506 results about "Human Induced Pluripotent Stem Cells" patented technology

The invention discloses application and a preparation method of exosome EGF-FGF-EXO containing double growth factors, the exosome EGF-FGF-EXO is uniform in particle size, high in loading efficiency and good in biological activity, the double growth factors comprise an epidermal growth factor EGF and a fibroblast growth factor FGF, and the epidermal growth factor EGF and the fibroblast growth factor FGF are added into the exosome EGF-FGF. The preparation method of the exosome EGF-FGF-EXO comprises preparation of an induced pluripotent stem cell (iPSC) exosome and loading of EGF-FGF, a recombinant epidermal growth factor (EGF) and a fibroblast growth factor (FGF) are loaded on the exosome from iPS-clone 12c, and the exosome product which has high biological activity and is used for treating nervous system diseases of neuroinflammation is prepared. The invention develops a brand new technical path for treatment of nervous system diseases, and in the fields of epilepsy, neurodegenerative diseases, brain injury and the like, the life quality of patients can be improved and important theoretical basis and technical support can be provided for precise medical treatment of nervous system diseases by precisely regulating and controlling neuroinflammation.

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 discloses a method for differentiating induced pluripotent stem cells into macrophages. The method comprises the following steps: S1, inducing pluripotent stem cells to form embryoid bodies; s2, differentiating the embryoid body into a monocyte with CD14 expression; s3, the mononuclear cells are differentiated into macrophages with CD14 / CD11b expression; and S4, polarizing the macrophages into M1 type macrophages and / or M2 type macrophages. According to the method for differentiating the induced pluripotent stem cells into the macrophages, the cells can be effectively induced to differentiate towards the macrophages, the induced pluripotent stem cells are subjected to directional differentiation culture in a short time to obtain the macrophages, and the generated macrophages express CD14 and CD11b and have remarkable phagocytic activity on yeast glycan. The method is of great significance to large-scale production of macrophages.

The invention relates to a method of modulating the expression of an endogenous gene in a somatic cell derived from an induced pluripotent stem cell (iPSC), comprising differentiating an engineered iPSC capable of stable or inducible expression of a CRISPR-related protein into a somatic cell prior to introduction of a guide RNA (gRNA) sequence. The invention also relates to the engineered iPSC, cells obtained by the method and uses thereof.

The invention relates to the technical field of image processing, in particular to an induced pluripotent stem cell density detection method, computer equipment and a medium, and the method comprises the following steps: acquiring an induced pluripotent stem cell image; presetting a local area of each pixel point; obtaining a local brightness performance value according to the gray level distribution of the pixel points in the local area; obtaining local boundary ambiguity according to the gradient distribution characteristics; obtaining local form regularity according to the uniformity characteristic of gray level distribution in a pixel point local area; obtaining a density comprehensive performance value of each pixel point; clustering the pixel points to obtain a preset number of clusters; and respectively enhancing each cluster by adopting histogram equalization to obtain an enhanced induced pluripotent stem cell image, and carrying out density detection on the enhanced induced pluripotent stem cell image. The invention aims to enhance the induced pluripotent stem cell image and improve the accuracy of the cell density detection result.

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 composition for beautifying, which comprises a blood platelet concentrated solution prepared by differentiation of human pluripotent induced stem cells (iPSC), and an additive for preparing a blood platelet suspension or gel, and is characterized in that the composition for beautifying comprises the following components: a blood platelet concentrated solution prepared by differentiation of human pluripotent stem cells (iPSC), and an additive for preparing the blood platelet suspension or gel. The invention aims to provide a functional platelet-rich composition which can be prepared in a large scale, is stable in batch and can be produced in a standardized manner in the aspect of promoting wound healing. According to the composition, the platelet concentrate and platelet-rich plasma (PRP) separated from blood in the composition are completely different in component, the component is stable, and the slow-release growth factor is beneficial to tissue repair.

