133 results about "Germ layer" patented technology

The invention belongs to the technical field of organoid culture, and particularly relates to a co-differentiated vascularized liver organoid and a construction method thereof. Aiming at the problems that the existing liver organ vascularization mostly adopts a co-culture mode, the operation process is complicated, the cell source heterogeneity is relatively high, the randomness is strong, the uniformity is poor and the like, the invention provides the construction method of the co-differentiated vascularized liver organ. The preparation method comprises the following steps: sequentially inducing and culturing directional endoderm cells, posterior preintestinal cells and hepatic endoderm cells, adding an S1 culture medium containing VEGF to induce and culture for 1-3 days, then adding an S2 culture medium containing VEGF, BMP4 and FGF2 to induce and culture for 3-5 days, and then adding an S3 culture medium containing VEGF to induce and culture for 14-20 days to obtain the co-differentiated vascularized liver organ. According to the method disclosed by the invention, the operation process is simplified, the experiment efficiency is improved, the liver organ which is rich in blood vessel network, high in maturity and of a blood vessel-bile duct structure is obtained, and a foundation is laid for large-scale production and clinical application.

The invention belongs to the technical field of organoid culture, and particularly relates to a co-differentiated vascularized islet organoid and a construction method thereof. Aiming at the problems that the existing pancreatic islet organ vascularization mostly adopts a co-culture mode, the operation process is long, the difficulty is high, the randomness is realized, the uniformity is poor and the like, the invention provides the construction method of the co-differentiated vascularized pancreatic islet organ. The method comprises the following steps of: sequentially inducing and culturing directional endoderm cells, original intestinal canal cells and posterior preintestinal cells, and adding a culture medium containing VEGF (vascular endothelial growth factor), BMP4 (bone morphogenetic protein 4) and FGF2 (fibroblast growth factor 2) for culturing in subsequent induced differentiation, so as to obtain the co-differentiated vascularized islet organ. Under the condition that exogenous endothelial cells are not added, the iPS cells are directionally differentiated to generate the vascularized islet organ containing beta, alpha and delta cells and vascular endothelial cells for the first time, the vascularized islet organ is more mature than non-vascularized islet organs and has a stronger insulin secretion function, and a new thought and an experimental basis are provided for realizing functional healing of diabetes mellitus.

The invention provides a construction method and application of a blood sinus networked liver organ. According to the method, pluripotent stem cells are co-differentiated into HAND1 < + > mesoderm and endoderm, a front intestine rear endoderm containing KDR < + > mesoderm progenitor cells is generated, a liver endoderm with CD34 < + > / CD31 < + > endothelial progenitor cells is generated, and the blood sinus networked liver organ is formed. The liver organ has a functional blood sinus endothelial network penetrating through the liver cluster, and has the characteristic functions of low-density lipoprotein uptake, human serum albumin removal, mediated secretion of blood coagulation factor 8 and the like. The method provides a new-generation highly bionic research tool for liver disease mechanism analysis, drug screening and regeneration repair.

Provided are: a method for producing pancreatic endoderm cells, which comprises a step for culturing pancreatic endoderm cells in a culture medium containing a ROCK inhibitor and KGF and / or EGF; and pancreatic endoderm cells produced by the method.

The present disclosure provides methods of producing a population of endodermal stem cells (EnSC), a population of EnSC-derived PP cells, a population of EnSC-derived EP cells, or regenerative pancreatic islet tissue (E-islets). The disclosure also relates to EnSC, EnSC-derived PP cells, EnSC-derived EP cells or regenerated pancreatic islet tissue obtained using the method, and therapeutic uses thereof.

The invention belongs to the technical field of organoid culture, and particularly relates to a pulmonary alveolar organoid with an endogenous vascular network and a construction method of the pulmonary alveolar organoid. Aiming at the problems that mesoderm and endoderm are difficult to induce at the same time, the operation process is long, the difficulty is high and the like when a vascularized alveolar organ is constructed by an existing method, the invention provides the construction method of the alveolar organ with the endogenous vascular network. The method comprises the following steps: simultaneously and directionally inducing and differentiating human induced pluripotent stem cells (hiPSC) into fixed endoderm and mesoderm, anterior intestine endoderm, abdominal side anterior intestine endoderm and lung progenitor cells by adopting a one-step method, and promoting endothelial cell proliferation in an endogenous induced differentiation process through a specific growth factor combination. A functional vascular network is formed; and vascularized alveolar organs are induced to be generated. According to the invention, a qi-blood barrier structure in which vascular endothelial cells and pulmonary epithelial cells are tightly connected is successfully reproduced, the gas exchange function of lung tissues can be simulated, and a new way is provided for construction of vascularized alveolar organs.

