Fibroblast regenerative cells

Abstract

To provide fibroblast regenerative cells as novel regenerative cells.SOLUTION: Disclosed are compositions, systems, and methods comprising regenerative fibroblasts, populations thereof, or subsets thereof possessing regenerative activity useful for treatment of various degenerative diseases. In one embodiment, the disclosure provides fibroblasts with enhanced proliferative potential based on enrichment for CD105 and / or CD117 markers. In one embodiment, fibroblasts possessing CD105 and / or CD117 markers are further enriched for the activity of rhodamine 123 efflux.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This application was filed on January 11, 2019, and is incorporated herein by reference in its entirety. Priority is claimed to filed U.S. Provisional Patent Application No. 62 / 791,207.

[0002] This disclosure relates generally to cell biology and medicine. In particular, it relates to regenerative medicine. The present disclosure relates to methods comprising fibroblasts, fibroblast populations and subpopulations with inherent regenerative activity. and compositions. [Background technology]

[0003] Regenerative medicine applied in the form of stem cell therapy holds the potential to treat many previously incurable diseases. There are many types of stem cells, and more need to be identified. They are generally divided into embryonic and adult types. Embryonic stem cells have the ability to proliferate, but The specific induction of differentiation is unknown. Embryonic stem cells can induce teratomas. This is a major obstacle to its clinical development. Adult stem cells, including those derived from amniotic fluid, have demonstrated regenerative potential in a variety of diseases and degenerative disorders. However, these cell types have limitations due to their availability, invasiveness of extraction, and in some cases, These drawbacks are overcome and the problem of carcinogenicity is also addressed during culture. There is a need for improved cells that do not suffer from karyotypic abnormalities and are suitable for various medical conditions. There is also a need for improved cell therapies for inflammatory bowel disease. The present disclosure provides methods for the treatment of inflammatory bowel disease that have superior activity to stem cells. By providing fibroblast regenerative cells, a novel regenerative cell that overcomes the limitations of stem cells, These fibroblast regenerative cells can be used for therapeutic purposes. Cut. Summary of the Invention

[0004] Embodiments of the present disclosure provide cell therapy and regenerative activity agents useful for treating various diseases, disorders, or conditions. Some embodiments include compositions, methods, and systems containing fibroblasts having the following structure: Morphologically, fibroblasts are enriched for at least one marker. In certain embodiments, the fibroblasts express CD105 and / or In some embodiments, the cells express the CD117 marker. Fibroblast regenerative cells bearing the D117 marker were characterized for rhodamine 123 efflux. In some embodiments, the fibroblast regenerative cells are further enriched for MHC class I. I, MHC class II, CD45, CD13, CD49c, CD66b, CD73, CD 105 or CD90.

[0005] In embodiments, fibroblast regenerative cells, populations, progeny or subsets of fibroblast regenerative cells. fibroblast regenerative cell conditioned medium, composition containing fibroblast regenerative cells, fibroblast regenerative cell Pharmaceutical preparations containing cells, kits containing fibroblast regenerative cells, kits containing a population of fibroblast regenerative cells The present invention includes compositions including cell banks containing the same.

[0006] In some embodiments, the isolated fibroblast regenerative cells are characterized by the marker CD105 and and / or expressing or selected for expressing the CD117 marker. In some embodiments, the cells also express Oct-4, CD-34, KLF-4, Nanog, So x-2, Rex-1, GDF-3, IL-10, Stella and / or any of them In some embodiments, the fibroblast regenerative cells also express a combination of rhodamine and erythritol. In some embodiments, the cells contain GD123 efflux activity relative to control cells. This includes enhanced expression of F-11.

[0007] In some embodiments of the present disclosure, fibroblast regenerative cells include, but are not limited to, , placental tissue, umbilical cord tissue, endometrial cells, Wharton's jelly, bone marrow, adipose tissue, or the like In some embodiments, the tissue is derived from tissue with regenerative properties, such as a mixture of regenerative fibroblasts. Cells can proliferate and differentiate into at least two of the germ layers: ectoderm, mesoderm, or endoderm. The fibroblast regenerative cells are autologous to the subject undergoing treatment. In some embodiments, the cells are plastically attached. In a further aspect, the cells may be cryopreserved.

[0008] Further aspects of the present disclosure include methods or methods comprising fibroblast regenerative cells and exogenous mitochondria. In some embodiments, the exogenous mitochondria are isolated from other cellular components. In some embodiments, the mitochondria are isolated and substantially purified. Fibroblast regeneration by polyethylene glycol-mediated fusion, or electroporation In some embodiments, the mitochondria are administered to living cells. To facilitate uptake of the drug, the drug may be encapsulated or treated with one or more drugs. Chondria may be autologous, syngeneic, allogeneic, or xenogeneic to the subject receiving treatment. In some embodiments, the mitochondria are derived from stem cells or They are derived from less differentiated cells than fibroblasts. In some methods, mitochondria are The ria are administered separately to the subjects.

[0009] The present disclosure also provides isolated cells that can proliferate and differentiate into ectoderm, mesoderm, or endoderm. and a population thereof, wherein the isolated fibroblast regenerative cells are: Oct-4, Nanog, IL-10, Sox-2, KLF4, c-Myc, Rex-1 , GDF-3, LIF receptor, CD105, CD117, CD344 or Stella express at least one of the following markers: MHC class I, MHC class II, and CD4 5. CD13, CD49c, CD66b, CD73, CD105, or CD90 They do not express at least one cell surface protein.

[0010] In certain embodiments, the cells, compositions, or formulations of the present disclosure include fibroblast regenerative cells, populations thereof, The cells may be administered with one or more other compounds that are not the cells, their progeny and / or their conditioned medium. The one or more other compounds may be present in the cells, compositions or formulations encompassed herein. In particular cases, the compound may be vitamin A, vitamin C, vitamin D , Vitamin E, Vitamin K, Folic Acid, Choline, Vitamin B1, Vitamin B2, Vitamin B5 , Vitamin B6, Vitamin B12, Biotin, Nicotinamide, Beta-Carotene, Coene Zyme Q, selenium, superoxide dismutase, glutathione peroxide, uridine, co Creatine Succinate, Pyruvate, Dihydroxyacetone), Acetyl-L-Carnitine, Alpha-lipoic acid, cardiolipin, omega fatty acids, lithium carbonate, lithium citrate, calcium In some embodiments, the compound may be one or more In some embodiments, the anti-inflammatory agent is alclofenac; Methasone dipropionate; algestone acetonide; alpha-amylase; alpha-lipoic acid; alpha -Tocopherol; Amcinafal; Amcinafide; Amfenac sodium; Aqua hydrochloride Miprilose; Anakinra; Anilorac; Anitrazafen; Apazone; Ascorbic Acid; Balsalazide disodium; Bendazac; Benoxaprofen; Benzydamine hydrochloride ;Bromelain;Properamol;Budesonide;Carprofen;Chlorogenic acid;Cyclo profen;synthazone;criprofen;clobetasol propionate;clobetasol Betasone butyrate; Clopirac; Cloticasone propionate; Cormetasone acetate; Cortodoxone; Deflazacort; Desonide; Desoximetasone; Dexamethasone dipropionate Methasone;Diclofenac potassium;Diclofenac sodium;Diflorasone diacetate diflumidone sodium; diflunisal; difluprednate; diphthalone; dimethicone Tyrsulfoxide; Drocinonide; Ellagic acid; Endrison; Enlimomab; Enolicum sodium; epirizole; etodolac; etofenamate; felbinac; fenamo fenbufen; fenclofenac; fenclorac; fendosal; fen Piparon; Fentiazac; Flazaron; Fluazacort; Flufenamic acid; Flumizo Flunisolide acetate; Flunixin; Flunixin meglumine, Fluocorticoid butyl; fluorometholone acetate; fluquazone; flurbiprofen; fluretofen; Fluticasone propionate; Furaprofen; Flobufen; Glutathione; Ha Rucinonide; Halobetasol propionate; Halopredone acetate; Hesperedin (hesperedin); ibufenac; ibuprofen; ibuprofen aluminum Ibuprofen piconol; Ilonidap; Indomethacin; Indomethacin sodium Indoprofen; Indoxol; Intrazol; Isoflupredone acetate; Isoflupredone Soxepac; Isoxicam; Ketoprofen; Lofemizole hydrochloride; Lomoxicam; Loteprednol etabonate; Lycopene; Sodium meclofenamate; Meclofenamic Acid;Meclorizone dibutyrate;Mefenamic acid;Mesalamine;Meseclazone;Methylprednisolone Donisolone suleptanate; Morniflumate; Nabumetone; Naproxen; Naproxen Naproxol; Nimazone; Oleuropein; Olsalazine sodium; Orgotein; Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyl hydrochloride pentosan polysulfate sodium; phenbutazone sodium glycerate; Pirfenidone; Piroxicam; Piroxicam cinnamate; Piroxicam olamine; Piroxicam Luprofen; Pycnogenol; Polyphenols; Prednazate; Preferon; Pro Rhodolic acid; Proquazone; Proxazole; Proxazole citrate; Quercetin; Suberatrol; Rimexolone; Romazarit; Rosmarinic acid; Rutin; Sarcorex; Salnacedin; Salsalate; Sanguinarium chloride; Seclazone; Sermetacin; Sudo Xicam; Sulindac; Suprofen; Talmetacin; Talniflumate; Talosale Tebufelone; Tenidap; Tenidap sodium; Tenoxicam; Tesicam; Tesi amide; tetrahydrocurcumin; tetridamine; thiopinac; tixocortol pivalate tolmetin; tolmetin sodium; triclonide; triflumidate; zidometabolite and zomepirac sodium. , wherein the compound is a growth factor, a cytokine, an antibody, an antibody fragment, and / or a compound having a molecular weight of e.g. 5000 Daltons. Bioactive compounds include, but are not limited to, organic molecules with a mass less than 1000 kJ / cm. Compounds mentioned elsewhere herein may also be administered simultaneously with the compositions of the present disclosure. Alternatively, the compound may be administered before and / or after the composition is administered to the subject. This may be done.

[0011] In some embodiments, the composition comprising fibroblasts may be, for example, embryonic stem cells, tissue-specific The method further comprises one or more types of stem cells, including induced pluripotent stem cells, mesenchymal stem cells, or induced pluripotent stem cells. The cells should be at least 1 x 10 2 , 1×10 6 , 1×10 9 , 1×10 10 , 1×10 12 , 1×10 14 The stem cells may be provided in an amount of 1000-150 ... The cells can be mesenchymal cells, embryonic stem cells, or differentiated cells. For example, stem cells can be, e.g., Myocytes, adipocytes, ectodermal cells, muscle cells, osteoblasts, chondrocytes, endothelial cells, fibroblasts , pancreatic cells, hepatic cells, bile duct cells, bone marrow cells, neuronal cells, or urogenital cells.

[0012] Also, embodiments include methods of providing cell therapy, methods of treating subjects who would benefit from cell therapy, Methods for treating a disease, disorder or condition, methods for treating cancer, methods for treating degenerative diseases, fibrosis Method for using fibroblast regenerative cells, method for enhancing the regenerative activity of fibroblasts, fibroblast regenerative cells Methods for generating a population of fibroblast regenerative cells, methods for selecting fibroblast regenerative cells, and identification of fibroblast regenerative cells Methods for isolating and culturing populations of regenerative fibroblasts, and methods for producing regenerative fibroblast cells The present invention includes a method for enriching fibroblast regenerative cells, a method for expanding fibroblast regenerative cells, or a combination thereof. The processes and embodiments discussed in this disclosure are contemplated as part of any of these methods. Additionally, compositions for use in any of these methods are also contemplated.

[0013] In certain embodiments, isolated fibroblast regenerative cells, populations thereof, their progeny, and / or and administering to the subject a composition containing the conditioned medium, thereby The present invention also includes a method for treating a condition, wherein the fibroblast regenerative cells are This results in increased regenerative activity.

[0014] The method may consist of, or consist essentially of, one or more of the following steps: administering an effective amount of fibroblast regenerative cells to a subject; and One or more additional compounds that enhance or increase or improve the regenerative activity of fibroblasts of the present disclosure Administering to a subject.

[0015] In some embodiments, the subject suffers from or develops a condition, disease, or disorder. At risk of or suspected of having a condition, disease or disorder. In this case, the subject may be undergoing one or more conventional treatments for the condition, disease or disorder. In further embodiments, the subject may be resistant to certain other treatments. The diseases or disorders include immune disorders, muscle disorders, hematopoietic disorders, liver disorders, angiogenesis disorders, pancreatic disorders, and heart diseases. Diseases, pulmonary diseases, neurological diseases, neuropathies, neurodegenerative diseases, muscular diseases, immune diseases, inflammatory-mediated diseases, Inflammation-mediated disorders, inflammation, ischemia, stroke, ischemic heart disease, liver failure, kidney failure, peripheral arterial disease, Pulmonary fibrosis, liver fibrosis, pancreatic fibrosis, diabetic limb, fibrosis, scar tissue formation, pathological apoptosis , diabetes, cirrhosis, hepatitis, osteoporosis, fractures, neurological damage, need for joint prostheses, Need for dermal stem cells, need for proliferation of pancreatic islet cells, need for increased insulin production, bone cells the need for increased bone cell formation; the need for increased bone cell function; the need for neoplastic disease In some embodiments, the cardiac or pulmonary disease may be atherosclerosis, pulmonary embolism, or a need for cell therapy. Arteriosclerosis, myocardial infarction (heart attack), cardiac infection, heart failure, ischemic heart failure, hypertension blood pressure (hypertension), or pulmonary hypertension, idiopathic pulmonary fibrosis, stroke, congenital heart disease (CHD), Congestive heart failure, angina pectoris, myocarditis, coronary artery disease, cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, Endocarditis, diastolic dysfunction, cerebrovascular disease, valvular disease, mitral valve prolapse, venous thromboembolism or arrhythmia In some embodiments, the neurological or muscular disease is multiple sclerosis (MS), spinal cord injury, or Spinal cord injury, muscular dystrophy (Becker or Duchenne), amyotrophic lateral sclerosis (A LS (Lou Gehrig's disease or classical motor neuron disease), autism, progressive bulbar palsy (progressive Pseudobulbar palsy, primary lateral sclerosis (PLS), progressive muscular atrophy, spinal muscular atrophy (SMA Type I - Werdnig-Hoffmann disease, SMA Type II, or SMA Type III -SMA including Kugelberg-Welander disease), Fazio-Londo disease, Kennedy disease ( Progressive Spinal and Bulbar Muscular Atrophy), Congenital SMA with Joint Contractures, and Post-Polio Syndrome (P In some embodiments, the immune disorder or inflammation-mediated disease is thyroiditis, pancreatic cancer, or thyroid cancer. Insulitis, multiple sclerosis, iridocyclitis, uveitis, orchitis, Addison's disease, myasthenia gravis, Rheumatoid arthritis, lupus erythematosus, immune overreaction, insulin-dependent diabetes mellitus, anemia, regeneration Aplastic anemia, hemolytic anemia, hepatitis, autoimmune hepatitis, scleritis, idiopathic thrombocytopenic purpura, Autoimmune diseases, gastrointestinal diseases, Crohn's disease, ulcerative colitis, inflammatory bowel disease, juvenile arthritis , scleroderma and systemic sclerosis, Sjögren's syndrome, undifferentiated connective tissue syndrome, antiphospholipidosis syndrome, vasculitis, polyarteritis nodosa, allergic granulomatosis, vasculitis, Wegener's granuloma Kawasaki disease, hypersensitivity vasculitis, Henoch-Schönlein purpura, Behçet's syndrome, hypersensitivity vasculitis Arteritis, giant cell arteritis, thromboangiitis obliterans, polymyalgia rheumatica, essential criolloarthritis Globulinemia, psoriasis vulgaris and psoriatic arthritis, diffuse fasciitis with eosinophilia, eosinophilia Diffuse fasciitis without cytosis, polymyositis and other idiopathic inflammatory myopathies, recurrent relapsing panniculitis, relapsing polychondritis, lymphomatous granulomatosis, erythema nodosum, ankylosing spondylitis, Unwanted immune and inflammatory reactions associated with Reiter's syndrome, inflammatory dermatitis, and arthritis, joints Rheumatism, inflammation accompanied by hypersensitivity and allergic reactions, systemic lupus erythematosus, collagen diseases, Inflammation associated with atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, blood vessels Inflammatory disorders, respiratory distress syndrome, cardiopulmonary disease, inflammation associated with peptic ulcers, liver fibrosis, liver cirrhosis, liver Diseases, thyroiditis and other glandular diseases, glomerulonephritis, kidney diseases, urinary tract diseases, otitis, ENT diseases dermatitis, skin diseases, periodontal disease, dental diseases, orchitis or epididymitis, infertility, testicular trauma, Immune-related testicular disease, placental dysfunction, placental insufficiency, habitual abortion, eclampsia, preeclampsia, immune-related maternal Gynecological diseases, inflammatory gynecological diseases, posterior uveitis, intermediate uveitis, anterior uveitis, Conjunctivitis, chorioretinitis, uveitis, optic neuritis, endophthalmitis (e.g., retinitis or cystoid macular edema) sympathetic ophthalmia, scleritis, retinitis pigmentosa, immune and inflammatory components of degenerative fundus disease, extraocular Inflammatory components of wounds, inflammation of the eye due to infection, proliferative vitreoretinopathy, acute ischemic optic neuropathy, excessive Scarring, immune response to ocular implants, inflammatory response to ocular implants, immune Virus-related eye diseases, inflammation-related eye diseases, autoimmune diseases, or diseases of the central nervous system (CNS) or other organs Inflammation associated with conditions or disorders, Parkinson's disease, complications associated with the treatment of Parkinson's disease, and / or Side effects include AIDS-associated dementia complex, HIV-associated encephalopathy, Devic's disease, Sydenham chorea, and Alzheimer's disease. Alzheimer's disease and other degenerative diseases, CNS conditions or disorders, inflammatory components of stroke, Streptococcus syndrome, immune and inflammatory components of psychiatric disorders, myelitis, encephalitis, subacute sclerosing panencephalitis, Encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy, Guillain-Barré syndrome Barre syndrome, Sydenham chorea, pseudotumor cerebri, Down syndrome, Huntington's disease, amyotrophic lateral sclerosis sclerosing, inflammatory component of CNS compression, inflammatory component of CNS compression, inflammatory component of CNS compression , CNS trauma, CNS infection, inflammatory component of muscular atrophy, inflammatory component of muscular dystrophy, conditions or disorders of the central and peripheral nervous system, post-traumatic inflammation, septic shock, infectious diseases, surgical Inflammatory complications, inflammatory complications of organ transplantation, side effects of surgery, side effects of organ transplantation, gene therapy Inflammatory complications of gene therapy, immune complications of gene therapy, inflammation-related side effects of gene therapy, gene therapy immune-related side effects of AIDS, inflammation associated with AIDS, humoral immune response, cellular immune response, monocytoproliferative The disease is a leukoproliferative disorder, leukemia, hypermonocytic or hyperlymphocytic disorder, or transplant rejection. In some embodiments, the transplant rejection is due to transplantation of natural cells, artificial cells, natural tissue, artificial tissue, bone marrow, After transplantation of an organ, pacemaker, cornea, or lens. In some embodiments, the neurodegenerative disease is Parkinson's disease. In some embodiments, the disease is a cardiac disease or a pulmonary disease. Diseases include atherosclerosis, myocardial infarction, cardiac infection, heart failure, ischemic heart failure, and hypertension. Pressure, pulmonary hypertension, idiopathic pulmonary fibrosis, stroke, congenital heart disease (CHD), congestive heart failure, stenosis Cardiopathy, myocarditis, coronary artery disease, cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, endocarditis, diastolic function The causes of these conditions are: stroke, cerebrovascular disease, valvular disease, mitral valve prolapse, venous thromboembolism, or arrhythmia.

