47 results about "Germ layer" patented technology

Described are small molecule cocktails that enable de novo derivation and long-term culture of bovine extraembryonic endoderm cells (bXENs). Methods of using the small molecule cocktails to generate bXENs are also described. Methods of using the bXENs to form blastocyst models or to maintain the stemness of bovine ESCs and prevent them from differentiation are also described.

A novel method of inducing or producing hepatocytes from human induced pluripotent stem cells at an unprecedented efficiency and functionality. The core of the invention is the use of experimentally discovered mRNAs at multiple critical differentiation decision points along a pluripotent to mesendoderm to endoderm to hepatocytes pathway in a previously unknown manner.

The invention relates to the technical field of biology, in particular to a method for efficiently obtaining human definitive endoderm cells in vitro, which comprises the following steps: S1, inoculating human pluripotent stem cells into matrigel, and performing maintenance culture for 16-24 hours by using a stem cell maintenance culture medium; s2, from Day-2 to Day-1, absorbing and abandoning the stem cell maintenance culture medium, adding a pretreatment culture medium for culture, and changing the pretreatment culture medium once every day; the pretreatment culture medium is a stem cell maintenance culture medium added with dimethyl sulfoxide; s3, carrying out Day0 to Day2, absorbing and abandoning the pretreatment culture medium, adding a definitive endoderm differential culture medium, carrying out induced culture, and changing the definitive endoderm differential culture medium once a day; the definitive endoderm differential culture medium is an RPMI1640 culture medium added with fetal calf serum and activin A; s4, Day3, obtaining the required human definitive endoderm cells, and solving the problems that the existing definitive endoderm differentiation efficiency and cell population purity are limited and are influenced by batch-to-batch variation.

The invention relates to the technical field of construction of conditional gene knockout models, and discloses a construction method of a mouse Oct4 conditional gene knockout model based on quadruple sgRNA collaborative targeting, which comprises the following steps: step 1, designing four sgRNAs, namely sgRNA1, sgRNA2, sgRNA3 and sgRNA4, in which the sgRNA1 and the sgRNA2 target 5'end and 3 'end non-coding regions of a second exon of an Oct4 gene, and the sgRNA3 and the sgRNA4 target structural domain conserved sequences; step 2, preparing an electrotransfection solution, wherein the electrotransfection solution is composed of Cas9mRNA, the four sgRNAs and an electrotransfection buffer solution; step 3, carrying out electroporation treatment on the fertilized eggs of the mice; step 4, culturing the fertilized eggs subjected to the electrotransformation treatment to a blastocyst stage; and 5, carrying out genotype identification on the cultured blastocyst. According to the scheme, the sgRNA combination and embryo electrotransfection system is optimized, the mouse Oct4 conditional gene knockout model is efficiently and accurately constructed, a reliable tool is provided for regulation mechanism research of Oct4 in mouse early embryo trigerm differentiation, and the medical transformation value of Oct4 is excavated.

Tissue-specific progenitor cells representing the endoderm, ectoderm, and mesoderm lineages are generated from pluripotent sources. Progenitor cell generation is accomplished by contacting pluripotent stem cells with tissue-specific exosomes, microRNAs, proteins, and peptides, obtained by stressing the tissue under in vitro conditions. In vitro-generated tissue organoids are used as a source of "differentiation factors" for generating individualized progenitor cells. For example, to generate lung progenitor cells, allogeneic stem cells are implanted into decellularized cadaveric lung tissue to form lung structures, which are then exposed to various cellular stressors and conditioned media to differentiate the pluripotent stem cells into progenitor cells. Extracellular matrix can be obtained from decellularized structures seeded with regenerating cells and used to generate organoids, which are then subjected to stress to produce differentiation factors.

Provided is an embryo model, which is obtained by means of self-assembly of induced pluripotent stem cells and induced hypoblast stem cells, wherein both the induced pluripotent stem cells and the induced hypoblast stem cells are obtained by means of somatic cell reprogramming. The self-assembly of the stem cells of two different lineages generated by means of somatic cell reprogramming forms an embryo model simulating human peri-gastrulation. The embryo model better reproduces the interaction and communication mechanism among cells of multiple lineages during human embryonic development by means of combining cells of different lineages. With the embryo model, by means of a gene editing tool, lineage tracing and gene regulatory network research can be performed on cells of different lineages, thereby enabling an in-depth understanding of the molecular regulatory mechanisms of early human embryonic development.

