WO2014201986A1 - 建立单克隆间充质干细胞的方法及其应用 - Google Patents

建立单克隆间充质干细胞的方法及其应用 Download PDF

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WO2014201986A1
WO2014201986A1 PCT/CN2014/079970 CN2014079970W WO2014201986A1 WO 2014201986 A1 WO2014201986 A1 WO 2014201986A1 CN 2014079970 W CN2014079970 W CN 2014079970W WO 2014201986 A1 WO2014201986 A1 WO 2014201986A1
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cells
cell
mesenchymal stem
reporter protein
stem cell
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PCT/CN2014/079970
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English (en)
French (fr)
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张文炜
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Zhang Wenwei
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the invention relates to the field of biomedicine.
  • the invention relates to methods of isolating stem cells and uses thereof. More specifically, the present invention relates to a method of isolating stem cells, a stem cell obtained by the method or a derivative thereof, and the use of the stem cell or a derivative thereof for the preparation of a medicament. Background technique
  • MSCs Mesenchymal stem cells
  • BM MSCs Mesenchymal stem cells isolated from bone marrow have been reported as pluripotent cells with high proliferative potential in vitro.
  • BM MSCs can differentiate into adipocytes (A), osteoblasts (0), chondrocytes (C), and vascular smooth muscle (V) lineage, but cannot differentiate into skeletal muscle cells, cardiomyocytes, hematopoietic cells, hepatocytes, or nerve cells.
  • pedigree This suggests that they can be used for cell therapy in many damaged tissues: bone, cartilage, central nervous cells, and myocardial tissue.
  • the main autologous stem cell transplantation technology is widely used in various clinical diseases.
  • the disadvantages are: the bone marrow source is inconvenient and easy to cause autologous damage; the peripheral blood source must rely on exogenous stimuli to make the stem cells reach a certain amount; although the fat source avoids the above disadvantages, there is tumorigenicity and serum in vitro expansion. The pathogenicity of use.
  • the present invention is directed to solving at least some of the above technical problems or at least providing a useful commercial option. To this end, it is an object of the present invention to provide a method for efficiently separating stem cells by which stem cells in a single cell form can be obtained, thereby obtaining a cluster of stem cells formed of a single cell.
  • the invention provides a method of isolating stem cells.
  • the method comprises: A) digesting an animal sample with collagenase to disperse cells contained in the animal sample to obtain a cell mixture comprising stem cells; B) nucleic acid encoding a reporter protein Molecular introduction of the cell mixture In at least a portion of a cell, wherein the nucleic acid molecule encoding a reporter protein is operably linked to a mesenchymal stem cell-specific promoter for expression of the reporter protein in mesenchymal stem cells; and C) utilizing FACS, Mesenchymal stem cells expressing the reporter protein are obtained by sorting, wherein the mesenchymal stem cells are in the form of single cells.
  • the nucleic acid molecule encoding the reporter protein since the nucleic acid molecule encoding the reporter protein is operably linked to the mesenchymal stem cell-specific promoter, the nucleic acid molecule encoding the reporter protein will contain the mesenchyme contained in the cell mixture.
  • Specific expression in stem cells allows mesenchymal cells that specifically express the reporter protein to be efficiently sorted by means of cell flow sorting to obtain mesenchymal stem cells in a single cell form.
  • the inventors have surprisingly found that stem cells obtained by the method of the present invention still have differentiation potential after subculture for several generations compared to conventionally obtained stem cells, and can detect up to 98% of specific markers.
  • the stem cell population obtained by the conventional method can only maintain the differentiation potential within 10 generations of subculture in vitro. When it is subcultured for more than 10 generations in vitro, the differentiation potential of the stem cells is basically lost, and the specific marker that can be detected is decreased. Up to 50%.
  • the aforementioned method may also have the following additional technical features:
  • the animal sample is at least one selected from the group consisting of liver tissue, adipose tissue, bone parenchyma, muscle, cord blood, umbilical cord, placenta, peripheral blood, pancreas, lung, menstrual blood, and pulp.
  • the animal sample is from fetal liver tissue and adult bone marrow tissue.
  • introducing a nucleic acid encoding a reporter protein into at least a portion of the cells of the mixture of cells comprises: culturing the mixture of cells with a liquid medium; introducing the construct into the adherent obtained by the culturing In the cell, wherein the construct comprises: a nucleic acid molecule encoding a reporter protein; and a mesenchymal stem cell-specific promoter, the nucleic acid molecule encoding the reporter protein being operably linked to a mesenchymal stem cell-specific promoter.
  • the nucleic acid molecule encoding the reporter protein can be efficiently introduced into the cell, and the nucleic acid molecule encoding the reporter protein can be efficiently obtained to obtain expression in the mesenchymal stem cell.
  • the method of introducing the construct into the adherent cells obtained by the culture is not particularly limited, and according to a specific example of the present invention, it can be transferred by electroporation, liposome, nanoparticle, PEI or calcium phosphate. At least one of the staining methods introduces the construct into the obtained adherent cells. Thereby, the desired construct can be efficiently introduced into the cells, and the transfection efficiency can be improved.
  • the Nanog-GFP plasmid is transfected into adherent cells using calcium phosphate transfection.
  • the liquid medium is alpha-strontium medium.
  • the medium may also add biological factors such as EGF or FGF2 to maintain optimal biological characteristics and differentiation potential of MSCs.
  • the type of reporter protein that can be employed is not particularly limited as long as it has detectable activity.
  • the reporter protein may be at least one selected from the group consisting of a photoprotein, a fluorescent protein, and an enzyme.
  • the reporter protein may be a protein capable of generating an optical signal A substance such as a photoprotein or a fluorescent protein such as green fluorescent protein or the like, or a reporter protein may be an enzyme capable of interacting with a substrate to produce a detectable signal.
  • the reporter protein can be monitored and the cells expressing the reporter protein can be sorted according to conventional methods.
  • the reporter protein may be at least one of green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), and luciferase.
  • GFP green fluorescent protein
  • EGFP enhanced green fluorescent protein
  • luciferase it is preferred that the reporter protein is an enhanced green fluorescent protein, whereby the fluorescent signal generated by the reporter protein can be detected more easily, thereby making the signal detection more sensitive, thereby, in the present invention
  • the reporter protein is at least one selected from the group consisting of a photoprotein, a fluorescent protein, and an enzyme.
  • the reporter protein is a green fluorescent protein.
  • operably refers to a functional linkage between a nucleic acid expression control sequence, such as a promoter, etc., and a nucleic acid sequence of interest, wherein when a suitable molecule, such as a transcriptional activating molecule, binds to an expression control sequence,
  • a suitable molecule such as a transcriptional activating molecule
  • the expression control sequence affects the transcription and/or translation of the nucleic acid corresponding to the target nucleic acid sequence.
  • a specific mesenchymal stem cell-specific promoter can be introduced into a mesenchymal cell directly by a construct for initiating transcription and expression of a nucleic acid molecule encoding a reporter protein, thereby The expression of the reporter protein in the obtained cells receiving the nucleic acid molecule is increased.
  • a mesenchymal stem cell-specific promoter which can be employed is a human NANOG promoter whose genome sequence is:
  • construct refers to a genetic vector comprising a specific nucleic acid sequence and capable of transferring a nucleic acid sequence of interest into a host cell for homologous recombination with the genome of the host cell to obtain recombination. cell.
  • the form of the construct is not particularly limited, and according to a specific example of the present invention, it may be at least one of a plasmid, a phage, an artificial chromosome, a cosmid, and a virus.
  • the construct is in the form of a plasmid.
  • the plasmid As a genetic vector, the plasmid has the advantages of simple operation, can carry a large fragment, and is convenient for handling and processing.
  • the form of the plasmid is also not particularly limited, and may be either a circular plasmid or a linear plasmid, i.e., either single-stranded or double-stranded. Those skilled in the art can make selections as needed.
  • the invention provides a stem cell or a derivative thereof, which is prepared by the method of any of the preceding claims. Since the stem cells obtained by the method described above are stem cells in the form of single cells, or a monoclonal cluster belonging to stem cells, they have the same genetic background and reduce their immunogenicity when used in animals. In addition, the stem cells or derivatives thereof obtained according to the examples of the present invention are of high purity; The group is single, which is convenient for future genome sequencing; it is not easy to differentiate or differentiate less than non-monoclonal cells, and it is easy to maintain the characteristics and potential of the original MSCs.
  • the invention provides the use of the aforementioned stem cells or derivatives thereof for the preparation of a medicament for the treatment of at least one selected from the group consisting of: blood system diseases, cardiovascular Disease, cirrhosis, nervous system disease, nervous system repair, knee joint meniscus partial resection injury repair, autoimmune disease, spinal cord injury, cerebral palsy, amyotrophic lateral sclerosis, systemic lupus erythematosus, systemic sclerosis, cloning Disease, stroke, diabetes, diabetic foot, scleroderma and cirrhosis.
  • blood system diseases cardiovascular Disease, cirrhosis, nervous system disease, nervous system repair, knee joint meniscus partial resection injury repair, autoimmune disease, spinal cord injury, cerebral palsy, amyotrophic lateral sclerosis, systemic lupus erythematosus, systemic sclerosis, cloning Disease, stroke, diabetes, diabetic foot, scleroderma and cirrhosis.
  • mesenchymal stem cells can be effectively treated, whereby the mesenchymal stem cells obtained by the method of the present invention can be more effectively treated based on the advantages of the aforementioned monoclonal mesenchymal stem cells of the present invention. effect.
  • the invention provides a pharmaceutical composition comprising: the stem cells described above or derivatives thereof; and a pharmaceutically acceptable excipient.
  • Treatment is included in the invention to prevent, alleviate, inhibit or cure a defect, dysfunction, disease or other deleterious process, including interference with therapy and/or a process caused by treatment.
  • stem cells are present in a composition that is suitable for administration, i.e., physiologically compatible.
  • compositions typically also include one or more buffers (eg, neutral buffered saline or phosphate buffered saline), carbohydrates (eg, glucose, mannose, sucrose, or dextran), mannitol, proteins, polypeptides, or amino acids.
  • buffers eg, neutral buffered saline or phosphate buffered saline
  • carbohydrates eg, glucose, mannose, sucrose, or dextran
  • mannitol proteins
  • polypeptides or amino acids.
  • glycine e.g, glutathione
  • an adjuvant such as aluminum hydroxide
  • Solutes, suspending agents, thickeners and/or preservatives may be present in a composition suitable for freezing or storage.
  • the purity of the cells used to administer to the subject is about 100%. In other embodiments, it is between 95% and 100%. In other embodiments, it is between 95% and 100%. Preferably, for mixtures with other cells, the percentage may be about 10%-15%, 15%-20% 20%-25% 25%-30% 30%-35% 35%-40%, 40% -45%, 45%-50%, 60%-70%, 70%-80%, 80%-90% or 90%-95%.