The invention belongs to the technical field of regenerative medicine, and particularly relates to a method for generating vascular endothelial cells by inducing pluripotent stem cells to differentiate in vitro. The method comprises the following steps: carrying out first culture on pluripotent stem cells in a differential medium I to obtain cells A; carrying out second culture on the cell A in a differential culture medium II, and differentiating and directionally forming an endoderm cell B; performing third culture on the endoderm cells B in a differential culture medium III to obtain differentiated cells C; carrying out fourth culture on the differentiated cells C in a differentiation culture medium IV, and differentiating to form differentiated cells D; carrying out fifth culture on the differentiated cells D in a differential culture medium V, and differentiating to obtain vascular endothelial cells; wherein the differential culture media I-V do not contain activin A, VEGF (vascular endothelial growth factor) and BMP4 (bone morphogenetic protein 4). The method can improve the differentiation efficiency of the IPS cells to the vascular endothelial cells, and has a wide prospect in the cell therapy industry.

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.

There is described herein a method of producing primitive streak cells for differentiation to definitive endoderm from a population of human pluripotent stem cells (hPSCs), the method comprising: differentiating a population of hPSCs to primitive streak cells in culture medium comprising Latrunculin A (LatA).

The invention provides a method for 3D differentiation of pluripotent stem cells into definitive endoderm cells by using a bioreactor, a differentiation medium and a differentiation factor A / B are also included, and the medium and the differentiation factor A / B comprise the following components: an MCDB131 medium, 2.44 mM glucose, 29.28 mM sodium bicarbonate, 2% of HSA, 1% of GlutaMAX, 250 [mu] M Vitamin C, 0.002% of ITS-X, 1% w / v of Polyethylene glycol (PEG), 1 mg / ml of Heparin Sodium salt, 100 ng / mL of Activin A and 3 [mu] M of CHIR99021. The method uses the bioreactor to differentiate the pluripotent stem cells into the stereotyped endoderm cells in a 3D suspension manner, is simple to operate and high in differentiation efficiency, provides technical support for scientific research and industrial large-scale production, and has a good application prospect.

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 a culture medium and a method for obtaining extraembryonic endoderm stem cells (XEN) from porcine blastocysts and embryonic stem cells (ESCs), and belongs to the technical field of biology. The invention develops a two-step induction method for directly converting pig ESCs into pig XEN without gene modification and two culture mediums 4FY and 2FYSC. The invention provides a high-quality model for exploring separation characteristics and intercellular communication of embryos and extraembryonic pedigree in early embryonic development of large animals, provides a good cell model for researching dynamic development of extraembryonic pedigree, and provides a high-quality material for regenerative medicine and agricultural breeding.

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.

The invention relates to the technical field of animal cell culture, in particular to a culture medium for improving in-vitro culture efficiency and quality of pig embryos and application of the culture medium. The invention provides a new application of taurine and a taurine-containing pig embryo in-vitro culture medium, the in-vitro culture medium can significantly improve the in-vitro culture efficiency of the pig embryo and the quality of a pig blastocyst, can significantly prolong the in-vitro culture time of the pig embryo to 35 days, can improve the pluripotent state of the pig embryo, and has a good application prospect. Differentiation of epiderm and hypoderm of a pig embryo can be promoted, and differentiation and development of an embryo pedigree as well as development of nerves and a heart can be improved. The taurine-containing pig embryo in-vitro culture medium provided by the invention is a novel efficient in-vitro culture medium, can promote high-quality development of a pig blastocyst, and can be used for later culture of the pig embryo. In conclusion, the invention provides the culture medium for in-vitro long-term culture of the pig embryo, and also develops a method for improving the development efficiency and the development quality of the in-vitro cultured pig embryo.

The invention discloses a method for efficiently and directionally inducing IPS to be differentiated into pancreatic beta cells. According to the method, through staged signal regulation and strict cell screening, the directionality and the overall efficiency of differentiation are remarkably improved. The whole process pays attention to cell quality control from the initial stage, and a stable foundation is laid for subsequent differentiation by screening iPS cells with uniform morphology and high expression of pluripotent markers. Progressive signal molecule regulation is adopted in each stage, and an accurate differentiation path is formed from Activin A gradient adjustment during endoderm induction, synergistic effect of FGF10 and retinoic acid in pancreatic precursor cell differentiation and Wnt signal inhibition during endocrine precursor cell induction, so that the non-specific differentiation tendency is effectively reduced. Meanwhile, cell enrichment steps of key nodes, such as SOX17 positive cell screening in an endoderm stage and Ngn3 magnetic bead sorting of endocrine precursor cells, further improve the purity of a target cell population and avoid interference of parenchyma cells on subsequent function maturation.

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.

The present disclosure relates to in vitro co-culture models and methods for generating such co-culture models. Co-culture models of this disclosure, and related methods, may include co- culturing or maintaining different cell types in a culture receptacle in a common culture medium. In a specific example, a co-culture model (and related methods) may include co- culturing or maintaining one, two, or more mesoderm lineage cell-types and at least one endoderm lineage cell-type in a culture receptacle in a common culture medium.

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.

