Abstract: The invention provides a method of differentiating human pluripotent stem cells to ectodermal cell by treating human pluripotent stem cells, which are dissociated single cells, and inducing differentiation to ectodermal cells under conditions where a ROCK(Rho-kinase) inhibitor is present in a culture medium in contact with the cells after dissociation of the human pluripotent stem cells.

Abstract: The present invention provides a method of obtaining aggregates containing a rostral hypothalamus tissue and a rostral head ectodermal tissue, a hypophysis precursor tissue and a hypophysis hormone producing cell, by using a serum-free medium (preferably substantially free of growth factor and insulins), forming homogeneous aggregates of stem cells from pluripotent stem cells such as ES cell and the like, which are plated at a high cell concentration, and subjecting the formed aggregates to floating-culture.

Abstract: The invention provides a method for producing a ciliary marginal zone stem cell induced to differentiate from a pluripotent stem cell, including either the following step (1) or step (2), or both of these steps: (1) a step of floating culturing cells obtained from a cell aggregate containing a ciliary marginal zone-like structure induced to differentiate from pluripotent stem cells, thereby obtaining a retinosphere; and (2) a step of collecting stage specific embryonic antigen-1 positive cells from cells obtained from a cell aggregate containing a ciliary marginal zone-like structure induced to differentiate from pluripotent stem cells.

Abstract: The invention provides a method for producing a cell aggregate containing a ciliary marginal zone-like structure by culturing a cell aggregate containing a retinal tissue 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 acting on the Wnt signal pathway for only a period before the appearance of a RPE65 gene expressing cell, followed by culturing the “cell aggregate in which a RPE65 gene expressing cell does not appear” thus obtained in a serum-free medium or serum-containing medium, each not containing a substance acting on the Wnt signal pathway and so on.

Abstract: The present invention provides a method for producing a retinal progenitor cell, including (1) a first step of subjecting pluripotent stem cells to floating culture in a serum-free medium to form an aggregate of pluripotent stem cells, and (2) a second step of subjecting the aggregate formed in step (1) to floating culture in a serum-free medium or serum-containing medium each being free of a substance acting on the Sonic hedgehog signal transduction pathway but containing a substance acting on the BMP signal transduction pathway, thereby obtaining an aggregate containing retinal progenitor cells.

Abstract: The present invention provides a method for producing a retinal progenitor cell, including (1) a first step of subjecting pluripotent stem cells to floating culture in a serum-free medium to form an aggregate of pluripotent stem cells, and (2) a second step of subjecting the aggregate formed in step (1) to floating culture in a serum-free medium or serum-containing medium each being free of a substance acting on the Sonic hedgehog signal transduction pathway but containing a substance acting on the BMP signal transduction pathway, thereby obtaining an aggregate containing retinal progenitor cells.

Abstract: The present invention provides a method for producing a retinal cell or a retinal tissue, including the following steps (1)-(3): (1) a first step of culturing pluripotent stem cells in the absence of feeder cells in a medium containing 1) a TGF? family signal transduction pathway inhibiting substance and/or a Sonic hedgehog signal transduction pathway activating substance, and 2) a factor for maintaining undifferentiated state, (2) a second step of culturing the cells obtained in the first step in suspension in a medium containing a Wnt signal transduction pathway inhibiting substance to form a cell aggregate, and (3) a third step of culturing the aggregate obtained in the second step in suspension in the presence or absence of a Wnt signal transduction pathway inhibiting substance in a medium containing a BMP signal transduction pathway activating substance to obtain an aggregate containing a retinal cell or a retinal tissue.

Abstract: The present invention provides a method for producing retinal cells or a retinal tissue, comprising the following steps (1)-(3): (1) a first step of culturing human pluripotent stem cells in the absence of feeder cells and in a medium comprising a factor for maintaining undifferentiated state, (2) a second step of culturing the pluripotent stem cells obtained in the first step in suspension in the presence of a Sonic hedgehog signal transduction pathway activating substance to form a cell aggregate, and (3) a third step of culturing the aggregate obtained in the second step in suspension in the presence of a 1) a BMP signal transduction pathway activating substance to obtain an aggregate containing retinal cells or a retinal tissue.

Abstract: The present invention provides a method for producing neural cells or a neural tissue, including the following steps (1)-(3): (1) a first step of culturing pluripotent stem cells in the absence of feeder cells and in a medium containing 1) a TGF? family signal transduction pathway inhibiting substance and/or a Sonic hedgehog signal transduction pathway activating substance, and 2) a factor for maintaining undifferentiated state, (2) a second step of culturing the cells obtained in the first step in suspension to form a cell aggregate, and (3) a third step of culturing the aggregate obtained in the second step in suspension in the presence or absence of a differentiation-inducing factor to obtain an aggregate containing neural cells or a neural tissue.

Abstract: The present invention provides a method of producing more mature telencephalon or a progenitor tissue thereof, in vitro, from mammalian pluripotent stem cells, comprising obtaining a telencephalon marker-positive aggregate by culturing an aggregate of pluripotent stem cells in suspension in the presence of a Wnt signal inhibitor and a TGF? signal inhibitor, and further culturing the telencephalon marker-positive aggregate in suspension under a high oxygen partial pressure condition. In one embodiment, the suspension culture under a high oxygen partial pressure condition is performed in the presence of a Wnt signal enhancer and a bone morphogenetic factor signal transduction pathway activating substance.

