Regenerative abscopal effects

Abstract

To provide systems, methods and compositions for regenerating one or more tissues in an individual at one or more sites in need of cell and / or tissue regeneration.SOLUTION: Embodiments of the disclosure encompass methods and compositions using fibroblasts for stimulating regeneration in a first tissue site in an individual, the methods comprising the step of administering at least one regenerative composition to a second tissue site, where the second tissue site comprises the same tissue type as the first tissue site in the individual. The first and second sites are at different locations in the individual in particular embodiments. Particular embodiments comprise administering one or more compositions to an individual at a different anatomical site than a site in need, such as a joint.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application is a continuation of U.S. Provisional Patent Application No. 62 / 757,764, filed November 9, 2018. This provisional application claims priority to US Provisional Patent Application No. 2005 / 0133636, filed on May 13, 2005, the entirety of which is incorporated herein by reference.

[0002] Embodiments of the present disclosure relate to at least one of the following: cell biology, molecular biology, cell therapy, physiology, and Including the field of medicine. [Background technology]

[0003] Stem cell therapy has made considerable progress over the past two decades. The original use of stem cells was for the treatment of blood disorders. This was related to the reconstitution of hematopoiesis in recipients after myeloablative treatment in the field. After the first successful clinical bone marrow transplant, many studies were conducted and it was found that bone marrow cells can be used to treat liver failure, heart failure, and other conditions. It has been demonstrated that this drug can be effective in treating diseases other than hematology, such as stroke and limb ischemia.

[0004] Local administration of stem cells induces regenerative effects at the site of administration, such as in joints, muscles, and other tissues. Unfortunately, under certain conditions, all tissues that require regeneration may be It is difficult to administer stem cells locally to tissues. In mice, stem cells are known to induce local re-expression of dystrophin; It is difficult to inject cells into every muscle in the body.

[0005] The present disclosure provides a solution to a long-standing need in the field of cell therapy using a new and efficient method. Provide a solution. Summary of the Invention

[0006] The present disclosure provides a method for the treatment of a patient's aging at one or more sites in need of cell and / or tissue regeneration. The present invention relates to systems, methods and compositions for regenerating one or more tissues. The method comprises administering one or more compositions to an individual at an anatomical site different from the site requiring regeneration. The composition is administered to a site that is compatible with the tissue type of the site requiring regeneration. When the tissue type is included, it can induce regeneration in the area where it is needed. The site of interest can be a joint, such as the left knee, and the administration site is the right knee. In the example shown, the right knee may or may not need regeneration, but Without being bound by theory, the composition is capable of inducing regeneration in the left knee. Particular embodiments may be characterized, for example, in muscle tissue, connective tissue, articular tissue, epithelial tissue, endometrial tissue, Dermal tissue, nerve tissue, adipose tissue, skin tissue, lung tissue, liver tissue, bladder tissue, kidney tissue, heart tissue In any tissue, such as stomach tissue, intestinal tissue, spinal cord tissue, eye tissue, fibrous tissue, omentum or bone marrow. The method may include administering a tissue that is made up of any one or more cell types. Administration of one or more regenerative compositions to any such tissue may provide regeneration in the same tissue type. may provide and / or induce regeneration at the site of administration, but may not provide and / or induce regeneration at the site of administration The present disclosure specifically encompasses regeneration at a site distinct from the site of administration.

[0007] In certain embodiments, administration includes systemic or local delivery of the composition, Administration may be to a specific site, and may or may not be by injection. The administration may, in some cases, be a single dose or may be administered over a specific period of time. Multiple doses, including 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses It may be a single dose.

[0008] In certain embodiments, the site in need of regeneration is a site that has a partial or complete loss of function. Partial loss of function may occur, such as partial loss of glomerular filtration in the kidney. This includes tissue that does not have the full functionality compared to healthy tissue of the same tissue type. Loss of function includes complete loss of normal function of that tissue type, for example complete renal failure. The loss may be, for example, cell death, tissue necrosis, atrophy, fibrosis, inflammation, fat deposition, degeneration, etc. in the tissue. Production of sex molecules, loss of elasticity, neurodegeneration, autoimmunity, complement activation, loss of cartilage, ligament rupture, muscle These may include tears, loss of connective tissue, and neoplasms.

[0009] In certain embodiments, the composition administered comprises at least one of cells, growth factors, , blood products, exosomes, miRNA, epigenetic acting compositions or the like The cells include fibroblasts or stem cells or a mixture thereof. Stem cells can be of any type or origin, including, but not limited to, stem cells. Stem cells can be of any lineage, Mesenchymal stem cells, pluripotent stem cells, hematopoietic stem cells, induced pluripotent stem cells, parthenogenetic stem cells or mesenchymal stem cells derived from a pluripotent source, or any other stem cells. The cells may, for example, help identify the cells being used or identify certain specific For example, the cells may express certain cell markers and / or may be capable of producing certain The cells may lack expression of a cell marker. The cells may be derived from, for example, bone marrow, peripheral blood, adipose tissue, mobilized peripheral blood, or other tissue. Peripheral blood, umbilical cord blood, Wharton's jelly, umbilical cord tissue, skeletal muscle tissue, subepithelial umbilical cord, endometrial tissue, menstrual From any tissue, such as menstrual blood, fallopian tube tissue, foreskin, placenta, ear lobe, omentum, or a combination thereof The cells may be from allogeneic or autologous or xenogeneic origin. The genetically acting composition may be a histone deacetylase inhibitor or a histone the therapeutic agent may be a steroid methyltransferase inhibitor, or a combination thereof.

