41 results about "Totipotent stem cell" patented technology

Non-embryonic blastomere-like totipotent stem cells are disclosed. Most preferably, such cells are obtained from various tissues of postnatal mammals (e.g., using tissue biopsied from the mammal), are smaller than 1 μm, have normal karyotype, and do not spontaneously differentiate in serum-free medium without differentiation inhibitors. These non-embryonic blastomere-like totipotent stem cells typically express CD66e, CEA-CAM-1 and telomerase, but do not typically express CD10, SSEA-1, SSEA-3, and SSEA-4. Such blastomere-like totipotent cells can be differentiated into ectodermal, mesodermal, or endodermal tissues, including placental tissues and germ cells. Moreover, when implanted into a mammal, such cells will not be teratogenic.

The invention provides a sub-totipotent stem cell of a human placenta. The sub-totipotent stem cell of the human placenta is derived from stripped human placenta amnion, aiming at the characteristics of cells contained by an epithelial layer and a mesenchyme layer of the amnion, a step-by-step separation method is adopted, pollution of the sub-totipotent stem cell of the placenta by the mesenchyme layer of the amnion is reduced to the greatest extent, and the stemness of the sub-totipotent stem cell of the human placenta is improved effectively. The method has the characteristics of low cost, broad application prospect and capacity of supplying a large quantity of stem cell resources to clinic and research. The invention further provides a method for preparing the sub-totipotent stem cell of the human placenta from the human placenta amnion and constructing a stem cell bank.

The invention relates to a method for extracting sub-totipotent stem cells from a chorion of a fetal surface of a placenta. The method comprises the steps of removing an amnion and stagnated blood from the placenta, and then repetitively washing the surface of the placenta to perform sterilization; shearing the chorion of the fetal surface in a glass utensil, removing placenta lobule tissues remained on the surface as much as possible, and shearing the chorion into small blocks; washing small tissue blocks by using a screen and a great amount of normal saline, and removing residual blood cells; performing tissue digestion: performing oscillatory digestion in a constant temperature shaker by using mixed enzymes; adding a proper amount of FBS for termination after digestion, performing filtration through a filter screen, and adding a great amount of normal saline to wash filter residues to obtain cells as many as possible; performing centrifugation, abandoning supernatant, adding saline water for washing and performing centrifugation to obtain mononuclear cells; performing magnetic bead sorting (OCT-4 positive, Nanog positive and STRO-1 negative) to obtain target cells. The invention further relates to the sub-totipotent stem cells obtained by adopting the method and pharmaceutical use thereof. The sub-totipotent stem cells have excellent characteristics.

The invention discloses a sub-totipotent stem cell from cut human umbilical cord or placenta, wherein the cell marker is CD151<+>OCT4<+>CD184<->; the cell grows adhering to the wall in a culture container and can differentiate to the body entoderm, mesoblast and ectoderm tissues. The invention also discloses a preparation method of the sub-totipotent stem cell and an application thereof for preparing drug for treating cell injuries or cell aging diseases, and an application thereof as a carrier cell of a gene treatment drug.

The invention discloses a preparation method of sub-totipotent stem cells. The method comprises the following steps: acquisition of tissues, disinfection, isolation, washing and shearing of the tissue, collagenase digestion, and acquisition of the sub-totipotent stem cells. The invention discloses a method for isolating sub-totipotent stem cells from umbilical cord, placenta amnion, chorion or decidua basalis tissues, and the obtained sub-totipotent stem cells have the advantages of uniformity, stability and short growth cycle.

