50 results about "Hepatic stem cell" patented technology

The subject invention a method for converting liver stem / progenitor cells to a pancreatic functional cell by transfecting said liver cells with a pancreatic development gene and / or by culturing with pancreatic differentiation factors. The resulting cells produce and secrete insulin protein in response to glucose stimulation.

Disclosed are compositions and methods for isolating, immortalizing and differentiating adult stem cells, for example, particular human clonal adult liver stem cells or adipose stem cells, including specialized cell culture media for the isolation and propagation of such stem cells. Also disclosed are methods of screening for toxicity, carcinogenicity and therapeutic activity using such stem cells and immortalized or differentiated derivatives thereof. In addition, methods of treatment using such stem cells and their differentiated or immortalized derivatives thereof are disclosed.

The invention discloses hepatic stem cell preserving solution and applications of the hepatic stem cell preserving solution. The hepatic stem cell preserving solution provided by the invention is prepared by fixing the volume of injection solution including 0.1 to 1g of human albumin, 2.60 to 4.97g of sodium chloride, 2.48 to 4.74g of sodium gluconate, 1.82 to 3.40g of sodium acetate, 0.18 to 0.35g of potassium chloride, 0.15 to 0.28g of magnesium chloride, and 0.4 to 0.7mL of heparin calcium based on effective dose by water to be reach to 100mL. The hepatic stem cell preserving solution provided by the invention is used for storing hepatic stem cells, and the survival rate of the hepatic stem cell is more than 85% and even more than 95% within 12 hours. The cell suspension which is obtained by floating the hepatic stem cells on the hepatic stem cell preserving solution can be used as the medicine for treating diabetes mellitus, and has the characteristics of being high in stability, good in curative effect, high in safety, being without toxic or side effect, convenient to store and transport, applicable to massive clinical use, and broad in application prospect. The hepatic stem cell preserving solution brings good news for diabetic patients, and provides a new way for clinical stem cell use.

Disclosed are: an induced hepatic stem cell useful for safety tests, toxicity tests, metabolism tests, drug interaction tests, anti-viral activity tests, screening tests for medicinal agents such as hyperlipemia therapeutic agents, hypertension therapeutic agents, pharmaceutical low-molecular-weight compound agents and pharmaceutical antibody agents, the screening for potential drug targets, the production of animal models, the production of hepatocyte-produced proteins, and regenerative medicine; a process for producing the cell; and applications of the cell. The induced hepatic stem cell is characterized by fulfilling at least the following requirements (1) to (3): (1) at least 15 genes selected from specific genes that are marker genes for embryonic stem cells are expressed in the cell; (2) the cell has properties of hepatocytes; and (3) the cell can be proliferatively cultured or sub-cultured for at least three days.

Hepatic progenitors comprise two populations of human hepatic stem cells, primitive and proximal hepatic stem cells, and two populations of committed progenitors, one for biliary cells and one for hepatocytes. Human primitive hepatic stem cells are a very small fraction of the liver cell population and give rise to proximal hepatic stem cells constituting a much larger fraction of the liver. Human proximal hepatic stem cells give rise to biliary and hepatocyte committed progenitors. Primitive and proximal stem cells are the primary stem cells for the human liver. Human primitive hepatic stem cells may be isolated by immunoselection from human livers or culturing human liver cells under conditions which select for a human primitive hepatic stem cell. Proximal hepatic stem cells may be isolated by immunoselection, or by culturing human liver cells under conditions which include a developmental factor. Proximal hepatic stem cells may also be isolated by culturing colonies comprising a primitive hepatic stem cell under conditions which include a developmental factor. Resulting compositions may be used for treating liver disorders and for producing bioartificial organs.

Isolated liver progenitor stem cells and cell populations of isolated liver progenitor stem cells are disclosed. The progenitor stem cells originate from adult liver, especially human adult liver. The isolated progenitor stem cells have uses in medicine, hepatology, inborn errors of liver metabolism transplantation, infectious diseases and liver failure. Methods of isolating these cells and their culture is described. The isolated cells are characterized before and after differentiation. Their use for transplantation and as animal models of human disease, toxicology and pharmacology is disclosed.