[0016] In some embodiments, the subject is diagnosed with increased hematopoiesis, increased liver activity, increased angiogenesis, inflammation, The ischemia can affect the cardiac tissue, lung tissue, and / or the immune system. The ischemia may be in tissue, kidney tissue, brain tissue, muscle tissue, limbs, or may be in a variety of forms, including stroke, ischemia, and the like. It may be associated with coronary heart disease, liver failure, kidney failure, and peripheral arterial disease. In some embodiments, the subject is diagnosed with stroke, pulmonary fibrosis, diabetic limb, ischemic heart disease, liver failure, kidney failure, or the like. , peripheral arterial disease, diabetes, liver failure, cirrhosis, liver or pancreatic fibrosis, or hepatitis, osteoporosis, fractures , have or are at risk of developing heart disease, lung disease or scar tissue formation. In embodiments, scar tissue formation or fibrosis is observed in pancreatic tissue, liver tissue, heart tissue, lung tissue, limb tissue, or the like. , in the liver, pancreas or kidneys.

[0017] In some embodiments, the subject is a patient with a disease characterized by the inhibition, reduction, decrease, control, or reversal of pathological apoptosis. In some aspects, the subject requires pancreatic function, liver function, bone cell function, insulin function, or the like. Those who need to improve their spirulina production, lung function, heart function, or who need a joint prosthesis or increased islet cell numbers, increased hepatocyte numbers, or increased insulin production; and / or In some embodiments, the subject is in need of an increased number of dermal stem cells and / or bone cells. or in need of endogenous dermal stem cell activation. be.

[0018] In some embodiments of the disclosed methods, the composition comprising fibroblast regenerative cells is administered parenterally. Introduced into a subject orally, transdermally, transmucosally, by implant, or by transplantation. For example, the composition may be administered intravenously, intramuscularly, intrathecally, intraarterially, intradermally, subcutaneously, intrapleurally, intracranially, intravenously, intramuscularly, intravenously ... It is administered intravenously, intraocularly or mucosally.

[0019] A further aspect of the present disclosure is an in vitro method for producing fibroblast regenerative cells or populations thereof. In this regard, the method comprises selecting and / or culturing fibroblasts that express CD117 and / or CD105. or expanding the fibroblasts, wherein the fibroblasts are, in at least some cases, , compared to control fibroblasts, which included fibroblasts that did not express CD117 and / or CD105. In certain embodiments, the method further comprises determining whether CD34 expression is increased. In another embodiment, the method further comprises selecting fibroblast regenerative cells. The method further includes selecting fibroblast regenerative cells for rhodamine 123 efflux activity. In some embodiments, the method further comprises administering to the subject an antibody against Oct-4, CD34, KLF-4, Nan og, Sox-2, Rex-1, GDF-3, Stella, IL-10 and their combinations and a combination of the fibroblast regeneration markers. In some additional embodiments, the method further comprises selecting for live cells. Selecting fibroblast regenerative cells with enhanced GDF-11 expression compared to control fibroblasts In some embodiments of the method, the fibroblast regenerative cells are selected from the group consisting of M MHC class I, MHC class II, CD45, CD13, CD49c, CD66b, CD 73, CD105, or CD90. In some embodiments, the method does not include preparing a therapeutically effective amount of fibroblast regenerative cells. a therapeutically effective amount of fibroblast regenerative cells, a population thereof, a progeny thereof, or a conditioned medium thereof; The method further includes providing the same to a subject in need thereof. The fibroblast regenerative cell population contains at least 0.01%-5% freshly extracted fibroblasts. include.

[0020] A further aspect of the present disclosure is to select fibroblasts that express CD117 and / or CD105. a step of obtaining fibroblast regenerative cells or a population thereof by selecting fibroblasts have increased regenerative activity compared to control fibroblasts); and fibroblast regenerative cells bFGF, FGF-8, SHH, or BDNF in combination with at least two of the above. The present invention relates to an in vitro method for forming or regenerating one or more nerve cells, comprising culturing the cells in a culture medium. do.

[0021] In another aspect, the present disclosure provides a method for selecting fibroblasts that express CD117 and / or CD105. obtaining fibroblast regenerative cells or a population thereof by selecting and expanding the cells; fibroblasts have increased regenerative activity compared to control fibroblasts; and Culturing the blast regenerative cells using hepatocyte growth factor (HGF) and / or FGF-4 The present invention relates to an in vitro method for forming hepatocytes, comprising:

[0022] In another aspect, the present disclosure provides a method for selecting fibroblasts that express CD117 and / or CD105. a step of obtaining fibroblast regenerative cells or a population thereof by selecting the fibroblasts increased regenerative activity compared to control fibroblasts); and and culturing the cells with vascular endothelial growth factor (VEGF) to form endothelial cells. In vitro methods.

[0023] The present disclosure also provides a method for selecting fibroblasts that express CD117 and / or CD105. obtaining fibroblast regenerative cells or a population thereof (wherein the fibroblasts are and (iii) the fibroblast regenerative cells have increased regenerative activity compared to osteoblasts; and (iv) the fibroblast regenerative cells are used to form bone. BMP4, VEGF, bFGF, stem cell factor (SCF), Flt3L , Hyper IL6, thrombopoietin (TPO) or erythropoietin (EPO) In vitro methods for forming hematopoietic cells, comprising culturing the cells using a combination of at least two of the following: Regarding the Toro method.

[0024] In certain embodiments of the above in vitro methods, the method comprises screening for CD34 expression. The method may further comprise the step of selecting fibroblast regenerative cells, and / or the method may further comprise the step of selecting fibroblast regenerative cells using Rhodamine 12. 3. The method further comprises selecting fibroblast regenerative cells for efflux activity. In one embodiment, the method is for detecting Oct-4, CD-34, KLF-4, Nanog, Sox -2, Rex-1, GDF-3, Stella, IL-10, and their combinations The fibroblast regenerative cells are characterized for at least one additional marker selected from the group consisting of: In some embodiments, the method further comprises selecting a fibroblast from a control fibroblast. and selecting fibroblast regenerative cells with enhanced GDF-11 expression. In a further embodiment of the above method, the fibroblast regenerative cells are MHC class I, MHC class II, MHC class III, ... C class II, CD45, CD13, CD49c, CD66b, CD73, CD105 or does not express at least one or more cell surface proteins of CD90.

[0025] Further aspects of the present disclosure include isolated fibroblasts, populations thereof, progeny thereof or conditions thereof. and / or a pharmaceutical preparation comprising the medium, wherein the fibroblasts are control fibroblasts. In some embodiments, the fibroblasts have increased regenerative activity compared to CD10 In some embodiments, the fibroblasts express the CD115 marker and / or the CD117 marker. further has rhodamine 123 efflux activity and / or further has Oct-4, CD-34 , KLF-4, Nanog, Sox-2, Rex-1, GDF-3, Stella, IL -10 and combinations thereof. Fibroblasts express enhanced GDF-11 expression compared to control cells. In some embodiments, the population is at least 1 x 10 2 , 1×10 6 , 1×10 9 , 1×10 10 , 1×1012 , 1×10 14 cells, or any amount in between In some embodiments, the composition comprises a NaCl solution, such as a 0.8% to 1% NaCl solution. in NaCl solution.

[0026] In some aspects of the present disclosure, the regenerative activity of fibroblast regenerative cells stimulates angiogenesis. In some embodiments, the angiogenesis is the ability to induce human umbilical vein endothelial cell (HUVEC) proliferation. In some embodiments, the angiogenesis further comprises the production of collateral blood vessels. , ischemic heart tissue, or ischemic limb tissue, or surrounding an occluded blood vessel.

[0027] The present disclosure further provides the above-described fibroblast regenerative cells, populations, compositions, progeny, conditioned medium or the like. The present invention provides a kit comprising any of the formulations.

[0028] In a further aspect, the present disclosure provides a regenerating cell that can proliferate and differentiate into ectoderm, mesoderm, or endoderm. A master cell bank is provided, comprising multiple packaged populations of fibroblasts. The isolated fibroblast regenerative cells express Oct-4, Nanog, Sox-2, and KLF4. , c-Myc, Rex-1, GDF-3, LIF receptor, CD105, CD117, CD 344, IL-10, and Stella, and I, MHC class II, CD45, CD13, CD49c, CD66b, CD73, CD 105, or CD90. In some embodiments, the packaged population is at least 1 x 10 2 Previous The present invention includes a cell of the present invention.

[0029] The present disclosure also relates to embodiments for methods for isolating a population of regenerative fibroblasts, The method provides tissue with regenerative activity; and the tissue is about 6-12 microns in size. The method includes enriching a population of fibroblast regenerative cells, wherein the fibroblast regenerative cells are Oct-4, Nanog, Sox-2, KLF4, c-Myc, Rex-1, GDF-3, LIF receptor At least one of CD105, CD117, CD344, IL-10, and Stella and express at least one of MHC class I, MHC class II, CD45, CD13, at least one of CD49c, CD66b, CD73, CD105, or CD90 In some embodiments, the method does not express a cell surface protein. Depleting cells from the population that express markers or MHC proteins, thereby In some embodiments, the cells to be depleted are selected from the group consisting of MH, MH-1, MH-2, MH-3, MH-4, MH-5, MH-6, MH-7, MH-8, MH-9, MH-10, MH-11, MH-12, MH-13, MH-14, MH-15, MH-16, MH-17, MH-18, MH-19, They express C class I, CD66b, glycophorin a, or glycophorin b. In this embodiment, the method optionally comprises operably linking a reporter gene or a selection gene to the target gene. The cells were transfected with a polynucleotide vector containing a stem cell-specific promoter. In some embodiments, the cell-specific promoter is Oct-4 , Nanog, Sox-9, GDF3, Rex-1, or Sox-2 promoter. In some embodiments, the method uses expression of a reporter gene or a selection gene. In some embodiments, the method further comprises enriching the population of regenerative fibroblasts. The method further comprises enriching the population of regenerative fibroblasts by flow cytometry. In some embodiments, the method further comprises: contacting the compound with a compound that is effective in proliferating and non-proliferating cells; selectively detectable); and further enriching the population of cells for proliferating cells. In some embodiments, the detectable compound is carboxyfluorescein diacetate. In some embodiments, the hydroxybenzoate is a succinimidyl ester, a succinimidyl ester, or an Aldefluor. The method further comprises culturing the cells under conditions to form tissue aggregates. In one embodiment, the method comprises collecting fibroblast regenerative cells under conditions that support cell proliferation. The method further comprises the step of culturing the group.

[0030] In some embodiments, the method further comprises the step of extracting granulocytes, T cells, B cells, and / or fibroblast regenerative cells. and further separating cell types such as cells, NK cells, red blood cells, or any combination thereof. In some embodiments, the separation of cell types is performed by cell depletion.

[0031] A further embodiment of the present disclosure relates to a method for identifying fibroblast regenerative cells, the method comprising: A vector containing a fibroblast-specific promoter linked to at least one selectable marker gene. a step of introducing a vector into cells, and a step of transducing a selection marker gene from a cell-specific promoter in the cells. and detecting the expression of a marker gene in the cells, thereby identifying fibroblasts. and identifying fibroblast regenerative cells, wherein the fibroblast regenerative cells are selected from MHC class I, MHC class II, CD44, CD45, CD13, CD34, CD49c, CD66b , CD73, CD105, and CD90. fibroblast regenerative cells express no expression of Oct-4, Nanog, Sox-2, Rex-3, At least one of GDF-3, Stella, FoxD3, IL-10, or Polycomb The fibroblasts express at least one embryonic transcription factor, and the fibroblasts express mesodermal, ectodermal and / or endodermal In some embodiments, the fibroblasts are capable of differentiation into CD13, C They do not express D44, CD90, or a combination thereof.

[0032] In some embodiments, the fibroblast-specific promoter is the Oct-4 promoter. tar, Nanog promoter, Sox-2 promoter, Rex-1 promoter, G DF-3 promoter, Stella promoter, FoxD3 promoter, Poly Combinatorial Repressor Complex 2 promoter, IL-10 promoter In some embodiments, the promoter is a CTCF promoter. The heterologous promoter is flanked by loxP sites.

[0033] In some embodiments, the method further comprises isolating fibroblast regenerative cells. In some embodiments, the fibroblast regenerative cells are derived from bodily fluids and / or mammalian tissues. In some embodiments, the body fluid is synovial fluid or blood. In some embodiments, the mammal is a human.

[0034] In some embodiments, the vector is a retroviral vector. In some cases, the selectable marker gene is a fluorescent protein such as green fluorescent protein (GFP). In some embodiments, the vector encodes a protein, but is not limited to a protein. , containing two selectable marker genes, which can be used to encode a fluorescent protein, a drug Proteins sensitive to selection, cell surface proteins, or any combination thereof This includes.

[0035] In a further aspect of the invention, a gene encoding a nucleotide sequence linked to at least one selectable marker gene is provided. A vector containing a promoter, such as a regenerative cell-specific promoter, is introduced into a population of fibroblasts. a step of injecting the regenerative cells (wherein the regenerative cells are MHC class I, MHC class II, CD44, CD 45, CD13, CD34, CD49c, CD73, CD105, and CD90 on the cell surface protein from a regenerative cell-specific promoter in fibroblast populations; expressing a selectable marker gene; and The present invention relates to a method for generating regenerative fibroblasts, which comprises detecting the expression of a gene.

[0036] In some embodiments, the method involves transfecting regenerating fibroblasts with the OCT-4 transcription factor. The method further comprises the step of transfecting the cells with fibroblasts, thereby enhancing the regenerative activity of the fibroblasts.

[0037] In some embodiments, the method further comprises fusing regenerative fibroblasts with cells having pluripotency. The method further comprises the step of culturing fibroblasts in a manner that produces fibroblasts with enhanced regenerative activity.

[0038] In some embodiments, the method comprises administering to a subject a subject that expresses CD105 and / or CD117. selecting fibroblasts; and selecting fibroblasts expressing CD105 and / or CD117. The method further includes transfecting the cells with a transgenic NANOG gene. In embodiments, the fibroblast regenerative cells further have rhodamine 123 efflux activity. In some embodiments, the fibroblast regenerative cells have enhanced expression of GDF-11 compared to a control. It is being done.

[0039] The use of one or more compositions may be employed in accordance with the methods described herein. The use of one or more compositions in the preparation of a medicament for treatment according to the methods described herein. Other embodiments are discussed throughout this application. Any embodiment described with respect to one aspect also applies to other aspects of this disclosure, and vice versa. is.

[0040] The foregoing describes the features and technical advantages of the present disclosure in order that the following detailed description may be better understood. have been outlined quite broadly. Additional features and advantages form the subject of the claims herein. The concepts and specific embodiments disclosed are intended to carry out the same objectives of the present design. It will be readily apparent to those skilled in the art that the present invention may be readily utilized as a basis for modifying or designing other structures for the purpose of It should be understood that such equivalent constructions are also within the scope of the appended claims. It should be understood by those skilled in the art that this does not depart from the spirit and scope of what is described. Further objects and advantages are set forth in the novel features believed characteristic of the design disclosed herein. The following, both as to organization and method of operation, when considered in conjunction with the accompanying drawings, It will be better understood from the description below. However, each figure is for purposes of illustration and explanation. It should be expressly understood that these are provided for purposes of illustration only and are not intended as a definition of the limits of the present disclosure. do. [Brief explanation of the drawings]

[0041] For a more complete understanding of the present disclosure, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which:

[0042] [Figure 1] Figure 1 shows enhanced expression of OCT-4 in CD105 purified fibroblasts: control (left bar), CD105 negative (middle bar), and CD105 positive (right bar).

[0043] [Figure 2] Figure 2 shows enhanced expression of NANOG in CD105 purified fibroblasts: control (left bar), CD105 negative (middle bar), and CD105 positive (right bar).

[0044] [Figure 3] Figure 3 shows enhanced expression of KLF-4 in CD105 purified fibroblasts (control, left bar; CD105 negative, middle bar; CD105 positive, right bar).

[0045] [Figure 4] Figure 4 shows enhanced expression of the immunoregulatory cytokine IL-10 by CD105-selected fibroblasts. Control is shown in the left bar, CD105-negative in the middle bar, and CD105-positive in the right bar.

[0046] Although various embodiments of the present disclosure have been shown and described herein, it is understood that such embodiments are not intended to be limiting. It will be apparent to those skilled in the art that the present invention is provided solely as a Numerous variations, modifications, and substitutions will occur to those skilled in the art. It will be appreciated that various alternatives to the configuration may be used. DETAILED DESCRIPTION OF THE INVENTION

[0047] [I.Definition] In accordance with long-standing patent law practice, the terms "a" and "an" are used throughout this specification, including the claims. When used in conjunction with "comprising," the word "comprising" means "one or more" Some embodiments of the disclosure may comprise one or more elements, method steps, and / or or method. The composition can be performed with respect to any other method or composition described herein and may be used in a different manner. It is contemplated that the embodiments may be combined.