The present invention provides a method for producing mesenchymal cells for production of mesenchymal cells for formation of a tooth, the method comprising: culturing totipotent stem cells in the presence of a differentiation inducer to produce a cell population after differentiation induction treatment, the cell population containing CD44-positive and CD29-positive cells or CD44-positive and CD 106-positive cells; and selecting, from the cell population after the differentiation induction treatment, the CD44-positive and CD29-positive cells or CD44-positive and CD 106-positive cells as the mesenchymal cells for the formation of the tooth. The present invention also provides a method for producing a tooth comprising: positioning, in a support carrier capable of retaining cells in a state of contacting therewith, a first cell mass substantially consisting of only either one of mesenchymal cells and epithelial cells and a second cell mass substantially consisting of only the other one of the mesenchymal cells and epithelial cells, the first and second cell masses being not mixed with each other but made to closely contact with each other; and culturing the first and second cell masses; wherein the mesenchymal cells comprise the mesenchymal cells for the formation of the tooth.

The invention discloses a human adipose sub-totipotent stem cell isolated-culture method and a stem cell bank establishing method. The human adipose sub-totipotent stem cell isolated-culture method includes the following steps of human adipose tissue collection, sub-totipotent stem cell separation and obtaining, sub-totipotent stem cell culture and proliferation, sub-totipotent stem cell cryopreservation, product quality control, stem cell bank establishing and stem cell revivifying and annual inspection. Mixed collagenase used by the method can effectively separate sub-totipotent stem cells from adipose tissues, is homogenous in tissue digestion, no obvious tissue residual is produced, the cell activity is good, wall adherence is quick, and the homogeneity is good. Cryopreservation liquid adopted by the method can effectively maintain the cell activity, a gradient cooling box is adopted to cryopreserve cells, the cell activity is not affected, and the human adipose sub-totipotent stem cell isolated-culture method is simple in operation, convenient and high in efficiency.

The invention discloses a method for separating and purifying totipotent stem cells from the tissues of early aborted fetuses of human beings, including the steps: a. a sample of the placenta tissue of the aborted fetus after the first pregnancy period is collected; b. the umbilical cord tissue or the intestine tissue of the fetus is obtained from the sample gained in the step a; c. cells are obtained from the umbilical cord tissue or the intestine tissue of the fetus gained in the step b; and d. markers of the embryonic stem cell, such as OCT-4, Nanog, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and the like which are obtained from the cells of the umbilical cord tissue or the intestine tissue of the fetus, are separated, purified and expressed, and can form the stem cell of an embryoid body. The cells obtained by the method of the invention are always in the state of undifferentiation. The invention also discloses the application of the stem cells in forming different human cells, such as cardiac muscles, nerves and islet beta cells or osseus cells. The method avoids the dispute over ethnics which needs to be faced with during the operation of obtaining the totipotent stem cells from the embryo, and more stem cells can be obtained than from blastocyst.

The invention provides a method for inducing the transformation of totipotent stem cells into mesenchymal stem cells. In the method, a simple culture method, a fibroblast medium and a monoclonal separation method are adopted to purify and obtain mesenchymal stem cells, the mesenchymal stem cell content is over 95 percent, and more than 109 mesenchymal stem cells can be obtained. The invention has the advantages that: (1) the cells obtained by the method have excellent boundary expansion performance, have properties that can be retained in 15 generation and can from bones, cartilage and muscle tendons; (2) the method for acquiring the cells of the invention is simple, high in efficiency and free from flow type, magnetic bead and the like sorting and purification methods and greatly reduces the cost of the transformation of totipotent stem cells into mesenchymal stem cells; and (3) the method is suitable to be used to repair of defects in tissues such as bone, muscle tendon, cartilage and skin and suitable to be used as seed cells in tissue engineering.

The invention provides a special culture medium and a special culture method for sub-totipotent stem cells. Enough stem cells can be obtained through one passage, and are stored. The culture medium contains no serum, and can be conveniently used. The special culture medium for the sub-totipotent stem cells includes 95% of DMEM / F12, 5% of a serum substitute, 5mg / ml of human albumin, 5.5[mu]g / ml of linoleic acid, 10ng / ml of a basic fibroblast growth factor, 2*10<-8>mol / ml of dexamethasone, 10ng / ml of a human platelet-derived growth factor, 10ng / ml of an epidermal growth factor, 50[mu]mol / L of beta-mercaptoethanol and 2mmol / L of L-glutamine. The culture medium contains no serum, has a good culture effect, and allows a required number of frozen sub-totipotent stem stems to be reached only through culturing one generation.

