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Provided is an in vitro method for differentiating pluripotent stem cells, comprising a step of exposing stem cells to an effective concentration of one or more inhibitors of TGFβ/Activin-Nodal signaling, one or more inhibitors of BMP signaling, one or more inhibitors of Wnt signaling, one or more inhibitors of MAPK/ERK kinase signaling, one or more inhibitors of FGF signaling, and one or more inhibitors of Notch signaling, such that a plurality of the cells differentiate and express one or more cortical neuron precursor markers.

Provided is a method for generating functional mature beta cells from human pluripotent stem cell-derived endocrine progenitors comprising the steps of (1) culturing the stem cell-derived endocrine progenitor cells in a medium comprising histone methyltransferase EZH2 inhibitor, TGF-beta signaling pathway inhibitor, Heparin and Nicotinamide in basal medium, to obtain INS+ and NKX6.1+ double positive immature beta cells and (2) culturing the beta cells obtained in step (1) with 12% knockout serum replacement and GABA , to obtain functional mature beta cells.

Provided is a method of generating intestinal cells, comprising steps of (i) generating definitive endoderm by culturing iPSCs in the presence of Activin A and Wnt3A, (ii) differentiating into hindgut by culturing the definitive endoderm in the presence of FGF4 and either Wnt3A or CHIR99021, (iii) collecting epithelial spheres or epithelial tubes, (iv) suspending the epithelial spheres or the epithelial tubes in Matrigel, and (v) culturing in the presence of CHIR99021, noggin and EGF to generate intestinal cells.

Provided is a method of generating functional neurons from stem cells, comprising contacting a population of stem cells in spin culture with at least one differentiation medium to induce the differentiation of at least one sphere in the spin culture into a functional neuron differentiation sphere (especially motor neuron differentiation sphere or a cortical neuron differentiation sphere).

Provided is a 3D neural tissue composition comprising a cerebral organoid exhibiting discrete brain regions comprising one or more sensory receptors and cells. Also provided is an in vitro method of producing the 3D neural tissue composition from human iPS cells.

Provided is an in vitro generated 3D neural organoid derived from a hiPS, which comprises a first region expressing retinal or cortical markers, and one or more additional neural regions each expressing markers of the brain stem, cerebellum, and/or spinal cord.

Provided is an in vitro method of producing microtissue from pluripotent stem cell-derived hepatocytes comprising culturing a cell suspension of said hepatocytes in the presence of one or more extracellular matrix proteins or fragments thereof in a culture container having an essentially cell non-adhesive bottom to obtain microtissue.

Provided is a method of producing iPSCs that are essentially free of reprogramming vectors, comprising steps of (a) introducing reprogramming vectors into a human somatic cell to produce the first cell population, wherein the reprogramming vectors comprises a viral origin of replication, an expression cassette encoding an iPSC reprogramming factor, a synthetic transcription factor, or both, and a suicide gene, (b) culturing the first cell population to effect expression of the reprogramming factor, the synthetic transcription factor, or both, to produce the second cell population, and (c) contacting the second cell population with a suicide gene substrate to produce iPSCs that are essentially free of reprogramming vectors.

Provided is a method for inducing differentiation of stem cells, comprising contacting a population of stem cells with effective amounts of one or more inhibitor of TGFβ/Activin-Nodal signaling and one or more activator of Wnt signaling, and further contacting said population of cells with an effective amount(s) of one or more molecule that induces vagal neural crest patterning for at least about 2 days to produce a population of differentiated cells that express one or more enteric neural crest lineage marker.

Provided is a method for detecting a pluripotent stem cell in an undifferentiated state, comprising analysing miRNA 142 expression in a PSC, wherein an increase of the expression level of miRNA 142 is indicative for an undifferentiated state, when compared to a differentiating or differentiated PSC.

Provided is a method for producing brown adipocytes comprising steps of; (a) providing a pluripotent stem cell-derived clonal embryonic progenitor cell line that expresses DI03, DLK1, ZIC2, SLC1A3 and SBSN but does not express COX7A1 and does not express one or more of HOXA5, IL13RA2, DLX5, CRABP1, NEFM, PRG4, and RBP1, (b) differentiating the said clonal embryonic progenitor cell line to a brown adipocyte lineage in the presence of a PPARγ agonist, and (c) screening the said differentiated cell line for the expression of UCP1 marker wherein the presence of the UCP1 marker identifies brown adipocytes.

Provided is a method for producing cells with a hepatic stellate cell phenotype, comprising steps of; (a) culturing pluripotent cells with about 1 ng/ml to about 200 ng/ml BMP4, (b) culturing the cells of step (a) with about 1 ng/ml to about 200 ng/ml FGF1 and about 1 ng/ml to about 200 ng/ml FGF3, (c)culturing the cells of step (b) with about 0.5 μΜ to about 50 μΜ retinol and about 10 μΜ to about 1 m M palmitic acid, and (d) isolating hepatic stellate cells produced by the differentiation protocol.

Provided is a method for preparing a composition of T cells from stem or progenitor cells. The method comprises a step of culturing a three-dimensional cell aggregate comprising: a selected population of stromal cells that express a Notch ligand and a selected population of stem or progenitor cells, wherein the culturing is performed in a serum-free medium comprising insulin, biotin, transferrin, and albumin.

Provided is a cell culture method of generating astrocytes from neuronal precursor cells (NPCs). The NPCs may be obtained from induced pluripotent stem cells.

Provided is a method for efficiently generating an induced pluripotent stem cell （iPSC) comprising steps of (i) providing a cell which is not a stem cell, (ii) arresting the cell cycle of the cell, and (iii) transforming the cell to generate the iPSC.