[0048] As used herein, the term "cell surface protein" refers to a protein that is expressed on the surface of a cell. refers to protein.

[0049] As used herein, the term "expansion" refers to cells that undergo terminal differentiation. refers to the proliferation of one or more cells that do not undergo cell division.

[0050] As used herein, the term "differentiation" refers to the progression of lineage differentiation. They represent pathways through which precursor or "progenitor" cells undergo stepwise physiological changes and acquire characteristic functions. This refers to the process of differentiation into specific cell types (e.g., nerve cells, muscle cells, endothelial cells, etc.). This occurs during cellular differentiation, which gradually leads to cell specification until full maturation, also known as "terminal differentiation." "Terminally differentiated cells" are cells that have committed to a specific lineage and reached the final stage of differentiation (i.e. , fully mature cells) and are "committed" or "differentiated" to become specific By "cells" is meant cells that express one or more markers or other characteristics of cells of a lineage of a certain type.

[0051] As used herein, the term "isolated" refers to a non-naturally occurring state outside the body. refers to a population of stem cells or daughter stem cells (e.g., isolated from or derived from the body). Biological samples include synovial fluid, blood (e.g., peripheral blood), or tissue. obtain.

[0052] As used herein, the term "purified" refers to "purified cells." The term "cell type" refers to the presence of other cell types that are isolated from the subject's body but are also obtained from the subject's body. "Substantially purified" means that the desired cells are at least 20%, more preferably at least 50%, even more preferably at least 75%, Preferably, this means that the concentration is at least 90%, or even 95% concentrated.

[0053] By "cell population" is meant a collection of at least 10 cells. Preferably, the population is small. At least 20 cells, more preferably at least 100 cells, and most preferably at least The stem cells of the present invention are capable of self-regeneration. They exhibit viability and can be expanded in culture to produce populations of billions of cells.

[0054] The "germ layers" are the three primary layers formed as a result of gastrulation in the early embryo: endoderm, mesoderm, and The germ layer of an embryo is the source from which all tissues and organs are derived. The endoderm is , e.g., the pharynx, esophagus, stomach, intestines and associated glands (e.g., salivary glands), liver, respiratory tract, and gastrointestinal tract It gives rise to the epithelial lining, pancreas, and lungs. The mesoderm also gives rise to, for example, smooth and striated muscle, connective tissue, It is the source of tissues, blood vessels, the cardiovascular system, blood cells, bone marrow, skeleton, reproductive organs, and excretory organs. Leaves contain, for example, the epidermis (top layer of the skin), the sensory organs, the brain, the spinal cord and all the outer components of the nervous system. It is the source of the entire nervous system, including the nervous system.

[0055] The term "pluripotency" in relation to the stem cells of the present invention refers to the differentiation of the three primitive germ layers (endodermal It refers to the ability of stem cells to give rise to all cells (e.g., mesoderm, and ectoderm).

[0056] As used herein, the term "allogeneic" refers to cells of the same species that are genetically distinct from the host's cells. refers to the cells of the

[0057] The term "autologous" as used herein refers to cells derived from the same subject The term "engraft" as used herein refers to the transfer of existing cells of a tissue. This refers to the process by which stem cells are incorporated into a target tissue in vivo through contact with the

[0058] "Not detectably expressed" means that the expression of the protein or gene is not detectable by standard methods. In the case of cell surface markers, expression can be measured by, for example, flow cytometry. By using a negative control (i.e., cells known to lack the antigen of interest) or an antibody A cutoff value obtained from a type control (measuring non-specific binding of the antibody to the cells) was used. Thus, cells that "do not detectably express" a marker can be measured. The gene expression was analyzed using standard PCR methods. Following the protocol, the presence of the mRNA can be visually detected on a standard agarose gel. If not, the gene is said to be "not detectably expressed."

[0059] Conversely, if a protein or gene can be detected, including by the same method, the cell is likely to contain the protein. A protein or gene is said to be "expressing" a gene.

[0060] The term "cultured expanded population" refers to cells whose numbers have been increased by cell division in vitro. "Cells" refers to a population of cells. The term can be applied to stem cell populations and non-stem cell populations alike.

[0061] The term "passaging" refers to the process of transferring a portion of cells from one culture vessel to a new one. Refers to the s.

[0062] The term "cryopreservation" refers to the preservation of cells for long-term storage in a cryoprotectant at low temperatures. Refers to...

[0063] The term "master cell bank" refers to a collection of cryopreserved cells. Such cell banks may contain stem cells, non-stem cells, and / or a mixture of stem cells and non-stem cells. .

[0064] As used herein, the terms "about" or "approximately" refer to a reference amount, level, or value. , 30, 25, 20, 25 for number, frequency, proportion, dimension, size, amount, weight or length Amount, level, value, number, or frequency that varies by 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% In certain embodiments, the numerical value may be preceded by "about." " or "approximately" means a range of 15%, 10%, 5%, or 1%. In relation to biological systems or processes, this term can mean within an order of magnitude of a value, preferably within 5 times, more preferably within 2 times. Unless otherwise specified, the term "about" means within an acceptable error range for a particular value.

[0065] As used herein, the term "activated fibroblast" refers to a cell that has undergone one or more One or more stimuli that can induce changes: metabolic, immunological, growth factor secretion, surface marker expression, and / or production of microvesicles.

[0066] As used herein, the term "activated immune cells" refers to cells that have undergone one or more changes in their function. One or more stimuli that can induce differentiation: metabolic, immunological, growth factor secretion, surface marker expression, and and / or microvesicle production.

[0067] The terms "administered" or "administering" as used herein refer to a composition Any method of providing a composition to an individual such that the composition has its intended effect on the patient. For example, one method of administration may be via a medical device such as a catheter, applicator gun, or syringe. Examples of mechanisms that may be used include, but are not limited to, indirect mechanisms using therapeutic devices. Typical methods of administration include direct mechanisms, e.g., local tissue administration, oral ingestion, transdermal patches, topical , inhalation, suppositories, etc.

[0068] As used herein, "allogeneic" refers to a gene derived from one or more individuals of the same species. or other antigens that are or may be immunologically incompatible in their natural setting. refers to tissues or cells derived from the body.

[0069] As used herein, the term "allograft" refers to the transfer of an organ from a donor to a recipient. refers to the transplantation of organs, tissues, and / or cells, where the donor and recipient are different individuals. The tissue transplanted by this method is called an allograft. These transplants are called allografts or transplants.

[0070] As used herein, the terms "allostimulatory" and and "alloreactive" refers to the reaction to the same antigen, i.e., "alloant" "igen," or stimulation and reaction of the immune system in response to cells expressing a foreign HLA haplotype It means response.

[0071] As used herein, the term "angiogenesis" refers to the growth of new blood vessels from pre-existing blood vessels. It refers to the physiological process that involves the growth of blood vessels, and the formation of blood vessels through the process of initiation of angiogenesis, i.e., the growth of blood vessels from pre-existing blood vessels. The formation of new blood vessels by dividing existing blood vessels, and the division of existing blood vessels (intussusception) This includes the formation of new blood vessels.

[0072] As used herein, the term "autoimmunity" refers to the destruction of an organism's own healthy cells and tissues. It refers to the immune response system against the virus.

[0073] As used herein, "autologous" refers to organisms derived from or derived from the same individual's body. Refers to tissues or cells transferred from a donor (i.e., autologous blood donation; autologous bone marrow transplant).

[0074] As used herein, the term "autologous transplant" refers to a transplant from a part of the body of an individual. Refers to the transplantation of organs, tissues, and / or cells in the same individual to another part of the body (i.e. (In this case, the donor and recipient are the same individual.) Such "autologous" procedures result in transplantation. The implanted tissue is an autograft or autotransplant. plant).

[0075] The term "biologically active" or "bioactive compound" refers to a compound that has a structural, regulatory or refers to any molecule that has a biochemical function. For example, biological activity can refer to, for example, protein activity. This can be determined by the restoration of wild-type growth in cells lacking the protein activity. Cells can be generated in many ways (i.e., point mutations and frameshift mutations, for example). complementation can be achieved by providing cells lacking a protein activity with the protein, its derivative, or a portion thereof. This can be achieved by transfecting the gene with an expression vector that expresses the gene. In this context, a fragment of a gene product (e.g., a protein) may be a fragment that does not retain the activity of the full-length gene product. If it does, it can be considered biologically active (or called functionally active). ) but it may be that the activity of the full-length gene product is reduced but still detectable. .

[0076] "Cell culture" refers to a living organism, whether quiescent, senescent, or (actively) dividing. In cell culture, cells are grown in an artificial in vitro system containing living cells at a suitable temperature, They are typically grown and maintained at a temperature of 37°C, typically in an atmosphere containing oxygen and CO2. Culture conditions can vary widely for each cell type, but for a particular cell type, Variations in conditions can result in the expression of different phenotypes. The most commonly variable factors in culture systems are The second component is the growth medium. Growth medium is determined by the concentration of nutrients, growth factors, and the presence of other components. The growth factors used to supplement the culture medium are derived from animal blood, such as calf serum. I often do this.

[0077] As used herein, the term "collateralization" is a vessel that cannot adequately supply its end organ or vascular bed, but instead is connected to the same end organ. Refers to the growth of a blood vessel or several blood vessels that serve an organ or vascular bed.

[0078] Throughout this specification, unless the context requires otherwise, the words "comprises" and "including" are used. means the inclusion of the specified step or element or elements, but the exclusion of other steps or elements or elements It is understood that "consists of" does not mean "consisting of" "Consisting of" means including and limited to the following: Indicates that the specified element is required or essential, and that no other element may be present. "Consisting essentially of" includes any element listed after the phrase, but does not include any element listed. Other elements that do not interfere with or contribute to the activity or function specified in the disclosure of the identified element It means that something is essentially limited to something. In the example, the listed elements are required or mandatory, while other elements are optional. It may be present or absent depending on whether it affects the activity or action of the listed element. It shows a good thing.

[0079] As used herein, the term "conditioned medium of fibroblast regenerative cells" refers to a medium that is in contact with cells. The cells produce factors that enter the medium, giving the medium therapeutic activity. It means to be given.

[0080] As used herein, the terms "drug," "agent," or "compound" refer to a substance that has a desired effect. Drugs or compounds are any pharmacologically active substance that can be administered to achieve a , synthetic or naturally occurring, non-peptides, proteins or peptides, oligonucleotides The nucleic acid may be a peptide, or a nucleotide (DNA and / or RNA), a polysaccharide, or a sugar.

[0081] A "growth factor" is a compound capable of stimulating cell proliferation and / or cell differentiation and / or cell migration. It can be a naturally occurring, endogenous or foreign protein, or a recombinant protein. obtain.

[0082] As used herein, the term "subject" or "individual" refers to a person who is admitted to a medical facility or refers to a human or animal that may be hospitalized or treated as an outpatient in a medical facility. An individual may receive one or more medical compositions via the internet. can include non-human animals of any age, and therefore includes both adults and juveniles (i.e. Therefore, the term "individual" includes any individual who is in need of medical treatment. Therefore, individuals may not use the product in support of clinical or basic science research. Participation in an experiment can be voluntary or involuntary. The term "subject" or "individual" refers to any living organism or animal that is the subject of a method or material, including mammals, e.g., humans, experimental animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cattle, sheep, domestic animals (e.g., dogs, cats, rodents); horses, and transgenic non-human animals.

[0083] As used herein, the term "ischemia" or "ischemic condition" refers to an ischemic event in an organ or part of the body. This condition is called inadequate blood supply to the arteries. Such conditions include cardiac ischemia, ischemic colitis, and mesenteric ischemia. , ischemic stroke, cerebral ischemia, renal ischemia, and limb ischemia.

[0084] "Mesenchymal stem cells" or "MSCs" are, for example, cells that (1) adhere to plastic, (2) They express CD73, CD90, and CD105 antigens, while CD14, CD34, and CD4 5, and HLA-DR negative, and (3) osteogenic, chondrogenic, and adipogenic In a specific embodiment, the mesenchymal stem cells are cells that have the differentiation potential of HLA-DR, A substantial increase (e.g., compared to baseline) in CD117, CD45, or a combination thereof As used herein, "mesenchymal stromal cells" refers to cells that do not express a level of IL-1 or IL-2 (greater than 50% of the total IL-1 or IL-2) that is higher than the level of IL-1 or IL-2. ” (also called “mesenchymal stem cells”) or “MSCs” are derived from bone marrow, adipose tissue, amniotic fluid, and the uterus. Membrane, trophoblast-derived tissue, umbilical cord blood, Wharton's jelly, placenta, amniotic tissue, pluripotent stem cells The term may be derived from any tissue, including but not limited to teeth. As previously mentioned, "mesenchymal stromal cells" or "MSCs" are C cells when first isolated from tissue. These cells are D34 positive but phenotypically and functionally similar to the cells described above. "MSC" as used herein includes NGF-R, PDGF-R, EGF-R, IGF- R, CD29, CD49a, CD56, CD63, CD73, CD105, CD106, CD140b, CD146, CD271, MSCA-1, SSEA4, STRO-1 and a cell surface marker selected from the list consisting of STRO-3, STRO-4, or any combination thereof Cells isolated from tissue using a fluorouracil filter and meeting ISCT criteria either before or after expansion As used herein, "mesenchymal stromal cells" or "MSCs" include those described in the literature. The cells that have been used include bone marrow stromal stem cells (BMSSCs), bone marrow-isolated multipotent adult progenitor cells ( MIAMI), Mesenchymal Adult Stem Cells (MASCS), MultiStem™, Pro chymal™, remestemcel-L, mesenchymal progenitor cells (MPC), dental pulp Stem cells (DPSC), PLX cells, PLX-PAD, Allostem(TM), Ast rostem(trademark), Ixmyelocel-T, MSC-NTF, NurOwn(commercial) ), Stemedyne(TM)-MSC, Stempeucel(TM), Stem peucelCLI, StempeucelOA, HiQCell, Hearticel lgram-AMI, Revascor(TM), Cardiorel(TM), Car tistem(trademark), Pneumostem(trademark), Promostem(trademark), Homeo-GH, AC607, PDA001, SB623, CX601, AC607, These include endometrial regenerative cells (ERCs) and adipose-derived stem and regenerative cells (ADRCs).

[0085] Throughout this specification, the terms "one embodiment," "embodiment," "particular embodiment," "related embodiment," "related" and "related" are used interchangeably. "some embodiments," "particular embodiments," "additional embodiments," or "further embodiments" Reference to a particular feature, configuration, or combination thereof described in connection with an embodiment is not intended to be limiting. or features are included in at least one embodiment of the present invention. The appearances of the foregoing phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, any particular feature, structure or characteristic may be incorporated into one or more embodiments. The components may be combined in any suitable manner.

[0086] As used herein, the term "pharmaceutically" or "pharmacologically acceptable" refers to an animal or human a molecule that does not produce adverse, allergic, or other untoward reactions when administered to a subject Refers to entities and compositions.

[0087] As used herein, the term "pharmaceutically acceptable carrier" includes water, ethanol, polyisoprene, ethanol ... alcohols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol) and suitable mixtures thereof, as well as vegetable oils, coatings, isotonic and absorption delaying agents. any suitable surfactant, including, but not limited to, liposomes, commercially available detergents, etc. and any solvent or dispersion medium. Supplementary biologically active ingredients may also be incorporated into such carriers. It can be embedded.

[0088] Refers to the occurrence of any symptoms in untreated subjects compared to treated subjects. "decreases," "inhibits," "reduces," "suppresses," "diminishes," "prevents," and grammatical equivalents The term "lower" (including "smaller" and "smaller") refers to the degree to which the The amount and / or magnitude of symptoms can be assessed as clinically relevant by any medically trained person. the amount and / or magnitude of symptoms in the treated subject by any amount recognized as In one embodiment, the amount and / or magnitude of symptoms in a treated subject is The amount and / or magnitude of symptoms in the treated subject is at least 10% lower than that in the treated subject and at least 2 5% lower, at least 50% lower, at least 75% lower, and / or at least 90% low.

[0089] A "therapeutic agent" has a "therapeutic effect" in modulating angiogenesis and / or wound healing. and the amount of therapeutic agent is determined based on the subject in need of modulation of angiogenesis (e.g., an animal model or human). and (iii) significantly modulating (i.e., increasing or decreasing) angiogenic activity when administered to a human subject (e.g., a human subject). An amount sufficient to induce this is said to be an "angiogenesis-modulating amount."

[0090] As used herein, the term "therapeutically effective amount" means "effective amount," "therapeutically effective use," "effective dose" and / or "effective amount" and mean the amount of a therapeutically effective dose that can be administered to an individual in need thereof. The amount of a compound that elicits the biological, cosmetic, or clinical response desired by a patient. Examples include: An effective amount is an amount sufficient to reduce the immunogenicity of a group of cells. An effective amount is sufficient to promote the formation of a blood supply sufficient to support the transplanted tissue. As another non-limiting example, an effective amount is an amount that induces new blood vessels and associated vasculature (vasculature formation). and / or the repair or replacement of existing blood vessels and associated vasculature. The amount is sufficient to promote remodeling. The appropriate effective amount to be administered will be readily apparent to those of skill in the art using the guidance provided herein. For example, an effective amount can be determined by in vitro and in vivo methods as described herein. Those skilled in the art will appreciate that the condition of an individual can be monitored throughout the course of treatment. and that an effective amount of the compound or composition disclosed herein administered is It will be appreciated that the frequency of the pulses may be adjusted according to the frequency of the pulses.

[0091] As used herein, the term "transplantation" means The process of taking living tissue or cells and transplanting them into another part of the body or into another body. He points to Seth.