The invention relates to a method for separating, amplifying, cryopreserving and reviving a placenta sub-totipotent stem cell. The method comprises the following steps: (1) collecting a sample; (2) separating the placenta sub-totipotent stem cell; (3) performing primary culture on the placenta sub-totipotent stem cell; (4) performing secondary multiplication culture on the placenta sub-totipotentstem cell; (5) cryopreserving; and (6) reviving. The placenta sub-totipotent stem cell separated by adopting the method has high purity, the primary cultured cell has high survival rate, the cryopreserved cell has low pollution rate, and the amplified cell quality of the revived placenta sub-totipotent stem cell can be remarkably improved.

The invention discloses a preparation method of a placenta sub-totipotent stem cell culture medium for skin care. Human placenta tissue is cut, smashed, degraded, screened and separated to obtain placenta sub-totipotent stem cells; then the placenta sub-totipotent stem cells are placed in a stem cell culture medium containing amino acid, non-essential amino acid and cell growth factors to be cultured; finally, collected supernate is filtered, small-molecular collagen peptide is added, and the placenta sub-totipotent stem cell culture medium can be obtained. The placenta sub-totipotent stem cell culture medium prepared through the method is high in active substance content and high in bioactivity, can care skin, can promote skin regeneration, and enhances metabolism of skin.

The invention discloses a method for induced differentiation of human placenta sub-totipotent stem cells to obtain cardiac muscle cells. The method comprises the following steps: inoculating human placenta sub-totipotent stem cells into a 24-pore plate containing a stem cell culture medium; after cell attachment, removing the stem cell culture medium; and adding a cardiac muscle cell culture medium for culture for 28 days, and changing the culture medium every 3 days. The stem cell culture medium comprises a DMEM culture medium, hydroxyethyl starch, human serum albumin, potassium bicarbonate, ethylenediamine tetraacetic acid sodium salt, sodium selenite, transferrin, hyaluronic acid, taurine, placental growth factors, fibroblast growth factors, glutathione, lipoic acid, astragalus polysaccharide, codonopsis pilosula polysaccharide and jujube polysaccharide. The cardiac muscle cell culture medium comprises an RPMI-1640 culture medium, trehalose, taurine, fibroblast growth factors, endothelial cell growth factors, potassium bicarbonate, amino acid chelated selenium, glutamine, progesterone, coenzyme I, lipoic acid, salvianolic acid B, polygonatum polysaccharide and ginsenoside.

The invention provides a preparation method of a medical dressing, which includes preparation of secreted PLGA sustained-release microspheres, preparation of a hydrogel, preparation of an electrospunmembrane, and preparation of a stem cell secretion medical dressing. In the invention, a combination of sub-totipotent stem cell secretin, PLGA sustained-release microspheres and hyaluronic acid hydrogel is used, and the electrospun membrane is used as a carrier to prepare the medical dressing, the sub-totipotent stem cell secretion is rich in various active factors, and has a good repair effect on wounds; Experiments show that the medical dressing prepared by the invention has a good healing effect on both acute and chronic wounds. The medical dressing can adhere to the wound well, and the sub-totipotent stem cell secretin in the PLGA sustained-release microspheres is slowly released, which greatly improves the utilization rate of the secretion and the durability, the PLGA slow-release microspheres contain a larger amount of subtotipotent stem cell secretions, which reduces the number of dressing changes, avoids secondary trauma during the dressing change, and facilitates wound repair.