A method and system are disclosed for differentiating non-human primate induced pluripotent stem cells (NHP iPSCs) into hepatocyte-like cells through a defined, multi-stage protocol. The method includes sequentially exposing NHP iPSCs to six distinct cell differentiation media over an approximate eighteen-day period. Each medium comprises defined combinations of growth factors, small molecules, and inhibitors that direct progressive lineage specification through anterior primitive streak, definitive endoderm, posterior foregut, liver bud progenitors, hepatoblasts, and hepatocyte-like cells. Concentration ranges and exposure durations are provided for each stage, ensuring reproducibility and reducing heterogeneity across cell populations. In some embodiments, treatment with a transforming growth factor beta (TGF-β ) receptor I inhibitor during early hepatoblast specification enhances lineage fidelity. The resulting hepatocyte-like cells are validated by immunostaining and western blotting for hepatic markers. Applications include preclinical drug metabolism, hepatotoxicity assessment, and in vitro disease modeling using non-human primate–derived systems.

The present application relates to the technical field of biology, and particularly discloses a method for culturing and obtaining foregut endoderm cell spheroids and application thereof. The method for culturing and obtaining foregut endoderm cell spheroids of the present application obtains foregut endoderm cell spheroids by inducing differentiation of stem cells on a culture surface with a PDA-matrigel coating. The culture method of the present application can ensure the differentiation efficiency of DE and AFE adherent cells and spheroids, promote the generation of AFE cell spheroids and the separation from the adherent cell layer, reduce the edge rolling of AFE adherent cells, prolong the production time of AFE cell spheroids, significantly improve the yield of AFE cell spheroids, and provide a basis for subsequent differentiation and culture of lung organoids.

The invention relates to methods of pluripotent stem cell (PSC) culture, PSC cultured in 3D suspension culture bioreactor systems, expansion of PSC in 3D suspension, and differentiation of 3D expanded PSC clusters into Definitive Endoderm (DE).

The invention provides a construction method of a thyroid organoid derived from human induced pluripotent stem cells, and belongs to the technical field of organoid culture. The construction method of the thyroid organ comprises the following steps: (1) separating human induced pluripotent stem cells into single cells, and transferring the single cells to a 2D matrigel working solution for incubation; (2) carrying out endoderm induced culture on the cells incubated in the step (1); (3) carrying out preintestinal endoderm induced culture on the cells obtained in the step (2); (4) carrying out thyroid gland directional induction culture on the cells obtained in the step (3); (5) carrying out thyroid maturation induction culture on the cells obtained in the step (4) to obtain thyroid organs; naI with the final concentration of 0.8-1.2 [mu] M is added during thyroid maturation induction culture. The thyroid organoid constructed by the method is high in maturity, can simulate the state of the in-vivo organoid more truly, and has good capability of secreting thyroid-related hormones at the same time.

While studies have been made on a method of selectively inducing differentiation from pluripotent stem cells into enterocyte-like cells, provided is an excellent and high-functionality enterocyte-like cell population that can be stably tested for multi-drug metabolism and permeability, and also provided an efficient method of producing cells. The method of inducing differentiation from pluripotent stem cells into enterocyte-like cells includes the following steps (1) and (2): (1) a step of inducing differentiation from pluripotent stem cells into definitive endoderm cells; and (2) a step of culturing the definitive endoderm cells in a system containing CHIR99021, followed by induction of differentiation into intestinal progenitor cells. A cell population of enterocyte-like cells is more effectively obtained by seeding and culturing enterocyte-like cells produced by the method of inducing differentiation, in a base material for organoid production or a base material for two-dimensional culture.

Provided herein are compositions of anorectal transition zone stem cells, to treat endodermal, ectodermal or mesodermal disease. Also provided are cellular compositions of the anorectal transition zone stem cells, in a pharmaceutically acceptable carrier.

The present invention relates to: a method for differentiating stem cell-derived hepatocytes by using fasudil and gelatin; and the like. The method for differentiating stem cell-derived hepatocytes, according to the present invention, induces, by means of fasudil, the differentiation of stem cells into endoderm in an early stage of differentiation of stem cells into hepatocytes, inhibits lipid droplet formation through high-viscosity gelatin coating of a culture container, activates mitochondrial functions, up-regulates the expression of endoderm genes, and down-regulates the expression of ectoderm and mesoderm genes, thereby improving the efficiency of differentiation of stem cells into hepatoblasts. In addition, low-viscosity gelatin is added to the medium in a differentiation and maturation (late) stage so as to suppress the accumulation of lipid droplets, mitochondria are activated to improve the efficiency of differentiation of hepatoblasts into hepatocytes, and the function of differentiated hepatocytes is enhanced to improve the efficiency of differentiation of stem cells into hepatocytes. Therefore, hepatocytes obtained using the method are expected to be effectively used as a cell therapeutic agent, a transplant, and the like for treating liver diseases.