  • the isolation/purity can be expressed in the form of a cell doubling in which the cells have pluried, for example, 10-20, 20-30, 30-40, 40-50 or more. The number of cells in a given volume can be determined by well-known and conventional methods and instruments.
  • the percentage of cells in a given volume of cell mixture can be determined by almost the same method. Cells can be easily counted manually or by using an automated cell counter. A given volume of a particular cell can be determined using specific staining and visual inspection and by automated methods using specific binding reagents (typically antibodies), fluorescent labels, and fluorescent activated cell sorters.
  • a dosage form for administering the cells for a given use is selected depending on a variety of factors. Among these factors, the species of the subject, the disease to be treated The nature of the disease, dysfunction or disease and its status and distribution in the subject, the nature of the other therapies and agents to be administered, the best route of administration, the persistence of the route, the dosing regimen and other The factors that are clear to the skilled person in the field.
  • a suitable carrier and other additives will depend on the exact route of administration and the nature of the particular formulation.
  • cell survival can be an important determinant of the efficacy of cell-based therapies. This is true for both primary and secondary treatments. Another concern arises when the target site is not suitable for cell seeding and cell growth. This may prevent therapeutic cells from reaching the site and/or transplanting there.
  • Various embodiments of the invention include measures to increase cell survival and/or overcome problems caused by vaccination and/or growth barriers.
  • Final formulation of the aqueous suspension of cells/medium generally involves adjusting the ionic strength of the suspension to isotonic (i.e., about 0.1-0.2) and adjusting the pH to physiological pH (i.e., about pH 6.8-7.5).
  • the final formulation typically also contains a liquid lubricant such as maltose, which must be tolerated by the body.
  • exemplary lubricant ingredients include glycerin, glycogen, maltose, and the like.
  • Organic polymer based materials such as polyethylene glycol and hyaluronic acid and non-fibrous collagen (preferably succinated collagen) can also be used as lubricants. These lubricants are commonly used to improve the injection capacity, run-up and dispersibility of the injected biomaterial at the injection site, and to reduce spiking by altering the viscosity of the composition.
  • the final formulation is to confine the cells to a pharmaceutically acceptable carrier. The cells are then placed in a syringe or other injection device for precise injection into the site of tissue defect.
  • the manner in which the cells are injected into the tissue defect site is not particularly limited, and may be various conventional injection methods in the art, and according to a specific embodiment of the present invention, a local injection needle injection or Intravenous injection.
  • injectable means that the formulation is administered in a syringe with a needle as low as 25 gauge under normal pressure and normal conditions, substantially without pushing. Pushing can cause the composition to spill from the syringe rather than being injected into the tissue.
  • needles as small as 27 (200 ⁇ >.) or even 30 (150 ⁇ >.) are required.
  • the maximum particle size that can be extruded through these needles is a complex function with at least the following parameters: maximum particle size, particle aspect ratio (length: width), particle stiffness, particle surface roughness, and factors affecting the viscosity between the particles, The viscoelasticity of the suspension, as well as the flow rate through the needle.
  • the rigid beads suspended in the Newtonian fluid species represent the simplest case, while the fibrous or branched particles in the viscoelastic fluid appear to be much more complicated.
  • the desired isotonicity of the composition can be achieved using sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes.
  • Sodium chloride is particularly preferred for use in buffers containing sodium ions.
  • the viscosity of the composition can be maintained at a selected level using a pharmaceutically acceptable thickening agent.
  • Methylcellulose is preferred because it is conveniently and economically available and easy to use.
  • Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The preferred concentration of the thickening agent will depend on the agent selected. It is important to use the amount to achieve the selected concentration.
  • the viscous composition is usually prepared by adding the thickener to a solution. Pharmaceutically acceptable preservatives or stabilizers can be used to increase the life of the cell/medium composition.
  • the stem cells can be formulated in a unit dosage injectable form, such as a solution, suspension or emulsion.
  • Pharmaceutical preparations suitable for cell injection are typically sterile aqueous solutions and dispersions.
  • the carrier for the injectable preparation may be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, a polyol (e.g., glycerin, propylene glycol, and liquid polyethylene glycol, and the like), and a suitable mixture thereof.
  • a polyol e.g., glycerin, propylene glycol, and liquid polyethylene glycol, and the like
  • One skilled in the art can readily determine the amount of cells and optional additives, vehicles and/or carriers in the compositions to be administered in the methods of the invention.
  • any additive (other than the cells) is present in a solution such as phosphate buffered saline in an amount of from 0.001 wt% to 50 wt%.
  • the active ingredient is present in the order of micrograms to milligrams, for example from about 0.0001 wt% to about 5 wt%, preferably from about 0.0001 wt% to about 1 wt%, most preferably from about 0.0001 wt% to about 0.05 wt%, or about 0.001 wt. % to about 20 wt%, preferably from about 0.01 wt% to about 10 wt%, most preferably from 0.05 wt% to about 5 wt%.
  • the cells are encapsulated for administration, particularly when encapsulation can enhance therapeutic efficacy or provide advantages in handling and/or shelf life.
  • encapsulation provides for the subject's immune response to further reduce the subject's immune response to the cells (usually not immunogenic or only weakly immunogenic in allogeneic transplantation) The barrier of the immune system, thereby alleviating any transplant rejection or inflammation that may occur as a result of administration of the cells.
  • Cells can be encapsulated by membranes and capsules prior to implantation.
  • the cells are individually encapsulated. In certain embodiments, many cells are encapsulated within the same membrane. In embodiments in which the cells are removed after implantation, a larger sized structure, such as encapsulating a plurality of cells in a single membrane, may provide a convenient method of recovery. A variety of materials can be used in various embodiments of microencapsulated cells.
  • polymer capsules include, for example, polymer capsules, alginate-poly-L-lysine-alginate microcapsules, poly-L-lysine alginate capsules, alginate capsules, polyacrylonitrile/polyvinyl chloride (PAN/PVC) hollow fiber and polyethersulfone (PES) hollow fiber.
  • PAN/PVC polyacrylonitrile/polyvinyl chloride
  • PES polyethersulfone
  • Certain embodiments incorporate cells into a polymer, such as a biopolymer or a synthetic polymer.
  • biopolymers include, but are not limited to, fibronectin, fibrin, fibrinogen, thrombin, collagen, and proteoglycans. Other factors such as the cytokines discussed above can also be added to the polymer.
  • cells can be added to the voids of the three-dimensional gel.
  • Large polymers or gels are usually implanted by surgery. Polymers or gels that can be formulated into sufficiently small particles or fibers can be administered by other conventional, more convenient, non-surgical routes.
  • the cells can be encapsulated in a device that can be implanted into a subject. Cells can be implanted into the liver or near the liver or elsewhere to replace or supplement liver function. Cells can also be implanted without the device, such as in existing liver tissue.
  • composition may be administered in dosages and according to techniques well known to those skilled in the medical and veterinary arts, depending on factors such as the age, sex, weight and condition of the particular patient, and the formulation (e.g., solid or liquid) to be administered.
  • Human or other Mammalian dosages can be derived by the skilled artisan in light of the present disclosure, the literature cited herein, and the knowledge in the art without undue experimentation.
  • the dosage of cells suitable for use in various embodiments of the invention depends on a variety of factors. It can vary considerably for different situations.
  • Parameters that determine the optimal dosing dose for primary and adjuvant therapy include some or all of the following: the disease to be treated and its stage; the species of the subject, their health, sex, age, weight, and metabolic rate; Immune activity; other therapies being administered; and possible complications predicted based on the subject's history or genotype.
  • the parameters may also include: whether the cells are isogenic, autologous, allogeneic or xenogeneic; their potency (specific activity); sites and/or distributions that are effective for the cells to be targeted And these properties of the site such as cell accessibility and/or cell implantability.
  • Other parameters include co-administration with other factors such as growth factors and cytokines.
  • the optimal dosage for a given situation also takes into account the manner in which the cell preparations are administered, the manner in which they are administered, and the extent to which the cells are localized to the target site after administration. Finally, the determination of the optimal dose must provide an effective dose that is neither below the threshold of the maximum benefit nor above the threshold of the dose-related detrimental effect exceeding the benefit of increasing the dose.
  • the optimal dose of cells is within the dosage range for autologous, mononuclear bone marrow transplantation.
  • the optimal dose for each administration may be from 10 4 to 10 8 cells/kg of recipient mass. In certain embodiments, the optimal dose per administration is from 10 5 to 10 7 cells/kg.
  • the optimal dose per administration is 5 X 10 5 ⁇ 5 X 10 6 cells/kg.
  • the aforementioned higher doses are similar to the doses of nucleated cells used for autologous mononuclear bone marrow transplantation. Some lower doses are similar to the amount of CD34+ used for autologous mononuclear bone marrow transplantation. It will be understood that a single dose may be delivered in one portion, in portions, or continuously over a period of time. The entire dose can also be delivered to one location or to several locations.
  • the cells can be administered in an initial dose and then maintained by re-administration.
  • the cells can be administered initially by one method and then administered by the same method or by one or more different methods.
  • the level can be maintained by continuing to administer the cells.
  • administer the cells at the beginning by intravenous injection and/or maintain their levels in the subject.
  • other modes of administration may be used depending on the condition of the patient and other factors discussed elsewhere herein. It should be noted that human subjects are generally treated longer than experimental animals, however, the length of treatment is usually proportional to the length of the disease process and therapeutic effect.
  • the cells are administered to the subject in one dose. In other embodiments, the cells are administered to the subject continuously in a series of two or more doses. Others administered to the cells in one dose, two doses, and/or more than two doses In some embodiments, the dosages may be the same or different and the intervals between administrations may be the same or different.
  • Cells can be administered at a variety of frequencies over a variety of different times. In certain embodiments, the cells are administered in less than one day. In other embodiments, they are administered over a period of 2, 3, 4, 5 or 6 days. In certain embodiments, the cells are administered once or more times per week over a period of weeks.
  • the cells are administered for a period of one to several months over a period of weeks. In various embodiments, the cells can be administered over a period of months. In other embodiments, the cells can be administered over a period of one or several years.
  • the length of treatment is proportional to the length of the disease process, the effect of the therapy used, and the condition and response of the subject to be treated.
  • the isolated mesenchymal stem cells may have the ability to be induced to differentiate to form at least one cell selected from the group consisting of osteoblasts, chondrocytes, adipocytes, fibroblasts, bone marrow stroma, bones Muscle, smooth muscle, myocardium, endothelial cells, epithelial cells, hematopoietic cells, glial cells, neuronal cells or oligodendrocyte types.