The present invention provides clonal pluripotent hepatic stem cells using flow cytometry and in vitro single-cell-based assays. These cells possess multilineage differentiation potential and self-renewing capability. These cells may be clonally propagated in culture, to continuously produce hepatocytes and cholangiocytes as descendants while maintaining primitive stem cells. When expanded cells are transplanted into recipient animals, they morphologically and functionally differentiated into hepatocytes and cholangiocytes, with reconstitution of hepatocyte and bile duct structures. Furthermore, these cells differentiated into pancreatic ductal and acinar cells or intestinal epithelial cells when transplanted into pancreas or duodenal wall. Thus, the self-renewing multipotent stem cells persist in the developing mouse liver and can be induced to become cells of other organs of endodermal origin under appropriate microenvironment, providing new insight into therapies for diseases of the digestive system.

Hepatic progenitors comprise two populations of human hepatic stem cells, primitive and proximal hepatic stem cells, and two populations of committed progenitors, one for biliary cells and one for hepatocytes. Human primitive hepatic stem cells are a very small fraction of the liver cell population and give rise to proximal hepatic stem cells constituting a much larger fraction of the liver. Human proximal hepatic stem cells give rise to biliary and hepatocyte committed progenitors. Primitive and proximal stem cells are the primary stem cells for the human liver. Human primitive hepatic stem cells may be isolated by immunoselection from human livers or culturing human liver cells under conditions which select for a human primitive hepatic stem cell. Proximal hepatic stem cells may be isolated by immunoselection, or by culturing human liver cells under conditions which include a developmental factor. Proximal hepatic stem cells may also be isolated by culturing colonies comprising a primitive hepatic stem cell under conditions which include a developmental factor. Resulting compositions may be used for treating liver disorders and for producing bioartificial organs.

The invention relates to the technical field of medical bioengineering, and provided with a specificity surface molecule marker CD63 of a hepatic stem cell. The invention further provided with application of the specificity surface molecule marker CD63 of the hepatic stem cell. A method that the molecule marker is used for quickly separating, culturing and amplifying the hepatic stem cell from a liver specifically comprises the steps that the specificity surface molecule marker CD63 of the hepatic stem cell is used to quickly separate the hepatic stem cell from prepared liver cell suspension through a flow cytometer. The hepatic stem cell of the CD63+ has the characters of self-renewing and bilateral differentiation out of a body, and has therapeutical effects on a damaged mouse liver. According to the specificity surface molecule marker CD63 of the hepatic stem cell and the application of the specificity surface molecule marker CD63 of the hepatic stem cell, the hepatic stem cell obtained by separation can be served as a seed cell for liver drug screening, systematization projection liver and hepatic disease cell therapy, and provides an ideal cell model for liver development and the cell biological property research of the hepatic stem cell.

The invention provides a mouse liver tumor cell line for highly expressing CD133 and a preparation method thereof. The mouse liver tumor cell line with high content of CD133+cell subpopulations is established by the following steps of: importing a cancer gene Hras into p53- / - mouse fetal liver cells, obtaining a single-cell clonal group derived from CD133+cells by utilizing a monoclonal patternmaking technology, and finally screening. The cell line LPC-H12 expresses related genes of various liver stem cells and the stem cells, namely Marker:CD133 and EpCAM, and has high in-vitro balling capacity and in-vitro tumorigenic capacity. The mouse liver tumor cell line provides a powerful tool for researching the action and the mechanism of CD133+liver cancer stem cell subpopulations in the occurrence and development process of liver cancer and screening medicaments used for liver tumor stem cells.

The present invention is directed to methods for readily propagating somatic liver stem cells. The methods comprise enhancing guanine nucleotide (GNP) biosynthesis, thereby expanding guanine nucleotide pools. This in turn conditionally suppresses asymmetric cell kinetics in the explanted cells. The methods of the invention include pharmacological methods and genetic methods. For example, the resulting cultured somatic liver stem cells can be used for a variety of applications including cell replacement therapies, gene therapies, drug discovery applications, and tissue engineering applications, such as the generation of artificial liver.

Isolated liver progenitor stem cells and cell populations of isolated liver progenitor stem cells are disclosed. The progenitor stem cells originate from adult liver, especially human adult liver. The isolated progenitor stem cells have uses in medicine, hepatology, inborn errors of liver metabolism transplantation, infectious diseases and liver failure. Methods of isolating these cells and their culture is described. The isolated cells are characterized before and after differentiation. Their use for transplantation and as animal models of human disease, toxicology and pharmacology is disclosed.