[0092] "Treatment," "treat," or "treating" means reducing the effects of a disease or condition. Treatment also refers to a method of alleviating the disease or condition itself, rather than merely the symptoms. Treatment can be any reduction from pre-treatment levels and may be used to treat a disease, condition, or may be, but is not limited to, complete ablation of the symptoms of the disease or condition. Thus, in the disclosed methods, "treatment" refers to the reduction in the severity of at least one symptom of a disease. 10%, 20%, 30%, 4% or 5% reduction in the severity of established disease or disease progression, including a reduction in severity of disease It can refer to a reduction of 0%, 50%, 60%, 70%, 80%, 90%, or 100%. For example, the disclosed methods for reducing the immunogenicity of cells may be used in combination with the same subject or control. There is a detectable decrease in the immunogenicity of the cells when compared to pre-treatment levels in a subject. A reduction is considered therapeutic if it is present. 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% or "Treatment" does not necessarily refer to a cure of a disease or condition. It is understood and contemplated herein that the term refers to an improvement in the outlook for a disease or condition, rather than an improvement in the prognosis of the disease or condition. In certain embodiments, treatment refers to a decrease in the severity or extent of at least one symptom, Alternatively or additionally, it may refer to a delay in the onset of at least one symptom.

[0093] The terms "select" or "select for expression" refer to the expression of one or more molecules specific to a cell. This means isolating specific cells from a heterogeneous population of other cells, for example by using a gene. do.

[0094] II. Cell proliferation Any cell encompassed herein can be cultured in any manner, including using a variety of in vitro systems. In one embodiment, the in vitro propagation means comprises one or more biosynthetic cells. The bioreactor used in the present disclosure includes a bioreactor that can be used to generate nutrient concentrations in vivo. and a culture process that can supply medium and oxygen at controlled concentrations and rates that mimic the Bioreactors have been commercially available for many years and are designed to provide a variety of Various types of bioreactors used in mammalian cell culture are used. Of these, most are designed to enable the production of high-density cultures of a single cell type. For use in the present invention, the proliferation of mesenchymal stem cells is The culture techniques and bioreactors used for propagation are adaptable for use with regenerative fibroblasts. Several methods are described in references 1 to 8.

[0095] In one embodiment, a typical application for these high density systems is to use fine The goal is to produce a conditioned medium produced by cells, e.g., monoclonal Packaging cell lines for antibody hybridoma production and viral vector production However, these applications may be limited to therapeutic applications, such as in some embodiments of the present disclosure. This differs from applications where the final product is the harvested cells themselves. When activated, it provides automatically regulated medium flow, oxygenation, and temperature and pH control. Generally, bioreactors allow for the production of large amounts of cells. Therefore, bioreactors are economical for labor. The most sophisticated bioreactors offer high throughput and minimize the chance of contamination during the process. Setup, growing, selection and harvesting, including minimal manual labor requirements and open processing steps Such a bioreactor would optimally allow for the preparation of a bioreactor according to the present invention. As intended, it is designed for use with a homogenous cell mixture or an aggregated cell population. Suitable bioreactors for use in the present invention include those described in U.S. Pat. 4, 5,985,653, U.S. Patent No. 6,238,908, U.S. Patent No. 5,51 No. 2,480, U.S. Pat. No. 5,459,069, U.S. Pat. No. 5,763,266, U.S. Pat. No. 5,888,807 and U.S. Pat. No. 5,688,687 (incorporated herein by reference). (incorporated by reference). Culture requires the control of several fundamental parameters: maintaining cell viability, proliferation, and A medium must be provided that allows for the differentiation and eventual storage of cell cultures. Typically, the various media are delivered to the cells by a pumping mechanism within the bioreactor. The medium is periodically replenished and replaced

[10] . Metabolic product quantification may also be performed

[11] . In the exchange process, by-products are removed from the culture. It is possible.

[0096] Growing cells or tissues also require a source of oxygen. Different cell types have different oxygen requirements. Therefore, a flexible and adjustable means for supplying oxygen to cells is a desirable component. is.

[0097] Depending on the particular culture, uniform distribution of the cell population and medium supply in the culture chamber may be achieved by: This can be an important process control [12, 13]. This can be effective when cell-cell interactions are not important. Examples of suspension culture systems include various tank reactor designs and gas permeable plastics. These include stick bags that do not require assembly into a three-dimensional structure or interstitial layers. or proximity to the feeder layer (such as most blood cell precursors or mature blood cells) For cells that do not require such a suspension design, the culture process can be Efficient cell harvesting upon completion is a key feature of an effective cell culture system One approach to producing cells as a product involves simple elution of the cell product. The resulting concentrate is easy to handle and can be washed in a commercially available closed-system cell washer designed for final washing. in a defined space without physical barriers for collection, resulting in a defined volume of cells. In a specific embodiment, the system is a pharmaceutically acceptable carrier, with or without a cell preservative compound, at least some of the components of the formulation can be added to the formulation and provide for efficient collection into suitable sterile packaging. In this case, the collection and packaging process may breach the sterile barrier of the culture chamber fluid pathway. In some embodiments, cell proliferation can be completed without disruption [15, 16]. This is done using microcarrier beads [17-23]. In an embodiment, the cells are cultured in a xeno-free medium, referred to as a "xeno-free" medium. In some embodiments, the xeno-free medium is platelet-free. They are produced using lysates or other defined conditions [24-26].

[0098] In some embodiments of the present disclosure, cells are is grown under hypoxic conditions

[27] .

[0099] [III. Mitochondria] In some embodiments of the present disclosure, the fibroblast regenerative cells, composition, or pharmaceutical preparation is administered exogenously. The effect of the present invention is enhanced by the introduction or co-administration of isolated, substantially pure mitochondria. Mitochondria can be fused by various means (e.g., lipid fusion, polyethylene glycol-mediated fusion, or The method of the present invention can be administered to fibroblast regenerative cells via electroporation. In another embodiment, mitochondrial delivery is performed to facilitate mitochondrial uptake into a subject. The doria may be encapsulated or treated with a drug. The method further comprises administering to the patient a vitamin A, Vitamin C, Vitamin D, Vitamin E, Vitamin K, Folic Acid, Choline, Vitamin B1, Vitamin Vitamin B2, Vitamin B5, Vitamin B6, Vitamin B12, Biotin, Nicotinamide, Beta-carotene, Coenzyme Q, Selenium, Superoxide Dismutase, Gluconate Calcium carbonate, uridine, creatine succinate, pyruvate, dihydroxyacetone), acetone L-carnitine, alpha-lipoic acid, cardiolipin, omega fatty acids, lithium carbonate, One or more agents selected from lithium enoate, calcium enoate, and mixtures thereof are added to the composition of the present invention. In any of the above embodiments, the cocktail described in Even though mitochondria may be homologous, allogeneic, or heterologous, For use in the methods, kits, and compositions of the present invention, The mitochondria can be obtained from any of the sources described herein. Means for achieving chondrial transfer are known in the art and are described in references 28-38. In some embodiments of the present disclosure, fibroblasts are stem cells or fibroblasts as described in reference 3. Mitochondria derived from less differentiated cells compared to the fibroblasts, such as those shown in Figures 9-50. All of the above references are incorporated herein by reference in their entirety. will be incorporated into

[0100] In certain embodiments, the method includes (i) separating mitochondria from other components of the cell. (ii) administering the isolated mitochondria to a subject to produce isolated, substantially pure mitochondria; and administering the isolated, substantially pure mitochondria, e.g., together with fibroblasts. In certain embodiments, the cells are progenitor cells or any of the cells described herein. Other cell types of interest.

[0101] [IV. Cell transplantation therapy] Any medical condition, disease, or disorder for which fibroblast regenerative cells are therapeutic or preventative. can be treated by the methods of the present disclosure.

[0102] As an example, autoimmune diseases are characterized by an overreaction of the immune system against endogenous tissues. The immune system mistakenly identifies endogenous tissues as foreign bodies that must be fought, resulting in a severe inflammatory response. This causes damage to the affected organs. The key distinguishing factor is the T lymphocyte or T cell. is "conjugated" in the thymus so that it only docks with endogenous cell surface molecules, so-called MHC molecules. They are "trained" and therefore tolerant of endogenous structures.

[0103] In autoimmune diseases, a group of T cells behave abnormally, preventing the body from defending itself against foreign molecules and organisms. In addition to still functioning, they now attack endogenous structures. When vital structures are affected, autoimmune diseases can become fatal. The immune system then directs defense against these structures, triggering cellular and humoral defense responses. However, autoantibodies are formed, which eventually causes the affected organ to fail. In general, it weakens the immune system and makes you more susceptible to all kinds of illnesses. , the recognition of foreign matter is also hindered, thereby effectively preventing the spread of degenerated cancer cells (for example). As the disease progresses, the immune system becomes less able to fight off infections, making the patient more susceptible to infections. The cells of the immune system destroy endogenous structures, while the body's repair mechanisms repair damaged organ parts. Generally, the wrong attack of this defense system is not cured. The case continues for life or until the target structure is completely destroyed.

[0104] Autoimmune diseases are treated according to the organ system affected. In this case, the basic principle of causal therapy is The treatment involves suppressing the activity of the immune system by administering immunosuppressants such as cortisone. Thanks to efforts to develop new drugs that specifically affect the mechanisms involved in disease events, These substances were characterized by multiple systemic side effects and interactions.

[0105] In one embodiment of the present disclosure, fibroblast regenerative cells are used to treat autoimmune or inflammatory disorders. In some embodiments of the present disclosure, the fibroblasts are administered to an individual for treatment. are cultured ex vivo and subjected to conditions that reduce their immunogenicity, and then is utilized to stimulate anti-inflammatory and / or immunomodulatory properties. and administering the cells to an individual in need thereof for the purpose of treating an autoimmune and / or inflammatory condition. It is directed towards how to do this.

[0106] Embodiments of the present disclosure involve modifying culture conditions to reduce the immunogenicity of fibroblasts. In this way, a means is provided for utilizing fibroblasts as allogeneic therapeutic cells. In this context, fibroblasts are preferred over sources with lower immunogenicity (e.g., placental fibroblasts, etc.). In another embodiment, the fibroblasts are extracted from, for example, ex vivo culture. , and are subjected to interferon gamma (IFN-γ), but this is not limited to the mechanism In certain embodiments, reducing immunogenicity includes reducing the immunogenicity of the antibody. This can be exemplified by inhibiting the ability of fibroblasts to induce autoreactive T cell responses. In certain embodiments of the present disclosure, these modified fibroblasts are universal donor -fibroblasts.

[0107] Embodiments of the present disclosure provide autologous or inflammatory cytokines for the treatment of inflammatory and / or autoimmune conditions. The present disclosure is directed to systems and methods for the use of fibroblasts, either allogeneic or allogeneic. The methods and compositions provide specific modified inflammatory and / or autoimmune conditions for the treatment of In particular, the cells include at least any type of fibroblast. or fibroblasts, as well as fibroblasts of various tissue origins that actively inhibit autoimmune processes. In one embodiment of the present disclosure, fibroblasts are used to treat autoimmune conditions. In certain embodiments, the present invention provides a method for the prevention and / or treatment of cancer. Fibroblasts may be used to treat inflammatory and / or autoimmune processes directly or indirectly. The route of administration, dosage, and frequency of administration are determined by the disease. as a function of the stage of the disease and the disease process, and is used in everyday medical practice. It can be optimized.

[0108] In one embodiment, allogeneic fibroblasts are grown in a non-manipulated manner (e.g., interferon). administered to an individual (without prior exposure to one or more specific factors, such as γ), but naturally characterized by immunomodulatory activity, such as pelvic fibroblasts or adipose tissue-associated fibroblasts. In another embodiment of the present disclosure, any fibroblast is selected from a source. The cells are cultured under conditions that induce retrodifferentiation to give fibroblasts an immature phenotype, Here, the immature phenotype correlates with enhanced anti-inflammatory and / or immunomodulatory capabilities. Fibroblasts were cultured in the presence of one or more histone deacetylase inhibitors, such as valproic acid. These cells can be cultured in vitro (Moon et al., 2008; Huang et al., 2011). In addition, 8-Br-cAMP (Wang et al., 2011); M-CSF treatment (Li et al., 20 16); exposure to revelecine (Li et al., 2016); and / or exposure to stem cell extracts ( Other means of inducing fibroblast dedifferentiation, such as Xiong et al., 2014, may also be useful in this study. In the context of the invention, the characterization of fibroblast dedifferentiation can be carried out using, for example, CXCR4, Assessment of extracellular markers such as VEGFR-2, CD34, and / or CD133, and for evaluation of intracellular markers such as SOX-2, NANOG, and / or OCT-4 Therefore, it can be done.

[0109] In one embodiment of the present invention, the fibroblasts are cultured in a manner that preserves the viability and proliferation capacity of the fibroblasts. For this purpose, the cells are cultured ex vivo using means known in the art. and providing modifications of known culture techniques to reduce recognition of fibroblasts by the patient's immune system. In one embodiment, the fibroblasts are cultured in a condition lacking xenogeneic components (e.g., fetal bovine fibroblasts). The xenogeneic component induces immunological responses, including the induction of antibody and T cell responses. are known to induce , 2000;Kadri et al., 2007;Forni et al., 1976;Lauer et al., 198 3) Many individuals have natural antibodies of the IgM isotype against components related to fetal bovine serum. (Irie et al., 1974) and after administration of cells grown in the presence of fetal bovine serum. It can cause rejection, inflammation, or anaphylaxis (Macy et al., 1989). In some embodiments, the present disclosure provides a method for reducing the immunogenicity of fibroblasts by using bovine fibroblasts. Fetal serum was replaced with human platelet-rich plasma, platelet lysate, cord blood serum, autologous serum, and / or This involves replacing the tissue in xeno-free medium with a defined cytokine mixture. Culturing procedures are known in the art for other cell types and are incorporated by reference. (Riordan et al., 2015).

[0110] Embodiments of the present disclosure provide methods and compositions comprising fibroblasts. Fibroblasts are cells that are capable of transporting tissue from the skin. Various tissues, such as skin, heart, blood vessels, bone marrow, skeletal muscle, liver, pancreas, brain, and / or foreskin They may be derived from organs, and may be obtained by biopsy (as appropriate) or autopsy. In some aspects, the cells may be of, for example, fetal, neonatal, adult origin, or a combination thereof. The present invention includes fibroblasts that can be derived from the

[0111] Various quality control measures are known in the art for practitioners of the present invention to administer cells clinically. Exemplary criteria for cell qualification include flow cytometry, viability, and and / or endotoxin content, as well as marker identification and the detection of microorganisms and mammals. Includes assessment of icoplasma contamination.

[0112] In one embodiment of the present disclosure, universal donor fibroblasts are used to treat autoimmune or inflammatory diseases. In some embodiments of the present disclosure, the cells are administered to an individual for the treatment of an infectious disorder. The cells are cultured ex vivo under conditions that reduce immunogenicity and stimulate anti-inflammatory and / or immunomodulatory properties. Further embodiments are directed to the use of the compounds of the present invention for the treatment of autoimmune and / or inflammatory conditions. The present invention is directed to a method of administering cells to an individual in need thereof.

[0113] V. VARIOUS EMBODIMENTS In accordance with the present disclosure, methods for providing cell therapy and treating subjects who would benefit from cell therapy are provided. In one embodiment, the method comprises the steps of: A population or culture of cells, progeny of fibroblast regenerative cells, or conditions of fibroblast regenerative cells Administering the medium to a subject in an amount sufficient to provide a benefit to the subject. In certain embodiments, the subject is in need of increasing, stimulating, inducing, promoting, enhancing or augmenting hematopoiesis. In a further non-limiting aspect, the subject is receiving a drug to increase, stimulate, promote, enhance or otherwise improve liver function or activity. is enhanced; requires the reduction, suppression, blocking, prevention, control or limitation of inflammation or autoimmunity; Alternatively, angiogenesis needs to be increased, stimulated, induced, promoted, enhanced, or enhanced. Thus, the disclosed methods increase, stimulate, induce, promote, augment or otherwise enhance hematopoiesis (in a deficient subject). to increase, stimulate, promote, enhance or increase liver function or activity; to reduce inflammation To reduce, lower, suppress, block, prevent, control, or limit (e.g., involving inhibition of inflammation) to a subject); and increasing, stimulating, inducing, promoting, augmenting or enhancing angiogenesis, For example, fibroblast regenerative cells can be used to induce angiogenesis (e.g., in a subject). by homing to ischemic tissue in the ischemic tissue) into a subject with ischemia (e.g., intravenously) It can be administered to treat many diseases, including stroke, ischemic heart disease, liver failure, kidney failure, and peripheral arterial disease. Furthermore, the disclosed methods can be used to treat tissue or organ (e.g., heart or lung) diseases associated with ischemia. and treating a subject having or at risk of having ischemia in a tissue, limb, or kidney. for treating a subject in need of or having fibrosis or inhibition of scar tissue formation; ;For treating a subject in need of inhibition, reduction, control or reversal of pathological apoptosis;Pancreatic or to treat a subject in need of increasing or improving liver function; To increase the number of cells or to increase insulin production; diabetes, liver failure, cirrhosis for treating a subject having or at risk of having liver fibrosis or pancreatic fibrosis, or hepatitis. for treating a subject in need of bone cells or bone cell function (e.g., increasing bone cell numbers, bone cell function, to increase, stimulate, induce, enhance or increase bone formation or bone cell function; osteoporosis, fractures or Patients with or at risk of having bone destruction or requiring a prosthesis in the joint and for treating animals requiring activation or stimulation of dermal stem cells or endogenous dermal stem cells. fibroblast regenerative cells, a population of fibroblast regenerative cells, or administering a plurality of cultures, progeny of fibroblast regenerative cells, or conditioned medium of fibroblast regenerative cells; Furthermore, the method of the present invention includes the steps of: Group or groups of cultures, progeny of fibroblast regenerative cells, or conditioned medium of fibroblast regenerative cells administered to a subject in need of increased or improved pulmonary or cardiac function (e.g., a patient with cardiac or pulmonary disease) The present invention also includes treating subjects with heart and lung disease (subjects with or at risk of having heart and lung disease). Non-limiting examples of diseases include atherosclerosis, myocardial infarction (heart attack), cardiac infection, cardiac heart failure, ischemic heart failure, high blood pressure (hypertension) or pulmonary hypertension, idiopathic pulmonary fibrosis, stroke, Congenital heart disease (CHD), congestive heart failure, angina, myocarditis, coronary artery disease, cardiomyopathy, dilated Cardiomyopathy, hypertrophic cardiomyopathy, endocarditis, diastolic dysfunction, cerebrovascular disease, valve disease, mitral valve prolapse, Includes venous thromboembolism or cardiac arrhythmia.