  • the present invention also provides a differentiated cell obtained from the mesenchymal cell, wherein the progeny cell can be bone, cartilage, adipocyte, fibroblast, bone marrow stroma, skeletal muscle, smooth muscle, cardiac muscle, endothelial cell, epithelial cell, endocrine cell , exocrine cells, hematopoietic cells, glial cells, neuronal cells or oligodendrocytes.
  • the progeny cell can be bone, cartilage, adipocyte, fibroblast, bone marrow stroma, skeletal muscle, smooth muscle, cardiac muscle, endothelial cell, epithelial cell, endocrine cell , exocrine cells, hematopoietic cells, glial cells, neuronal cells or oligodendrocytes.
  • the differentiated progeny cells may be skin epithelial cells, hepatic epithelial cells, pancreatic epithelial cells, pancreatic endocrine or islet cells, pancreatic exocrine cells, intestinal epithelial cells, renal epithelial cells or epithelial-related structures.
  • Cells or their differentiated progeny can be used to correct genetic diseases, degenerative diseases, cardiovascular diseases, metabolic storage diseases, neurological diseases or cancer processes. They can be used to produce gingival-like materials for the treatment of periodontal disease. They can be used to form skin epithelial tissue derived from cells useful for skin grafting and orthopedic surgery. They can be used, for example, to strengthen muscles in the penis or heart.
  • Optic nerve cells can be used to treat blindness caused by, but not limited to, optic nerve diseases caused by, but not limited to, macular degeneration, diabetic retinopathy, glaucoma, retinitis pigmentosa.
  • the cells or cardiomyocytes derived from the cells can be used to treat heart disease including, but not limited to, myocarditis, cardiomyopathy, heart failure, damage caused by a heart attack, hypertension, atherosclerosis, and heart valve insufficiency. . They can also be used to treat diseases involving CNS defects or injuries. Furthermore, the stem cells or their neuronal differentiated progeny cells can be used to treat diseases involving neurological deficits or degeneration, including but not limited to stroke, Alzheimer's disease, Parkinson's disease, Huntington's disease, AIDS-related dementia, spinal cord A metabolic disease that affects the brain or other nervous tissues.
  • Cells or their differentiated progeny can be used to support the growth and differentiation of other cell types in vivo or in vitro, including but not limited to hematopoietic cells, pancreatic islet cells or beta cells, hepatocytes, and the like.
  • the cells or differentiated cartilage progeny can be used to treat joint or cartilage diseases including, but not limited to, cartilage tears, cartilage thinning, osteoarthritis.
  • the cells or their differentiated osteoblast progeny can be used to improve the process of detrimental effects on bone, including but not limited to fractures, fracture nonunion, osteoarthritis, Bone "holes" caused by tumors scattered to the bone (eg, prostate cancer, breast cancer, multiple myeloma, etc.).
  • suitable growth factors, chemokines, and cytokines cells can be induced to differentiate into several lineages, including, for example, a variety of cells with a mesodermal phenotype, cells with a neuroectodermal phenotype (glial cells, less Microglia and neurons) and cells with a blastoderm phenotype.
  • osteoblasts include osteoblasts, chondrocytes, adipocytes, cartilage and bone, skeletal muscle, smooth muscle, myocardium, endothelial cells, hematopoietic cells, stromal cells, neuronal cells, and epithelial cells.
  • Cells that have been induced to differentiate to form osteocytes can be found in osteoporosis, Paget's disease, fractures, osteomyelitis, osteonecrosis, achondroplasia, osteogenesis imperfecta, hereditary multiple osteochondroma , multiple epiphyseal dysplasia, Marian's syndrome, mucopolysaccharidosis, neurofibromatosis or scoliosis for cell therapy or for tissue regeneration, local deformity, spina bifida, hemi-vertebral Reconstruction of the body or fusion vertebrae, limb malformation, reconstruction of tumor injury tissue, and reconstruction after infection (eg, otitis media).
  • osteoporosis Paget's disease, fractures, osteomyelitis, osteonecrosis, achondroplasia, osteogenesis imperfecta, hereditary multiple osteochondroma , multiple epiphyseal dysplasia, Marian's syndrome, mucopolysaccharidosis, neuro
  • Cells that have been induced to differentiate to form chondrocytes can be used for age-related diseases or injuries, tissue regeneration in exercise-related injuries, or specific diseases such as rheumatoid arthritis, psoriatic arthritis, and Reiter's arthritis. ;), ulcerative colitis, Crohn's disease;, ankylosing spondylitis, cell therapy or tissue regeneration in osteoarthritis; reconstruction of the external ear; reconstruction of the nose; and reconstruction of the cartilage.
  • Cells that have been induced to differentiate to form adipocytes can be used for reconstruction or remodeling of cosmetic surgery, including, but not limited to, breast reconstruction after mastectomy, tissue loss due to other procedures such as removal of tumor from the face or hand. Plastic surgery, breast augmentation and wrinkle removal.
  • Fibroblasts can be used in cell therapy or tissue repair to promote wound healing or to provide connective tissue supports, such as stents for cosmetic surgery.
  • Cells that have been induced to differentiate to form skeletal muscle cells can be used for Duchenne muscular dystrophy, Beckermus dystrophy, myotonic dystrophy, tissue repair in the treatment of musculoskeletal disorders, and Reconstructive surgery to repair skeletal muscle damage.
  • Cells that have been induced to differentiate to form smooth muscle cells can be used for cell therapy or tissue repair in the treatment of gastrointestinal dysplasia such as esophageal atresia, intestinal atresia, and intussusception, as well as tissue replacement after intestinal infarction or colostomy surgery.
  • Smooth muscle cells can also be used for bladder or uterine reconstruction, neovascularization, repair of vascular damage caused by, for example, atherosclerosis or aneurysms.
  • Smooth muscle precursor cells glomerular cells
  • Smooth muscle precursors can be used as an in vitro model of glomerular disease or for cell therapy or tissue regeneration in diabetic neuropathy. Smooth muscle precursors can also be used to repair dense plaques in distal convoluted tubules or para-glomerular tissues.
  • Cardiomyocytes can be used to treat cell therapy or tissue repair after myocardial infarction, with congestive heart failure, during valve replacement, caused by congenital heart abnormalities, or by cardiac tissue damage caused by cardiomyopathy or endocarditis.
  • Microglia can be used to treat spinal cord injury and neurodegenerative disorders such as Huntington's disease, Parkinson's disease, multiple sclerosis and Alzheimer's disease, as well as infectious diseases affecting the central nervous system. Repair of damaged tissue during the process.
  • Microglial cells that are genetically altered to produce cytokines can also be used for transplantation to treat infectious diseases in the central nervous system where the channel is restricted by the blood-brain barrier.
  • Glial cells can also be produced by multiple sclerosis, muscle atrophy Lateral cord sclerosis and brain cancer caused by regeneration of nerve tissue after stroke and regeneration of growth factor or growth factor inhibitor after spinal cord injury.
  • Stromal cells Stromal cells can be used as transplanted cells for bone marrow replacement and bone marrow transplantation after chemotherapy.
  • Endothelial cells can be used to treat Factor VIII deficiency and for neovascularization. Endothelial cells can also provide in vitro models of inhibition of tumors using angiogenesis inhibitors, as well as in vitro models of vasculitis, hypersensitivity, and coagulopathy.
  • Hematopoietic cells Hematopoietic cells can be used to proliferate bone marrow after high-dose chemotherapy.
  • Hematopoietic cells derived from the aggregated cells can be further differentiated to form blood cells for storage in a blood bank, alleviating the problem of insufficient blood transfusion.
  • Microglia can be used to treat spinal cord injury and neurodegenerative disorders such as Huntington's disease, Parkinson's disease, multiple sclerosis and Alzheimer's disease, as well as damage tissue in infectious diseases affecting the central nervous system. repair.
  • Microglial cells that are genetically altered to produce cytokines can also be used for transplantation to treat infectious diseases in the central nervous system whose channels are restricted by the blood-brain barrier.
  • Glial cells can also produce growth factors or growth factor inhibitors that are regenerated by post-stroke nerve tissue and regenerated after spinal cord injury caused by multiple sclerosis, amyotrophic lateral sclerosis, and brain cancer.
  • Cells that are induced to form oligodendrocytes and astrocytes, for example, can be used for transplantation into demyelinated tissues, especially the spinal cord, where their function is to add myelination to peripheral nerve tissue.
  • the cells can also be used in cell replacement therapy and/or gene therapy to treat congenital neurodegenerative disorders or storage disorders such as mucopolysaccharidosis, leukoaraiosis (spheroidal leukodystrophy, canadian disease) Canavan's disease)), fucoidemia, GM2 gangliosidosis, Niemann-Pick;, Sanfilippo syndrome, Wolman's disease ) and Tay Sachs; They can also be used in traumatic diseases such as stroke, central nervous system hemorrhage and central nervous system trauma; for peripheral nervous system diseases such as spinal cord injury or syringomyelia; for retinal diseases such as retinal detachment, macular degeneration and other degenerative retinal diseases , as well as diabetic retinopathy.
  • congenital neurodegenerative disorders or storage disorders such as mucopolysaccharidosis, leukoaraiosis (spheroidal leukodystrophy, canadian disease) Canavan's disease)), fuco
  • Ectoderm Epithelial Cells Cells can be used in cell replacement therapy and/or gene therapy to treat or alleviate symptoms of skin diseases such as hair loss, skin defects such as skin burns and albinism. Epithelial cells can be used in cell replacement therapy and/or gene therapy to treat or ameliorate the symptoms of several organ diseases.
  • the cells can be used to treat or alleviate congenital liver diseases, for example, storage diseases such as mucopolysaccharidosis, leukodystrophy, GM2 gangliosidosis; bilirubin increases lesions, such as Kegler - Crigler-Najjar syndrome; ammonia diseases such as congenital disorders of the urea cycle such as ornithine decarboxylase deficiency, citrullinemia and arginine succinate; amino acids and organic acids Congenital disorders such as phenylketonuria, hereditary tyrosinemia and alpha antitrypsin deficiency; and coagulopathy such as factor VIII and IX deficiency.
  • storage diseases such as mucopolysaccharidosis, leukodystrophy, GM2 gangliosidosis
  • bilirubin increases lesions, such as Kegler - Crigler-Najjar syndrome
  • ammonia diseases such as congenital disorders of the urea
  • the cells can also be used to treat acquired liver disease caused by viral infection.
  • the cells can also be used in vitro, for example to produce an artificial liver, to produce coagulation factors, and to produce proteins or enzymes produced by liver epithelial cells.