The invention discloses hepatic stem cell preserving solution and applications of the hepatic stem cell preserving solution. The hepatic stem cell preserving solution provided by the invention is prepared by fixing the volume of injection solution including 0.1 to 1g of human albumin, 2.60 to 4.97g of sodium chloride, 2.48 to 4.74g of sodium gluconate, 1.82 to 3.40g of sodium acetate, 0.18 to 0.35g of potassium chloride, 0.15 to 0.28g of magnesium chloride, and 0.4 to 0.7mL of heparin calcium based on effective dose by water to be reach to 100mL. The hepatic stem cell preserving solution provided by the invention is used for storing hepatic stem cells, and the survival rate of the hepatic stem cell is more than 85% and even more than 95% within 12 hours. The cell suspension which is obtained by floating the hepatic stem cells on the hepatic stem cell preserving solution can be used as the medicine for treating diabetes mellitus, and has the characteristics of being high in stability, good in curative effect, high in safety, being without toxic or side effect, convenient to store and transport, applicable to massive clinical use, and broad in application prospect. The hepatic stem cell preserving solution brings good news for diabetic patients, and provides a new way for clinical stem cell use.

The invention relates to a method for large scale preparation of liver stem cells, and uses of the liver stem cells in the fields of cell transplantation, stem cell culture, amplification, directional differentiation and tissue formation. The preparation method of the liver stem cells includes the following steps: extracting the liver stem cells from animal or human liver tissues through using a mechanical method, enzymatic method and mechanical-enzymatic method and density gradient centrifuge method combined technology; and carrying out in-vitro large scale culture, amplifying, carrying out liver stem cell identification, and freezing the liver stem cells for later use. The liver stem cell preparation efficiency of the method disclosed in the invention is about 100-10000 times higher than that of conventional methods, and the method can be used in tissue engineering and the production of liver stem cell preparations or stem cell drugs in order to provide a new method for the treatment of human diseases.

A method of repairing diseased or dysfunctional pancreas or liver is provided. The method involves preparation of a suspension of stem cells and / or progenitor cells such as biliary tree stem cells, hepatic stem cells, pancreatic stem cells or their descendants, committed progenitor cells, from healthy tissue of the patient or of the biliary tree of a non-autologous donor and engrafting the cells into the wall of bile ducts near to the organ to be treated. The graft consists of stem cells or progenitors that are admixed with biomaterials and, optionally, with cytokines and / or native epithelial-mesenchymal cells appropriate for the maturational lineage stage of the cells to be engrafted. The cells are specifically introduced to the hepato-pancreatic common duct of the subject for treatment of pancreatic conditions or to the bile duct wall near to the liver for treatment of liver conditions and allowed to migrate to the pancreas or to the liver and expand and then rebuild part or the entirety of the diseased or dysfunctional organ.

The invention is directed to methods for culturing cells so that the cells are induced to differentiate into cells that express a hepatic stellate phenotype and cells that express a hepatic sinusoidal endothelial phenotype. The invention is also directed to cells produced by the methods of the invention. The cells are useful, among other things, for treatment of liver deficiency, liver metabolism studies, and liver toxicity studies.

The invention relates to the technical field of biomedical engineering. Liver transplantation is the only effective means that can be employed for treating liver diseases in late stage at present and is greatly limited in clinic use due to lack of liver sources. Hepatic stem cells have a self-updating capacity and a bidirectional differentiation capacity of differentiating into hepatocytes and cholangiocytes and therefore can provide an infinite number of donor cells for hepatocyte transplantation treatment in theory. The method for the transdifferentiation of fibroblasts into hepatic stem cells, which is provided by the invention, is to reprogram fibroblasts into hepatic stem cells which have the unique self-updating and bidirectional differentiation capacities of hepatic stem cells by using three transcription factors, namely c-Jun, Foxa2 and Hnf1beta. The inducible hepatic stem cells prepared by the method can be used as ideal cell sources for cell treatment of acute hepatic failure and liver diseases in medium and late stages, can be used as seed cells for medicine screening and tissue engineering livers and can provide an ideal research platform for researching cell biological characteristics of hepatic stem cells, liver development and the like.

