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Provided is a method for producing mammalian insulin-secreting cells, comprising steps of (i) culturing mammalian stem cells which may be iPS cells in a first medium comprising an endoderm-inducing factor, thereby differentiating the cells into endoderm cells, and (ii) culturing the endoderm cells from (i) in a second medium comprising an endocrine-inducing factor, thereby differentiating the cells into endocrine cells; wherein the mammalian stem cells are not exposed to KGF prior to differentiation into endoderm cells.

Provided is a method for preparing human pluripotent stem cell (hPSC)-derived functional cardiomyocytes through cell differentiation and maturation, comprising steps of (i) culturing hPSC in a Matrigel-coated culture medium, (ii) inducing the differentiation of hPSC in step (i) to mesoderm in a mesodermal induction medium containing insulin-free B27 and a wnt signaling activator, (iii) differentiating the cells in step (ii) in a differentiation medium containing insulin-free B27, (iv) inducing myocardial differentiation of the cells in step (iii) in a myocardial differentiation inducing medium containing insulin-free B27 and a wnt signaling regulator, and (v) maturing the differentiated cardiomyocytes in step (iv) into functional cardiomyocytes by culturing in a myocardial maturation medium containing vitamin A-free B27, IL-15, and NGF.

Present invention provides a novel and efficient method for differentiating pluripotent stem cells into intestinal epithelial cells, comprising steps of (1) differentiating the pluripotent stem cells into intestinal stem cell-like cells and (2) differentiating the intestinal stem cell-like cells obtained in step (1) into intestinal epithelial cell-like cells by using an MEK1 inhibitor, a DNA methylation inhibitor, a TGFβ receptor inhibitor, EGF and a cAMP activator.

Provided is a method for megakaryocyte production, comprising steps of (i) expanding pluripotent stem cells under low adherent or non-adherent conditions and under agitation wherein expanded pluripotent stem cells form self-aggregating spheroids, (ii) differentiating the pluripotent cells in a first culture medium into hemogenic endothelial cells, and (iii) differentiating the hemogenic endothelial cells in a second culture medium into megakaryocytic progenitors.

Provided is a culture medium for inducing corneal endothelium replacement cells from iPS cells, which is characterized by containing insulin-like growth factor and a STAT3 activator, and not containing bFGF or a ROCK inhibitor. Also provided is a method for producing corneal endothelium replacement cells from iPS cells by using said culture medium.

Provided is a method for obtaining functional liver cells (or progenitor cells) or functional small-intestinal epithelial cells (or progenitor cells), comprising a step of culturing an isolated cell population including liver cells (or progenitor cells) or small-intestinal epithelial cells (or progenitor cells) which may be differentiated from iPS cells, in the presence of an antibiotic.

Described herein are methods and compositions related to use of cranium-specific iPSC-derived cells that are demonstrated as capable of revitalizing structural allografts. Specifically, coating cranial allografts with mesenchymal stem cells derived from iPSC-derived neural crest cells provides an effective therapeutic avenue for cranial defects.

Provided is a method for producing oligodendrocyte progenitor cells (OPCs) comprising (i) a step of introducing into pluripotent stem cells at least four transcription factors selected from (a) OLIG2, (b) SOX9, (c) NKX6.1, (d) NKX6.2 and (e) OCT4, and (ii) a step of culturing the pluripotent stem cells to produce a population of cells comprising OPCs.

Provided is a method for producing myotube from pluripotent stem cells efficiently, comprising (a) a step of differentiating pluripotent stem cells to myogenic progenitors, and (b) a step of terminally differentiating the myogenic progenitors from (a) into myotubes in the presence of at least one gamma secretase inhibitor.

Provided is a method for manufacturing an organ bud using (i) vascular cells, (ii) mesenchymal cells, and (iii) tissue or organ cells, wherein each of (i), (ii), and (iii) has been induced from human pluripotent stem cells. In a preferred embodiment, (iii) is a TBX3 and ADRA1B positive human iPSC-derived hepatocyte and the manufactured organ bud is a liver bud.

Provided is an in vitro method to increase the myogenic potential of an iPSC derived mesodermal progenitor cell by use of a miRNA or a mimic of miR-132, miR-424, and miR-146b, and an inhibitor of miR-34c-5p, an inhibitor of miR-34c-3p, and an inhibitor of miR-362.

Provided is a method for reducing the number of pluripotent stem cells in a cell population comprising pluripotent stem cells and differentiated cells (preferably cardiomyocytes) derived from the pluripotent stem cells comprising a step of contacting the cell population with a compound of the specified formulae. In a preferred embodiment, the compound is N, N'-di(2-hydroxybenzyl)-1,2-diaminocyclohexane.