  • the epithelial cells can also be used in cell replacement therapy and/or gene therapy to treat or alleviate the symptoms of biliary diseases such as biliary cirrhosis and biliary atresia.
  • the epithelial cells can also be used in cell replacement therapy and/or gene therapy to treat or ameliorate symptoms of pancreatic disorders such as pancreatic atresia, pancreatitis, and alpha antitrypsin deficiency.
  • pancreatic epithelial cells and nerve cells when pancreatic epithelial cells and nerve cells are available, ⁇ cells can be produced. These cells can be used for the treatment of diabetes (subcutaneous implantation or intracavitary or intrahepatic implantation;). In addition, the epithelial cells are also It can be used in cell replacement therapy and/or gene therapy to treat or alleviate symptoms of intestinal epithelial disorders such as intestinal atresia, inflammatory bowel disease, intestinal infarction, and bowel resection.
  • Cells can be used for tissue repair: Cells can also be used for tissue repair. Cells can be implanted into the bone to enhance the repair process to strengthen the weakened bone or re-cover the joint. Chondrocytes can be injected into the joint to re-cover the articular cartilage.
  • Caplan et al. (U.S. Patent 5,855,619) describes a biological matrix implant comprising a shrink gel matrix into which mesenchymal hepatocytes have been added.
  • the implant is intended to repair tissue defects, especially tendons, ligaments, meniscus or muscle damage.
  • cartilage can be formed by adding chondrocytes to a peripheral region of a porous, three-dimensional scaffold made of, for example, collagen, synthetic polyglycolic acid fibers, or synthetic polylactic acid fibers.
  • the inventors have demonstrated that the cells of the invention differentiate to form chondrocytes, for example, which may be deposited in or around collagen, synthetic polyglycolic acid, synthetic polylactic acid or other scaffolding materials to provide an implant to facilitate tissue repair.
  • Figure 1 shows the analysis of transfected MSCs cells by flow cytometry according to an embodiment of the present invention.
  • FIG. 2 shows the expression of GFP in transfected MSCs cells observed by fluorescence microscopy according to an embodiment of the present invention
  • Figure 3 shows an optical microscopic view of monoclonal MSCs cultured in 96-well plates after sorting according to an embodiment of the present invention
  • Figure 4 is a graph showing the expression of VIMENTIN gene (4A), ⁇ -SMA gene (4B) and P CNN1 gene (4C) in transfected MSCs cells by fluorescence microscopy according to an embodiment of the present invention.
  • the different tissues derived from the animals were separately digested with collagenase. After centrifugation, the supernatant was removed, and the collected cells were washed twice with PBS and then cultured in complete medium. The MSCs adhered rapidly and 24 hours later. The fluid was exchanged and the remaining suspended cells were removed to obtain all adherent cells.
  • the complete medium includes ⁇ - ⁇ medium, 10% fetal bovine serum.
  • the newly isolated adherent cells of Example 1 were transfected with the invitrogen liposome 2000 transfection kit as follows:
  • Example 1 The newly isolated adherent cells in Example 1 were cultured for 24 hours, and the culture was continued after the exchange.
  • the cells were cultured in an antibiotic-free medium before transfection, and the cells were fused 70%-80%, and the plasmid containing the Nanog promoter was
  • the control plasmid PRRL.sin.PGK.GFP
  • liposome (lipofectin regeant, LR) reagent is cationic liposome N-[l,2,3-Dioleyoxy Propyl]-n, n, n-Trimethylammonium Chi oride (DOTMA) and Dioleoyl photidye-
  • DOPE thanolamine
  • FIG. 1 shows the results of transfection of the cells obtained from Example 1 transfected with the human NANOG promoter sequence and GFP under a fluorescence microscope green polarizer.
  • the transfected adherent cells were transferred from the culture dish with PBS, and after washing, they were detected by a FACS flow cytometer under an argon laser at 488 nm.
  • the Cell-Quest software (Weasel V2.3.1) was used to analyze 10,000 cells in the sample, and cells expressing the reporter protein GFP were successfully collected.
  • the individual cells after sorting were cultured in a 96-well plate at 37 ° C and 5% C0 2 in the above-mentioned complete medium, and the liquid was changed every 3 to 5 days. It was observed that the early single cloned cells grew slowly, but Cell after one week After presenting the colony group, it grows rapidly and grows like a typical mesenchymal stem cell, which is fusiform, vortex-like, and radial growth. The results are shown in Fig. 3.