[0010] Embodiments of the present disclosure include a method of stimulating regeneration at a first tissue site in an individual (first tissue The method includes the step of: At least one cell line comprising dedifferentiated fibroblasts and / or dedifferentiated fibroblasts and optionally stem cells and administering the regenerative composition to a second tissue site, the second tissue site being a first tissue site of the individual. The first tissue site and the second tissue site are from different tissue types in the individual's body. The administration of the composition may be by systemic injection, local injection, systemic delivery and / or local delivery. The administration of the composition may include at least one administration or 2, 3, 4, 5, 6, 7, 8 In a specific embodiment, the first set may include 9, 10 or more administrations. The tissue is responsible for the following: cell death, tissue necrosis, atrophy, fibrosis, inflammation, fat deposition, and degenerative molecular changes in that tissue. generation, loss of elasticity, neurodegeneration, autoimmunity, complement activation, cartilage loss, ligament rupture, muscle rupture, Partial or complete loss of function, including loss of vascular function, loss of synovial tissue, neoplasms, or a combination thereof There is a complete loss of function. The tissues are muscle tissue, connective tissue, epithelial tissue, endothelial tissue, nervous tissue, and adipose tissue. Fat tissue, skin tissue, lung tissue, liver tissue, bladder tissue, kidney tissue, heart tissue, stomach tissue, intestinal tissue, spinal tissue Tissues including medullary tissue, ocular tissue, fibrous tissue, omentum, lymphatic tissue, bone marrow, or combinations thereof In a specific case, the tissue is an endothelial cell. , epithelial cells, skin cells, endodermal cells, mesodermal cells, fibroblasts, bone cells, chondrocytes, Neural killer cells, dendritic cells, liver cells, pancreatic cells, interstitial cells, salivary gland mucous cells, salivary glands Serous cells, von Ebner's gland cells, mammary gland cells, lacrimal gland cells, ear canal gland cells, eccrine sweat gland dark cells Cells, eccrine clear cells, apocrine clear cells, Molar gland cells, sebaceous gland cells, Bowman's gland cells vesicles, Brunner's gland cells, seminal vesicle cells, prostate cells, bulbourethral gland cells, Bartholin's gland cells, Litho gland cells, endometrial cells, isolated goblet cells, mucosal cells of the stomach wall, gastric gland zymogen cells, gastric gland acidogen cells Secretory cells, pancreatic acinar cells, Paneth cells, type II alveolar cells, Clara cells, growth hormone-producing cells cells, lactotrophs, thyrotrophs, gonadotrophs alveolar cells, adrenocorticotropic hormone-producing cells, cells of the intermediate lobe of the pituitary gland, magnocellular neurosecretory cells, intestinal cells, Airway cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, chief parathyroid cells, eosinophilic cells, Adrenal cells, chromaffin cells, Leydig cells, theca interna cells, luteal cells, granulosa luteal cells Follicle, theca luteal cells, juxtaglomerular cells, macula densa cells, perivascular pole cells, mesangial cells, blood Vascular endothelial fenestrated cells and lymphatic endothelial fenestrated cells, vascular endothelial continuous cells and lymphatic endothelial continuous cells cells, vascular endothelial splenocytes and lymphatic endothelial splenocytes, synovial cells, serosa cells (peritoneal cavity, pleural cavity and the pericardial cavity), squamous cells, columnar cells, dark cells, vestibular membrane cells (endolymphatic space of the ear endolymphatic space of the ear), basal cells of the stria vascularis, marginal cells of the stria vascularis (lining of the endolymphatic space of the ear), Claudius cells, Boettcher cells, choroid plexus cells, pia arachnoid squamous epithelial cells, pigmented ciliary epithelial cells, Non-pigmented ciliary epithelial cells, corneal endothelial cells, PEG cells, airway ciliated cells, fallopian tube ciliated cells, uterus Endometrial ciliated cells, reticulum ciliated cells, efferent ductal ciliated cells, ciliated ependymal cells, epidermal keratinocytes, Epidermal basal cells, fingernail and toenail keratinocytes, nail bed basal cells, medullary hair stem cells, skin Hair stem cells, cuticle hair stem cells, cuticle hair root sheath cells, hair root sheath cells of the Huxley layer, Root sheath cells of the stratum corneum, outer root sheath cells, hair matrix cells, surface epithelial cells of stratified squamous epithelium, basal epithelium cells, urinary epithelial cells, inner hair cells of the organ of Corti in the ear, outer hair cells of the organ of Corti in the ear, olfactory epithelium basal cells of the epidermis, cold-sensitive primary sensory neurons, heat-sensitive primary sensory neurons, and the epidermis olfactory receptor neurons, pain-sensitive primary sensory neurons, photoreceptor rod cells, photoreceptor blue cells Color-sensitive cone cells, photoreceptor green-sensitive cone cells, photoreceptor red-sensitive cone cells, specific receptor cells Captive primary sensory neurons, Touch-sensitive primary sensory neurons, Type I carotid body cells, Type II Carotid body cells (blood pH sensor), type I hair cells in the vestibular apparatus of the ear (acceleration and gravity), Type II hair cells of the vestibular apparatus of the ear, type I taste bud cells, cholinergic neurons, adrenergic neurons Neuropathic neurons, peptidergic neurons, inner column cells of the organ of Corti, outer column cells of the organ of Corti, The inner supporting cells of the organ of Corti, the outer supporting cells of the organ of Corti, the border cells of the organ of Corti, and the Hensen's cells of the organ of Corti. Alveolar vesicles, vestibular supporting cells, taste bud supporting cells, olfactory epithelium supporting cells, Schwann cells, satellite cells, intestinal gliomas astrocytes, neurons, oligodendrocytes, spindle neurons, anterior lens epithelium Cells, crystallin-containing lens fiber cells, hepatocytes, adipocytes, white adipocytes, brown adipocytes Cells, liver lipid cells, renal glomerular parietal cells, renal glomerular podocytes, renal proximal tubule brush border cells, hepatocytes Cells in the thin part of the kidney loop, renal distal tubule cells, renal collecting duct cells, type I pneumocytes, pancreatic duct cells, Smooth muscle duct cells, duct cells, intestinal brush border cells, exocrine gland striated duct cells, gallbladder epithelial cells, testis Efferent duct non-ciliated cells, epididymal principal cells, epididymal basal cells, ameloblastic epithelial cells, crescent Face epithelial cells, interdental epithelial cells of the organ of Corti, loose connective tissue fibroblasts, keratocytes, tendon fibroblasts Cells, bone marrow reticular tissue fibroblasts, non-epithelial fibroblasts, pericytes, nucleus pulposus cells, cementoblasts Cementoblast / cementocytes, odontoblasts Cells, odontocytes, chondrocytes of hyaline cartilage, cartilage cells of fibrocartilage Bone cells, chondrocytes of elastic cartilage, osteoblasts, osteocytes, osteoclasts, osteocyte precursor cells, vitreous cells Alveolar stellate cells, stellate cells (ear), hepatic stellate cells (Ito cells), pancreatic stellate cells, red skeletal muscle cells, white skeletal muscle cells , intermediate skeletal muscle cells, muscle spindle nuclear bag cells, muscle spindle nuclear chain cells, satellite cells, normal cardiac muscle cells, Nodular cardiomyocytes, Purkinje fiber cells, smooth muscle cells, myoepithelial cells of the iris, myoepithelium of exocrine glands Cells, reticulocytes, megakaryocytes, monocytes, connective tissue macrophages. epithelial Langerhans cells, Dendritic cells, microglial cells, neutrophils, eosinophils, basophils, mast cells, helper T cells , suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, melanocytes, retinal pigment epithelial cells, oogonia / oocytes, sperm cells, spermatozoa cells, spermatogonia, sperm, follicular cells, Sertoli cells, thymic epithelial cells, interstitial kidney cells and The present invention includes one or more cells selected from the group consisting of these combinations.

[0011] Any regenerative composition may be used, including AM, Ang, BMP, BDNF, EGF, Epo, FGF, G NDF, G-CSF, GM-CSF, GDF-9, HGF, HDGF, IGF, migration stimulation Factors, GDF-8, GDF-11, GDF-15, MGF, NGF, PlGF, PDGF , Tpo, TGF-alpha, TGF-beta, TNF-alpha, VEGF, Wnt-ta Protein, Interleukin, IL-1 alpha, IL-1 beta, IL-1F1, IL -1F2, IL-1F3, IL-1F4, IL-1F5, IL-1F6, IL-1F7, IL-1F8, IL-1F9, IL-2, IL-3, IL-4, IL-5, IL-6, I L-7, IL-8, IL-9, IL-10, IL-11, IL-12, 35kDa Alf a subunit, IL-12, 40 kDa beta subunit, IL-13, IL-14 , IL-15, IL-16, IL-17A, IL-17B, IL-17C, IL-17D , IL-17E, IL-17F isoform 1, IL-17F isoform 2, IL -18, IL-19, IL-20, IL-21, IL-22, IL-23 p19 subyu nit, IL-23 p40 subunit, IL-24, IL-25, IL-26, IL -27B, IL-27-p28, IL-28A, IL-28B, IL-29, IL-30 , IL-31, IL-32, IL-33, IL-34, IL-35, IL-36 Alpha , soluble receptor for IL-36 beta, IL-36 gamma, interferon ( IFN), IFN-alpha, IFN-beta, IFN-gamma, IFN-lambda 1, I FN-lambda 2, IFN-lambda 3, IFN-K, IFN-epsilon, IFN-kappa IFN-tau, IFN-delta, IFN-zeta, IFN-omega, and IFN-v Soluble receptors for insulin, proinsulin, and insulin receptors Growth factors such as leptin (LEP) and combinations thereof. It may include at least one growth factor.

[0012] In certain embodiments, the regenerative composition comprises platelet-rich plasma, which may include platelet lysate. The platelet-rich plasma may be derived from peripheral blood, umbilical cord blood, or a mixture thereof. In one embodiment, the regenerative composition comprises at least one regenerative cell, such as stem cells and / or fibroblasts. It contains one or more exosomes derived from live cells. Fibroblasts are found in the foreskin, adipose tissue, and fetal tissue. Tissue selected from the group consisting of disc, ear lobe, omentum, Wharton's jelly and combinations thereof It can be derived from the origin.

[0013] When the regenerative composition includes one or more exosomes, the exosomes are about 30 These exosporins may range in size from about 2 nm to 200 nm, including sizes up to 150 nm. The lipidosome is made up of phospholipids, phosphatidylserine, phosphatidylinositol, and phosphatidylinositol. Zycoline, sphingomyelin, ceramide, glycolipid, cerebroside, steroid, chole At least one lipid selected from the group consisting of sterols and combinations thereof These exosomes may contain at least one lipid raft. In embodiments, the exosomes contain one or more antigenic markers on the surface of the exosome. The markers include CD9, CD63, CD81, ANXA2, ENO1, HSP90AA1, EEF1A1, YWHAE, SDCBP, PDCD6IP, ALB, YWHAZ, EEF2, ACTG1, LDHA, HSP90AB1, ALDOA, MSN , ANXA5, PGK1, CFL1, and combinations thereof. do.

