to form an aggregate of pluripotent stem cells, and
(B) performing floating culture of the aggregate formed in step (A) in a serum-free medium or serum-containing medium each free of a substance capable of enhancing signal transduction mediated by Sonic hedgehog and containing a substance capable of enhancing signal transduction mediated by BMP to produce a cell aggregate comprising a retinal tissue,
followed by performing step (i) or (ii):
(i) culturing a cell aggregate comprising a retinal tissue as produced in step (B) in which Chx10 positive cells are present in a proportion of 20% or more and 100% or less of the tissue in a serum-free medium or serum-containing medium each containing a substance capable of enhancing signal transduction mediated by Wnt and a substance inhibiting the FGF signal pathway for only a period before the appearance of a RPE65 gene-expressing cell or until RPE65 positive cells appear in the retinal tissue at a ratio of about 1% or less, followed by culturing the resulting cell aggregate in which a RPE65 gene-expressing cell does not appear or RPE65 positive cells are present in the retinal tissue at a ratio of about 1% or less in a serum-free medium or serum-containing medium each free of a substance capable of enhancing signal transduction mediated by Wnt, thereby obtaining a cell aggregate comprising a ciliary marginal zone-like structure induced to differentiate from pluripotent stem cells, or
(ii) culturing a cell aggregate comprising a retinal tissue as produced in step (B) in which Chx10 positive cells are present in a proportion of 20% or more of the tissue in a serum-free medium or serum-containing medium each containing a substance capable of enhancing signal transduction mediated by Wnt for only a period before the appearance of a RPE65 gene-expressing cell, followed by culturing the resulting cell aggregate in which a RPE65 gene-expressing cell does not appear in a serum-free medium or serum-containing medium each free of a substance capable of enhancing signal transduction mediated by Wnt, thereby obtaining a cell aggregate comprising a ciliary marginal zone-like structure induced to differentiate from pluripotent stem cells;
followed by performing step (1):
(1) dispersing (a) the cell aggregate comprising a ciliary marginal zone-like structure generated in step (i) or (ii), or (b) a ciliary marginal zone-like structure separated from the cell aggregate comprising a ciliary marginal zone-like structure generated in step (i) or (ii), followed by floating culturing the resulting dispersed cells at 1×102 cells/ml to 1×106 cells/ml in a serum-free medium, thereby obtaining a retinosphere comprising a ciliary marginal zone stem cell,
wherein the ciliary marginal zone stem cell is stage specific embryonic antigen-1 positive, Rax gene positive, Chx10 gene positive, Rdh10 gene positive, Otx1 gene positive, Crx gene negative, and β-III tubulin gene negative and non-pigmented,
wherein the pluripotent stem cell is a human pluripotent stem cell, and
wherein the retinosphere contains 40% or more of Rax positive and stage specific embryonic antigen-1 positive cells and is free of pigment deposition.
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