  • the differentiated cells were cultured in Chinese called culture chamber slides (B&D).
  • the fused cell layer was fixed with a 4% (vol/vol) paraformaldehyde solution dissolved in phosphate buffered saline (PBS) for 15 minutes at room temperature.
  • PBS phosphate buffered saline
  • the paraformaldehyde solution was discarded and washed and added to the cells to dissolve in PBS.
  • 0.1% Triton 100X and incubated for 10 minutes at room temperature for protein expression in the nucleus.
  • the cytoskeletal protein was fixed with methanol at -20 ° C for 5 minutes.

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Abstract

本发明提供一种分离间充质干细胞的方法，包括：A）利用胶原酶对动物样本进行消化，分散所述动物样本中所包含的细胞，以便获得包含干细胞的细胞混合物；B）将编码报告蛋白的核酸分子引入所述细胞混合物的至少一部分细胞中，其中，所述编码报告蛋白的核酸分子与间充质干细胞特异性启动子可操纵地相连，以便在间充质干细胞中表达所述报告蛋白；以及C）利用FACS，分选获得表达所述报告蛋白的间充质干细胞，其中，所述间充质干细胞呈单个细胞的形式。

Description

建立单克隆间充质干细胞的方法及其应用
技术领域
本发明涉及生物医学领域。 具体的， 本发明涉及分离干细胞的方法及其用途。 更具体 的， 本发明涉及分离干细胞的方法， 利用该方法所得到的干细胞或其衍生物， 以及该干细 胞或其衍生物在制备药物中的用途。 背景技术
间充质干细胞（MSCs)最近受到了广泛关注， 因为其具有有利于移植的性质， 即其具 有多潜能和非免疫原性。 已经报道了可以从成人骨髓和胎儿组织分离获得间充质干细胞 (MSCs)。 从骨髓分离的 MSCs (BM MSCs) 已经被报道为在体外具有高增殖潜能的多潜 能细胞。 BM MSCs能够分化为脂肪细胞 (A)、成骨细胞 (0)、软骨细胞 (C)以及血管平滑肌 (V) 谱系， 但不能分化成骨骼肌细胞、 心肌细胞、 造血细胞、 肝细胞或神经细胞谱系。 这暗示 了他们可以用于众多受损组织的细胞治疗： 骨骼、 软骨、 中枢神经细胞和心肌组织。
首先， 从间充质干细胞的体外分离方法上来看， 主要有贴壁法， 密度梯度离心法， 流 式细胞术和免疫磁珠分离法。 前两种方法的缺点主要在于细胞纯度较低； 后两种方法的共 同缺点在于 1 )没有真正特异性的细胞表面标记； 2)对细胞活性有较大影响， 易造成 MSCs 的损伤， 出现增殖缓慢等问题； 3 )操作复杂， 价格昂贵， 一般仅限于实验室应用。 这些存 在的技术瓶颈问题在很大程度上限制了 MSCs的临床应用与发展。 然而， 目前还没有能够 分离单克隆间充质干细胞的方法。 其次， 从间充质干细胞临床运用角度来看， 目前主要开 展的自体干细胞移植技术在临床各类疾病中得到广泛运用。 但缺点在于： 骨髓来源取材不 便， 易造成自体损伤； 外周血来源必须借助外源性剌激， 使干细胞达到一定数量； 脂肪来 源虽避免了上述劣势， 但存在体外扩增的致瘤性及血清使用的致病性。
因此， 建立单克隆间充质干细胞的方法有待于进一步地改进。 发明内容
本发明旨在至少在一定程度上解决上述技术问题之一或至少提供一种有用的商业选 择。 为此， 本发明的一个目的在于提出一种能够有效分离干细胞的方法， 利用该方法能够 获得单个细胞形式的干细胞， 进而可以获得由单个细胞形成的干细胞单克隆集群。
在本发明的第一方面， 本发明提出了一种分离干细胞的方法。 根据本发明的实施例， 该方法包括： A) 利用胶原酶对动物样本进行消化， 以便分散所述动物样本中所包含的细 胞， 以便获得包含干细胞的细胞混合物； B)将编码报告蛋白的核酸分子引入所述细胞混合 物的至少一部分细胞中， 其中， 所述编码报告蛋白的核酸分子与间充质干细胞特异性启动 子可操纵地相连， 以便在间充质干细胞中表达所述报告蛋白； 以及 C)利用 FACS, 分选获 得表达所述报告蛋白的间充质干细胞， 其中， 所述间充质干细胞呈单个细胞的形式。
根据本发明实施例的方法， 由于编码报告蛋白的核酸分子与间充质干细胞特异性启动 子可操纵地相连， 因此， 该编码报告蛋白的核酸分子将会在细胞混合物中所包含的间充质 干细胞中得到特异性表达， 从而利用细胞流式分选手段， 可以对特异性表达报告蛋白的间 充质细胞进行有效分选， 从而得到单个细胞形式的间充质干细胞。 发明人惊奇地发现， 利 用本发明实施例的方法获得的干细胞与常规获得的干细胞相比， 在体外传代培养几十代后 的仍然具有分化潜能， 且可以检测到高达 98%的特异性标志物， 而利用常规方法获得的干 细胞群体只能在体外传代培养 10代以内而保持分化潜能， 当其在体外传代培养超过 10代 后该干细胞的分化潜能基本丢失， 可以检测到的特异性标志物下降到 50%。
根据本发明的实施例， 前述方法还可以具有下列附加技术特征：
在本发明的一个实施例中， 所述动物样本为来自选自肝脏组织、 脂肪组织、 骨实质、 肌肉、 脐带血、 脐带、 胎盘、 外周血、 胰腺、 肺脏、 经血和牙髓的至少一种。 可选的， 所 述动物样本为来自胎儿肝脏组织及成人骨髓组织。 从而可以有效地提高分离间充质干细胞 的效率。
在本发明的一个实施例中， 将编码报告蛋白的核酸引入所述细胞混合物的至少一部分 细胞中包括： 利用液体培养基对所述细胞混合物进行培养； 将构建体引入所述培养获得的 贴壁细胞中， 其中， 所述构建体包括： 编码报告蛋白的核酸分子； 以及间充质干细胞特异 性启动子， 所述编码报告蛋白的核酸分子与间充质干细胞特异性启动子可操纵地相连。 由 此， 可以有效地将编码报告蛋白的核酸分子引入到细胞中， 并且可以使得编码报告蛋白的 核酸分子能够有效地在间充质干细胞中获得表达。 根据本发明的实施例， 将构建体引入所 述培养获得的贴壁细胞中的方法不受特别限制， 根据本发明的具体事例， 可以通过电转、 脂质体、 纳米颗粒、 PEI或磷酸钙转染法的至少一种将所述构建体引入所获得的贴壁细胞。 由此， 可以有效地将目的构建体引入到细胞中， 提高转染效率。 根据本发明的一个实施例， 利用磷酸钙转染法将 Nanog-GFP质粒转染到贴壁细胞中。
在本发明的一个实施例中， 所述液体培养基为 α-ΜΕΜ培养基。 由此， 可以进一步提高 后续分离间充质干细胞的效率。 根据本发明的实施例， 该培养基还可以添加 EGF或 FGF2 等生物因子， 从而维持 MSCs的最佳生物学特性及分化潜能。
根据本发明的实施例， 可以采用的报告蛋白的类型不受特别限制， 只要其具有可检测 的活性即可。 根据本发明的一些实施例， 报告蛋白可以为选自发光蛋白、 荧光蛋白、 酶的 至少一种。 具体地， 根据本发明的实施例， 报告蛋白可以是一种能够产生光学信号的蛋白 质例如发光蛋白或者荧光蛋白例如绿色荧光蛋白等， 或者报告蛋白可以是一种能够与底物 相互作用以产生可检测信号的酶。 由此， 可以根据常规的方法， 对报告蛋白进行监测和对 表达报告蛋白的细胞进行分选。 根据本发明的一些具体示例， 优选地， 报告蛋白可以为绿 色荧光蛋白 （GFP)、 增强型绿色荧光蛋白 （EGFP) 以及荧光素酶的至少一种。 根据本发 明的一个具体示例， 优选报告蛋白为增强型绿色荧光蛋白， 由此， 报告蛋白产生的荧光信 号能够更容易地被检测到， 从而使信号检测更加灵敏， 由此， 在本发明的一个实施例中， 所述报告蛋白为选自发光蛋白、 荧光蛋白、 酶的至少一种。 可选的， 所述报告蛋白为绿色 荧光蛋白。
在本发明中所使用的术语"可操纵地"指的是核酸表达控制序列例如启动子等与目标核 酸序列之间的功能连接， 其中当合适的分子例如转录活化分子与表达控制序列结合时， 表 达控制序列影响着对应于目标核酸序列的核酸的转录和 /或翻译。 由此， 根据本发明的实施 例， 可以直接通过构建体在间充质细胞中引入特定的间充质干细胞特异性启动子用于启动 编码报道蛋白的核酸分子的转录和表达， 由此， 可以提高所获得的接受该核酸分子的细胞 中报告蛋白的表达。 根据本发明的具体示例， 可以采用的间充质干细胞特异性启动子为人 NANOG启动子， 其基因组序列为：
Figure imgf000004_0001
GCCTCATGTTATTAT-3
在本发明中所使用的术语"构建体"是指这样的一种遗传载体， 其包含特定核酸序列， 并且能够将目的核酸序列转入宿主细胞中与宿主细胞的基因组发生同源重组以获得重组细 胞。 根据本发明的实施例， 构建体的形式不受特别限制， 根据本发明的具体示例， 其可以 为质粒、 噬菌体、人工染色体、粘粒（Cosmid)、病毒的至少一种。根据本发明的具体示例， 构建体呈质粒的形式。 质粒作为遗传载体， 具有操作简单， 可以携带较大片段的性质， 便 于操作和处理。 质粒的形式也不受特别限制， 既可以是环形质粒， 也可以是线性质粒， 即 可以是单链的， 也可以是双链的。 本领域技术人员可以根据需要进行选择。
在本发明的第二方面， 本发明提出了一种干细胞或其衍生物， 所述干细胞是通过前面 任一项所述的方法制备的。 由于利用前面所述的方法所得到的干细胞是单细胞形式的干细 胞， 或者属于干细胞的单克隆集群， 因而其具有相同的遗传背景， 降低了其在用于动物体 时的免疫原性。 另外， 利用根据本发明的实施例所获得的干细胞或其衍生物纯度高； 基因 组单一性，便于今后基因组测序;不易分化或分化程度较非单克隆细胞低，易保持原有 MSCs 的特性与潜能。
因而， 在本发明的第三方面， 本发明提出了前面所述的干细胞或其衍生物在制备药物 中的用途， 所述药物用于治疗选自下列的至少一种： 血液系统疾病、 心血管疾病、 肝硬化、 神经系统疾病、 神经系统修复、 膝关节半月板部分切除损伤修复、 自身免疫性疾病、 脊髓 损伤、 脑瘫、 肌萎缩侧索硬化症、 系统性红斑狼疮、 系统性硬化症、 克隆氏病、 中风、 糖 尿病、 糖尿病足、 硬皮病和肝硬化。 这些疾病已经被证实， 可利用间充质干细胞进行有效 治疗， 从而利用根据本发明方法所得到的间充质干细胞， 基于前述本发明单克隆间充质干 细胞的优点， 将能够获得更有效地治疗效果。