The invention belongs to the field of stem cell biology, and relates to pedigree specific differentiation of human pluripotent or embryonic stem cells, in particular to a mesenchymal stem cell induced differentiation culture medium and application thereof. The mesenchymal stem cell induced differentiation culture medium is used for directly inducing and differentiating human induced pluripotent stem cells / or embryonic stem cells into mesenchymal stem cells, the culture medium comprises a mesenchymal stem cell basic culture medium and an additive, and the additive comprises the following components: 2-10 [mu] M of a TGF-beta inhibitor, 2-10 [mu] M of a GSK3 beta inhibitor and 5-10 [mu] M of a Rock inhibitor. By adopting the induced differentiation culture medium provided by the invention, human induced pluripotent stem cells / or embryonic stem cells can be directly induced and differentiated into mesenchymal stem cells, and the obtained mesenchymal stem cells have the advantages of infinite sources, stable quality, high homogeneity and the like; and the human induced pluripotent stem cells / or embryonic stem cells can be directly and directionally differentiated into the mesenchymal stem cells in a relatively short time, so that the culture time is shortened, and the production cost is reduced.

The invention discloses a method for differentiating induced pluripotent stem cells into megakaryocytes and platelets, and relates to the technical field of induced pluripotent stem cell directional differentiation, and the method comprises the following steps: inoculating an iPSC single cell into a culture vessel, and carrying out adherent culture through a culture solution; after the cell adheres to the wall and before cloning is not formed, the liquid is changed into a first induction culture liquid, and the cell is induced and differentiated into a mesoderm cell; changing the solution into a second induction culture solution to induce and differentiate the mesoderm cells into hematopoietic endothelial cells; collecting the suspension cells, culturing the suspension cells in an amplification culture solution, and enriching megakaryocytes; inducing megakaryocyte polyploidy through a third induction culture solution; and culturing the polyploidy megakaryocyte through a fourth induction culture solution to promote platelet release. The method for inducing the pluripotent stem cells to differentiate into megakaryocytes and platelets is convenient and rapid, the megakaryocytes and platelets can be obtained through iPSC in a short time, and feeder cells, serum and additional gene editing are not needed; meanwhile, the yield is high.

The present invention discloses a method for enhancing cardiomyocyte production from induced pluripotent stem cells (iPSCs) by adjusting the culture medium exchange process during the transition from WNT activation to inhibition for differentiation. Instead of completely replacing the culture medium, this innovative technique involves partially changing the medium, which allows for a controlled exposure to WNT activators concurrent with the introduction of WNT inhibitors. This key modification in the protocol markedly increases the consistency and efficiency of cardiomyocyte generation, reducing variability between batches. By fine-tuning the medium exchange process, this method streamlines the creation of cardiomyocytes for use in regenerative medicine, emphasizing the critical adjustment phase within the culture conditions.

The invention discloses a fluorescent quantitative PCR (polymerase chain reaction) detection method and a detection kit for detecting differentiation potential of induced pluripotent stem cells. The detection method comprises the following steps: S1, preparing an embryoid body by utilizing iPSC (induced pluripotent stem cells); s2, extracting RNA (Ribonucleic Acid) of the iPSC and the embryoid body; s3, respectively carrying out reverse transcription on the iPSC and RNA of an embryoid body to obtain cDNA; s4, establishing a fluorescent quantitative PCR reaction system by adopting the primer composition in claim 1; s5, carrying out fluorescent quantitative PCR (Polymerase Chain Reaction) detection on the cDNA in S3; and S6, analyzing a result. The detection kit disclosed by the invention can be used for rapidly and accurately evaluating the differentiation potential of the induced pluripotent stem cells, and is suitable for large-scale cell differentiation research and clinical application.

Aspects of the present disclosure provide engineered induced pluripotent stem cell (iPSC)-derived mesenchymal stromal cells (EiMSCs) that exhibit persistent transgene expression. Persistent transgene expression may be achieved through use of an engineered chromatin opening sequence (ECOS). The engineered EiMSCs of the present disclosure can comprise a therapeutic polypeptide comprising one or more cytokines for the treatment of a metabolic disease, a proliferative disease, an autoimmune disease, a neurological disease, or any combination thereof.