[0114] Additionally, the methods of the present disclosure may be used in patients with a neurological or muscular disease or disorder, or fibroblast regenerative cells, fibroblast regenerative cells, Populations or cultures of fibroblast regenerative cells, progeny of fibroblast regenerative cells, or conditions of fibroblast regenerative cells Non-limiting examples of neurological and muscular diseases and disorders include multiple sclerosis, MS, spinal cord injury, muscular dystrophy (Becker or Duchenne), amyotrophic lateral sclerosis ALS (Lou Gehrig's disease or classical motor neuron disease), autism, progressive Pseudobulbar palsy (progressive bulbar atrophy), pseudobulbar palsy, primary lateral sclerosis (PLS), progressive muscle atrophy, Spinal muscular atrophy (SMA type I - Werdnig-Hoffmann disease, SMA type II, or S SMA type III (including Kugelberg-Welander disease), Fazio-Londo disease , Kennedy's disease (progressive spinal and bulbar muscular atrophy), congenital SMA with joint contractures, and post-partum Examples include Rio syndrome (PPS).

[0115] In some embodiments, the methods of the present disclosure also include the treatment of thyroiditis, insulitis, multiple sclerosis, ipsilateral thyroiditis, and thyroid cancer. Colorcyclitis, uveitis, orchitis, Addison's disease, myasthenia gravis, rheumatoid arthritis, lupus erythema Toxicosis, immune hyperreaction, insulin-dependent diabetes mellitus, anemia (aplastic, hemolytic), hepatitis , autoimmune hepatitis, scleritis, idiopathic thrombocytopenic purpura, gastrointestinal diseases (e.g., cholecystectomy) ulcerative colitis, inflammatory bowel disease), juvenile arthritis, scleroderma and systemic sclerosis, Sharp Glenn's syndrome, undifferentiated connective tissue syndrome, antiphospholipid syndrome, vasculitis (polyarteritis nodosa) , allergic granulomatosis and vasculitis, Wegener's granulomatosis, Kawasaki disease, hypersensitivity vasculitis, Heno Behcet's syndrome, Takayasu's arteritis, giant cell arteritis, occlusion thromboangiitis), polymyalgia rheumatica, essential (mixed) cryoglobulinemia, Plaque psoriasis and psoriatic arthritis, diffuse fasciitis with or without eosinophilia, polymyositis and other idiopathic inflammatory myopathies, relapsing panniculitis, relapsing polychondritis, lymphoma Granulomatosis, erythema nodosum, ankylosing spondylitis, Reiter's syndrome, inflammatory dermatitis, rheumatoid arthritis Unwanted immune and inflammatory responses, hypersensitivity and allergic reactions associated with arthritis, including Inflammation, systemic lupus erythematosus, collagen disease, inflammation associated with atherosclerosis, arteriosclerosis arterial hypertension, atherosclerotic heart disease, reperfusion injury, vascular inflammatory disorders, respiratory distress syndrome, or other conditions cardiopulmonary disease, inflammation associated with peptic ulcers, liver fibrosis, cirrhosis or other liver diseases, thyroiditis, other glandular diseases, glomerulonephritis or other kidney diseases and urinary diseases, otitis or other ear, nose and throat diseases Throat diseases, dermatitis or other skin diseases, periodontal or other dental diseases, orchitis or testicular epiphysitis, infertility, testicular trauma or other immune-related testicular diseases, placental dysfunction, placental insufficiency, Inertial abortion, eclampsia, pre-eclampsia or other immune and / or inflammation-related gynecological diseases, posterior gravida Uveitis, intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveitis, optic neuritis , endophthalmitis (e.g., retinitis or cystoid macular edema), sympathetic ophthalmia, scleritis, retinitis pigmentosa Disease, immune and inflammatory components of degenerative fundus disease, inflammatory components of ocular trauma, infection-induced ocular inflammation, proliferation vitreoretinopathy, acute ischemic optic neuropathy, excessive scarring, e.g., after glaucoma filtering surgery, Immune and / or inflammatory responses to implants and other immune and inflammatory-related Eye diseases, autoimmune diseases or central nervous system (CNS) or other organ, immune, and / or inflammatory diseases Inflammation associated with conditions or disorders in which suppression of the disease would be beneficial, Parkinson's disease, treatment of Parkinson's disease Complications and / or side effects associated with AIDS-related dementia complex, HIV-associated encephalopathy, Devic's disease , Sydenham's chorea, Alzheimer's disease and other degenerative diseases, CNS conditions or disorders, brain Inflammatory components of stroke, post-polio syndrome, immune and inflammatory components of psychiatric disorders, myelitis, encephalitis, Subacute sclerosing panencephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy ulcerative colitis, Guillain-Barré syndrome, Sydenham chorea, pseudotumor cerebri, Down syndrome, Hunchback syndrome CNS trauma or CNS sensitivity. Inflammatory components of infections, muscular atrophies and dystrophies, immune and anti-inflammatory effects of the central and peripheral nervous system Associated diseases, conditions or disorders, such as post-traumatic inflammation, septic shock, infectious diseases, surgery or inflammatory complications or side effects of organs and / or immune complications or side effects of gene therapy for immune or inflammatory disorders or diseases, such as the effects of infection with a virus carrier or to suppress or inhibit the inflammatory, humoral and / or cellular immune responses associated with AIDS. to treat or ameliorate monocyte or leukoproliferative disorders; e.g., monocytes or to reduce the amount of lymphocytes; or to reduce the amount of natural or artificial cells, tissues, and organs Organs (liver, kidney, heart, lung, cornea, bone marrow, organs, lens, pacemaker, natural or fibroblasts to prevent or treat graft rejection during transplantation of artificial skin tissue, etc. Regenerative cells, one or more populations of fibroblast regenerative cells, or cultures of fibroblast regenerative cells , progeny of fibroblast regenerative cells, or conditioned medium of fibroblast regenerative cells.

[0116] Further, the method includes the step of: fibroblast regenerative cells; one or more populations of fibroblast regenerative cells; Cultures of fibroblast regenerative cells, progeny of fibroblast regenerative cells, or conditioned medium of fibroblast regenerative cells. , including administering to a subject in need of stimulating, increasing, inducing, increasing, or enhancing immune tolerance. Such methods may stimulate, increase, induce, augment, or enhance immune tolerance, thereby Treating autoimmune disorders.

[0117] Additionally, the methods of the present disclosure include the use of fibroblast regenerative cells, one or more populations of fibroblast regenerative cells, Cultures of fibroblast regenerative cells, progeny of fibroblast regenerative cells, or conditions of fibroblast regenerative cells The subject medium is adapted to inhibit, reduce, decrease, block, or inhibit immunological rejection of transplantation, transplant fibrosis, or graft failure. Such methods include administering to a subject in need of prevention, control, or limitation. , inhibiting, reducing, decreasing, blocking, preventing, controlling or immunological rejection of transplant fibrosis or transplant failure This may limit the amount of blood flow to the tissue, thereby facilitating acceptance of the graft or transplant by the subject.

[0118] Additionally, the disclosed methods may include the use of fibroblast regenerative cells, one or more populations of fibroblast regenerative cells, or The present invention relates to a culture of fibroblast regenerative cells, the progeny of fibroblast regenerative cells, or the progeny of fibroblast regenerative cells. administering the conditioned medium to treat a subject in need of treatment for melanoma.

[0119] The fibroblast regenerative cells can be administered to the subject by any route appropriate to the treatment method or protocol. Specific non-limiting examples of routes of administration and delivery include parenteral, e.g. For example, intravenous, intramuscular, intrathecal (intraspinal), intraarterial, intradermal, subcutaneous, intrapleural, transdermal (local) , transmucosal, intracranial, intraocular, mucosal, implant and transplant.

[0120] The fibroblast regenerative cells of the present disclosure can be autologous to the subject. The stem cells used in (or to produce conditioned medium) are treated according to this method. Fibroblast regenerative cells, fibroblasts, derived from cells from the subject. one or more populations of fibroblast regenerative cells or cultures of fibroblast regenerative cells, progeny of fibroblast regenerative cells Alternatively, the conditioned medium of the fibroblast regenerative cells may be allogeneic with respect to the subject, i.e., The stem cells to be used (or to produce conditioned medium) are treated according to this method. The cells may be obtained or derived from cells from a subject different from the subject.

[0121] In one embodiment of the present disclosure, proliferation and differentiation of at least two of the ectoderm, mesoderm, or endoderm is achieved. They are capable of differentiation and adhere to plastic, and are more resistant to fibroblasts than unselected fibroblasts. A fibroblast population having enhanced proliferation activity due to the inclusion of a fibroblast gene and expressing one or more of the following genes: and various fibroblast populations are disclosed: Oct-4, KLF-4, Nanog, CD1 17, Sox-2, CD34, Rex-1, GDF-3, CD105, IL-10 and S In another embodiment, the cells are cryopreserved and the population is stored in a container (e.g., a vial, A syringe, or a bag or other container suitable for intravenous delivery of cells into a human or animal. or other container suitable for localized delivery to a site within a human or animal. So the population is at least 1 × 10 2 , 1×10 6 , 1×10 9 , 1×10 10 , 1×1 0 12 , 1×10 14 In some embodiments, the amount is 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×1 0 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1× 10 15 , 1×10 16 , 1×10 17 , 1×10 18 , 1×10 19 , 1×10 20 , 1×10 25 , 1×10, 1×10 35 , 1×10 40 , 1×10 45 , 1×10 50 , 1×10 55 , 1×10 60 , 1×10 65 , 1×10 70 , 1×10 75 , 1×10 8 0 , 1×10 85 , 1×10 90 , 1×10 95 , 1×10 100 or any amount in between is.

[0122] In another embodiment, the population is contained in a 0.9% NaCl solution. The group expresses a bioactive compound (e.g., a growth factor, cytokine, antibody or fragment thereof). Alternatively, the population further comprises organic molecules with a mass of less than 5,000 daltons. The present invention provides a method for producing multiple cryopreserved, individually packaged collections of isolated adult stem cells. a master cell bank comprising populations of at least 1 x 10 or more of the preceding embodiment; The cells derived from the present invention can differentiate into various tissues, for example, The invention provides a method for generating neurons, the method comprising: In one embodiment, the method comprises contacting fibroblasts selected / cultured for enhancement of The conditions include culturing the cells with bFGF, FGF-8, SHH, and BDNF. In another aspect, the present invention provides a method for generating hepatocytes, comprising: In one embodiment, the method comprises culturing regenerative cells derived from blast cells. The conditions include culturing the cells with hepatocyte growth factor (HGF) and FGF-4. In another embodiment of the disclosure, a method for forming endothelial cells is provided, comprising: In one embodiment, the conditions include culturing fibroblasts under conditions of VEG This involves culturing cells with F.

[0123] In another embodiment, a method for generating hematopoietic cells comprises culturing fibroblasts under hematopoietic cell generating conditions. Provided herein are methods comprising culturing alveolar regenerative cells. Conditions included bone morphogenetic protein-4 (BMP4), VEGF, bFGF, stem cell factor (SCF), and F), Flt3L, HyperIL6, thrombopoietin (TPO) and erythropoietin This involves culturing cells with EPO.

[0124] In another embodiment of the present invention, a means for treating cardiovascular disease in a subject is disclosed, The method involves administering a sufficient amount of fibroblast regenerative cells, or directed or directed fibroblast regenerative cells, to treat the disease. In one embodiment, the cardiovascular disease is , myocardial infarction, congestive heart failure, ischemic cardiomyopathy and coronary artery disease. The present invention provides a method for increasing angiogenesis in a subject, the method comprising: fibroblast-derived regenerative cells, or directed or differentiated cells thereof, in a sufficient amount to In one embodiment, the subject is a patient suffering from type II diabetes. In another embodiment, the subject has cardiovascular disease. The present invention provides a method for treating a neurological disorder in a subject, the method comprising: fibroblast-derived regenerative cells or their committed or differentiated forms in an amount sufficient for In one embodiment, the neurological disorder is a neurodegenerative disease. disease (e.g., Parkinson's disease, Alzheimer's disease, or Huntington's disease) or neurological damage It's a wound.

[0125] In another embodiment, the present disclosure provides a population of at least about 3, 4, 5, 6, 7, 8, 9, 10 Expand the fibroblast regenerative cell population, including passage 15, 20, 30, or 40 times In another embodiment, the present invention provides a process for the production of isolated stem cells. A process for differentiating isolated fibroblast regenerative cells under conditions sufficient to induce differentiation. Provide Seth.

[0126] In various embodiments of the above aspects or other aspects elucidated herein, the basis for selection Antigens used as antibodies are CD105, CD117 and / or CD34. In some embodiments, the cells are substantially purified cells (e.g., at least about 20%, 25% , 30%, 40%, 50%, 75%, 80% or more of the cells of the present invention). In some embodiments, the cells are isolated from a mammal (e.g., a human). , isolated from an adult mammal. In yet other embodiments, the cell comprises a heterologous nucleic acid sequence.

[0127] In another embodiment, the present disclosure provides a method for the production of cells capable of proliferation and differentiation into ectoderm, mesoderm, and endoderm. isolated (e.g., purified or substantially purified) fibroblast regenerative cells capable of , Oct-4, Nanog, Sox-2, KLF4, c-Myc, Rex-1, GDF- 3, LIF receptor, CD105, CD117, CD344, IL-10, and Stell a) expressing at least one (e.g., at least 2, 4, 5, or 6) of the MHC class a Class I, MHC class II, CD45, CD13, CD49c, CD66b, and CD9 At least one of 0 (e.g., at least 2, 3, 4, 5, 6, 7, 8, or all In another aspect, the present disclosure provides a method for the production of a cell surface protein comprising: The population is characterized as a population of cells containing either one of the two previous aspects (e.g., embryo-like cells). At least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% of the cells in the body The population may further comprise 80%, 90%, 95% or 99% of the total population. In another embodiment, the compound may be part of a composition comprising: The population of cells may be stem cells (e.g., any cell type known in the art, such as mesenchymal or embryonic stem cells). any) or differentiated cells (e.g., myocytes, adipocytes, ectodermal cells, muscle cells, osteoblasts , chondrocytes, endothelial cells, fibroblasts, pancreatic cells, hepatocytes, bile duct cells, bone marrow cells, nerve cells, and any of those described herein, including urogenital cells. Such cells may be autologous or allogeneic to the stem cells of the present invention. Good too.

[0128] In another aspect, the disclosure features a method for isolating a population of regenerative fibroblasts. The method includes the steps of (a) providing tissue having regenerative activity; (b) providing tissue having a size of about 6-12 mm; (c) enriching the population of cells by detecting a stem cell surface marker; or a population expressing an MHC protein (e.g., any of those described herein). The method may include depleting cells from the culture medium, thereby isolating a population of stem cells. Step (c) is to obtain MHC class I, CD66b, glycophorin a, or glycophorin B. The method may further comprise (d) depleting cells expressing phosphob. In another embodiment, the method further comprises (d) providing a reporter gene a stem cell-specific promoter (e.g., Oct- 4. Nanog, Sox-9, GDF3, Rex-1, or Sox-2 promoter, or is any promoter described herein) to cells. and (e) using the expression of a reporter gene or a selection gene. Further characterize the population for regenerative fibroblasts (e.g., using flow cytometry). In another embodiment, the method further comprises (d) concentrating the cells. A detectable compound (e.g., carboxyfluorescein diacetate, succinimide) contacting the compound with methyl ester or aldefluor (this compound (e) the compound is selectively detectable in proliferating and non-proliferating cells; and In another embodiment, the method further comprises enriching the cell population for cells. The method further comprises culturing the cells under conditions to form tissue aggregates. From fibroblast regenerative cells, granulocytes, T cells, B cells, NK cells, red blood cells, or their The method further includes separating (e.g., by cell depletion) cell types, such as any combination thereof. The method involves culturing a population of fibroblast regenerative cells under conditions that support cell proliferation. In any of the embodiments of this aspect of the invention, the cells may further comprise the step of: They can also be cryopreserved.

[0129] The disclosure also features cells produced by any of the above methods. Also provided is a method for identifying fibroblast regenerative cells, comprising: A step of introducing a vector containing a cell-specific promoter linked to a marker gene into cells. wherein the fibroblast regenerative cells are MHC class I, MHC class II, CD44, CD45, CD13, CD49c, CD66b, CD73, CD105, and CD90 At least one of (e.g., at least 2, 3, 4, 5, 6, 7, 8, or all) do not express cell surface proteins and can differentiate into mesoderm, ectoderm, and endoderm; At least one embryonic transcription factor (e.g., Oct-4, Nanog, Sox-2, Rex -1, GDF-3, Stella, FoxD3, Polycomb) expressing a selectable marker gene from a cell-specific promoter in the stem cells; and detecting the expression of a marker gene in the stem cells, thereby identifying the cells. The method further comprises isolating fibroblast regenerative cells. Fibroblasts can be derived from mammalian body fluids (e.g., synovial fluid or blood) as well as tissues. Preferably, the mammal is a human. In certain embodiments, the cells express CD13, CD 44, and does not express CD90.

[0130] In various embodiments, the fibroblast-specific promoter is the Oct-4 promoter. , Nanog promoter, Sox-2 promoter, Rex-1 promoter, GDF -3 promoter, Stella promoter, FoxD3 promoter, Polyco The promoter is the mb repressor complex 2 promoter, or the CTCF promoter. In another embodiment, the fibroblast-specific promoter is flanked by loxP sites. In another embodiment, the vector is a retroviral vector. Marker genes encode fluorescent proteins such as green fluorescent protein (GFP). In some embodiments, enhanced regenerative activity has been described for mesenchymal stem cells, but fibroblasts Transfection of fibroblasts with OCT-4, which is applied to cells, This results in enhanced bioactivity (Wang, KH, et al. Upregulation of Nanog and Sox-2 g enes following ectopic expression of Oct-4 in amniotic fluid mesenchymal stem ce lls. Biotechnol Appl Biochem, 2015. 62(5): p. 591-7, incorporated herein by reference. (Can be).