为此， 在本发明的第四方面， 本发明提出了一种药物组合物， 其特征在于， 包括： 前 面所述的干细胞或其衍生物； 以及药物上可以接受的赋形剂。
"治疗 "在本发明中包括预防、 缓解、 抑制或治愈缺陷、 功能障碍、 疾病或其他有害过 程， 包括干扰治疗和 /或由治疗引起的过程。
由本文所述方法产生的单个形式的细胞或单克隆干细胞都可用于临床以处理受试者。 "受试者"是脊椎动物， 优选哺乳动物， 更优选人。 哺乳动物包括但不限于人、 家畜、 竞技 动物和宠物。 需要用本发明的方法治疗的受试者包括由于物理损伤或疾病有关的损伤而患 有功能丧失的受试者。 因此， 可将它们制剂成药物组合物。 因此， 在某些实施例中， 干细 胞存在于适于给药即生理上相容的组合物中。 因此， 组合物通常还包括一种或多种缓冲剂 (例如中性缓冲盐水或磷酸缓冲盐水)、 碳水化合物 (例如葡萄糖、 甘露糖、 蔗糖或葡聚糖)、 甘露醇、 蛋白、 多肽或氨基酸例如甘氨酸、 抗氧化剂、 抑菌剂、 螯合剂例如 EDTA或谷胱 甘肽、 佐剂 (例如氢氧化铝)、 使所述制剂与接体的血液相比等渗、 低渗或稍微高渗的溶质、 助悬剂、 增稠剂和 /或防腐剂。 在其他实施例中， 细胞可以存在于适于冷冻或储藏的组合物 中。 在许多实施例中， 用于给予受试者的细胞的纯度为约 100%。 在其他实施例中， 其为 95%-100%。 在其他实施方式中， 其为 95%-100%。 优选地， 对于与其他细胞的混合物， 所 述百分比可为约 10%-15%、 15%-20% 20%-25% 25%-30% 30%-35% 35%-40%、 40%-45%、 45%-50%、 60%-70%、 70%-80%、 80%-90%或 90%-95%。 或者， 分离 /纯度可以细胞倍增的 形式表示， 其中细胞已发生例如 10-20、 20-30、 30-40、 40-50或更多倍的细胞倍增。 给定 体积的细胞数量可通过广为人知的和常规的方法和仪器测定。 给定体积的细胞混合物中的 细胞的百分数可通过几乎一样的方法测定。 可手动或通过使用自动细胞计数器容易地对细 胞进行计数。 给定体积的特定细胞可使用特异性染色和目测以及通过使用特异性结合试剂 (一般为抗体)、荧光标记和荧光激活细胞分选器的自动化方法而测定。倚赖多种因素选择用 于为给定用途给予所述细胞的剂型。 在这些因素中， 重要的是受试者的物种， 待治疗的病 症、 功能障碍或疾病的性质及其在受试者中的状态和分布， 其他待给予的疗法和试剂的性 质， 给药的最佳途径， 经过该途径的残存性， 给药方案以及其他本领域技术人员明了的因 素。 例如， 具体地， 合适载体和其他添加剂的选择会倚赖给药的确切途径和特定剂型的性 质。 例如， 细胞存活可为基于细胞的疗法的效力的一个重要决定因素。 这对于主要治疗和 辅助治疗都是成立的。 当目标位点不适于细胞接种和细胞生长时会产生另一个担心。 这可 能会阻止治疗性细胞到达该位点和 /或移植到那里。 本发明的多个实施例包括增加细胞存活 和 /或克服因接种和 /或生长屏障而引起的问题的措施。 细胞 /培养基的水性悬液的最终配制 一般包括将所述悬液的离子强度调节到等渗 (即约 0.1-0.2)以及将 pH调节到生理 pH (即约 pH 6.8-7.5)。 所述最终制剂通常还含有液体润滑剂例如麦芽糖， 其必须为身体所耐受。 示例性 润滑剂成分包括甘油、 糖原、 麦芽糖等。 基于有机聚合物的材料， 例如聚乙二醇和透明质 酸以及非纤维状胶原 (优选琥珀酸化的胶原)，也可用作润滑剂。这些润滑剂通常用于在注射 位置上改善所注射生物材料的注射能力、 润透性和分散性， 以及通过改变所述组合物的粘 性来降低推动力 (spiking)。最后的配制是将所述细胞限定在可药用载体中。随后将所述细胞 置于注射器或其他注射装置中以精确注射到组织缺损的位点上。 根据本发明的实施例， 将 所述细胞注射到组织缺损位点的方式不受特别限制， 可以是本领域的各种常规注射方式， 根据本发明的具体实施例， 可以为局部注射针管注射或静脉回输注射。 术语"可注射 "指所 述制剂基本不需推动即可在正常压力和正常条件下以带有低至 25号针头的注射器中给予。 推动可导致组合物从注射器溢出而非注射到组织中。 对于这一精确布置， 需要细至 27 号 (200μΙΧ>.)甚至 30号 (150μΙΧ>.)的针头。 可通过这些针头挤出的最大颗粒大小是至少具有以 下参数的复杂函数： 颗粒最大尺寸、 颗粒长宽比 (长： 宽)、 颗粒刚性、 颗粒表面粗糙度和影 响颗粒之间粘性的有关因素、 悬浮液的粘弹性， 以及通过针头的流速。 悬浮于牛顿流体种 的刚性圆珠代表最简单的情况，而在粘弹性流体中的纤维性或有分支的颗粒似乎复杂得多。 所述组合物的理想等渗性可使用氯化钠或其他可药用剂例如右旋糖、 硼酸、 酒石酸钠、 丙 二醇、 或其他无机或有机溶质实现。 氯化钠特别优选用于含有钠离子的缓冲液。 如果需要， 可使用可药用的增稠剂将所述组合物的粘性保持在选定水平上。 优选甲基纤维素， 因为它 可方便和经济地获得并且易于使用。 其他合适的增稠剂包括例如黄原胶， 羧甲基纤维素， 羟丙基纤维素， 卡波姆等。 所述增稠剂的优选浓度取决于所选择的试剂。 重要的是使用量 可实现选定的浓度。 粘性组合物通常是通过在溶液中加入所述增稠剂而制备的。 可药用的 防腐剂或稳定剂可用于增加细胞 /培养基组合物的寿命。 如果加入所述防腐剂， 那么选择不 影响所述细胞生活力或效力的组合物完全在本领域技术人员的知识范围之内。 本领域技术 人员会认识到所述组合物的成分在化学上应为惰性的。 这在化学和药学原理方面不对本领 域技术人员构成问题。 可使用本公开内容提供的信息、 本文引用的和本领域通常可获得的 文献，参考教科书或通过简单实验 (不包括过度试验)可以容易地避免问题。无菌可注射溶液 可通过以下方式制备： 根据需要， 将用于实现本发明的细胞与不同量的其他成分掺入所需 量的合适溶剂中。
在某些实施例中， 干细胞可被制成单位剂量的可注射形式， 例如溶液、 悬液或乳液。 适于细胞注射的药物制剂通常为无菌水性溶液和分散体系。 用于可注射制剂的载体可为溶 剂或分散介质， 含例如水、 盐水、 磷酸缓冲盐水、 多元醇 (例如甘油、 丙二醇和液体聚乙二 醇等)以及它们的合适混合物。本领域技术人员可容易地确定将在本发明方法中给予的组合 物中的细胞和可选添加剂、 媒介 (vehicle)和 /或载体的量。 通常， 任何添加剂 (除所述细胞以 外)都以 0.001 wt%-50wt%的量存在于溶液例如磷酸缓冲盐水中。所述活性组分以微克至毫 克量级存在，例如约 0.0001 wt%到约 5wt%，优选约 0.0001 wt%到约 1 wt%，最优选约 0.0001 wt%到约 0.05 wt%, 或者约 0.001 wt%到约 20 wt%, 优选约 0.01 wt%到约 10 wt%， 最优选 0.05 wt%到约 5 wt%。在某些实施例中， 将细胞封装以给药， 特别是当胶囊化可提高治疗效 力时或者提供处理和 /或贮藏寿命上的优点时。 在某些实施例中， 当胶囊化增加细胞介导的 免疫抑制的效力时， 它也因此降低对免疫抑制药物治疗的需要。 此外， 在某些实施例中， 胶囊化提供可进一步降低受试者对所述细胞 （通常在同种异体移植中不是免疫原性的或者 只是微弱免疫原性的） 的免疫反应的针对受试者免疫系统的屏障， 从而减轻由于给予所述 细胞而可能发生的任何移植排斥或炎症。 细胞可在植入前通过膜以及胶囊进行封装。 应考 虑到， 可使用可用于细胞胶囊化的多种方法中的任意一种。 在某些实施例中， 细胞被单独 封装。 在某些实施例中， 许多细胞被封装在同一膜内。 在其中所述细胞在植入后被取出的 实施例中， 一个例如在单个膜内封装许多细胞的较大尺寸的结构可提供方便的回收方法。 在微囊化细胞的多种实施例中可使用多种材料。 这些材料包括例如聚合物胶囊， 藻酸盐-聚 -L-赖氨酸-藻酸盐微胶囊、 聚 -L-赖氨酸藻酸钡胶囊、 藻酸钡胶囊、 聚丙烯腈 /聚氯乙烯 (PAN/PVC)中空纤维以及聚醚砜 (PES)中空纤维。 某些实施例将细胞加入聚合物中， 例如生 物聚合物或合成聚合物。 生物聚合物的实例包括但不限于纤维连接蛋白、 纤维蛋白、 纤维 蛋白原、 凝血酶、 胶原和蛋白多糖。 其他因子例如上面讨论的细胞因子也可被加入所述聚 合物中。 在其他实施例中， 细胞可被加入三维凝胶的空隙中。 大的聚合物或凝胶通常通过 手术植入。 可配制成足够小的颗粒或纤维的聚合物或凝胶可通过其他常规的、 更方便的、 非手术的途径给予。 具体地， 对于治疗肝脏缺陷的情况， 所述细胞可被封装在一个可植入 受试者的装置中。 细胞可被植入到肝脏中或肝脏附近或其他位置以取代或补充肝脏功能。 细胞也可不在装置中而植入， 例如现有的肝脏组织中。
组合物可根据诸如具体患者的年龄、 性别、 体重和病症， 以及将给药的制剂 （例如固 体或液体） 等因素， 按剂量并通过医药和兽医领域技术人员公知的技术给药。 人类或其他 哺乳动物的剂量可由技术人员根据本公开内容、 本文引用的文献以及本领域的知识， 不需 过度实验而得出。 适用于本发明多个实施例的细胞剂量取决于多种因素。 其对于不同的情 况可发生相当大的变动。 可确定主要和辅助治疗的最佳给药剂量的参数包括下面的一些或 全部： 要治疗的疾病及其阶段； 受试者的物种、 它们的健康、 性别、 年龄、 体重和代谢速 率； 受试者的免疫活性； 其他正在给予的疗法； 以及根据受试者的历史或基因型预测的可 能并发症。 所述参数还可包括： 所述细胞是同基因的、 自体的、 同种异体的还是异种的； 它们的效能 (具体活性)； 使所述细胞有效而必须靶向的位点和 /或分布； 以及所述位点的这 些特性例如细胞的可达性和 /或细胞的植入性。 其他参数包括与其他因子 （例如生长因子和 细胞因子） 的共给药。 给定情况下的最佳剂量还要考虑将细胞制剂的方式、 给予它们的方 式、 给药后细胞定位到靶标位点的程度。 最后， 最佳剂量的确定必须提供这样的有效剂量， 即所述剂量既不低于最大有益效果的阈值也不高于剂量相关的有害作用超过增加剂量的益 处的阈值。 对于某些实施例， 细胞的最佳剂量处在用于自体、 单核骨髓移植的剂量范围内。 对于非常纯的细胞制剂， 在不同实施例中， 每次给药的最佳剂量可为 104 〜 108个细胞 /kg 接受者质量。 在某些实施例中， 每次给药的最佳剂量为 105 ~ 107 个细胞 /kg。 在许多实施 例中， 每次给药的最佳剂量为 5 X 105 ~ 5 X 106 个细胞 /kg。 用于参考， 前述较高的剂量与 用于自体单核骨髓移植的成核细胞的剂量相似。 某些较低的剂量与用于自体单核骨髓移植 的 CD34+的数量相似。 应理解， 单份剂量可一次、 分份或在一段时间内连续递送。 也可将 整份剂量递送到一个位置或分散到若干个位置。