The invention discloses an inducing and culturing system-establishing method for totipotent stem cell quteASCs of mice or human beings. The materials are early embryo of mice growing for 3-8 days (obtained through in-vitro fertilization or collected from the uterus of a parent body), and the early embryo of the human beings growing for 3-14 days (obtained through in-vitro fertilization or collected from the uterus of a parent body). The inducing method comprises the following two key technical processes: 1, carrying out inducing to establish excess type stem cells ASCs, wherein the key inducing factor is AF; and 2, carrying out further inducing to complete the system establishing technological process of the novel pleuripotent stem cells quteASCs, wherein the key inducing factor is ABCL. According to the invention, through the specific inducing method, the novel stem cells quteASCs of the mice or the human beings are successfully obtained through the specific inducing method, and the totipotency of the novel stem cells quteASCs of the mice or the human beings for developing to the individual tissue of animal or the placenta of the animal is verified through the experimental means.

The invention discloses a method for separating and purifying human amniotic fluid stem cells. The method is to use an embryo antigen-4 at a special stage as a screening marker to separate and purify the human amniotic fluid stem cells which have homogeneous characteristics and are positively expressed by the embryo antigen-4 at the special stage from the human amniotic fluid stem cells which are adherently cultured. Experimental results show that the method can obtain the human amniotic fluid stem cells which have homogeneous characteristics, good capacities of proliferation and inductive differentiation, and cell ratio of G0 stage to G1 stage cells of more than 90 percent, and can express specificity markers of human totipotent stem cells such as the embryo antigen-4, Nanog and Oct-4 at the special stage. The invention provides a new way to obtain the human amniotic fluid stem cells, and lays a foundation for the further application of the human amniotic fluid stem cells.

The invention discloses a sub-totipotent stem cell from cut human umbilical cord or placenta, wherein the cell marker is CD151<+>OCT4<+>CD184<->; the cell grows adhering to the wall in a culture container and can differentiate to the body entoderm, mesoblast and ectoderm tissues. The invention also discloses a preparation method of the sub-totipotent stem cell and an application thereof for preparing drug for treating cell injuries or cell aging diseases, and an application thereof as a carrier cell of a gene treatment drug.

The invention provides a totipotent stem cell culture method. A polypeptide which is artificially synthesized is taken as an extracellular matrix and spread on the surface of a cell culture dish, totipotent stem cells are inoculated to the surface of the culture dish and injected into a culture medium without a feeder layer, and the totipotent stem cells after inoculation are used for culture and passage. According to the method provided by the invention, the amplification of the totipotent stem cells can be supported, and the cell content of Oct4+ cells can be always kept above 80%. Through at least 40 days of culture (six passages and about 40 times of cell doubling), the cells can still realize high expression of various markers Oct4 of the totipotent stem cells, and after the amplification culture, the karyotype can still be kept consistent.

Provided are a sub-totipotent stem cell product and apparent hereditary modifying label thereof, a method for inducing the generation of the sub-totipotent stem cell product and identification for the apparent hereditary modifying label of the differentiated potential of stem cells. Also provided is a use of a histone modifying state of a sub-totipotent gene and / or differentiated related gene to predict the apparent hereditary modifying label of the differentiated potential of stem cells.

The invention discloses a mouse embryonic stem cell (ESC) induction culture solution for improving 2C-like cells and a mouse ESC induction culture method. The method comprises the steps that: first, fetal bovine serum is removed from a mouse ESCs culture system, so that mouse ESCs still maintain original pluripotency characteristics; and second, retinoic acid (RA) is utilized for increasing the proportion of the 2C-like cells in the mouse ESCs in a culture solution without containing the fetal bovine serum, and the result is increased from 0.2% of a control group to 3.3%. The 2C-like cell proportion in the mouse ESCs is increased by using a specific induction method, the 2C-like gene expression level and protein level of the mouse ESCs in the culture solution without containing the fetal bovine serum are verified by experimental means, and a new thought is provided for research of mammal totipotent stem cells.