[0014] When the regenerative composition includes one or more fibroblasts, the fibroblasts may be used to regenerate the foreskin, fat tissue, and / or the like. tissue, placenta, ear lobe, adipose tissue, omentum, Wharton's jelly and combinations thereof The fibroblasts may be derived from any suitable source, such as from a tissue source selected from the following: NOG, OCT-4, SSEA-4, stem cell factor receptor and their combinations Any of the fibroblasts may express at least one marker selected from the group consisting of: The regenerative compositions may also include stem cells.

[0015] When the regenerative composition includes one or more stem cells, the stem cells may be of any type. Examples include hematopoietic stem cells, embryonic stem cells, parthenogenetic stem cells, induced pluripotent stem cells, etc. Cells, stem cells derived from somatic cell nuclear transfer, stem cells derived from cytoplasmic transfer, stimulus-induced pluripotency acquisition and and combinations thereof. The hematopoietic stem cells that may be used are capable of multilineage reconstitution in immunocompromised hosts. These hematopoietic stem cells express c-kit, Sca-1, CD34, CD133 and other The present invention relates to a method for expressing at least one of the proteins selected from the group consisting of the above combinations. In some cases, the hematopoietic stem cells express CD38, CD14, CD16, CD56, or Hematopoietic stem cells lack expression of one or more lineage markers, such as chromatin, chromatin, nuclein, or a combination thereof. - kit expression may be positive, Sca-1 expression may be positive, and / or the system Hematopoietic stem cells are derived from peripheral blood, mobilized peripheral blood, bone marrow, umbilical cord blood, and From the group consisting of adipose stromal vascular cells, those derived from progenitor cells, and combinations thereof In a specific embodiment, the hematopoietic progenitor cells are derived from pluripotent stem cells. The stem cells, when used, are mesenchymal stem cells, including mesenchymal stem cells, which are plastic-adherent. Mesenchymal stem cells may contain CD73, CD90, CD105 and their related genes. The mesenchymal stem cells may express a marker selected from the group consisting of CD1 4. A marker selected from the group consisting of CD45, CD34 and a combination thereof In a specific embodiment, the mesenchymal stem cells are derived from bone marrow, peripheral blood, adipose tissue, or the like. , Mobilized peripheral blood, Umbilical cord blood, Wharton's jelly, Umbilical cord tissue, Skeletal muscle tissue, Subepithelial umbilical cord, Endometrium tissue, menstrual blood, fallopian tube tissue, and combinations thereof. Mesenchymal stem cells express oxidized low-density lipoprotein receptor 1, chemokine receptor 1, and ligand 3, granulocyte chemotactic protein, and combinations thereof. In a specific case, the umbilical cord tissue may be derived from umbilical cord tissue expressing a marker that is Mesenchymal stem cells express CD117, CD31, CD34, CD45, and combinations of these. The mesenchymal stem cells derived from umbilical cord tissue do not express a marker selected from the group consisting of In some cases, higher levels of interleukin 8 and / or may express reticulon 1. In a specific embodiment, mesenchymal stem cells derived from umbilical cord tissue The cells express CD10, CD13, CD44, CD73, CD90 and combinations thereof. The cells from the umbilical cord tissue express a marker selected from the group consisting of: MCP-1, MIP1 beta, IL-6, IL-8, GCP-2, HGF, KGF, FG F, HB-EGF, BDNF, TPO, RANTES, TIMP1, and combinations thereof The umbilical cord tissue-derived cells may be selected from the group consisting of TRA1-60, TRA1- 81, SSEA3, SSEA4, NANOG, and combinations thereof Umbilical cord tissue-derived mesenchymal stem cells can self-renew and express markers that are selective for the proliferation and differentiation of umbilical cord tissue. and isolating the umbilical cord tissue from a substantially blood-free umbilical cord tissue capable of being expanded. The cells derived from umbilical cord tissue may stain positive for alkaline phosphatase. In a specific embodiment, the cord tissue derived mesenchymal stem cells have been cultured for at least 20 cycles. Mesenchymal stem cells derived from cord tissue maintain normal karyotypes during passage. In some cases, the umbilical cord tissue derived mesenchymal stem cells are identified as CD10, CD13, CD 44, CD73, CD90, PDGFr-alpha, PD-L2, HLA-A,B,C and and combinations thereof. Mesenchymal stem cells express CD31, CD34, CD45, CD80, CD86, CD117, and CD 141, CD178, B7-H2, HLA-G, HLA-DR,DP,DQ and their The bone marrow may not express one or more markers selected from the group consisting of a combination of: Marrow-derived mesenchymal stem cells express markers LFA-3, ICAM-1, and PECAM- 1. P-selectin, L-selectin, CD49b / CD29, CD49c / CD29, CD49d / CD29, CD29, CD18, CD61, 6-19, thrombomodulin , telomerase, CD10, CD13, CD34, CD56, CD117, integrin beta, and combinations thereof. The bone marrow mesenchymal stem cells may be selected from the group consisting of C Bone marrow mesenchymal stem cells express CD2, CD5, CD14, and CD10. Do not express at least one of D19, CD33, CD45 and / or DRII Bone marrow mesenchymal stem cells express CD13, CD34, CD56, CD90, and CD1 In a specific embodiment, the IL-17 and / or Nestin can be expressed. , Bone marrow derived mesenchymal stem cells include mesenchymal stem cell progenitors. Mesenchymal progenitor cells are The present invention relates to a method for the preparation of a bone marrow mesenchymal stem cell population enriched for TRO-1 expressing cells. In some embodiments, the mesenchymal precursor cells express both STRO-1 and VCAM-1. STRO-1 expressing cells express CBFA-1, type II collagen, PPAR. NMDA 2, osteopontin, osteocalcin, parathyroid hormone receptor, leptin , H-ALBP, aggrecan, Ki67, glycophorin A, and their combinations In a specific embodiment, at least one marker selected from the group consisting of: In this study, bone marrow mesenchymal stem cells express at least one of CD14, CD34, and / or CD45. STRO-1 expressing cells lack expression of at least one of the following: VCAM-1, TKY- 1, CD146, STRO-2, and combinations thereof. In a specific embodiment, the skeletal muscle stem cells may be positive for CD13, CD3 4. A marker selected from the group consisting of CD56, CD117, and combinations thereof Skeletal muscle mesenchymal stem cells may not express CD10. In this state, skeletal muscle mesenchymal stem cells express CD2, CD5, CD14, CD19, CD33, Does not express at least one of CD45 and / or DRII. mesenchymal stem cells may be used, which are expressed by the following markers: CD29, CD73, CD90, CD166, SSEA4, CD9, CD44, CD146, CD105 and combinations thereof The subepithelial umbilical cord derived mesenchymal stem cells may have a marker selected from the group consisting of CD45 , CD34, CD14, CD79, CD106, CD86, CD80, CD19, CD1 17, Stro-1, HLA-DR and combinations thereof In a specific embodiment, the subepithelial umbilical cord-derived mesenchymal Stem cells are CD29, CD73, CD90, CD166, SSEA4, CD9, and CD44 In particular, the IL-161 cells express at least one of CD146 and / or CD105. In this case, mesenchymal stem cells derived from the subepithelial umbilical cord express CD45, CD34, CD14, and CD7 9, CD106, CD86, CD80, CD19, CD117, Stro-1 and / or or HLA-DR. Mesenchymal stem cells derived from subepithelial umbilical cord may be positive for SOX2 and / or OCT4.

[0016] In certain embodiments, the regenerative composition comprises one or more fibroblast-derived apoptotic microenvironments. The regenerative composition may include fibroblast-derived miRNA; iRNAs can be contained in exosomes; miRNAs derived from fibroblasts can be used to induce apoptosis miRNAs contained in the body; and / or derived from fibroblasts circulate in plasma. In specific embodiments, one or more remote playback effects are enhanced. In certain embodiments, this includes one or more histone deacetylase inhibitors; one or more DNA markers; Systemic administration of one or more epigenetically active compositions, such as taurate transferase inhibitors. This is achieved by administration (e.g., at the site of administration of the regenerative composition or at a different site). do.