The invention discloses a cell sphere gathering culture medium. The cell sphere gathering culture medium comprises a culture area and a fence connected to the periphery of the culture area. The culture area is provided with multiple sphere gathering holes, wherein the lower half portions of the sphere gathering holes are spherical-segment-shaped cavities. The invention further discloses a medium die which comprises a first platy die plate and a second platy die plate, wherein the first platy die plate and the second platy die plate are mutually nested to form a cell sphere gathering culture medium pouring forming cavity. The first die plate is internally provided with a boss and a groove formed adjacent to the boss in the circumferential direction. Multiple protruding points are arranged on the surface of the boss, wherein the upper half portions of the protruding points are spherical segment bodies. A through hole matched with the outer contour of the outer side groove wall of the groove is formed in the second die plate. The invention further discloses a method for conducting animal cell in-vitro three-dimensional sphere gathering culture by utilizing the cell sphere gathering culture medium. The in-vitro three-dimensional culture method for the hepatic cell is relatively low in cost, controllable in scale and simple in process, and the culture effect is greatly superior to that of two-dimensional plate culture.

This invention provides methods and systems for identifying and typing toxicity of chemical compositions, as well as for screening new compositions for toxicity. The invention involves detecting alterations in gene or protein expression and hence establishing molecular profiles in isolated mammalian LSCs contacted with various chemical compositions of known and unknown toxicities, and correlating the molecular profiles with toxicities of the chemical compositions.

The present invention relates to an induced hepatic stem cell defined as follows, a process for production thereof, and applications of the cell, which are useful in safety tests, toxicity tests, metabolism tests, drug interaction tests, antiviral activity tests, screening tests for pharmaceuticals such as hyperlipidemic therapeutics, hypertension therapeutics, low-molecular weight compound medicaments, and antibody medicaments, screening for targets in drug discovery, preparation of animal models, production of hepatocyte-produced proteins, and in regenerative medicine. The induced hepatic stem cell of the present invention is characterized by at least satisfying the following requirements (1)-(3): (1) it expresses at least 15 genes as selected from the group of the genes which are marker genes for an embryonic stem cell; (2) it has properties of a hepatocyte; and (3) it can be subjected to expansion culture or passage culture for at least 3 days.

The invention provides a method for inducing adult liver stem cells for transforming to high metastatic liver cancer cells and the stable liver cancer cells screened by the method. The method comprises the following steps: S1: performing amplification, culture and preservation of mice adult liver stem cells; S2: employing a gene transfection technology for transferring a lentiviral vector containing the Notch1 gene into the mice adult liver stem cells to obtain the mice adult liver stem cells for stable over-expression of the Notch1; S3: using the mice adult liver stem cells for stable over-expression of Notch1, employing a selective condition culture method for culture, performing in-vitro further differentiation, screening the liver cancer cells having strong and stable tumorigenic capability; and S4: employing the screened liver cancer cells, through an in-situ liver transplantation tumor model having same species, liver cancer is formed on liver, double-lung metastasis tumor is spontaneously formed, so that the high metastatic liver cancer cells can be obtained. The screened stable liver cancer cells by the method of the invention have the advantages of short tumor formation time and good tumorigenicity, and have lung metastasis capability.

A method of repairing diseased or dysfunctional pancreas or liver is provided. The method involves preparation of a suspension of stem cells and / or progenitor cells such as biliary tree stem cells, hepatic stem cells, pancreatic stem cells or their descendants, committed progenitor cells, from healthy tissue of the patient or of the biliary tree of a non-autologous donor and engrafting the cells into the wall of bile ducts near to the organ to be treated. The graft consists of stem cells or progenitors that are admixed with biomaterials and, optionally, with cytokines and / or native epithelial-mesenchymal cells appropriate for the maturational lineage stage of the cells to be engrafted. The cells are specifically introduced to the hepato-pancreatic common duct of the subject for treatment of pancreatic conditions or to the bile duct wall near to the liver for treatment of liver conditions and allowed to migrate to the pancreas or to the liver and expand and then rebuild part or the entirety of the diseased or dysfunctional organ.

This application describes liver stem cells (LSC), and differentiated hepatocytes, cholangiocytes, and 3D cellular structures derived therefrom. Methods for producing LSC and mature, differentiated hepatocytes and cholangiocytes in culture are provided. Also provided are cell culture systems and cell culture media for producing a homogenous population of liver stem cells that remain in an undifferentiated state over multiple passages in culture. The LSC and methods are useful for producing homogenous populations of hepatocytes and cholangiocytes for downstream applications. The LSC can be transplanted into subjects to treat liver diseases.