The invention relates to a method for extracting sub-totipotent stem cells from a chorion of a fetal surface of a placenta. The method comprises the steps of removing an amnion and stagnated blood from the placenta, and then repetitively washing the surface of the placenta to perform sterilization; shearing the chorion of the fetal surface in a glass utensil, removing placenta lobule tissues remained on the surface as much as possible, and shearing the chorion into small blocks; washing small tissue blocks by using a screen and a great amount of normal saline, and removing residual blood cells; performing tissue digestion: performing oscillatory digestion in a constant temperature shaker by using mixed enzymes; adding a proper amount of FBS for termination after digestion, performing filtration through a filter screen, and adding a great amount of normal saline to wash filter residues to obtain cells as many as possible; performing centrifugation, abandoning supernatant, adding saline water for washing and performing centrifugation to obtain mononuclear cells; performing magnetic bead sorting (OCT-4 positive, Nanog positive and STRO-1 negative) to obtain target cells. The invention further relates to the sub-totipotent stem cells obtained by adopting the method and pharmaceutical use thereof. The sub-totipotent stem cells have excellent characteristics.

The invention discloses a device for separating and purifying placenta totipotent stem cells. The device comprises an upper sealing cover, a temperature and humidity controller, a door, a box, a vibrating centrifugal tube, a bottom sealing plate, a purifying beaker, a fixed slide rail, a fixed base, a limiting fixed block, a data connecting socket, a filter screen and a connecting hinge. The device has the beneficial effects: the temperature and humidity controller is capable of guaranteeing suitability of inner separating and purifying environment and guaranteeing activity of placenta totipotent stem cells; the vibrating centrifugal tube is capable of preventing solution splashing and leakage caused by centrifugation and guaranteeing smooth proceeding of centrifugation; the vibrating centrifugal tube can be conveniently mounted and dismounted; the purifying beaker is convenient for separation and purification operations which can be finished in one device; the purifying beaker can beconveniently taken out and fixed and is capable of preventing the purifying solution from splashing and leakage; the fixed slide rail is capable of guaranteeing smoothness and stability of operation and is convenient for purification operation; constitutional units of the filter screen are in same size and shape and are fixed above the bottom sealing plate in the manner of slots; working efficiency is increased; operation steps are simplified; mounting, dismounting and replacement are convenient.

The present invention relates to 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 the TGF-beta superfamily withthe ability to inhibit stem cell differentiation, its use in the culture of human pluripotent stem cells, a cell culture system comprising human pluripotent stem cells and the chemically defined medium, as well as a kit for proliferation and / or maintenance of human pluripotent stem cells.

The invention discloses a separation method of human placenta sub-totipotent stem cells. The separation method comprises the following steps: S1, preparing an amniotic membrane sample; S2, preparing an amniotic membrane specimen; S3, performing enzymolysis; a scheme is short in technological process and simple in workstage, a partition amniotic membrane stripping way is adopted in the step of preparing the amniotic membrane specimen, and the amniotic membrane is not easy to break; on the one hand, the regular amniotic membrane is convenient for subsequent flushing, blood coagulation and jellyremoval, and primary enzymolysis and secondary enzymolysis work; on the other hand, the partition amniotic membrane stripping way can ensure that the amniotic membrane is totally taken out, and ensures the yield of the human placenta sub-totipotent stem cells. According to the method, the amniotic membrane which is stripped in partition is regular and tidy and is convenient to clean, the blood coagulation and jelly on the amniotic membrane are cleaned out, and the enzymolysis effects are ensured; the cleaned amniotic membrane specimen is subjected to the enzymolysis twice, parenchyma cells onthe amniotic membrane are removed in the primary enzymolysis, so that a Ficoll lymphocyte separating medium is not needed for separation, and the human placenta sub-totipotent stem cells with relatively high purity can also be obtained.