This invention discloses the application of the Src kinase inhibitor PP2 in improving the differentiation efficiency of pluripotent stem cells into germ cells, belonging to the field of bioengineering technology. The specific culture method is as follows: Human embryonic stem cells are prepared into a single-cell suspension and seeded into a cell culture plate; the cell culture plate is placed in a cell culture incubator for culture, and then the culture medium is replaced with the first-stage differentiation medium for inducing human embryonic stem cells to differentiate into epiblast cells for continued culture; the cell culture medium for epiblast cells is aspirated and replaced with the second-stage differentiation medium for inducing epiblast cells to differentiate into primordial germ cell-like cells, and the plate is placed in a cell culture incubator for culture. Then, the Src kinase inhibitor PP2 is added to the culture medium at a final concentration ranging from 2.5 to 10 μM, and culture continues. This invention significantly improves differentiation efficiency and provides more optimized experimental conditions for exploring the developmental mechanism of germ cells through a primordial germ cell-like cell induction system.

The present invention relates to an in vitro human embryo organoid at a post-gastrulation stage of development. Methods of forming the organoid and derivatives therefrom are described as well as their uses. The in vitro human embryo organoid at a post-gastrulation stage of development demonstrates early human development that recapitulates the formation of three germ layers, and hPGCLCs without exogenous BMP signalling.

The present invention belongs to the technical field of stem cells. Provided are a culture medium for induced hypoblast stem cells and the use thereof. Provided is a culture medium for induced hypoblast stem cells. The components of the culture medium comprise platelet-derived growth factor AA, a leukemia inhibitory factor, fibroblast growth factor 4, GSK-3α / β inhibitor CHIR99021, TGF-β type I receptor inhibitor A83-01, and bone morphogenetic protein 4. The culture medium for induced hypoblast stem cells can accurately regulate the activity of key signaling pathways such as NODAL, BMP, WNT, FGF, and JAK / STAT in the process of somatic cell reprogramming, and ensure that the properties of hypoblast stem cells are maintained in the process of somatic cell reprogramming to support the self-renewal and hypoblast lineage induction of somatic cells in the reprogramming process, thereby realizing the fate transition of somatic cells to hypoblast stem cells.

This disclosure provides thymocytes, thymic organoids, and animal models comprising such thymocytes and thymic organoids. It also provides methods for inducing the differentiation of pluripotent stem cells into endoderm (DE), anterior foregut endoderm (AFE), ventral pharyngeal endoderm (VPE), thymic epithelial precursor (TEP) cells, thymic epithelial cells (TEC), and thymic organoids in vitro and in vivo.

Disclosed is an in-vitro protocol for differentiating human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESC) to give rise to a definitive endoderm, followed by progression into anteriorized foregut endoderm that has the ability to give rise to both proximal and distal lung epithelial cells. The protocol not only offers great opportunities for the study of human development but also have tremendous potential for future clinical cell-based therapies. The protocol outlined here is used to differentiate hiPSCs into lung epithelial cell types through a process that faithfully recapitulates the stepwise events observed in-vivo. The was followed with the working cell bank of an hiPSC line made under current Good Manufacturing Practice (cGMP) conditions, a necessary step for the future clinical application of these cells.

Embodiments of the present disclosure relate to novel compositions and methods for generating thymocytes. In some embodiments, thymocytes can be differentiated from pluripotent stem cells (PSCs), anterior primitive streak (APS) cells, definitive endoderm (DE) cells, anterior foregut endoderm (AFE) cells, pharyngeal endoderm (PE) cells, ventral pharyngeal endoderm (VPE) cells, and third pharyngeal pouch endoderm (TPPE) cells using the compositions and methods disclosed herein. In certain embodiments, thymocytes generated by the compositions, systems, and methods disclosed herein can be used to treat health conditions.

Disclosed is an in-vitro protocol for differentiating human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESC) to give rise to a definitive endoderm, followed by progression into anteriorized foregut endoderm that has the ability to give rise to both proximal and distal lung epithelial cells. The protocol not only offers great opportunities for the study of human development but also have tremendous potential for future clinical cell-based therapies. The protocol outlined here is used to differentiate hiPSCs into lung epithelial cell types through a process that faithfully recapitulates the stepwise events observed in-vivo. The was followed with the working cell bank of an hiPSC line made under current Good Manufacturing Practice (cGMP) conditions, a necessary step for the future clinical application of these cells.