Provided is a method for generating retinal pigment epithelial (RPE) cells comprising steps of (a) culturing a cell population of undifferentiated human pluripotent stem cells on an adherent surface in a medium comprising a differentiating agent to obtain differentiating cells and (b) culturing said differentiating cells on said adherent surface in a medium which comprises one or more members of the TGFβ superfamily to obtain RPE cells.

Provided is a method for producing hepatocytes comprising a step of plating human pluripotent stem cells on a cell culture substrate comprising (i) a first laminin which is laminin-521 and (ii) a second laminin selected from the group consisting of laminin-111 and laminin-221.

Provided is a method of generating hematopoietic lineage cells comprising a step of directing differentiation of iPSCs to hematopoietic lineage cells. The directing differentiation comprises steps of (i) contacting iPSCs with a composition comprising a BMP pathway activator, and optionally bFGF, to obtain mesodermal cells and (ii) contacting the mesodermal cells with a composition comprising a BMP pathway activator, bFGF, and a WNT pathway activator, to obtain mesodermal cells having definitive hemogenic endothelium potential which are capable of providing hematopoietic lineage cells, wherein steps (i) and (ii) are without a step of forming embryoid bodies.

Provided is a method of culturing cells whereby the cells mimic the structure and function of the blood brain barrier, comprising steps of (a) providing a fluidic device comprising a membrane that comprises a top surface and a bottom surface, (b) seeding cells those may be selected from the group consisting of stem cell-derived cells, cells differentiated from stem cells and primary cells on said bottom surface, and (c) culturing said seeded cells under conditions that support the maturation of brain microvascular endothelial cells. The method may further comprise a step of (d) seeding stem cell-derived cells or cells differentiated from stem cells on said top surface and culturing said top surface seeded cells under conditions that support the maturation of astrocytes or neurons.

Provided is a method for obtaining human pre-otic epithelial cells comprising steps of (a) culturing human pluripotent stem cell aggregates in a culture medium comprising a BMP and a TGFβinhibitor for about 8 to 10 days, (b) further culturing the cultured aggregates of (a) in the presence of a FGF and a BMP inhibitor for about 4 days, and (c) contacting the further cultured aggregates of (b) to a Wnt agonist for about 4 days; whereby cells within the contacted aggregates differentiate into pre-otic epithelial cells.

Provided is a method for obtaining a three-dimensional multilayered skin composition comprising steps of (a) culturing human pluripotent stem cell aggregates in a culture medium comprising BMP4 and a TGFβinhibitor for about 8 to 10 days; whereby non-neural epithelium forms within the aggregates, (b) embedding the cultured aggregates of (a) in a porous substrate comprising extracellular matrix components, and (c) culturing the embedded aggregates of (b) for at least about 25 to 120 days under conditions that promote self-assembly of cells within the embedded aggregates into three-dimensional, multilayered composition comprising an epidermal layer, a dermal layer, and a plurality of cells capable of forming a functional hair follicle.

Provided is a method for producing virus-receptive pluripotent stem cell (PSC)-derived hepatocytes comprising steps of (a) obtaining PSC-derived hepatocytes, and (b) culturing the PSC-derived hepatocytes in media comprising cAMP and a Janus kinase inhibitor, thereby producing virus-receptive hepatocytes.

Provided is a method for producing immune cells comprising steps of (a) obtaining pluripotent stem cells (PSCs) reprogrammed from a population of somatic cells, (b) differentiating the PSCs to hematopoietic precursor cells (HPCs), and (c) culturing the HPCs under conditions to promote immune cell differentiation, thereby producing immune cells. In certain aspects, the above step (b) specifically comprises sequential steps of culturing the cells in a media comprising BMP4, FGF2 and VEGF, and in a media comprising IL-3 and Flt3 ligand.

Provided is a method for producing hematopoietic precursor cells (HPCs) from pluripotent stem cells (PSCs) comprising steps of (a) providing PSCs comprising at least one expression construct encoding hematopoietic precursor programming genes comprising ETS/ERG, GATA2, and HOXA9, and (b) culturing the PSCs under conditions such that the hematopoietic precursor programming genes are expressed, thereby producing HPCs.

Provided is a method for providing an enriched population of retinal pigment epithelial cells from a starting cell population including those derived from pluripotent stem cells by removing the cells positive for CD24, the cells positive for D56, and/or the cells positive for CD90.

Provided is a method for differentiating human pluripotent stem cells into multipotent nephron progenitor cells (NPCs) that can form nephron-like structures. The method enables generation of SIX2+SALL1+WT1+PAX2+ NPCs.

Provided is a method for inducing differentiation of monolayer cardiac cells from pluripotent stem cells, comprising steps of (i) culturing the pluripotent stem cells in a medium containing at least two GSK3 inhibitors and (ii) culturing the cells obtained in (i) in a medium containing one or more WNT signaling inhibitor(s).

Provided is a method for differentiating a human pluripotent stem cell into a cardiomyocyte cell comprising a step of contacting the human pluripotent stem cell with a small molecule compound XAV-939 and a second small molecule compound IWP-L6 or C59.

Provided is a non-invasive method for assessing quality of a pluripotent stem cell in culture comprising steps of assessing cell-free nucleic acids in the supernatant of the cell culture and determining the presence of at least one selected hyper-recurrent sequence in the nucleic acids.

Provided is a stem cell manufacturing system comprising a sending channel through which a solution containing cells flows, an apparatus which is connected to the sending channel and transfers a pluripotency inducer into the cells to produce cells harboring the inducer, and an apparatus which cultures the cells harboring the inducer to produce cell clusters consisting of stem cells.