在各种实施例中， 细胞可以一个初始剂量给药， 然后通过再次给药保持。 细胞可在开 始通过一种方法给药， 然后通过相同方法或者一种或多种不同方法给药。 水平可通过继续 给予所述细胞而保持。 各种实施例通过静脉注射在开始时给予所述细胞和 /或保持它们在受 试者中的水平。 在多种实施例中， 可根据患者的病情和本文其他地方讨论的其他因素使用 其他给药形式。 应注意， 人类受试者接受治疗的时间通常长于实验动物， 然而， 治疗的长 度通常与疾病过程和治疗效果的长度成比例。本领域技术人员会考虑使用在人和 /或动物 (诸 如大鼠、 小鼠、 非人灵长类等)中实行的其他方法的结果， 以确定用于人的合适剂量。 基于 这些考虑并根据本公开内容和现有技术所提供的指导的， 这种确定可使技术人员不需过度 实验而确定剂量。 初始给药和再次给药或连续给药的合适方案可以都相同或者可以是变化 的。 合适方案可由技术人员根据本公开内容、 本文引用的文献以及本领域的知识， 不需过 度实验而得出。 治疗的剂量、 频率和持续时间取决于许多因素， 包括疾病的性质、 受试者 以及可能给予的其他疗法。 因此， 多种方案可用于给予所述细胞 /培养基。在某些实施例中， 将细胞以一个剂量给予受试者。 在其他一些实施例中， 将细胞以一系列的两个剂量或多个 剂量连续给予受试者。 在其中以一个剂量、 两个剂量和 /或多于两个的剂量给予细胞的其他 一些实施例中， 剂量可相同或不同， 并且给药之间的间隔可相同或不同。 细胞可在各种不 同时间内以多种频率给药。 在某些实施例中， 细胞在小于 1天的时间内给药。 在其他实施 例中， 它们在 2、 3、 4、 5或 6天的时间内给药。 在某些实施例中， 细胞在数周的时间内以 每周一次或多次给药。 在其他实施例中， 细胞在数周的时间内给药持续一到数月。 在多种 实施例中， 细胞可在数月的时间内给药。 在其他实施例中， 细胞可在一或数年的时间内给 药。 通常治疗长度是与疾病过程的长度、 所用疗法的效果以及要治疗的受试者的情况和反 应成比例的。
根据本发明的实施例， 所分离的间充质干细胞可具有被诱导分化以形成至少一种选自 以下的细胞的能力： 成骨细胞、 软骨细胞、 脂肪细胞、 成纤维细胞、 骨髓基质、 骨骼肌、 平滑肌、 心肌、 内皮细胞、 上皮细胞、 造血细胞、 神经胶质细胞、 神经元细胞或少突胶质 细胞类型。 本发明还提供从上述间充质细胞获得的分化细胞， 其中其后代细胞可为骨、 软 骨、 脂肪细胞、 成纤维细胞、 骨髓基质、 骨骼肌、 平滑肌、 心肌、 内皮细胞、 上皮细胞、 内分泌细胞、 外分泌细胞、 造血细胞、 神经胶质细胞、 神经元细胞或少突胶质细胞。 所述 分化后代细胞可为皮肤上皮细胞、 肝上皮细胞、 胰腺上皮细胞、 胰腺内分泌细胞或岛细胞、 胰腺外分泌细胞、 肠上皮细胞、 肾上皮细胞或上皮相关结构。 细胞或其分化后代可用于纠 正遗传疾病、 退化性疾病、 心血管疾病、 代谢贮积病、 神经疾病或癌症过程。 它们可用于 产生用于治疗牙周病的齿龈样材料。 它们可用于形成皮肤上皮组织， 所述皮肤上皮组织源 自可用于皮肤移植和整形手术的细胞。 它们可用于例如在阴茎或心脏中强化肌肉。 它们可 用于产生治疗用体外血， 或产生人造血细胞和 /或人用出生前或出生后动物血。 它们可用作 治疗剂以例如在患者从癌症治疗、 自身免疫疾病治疗的化疗或放疗恢复中起辅助作用， 以 引起接受者的耐受性。 它们可用于治疗 AIDS或其他传染病。 视神经细胞可用于治疗由包 括但不限于视神经疾病导致的失明， 所述视神经疾病由包括但不限于黄斑变性、 糖尿病性 视网膜病变、 青光眼、 视网膜色素变性导致。 所述细胞或源自所述细胞的心肌细胞可用于 治疗心脏病， 包括但不限于心肌炎、 心肌病、 心力衰竭、 由心脏病发作导致的损伤、 高血 压、 动脉粥样硬化和心脏瓣膜功能不全。 它们还可用于治疗涉及 CNS缺陷或损伤的疾病。 此外， 所述干细胞或其神经元分化后代细胞可用于治疗涉及神经缺陷或退化的疾病， 包括 但不限于中风、 阿尔茨海默氏症、 帕金森氏病、 亨廷顿氏病、 艾滋病相关痴呆、 脊髓损伤、 影响脑部或其他神经组织的代谢性疾病。 细胞或它们的分化后代例如基质细胞可用于支持 其他细胞类型在体内或体外的生长和分化， 所述其他细胞类包括但不限于造血细胞、 胰腺 岛细胞或 β细胞、 肝细胞等。 所述细胞或分化软骨后代， 可用于治疗关节或软骨疾病， 包 括但不限于软骨撕裂、 软骨变薄、 骨关节炎。 此外， 所述细胞或它们的分化成骨细胞后代 可用于改善对骨具有有害效果的过程， 包括但不限于骨折、 骨折不愈合、 骨关节炎， 由扩 散到骨的肿瘤 (例如前列腺癌、 乳腺癌、 多发性骨髓瘤等)导致的骨"洞"。 使用合适的生长因 子、 趋化因子和细胞因子， 细胞可被诱导以分化形成若干谱系， 包括例如多种具有中胚层 表型的细胞、具有神经外胚层表型的细胞 (神经胶质细胞、少突胶质细胞和神经元)和具有内 胚层表型的细胞。 这些包括成骨细胞、 软骨细胞、 脂肪细胞、 软骨和骨、 骨骼肌、 平滑肌、 心肌、 内皮细胞、 造血细胞、 基质细胞、 神经元细胞和上皮细胞。 已被诱导分化以形成骨 细胞的细胞可在骨质疏松、 佩吉特氏病 (Paget's disease)^ 骨折、 骨髓炎、 骨坏死、 软骨发 育不全、 成骨不全症、 遗传性多发性骨软骨瘤、 多发性骨骺发育不良、 玛丽安氏综合征 (Marian's syndrome)^ 粘多糖贮积症、 神经纤维瘤病或脊柱侧弯中用作细胞疗法或者用于 组织再生， 局部畸形、 脊柱裂、 半椎体或融合椎骨、 肢体畸形的重建手术， 肿瘤损伤组织 的重建以及感染 (如中耳炎)后的重建。已被诱导分化以形成软骨细胞的细胞可用于与年龄有 关的疾病或损伤、 运动相关损伤中的组织再生， 或特定疾病例如类风湿关节炎、 银屑病关 节炎、 赖特关节炎 (Reiter's arthritis；)、 溃疡性结肠炎、 克罗恩病 (Crohn's disease；)、 强直性脊 柱炎、 骨关节炎中的细胞疗法或者组织再生； 外耳的重建手术； 鼻的重建手术； 以及环状 软骨的重建手术。 已被诱导分化以形成脂肪细胞的细胞可用于重建或整容手术的重塑， 包 括但不限于， 乳房切除术后的乳房重建、 对因其他手术例如从脸或手上切除肿瘤造成的组 织丧失进行整形、 隆胸和去皱。 还可用于 II型糖尿病的治疗。 因此衍生的脂肪细胞还可提 供用于研究脂肪调节的有效体外模型系统。 成纤维细胞： 源自所述细胞的成纤维细胞可用 于细胞疗法或组织修复以促进伤口愈合或提供结缔组织支撑物， 例如整容手术的支架。 已 被诱导分化以形成骨骼肌细胞的细胞可用于杜氏肌营养不良症 (Duchenne muscular dystrophy) 贝克肌营养不良症 (Beckermuscular dystrophy) 强直性肌营养不良症、 骨骼肌 肉病变的治疗中的组织修复， 以及修复骨骼肌损伤的重建手术。 已被诱导分化以形成平滑 肌细胞的细胞可用于胃肠系统发育异常例如食道闭锁、 肠闭锁和肠套叠的治疗中的细胞疗 法或组织修复， 以及肠梗塞或结肠造口手术后的组织替换。 平滑肌细胞还可用于膀胱或子 宫重建、 新血管形成、 由例如动脉粥样硬化或动脉瘤造成的血管损伤的修复。 平滑肌前体 细胞 (肾小球膜细胞)可用作肾小球疾病的体外模型或用于糖尿病性神经病中的细胞疗法或 组织再生。 平滑肌前体还可用于修复远曲小管或肾小球旁组织的致密斑。 心肌细胞可用于 治疗心肌梗塞后的、 伴随充血性心力衰竭的、 瓣膜置换过程中的、 由先天性心脏异常引起 的或者由心肌病或心内膜炎引起的心脏组织损伤的细胞疗法或组织修复。 小神经胶质细胞 可用于治疗脊髓损伤和神经退化性障碍例如亨廷顿氏症 (Huntington's disease) 帕金森氏 症、 多发性硬化症和阿尔茨海默氏症， 以及用于影响中枢神经系统的传染病过程中损伤组 织的修复。 被遗传改变以产生细胞因子的小神经胶质细胞还可用于移植以治疗通道因血脑 屏障被限制的中枢神经系统中的传染病。 神经胶质细胞还可产生由多发性硬化症、 肌萎缩 侧索硬化症和脑癌引起的中风后神经组织再生以及脊髓损伤后再生的生长因子或生长因子 抑制因子。 基质细胞： 基质细胞可用作化疗后骨髓置换和骨髓移植的移植细胞。 内皮细胞 可用于治疗因子 VIII缺陷和用于新血管形成。 内皮细胞还可提供使用血管形成抑制因子抑 制肿瘤的体外模型， 以及血管炎、 超敏反应和凝血障碍的体外模型。 造血细胞： 造血细胞 可用于在高剂量化疗后再增殖骨髓。 源自所述聚集体细胞的造血细胞可进一步分化以形成 血细胞， 以贮存在血库中， 缓解输血血液不足的问题。 小神经胶质细胞可用于治疗脊髓损 伤和神经退化性障碍例如亨廷顿氏症、 帕金森氏症、 多发性硬化症和阿尔茨海默氏症， 以 及对影响中枢神经系统的传染病中损伤组织的修复。 被遗传改变以产生细胞因子的小神经 胶质细胞还可用于移植， 以治疗通道因血脑屏障被限制的中枢神经系统中的传染病。 神经 胶质细胞还可产生由多发性硬化症、 肌萎缩侧索硬化症和脑癌引起的中风后神经组织再生 以及脊髓损伤后再生的生长因子或生长因子抑制因子。 被诱导形成少突胶质细胞和星形胶 质细胞的细胞， 例如， 可用于移植到脱髓鞘组织尤其是脊髓中， 在那里它们的功能是为周 围神经组织加髓鞘。 所述细胞还可用于细胞置换疗法和 /或基因疗法以治疗先天性神经退化 障碍或贮积障碍例如粘多糖贮积症、 脑白质营养不良 (球状细胞脑白质营养不良、 卡纳文氏 症 (Canavan's disease))、岩藻糖血症、 GM2神经节苷脂沉积症、尼曼-皮克病 (Niemann-Pick；)、 圣菲利波综合征 (Sanfilippo syndrome) 沃尔曼氏病 (Wolman's disease)和泰-萨克斯病 (Tay Sachs；)。它们还可用于创伤性疾病例如中风、 中枢神经系统出血和中枢神经系统创伤； 用于 周围神经系统疾病例如脊髓损伤或脊髓空洞症； 用于视网膜疾病例如视网膜脱离、 黄斑变 性和其他退化性视网膜疾病， 以及糖尿病性视网膜病变。 外胚层上皮细胞： 细胞可用于细 胞置换疗法和 /或基因疗法中， 以治疗或缓解皮肤病例如脱发、 皮肤缺损如皮肤烧伤和白化 病的症状。 上皮细胞可用于细胞置换疗法和 /或基因疗法中， 以治疗或缓解若干器官病的症 状。 所述细胞可用于治疗或缓解先天性肝脏疾病， 例如， 贮积病如粘多糖贮积症、 脑白质 营养不良、 GM2 神经节苷脂沉积症； 胆红素增加病变， 例如克里格勒-纳贾尔综合征 (Crigler-Najjar syndrome); 氨病， 例如尿素循环的先天性障碍如鸟氨酸脱羧酶缺乏症、 瓜 氨酸血症和精氨酸琥珀酸尿症； 氨基酸和有机酸的先天性障碍， 如苯丙酮尿症， 遗传性酪 氨酸血症和 α抗胰蛋白酶缺乏症； 以及凝血障碍例如因子 VIII和 IX缺乏症。 