[0017] The foregoing has been a summary of the features and techniques of the present disclosure in order to better understand the detailed description that follows. The technical advantages have been outlined quite broadly. Additional features and advantages which form the subject of the claims herein. The disclosed concepts and specific embodiments are within the scope of the present invention. It can be readily used as a basis for modifying or designing other structures to achieve the intended purpose. Those skilled in the art will recognize that such equivalent constructions are set forth in the appended claims. It will be readily understood by those skilled in the art that such modifications will not depart from the spirit and scope of the present invention. The present invention is disclosed herein, both as to construction and method of operation, together with other objects and advantages thereof. The novel features believed to be unique to the concept will be better understood from the following description. .

[0018] While various embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such It should be apparent that the above embodiments are provided by way of example only. Numerous variations, modifications, and substitutions will be apparent to those skilled in the art. It should be understood that various alternatives to the embodiments of the present disclosure may be used. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] In the detailed disclosure that follows and in the specification more broadly, reference is made herein to All patents, patent applications, patent publications, technical publications, scientific publications and other references published To the extent that they do not contradict the teachings herein, the references are incorporated herein by reference in their entireties. All technical details used herein should be understood to be incorporated herein by reference. Technical and scientific terms, unless otherwise defined herein, have the meanings commonly understood by those of ordinary skill in the art. It is important to the understanding of this disclosure to note that the term "flavor" is intended to have the same meaning as "taste." The methods used herein are also known to those skilled in the art unless otherwise stated. Of course, specific components and / or conditions may vary and may not all be consistent with the teachings of this disclosure. It is also understood that the teachings are not limited to the specific embodiments and methods described below. Further, the terminology used herein is for the purpose of describing particular embodiments of the present disclosure only. These are used interchangeably and are not intended to be limiting in any way.

[0020] I. Definition

[0021] In accordance with longstanding patent law practice, the words "a" and "an" are used in conjunction with the term "including." As used herein (including the claims), the term "one or more" refers to the term "one or more" as used herein. Some embodiments include one or more elements, method steps and / or methods of the present disclosure. Any method or composition described herein may consist of or consist essentially of The method may be performed in accordance with any other method or composition described herein. And it is contemplated that different embodiments may be combined.

[0022] As used herein, the terms "or" and "and / or" refer to multiple constructs. Used to describe constituent elements in combination or exclusive of each other. For example, "x, y "and / or z" means "x" only, "y" only, "z" only, "x, y and z", "(x and y) or z", "x or (y and z)" or "x or y or z" It is to be noted that x, y, or z may be specifically excluded from an embodiment. It is definitely planned.

[0023] As used herein, the word "comprising" means "including "including," "having," "containing" or "characterized by." These terms are inclusive and open-ended. and does not exclude additional, unrecited elements or method steps. In this case, the phrase "consisting of" does not include any element, step or component not specified in the claim. This phrase also excludes components. If a phrase is used, it limits only the elements listed in that clause and does not include other elements. According to this disclosure, the phrase "consisting essentially of" is not excluded from the entire claim. In addition to the materials or steps specified, the essential characteristics and novelty of the claimed subject matter are Limit the scope of the claims to materials or steps that do not materially affect properties.

[0024] Throughout this specification, the terms "one embodiment," "an embodiment," "particular embodiment," "particular embodiment," "particular "Related embodiments," "certain embodiments," "additional embodiments," or "further embodiments" Reference to "an embodiment" or a combination thereof is intended to mean that the embodiment is described in relation to that embodiment. It is contemplated that a particular feature, structure, or characteristic described herein may be included in at least one embodiment of the present invention. Accordingly, the appearance of the foregoing phrases in various places throughout this specification In addition, the following descriptions may not necessarily all refer to the same embodiment. The features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. do.

[0025] As used herein, the terms "cell culture" and "culturing of cells" refer to a specific In one embodiment, the maintenance of cells, including human cells, human-derived cells, and animal cells, in vitro is performed. The cells may be fibroblasts, stem cells or a mixture of both. It could be.

[0026] As used herein, the term "cell culture medium" refers to a medium for culturing cells in vitro. For some cell types, the medium helps the cells grow in culture. The medium according to the present disclosure may be sufficient to culture the cells. It may contain one or more nutrients, such as calcium and / or inorganic ions. (e.g., phenol red), sodium pyruvate, some vitamins, free fatty acids , antibiotics, antioxidants and / or trace elements. When culturing the stem cell or mesenchymal stem cells or stem cell-like cells according to the present disclosure, Prior to incubation, the medium was, for example, Iscove's Modified Dulbecco's Medium (IMDM), alpha-MEM, or Dulbecco's Medium (DMEM). Any standard medium such as Becko's Modified Eagle Medium (DMEM), RPMI medium, and McCoy's medium Once the cells have been reprogrammed, they may be cultured in a medium suitable for use in certain embodiments. In some embodiments, the cells can be cultured in, for example, embryonic stem cell medium.

[0027] As used herein, the term "derived from" refers to a desired product derived from a particular source. It refers to obtaining a composition from its source so as to at least partially retain its properties. For the purpose of the present invention, fibroblasts are isolated and obtained from any suitable tissue, such as, for example, adipose tissue. The isolated fibroblasts are derived from fibroblasts found in adipose tissue. The isolated fibroblasts in the provided example at least partially retain their characteristics. Alveoli are described as fibroblasts derived from adipose tissue.

[0028] As used herein, the term "dedifferentiate" or "dedifferentiation" refers to, for example, the dedifferentiation of a system. Lineage-committed cells, myoblasts or osteoblasts , reverse their lineage commitment, e.g., to progenitor or precursor cells, multipotent stem cells or pluripotent This refers to the process of becoming a sexual stem cell. Dedifferentiated cells are cells that have not yet become lineage-committed, e.g. can be identified by loss of associated patterns of gene expression and cell surface protein expression In some embodiments, de-differentiated cells are cells that have a cell type similar to that of a previous developmental point. An example of dedifferentiation is the differentiation of cells in wounds and healing. Dedifferentiation is a temporary loss of epithelial cell characteristics. Dedifferentiation can occur to various degrees. In wound healing, for example, dedifferentiation occurs only slightly, after which the cells revert to recognizable epithelium. For example, a cell that has been significantly dedifferentiated resembles a stem cell. Can the cells remain dedifferentiated and proliferate as dedifferentiated cells; can the cells remain dedifferentiated and proliferate as dedifferentiated cells? Can the cell redifferentiate along a developmental pathway; or can the cell redifferentiate along a developmental pathway different from that along which it previously dedifferentiated? In the context of the present disclosure, dedifferentiated mesenchymal stem cells can redifferentiate along other They have high plasticity and the ability to differentiate or re-differentiate into mesenchymal stem cells. The dedifferentiated state of the treated cells was possibly characterized by alkaline phosphatase (ALP), OC T4, SOX2, human telomerase reverse transcriptase (hERT), SSEA-4 and their The combination of The cells can be verified by, for example, positive staining for alkaline phosphatase. That is, reprogramming genes (e.g., OCT4, Nanog, or Sox-2 The somatic cells transfected with ) are treated with a dedifferentiation agent such as valproic acid, and then the dedifferentiation process is The initial process of colony formation in the culture medium is alkaline phosphatase staining (AP staining). The cells were then visualized by immunofluorescence (IF) using Oct4 antibody. The expression of ct4 is verified. In some cases, the fibroblasts are Before being used in a therapeutic application, the cells are dedifferentiated into another cell type.

[0029] As used herein, the term "differentiate" or "differentiation" refers to the differentiation of a precursor or progenitor cell. Progenitor cells, e.g., stem cells, can give rise to specific cell types, e.g., osteoblasts, or cells of a specific tissue type (e.g., For example, skeletal muscle, vascular smooth muscle, pericytes or vascular endothelium), or cells of a particular phenotype. The process of differentiation into cells (e.g. osteogenic, adipogenic or chondrogenic differentiation) Differentiated cells are characterized by patterns of gene expression and cell surface protein expression. It can be identified.

[0030] As used herein, the term "express" refers to the expression of specific proteins, markers and When referring to cells expressing a gene and / or a gene, it is understood that those cells Contains specific proteins, markers, and / or transcribed RNA of that specific gene It means to contain or comprise. , the particular gene may have already been transcribed or may be actively transcribing. , and have translated the mRNA that codes for that particular protein and / or marker. In certain embodiments, the cell may be capable of detecting or actively translating If a particular protein, marker and / or gene is present in sufficient quantity, that particular target protein, marker and / or gene may be detected. The protein, marker and / or gene may be expressed or determined to be expressed. Western blot, flow cytometry, mass spectrometry, PCR, qPCR, RT-q PCR, Southern blotting, ELISA and / or designed kits For example, methods known in the art for detecting proteins, markers and / or genes are Conversely, cells may be said to lack expression or to express a particular protein. When the protein, marker and / or gene is not expressed, a detectable amount of the specific proteins, markers and / or genes may be absent.