The invention discloses a biconvex crystalline lens organ relying on stiffness-controllable hydrogel external directional induction as well as a construction method and application thereof. A crystalline lens organ model provided by the invention is jointly realized by virtue of stiffness-controllable hydrogel and a composite growth factor; and the pluripotent stem cells are rapidly and directionally induced into crystalline lens organs with high quality in vitro. According to the technical scheme, the method comprises the following steps: (1) directionally inducing and differentiating the pluripotent stem cells into primary ectoderm cell clusters; (2) preparing hydrogel with specific rigidity and carrying out surface cell adhesion treatment; (3) planting the primary ectoderm cells on the cleaned polyacrylamide hydrogel and inducing the primary ectoderm cells into a neural ectoderm cell cluster; and (4) inducing the neural ectoderm cells to directionally differentiate into mature crystalline lens organs. The three-dimensional lens organ-like model obtained by the method has a sphere-like biconvex morphology with a top surface and a bottom surface, is transparent and has structures such as capsule membranes, lens epithelial cells and lens fibrocytes, and the bottom surface is also wrapped by a complete lens capsule membrane. Compared with the prior art, the method realizes the formation of a complete capsule film on the bottom surface and a sphere-like form. The obtained crystalline lens organ can be used for research on crystalline lens embryonic development, research on cataract pathogenesis and research, development and screening of cataract drugs.

Materials, methods, and systems are provided for cell redifferentiation and expansion of induced pluripotent stem cell (iPSC)-derived hematopoietic stem cells (iHSCs) without the use of serum or additional cells.

The invention provides a preparation method and a kit for reprogramming induced pluripotent stem cells of PBMC (peripheral blood mononuclear cells), particularly provides PBMC reprogramming based on mRNA-LNP (peripheral blood mononuclear cells), and more particularly relates to a preparation method for reprogramming induced pluripotent stem cells of peripheral blood mononuclear cells. The preparation method comprises the following steps: preparing a nano liposome mRNA (messenger ribonucleic acid) cocktail-LNP; according to the method, the nano-liposome mRNA-LNP is used for inducing peripheral blood mononuclear cells to reprogram, so that reprogrammed induced pluripotent stem cells are obtained, and an iPSCs reprogramming technology which is low in cost, low in toxicity, efficient, stable, repeatable and safer is realized. The invention also provides a reprogramming kit and a preparation method of the induced pluripotent stem cells.

The present invention provides methods of producing in vitro derived neutrophils or macrophages in xenogen- and serum-free conditions from pluripotent stem cells and in vitro derived populations of neutrophils and macrophages. Methods of treatment using in vitro derived neutrophils or macrophages are also contemplated.

The invention provides cell culture conditions for culturing stem cells, including feeder-free conditions for generating and culturing human induced pluripotent stem cells (iPSCs). More particularly, the invention provides a culture platform that allows long-term culture of pluripotent cells in a feeder-free environment; reprogramming of cells in a feeder-free environment; single-cell dissociation of pluripotent cells; cell sorting of pluripotent cells; maintenance of an undifferentiated status; improved efficiency of reprogramming; and generation of a naïve pluripotent cell.

The present invention relates to an induced pluripotent stem cell (iPSC) or a somatic cell derived from an iPSC by forward programming or directed differentiation constitutively expressing an alternative splice form of Lamin A. The cells comprise an expression cassette comprising a coding sequence for the alternative Lamin A splice form operably linked to a cumate operator (CuO) sequence and a constitutive promoter. Also provided is the use of the cells described herein in a method of identifying a gene or combination of genes involved in the reversal of an ageing phenotype or in the maintenance of a non-aged phenotype. Methods of rejuvenating somatic cells, treating diseases or disorders associated with ageing, and modulating ageing (e.g. age-modulating methods and uses), including in vivo, are also provided, said methods comprising modulating the expression and / or activity of the gene / combination of genes identified herein.

The invention relates to the technical field of preparation of organoids, and discloses a method for regulating and controlling human induced pluripotent stem cells to differentiate into kidney organoids, which comprises the following steps: resuspending a cell cluster formed by human induced pluripotent stem cells by using a basic differentiation culture solution containing a GSK-3beta inhibitor, inoculating the cell cluster into an ultralow adsorption cell culture plate, and starting to culture, marking the time as the 0th hour of differentiation; and after culturing for 70-76 hours, replacing the basic differentiation culture solution containing the GSK-3beta inhibitor with a basic differentiation culture solution containing a Knockout TM serum substitute (KSR), and continuing to carry out differentiation culture. By adopting the method disclosed by the invention, the human induced pluripotent stem cells can be efficiently, quickly, simply, conveniently and stably subjected to directional induced differentiation into kidney organs containing podocytes, renal tubules and other structures, and the time for constructing a kidney in-vitro model can be shortened, so that the research on kidney diseases, kidney development and the like can be greatly assisted; and a powerful platform is provided for kidney toxicity assessment.