Abstract: The present invention provides a production method of a cell aggregate containing an anterior eye segment tissue or a partial structure thereof, or a precursor tissue thereof, including culturing pluripotent stem cell aggregates in suspension in the presence of a bone morphogenic factor signal transduction pathway activating substance to induce self-organization of an anterior eye segment tissue or a partial structure thereof, or a precursor tissue thereof. In one embodiment, the bone morphogenic factor signal transduction pathway activating substance is BMP4. In one embodiment, the suspension culture is performed entirely or partially in the presence of a fibroblast growth factor. The produced cell aggregate can further contain a neural retinal tissue.

Abstract: The invention provides a method of cryopreserving a tissue derived from a pluripotent stem cell, including the steps of (1) bringing a tissue derived from a pluripotent stem cell into contact with a cell protection solution that contains sulfoxide and chain polyol, (2) maintaining, in a cryopreservation solution, the pluripotent stem cell-derived tissue that was brought into contact with the cell protection solution in the first step, and (3) cryopreserving, in the presence of a coolant, the pluripotent stem cell-derived tissue that was maintained in the cryopreservation solution.

Abstract: The present invention provides a method for producing retinal cells or a retinal tissue, comprising the following steps (1)-(3): (1) a first step of culturing human pluripotent stem cells in the absence of feeder cells and in a medium comprising a factor for maintaining undifferentiated state, (2) a second step of culturing the pluripotent stem cells obtained in the first step in suspension in the presence of a Sonic hedgehog signal transduction pathway activating substance to form a cell aggregate, and (3) a third step of culturing the aggregate obtained in the second step in suspension in the presence of a 1) a BMP signal transduction pathway activating substance to obtain an aggregate containing retinal cells or a retinal tissue.

Abstract: The present invention provides a method for producing neural cells or a neural tissue, including the following steps (1)-(3): (1) a first step of culturing pluripotent stem cells in the absence of feeder cells and in a medium containing 1) a TGF? family signal transduction pathway inhibiting substance and/or a Sonic hedgehog signal transduction pathway activating substance, and 2) a factor for maintaining undifferentiated state, (2) a second step of culturing the cells obtained in the first step in suspension to form a cell aggregate, and (3) a third step of culturing the aggregate obtained in the second step in suspension in the presence or absence of a differentiation-inducing factor to obtain an aggregate containing neural cells or a neural tissue.

Abstract: The present invention provides a method of producing more mature telencephalon or a progenitor tissue thereof, in vitro, from mammalian pluripotent stem cells, comprising obtaining a telencephalon marker-positive aggregate by culturing an aggregate of pluripotent stem cells in suspension in the presence of a Wnt signal inhibitor and a TGF? signal inhibitor, and further culturing the telencephalon marker-positive aggregate in suspension under a high oxygen partial pressure condition. In one embodiment, the suspension culture under a high oxygen partial pressure condition is performed in the presence of a Wnt signal enhancer and a bone morphogenetic factor signal transduction pathway activating substance.

Abstract: The present invention provides a method for producing a cell aggregate containing a ciliary marginal zone-like structure, including a step of culturing a cell aggregate containing a retinal tissue 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 acting on the Wnt signal pathway and a substance inhibiting the FGF signal pathway for only a period before the appearance of a RPE65 gene expressing cell, followed by culturing the “cell aggregate in which a RPE65 gene-expressing cell does not appear” thus obtained in a serum-free medium or serum-containing medium each free of a substance acting on the Wnt signal pathway and so on. According to the production method of the present invention, a ciliary marginal zone-like structure can be produced with high efficiency.

Abstract: The invention provides a method for producing a retinal tissue by (1) subjecting pluripotent stem cells to floating culture in a serum-free medium containing a substance inhibiting the Wnt signal pathway to form an aggregate of pluripotent stem cells, (2) subjecting the aggregate to floating culture in a serum-free medium containing a basement membrane preparation, and then (3) subjecting the aggregate to floating culture in a serumcontaining medium. The invention also provides a method for producing an optic-cup-like structure, a method for producing a retinal pigment epithelium, and a method for producing a retinal layer-specific neural cell.

Abstract: The present invention aims to provide a method for suppressing differentiation of ganglion cell, amacrine cell, horizontal cell and/or bipolar cell in a neural retina tissue containing photoreceptor precursor and/or photoreceptor, and the like. A method for suppressing differentiation of a ganglion cell, an amacrine cell, a horizontal cell and/or a bipolar cell in a neural retinal tissue containing a photoreceptor precursor and/or a photoreceptor, including a step of culturing a retinal tissue comprising a neural retinal progenitor cell and in any stage between a differentiation stage immediately after emergence of a ganglion cell and a differentiation stage where emergence rate of a cone photoreceptor precursor reaches maximum in a medium containing a thyroid gland hormone signal transduction pathway agonist.

Abstract: Provided is a method of inducing the differentiation of a stem cell into nerve progenitor cells, comprising the step (1) of forming homogenous aggregates of stem cells in a serum-free medium (1) and the step (2) of suspension-culturing the homogenous aggregates of stem cells in the presence of a basement membrane reference standard.

Abstract: The invention provides a method for producing a retinal tissue by (1) subjecting pluripotent stem cells to floating culture in a serum-free medium containing a substance inhibiting the Wnt signal pathway to form an aggregate of pluripotent stem cells, (2) subjecting the aggregate to floating culture in a serum-free medium containing a basement membrane preparation, and then (3) subjecting the aggregate to floating culture in a serum-containing medium. The invention also provides a method for producing an optic-cup-like structure, a method for producing a retinal pigment epithelium, and a method for producing a retinal layer-specific neural cell.