[0031] As used herein, the term "homologous tissue" refers to tissues that are of the same type and have the same function. Homologous tissues in an individual include, for example, , left knee and right knee, left kidney and right kidney, left lung and right lung, any vertebra and any other vertebra, any intervertebral disk and any other across all discs, left and right eyes, left and right calves, or in different anatomical locations within an individual. Homologous tissues include skeletal muscle tissue, cardiac muscle tissue, and any other tissue of the same tissue type. The term also includes muscle types such as fibrous and smooth muscle tissue. For example, the skeletal muscle tissue of the arm is connected to the femoral bone. For convenience, homologous tissue refers to homologous cells, such as hematopoietic cells of various lineages. It also refers to the type.

[0032] As used herein, the term "reprogramming" or "remodeling" refers to For example, DNA methylation, histone methylation, and histone acetylation are involved in cellular epigenetic Changes in tick markers or transcription factor signaling systems, e.g., Oct4 In particular, the reprogramming of the present disclosure generally refers to the activation of a gene by inducing The embodiment of the invention provides at least one dedifferentiated and / or rejuvenated cell. Certain embodiments provide for the administration of cells having characteristics of multipotent stem cells, particularly pluripotent stem cells. Therefore, the cells to be reprogrammed already possess the phenotype of multipotency or pluripotency. In the case of cells that undergo reprogramming, the present disclosure provides a method for reprogramming the cells for long periods of time. The cells can be maintained in a multipotent or pluripotent state. In a specific embodiment, the cells have the properties of pluripotency or multipotency, prior to administration, The cells may be passaged for a sustained period of time. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2 Passaged 1, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more times The cells can maintain a normal karyotype throughout passage. When cells are in a senescent or differentiated state, they undergo dedifferentiation and multiplication. In certain embodiments, a multipotent cell can be a multipotent stem cell or a pluripotent stem cell. The cells can be reprogrammed to become competent cells.

[0033] As used herein, the term "stem cell" refers to a cell that is capable of differentiating into one or more cell types. Any pluripotent or multipotent cell or progenitor or precursor cell capable of self-renewal. Refers to somatic cells. Stem cells can differentiate into one cell type or more than one cell type and can be used indefinitely. Stem cells may have the proliferation ability of osteoblasts or mesenchymal cells (e.g., bone, cartilage, Stem cells that can differentiate into fat, muscle, stroma (including hematopoietic supporting stroma) and tendon cells. The cells may be included.

[0034] As used herein, the term "transfection" refers to the introduction of foreign nucleotides into a The gene sequence is preferably introduced into cells by viral or non-viral methods. In the embodiment of the present disclosure, the term "gene delivery method" refers to the introduction of foreign DNA / RNA into a host. / Proteins are synthesized by transient transfection of an expression vector encoding a polypeptide of interest. The foreign DNA / RNA / protein can be introduced into a cell by injection, As used herein, the term "antibody" refers to a molecule that is introduced into a cell and then gradually eliminated by the cell during mitosis. When used herein, the term "transient transfection" refers to the introduction of an expression vector and The polypeptide encoded by the vector is expressed either in the genome or intracellularly in the host cell. They are not permanently integrated into the host cell and therefore cannot be transferred to the host cell or its descendants. Proteins, polypeptides or other compounds can also be eliminated by the In certain embodiments, the antibody can be delivered into a cell using transfection methods. The cells of the present disclosure can be transfected.

[0035] II. General Methods

[0036] To induce regeneration of one or more homologous tissues at different anatomical locations, at least A single regenerative composition is administered to one or more tissues, followed by systemic (or localized) and tissue-specific Means, methods and compositions useful for inducing a specific regenerative effect are disclosed. The administration of at least one regenerative composition to the tissue to be stimulated comprises administering the regenerative composition to the tissue. It is capable of inducing a systemic effect resulting in the regeneration of non-administered tissues that are homologous to the tissue. In some embodiments, the present disclosure provides a method for treating a proximal or distal disc after administration of a regenerative composition to the disc. In a specific embodiment of the present disclosure, the regeneration of homologous tissues such as the kidney or distal vertebral disc is Bilateral tissues, such as an organ, joint, or set of joints, each affecting the kidneys in different parts of the body. As a result of administration of the regenerative agent to the organ or joint, the organ or joint is induced to regenerate. In some cases, the regeneration process occurs at a location distal to the site of administration. Such embodiments may be amplified by administration of one or more agents capable of inducing stem cell mobilization. It is possible.

[0037] The present disclosure provides a method for treating a regenerative disorder comprising administering one or more regenerative compositions to a patient at a site distal to the region of the body to which the regenerative compositions are administered. In a specific embodiment of the present disclosure, one or more fibroblasts are provided. The cells or any of the cells of the present disclosure may be administered to an area of ​​the body as at least part of a regenerative composition. The therapeutic agent may be administered to at least one distal body region, thereby inducing regeneration in at least one distal body region. The regenerative composition comprises fibroblasts and / or dedifferentiated fibroblasts and optionally stem cells. It may comprise cells.

[0038] In particular, administration of the regenerative composition is performed in an area of ​​the body that is homologous to the area in which regeneration is desired. In certain embodiments, the tissue to which the regenerative composition is administered undergoes regeneration and the regenerative composition Homologous tissue to which the substance was not administered also undergoes regeneration.

[0039] In certain embodiments, the one or more regenerative compositions are administered to a first tissue and / or a second tissue of the individual's body. The first tissue and / or organ site is not the same tissue and / or organ site as the organ, but is regenerating a second tissue and / or organ that is a similar type of tissue and / or organ to the organ; one or more regenerative drugs are provided to the first tissue and / or organ for the purpose of The delivery of the biocomposition results in regeneration of the first tissue and in the exact same tissue location as the first tissue. In some cases, regeneration of a second tissue can occur that is not a primary tissue but is the same type of tissue as the first tissue.

[0040] The regenerative composition may be administered locally (e.g., administered to tissue homologous to the tissue to be regenerated). They can be administered intracellularly (e.g., intracellularly) or systemically (e.g., to the tissue (or tissues) to be regenerated, such as blood cells). Administration may be by, for example, injecting the regenerative composition into the cytoplasm (e.g., where the cell type is located throughout the body). The delivery may be by injection using a sedative or, by way of example only, by implantation of the regenerative composition at the delivery site. Delivery may also be by mail or the like.