Provided is a method for producing induced pluripotent stem cells by which induced pluripotent stem cells having high undifferentiated and differentiated capabilities can be easily obtained. This method for producing induced pluripotent stem cells comprises: a step for introducing a reprogramming factor into somatic cells; and a step for culturing the somatic cells into which the reprogramming factor is introduced in the presence of a cytoskeleton material containing a peptide-containing polyvinyl acetal resin.

Described herein is a microphysiological system for models of disease. Specifically, induced pluripotent stem cells (iPSCs) and iPSC-derived cells, including those obtained from disease patients, are seeded onto microfluidic “chip” devices to study cellular development and disease pathogenesis. Herein, neurodegenerative disease modeling, including Parkinson's Disease (PD) is shown to reproduce key PD pathology in a vascularized human model that contains neurons relating to PD pathology. Such compositions and methods are used for research for PD biomarkers, patient screening for PD risk assessment, and therapeutic discovery and testing. A panel of biomarkers are generated through analysis of living PD-chips by neural activity, whole transcriptomic, proteomic, and metabolomic analysis, and functional enzyme tests of media and tissue. Introducing therapeutics through a vasculature channel, coupled with blood brain barrier penetration studies can be assessed for efficacy in the human neural cells present in the PD-Chip.

The invention discloses a multiplex fluorescent quantitative PCR detection composition, method and kit for detecting induced pluripotent stem cell residues in differentiated cells, the detection method is non-diagnostic or therapeutic, and comprises the following steps: S1, extracting an RNA template of a sample to be detected; s2, removing genome DNA and carrying out reverse transcription on RNA; s3, establishing a fluorescent quantitative PCR reaction system by adopting the detection composition in claim 1; s4, carrying out multiple fluorescent quantitative PCR (Polymerase Chain Reaction) detection; and S5, analyzing the result of the to-be-detected sample. The kit can be used for accurately and quickly detecting iPSC residues in a sample.

The invention provides a method for directionally inducing and differentiating human induced pluripotent stem cells into IMRC and purifying the IMRC to obtain IMRC-EV, and relates to the technical field of stem cells. According to the method for directionally inducing and differentiating the human induced pluripotent stem cells into the IMRC and purifying the IMRC to obtain the IMRC-EV, the child prepuce tissue-derived mesenchymal stem cells are used as seed cells for inducing the human induced pluripotent stem cells, so that the problem that the existing IMRC-EV is generally obtained from human embryo-derived stem cells, so that the source is limited is solved. Meanwhile, the method for preparing the IMRC-EV through induction of the human induced pluripotent stem cells is obtained through selection of a differential culture medium and an amplification culture medium, and then a basis is provided for research, development and clinical transformation of iPSC-IMRC-EV drugs.

The present invention provides isolated human breast milk exosomes and compositions thereof. The invention also provides an in vitro method for producing the exosome, comprising generating lactating cells derived from human induced pluripotent stem cells (hiPSCs), expressing a human milk-like product from the lactating cells, and purifying the exosome from the human milk-like product.

A microfluidic 3D-printed hydrogel based on fish liver decellularized extracellular matrix for liver regeneration and a preparation method. The hydrogel is prepared by means of combining fish liver decellularized extracellular matrix (dECM) and gelatin methacryloyl (GelMA), and loading induced pluripotent stem cell-derived hepatocytes (iPSC-heps) for liver regeneration. The microfluidic 3D-printed hydrogel based on fish liver decellularized extracellular matrix exhibits excellent biocompatibility, retains intact endogenous growth factors, maintains the biological activity of cells, ensures the effective encapsulation of the cells, and facilitates the robust functional expression of the iPSC-heps. After in-vivo transplantation, the survival rate and liver function of mice with acute liver failure are significantly improved, and liver regeneration and repair are promoted.

