Provided herein are systems and methods for identifying cell-specific differentiation markers. In particular, provided herein are systems and methods for generating induced pluripotent cells (IPS) from human cells, differentiating the IPS into differentiated cells, and identifying differentiation specific markers.

A method for inducing differentiation into pancreatic α cells includes: a step (a) of culturing endodermal cells, which have been induced to differentiate from pluripotent stem cells, in the presence of a bone morphogenetic protein (BMP) signaling inhibitor, and retinoic acid or a retinoic acid analog to induce differentiation into primitive gut tube (PGT) cells; a step (b) of culturing the primitive gut tube (PGT) cells to induce differentiation into pancreatic endocrine precursor (EP) cells; and a step (c) of culturing the pancreatic endocrine precursor (EP) cells to induce differentiation into pancreatic α cells, in which the step (b) and the step (c) are performed in the absence of ascorbic acid.

The present invention provides a technique that serves as a platform for inducing human organ cells at a low cost, stably and in a large quantity. A cell inducible after differentiating pluripotent stem cells and then passaging the resultant cells at least once or more times, which is negative for undifferentiated (pluripotent) cell markers NANOG, OCT4, MYC and LIN28A, negative for endoderm cell markers CXCR4, CER1, HHEX and GATA4, positive for intestinal endoderm cell markers CDX2 and HOXB9, negative for a mesenchymal cell marker brachyury (T), negative for a pancreatic cell marker PDX1, and capable of differentiating into at least a hepatocyte, a pancreatic cell and an intestinal cell. Also provided are methods of preparing and amplifying the above cells; a method of preparing organ cells using the above cells; and a method of constructing a working cell bank for preparing organ cells, comprising cryopreserving the above cells.

Alveolar-like macrophages and a method for generating alveolar-like macrophages from hemangioblasts is provided. The method comprises the steps of: i) culturing the hemangioblasts in a hematopoietic-inducing medium comprising vascular endothelial growth factor (VEGF), stem cell factor (SCF) and interleukin-3 (IL-3) for a sufficient period of time to generate macrophages, and ii) culturing the macrophages in an alveolar macrophage-inducing medium comprising granulocyte macrophage colony stimulating factor (GM-CSF), and optionally macrophage colony stimulating factor (M-CSF), under suitable conditions and for a sufficient period of time to yield alveolar-like macrophages.

A method for making an at least double layered cell aggregate and/or an artificial blastocyst, and/or a further-developed blastoid termed blastoid, by forming a double layered cell aggregate from at least one trophoblast cell and at least one pluripotent and/or totipotent cell, and culturing the aggregate to obtain an artificial blastocyst having a trophectoderm-like tissue that surrounds a blastocoel and an inner cell mass-like tissue. The cell aggregate can be formed from toti- or pluripotent stem cell types, or induced pluripotent stem cell types, in combination with trophoblast stem cells. Formation of a blastoid can be achieved by culturing the cell aggregate in a medium preferably comprising one or more of a Rho/ROCK inhibitor, a Wnt pathway modulator, a PKA pathway modulator, a PKC pathway modulator, a MAPK pathway modulator, a STAT pathway modulator, an Akt pathway modulator, a Tgf pathway modulator and a Hippo pathway modulator. Also, a method for growing an at least double layered cell aggregate into an artificial blastocyst, and into a further-developed blastoid, a foetus or a live animal and an in vitro cell culture comprising the mentioned compounds and/or cell aggregates.

A human immature endocrine cell population and methods for making an immature endocrine cell population are provided. Specifically, immature beta cells and methods for production of immature beta cells are described. Immature beta cells co-express INS and NKX6.1 and are uni-potent and thereby develop into mature beta cells when implanted in vivo. The mature beta cells in vivo are capable of producing insulin in response to glucose stimulation.

The invention relates to the use of CD34 as a cell surface marker to detect sinoatrial node-like pacemaker cells (SANLPCs) in a population of cells and to generate cell preparations highly enriched for SANLPCs. Also provides herein are methods of using SANLPC-enriched cell preparations for cardiac cell therapy.

One or more type 7 long terminal repeat (LTR7) nucleic acid sequences of type H human endogenous retroviruses (HERVH) (“LTR7/HERVH nucleic acid sequences”) can be used for identifying primate naïve pluripotent stem cells. LTR7/HERVH-associated transcription can be used as a marker for primate naive pluripotent stem cells. A reporter construct that includes LTR7/HERVH nucleic acid sequences can be used for optimizing culture conditions for naïve primate pluripotent stem cells. A cell growth medium can be used for cultivation of primate naive pluripotent stem cells, which can exhibit elevated levels of LTR7/HERVH-associated transcription in comparison to control cells.

Disclosed herein are cell cultures comprising definitive endoderm cells and methods of producing the same. Also disclosed herein are cell populations comprising substantially purified definitive endoderm cells as well as methods for enriching, isolating and purifying definitive endoderm cells from other cell types.

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 TGFβ 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.