[0041] The tissue to which the regenerative composition is administered or to be regenerated can be any tissue type. For example, the tissue may be muscle tissue, connective tissue, epithelial tissue, endothelial tissue, nervous tissue, adipose tissue, skin tissue, etc. tissue, lung tissue, liver tissue, bladder tissue, kidney tissue, heart tissue, stomach tissue, intestinal tissue, spinal cord tissue, eye tissue The tissue may be endothelial, epithelial, fibrous tissue, bone marrow, or a combination thereof. Cells, skin cells, endodermal cells, mesodermal cells, fibroblasts, bone cells, chondrocytes, natural killer cells, Lar cells, dendritic cells, liver cells, pancreatic cells, interstitial cells, salivary gland mucous cells, salivary gland serous cells , von Ebner's gland cells, mammary gland cells, lacrimal gland cells, ear canal gland cells, eccrine sweat gland dark cells, Phosphorycin clear cells, apocrine sweat gland cells, Molar gland cells, sebaceous gland cells, Bowman gland cells, Tunnel gland cells, seminal vesicle cells, prostate cells, bulbourethral gland cells, Bartholin's gland cells, Little gland cells , endometrial cells, isolated goblet cells, mucosal cells of the stomach wall, gastric gland zymogen cells, gastric gland acid-secreting cells, Pancreatic acinar cells, Paneth cells, type II alveolar cells, Clara cells, growth hormone-producing cells, mammary glands Thyrotropes, thyrotropes, gonadotropes, adrenal glands Corticotropic hormone-producing cells, cells of the intermediate lobe of the pituitary gland, magnocellular neurosecretory cells, intestinal cells, airway cells , thyroid epithelial cells, parafollicular cells, parathyroid cells, chief parathyroid cells, eosinophilic cells, adrenal cells , chromaffin cells, Leydig cells, theca interna cells, luteal cells, granulosa luteal cells, follicles Membrane luteal cells, juxtaglomerular cells, macula densa cells, perivascular pole cells, mesangial cells, vascular endothelial cells Fenestrated cells and lymphatic endothelial fenestrated cells, vascular endothelial continuous cells and lymphatic endothelial continuous cells, blood Vascular and lymphatic endothelial splenocytes, synovial cells, serous cells (peritoneal, pleural and peripheral cavities) lining of the heart chambers), squamous cells, columnar cells, dark cells, vestibular membrane cells (lining of the endolymphatic cavity of the ear) , basal cells of the stria vascularis, marginal cells of the stria vascularis (inner wall of the endolymphatic space of the ear), Claudius cells, Cher cells, choroid plexus cells, pia arachnoid squamous epithelial cells, pigmented ciliary epithelial cells, non-pigmented Ciliary epithelial cells, corneal endothelial cells, peg cells, airway ciliated cells, fallopian tube ciliated cells, endometrial cilia Cells, reticulum ciliated cells, efferent ductal ciliated cells, ciliated ependymal cells, epidermal keratinocytes, basal epidermis Cells, fingernail and toenail keratinocytes, nail bed basal cells, medullary hair stem cells, cortical hair shaft cells follicles, cuticular hair stem cells, cuticular hair root sheath cells, hair root sheath cells in Huxley's layer, hair in Henle's layer Root sheath cells, outer root sheath cells, hair matrix cells, surface epithelial cells of stratified squamous epithelium, basal cells of epithelium, secretory cells Urinary epithelial cells, inner hair cells of the organ of Corti, outer hair cells of the organ of Corti, basal cells of the olfactory epithelium alveoli, cold-sensitive primary sensory neurons, heat-sensitive primary sensory neurons, Merkel cells of the epidermis, Olfactory receptor neurons, pain-sensitive primary sensory neurons, photoreceptor rod cells, photoreceptor blue-sensitive Cone cells, photoreceptor green-sensitive cone cells, photoreceptor red-sensitive cone cells, proprioceptive primary Sensory neuron, touch-sensitive primary sensory neuron, type I carotid body cell, type II carotid body cell Somatic cells (blood pH sensor), type I hair cells of the vestibular apparatus of the ear (acceleration and gravity), vestibular Type II hair cells of the sacrum, type I taste bud cells, cholinergic neurons, adrenergic neurons vesicles, peptidergic neurons, inner column cells of the organ of Corti, outer column cells of the organ of Corti, inner Supporting cells, outer supporting cells of the organ of Corti, border cells of the organ of Corti, Hensen cells of the organ of Corti, vestibular Organ supporting cells, taste bud supporting cells, olfactory epithelium supporting cells, Schwann cells, satellite cells, enteric glial cells, Astrocytes, neurons, oligodendrocytes, spindle neurons, prelental epithelial cells, Listerin-containing lens fiber cells, hepatocytes, adipocytes, white adipocytes, brown adipocytes, liver Lipid cells, renal glomerular parietal cells, renal glomerular podocytes, renal proximal tubule brush border cells, Henle's loop thin part of the kidney, renal distal tubule cells, renal collecting duct cells, type I pneumocytes, pancreatic duct cells, smooth muscle conductor cells Duct cells, duct cells, intestinal brush border cells, exocrine gland striated duct cells, gallbladder epithelial cells, testicular efferent duct non- Ciliated cells, epididymal principal cells, epididymal basal cells, ameloblastic epithelial cells, semilunar epithelial cells alveolar cyst, interdental epithelial cells of the organ of Corti, loose connective tissue fibroblasts, keratocytes, tendon fibroblasts, bone Medullary reticular fibroblasts, non-epithelial fibroblasts, pericytes, nucleus pulposus cells, cementoblasts / cementoblasts odontoblasts, odontocytes, hyaline cartilage chondrocytes, fibrocartilage chondrocytes, elastic Chondrocytes of cartilage, osteoblasts, osteocytes, osteoclasts, osteoprogenitor cells, vitreous cells, astrocytes (ear), hepatic stellate cells (Ito cells), pancreatic stellate cells, red skeletal muscle cells, white skeletal muscle cells, intermediate skeletal Myocytes, muscle spindle nuclear bag cells, muscle spindle nuclear chain cells, satellite cells, normal myocardial cells, nodular myocardial cells alveolar cysts, Purkinje fiber cells, smooth muscle cells, myoepithelial cells of the iris, myoepithelial cells of exocrine glands, reticular Erythrocytes, megakaryocytes, monocytes, connective tissue macrophages. epithelial Langerhans cells, dendritic cells, Microglial cells, neutrophils, eosinophils, basophils, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells Follicles, melanocytes, retinal pigment epithelial cells, oogonia / oocytes, spermatids, spermatocytes, spermatogonia Cells, sperm, follicular cells, Sertoli cells, thymic epithelial cells, interstitial kidney cells, or combinations thereof The cells may be composed of any cell type, including any combination of cells.

[0042] Particular buffers, media, reagents, cells, culture conditions, etc., or any subclass thereof References to are not intended to be limiting and are intended to include any relevant examples that one of ordinary skill in the art would recognize as being of interest. It is understood that any such material or discussion is to be construed as including its value in the particular context in which it is presented. For example, it may be possible to substitute one buffer system or culture medium for another. and often serves the same purpose as the proposed intended use of the method, material or composition. In a specific embodiment, the cells are cultured in a culture vessel. Cell culture medium (especially suitable for culturing the cells and intended for culturing the cells) In a cell culture system containing a cell culture medium supplemented with one or more substances, The cells are cultured to confer the ability to induce a sexually acting regenerative effect.

[0043] In a specific embodiment, the genetic regulators are utilized to induce fibroblast and / or or cells, such as stem cells, for use in the methods and regenerative compositions of the present disclosure. The gene regulators include SOX2, NANO The reprogramming agents that cause dedifferentiation can also be used. It can be used in combination with or as a replacement for gene regulatory elements. Examples of reprogramming agents include at least valproic acid, trichostatin, and the like. According to the disclosure provided herein, at least one stably expressing another gene regulatory factor (e.g., in the absence of a reprogramming agent) ) for a period of time sufficient to culture the cells under culture conditions that support transformation of the desired cells. Embodiments that specify a sufficient period of time to be maintained in are within the skill of one of ordinary skill in the art. The sufficient or appropriate amount of time may vary according to a variety of factors, including: The specific type and epigenetic characteristics of the cells, e.g., the cell type of the starting cell and the desired cell type, the drug substance; the amount of starting material, e.g., the number of cells to be transformed; the amount of reprogramming agent and and type; genetic regulators (one or more agents that regulate gene expression); culture conditions; and / or the presence of a compound that accelerates reprogramming, e.g., cell cycle progression. Compounds that enhance, alter epigenetic states and / or cell viability In various embodiments, the presence of a compound that increases the reactivity of the Stably expressing at least one gene regulatory element in the absence of a programming agent. The time required to do this is about 1 day, about 2-4 days, about 4-7 days, about 1-2 weeks, about 2-3 In various embodiments, the cells are transformed with the desired and maintained in culture conditions that support stable expression of multiple second genes for a sufficient period of time. is about 1 day, about 2-4 days, about 4-7 days or about 1-2 weeks, about 2-3 weeks, about 3- In a specific embodiment, the transformation At the end of the period, the number of desired transformed cells will be greater than or equal to the number of cells of the first type initially provided. The amount of the cell is substantially equivalent to or greater than the amount of the cell.

[0044] For the purposes of this disclosure, the standard growth conditions used herein are, in some embodiments, The cells were cultured at approximately 37°C in a standard atmosphere containing approximately 5% CO2. The relative humidity may be maintained at about 100%. The above conditions are effective for the culture. Such conditions may vary, for example, with changes in temperature, CO2, relative humidity, oxygen, and growth medium. Those skilled in the art will recognize the options available in the art for culturing cells, such as by modifying the I want to understand that it is possible.