Provided is a method for producing a cell mass including pituitary tissue, comprising steps of (1) suspension-culturing pluripotent stem cells in the presence of a Wnt inhibitor and forming an aggregate of cells, and (2) suspension-culturing the aggregate obtained in step (1) in the presence of a BMP agonist and a sonic hedgehog agonist and obtaining a cell mass including pituitary tissue.

Provided is a method for efficiently preparing a population of iPS cells, comprising a step of expressing one or more Yamanaka factors selected from Oct3/4, Sox2, Klf4, Myc, Nanog and Lin28, and reducing the amount and/or activity of Menin protein (a putative tumor suppressor coded by Men1 gene) in a population of target cells.

Provided is a method for preparing hepatocytes or cells that can be differentiated into hepatocytes from pluripotent stem cells, comprising steps of (1) culturing pluripotent stem cells in a medium containing an activin receptor-like kinase-4,7 activator, (2) culturing the cells obtained in (1) in a medium containing an osteogenic factor and FGF, (3) culturing the cells obtained in (2) in a medium containing a HGF receptor activator and an oncostatin M receptor activator, and (4) culturing the cells obtained in (3) and obtaining hepatocytes or cells that can be differentiated into hepatocytes, wherein at least one of steps (2), (3), and (4) involves culturing the cells on a high-density collagen gel membrane. The method is usuful for the purpose of obtaining mature hepatocytes that are similar in many respects to primary cultured hepatocytes.

Provided is a method for producing a genetically diverse regenerated T cell colony via iPS cells, comprising steps of (1) concentrating a T cell colony that has tropism toward a target tissue or antigen from a sampled genetically diverse T cell colony, (2) reprogramming the concentrated T cell colony in step (1) into iPS cells and culturing the iPS cells while maintaining genetic diversity, and (3) producing a genetically diverse regenerated T cell colony from the cultured iPS cells in step (2).

Provided is a spherical cell aggregate comprising a core section including a neural retina and a cover section continuously or non-continuously covering at least a portion of the surface of the core section, which is characterized in that (1) in the neural retina, a neural retina layer including at least a visual cell layer is formed, the visual cell layer includes at least one type of cells selected from the group consisting of visual cells, visual cell precursor cells, and retina precursor cells, and cells included in the visual cell layer are continuously present in a direction tangential to the surface of the core section, (2) the cover section includes retinal pigment epithelial cells that are in contact with each other, (3) the cell aggregate does not include the crystalline lens, the vitreous body, the cornea, or blood vessels, and (4) the retinal pigment epithelial cells and the neural retina layer do not together form a continuous epithelial structure; wherein the core section including neura and the retinal pigment epithelial cells of the cover section may independently be derived from pluripotent stem cells and brought in contact together to form the spherical cell aggregate of the present invention.

Provided is a method for making stem cell-derived cardiomyocytes, comprising a step of contacting stem cell derived cardiomyocytes with a nanopatterned substrate, thyroid hormone T3, and a Let7i microRNA, wherein a transition of the stem cell-derived cardiomyocytes from a fetal phenotype to a more mature phenotype that more closely resembles that of adult cardiomyocytes is promoted.

Provided is a method for inducing differentiation of iPSCs and/or ESCs (SCs) into neural progenitor cells (NPCs) comprising steps of (1) plating SCs on a coated, non-tissue treated, culturing vessel in medium, (2) transfecting the SCs with mRNA encoding one or more cell-fate factors (in a preferred embodiment, selected from the group consisting of Ngn2, Pax6, Sox2, Brn2 and FoxO family factors) as individual mRNA or combined mRNA, and (3) culturing said SCs of step (2) until the cells differentiate into NPCs. Further provided is a method for generating oligodendrocyte progenitor cells (OPCs) from NPCs comprising steps of (4) plating NPCs on coated cell culture vessels in medium, and (5) transfecting the cells of step (4) with mRNA encoding one or more cell-fate factors (in a preferred embodiment, selected from the group consisting of 01igo2, NKX2.2, SOX10, and Oligol) as individual mRNA or combined mRNA to differentiate NPCs into OPCs.

Provided is a method for producing induced alveolar epithelial type 2 cells (iAEC2s), comprising a step of contacting a KX2-1+ lung epithelial progenitor cell (e.g., those derived from an iPS cell) with an agonist of Wnt/beta-catenin signaling, a corticosteroid and an agonist of cyclic AMP or the cyclic AMP pathway.