所述细胞还 可用于治疗由病毒感染导致的获得性肝病变。所述细胞还可用于体外， 例如生成人工肝脏， 产生凝血因子以及产生由肝脏上皮细胞生成的蛋白或酶。 所述上皮细胞还可用于细胞置换 疗法和 /或基因疗法， 以治疗或缓解胆病例如胆汁性肝硬化和胆道闭锁的症状。 所述上皮细 胞还可用于细胞置换疗法和 /或基因疗法， 以治疗或缓解胰腺疾病例如胰腺闭锁、 胰腺炎和 α抗胰蛋白酶缺乏症的症状。 此外， 当可得到胰腺上皮细胞、 神经细胞时， 可生成 β细胞。 这些细胞可用于糖尿病的治疗 (皮下植入或者胰腺内或肝脏内植入；)。此外，所述上皮细胞还 可用于细胞置换疗法和 /或基因疗法， 以治疗或缓解肠道上皮病症例如肠道闭锁、炎性肠病、 肠梗塞和肠切除的症状。 细胞可用于组织修复： 细胞还可用于组织修复。 可将细胞植入到 骨中以增强所述修复过程， 以强化变弱的骨或重新覆盖关节。 可将软骨细胞注射到关节中 以重新覆盖关节软骨。 Caplan等人 (美国专利 5,855,619 )描述了一种生物基质植入物， 包括 其中已加入间叶肝细胞的收缩凝胶基质。 所述植入物旨在修复组织缺陷， 尤其是肌腱、 韧 带、 半月板或肌肉的损伤。 例如， 软骨可通过将软骨细胞加入到由例如胶原、 合成聚乙醇 酸纤维或合成聚乳酸纤维制成的多孔、 三维支架的周边区域而形成。 发明人已经证明， 本 发明的细胞分化以形成软骨细胞， 例如， 其可沉积在胶原、 合成聚乙醇酸、 合成聚乳酸或 其他支架材料中或周围以提供植入物以促进组织修复。
根据本发明实施例的建立单克隆间充质干细胞的方法的有益效果如下：
1. 因具有相同的遗传背景， 降低了其在用于动物体时的免疫原性；
2. 纯度极高、 细胞基因组单一性； 易于今后基因组的测序；
3. 不易分化或分化程度较非单克隆细胞低， 易保持原有 MSCs的特性与多功能分化潜 能； 和
4. 可体外长期培养、 大量扩增以满足临床需求， 致瘤性低。
本发明的附加方面和优点将在下面的描述中部分给出， 部分将从下面的描述中变得明 显， 或通过本发明的实践了解到。 附图说明
图 1 显示了根据本发明实施例的利用流式细胞仪分析经过转染的 MSCs 细胞中的
Nanog启动子转染效率；
图 2显示了根据本发明实施例的利用荧光显微镜观察经过转染的 MSCs细胞中 GFP的 表达；
图 3显示了根据本发明实施例的经过分选后培养于 96孔板的单克隆 MSCs的光学显微 镜观察图； 以及
图 4 显示了根据本发明实施例的利用荧光显微镜观察经过转染的 MSCs 细胞中 VIMENTIN基因 （4A)、 α-SMA基因 （4B ) 禾 P CNN1基因 （4C) 的表达情况。 具体实施方式
下面详细描述本发明的实施例， 需要说明的是下面的实施例， 仅仅是为了说明本发明， 而不以任何方式限制本发明。 另外， 在下面实施例中所采用的试剂和材料也均是市售可得 的， 如果未明确说明， 则所采用的方法和条件， 也均按照公知的方法和条件进行相关处理。 实施例 1细胞分离和培养
将分别来源于动物的不同组织， 利用胶原酶进行消化， 离心后， 去除上清液后， 将收 集的细胞用 PBS洗涤 2次后用完全培养基进行培养， MSCs贴壁迅速， 24小时后进行换液， 去除其余悬浮的细胞， 从而获得所有的贴壁细胞。 其中完全培养基包括 α-ΜΕΜ培养液， 10%胎牛血清。 实施例 2细胞转染
采用 invitrogen脂质体 2000转染试剂盒转染实施例 1中新分离得到的贴壁细胞， 具体 如下：
将实施例 1 中新分离得到的贴壁细胞培养 24小时， 换液后继续培养， 在转染前使用 无抗生素培养基培养， 待细胞融合 70%-80%， 将含有 Nanog 启动子的质粒与对照质粒 (PRRL.sin.PGK.GFP) 分别溶于 50微升无血清的 Opti—MEM I培养液中， 混合均匀。 将 适量脂质体 （脂质体 Clipofectin regeant, LR)试剂是阳离子脂质体 N-[l,2,3-Dioleyoxy Propyl]-n,n,n-Trimethylammonium Chi oride(DOTMA)禾口 Dioleoyl photidye-thanolamine(DOPE) 的混合物 [1 : l(w/w)] )溶于 50微升无血清的 Opti—MEM I培养液中， 混合均匀。 在室温 下孵育 5分钟。 然后分别将 NANOG质粒， 对照质粒与脂质体混合（总体积 100微升）。 轻 轻混合均匀在室温下孵育 25分钟后， 在 24孔板中每孔加入 100微升的复合物以及适量培 养基。在加完复合物 4~6小时后将培养基更换为完全培养基，并将细胞继续在 37度 5% C02 培养箱中培养 48个小时后， 利用 FACS测量转染效率， 结果见图 1， 图 2。
从图 1中的结果可以看出， 对照组质粒显示出的转染效率为 56.8%， 间充质干细胞表 达的阳性率为 34.4%。 通过流式细胞仪检测， 可以确认根据本发明的方法可以成功将含有 人 NANOG启动子序列转染进入细胞， 且转染效率良好， 可以用于后续实验。 图 2显示了 从实施例 1获得的细胞经转染人 NANOG启动子序列和 GFP在荧光显微镜绿色偏光镜下的 转染效果结果。 实施例 3细胞分选
利用胰酶消化离心后，用 PBS将转染成功后的贴壁细胞从培养皿上转移下来， 经过清 洗后， 用 FACS流式细胞仪在 488纳米下氩激光下进行检测。 利用 Cell-Quest软件 (Weasel V2.3.1)对样品中的 10,000个细胞进行分析， 收集成功表达报告蛋白 GFP的细胞。
将分选后的单个细胞采用上述完全培养基在 96孔板中在 37摄氏度 5% C02条件下进 行培养， 每 3~5天换液一次， 由观察可知早期单个克隆细胞生长较为缓慢， 但一周后细胞 呈现集落群后， 生长迅速， 呈典型的间充质干细胞样生长， 呈梭状、 漩涡样、 放射状生长， 结果见图 3。
实施例 4免疫荧光
将分化好的细胞培养于中文名为培养腔室玻片 (B&D)。 融合的细胞层用溶解在磷酸缓 冲液 （PBS) 的 4% (体积 /体积） 多聚甲醛溶液在室温下进行固定 15分钟， 弃去多聚甲醛 溶液后经过清洗向细胞中加入溶解在 PBS中的 0.1% Triton 100X并在室温下孵育 10分钟以 便在细胞核中进行蛋白表达。细胞骨架蛋白用甲醇在 -20摄氏度条件下固定 5分钟。然后将 载有固定好的细胞的玻片分别与鼠抗人波形蛋白 VIMENTIN抗体、鼠抗人 α-平滑肌肌动蛋 白 (α-SMA)抗体、 鼠抗人钙调理蛋白 (CNN1)抗体在室温下孵育 1小时， 分别倾去抗体后对 玻片进行清洗后用对应的 Alexa-448结合的羊抗鼠、 羊抗兔和鼠抗羊抗体继续孵育 2分钟。 染色后， 用 Vecta-shield+DAPI (Vector)进行封片， 并用 Imager A 1 显微镜 （Carl Zeiss)进行 观察。 由图 4结果显示， 间充质干细胞中 VIMENTIN、 α-SMA和 CNN1蛋白的表达均为阳 性， 其中 VIMENTIN蛋白 100%表达， 其也可作为 MSCs蛋白表达的阳性对照。 在本说明书的描述中， 参考术语"一个实施例"、 "一些实施例"、 "示例"、 "具体示例"、 或"一些示例"等的描述意指结合该实施例或示例描述的具体特征、 结构、 材料或者特点包 含于本发明的至少一个实施例或示例中。 在本说明书中， 对上述术语的示意性表述不一定 指的是相同的实施例或示例。 而且， 描述的具体特征、 结构、 材料或者特点可以在任何的 一个或多个实施例或示例中以合适的方式结合。
尽管上面已经示出和描述了本发明的实施例， 可以理解的是， 上述实施例是示例性的， 不能理解为对本发明的限制， 本领域的普通技术人员在不脱离本发明的原理和宗旨的情况 下在本发明的范围内可以对上述实施例进行变化、 修改、 替换和变型。

Claims

权利要求书
1、 一种分离间充质干细胞的方法， 其特征在于， 包括：
A)利用胶原酶对动物样本进行消化， 以便分散所述动物样本中所包含的细胞， 以便获 得包含干细胞的细胞混合物；
B ) 将编码报告蛋白的核酸分子引入所述细胞混合物的至少一部分细胞中， 其中， 所 述编码报告蛋白的核酸分子与间充质干细胞特异性启动子可操纵地相连， 以便在间充质干 细胞中表达所述报告蛋白； 以及
C) 利用 FACS, 分选获得表达所述报告蛋白的间充质干细胞， 其中， 所述间充质干细 胞呈单个细胞的形式。
2、 根据权利要求 1所述的方法， 其特征在于， 所述动物样本为来自选自肝脏组织、 脂 肪组织、 骨实质、 肌肉、 脐带血、 脐带、 胎盘、 外周血、 胰腺、 肺脏、 经血和牙髓的至少 一种。
3、 根据权利要求 2所述的方法， 其特征在于， 所述动物样本为来自胎儿肝脏组织及成 人骨髓。
4、 根据权利要求 2所述的方法， 其特征在于， 将编码报告蛋白的核酸引入所述细胞混 合物的至少一部分细胞中包括：
利用液体培养基对所述细胞混合物进行培养；
将构建体引入所述培养获得的贴壁细胞中，
其中，
所述构建体包括：
编码报告蛋白的核酸分子； 以及
间充质干细胞特异性启动子， 所述编码报告蛋白的核酸分子与间充质干细胞特异性启 动子可操纵地相连。
5、 根据权利要求 4所述的方法， 其特征在于， 所述液体培养基为 α-ΜΕΜ培养基。
6、 根据权利要求 4所述的方法， 其特征在于， 所述报告蛋白为选自发光蛋白、 荧光蛋 白、 酶的至少一种。
7、 根据权利要求 6所述的方法， 其特征在于， 所述报告蛋白为绿色荧光蛋白。
8、 根据权利要求 4所述的方法， 其特征在于， 所述间充质干细胞特异性启动子为人 NANOG启动子。
9、 根据权利要求 4所述的方法， 其特征在于， 所述构建体呈质粒、 噬菌体、 人工染色 体、 粘粒、 病毒的至少一种的形式。
10、 根据权利要求 1所述的方法， 其特征在于， 进一步包括对所得到的单个细胞形式 的间充质干细胞进行培养， 以便获得干细胞单克隆集群。
11、 一种干细胞或其衍生物， 所述干细胞是通过权利要求 1-10任一项所述的方法制备 的。
12、 权利要求 11所述的干细胞或其衍生物在制备药物中的用途， 所述药物用于治疗选 自下列的至少一种： 血液系统疾病、 心血管疾病、 肝硬化、 神经系统疾病、 神经系统修复、 膝关节半月板部分切除损伤修复、 自身免疫性疾病、 脊髓损伤、 脑瘫、 肌萎缩侧索硬化症、 系统性红斑狼疮、 系统性硬化症、 克隆氏病、 中风、 糖尿病、 糖尿病足、 肝硬化和硬皮病。
13、 一种药物组合物， 其特征在于， 包括：
权利要求 11所述的干细胞或其衍生物； 以及
药物上可以接受的赋形剂。
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