Disclosed herein are methods for utilizing and forming suspension cultures of pluripotent stem cells (PSCs), and differentiated cells, spheroids, and organoids derived from PSCs (e.g., with industrial efficiency and / or scalability), as well as compositions comprising them. These methods can be carried out in suspension culture without the use of a basement membrane matrix during the maintenance and expansion of PSCs, and during the differentiation of PSCs into differentiated cells and organoids, such as endoderm (DE), hindgut spheroids (HGS), and intestinal organoids (IO). In some embodiments, the methods may be xenofree and may be carried out in accordance with Good Manufacturing Practices (GMP). Also disclosed herein are methods for controlling the polarity of epithelial cells in IOs, such that the apical layer is oriented outside the organoid, or alternatively inside the organoid. The use of methods and compositions for transplantation and therapeutic purposes is also disclosed.

The invention relates to the use of a laminin (LN) matrix for hepatic differentiation. The invention also relates to a method for inducing hepatic differentiation comprising the steps of: (i) providing a population of human totipotent cells, (ii) culturing the population on a laminin-coated support in an endoderm stimulation medium to produce a population of human DE cells, (iii) culturing said population of human DE cells on a laminin-coated support in a hepatic stimulation medium to produce a population of human hepatoblast-like cells, and (iv) optionally culturing said population of human hepatoblast-like cells on a laminin-coated support in a hepatic maturation medium to produce a population of human hepatocyte-like cells.The invention further relates to a population of human hepatoblast-type cells or human embryonic hepatocyte-type cells obtained by the method of the invention. The invention further relates to a population of human hepatoblast-type cells expressing HNF4 α and expressing substantially AFP for use in a method of treating the human body.

Provided are a stem cell-derived gastruloid model, method for constructing same, and use thereof. The gastruloid model is constructed in vitro from human pluripotent stem cells, and simulated the biological events of early embryonic development and the key structures of the embryo to a certain extent, such as the development of primitive endoderm, the establishment of bilaminar germ disc, the appearance of the amniotic cavity and amniotic cells, and the appearance of the primitive streak and the like, which has been verified at both the protein level and the transcriptome level, and the key features of embryos from the peri-implantation period to the gastrula period could be well reproduced. This model can be induced in batches as a drug screening model for early embryos and environmental toxicants and the like, thereby providing safety testing for the use of drugs for certain clinical patients in early pregnancy and so on.

This invention discloses a method for inducing blastocysts to form pluripotent embryonic stem cells, thereby constructing a new pluripotent embryonic stem cell line with highly efficient chimeric fetal capacity, which can completely replace the function of epiblast cells in blastocysts. This provides a new model for studying the epigenetic mechanism and transcriptional status of embryonic stem cell pluripotency, and has great application potential for studying mammalian development and generating mouse models.

The present invention relates to methods of efficiently generating pancreatic endoderm from human pluripotent stem (PS) cell derived human definitive endoderm. The present invention also relates to pancreatic endoderm cells obtained by the methods of the invention. Finally, the present invention relates to culture medium and composition comprising a RAR antagonist and uses of said RAR antagonist in the induction of pancreatic endoderm cells. The present invention provides a more homogenous and synchronised pancreatic cell population, with increased efficiency.

The invention discloses a method for preparing intestinal organs from human induced pluripotent stem cells, and belongs to the technical field of biomedicine. A related culture differentiation system is optimized, stem cells are efficiently induced to be sequentially differentiated into endoderm cells and midgut and posterior gut cells, and finally intestinal organs with a complete intestinal tissue structure are formed. The method comprises the following key steps: amplifying stem cells by using a special culture medium, taking 3-5 generations of cells, and carrying out synergistic induction on the cells by Activin A and Wnt3a to differentiate into an endoderm; fGF-4, CHIR99021 and Y-27632 are used for promoting the formation of middle and posterior intestine spheres and the maturation of precursor cells; and finally, forming the organoid in a Matrigel three-dimensional scaffold and a compound factor culture medium of EGF (Epidermal Growth Factor), Noggin, RSPO1, N2 and B27. The method can be used for intestinal development mechanism research, disease model construction, drug screening and regenerative medicine, and particularly can be used for establishing a model by utilizing the specific stem cells of the patient in personalized medicine, so that precise drug testing and treatment scheme optimization are realized.

The present invention relates to a method for accelerated and cost-effective generation of insulin-producing beta cells from pluripotent stem cells using a fully defined, small molecule-based differentiation protocol. Further, the present invention relates to a method for obtaining pancreatic endoderm cells from posterior foregut cells. The use of the cells obtained with the method according to the invention is described as well.

The present invention provides: a method for producing pancreatic endoderm cells, the method comprising a step of culturing pancreatic endoderm cells in a culture medium containing a ROCK inhibitor, and KGF and / or EGF; and pancreatic endoderm cells produced by said method.