Provided is a method for preserving neural tissue (e.g., those derived from pluripotent stem cells) for several hours to several weeks without freezing at a temperature of 8°C to 30°C, a preservative solution therefor, a method for transporting neural tissue by using said method and said preservative solution, and a transplant composition. In a preferred embodiment, the preservative solution has a potassium ion concentration of more than 0 mM and less than 115 mM.

Provided is a method for inducing differentiation of pluripotent stem cells into enterocyte-like cells, comprising steps of (1) differentiating pluripotent stem cells into endodermal cells, (2) culturing the endodermal cells obtained in (1) in a medium containing LY2090314 to differentiate them into intestinal progenitor cells, and (3) culturing the intestinal progenitor cells obtained in (2) in a medium containing Wnt3a and EGF (optionally further containing two or more factors selected from a group consisting of a p38 MAPK inhibitor, IGF-1, R-spondin, Noggin, and dexamethasone) to differentiate them into enterocyte-like cells.

Provided is a method for inducing peripheral sensory neuronal (PSN) cells from stem cells (e.g. iPS cells) comprising steps of (i) culturing the stem cells in a 3N medium with at least one SMAD inhibitor in a first culture vessel, (ii) culturing the primary differentiated cells obtained in (i) in a second culture vessel coated with polyornithine/laminin in a 3N medium supplemented with at least one mitogen, then in a 3N medium supplemented with at least one neurotrophic factor, at least one differentiation inductor and at least one cell transduction inductor, and (iii) culturing the secondary differentiated cells obtained in (ii) in a conditioned medium from human neonatal epidermal keratinocytes.

Provided is a method for removing undifferentiated pluripotent stem cells by using cardiac glycosides (CGs), of which chemical structures in the preferred embodiments are herein disclosed.

Provided is a method for predicting the differentiation potency of undifferentiated iPS cells into cartilage cells using indices of the increase and the decrease, respectively, in the expression levels of marker genes defined as genes that increase and those defined as genes that decrease in the undifferentiated iPS cells having said potency.

Provided is a chemically defined medium for eukaryotic cell culture, comprising water, at least one carbon source, one or more vitamins, one or more salts, one or more fatty acids, one or more buffer components, selenium, at least one substance of the group of Functional Inhibitors of Acid Sphingomyelinase (FIASMAs) and at least one polypeptide of TGF-β superfamily. The medium according to this invention enables continuous proliferation and serial passageability of human pluripotent stem cells.

Provided are a population of differentiated cells which produce insulin in response to glucose or GLP-1 obtained from ES cells or iPS cells, and a method for producing said cell population. The method is characterized by stepwise consecutive introduction into differentiating cells, accrding to specific differentiation stages, of three vectors comprising, respectively, nucleic acids encoding a PDX1 polypeptide, that encoding an NGN3 polypeptide, and that encoding a MAFA polypeptide.

Provided is a method for generating a microglia-like cell comprising steps of (i) differentiating an iPS cell (or, a hematopoietic stem cell or a CD34+ cell) into a monocyte, and (ii) differentiating the monocyte into a microglia-like cell, wherein the step (i) comprises contacting the cell with M-CSF, hSCF, Flt-3, hIL-3, or combinations thereof, and step (ii) comprises contacting the monocyte with GM-CSF and IL-34.

The present invention provides a method for efficiently and constantly producing CD4/CD8 double-positive cells (that may further be induced into CD8 single-positive cells) comprising steps of (1) separating from a cell population containing hematopoietic progenitor cells, cells that express one or more molecules selected from a group consisting of CD24、CD62L、CD90、CD143、CD263、Notch3、CD32、CD39、CD49a、CD164、CD317、CD200、CD218a、CD7、CD144、CD56、CD226、CD262, and CD325, and/or cells that do not express one or more molecules selected from a group consisting of CD49f、CD51、CD102、CD42b、CD61、CD62P、CD69、CD102, and CD156c, and (2) differentiating the cells obtained in (1) to CD4/CD8 double-positive cells.

Provided are a method for producing neural crest cells from pluripotent stem cells (e.g. iPS cells) comprising a step of culturing in a medium containing FGF2, retinoic acid and BMP4, and a method for producing sympathetic neurons from the neural crest cells comprising steps of culturing in a medium containing a cytokine and BMP4 to produce sympathetic neuron precursors, and then culturing in a medium containing a neurotrophic factor to thereby produce sympathetic neurons.