The application belongs to the technical field of biology, and discloses a kit for inducing stem cells to differentiate into megakaryocytes and / or platelets and application thereof. The kit can induce stem cells to differentiate into megakaryocytes and / or platelets without introducing exogenous cells and exogenous genes. The kit can highly simulate the generation process of megakaryocytes and platelets in vivo, and can induce stem cells into mesendoderm cells, hematopoietic endothelial cells and megakaryocyte-erythroid progenitor cells in sequence. The megakaryocyte-erythroid progenitor cells further develop into megakaryocytes with a multiploid characteristic, and finally produce stem cell-derived platelets with similar functions to natural platelets. The stem cells have the potential for unlimited proliferation, and can be massively expanded in vitro, which provides an unlimited cell source for the in vitro preparation of platelets.

The present invention belongs to the field of organoid culture technology, and specifically relates to a co-differentiated vascularized pancreatic islet organoid and a method for constructing the same. In view of the problems that the existing pancreatic islet organoid vascularization mostly adopts the co-culture method, which has a long operation process, is difficult, and has randomness and poor uniformity, the present invention provides a method for constructing a co-differentiated vascularized pancreatic islet organoid, which adopts human induced pluripotent stem cells hiPSCs, and sequentially induces and cultures them into directed endoderm cells, primitive intestinal tube cells and posterior foregut cells. In the subsequent induced differentiation, a culture medium containing VEGF, BMP4 and FGF2 is added for culture to obtain co-differentiated vascularized pancreatic islet organoid. For the first time, the present invention directs the differentiation of iPS cells to produce vascularized pancreatic islet organoids containing β, α, δ cells and vascular endothelial cells without adding exogenous endothelial cells. It is more mature than non-vascularized pancreatic islet organoids and has a stronger insulin secretion function, providing new ideas and experimental basis for achieving functional cure of diabetes.

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 discloses a method for identifying a pluripotent state of a cattle epidermal layer, and achieves the technical effect of identifying the pluripotent state of a cattle embryo, especially the pluripotent state of the cattle epidermal layer. The invention specifically discloses gene markers for different pluripotent stages and different developmental stages, and the pluripotent and developmental stages of to-be-detected bovine embryonic cells or cell clusters can be rapidly identified by using the gene markers for different pluripotent stages and different developmental stages of bovine embryos.

Embodiments of the instant disclosure relate to novel compositions and methods for generating thymic cells. In some embodiments, thymic cells can be differentiated from pluripotent stem cells (PSC), 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, thymic cells generated by composition, systems and methods disclosed herein can be used to treat a health condition.

The present disclosure provides thymic cells, thymic organoids, and animal models comprising such thymic cells and thymic organoids. The present disclosure also provides methods for inducing, in vitro and in vivo, differentiation of pluripotent stem cells into definitive endoderm (DE), anterior and anterior intestinal endoderm (AFE), ventral pharyngeal endoderm (VPE), thymic epithelial progenitor (TEP) cells, thymic epithelial cells (TEC), and thymic organoid.

Methods and compositions for generating human pancreatic beta cells and progenitor cells thereof from human pluripotent stem cells using chemically defined media are disclosed. The method can preliminarily generate FOXA2 + HNFlb + dorsal anterior intestinal endoderm cells (DFECs), and according to the provided method, the cells can be further differentiated into human PTF1A + PDX1 + pancreatic progenitor cells (PPCs) and human INS + PDX + pancreatic beta cells (PBCs). The invention also provides a culture medium, an isolated cell population and a kit.

In a first embodiment, a method for producing fetal and / or neonatal hepatocyte-like cells (fnHLCs) is disclosed. The method includes producing a definitive endoderm cell population by a stage 1 process, producing a hepatoblast-like progenitor cell population from the definitive endoderm cell population by a stage 2 process, and producing a fetal and / or neonatal hepatocyte-like cell population from the hepatoblast-like progenitor cell population by a stage 3 process. In another embodiment a method for screening a target is disclosed. The method includes co-culturing and incubating the target with a fetal and / or neonatal hepatocyte-like cell (fnHLC), retrieving a sample of a fnHLC after the co-culturing and incubating and determining one or more characteristics of the fnHLC based on analysis of the sample of the fnHLC.

Human induced pluripotent stem cells (hiPSCs) possess tremendous potential for tissue regeneration and banking hiPSCs by cryopreservation for their ready availability is crucial to their widespread use. However, contemporary methods for hiPSC cryopreservation are associated with both limited cell survival and high concentration of toxic cryoprotectants and / or serum. The latter may cause spontaneous differentiation and introduce xenogeneic factors, which may compromise the quality of hiPSCs. Here, sand from nature is discovered to be capable of seeding ice above −10° C., which enables cryopreservation of hiPSCs with no serum, minimized cryoprotectant, and high cell survival. Furthermore, the cryopreserved hiPSCs retain high pluripotency and functions judged by the pluripotency marker expression, cell cycle analysis, and capability of differentiation into the three germ layers. This unique sand-mediated cryopreservation method may greatly facilitate the convenient and ready availability of high-quality hiPSCs and probably many other types of cells / tissues for the emerging cell-based translational medicine.