The invention relates to the field of stem cells and regenerative medicine, and particularly relates to neural crest cell culture fluid, a preparation method of neural crest mesenchymal stem cells and an application of the neural crest mesenchymal stem cells. Experimental results of the invention show that mesenchymal stem cells of the neural crest stem cell lineage, induced and differentiated by human totipotent stem cells, can obviously improve neurological dysfunctions, reduce an area of an ischemic region, inhibit immune reactions and promote endogenous repairs of brain tissues in a neonatal rat hypoxic-ischemic model, thereby proving that human neural crest-derived mesenchymal stem cells have effects of significantly improving the inflammatory reaction of neonates with ischemic and anoxic encephalopathy and effectively promoting the neural function recovery. The invention also provides a test index for identifying the activity of the neural crest mesenchymal stem cells.

The invention provides a special culture medium and a special culture method for sub-totipotent stem cells. Enough stem cells can be obtained through one passage, and are stored. The culture medium contains no serum, and can be conveniently used. The special culture medium for the sub-totipotent stem cells includes 95% of DMEM / F12, 5% of a serum substitute, 5mg / ml of human albumin, 5.5[mu]g / ml of linoleic acid, 10ng / ml of a basic fibroblast growth factor, 2*10<-8>mol / ml of dexamethasone, 10ng / ml of a human platelet-derived growth factor, 10ng / ml of an epidermal growth factor, 50[mu]mol / L of beta-mercaptoethanol and 2mmol / L of L-glutamine. The culture medium contains no serum, has a good culture effect, and allows a required number of frozen sub-totipotent stem stems to be reached only through culturing one generation.

The present invention provides a method for producing mesenchymal cells for production of mesenchymal cells for formation of a tooth, the method comprising: culturing totipotent stem cells in the presence of a differentiation inducer to produce a cell population after differentiation induction treatment, the cell population containing CD44-positive and CD29-positive cells or CD44-positive and CD106-positive cells; and selecting, from the cell population after the differentiation induction treatment, the CD44-positive and CD29-positive cells or CD44-positive and CD106-positive cells as the mesenchymal cells for the formation of the tooth. The present invention also provides a method for producing a tooth comprising: positioning, in a support carrier capable of retaining cells in a state of contacting therewith, a first cell mass substantially consisting of only either one of mesenchymal cells and epithelial cells and a second cell mass substantially consisting of only the other one of the mesenchymal cells and epithelial cells, the first and second cell masses being not mixed with each other but made to closely contact with each other; and culturing the first and second cell masses; wherein the mesenchymal cells comprise the mesenchymal cells for the formation of the tooth.

The embodiment of the invention discloses a graft capable of reducing incidence of GVHD, which is characterized in that the graft is obtained by mixed culture of placenta sub-totipotent stem cells andumbilical cord blood hematopoietic stem cells; the placenta sub-totipotent stem cells and the umbilical cord blood hematopoietic stem cells are co-cultured to prepare the graft, and the graft is transfused back, so that the greening and morbidity degree of GVHD can be effectively reduced.

The invention provides a preparation method and application of decidua parietal subtotipotent stem cells, and relates to the technical field of cell engineering. The preparation method of the present invention comprises the following steps: pretreatment: cleaning the parietal decidua group, centrifuging, and removing the supernatant; digestion: cutting the parietal decidua tissue into pieces, adding tissue digestion liquid for digestion, and separating the supernatant and precipitation after digestion , resuspending the precipitate to obtain a suspension; the tissue digestion solution includes Tryple enzyme, normal saline, type I collagenase and type II collagenase, the volume concentration of the Tryple enzyme is 40% to 60%, and the volume concentration of type I collagenase The mass concentration is 0.8-1.2mg / mL, and the mass concentration of type II collagenase is 0.8-1.2mg / mL; Separation: add lymphocyte separation liquid to the suspension, centrifuge, separate the mononuclear cells after layering, centrifuge, Keep the precipitate, add culture medium, and mix well to obtain parietal decidua subtotipotent stem cell separation medium. The invention can successfully extract subtotipotent stem cells from parietal decidua tissue, and has good cell activity and strong differentiation ability.