The disclosure is directed to methods of generating induced pluripotent Afrotheria species stem cells by chemically reprogramming primary Afrotheria species cells. In certain embodiments, the methods also include selecting for the induced pluripotent elephant stem cells. Further, the cells are transfected with at least (i) C4, (ii) CS, (iii) C6, and (iv) SV40LT.

The invention relates to the technical field of organoid construction, and discloses a heart organoid construction method which comprises the following steps: preparing a culture medium I containing CD lipid, ITS-X, L-AA2P, alpha-MTG, insulin, FGF2 and a GSK-3beta inhibitor; preparing a culture medium II containing CD lipid, ITS-X, L-AA2P, alpha-MTG, insulin, FGF2 (fibroblast growth factor 2) and VEGFA (vascular endothelial growth factor A); the method comprises the following steps: digesting a human induced pluripotent stem cell mass, suspending the human induced pluripotent stem cell mass in a culture medium I, inoculating the human induced pluripotent stem cell mass into an ultra-low adsorption cell culture plate with a U-shaped hole bottom, culturing for 36-40 hours, removing the culture medium I, adding a culture medium II, and culturing until a heart organ is formed. In the method provided by the invention, two culture media are sequentially used for performing two-stage differentiation culture, namely, the heart organoid can be constructed from the human induced pluripotent stem cells dispersed after digestion, so that the construction process of the heart organoid is simplified, the construction time is shortened, and the operation difficulty is reduced.

The invention discloses a preparation method and application of mesenchymal stem cells based on neural crest organics.The preparation method comprises the following steps that 1, human induced pluripotent stem cells are added into digestive juice, and after digestion, a culture medium is added for dilution; 2, collecting cells, centrifugally separating the cells, removing supernatant, suspending the cells in a culture medium, and adjusting the size of a cell block to 50-80 microns; 3, culturing the cells, and then carrying out induced differentiation on neural crest organs; 4, digesting the neural crest organs into single cells, transferring the single cells into a serum-free mesenchymal stem cell culture medium for adherent culture, and then performing amplification to obtain the mesenchymal stem cells. According to the invention, the induced pluripotent stem cells ihPSCs3D are induced and differentiated to obtain neural crest organs, the neural crest-derived mesenchymal stem cells are obtained, and the obtained NCC-MSCs have improvement and repair effects on pancreatitis.

Injectable compositions and methods of preparation, as well as therapeutic uses, of induced pluripotent stem cell (iPSC)-derived notochordal cell (iNC)-loaded microgels are provided. Microfluidic on-chip platform can be utilized to prepare microgels (or microgel particles / spheres) formed from block copolymers that exhibit reverse thermal gelation, so as to encapsulate iNCs. Also provided are preconditioned iNC-loaded microgels and iNCs in bulk hydrogel. Cell purity, identity, viability, sterility, and the stability of microencapsulated iNCs have been evaluated. Safety and efficacy of the compositions as therapeutic candidates has been tested via intradiscal injection in animal models of intervertebral disc (IVD) degeneration and discogenic low back pain. Biobehavioral testing, MRI, and immunohistochemical analyses were utilized to evaluate the regenerative potential and reproducibility of the compositions as therapeutic candidate. Single cell RNA sequencing of the treated IVDs may also reveal mechanism of action of the compositions.

The invention discloses a construction method of a human microglial cell model carrying ENTPD1 gene mutation, and the human microglial cell model is prepared by the following steps: extracting PBMC from venous blood of a hereditary spasmodic paraplegia patient, then electrically transforming four factors of Oct4, Sox2, Klf4 and cMyc, and culturing in a Matrigel-coated culture dish for 15-20 days to obtain induced pluripotent stem cells; the induced pluripotent stem cells can form a unique human microglial cell model, and the human microglial cell model and the induced pluripotent stem cells are co-cultured to obtain an organoid, so that the analysis of a pathogenic molecular mechanism behind a disease risk gene is greatly facilitated, the defects of a rodent-derived microglial cell model are complemented, and the development of the rodent-derived microglial cell model is promoted. And the influence of the ENTPD1 on the related functions of the microglial cells is further discussed.