[0045] In some embodiments, a first muscle location may be assigned to a second muscle that is not identical, such as the same muscle type. In another embodiment, one or more regenerative compositions are administered to regenerate two muscle locations. , one or more regenerations for the purpose of regenerating a second, non-identical muscle position at the first joint position. In certain cases, a second, non-identical connective tissue composition is administered to the first connective tissue site. One or more regenerative compositions are administered to regenerate the location of the grafted tissue. One or more joint tissues may be inserted into one joint tissue location for the purpose of regenerating a second, non-identical joint tissue location. In some embodiments, the regenerative composition is administered to the location of the first epithelial tissue. One or more regenerative compositions are administered to regenerate the location of the missing second epithelial tissue. In certain cases, a second, non-identical endothelial tissue location is regenerated at the location of the first endothelial tissue. In some embodiments, one or more regenerative compositions are administered to a first nerve tissue location. and administering one or more regenerative compositions to the subject for the purpose of regenerating a second, non-identical location of neural tissue. In certain embodiments, a second, non-identical adipose tissue is added to the location of the first adipose tissue. One or more regenerative compositions are administered to regenerate the location of the first epidermis. The skin tissue location is provided with one or more regenerative tissues for the purpose of regenerating a second, non-identical skin tissue location. In certain embodiments, a second, non-identical composition is administered to the first lung tissue location. In one embodiment, one or more regenerative compositions are administered to regenerate the location of lung tissue in the lung. In this case, one or more liver tissues are inserted into the location of the first liver tissue in order to recreate the location of a second, non-identical liver tissue. In certain embodiments, the first bladder tissue is administered at a location where the regenerative composition is not identical. One or more regenerative compositions are administered to regenerate the location of the missing second bladder tissue. In some cases, the purpose of regenerating a second, non-identical renal tissue location in the location of the first renal tissue is to In a specific embodiment, the one or more regenerative compositions are administered to the first cardiac tissue location. and administering one or more regenerative compositions to regenerate a second, non-identical cardiac tissue location. In a specific case, a second, non-identical stomach tissue location is regenerated at the location of the first stomach tissue. In some embodiments, one or more regenerative compositions are administered to the first intestinal tissue. At that location, one or more regenerative compositions are administered for the purpose of regenerating a second, non-identical location of intestinal tissue. In some cases, a second, non-identical spinal tissue is administered at the location of the first spinal tissue. In one particular embodiment, one or more regenerative compositions are administered to regenerate the location of the tumor. The present invention relates to a method for regenerating a second, non-identical ocular tissue location by administering one or more regenerative compositions to a first ocular tissue location. In certain embodiments, a second, non-identical composition is administered to the location of the first fibrous tissue. One or more regenerative compositions are administered to regenerate the location of the two fibrous tissues. In this study, one or more bone tissues were implanted in the first bone tissue location with the aim of regenerating a second, non-identical bone tissue location. The above regenerative composition is administered.

[0046] III. Growth Factors and Platelet-Rich Plasma

[0047] In certain embodiments, the regenerative composition comprises at least one growth factor, The factors may be included in the composition along with the fibroblasts and / or stem cells. As used herein, a growth factor is a substance that promotes cell growth. Any compound, molecule, chemical, protein, peptide, nucleic acid, lipid or other substance that can stimulate In certain embodiments, growth factors include those that have the ability to induce regeneration. Examples include known molecules, peptides and / or proteins that can M, Ang, BMP, BDNF, EGF, Epo, FGF, GNDF, G-CSF, GM -CSF, GDF-9, HGF, HDGF, IGF, migration stimulating factor, GDF-8, GDF -11, GDF-15, MGF, NGF, PlGF, PDGF, Tpo, TGF-alpha α, TGF-beta, TNF-alpha, VEGF, Wnt proteins, interleukin-1 (IL-1) IL-1 alpha, IL-1 beta, IL-1F1, IL-1F2, IL-1F3, IL-1F4, IL-1F5, IL-1F6, IL-1F7, IL-1F8, IL-1F 9, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL- 9, IL-10, IL-11, IL-12, 35kDa alpha subunit, IL-1 2, 40 kDa beta subunit, IL-13, IL-14, IL-15, IL-16 , IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-1 7F isoform 1, IL-17F isoform 2, IL-18, IL-19, IL -20, IL-21, IL-22, IL-23 p19 subunit, IL-23 p4 0 subunit, IL-24, IL-25, IL-26, IL-27B, IL-27-p 28, IL-28A, IL-28B, IL-29, IL-30, IL-31, IL-32 , IL-33, IL-34, IL-35, IL-36 alpha, IL-36 beta, IL Soluble receptor for -36 gamma, interferon (IFN), IFN-alpha IFN-β, IFN-γ, IFN-lambda 1, IFN-lambda 2, IFN- Lambda 3, IFN-K, IFN-epsilon, IFN-kappa, IFN-tau, IFN -delta, soluble receptor for IFN-zeta, IFN-omega, and IFN-v, insulin, proinsulin, receptor for insulin, leptin (LEP) or Other growth factors may also be used. The growth factors may be, for example, Recombinant protein production, isolation from plasma or other cellular sources, peptide synthesis, and other related art The amount of growth factor required for administration can be determined by one of skill in the art. and may be in an amount sufficient to induce regeneration.

[0048] In a specific embodiment, the regenerative composition is administered in combination with fibroblasts and / or stem cells. Platelet rich plasma may or may not be included in the composition. The origin (e.g., autologous, allogeneic, syngeneic, xenogeneic or Plasma can be obtained from a variety of sources, including peripheral blood or umbilical cord blood. Plasma may be derived from any blood source, including blood plasma. Plasma may be manipulated to be enriched for platelets, Platelet-rich plasma contains a higher level or concentration of platelets than normal unmanipulated plasma. The concentration of platelets or growth factors in platelet-rich plasma is controlled by the 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times Any method of concentrating plasma to produce platelet-rich plasma (e.g. For example, centrifugation may be used. In some embodiments, platelets or platelet-rich The plasma is further manipulated to obtain a platelet lysate. Any method for obtaining a sufficient amount of blood may be used, including, for example, centrifugation or apheresis. The platelet lysate may be prepared by any method, such as freeze-thaw cycling, to remove platelets or polyclonal antibodies. It can be obtained from platelet melt.

[0049] IV. Stem cells

[0050] In a specific embodiment, the regenerative composition comprises one or more stem cells comprising at least mesenchymal stem cells. In some embodiments, "mesenchymal stem cells" or "MSCs" include (1) Adherence to plastic; (2) CD73, CD90, and CD10 5 antigens or a combination thereof, but not including CD14, CD34, CD45 and H (3) do not express LA-DR or a combination thereof; and The term refers to cells that have the ability to differentiate into the adipogenic lineage and the adipogenic lineage [1, 2]. In this study, MSCs are CD34 positive when first isolated from tissues (which is natural may lose CD34 expression over time), but are phenotypically and functionally similar to the cells described above. MSCs contain similar cells that express NGF-R, PDGF-R, EGF-R, and IGF- R, CD29, CD49a, CD56, CD63, CD73, CD90, CD105, C D106, CD140b, CD146, CD271, MSCA-1, SSEA4, STR A cell surface marker selected from the group consisting of STRO-1, STRO-3, and combinations thereof. Cells isolated from tissue using a marker (in a specific embodiment, the cells are CD7 3 positive CD90 positive and CD105 positive cells) before or after expansion ternational Society for Cellular Therapy Other cells with mesenchymal-like properties may also be included in the classification of cells that meet the criteria for mesenchymal stem cell transplantation (ISCT). Mesenchymal stem cells are included in the definition of "mesenchymal stem cells," but the cells have at least one of the following: The conditions are: a) regenerative activity; b) production of growth factors; and c) a healing response, either directly or intrinsically. As used herein, "mesenchymal stem cell proliferation" refers to the ability to induce proliferation of cells through induction of endogenous host repair mechanisms. "Mesenchymal stromal cells" or mesenchymal stem cells may be used interchangeably. MSCs can be used to differentiate into any tissue. These tissues include bone marrow [3-7], adipose tissue [8, 9], sheep Water [10, 11], endometrium [12-15], trophoblast-associated tissue

[16] , human chorionic villus trophoblast

[17] , umbilical cord blood

[18] , Wharton's jelly[19-21], umbilical cord tissue

[22] , placenta

[23] , amniotic tissue [24-26], and those derived from pluripotent stem cells [27 -31], peripheral blood, mobilized peripheral blood, umbilical cord blood, skeletal muscle, subepithelial umbilical cord tissue, menstrual blood, fallopian tube tissue, These include, but are not limited to, teeth or combinations thereof.