[0131] In another embodiment, the pluripotent cells are fused with fibroblasts to increase regenerative activity. Enhanced fibroblasts are artificially generated. Furthermore, fusion of fibroblast regenerative cells with other cells is also possible. is described in the following document, in which the inventors have demonstrated that the compound confers enhanced regenerative activity: We aim to provide guidelines for applying this method to fibroblasts (Xu, D., et al., Cocu lturing embryonic stem cells with damaged hepatocytes leads to restoration of da mage and high frequency of fusion. Cell Mol Biol (Noisy-le-grand), 2009. 55 Supp l: p. OL1186-99, incorporated herein by reference).

[0132] In another embodiment, transfection with cell-permeable NANOG

[53] is performed first. applied to fibroblasts selected for expression of CD105 and / or CD117 ( For example, Peitz, M., et al., Cell-permeant recombinant Nanog protein promotes plur ipotency by inhibiting endodermal specification. Stem Cell Res, 2014. 12(3): p. 680-9, incorporated herein by reference).

[0133] In yet another embodiment, a vector containing two selectable marker genes is utilized. In certain embodiments, the two selectable marker genes are a fluorescent protein and In yet another embodiment, the selectable The marker gene encodes a cell surface protein. A vector containing a regenerative cell-specific promoter linked to one selectable marker gene. introducing a vector into a population of fibroblasts, wherein the regenerative cells are Class I, MHC class II, CD44, CD45, CD13, CD34, CD49c, C fibroblasts, which do not express the cell surface proteins CD73, CD105, and CD90; expressing a selectable marker gene from a regenerative cell-specific promoter in the population. and a method comprising the step of detecting the expression of a marker gene in regenerating fibroblasts. The present invention provides fibroblast regenerative cells isolated by the method.

[0134] In one embodiment, the present disclosure provides fibroblast regenerative cells of the present invention for use in ectoderm, mesoderm and endoderm. to a cell lineage of an embryonic layer selected from the group consisting of neural, glial, chondroblastic, osteoblastic, and / or blast cells, adipocytes, hepatocytes, muscle cells (e.g., smooth or skeletal muscle), cardiac cells, pancreatic cells, The present invention provides methods for differentiation into specific cell types, including, but not limited to, lung cells and endothelial cells. To provide.

[0135] In some embodiments of the present disclosure, the fibroblast regenerative cells are selected from the group consisting of alclofenac; Alclometasone propionate; Algestone acetonide; α-amylase; α-lipoic acid ;α-Tocopherol;Amcinafal;Amcinafide;Amfenac sodium;Salt Amiprilose; Anakinra; Anilorac; Anitrazafen; Apazone; Ascor Balsalazide disodium; Bendazac; Benoxaprofen; Benzida hydrochloride amine; bromelain; broperamol; budesonide; carprofen; chlorogenic acid; Cloprofen; Synthazone; Cliprofen; Clobetasol propionate; Clobetasone butyrate; Clopirac; Cloticasone propionate; Colmetazo acetate Cortodoxone; Deflazacort; Desonide; Desoximetasone; Dipropionate Diclofenac potassium; Diclofenac sodium; Diflorazone acetate, diflumidone sodium, diflunisal; difluprednate; diphthalone; Dimethyl sulfoxide; Drocinonide; Ellagic acid; Endrison; Enlimomab; Enoli Cam sodium; Epirizole; Etodolac; Etofenamate; Felbinac; Fena Fenclofenac; Fenclorac; Fendosal; Fen Empiroparon; Fentiazac; Flazaron; Fluazacort; Flufenamic acid; Flu Mizole; Flunisolide acetate; Flunixin; Flunixin meglumine, Fluoco Rutin butyl; Fluorometholone acetate; Fluquazone; Flubiprofen; Fluretofen Fluticasone propionate; Furaprofen; Flobufen; Glutathione ;Halcinonide;Halobetasol propionate;Halopredone acetate;Hesperede Hesperedin; Ibufenac; Ibuprofen; Ibuprofen aluminum ibuprofen piconol; ironidap; indomethacin; indomethacinna Thorium; Indoprofen; Indoxol; Intrazol; Isoflupredone acetate ;Isoxepac;Isoxicam;Ketoprofen;Lofemizole hydrochloride;Lomoxica Loteprednol etabonate; Lycopene; Sodium meclofenamate; Meclofe Mefenamic acid;Meclorizone dibutyrate;Mefenamic acid;Mesalamine;Meseclazone;Methyl Prednisolone suleptanate; morniflumate; nabumetone; naproxen; naproxen Naproxen sodium; Naproxol; Nimazon; Oleuropein; Olsalazine sodium Orgothein; Orpanoxin; Oxaprozin; Oxyphenbutazone; Parahydrochloride Nilin; Pentosan polysulfate sodium; Phenbutazone sodium glycerol Piroxicam; Piroxicam cinnamate; Piroxicam olamine ;Pirprofen;Pycnogenol;Polyphenols;Prednazate;Priferon ;Prodolic acid;Proquazone;Proxazole;Proxazole citrate;Quercetin ;Resveratrol;Rimexolone;Romazarit;Rosmarinic acid;Rutin;Sarcorec s;sarnasedin;salsalate;sanguinarium chloride;seclazone;cermetacin; Sudoxicam; Sulindac; Suprofen; Talmetacin; Talniflumate; Talosa Late; Tebufelone; Tenidap; Tenidap sodium; Tenoxicam; Tesicam; Tessimide; Tetrahydrocurcumin; Tetridamine; Tiopinaco; Tixocorticol Valate; Tolmetin; Tolmetin sodium; Triclonide; Triflumidate; Zide together with one or more drugs with anti-inflammatory properties, including methacin; zomepirac sodium It is administered.

[0136] In some embodiments, pharmaceutical formulations comprising fibroblast regenerative cells are provided. Pharmaceutical formulations and delivery systems suitable for the compositions and methods are known in the art (see, e.g., Remingo n: The Science and Practice of Pharmacy (2003) 20.sup.th ed., Mack Publishing Co ., Easton, Pa.; Remington's Pharmaceutical Sciences (1990) 18.sup.th ed., Mack P publishing Co., Easton, Pa.; The Merck Index (1996) 12.sup.th ed., Merck Publishing ng Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (199 3), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmace utical Calculations (2001) 11.sup.th ed., Lippincott Williams & Wilkins, Baltimo re, Md.; and Poznansky et al., Drug Delivery Systems (1980), RL Juliano, ed., Oxford, NY, pp. 253-315.

[0137] [VI. Kit] Certain aspects of the present disclosure also include compositions of the present disclosure or compositions for carrying out the methods of the present disclosure. In some embodiments, the kit comprises fibroblast regenerative cells, It may be used to provide the population, its progeny, or its conditioned medium. In the present invention, the kit includes one or more reagents for producing and / or identifying fibroblast regenerative cells. Including medicines.

[0138] The kit may contain the components in a container such as a tube, bottle, vial, syringe, or other suitable container means. , may include components that may be individually packaged or disposed in a container.

[0139] Individual components may also be provided in the kit in concentrated amounts; in some embodiments In a solution, the components are provided individually at the same concentration as they are in solution with the other components. The concentrations of the components are , 1×, 2×, 5×, 10×, or 20× or more.

[0140] In certain aspects, negative and / or positive control agents are included in some kit embodiments. Control molecules can be used to verify the enhanced regenerative activity of fibroblasts. can.

[0141] The kit may include a container having a label. Suitable containers include, for example, bottles, vials, Containers can be made from a variety of materials, including glass and plastic. The container can hold a composition containing probes useful for prognostic or non-prognostic applications such as those described above. The label on the container may indicate that the composition is used for a particular prognostic or non-prognostic application. and also for either in vivo or in vitro use as described above. The kit may include the above-mentioned containers, buffers, diluents, filters, needles, syringes, and package inserts containing instructions for use, The container may also contain one or more other containers containing different materials. [Example]

[0142] The following examples are included to demonstrate certain non-limiting aspects of the disclosure. The techniques disclosed in the examples are believed to function well in the practice of the disclosed subject matter. It should be understood by those skilled in the art that the present invention represents technology discovered by the inventors. However, those skilled in the art will appreciate that, in light of this disclosure, many variations may be apparent to those skilled in the art in the specific embodiments disclosed. and similar or similar modifications may be made without departing from the spirit and scope of the disclosed subject matter. It should be understood that similar results can still be obtained.

[0143] Example 1: Enhanced expression of OCT-4 in CD105-purified fibroblasts Foreskin-derived fibroblasts were cultured at the American Type Culture Collection (ATC). ion (ATCC) and grown in Optimem medium containing 10% fetal bovine serum. Magnetically activated cell sorting (MACS) was used to separate CD105-positive and -negative cells. Sex cells were isolated.

[0144] Briefly, a suspension of fibroblasts was obtained from cultured cells by trypsinization. Wash once with 1× PBS and then with MACS buffer (0.5% fetal bovine serum (FBS; catalogue number 201401401). No. SH30087.01; HyClone; GE Healthcare Life e Sciences, Logan, UT, USA) and 2 mM ethylenediaminetetraacetic acid. The cells were resuspended in 1x PBS containing 1x PBS, pH 7.2. The cell suspension was filtered using a filter. The cells were diluted to 10 mL in MACS buffer. 7 Resuspend at 80 μl cells / The suspension was coated with microbeads (1:20) directly conjugated to mouse anti-human CD105 antibody. 0; Catalog No. MCA1557; Bio-Rad Laboratories, In c., Hercules, CA, USA) and rotate in the dark. After washing with 1x PBS, the cells were incubated at 4°C for 15 minutes. The cells were resuspended in MACS buffer and applied to a LS+ / VS+ separation column. The device was removed and the cells were flushed with MACS buffer. D105+ cells were harvested by centrifugation at 300 xg for 10 minutes at 4°C.

[0145] Expression of OCT-4, a marker of stem cell potential, was evaluated for intracellular OCT-4 protein. Flow cytometry was used to evaluate the effect of mouse cells. Anti-OCT-4 (ab91194, Abcam, Cambridge, MA; 1:200 The secondary antibody was allophycocyanin anti-mouse IgG ( A-865, Life Technologies, Grand Island, NY; 1:1000).

[0146] As can be seen in Figure 1, cells selected for CD105 expression showed no significant difference in OCT-4 expression. The present was strengthened.

[0147] [Example 2. Enhanced expression of NANOG in CD105-purified fibroblasts] Foreskin-derived fibroblasts were obtained from ATCC and cultured in Optime medium containing 10% fetal bovine serum. The cells were grown in 100% methylcellulose medium. CD105-positive cells were identified using magnetic activated cell sorting (MACS). Separation of viable and viable cells was performed.

[0148] Briefly, a suspension of fibroblasts was obtained from cultured cells by trypsinization. Wash once with 1× PBS and then with MACS buffer (0.5% fetal bovine serum (FBS; catalogue number 201401401). No. SH30087.01; HyClone; GE Healthcare Life Sciences, Logan, UT, USA) and 2 mM ethylenediaminetetraacetic acid; The cells were resuspended in 1x PBS containing pH 7.2. A nylon mesh (30 μm pores) was used. The cell suspension was filtered using a filter. The cells were diluted to 10 7 Resuspend at 80 μl cells / and directly conjugated mouse anti-human CD105 antibody (1:200; catalog no. MCA1557; Bio-Rad Laboratories, Inc., Hercules, CA, U. Mix with 20 μl of microbeads (SA) and incubate at 4°C for 15 min in the dark using a rotator. After washing with 1x PBS, DDFCs-CD105 cells were incubated with 1x PBS. The sample was resuspended in buffer and processed on a LS+ / VS+ separation column. The column was removed from the magnetic device. The cells were then removed and flushed with MACS buffer. The pellet was collected by centrifugation at 300 xg for 10 minutes at 4°C.

[0149] Expression of Nanog, a marker of potency, was measured by Abcam (Cambridge, MA). Flow cytometry mouse polyclonal antibody against human Nanog was purchased from It was evaluated using.

[0150] As can be seen in Figure 2, in cells selected for CD105 expression, NANO The expression of G was increased.

[0151] [Example 3. Enhanced expression of KLF-4 in CD105 purified fibroblasts] Foreskin-derived fibroblasts were obtained from ATCC and cultured in Optime medium containing 10% fetal bovine serum. The cells were grown in 100% methylcellulose medium. CD105-positive cells were identified using magnetic activated cell sorting (MACS). Separation of positive and negative cells was performed.

[0152] Briefly, a suspension of fibroblasts was obtained from cultured cells by trypsinization. Wash once with 1× PBS and then with MACS buffer (0.5% fetal bovine serum (FBS; catalogue number 201401401). No. SH30087.01; HyClone; GE Healthcare Life Sciences, Logan, UT, USA) and 2 mM ethylenediaminetetraacetic acid; The cells were resuspended in 1x PBS containing pH 7.2. A nylon mesh (30 μm pores) was used. The cell suspension was filtered using a filter. The cells were diluted to 10 7 Resuspend at 80 μl cells / and directly conjugated mouse anti-human CD105 antibody (1:200; catalog no. MCA1557; Bio-Rad Laboratories, Inc., Hercules, CA, U. Mix with 20 μl of microbeads (SA) and incubate at 4°C for 15 min in the dark using a rotator. After washing with 1x PBS, DDFCs-CD105 cells were incubated with 1x PBS. The sample was resuspended in buffer and processed on a LS+ / VS+ separation column. The column was removed from the magnetic device. The cells were then removed and flushed with MACS buffer. The pellet was collected by centrifugation at 300 xg for 10 minutes at 4°C.

[0153] Expression of Nanog, a marker of potency, was measured using Abcam (Cambridge, MA). Flow cytometry mouse polyclonal antibody against human KLF-4 was purchased from It was evaluated using.

[0154] As can be seen in Figure 3, in cells selected for CD105 expression, KLF- 4 expression was increased.

[0155] Example 4. Expression of immunoregulatory cytokine IL-10 by CD105-selected fibroblasts Current Enhancement] Foreskin-derived fibroblasts were obtained from ATCC and cultured in Optime medium containing 10% fetal bovine serum. The cells were grown in 100% methylcellulose medium. CD105-positive cells were identified using magnetic activated cell sorting (MACS). Separation of positive and negative cells was performed.

[0156] Briefly, a suspension of fibroblasts was obtained from cultured cells by trypsinization. Wash once with 1× PBS and then with MACS buffer (0.5% fetal bovine serum (FBS; catalogue number 201401401). No. SH30087.01; HyClone; GE Healthcare Life Sciences, Logan, UT, USA) and 2 mM ethylenediaminetetraacetic acid; The cells were resuspended in 1x PBS containing pH 7.2. A nylon mesh (30 μm pores) was used. The cell suspension was filtered using a filter. The cells were diluted to 10 7 Resuspend at 80 μl cells / and directly conjugated mouse anti-human CD105 antibody (1:200; catalog no. MCA1557; Bio-Rad Laboratories, Inc., Hercules, CA, U. Mix with 20 μl of microbeads (SA) and incubate at 4°C for 15 min in the dark using a rotator. After washing with 1x PBS, DDFCs-CD105 cells were incubated with 1x PBS. The sample was resuspended in buffer and processed on a LS+ / VS+ separation column. The column was removed from the magnetic device. The cells were then removed and flushed with MACS buffer. The pellet was collected by centrifugation at 300 xg for 10 minutes at 4°C.

[0157] Cells were cultured with allogeneic lymphocytes at a 1:1 ratio for 72 hours, and IL-10 production was measured by ELISA. (R&D Systems). As shown in Figure 4, the CD105 selection line Increased production of IL-10 was observed in fibroblasts.

[0158] Although the present disclosure and its advantages have been described in detail, the scope of the present invention is defined by the appended claims. Various changes, substitutions, and alterations may be made herein without departing from the spirit and scope of the present design. It is understood that the scope of the present application is limited to the and no limitation to particular embodiments of the process, machine, manufacture, composition of matter, means, methods or steps. As will be readily apparent from this disclosure, those skilled in the art will recognize that the perform substantially the same function or achieve substantially the same result as the corresponding embodiment(s) disclosed herein. Any now existing or later developed process, machine, manufacture, composition of matter, or means, Methods or processes may be utilized in accordance with the present disclosure. The scope of this application includes within its scope such process, machine, manufacture, composition of matter, means, methods, or steps. It is intended to include processes.

[0159] [References] All publications mentioned in this specification are indicative of the level of ordinary skill in the art to which this invention pertains. All publications are specifically and individually indicated to be incorporated by reference as if each individual publication were incorporated by reference. and are incorporated by reference herein to the same extent as if separately set forth. 1. Jossen, V., et al., Manufacturing human mesenchymal stem cells at clinical scale: process and regulatory challenges. Appl Microbiol Biotechnol, 2018. 102( 9): pp. 3981-3994. 2. Sher, N. and R. Ofir, Placenta-Derived Adherent Stromal Cell Therapy for H ematopoietic Disorders: A Case Study of PLX-R18. Cell Transplant, 2018. 27(1): p . 140-150. 3. Bunpetch, V., et al., From "Bench to Bedside": Current Advancement on Larg e-Scale Production of Mesenchymal Stem Cells. Stem Cells Dev, 2017. 26(22): p. 1 662-1673. 4. 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Claims

1. 1. A method of treating a medical condition in a subject, comprising: Isolated fibroblast regenerative cells, populations thereof, progeny thereof, and / or conditioned medium thereof. administering the composition to a subject; wherein the fibroblast regenerative cells have increased regenerative activity compared to control fibroblasts. 。

2. The isolated fibroblast regenerative cells or population thereof are characterized by the CD105 marker and / or C The method of claim 1, wherein the D117 marker is expressed.

3. 10. The method of claim 1, wherein the fibroblast regenerative cells further have rhodamine 123 efflux activity.

2. The method according to claim 2.

4. The fibroblast regenerative cells are selected from the group consisting of Oct-4, CD-34, KLF-4, Nanog, and So x-2, Rex-1, GDF-3, IL-10, Stella and combinations thereof The method of claim 1, wherein the vector expresses at least one additional marker selected from the group consisting of: Any of the methods described above.

5. The fibroblast regenerative cells have enhanced expression of GDF-11 compared to control cells. The method according to any one of claims 1 to 4.