The invention provides a method for preparing hepatic stem cell decorated by an insulin gene, application of the method to preparation of a diabetes preparation and a preparation comprising the hapatic stem cell decorated by the insulin gene. The invention also discloses the method for preparing the hepatic stem cell decorated by the insulin gene and the application of the method. By the preparation prepared from the hapatic stem cell decorated by the insulin gene, which is provided by the invention, pancreatic island beta cell damaged in the diabetes can be repaired in vivo to secrete insulin so as to provide the possibility of overcoming the defect that the diabetes depends on insulin therapy and radically treating the diabetes.

The invention discloses an artificial polypeptide capable of inducing bone mesenchymal stem cells to differentiate into hepatic cells and a biological product of such artificial polypeptide. The artificial polypeptide is a polypeptide containing an amino acid sequence shown in formula (IV): LGENQPDAK(Xa)PCFQEDPMA(Xb)GTDCTLMEIWN, (IV), wherein each of Xa and Xb is selected from M, Y, L, V, W or E. Professionals in the field know that a strong ability to induce the bone mesenchymal stem cells to differentiate into the hepatic cells is achieved in case of combination of acidic fibroblast growth factor, hepatocyte growth factor and oncostatin M, and after induced culture for 14 days, alpha fetal protein mRNA and albumin mRNA in the cells are in remarkably positive expression. It is proven that the artificial polypeptide with the same effect is capable of inducing the bone mesenchymal stem cells to differentiate into the hepatic cells, and accordingly the artificial polypeptide can be prepared into the biological product for inducing the bone mesenchymal stem cells to differentiate into the hepatic cells.

The invention discloses an induction method of Lgr5<+> (leucine-rich repeat-containing g-protein coupled receptor 5<+>) hepatic stem cells, and application thereof in detection. According to the induction method of the Lgr5<+> hepatic stem cells, hepatic injury is recovered through induction of the Lgr5<+> hepatic stem cells by HGF and / or Rspo1; by means of self-renewal and multifunctional differentiating capacity of endogenous stem cells of tissue and organs, the endogenous stem cells of the in-vivo tissue and organs are stimulated and activated directly by soluble molecules, so that self-healing / regeneration of the tissue and the organs is promoted. It is verified that HGF and / or Rspo1 have / has an induction effect on the humanLgr5<+> hepatic stem cells.

The invention discloses a method for inducing fetal hepatic stem cells to differentiate into mature hepatic cells in a three-step sequential mode, and belongs to the technical field of biology. The method comprises the following steps that (1) the fetal hepatic stem cells are obtained and identified, specifically, (a) the fetal hepatic steam cells are separated and amplified, and (b) the fetal hepatic stem cells are identified; (2) the fetal hepatic steam cells are induced to differentiate, specifically, (a) a tissue suspension prepared from normal hepatic tissue which is ground and homogenized is collected on dry ice at a low temperature, supernatant is collected after centrifuging and added into a Williams'E cell culture medium, and the fetal hepatic stem cells in the step (1) are cultivated to obtain hepatic progenitor cells, (b) the cells obtained in the step (a) are continuously cultivated through the Wiliams'E culture medium added with a Notch signal channel inhibitor to obtain hepatocyte-like cells, and (c) the cells obtained in the step (b) are cultivated through a rapid cell expansion culture medium added with HGF to obtain functional hepatic cells. Through the method, thefetal hepatic stem cells obtained early can be induced into the functional hepatic cells rapidly, accurately and efficiently.

This invention provides methods and systems for identifying and typing toxicity of chemical compositions, as well as for screening new compositions for toxicity. The invention involves detecting alterations in gene or protein expression and hence establishing molecular profiles in isolated mammalian LSCs contacted with various chemical compositions of known and unknown toxicities, and correlating the molecular profiles with toxicities of the chemical compositions.

Methods of using hepatic progenitors in treating liver dysfunction are provided. More particularly, methods of using hepatic progenitor cells, including hepatic stem cells, in treating liver dysfunction in the absence of immune-suppressing amounts of an immunosuppressant.