6. 6. The method according to claim 1, wherein the fibroblast regenerative cells are derived from tissues with regenerative properties. The method described above.

7. The fibroblast regenerative cells are derived from placental tissue, umbilical cord tissue, endometrial cells, Wharton's jelly, 7. The method according to claim 1, wherein the cells are derived from bone marrow or adipose tissue.

8. The method according to any one of claims 1 to 7, further comprising the step of adding exogenous mitochondria to the composition. The method described above.

9. The exogenous mitochondria are isolated from other cellular components and are substantially pure. Item 9. The method according to item 8.

10. The mitochondria are fused by lipid fusion, polyethylene glycol-mediated fusion or electroporation.

10. The method according to claim 8, wherein the fibroblast regenerative cells are administered by injection. How to post.

11. The mitochondria promote the uptake of the mitochondria into the fibroblasts. The method according to any one of claims 8 to 10, wherein the compound is encapsulated or treated with a drug for the purpose of 。

12. The composition contains vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, folic acid , choline, vitamin B1, vitamin B2, vitamin B5, vitamin B6, vitamin B12 , Biotin, Nicotinamide, Beta-Carotene, Coenzyme Q, Selenium, Super Oxide Cholesterol dismutase, glutathione peroxide, uridine, creatine succinate, pyruvate, Dihydroxyacetone), acetyl-L-carnitine, α-lipoic acid, cardiolipin, Mega fatty acids, lithium carbonate, lithium citrate, calcium, or any combination thereof The method of any one of claims 1 to 11, further comprising combining.

13. The mitochondria may be autologous, homologous, or allogeneic. The method according to any one of claims 8 to 12, which is a mitochondrion or a heterologous mitochondrion. Law.

14. The mitochondria are derived from stem cells or are reduced in number compared to the fibroblast regenerative cells. The method according to any one of claims 8 to 13, wherein the cells are derived from differentiated cells.

15. 2. The method of claim 1 further comprising administering the isolated substantially pure mitochondria to a subject.

15. The method according to any one of 1 to 14.

16. i) Separating mitochondria from other components of the cell to produce isolated, substantially pure mitochondria. (ii) producing isolated and substantially pure mitochondria in said subject; The method of any one of claims 1 to 15, further comprising administering a chondriac.

17. The regenerative fibroblasts proliferate and differentiate into at least two of the ectoderm, mesoderm, or endoderm. The method of claims 1 to 16, wherein the

18. The condition is an immune disorder, a muscle disorder, a hematopoietic disorder, a liver disorder, an angiogenesis disorder, a pancreatic disorder, or a heart disease. , Pulmonary diseases, Neurological diseases, Neuropathy, Neurodegenerative diseases, Muscle disorders, Muscle diseases, Immune diseases, Inflammation-mediated Disease, inflammatory-mediated disorders, inflammation, ischemia, stroke, ischemic heart disease, liver failure, kidney failure, peripheral arteries Disease, pulmonary fibrosis, liver fibrosis, pancreatic fibrosis, diabetic limb, fibrosis, scar tissue formation, pathological apoptosis ulcers, diabetes, cirrhosis, hepatitis, osteoporosis, fractures, neurological damage, need for joint prostheses the need for dermal stem cells, the need for proliferation of pancreatic islet cells, the need for proliferation of liver cells, insulin The need for increased bone production, the need for bone cells, the need for increased bone cell formation, and the need to enhance bone cell function. The method according to any one of claims 1 to 17, wherein the need for a drug or a cell therapy is 。

19. The heart disease or the lung disease is atherosclerosis, myocardial infarction (heart attack), cardiac Infection, heart failure, ischemic heart failure, high blood pressure (hypertension) or pulmonary hypertension, idiopathic pulmonary fibrosis, Stroke, congenital heart disease (CHD), congestive heart failure, angina, myocarditis, coronary artery disease, myocardial Dilated cardiomyopathy, hypertrophic cardiomyopathy, endocarditis, diastolic dysfunction, cerebrovascular disease, valve disease, mitral 19. The method of claim 18, wherein the condition is valvular prolapse, venous thromboembolism, or arrhythmia.

20. The neurological disease or the muscular disease is multiple sclerosis (MS), spinal cord injury, muscular dystrophy (Becker type or Duchenne type), amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease) or classical motor neuron disease), autism, progressive bulbar palsy (progressive bulbar atrophy), pseudobulbar palsy, Primary lateral sclerosis (PLS), progressive muscular atrophy, spinal muscular atrophy (SMA type I - Weld Nich-Hoffmann disease, SMA type II, or SMA type III - Kugelberg-Weller SMA (Schizophrenia including Bender's disease), Fazio-Londo disease, Kennedy disease (progressive spinal and bulbar muscular atrophy) , congenital SMA with joint contractures, and post-polio syndrome (PPS). The method described.

21. The immune disorder or inflammatory mediated disorder or immune disease or inflammatory mediated disease is thyroiditis, insulitis, Multiple sclerosis, iridocyclitis, uveitis, orchitis, Addison's disease, myasthenia gravis, arthritis Equine, lupus erythematosus, immune hyperreaction, insulin-dependent diabetes mellitus, anemia, aplastic Anemia, hemolytic anemia, hepatitis, autoimmune hepatitis, scleritis, idiopathic thrombocytopenic purpura, autoimmune Infectious diseases, gastrointestinal diseases, Crohn's disease, ulcerative colitis, inflammatory bowel disease, juvenile arthritis, scleroderma and systemic sclerosis, Sjogren's syndrome, undifferentiated connective tissue syndrome, antiphospholipid syndrome, Vasculitis, Polyarteritis nodosa, Allergic granulomatosis, Vasculitis, Wegener's granulomatosis, River Saki disease, hypersensitivity vasculitis, Henoch-Schönlein purpura, Behçet's syndrome, Takayasu's artery disease Arteritis, giant cell arteritis, thromboangiitis obliterans, polymyalgia rheumatica, essential cryoglobulinemia Phosphatemia, psoriasis vulgaris and psoriatic arthritis, diffuse fasciitis with eosinophilia, eosinophilia Diffuse fasciitis without inflammatory bowel disease, polymyositis and other idiopathic inflammatory myopathies, recurrent idiopathic fasciitis Panniculitis, relapsing polychondritis, lymphomatous granulomatosis, erythema nodosum, ankylosing spondylitis, leiter - syndrome, inflammatory dermatitis, unwanted immune and inflammatory reactions associated with arthritis, rheumatoid arthritis Inflammation associated with hypersensitivity and allergic reactions, systemic lupus erythematosus, collagen diseases, atherosclerosis inflammation associated with atherosclerosis, arteriosclerosis, atherosclerosis, atherosclerotic heart disease, reperfusion injury, vascular inflammatory Disorders, respiratory distress syndrome, cardiopulmonary disease, inflammation associated with peptic ulcers, liver fibrosis, cirrhosis, liver disease, Thyroiditis and other glandular diseases, glomerulonephritis, kidney diseases, urinary diseases, otitis, otorhinolaryngological diseases, skin diseases Dermatitis, skin diseases, periodontal disease, dental diseases, orchitis or epididymitis, infertility, testicular trauma, immune system disorders Testicular disease, placental dysfunction, placental insufficiency, habitual miscarriage, eclampsia, preeclampsia, immune-related gynecological diseases Inflammatory gynecological diseases, posterior uveitis, intermediate uveitis, anterior uveitis, conjunctivitis , chorioretinitis, uveitis, optic neuritis, endophthalmitis (e.g., retinitis or cystoid macular edema) , sympathetic ophthalmia, scleritis, retinitis pigmentosa, immune and inflammatory components of degenerative fundus disease, inflammation of ocular trauma Infectious components, ocular inflammation due to infection, proliferative vitreoretinopathy, acute ischemic optic neuropathy, excessive scarring Immune response to ocular implants, Inflammatory response to ocular implants, Immune-related eye disease, inflammation-related eye disease, autoimmune disease, or central nervous system (CNS) or other organ conditions or Inflammation associated with Parkinson's disease, complications and / or side effects associated with Parkinson's disease treatment AIDS-related dementia complex, HIV-associated encephalopathy, Devic's disease, Sydenham's chorea, Alzheimer's disease Immersion and other degenerative diseases, CNS conditions or disorders, inflammatory components of stroke, post-stroke Rio syndrome, immune and inflammatory components of psychiatric disorders, myelitis, encephalitis, subacute sclerosing panencephalitis, cerebrospinal inflammation, acute neuropathy, subacute neuropathy, chronic neuropathy, Guillain-Barré syndrome, Sydenham chorea, pseudotumor cerebri, Down syndrome, Huntington's disease, amyotrophic lateral sclerosis Inflammatory component of CNS compression Inflammatory component of CNS compression Inflammatory component of CNS compression CNS CNS trauma infection of S, inflammatory component of muscular atrophy, inflammatory component of muscular dystrophy, central and Peripheral nervous system conditions or disorders, post-traumatic inflammation, septic shock, infectious diseases, inflammatory processes of surgery Complications, inflammatory complications of organ transplants, side effects of surgery, side effects of organ transplants, inflammation in gene therapy Sexual complications, immune complications of gene therapy, inflammation-related side effects of gene therapy, immune complications of gene therapy Related side effects, AIDS-associated inflammation, humoral immune response, cellular immune response, monoproliferative disorders, leukoproliferative disorders, leukemia, hypermonocytic or hyperlymphocytic disorders, or transplant rejection.

18. The method according to claim 18.

22. The transplant rejection reaction may be caused by the use of natural cells, artificial cells, natural tissue, artificial tissue, bone marrow, organs, or parts of bone marrow.

22. The method of claim 21, after transplantation of a cornea, lens, or lens.

23. 23. The method of claim 22, wherein the organ is the liver, kidney, heart, or lung.

24. The neurodegenerative disease is Parkinson's disease, Alzheimer's disease, or Huntington's disease.

20. The method of claim 18.

25. The subject is treated with an anti-inflammatory drug, such as an anti-inflammatory drug, for example, an anti-inflammatory drug, or an anti-inflammatory drug, such as .... The method according to any one of claims 1 to 24, which requires control.

26. The method of any one of claims 1 to 25, wherein the subject has ischemia.

27. 27. The method of claim 26, wherein the ischemia is in cardiac tissue, pulmonary tissue, renal tissue, or a limb. The method described.

28. The ischemia is associated with stroke, ischemic heart disease, liver failure, kidney failure, and peripheral arterial disease.

28. The method according to claim 26 or 27.

29. The subject is a patient suffering from stroke, pulmonary fibrosis, diabetic limb, ischemic heart disease, liver failure, renal failure, peripheral arterial disease, or the like. heart disease, diabetes, liver failure, cirrhosis, liver or pancreatic fibrosis, or hepatitis, osteoporosis, fractures, heart any one of claims 1 to 28, having or at risk of developing a lung disease or pulmonary disease The method described in section.

30. The heart disease or the lung disease is atherosclerosis, myocardial infarction, cardiac infection, heart failure Allergies, ischemic heart failure, hypertension, pulmonary hypertension, idiopathic pulmonary fibrosis, stroke, congenital heart disease (CH) D) Congestive heart failure, angina pectoris, myocarditis, coronary artery disease, cardiomyopathy, dilated cardiomyopathy, hypertrophic heart Myopathy, endocarditis, diastolic dysfunction, cerebrovascular disease, valvular disease, mitral valve prolapse, venous thromboembolism, or 30. The method of claim 29, wherein the arrhythmia is arrhythmia.

31. The subject has or is at risk of developing fibrosis or scar tissue formation. The method of any one of claims 1 to 30.

32. The scar tissue formation or the fibrosis is in pancreatic tissue, liver tissue, heart tissue, lung tissue, limbs, 32. The method of claim 31, wherein the site is in the liver, pancreas, or kidney.

33. The subject is in need of inhibition, reduction, decrease, control, or reversal of pathological apoptosis.

33. The method according to any one of claims 1 to 32.

34. The subject has improved pancreatic function, liver function, bone cell function, insulin production, pulmonary function, and cardiac function. or a joint prosthesis. The method described in

35. The subject has an increased number of pancreatic islet cells, an increased number of liver cells, an increased amount of insulin production; an increased number of bone cells The method of any one of claims 1 to 34, wherein an increase in

36. The subject is in need of dermal stem cells or in need of activation of endogenous dermal stem cells. The method according to any one of claims 1 to 35,

37. 37. The method of claim 1, wherein the subject is administered one or more anti-inflammatory agents in addition to the composition. Any of the methods described above.

38. 38. The method of claim 37, wherein the anti-inflammatory agent is administered simultaneously with the composition.

39. 38. The method of claim 37, wherein the anti-inflammatory agent is administered before and / or after the composition. 。

40. The anti-inflammatory agent is selected from the group consisting of alclofenac, alclometasone dipropionate, and algestone. Acetonide; α-amylase; α-lipoic acid; α-tocopherol; Amcinafal; Am Sinafide; Amfenac sodium; Amiprilose hydrochloride; Anakinra; Anilorac ; Anitrazafen; Apazone; Ascorbic acid; Balsalazide disodium; Bendaza Benoxaprofen; Benzydamine hydrochloride; Bromelain; Properamol; Budesonide Nido; Carprofen; Chlorogenic acid; Cycloprofen; Synthazone; Cliprofen ; Clobetasol propionate; Clobetasone butyrate; Clopirac; Pro Cloticasone dopionate; Cormethasone acetate; Cortodoxone; Deflazacort; Desonil Dexamethasone; Dexamethasone dipropionate; Diclofenac potassium; Diclofenac Lofenac sodium; diflorasone diacetate, diflumidone sodium, difluni Monkey; Difluprednate; Diphthalone; Dimethyl sulfoxide; Drocinonide; Ellag acid; endrisone; enlimomab; enolicam sodium; epirizole; etodolac; Etofenamate; Felbinac; Fenamol; Fenbufen; Fenclofenac ; Fenclorac; Fendosal; Fenpipalon; Fentiazac; Flazaron Fluazacort; Flufenamic acid; Flumizole; Flunisolide acetate; Fluniki Syn; Flunixin meglumine, Fluocortin butyl; Fluorometholone acetate; Fluc Azone; Fluriprofen; Fluretofen; Fluticasone propionate; Flu Profen; Flobufen; Glutathione; Halcinonide; Halobetasol propionate Halopredone acetate; Hesperedin; Ibufenac ; Ibuprofen; Ibuprofen aluminum; Ibuprofen piconol; Ironida Indomethacin; Indomethacin sodium; Indoprofen; Indoxol ; Intrazol; Isoflupredone acetate; Isoxepac; Isoxicam; Ketoprofen lofemizole hydrochloride; lomoxicam; loteprednol etabonate; lycopene; Meclofenamic acid sodium; Meclofenamic acid; Mechlorizone dibutyrate; Mefena music acid; Mesalamine; Meseclazone; Methylprednisolone suleptanate; Morniflume Naproxen; Naproxen sodium; Naproxen; Nimazon ;Oleuropein;Olsalazine sodium;Orgotein;Orpanoxin;Oxap Rosin; Oxyphenbutazone; Paranyline hydrochloride; Pentosan polysulfate sodium phenbutazone sodium glycerate; pirfenidone; piroxicam; piroxicam Camcinnamate; Piroxicam olamine; Pirprofen; Pycnogenol; Polyf phenol; prednazate; preferon; prodolic acid; proquazone; proxazol Proxazole citrate; Quercetin; Resveratrol; Rimexol; Romazari Rosmarinic acid; Rutin; Sarcorex; Salnacedin; Salsalate; Sulfonyl chloride Inarium; Seclazon; Sermetacin; Sudoxicam; Sulindac; Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelon; Tenidap; Tenidap Tetris sodium; Tenoxicam; Tesicam; Tesimide; Tetrahydrocurcumin; Tetrida amine; tiopinac; tixocortol pivalate; tolmetin; tolmetin sodium triclonide; triflumidate; zidometacin; zomepirac sodium 40. The method according to any one of claims 37 to 39.

41. The method of any one of claims 1 to 40, wherein the composition further comprises a biologically active compound. 。

42. The bioactive compound may be a growth factor, a cytokine, an antibody, an antibody fragment, a 5000 dalton 42. The method of claim 41, wherein the organic molecule has a mass of less than 1000 uM.

43. The composition may be administered parenterally, transdermally, transmucosally, by implant, or by transplantation. The method of any one of claims 1 to 42, wherein the compound is introduced into the subject by

44. The composition is administered to the subject intravenously, intramuscularly, intrathecally, intra-arterially, intradermally, subcutaneously, intrapleurally, 44. The method of claim 43, wherein the administration is intracranially, intraocularly, or mucosally.

45. 45. The method of claim 1, wherein the fibroblast regenerative cells are allogeneic to the subject. The method described above.

46. 10. The method of claim 1, wherein the fibroblast regenerative cells or population thereof are autologous to the subject.

45. A method according to any one of claims 1 to 44.

47. 47. The method according to claim 1, wherein the fibroblast regenerative cells are plastic-adherent. The method described.

48. The fibroblast regenerative cells, populations thereof, or progeny thereof are cryopreserved prior to the administering step.

47. The method according to any one of claims 1 to 46.

49. The method of any one of claims 1 to 48, wherein the subject is an animal.

50. The method of any one of claims 1 to 49, wherein the composition further comprises stem cells.

51. The population is at least 1 x 10 ２ , 1 x 10 ６ , 1 x 10 ９ , 1 x 10 １０ , 1x1 0 １２ , 1 x 10 １４ 51. The method of claim 1, comprising administering to a subject a stem cell comprising administering to said ... The method according to any one of the preceding claims.

52. 52. The method of claim 50 or 51, wherein the stem cells are mesenchymal cells, embryonic stem cells or differentiated cells. How to do it.

53. The stem cells may be myocytes, adipocytes, ectodermal cells, muscle cells, osteoblasts, chondrocytes, endothelial cells, cells, fibroblasts, pancreatic cells, hepatocytes, bile duct cells, bone marrow cells, nerve cells, or urogenital cells The method according to any one of claims 50 to 52, wherein the cell is a cell.

54. The fibroblast regenerative cells are selected from MHC class I, MHC class II, CD45, and CD13 , CD49c, CD66b, CD73, CD105, or CD90 The method according to any one of claims 1 to 53, wherein the cell surface protein is not expressed.

55. 1. An in vitro method for producing fibroblast regenerative cells or populations thereof, comprising: Selecting and / or expanding fibroblasts that express CD117 and / or CD105 and The method, wherein the fibroblasts have increased regenerative activity compared to control fibroblasts.

56. further comprising selecting the fibroblast regenerative cells for expression of CD34.

56. An in vitro method according to paragraph 55.

57. further comprising selecting the blast regenerative cells for rhodamine 123 efflux activity.

56. The in vitro method of claim 55.

58. The fibroblast regenerative cells are selected from Oct-4, CD-34, KLF-4, Nanog, and So At least one selected from the group consisting of Rex-2, Rex-1, GDF-3 and Stella 56. The in vitro method of claim 55, further comprising selecting for one additional marker. method.

59. The fibroblast regenerative cells were examined for enhanced expression of GDF-11 compared to control fibroblasts.

59. The in vitro method according to any one of claims 55 to 58, further comprising the step of selecting.

60. The fibroblast regeneration is characterized by the presence of MHC class I, MHC class II, CD45, CD13, C At least one of CD49c, CD66b, CD73, CD105, and CD90 60. The in vitro method according to any one of claims 55 to 59, wherein the method does not express a cell surface protein of method.

61. 61. The method according to any one of claims 55 to 60, wherein the fibroblasts are derived from tissues with regenerative properties. The in vitro method described.

62. The fibroblasts may be derived from placental tissue, umbilical cord tissue, endometrial cells, Wharton's jelly, bone marrow, or 62. The in vitro method of any one of claims 55 to 61, wherein said cell is derived from adipose tissue.

63. Claims 55-62, wherein the population comprises 0.01-5% freshly extracted fibroblasts.

1. The in vitro method according to any one of the preceding claims.

64. preparing a therapeutically effective amount of the fibroblast regenerative cells; and The therapeutically effective amount of fibroblast regenerative cells, a population thereof, a progeny thereof, or a conditioned medium thereof is administered to the The process of providing it to those who need it 64. The in vitro method of any of claims 55 to 63, further comprising:

65. 1. An in vitro method for forming neural cells, comprising: By selecting fibroblasts that express CD117 and / or CD105, fibroblasts can be A process for obtaining fibroblast regenerative cells or a population thereof, wherein the fibroblasts are differentiated from control fibroblasts. and The fibroblast regenerative cells are treated with at least one of bFGF, FGF-8, SHH, or BDNF. a step of culturing using at least two combinations of An in vitro method comprising:

66. 1. An in vitro method for forming hepatocytes, comprising: By selecting and expanding fibroblasts expressing CD117 and / or CD105, obtaining fibroblast regenerative cells or a population thereof by have increased regenerative activity compared to blast cells; and The fibroblast regenerative cells were cultured using hepatocyte growth factor (HGF) and / or FGF-4. Nurturing process An in vitro method comprising:

67. 1. An in vitro method for forming endothelial cells, comprising: fibroblasts by selecting fibroblasts that express CD117 and / or CD105 A process for obtaining cell regeneration cells or a population thereof, wherein the fibroblasts have a higher cellular regeneration rate than control fibroblasts. and Culturing the fibroblast regenerative cells with vascular endothelial growth factor (VEGF) An in vitro method comprising:

68. 1. An in vitro method for forming hematopoietic cells, comprising: fibroblasts by selecting fibroblasts that express CD117 and / or CD105 A process for obtaining cell regeneration cells or a population thereof, wherein the fibroblasts have a higher cellular regeneration rate than control fibroblasts. and The fibroblast regenerative cells were treated with bone morphogenetic protein-4 (BMP4), VEGF, bFGF , stem cell factor (SCF), Flt3L, hyper IL6, thrombopoietin (TPO) or The method comprises culturing the cells using a combination of at least two of the following erythropoietin (EPO): Process An in vitro method comprising:

69. 10. The method of claim 1, further comprising selecting the fibroblast regenerative cells for CD34 expression.

69. An in vitro method according to any one of 65 to 68.

70. selecting the fibroblast regenerative cells for rhodamine 123 efflux activity; 70. The in vitro method of any of claims 65 to 69, comprising:

71. The fibroblast regenerative cells are selected from Oct-4, CD-34, KLF-4, Nanog, and So x-2, Rex-1, GDF-3, IL-10, Stella and combinations thereof The method further comprises selecting for at least one additional marker selected from the group consisting of: The in vitro method according to any one of claims 65 to 70.

72. The fibroblast regenerative cells were cultured to have enhanced expression of GDF-11 compared to control fibroblasts. The method according to any one of claims 65 to 71, further comprising the step of selecting for In vitro method.

73. The fibroblast regenerate comprises MHC class I, MHC class II, CD45, CD13, At least one of CD49c, CD66b, CD73, CD105, or CD90 The immunoglobulin according to any one of claims 65 to 72, which does not express any of the above cell surface proteins. In vitro method.

74. isolated fibroblasts, populations thereof, progeny thereof, or conditioned medium thereof, A composition wherein the fibroblasts have increased regenerative activity compared to control fibroblasts.

75. The fibroblasts express the CD105 marker and / or the CD117 marker.

75. The composition of claim 74.

76. 76. The method of claim 74 or 75, wherein the fibroblasts further have rhodamine 123 efflux activity. The composition described above.

77. The fibroblasts express Oct-4, CD-34, KLF-4, Nanog, Sox-2, At least one additional protein selected from the group consisting of Rex-1, GDF-3, and Stella.

77. The composition of any of claims 74 to 76, wherein the composition expresses an additional marker.

78. The fibroblasts have enhanced expression of GDF-11 compared to control cells.

74. The composition according to any one of claims 74 to 77.

79. 79. The method according to any one of claims 74 to 78, wherein the fibroblasts are derived from tissues with regenerative properties. The composition described.

80. The fibroblasts may be derived from placental tissue, umbilical cord tissue, endometrial cells, Wharton's jelly, bone marrow, or The composition of any one of claims 74 to 79, wherein the composition is derived from adipose tissue.

81. The regenerative cells of the fibroblasts, their populations or progeny, or the conditioned medium thereof are cryopreserved. The composition according to any one of claims 74 to 80,

82. The population is at least 1 x 10 ２ , 1 x 10 ６ , 1 x 10 ９ , 1 x 10 １０ , 1x1 0 １２ , 1 x 10 １４ 82. The method of claim 74, wherein the stem cells are present in an amount of 100 or any amount therebetween. The composition described in any one of the above.

83. 83. The composition of any one of claims 74 to 82, further comprising a biologically active compound.

84. The bioactive compound may be a growth factor, a cytokine, an antibody, an antibody fragment, a 5000 dalton 84. The composition of claim 83, wherein the organic molecule has a mass of less than 1000 .mu.m.

85. 85. The composition of any of claims 74 to 84, wherein the composition is in a NaCl solution.

86. The composition of claims 74-84, wherein the composition is in a 0.8% to 1% NaCl solution. 。

87. 87. The composition of any one of claims 74 to 86, further comprising exogenous mitochondria.

88. The exogenous mitochondria are isolated from other cellular components and are substantially pure. Item 88. The composition according to item 87.

89. The mitochondria are fused by lipid fusion, polyethylene glycol-mediated fusion or electroporation.

89. The method according to claim 87, wherein the fibroblasts are administered by injection. composition.

90. The mitochondria promote the uptake of mitochondria into the fibroblasts. The composition according to any one of claims 87 to 89, which is encapsulated or treated with a drug. 。

91. (blank)

92. (blank)

93. (blank)

94. (blank)

95. (blank)

96. (blank)

97. (blank)

98. (blank)

99. (blank)

100. Vitamin A, Vitamin C, Vitamin D, Vitamin E, Vitamin K, Folic Acid, Choline, Vitamin Vitamin B1, Vitamin B2, Vitamin B5, Vitamin B6, Vitamin B12, Biotin, Cochinamide, beta-carotene, coenzyme Q, selenium, superoxide dismutase Ze, glutathione peroxide, uridine, creatine succinate, pyruvate, dihydroxybenzoate cetone), acetyl-L-carnitine, alpha-lipoic acid, cardiolipin, omega fatty acids, charcoal The claimed invention further comprises lithium citrate, lithium citrate, calcium, or a combination thereof. The composition according to any one of claims 87 to 90.

101. The mitochondria may be homologous, allogeneic or heterologous.

92. The composition of any one of claims 87 to 91, which is a mitochondrial.

102. The mitochondria are derived from stem cells or cells that are less differentiated compared to fibroblasts. The composition of any one of claims 87 to 101, derived from

103. 103. The method according to any one of claims 74 to 102, wherein the regenerative activity is the ability to stimulate angiogenesis. The composition described above.

104. 11. The method of claim 10, wherein the angiogenesis further comprises stimulating human umbilical vein endothelial cell (HUVEC) proliferation.

3. The composition described in 3.

105. 105. The composition of claim 103 or 104, wherein the angiogenesis comprises the production of collateral blood vessels.

106. 106. The composition of claim 105, wherein the collateral vessel is in ischemic cardiac tissue.

107. 106. The composition of claim 105, wherein the collateral vessel is in ischemic limb tissue.

108. 106. The composition of claim 105, wherein the collateral vessel surrounds an occluded blood vessel.

109. A kit comprising the composition of any one of claims 74 to 108.

110. A pharmaceutical formulation comprising the composition of any one of claims 74 to 109.

111. In vitro isolated cells that can proliferate and differentiate into ectoderm, mesoderm, or endoderm Fibroblast regenerative cells or populations thereof, The isolated fibroblast regenerative cells contain Oct-4, Nanog, Sox-2, KLF 4, c-Myc, Rex-1, GDF-3, LIF receptor, CD105, CD117, C expressing at least one marker of D344, IL-10 or Stella; MHC class I, MHC class II, CD45, CD13, CD49c, CD66b, CD 73, CD105 or CD90. Not an in vitro isolated fibroblast regenerative cell or population thereof.

112. A plurality of regenerating fibroblasts that can proliferate and differentiate into ectoderm, mesoderm or endoderm.

1. A master cell bank comprising a packaged population, The isolated fibroblast regenerative cells express Oct-4, Nanog, Sox-2, KLF4, c-Myc, Rex-1, GDF-3, LIF receptor, CD105, CD117, CD3 44, IL-10, and Stella; MHC class I; MHC class II, CD45, CD13, CD49c, CD66b, CD73, CD10 and a master cell line that does not express at least one cell surface protein selected from the group consisting of CD5 and CD90. Lebank.

113. Each packaged population contains at least 1 x 10 ２ or more of the above aspects of the cell 113. The master cell bank of claim 112, comprising:

114. 1. A method for isolating a population of regenerative fibroblasts, comprising: Providing tissue with regenerative activity; and enriching a population of cells having a size of about 6 to 12 micrometers, Cell regeneration cells express Oct-4, Nanog, Sox-2, KLF4, c-Myc, Re x-1, GDF-3, LIF receptor, CD105, CD117, CD344, IL-10 and Stella, and expressing at least one of MHC class I, MHC class II , CD45, CD13, CD49c, CD66b, CD73, CD105 or CD90 not expressing at least one cell surface protein of A method comprising:

115. Depleting cells from a population that express stem cell surface markers or MHC proteins, 115. The method of claim 114, optionally comprising isolating the population of stem cells.

116. The cells to be depleted are selected from the group consisting of MHC class I, CD66b, glycophorin a, and glycophorin B. The method of claim 115, wherein folin b is expressed.

117. 115. The method of claim 114, optionally comprising cryopreserving the cells.

118. Optionally, a stem cell-specific promoter operably linked to a reporter gene or a selection gene. transfecting said cells with a polynucleotide vector comprising a vector; The method of claim 114.

119. The cell-specific promoter is selected from the group consisting of Oct-4, Nanog, Sox-9, GDF3, R The method of claim 118, wherein the promoter is Sox-1, Sox-2, or Sox-3.

120. Enriching the population of regenerative fibroblasts using expression of a reporter gene or a selection gene 120. The method of any one of claims 114 to 119, further comprising the step of:

121. further comprising enriching the population of regenerative fibroblasts by flow cytometry.

121. The method according to any one of claims 114 to 120.

122. contacting said cells with a detectable compound that enters said cells, said compound is selectively detectable in proliferating and non-proliferating cells; and enriching the cell population for the expanded cells The method of any one of claims 114 to 121, comprising:

123. The detectable compound is carboxyfluorescein diacetate, succinimidyl ester, or aldefluor. Law.

124. 114 to 116, further comprising culturing the cells under conditions to form tissue aggregates.

123. A method according to any one of claims 123 to 123.

125. From the fibroblast regenerative cells, granulocytes, T cells, B cells, NK cells, red blood cells or the like 125. The method of claim 114, further comprising separating cell types such as any combination of: Any of the methods described above.

126. 126. The method of claim 125, wherein the separation of cell types is performed by cell depletion.

127. and culturing said population of fibroblast regenerative cells under conditions that support proliferation of said cells. The method of any one of claims 114 to 126, further comprising:

128. 128. The method of any of claims 114 to 127, wherein the cells are cryopreserved.

129. A cell produced by the method of any one of claims 114 to 128.

130. 1. A method for identifying fibroblast regenerative cells, comprising: A fibroblast-specific promoter linked to at least one selectable marker gene. introducing a vector comprising the above into a cell; expressing the selectable marker gene from the cell-specific promoter in the cell; a step of causing and, Detecting expression of the marker gene in the cells, thereby identifying fibroblast regenerative cells. a step of: Including, The fibroblast regenerative cells are selected from MHC class I, MHC class II, CD44, CD45 , CD13, CD34, CD49c, CD66b, CD73, CD105 and CD90 does not express at least one of the cell surface proteins; The fibroblast regenerative cells are selected from the group consisting of Oct-4, Nanog, Sox-2, Rex-1, GD At least one of F-3, Stella, FoxD3, or Polycomb expressing embryonic transcription factors, and the fibroblast regenerative cells are mesoderm, ectoderm, and / or endoderm. A method capable of differentiating into leaves.

131. 131. The method of claim 130, further comprising isolating the fibroblast regenerative cells.

132. 130 or 131. The method of claim 130, wherein the fibroblast regenerative cells are derived from body fluids or mammalian tissue.

31. The method according to claim 31.

133. 133. The method of claim 132, wherein the bodily fluid is synovial fluid or blood.

134. 133. The method of any one of claims 130 to 132, wherein the mammal is a human.

135. The fibroblasts do not express CD13, CD44, CD90, or a combination thereof. The method according to any one of claims 130 to 134.

136. The fibroblast-specific promoter is an Oct-4 promoter, a Nanog promoter, or the like. ter, Sox-2 promoter, Rex-1 promoter, GDF-3 promoter, S tella promoter, FoxD3 promoter, Polycomb Repress or Complex 2 promoter, or CTCF promoter. 0-135. The method of any one of claims 0-135.

137. 130 to 135, wherein the fibroblast-specific promoter is flanked by loxP sites.

136. A method according to any one of claims 136 to 136.

138. 138. A method according to any one of claims 130 to 137, wherein the vector is a retroviral vector. The method described.

139. Claims 130 to 13, wherein the selectable marker gene encodes a fluorescent protein 9. The method according to any one of claims 8 to 8.

140. 140. The method of claim 139, wherein the fluorescent protein is green fluorescent protein (GFP). method.

141. The regenerating fibroblasts are transfected with the OCT-4 transcription factor, thereby The method according to any one of claims 130 to 140, further comprising the step of enhancing the regenerative activity of fibroblasts. How to post.

142. The regenerative fibroblasts are fused with pluripotent cells, thereby enhancing regenerative activity.

141. The method according to any one of claims 130 to 140, further comprising the step of generating fibroblasts. How to do it.

143. Selecting fibroblasts that express CD105 and / or CD117; and The fibroblasts expressing CD105 and / or CD117 are transfected with the NANOG gene. Transfecting step 143. The method of any one of claims 130 to 142, further comprising:

144. Any of claims 130 to 144, wherein the vector is two selectable marker genes. The method according to any one of the preceding claims.

145. The two selectable marker genes are sensitive to fluorescent proteins and drug selection. The method of claim 144, wherein the protein is a protein selected from the group consisting of:

146. 10. The method of claim 1, wherein one of the selectable marker genes encodes a cell surface protein.

44. The method according to claim 44.

147. 130. The fibroblast regenerative cells further have rhodamine 123 efflux activity.

147. The method of any one of claims 1 to 146.

148. The fibroblast regenerative cells have enhanced expression of GDF-11 compared to control cells.

148. The method of any one of claims 130 to 147.

149. Claims 130-148, wherein the fibroblast regenerative cells are derived from tissue with regenerative properties.

10. The method according to any one of the preceding claims.

150. The fibroblast regenerative cells are derived from placental tissue, umbilical cord tissue, endometrial cells, Wharton's jelly, 151. The method of any one of claims 130 to 150, wherein the cell membrane is derived from bone marrow or adipose tissue.

151. 1. A method for generating regenerative fibroblasts, comprising: A regenerative cell-specific promoter linked to at least one selectable marker gene into a population of fibroblasts, wherein the regenerative cells are Class I, MHC class II, CD44, CD45, CD13, CD34, CD49c, C not expressing CD73, CD105, and CD90 cell surface proteins; In the fibroblast population, the selectable promoter is selected from the regenerative cell-specific promoter. expressing a marker gene; and detecting the expression of the marker gene in the regenerated fibroblasts. A method comprising:

152. The selectable marker gene is a fluorescent protein, a protein sensitive to drug selection, or The method of claim 151, wherein said gene encodes a cell surface protein.

153. Transfecting the regenerated fibroblasts with a nucleic acid encoding the OCT-4 transcription factor; 151 or 152, further comprising the step of: thereby enhancing the regenerative activity of said fibroblasts.

52. The method according to claim 52.

154. The regenerative fibroblasts are fused with pluripotent cells, thereby enhancing regenerative activity.

154. The method of any one of claims 151 to 153, further comprising the step of generating the fibroblasts. The method described.

155. Selecting fibroblasts that express CD105 and / or CD117; and transfecting said fibroblasts with a permeable NANOG gene.

152. The method of claim 151, further comprising:

156. Fibroblast regeneration isolated by the method of any one of claims 151 to 155. living cells.

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