[0051] Additionally, as used herein, in some circumstances, MSCs may refer to In the field, bone marrow stromal stem cells (BMSSCs)

[32] , adult multipotent stem cells isolated from bone marrow, MIAMI cells [33, 34], and multipotent adult progenitor cells (MAPCs) [35- 38], MultiStem®, Prochymal [39-43], Remes Temcell-L

[44] , mesenchymal progenitor cells (MPC)

[45] , dental pulp stem cells (DPSC)

[0046] , PLX cells

[47] , Ixmyelocel-T

[48] , NurOwn (trademark )

[49] , Stemedyne™-MSC, Stempeucel® [50,51], HiQCell, Hearticellgram-AMI, Revas cor®, Cardiorel®, Cartistem® ), Pneumostem®, Promostem®, Homeo -GH, AC607, PDA001, SB623, CX601, AC607, Endometrial Re These include cells described as endogenous regenerative cells (ERCs) and adipose-derived regenerative stem cells (ADRCs)

[52] . do.

[0052] MSCs can be expanded and used by administration itself or to obtain conditioned medium. The cells may be cultured in a growth medium. Growth medium refers to a medium sufficient for the culture of cells derived from the umbilicus. In particular, one of the media for culturing the cells of the present disclosure is Dulbecco's modified basic In some embodiments, the culture medium comprises a DMEM. The medium was DMEM-low glucose (also abbreviated herein as DMEM-LG). DMEM-low glucose contains 1%, 5%, 10%, 15% or 20% (v / v) glucose. fetal serum; antibiotic / antimycotic solution, e.g., about 100 units / milliliter Penicillin at a suitable concentration; Penicillin at a suitable concentration may be about 100 milligrams per milliliter. Streptomycin; a suitable concentration may be about 0.25 micrograms / milliliter Amphotericin B; 2-mercaptoethanol at a suitable concentration which may be 0.001% (v / v); or a combination thereof.

[0053] Mesenchymal stem cells can be derived from the embryonic mesoderm or from adult bone marrow and, for example, peripheral blood, adipose tissue. Adipose tissue, mobilized peripheral blood, skeletal muscle tissue, endometrial tissue, menstrual blood, fallopian tube tissue, and other They can be isolated from adult tissues. They can differentiate and become, for example, muscle, bone, cartilage, fat, bone marrow tissue. The mesoderm can give rise to any tissue, including cardiac muscle, smooth muscle, or endothelium. They can also differentiate into visceral mesoderm, which can give rise to blood islands composed of hematopoietic progenitor cells and hematopoietic progenitor cells. The differentiation potential of mesenchymal stem cells has been investigated using the best-characterized mesenchymal stem cells (Pitte nger, et al. Science (1999) 284: 143-147 and the United States Patent No. 5,827,740 (SH2+, SH4+, CD29+, CD44+, CD71 , +CD90+, CD106+, CD120a+, CD124+, CD14-, CD34 - and / or CD45- stem cells) (see The present disclosure encompasses the use of a variety of mesenchymal stem cells.

[0054] In certain embodiments, the MSCs are derived from umbilical cord tissue, umbilical cord blood, Wharton's jelly, subepithelial umbilical cord tissue, Or a combination of these. Means for this have been previously disclosed and are incorporated by reference [18,21,53-57]. The term "umbilical tissue-derived cells (UTC)" refers to the cells described in, e.g., U.S. Pat. No. 7,510,873, U.S. Pat. US Patent No. 7,413,734, US Patent No. 7,524,489 and US Patent No. 560,276. UTC refers to cells such as those described in human, laminar The mammalian origin may be any mammalian origin, including rats, primates, pigs, etc. In an embodiment, the UTC is derived from the human umbilicus. Cells derived from the umbilicus include fibroblasts, mesenchymal Short stature compared to human cells such as stem cells or iliac crest bone marrow cells; homeobox 2; heat shock chemokine (CXC motif) ligand 12 (interstitial cell Cell-derived factor 1); Elastin (supravalvular aortic stenosis, Williams-Beuren syndrome); H Omo sapiens mRNA;cDNA DKFZp586M2022 (clone DKFZp586M2022 derived); mesenchymal homeobox 2 (growth arrest specific homeobox sine oculis homeobox homolog 1 (Drosophila); Stalin, alpha B; disheveled-related activator of morphogenesis 2; DKFZP 586B2420 protein; similar to neuralin 1; tetranec src homology 3 (SH3) and cysteine Cholesterol 25-hydroxylase; runt-related transcription factor 3; In IL-11 receptor, alpha; procollagen C-endopeptidase enhancer frizzled homolog 7 (Drosophila); hypothetical gene BC00896 7; collagen, type VIII, alpha 1; tenascin C (hexabrachion xabrachion);iroquois homeobox protein 5;Hephaestus hephaestin; integrin, beta 8; synaptic vesicle glycoprotein 2 ;Neuroblastoma, tumor suppressor 1;Insulin-like growth factor binding protein 2, 36 kDa ; Homo sapiens cDNA FLJ12280fis, clone MAMMA 1001744; Cytokine receptor-like factor 1; Medium / small conductance calcium activation Activated potassium channel, subfamily N, member 4; integrin, beta 7; PD Transcriptional coactivator containing Z-binding motif (TAZ); sine oculis homeo box homolog 2 (Drosophila); KIAA1034 protein; synaptic vesicle Membrane junction protein 5 (myobrevin); EGF-containing fibulin -like extracellular matrix protein 1; early growth response 3; distal-less less) Homeobox 5; Hypothetical protein FLJ20373; Aldo-keto reductase Enzyme family 1, member C3 (3-alpha hydroxysteroid dehydrogenase, type II); biglycan; transcriptional coactivator containing PDZ-binding motifs (TAZ) ;fibronectin 1;proenkephalin;integrin, beta-like 1 (EGF-like repeat Homo sapiens mRNA full-length insert cDNA clone EUROIMAGE1968422; EphA3; KIAA0367 protein; Tritium diuretic peptide receptor C / guanylate cyclase C (atrial natriuretic peptide receptor C) Petidroreceptor C);Hypothetical protein FLJ14054;Homo sapiens mRNA; cDNA DKFZp564B222 (derived from clone DKFZp564B222 BCL2 / adenovirus E1B 19 kDa interacting protein 3-like; AE binding protein 1; and cytochrome c oxidase subunit VIIa polypeptide 1 ( The mice with 10-fold lower expression of genes for one or more of the following:

[0055] Furthermore, these isolated human umbilical cord-derived cells express interleukin-8; reticulocyte-associated Chemokine (CXC motif) ligand 1 (Melonoma growth stimulating activity, Alpha α); chemokine (CXC motif) ligand 6 (granulocyte chemotactic protein 2); mokine (CXC motif) ligand 3; and tumor necrosis factor, alpha-induced tandem express at least one gene for at least one of the proteins 3, and the expression The development of human cells such as fibroblasts, mesenchymal stem cells, iliac crest bone marrow cells or placenta-derived cells. These cells express MCP-1, MIP1 beta, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, RANTES The cells may also secrete factors or proteins, including TIMP1, TIMP2, or combinations thereof. These cells include, for example, TRA1-60, TRA1-81, SSEA3, SSEA 4. may express specific markers, including NANOG, or a combination thereof. These cells are capable of self-renewal and expansion in culture and of differentiating into cells of other phenotypes. It has the potential to do so.

[0056] A method is provided for obtaining cord tissue mesenchymal stem cells from human umbilical tissue. The cells are cultured may be capable of self-renewal and expansion and may have the potential to differentiate into cells of other phenotypes. The method includes: (a) obtaining human umbilical tissue; (b) culturing the tissue to generate a cell that is capable of self-renewal and expansion in culture; 3. Remove substantially all blood so that there is no excess blood present. (c) obtaining umbilical tissue; and (b) treating the tissue by mechanical or enzymatic treatment or both. (d) dissociating the tissue in a culture medium; and (e) culturing the tissue. Human cells capable of self-renewal and expansion and have the potential to differentiate into cells of other phenotypes. The method includes providing growth conditions for growing cells derived from the umbilicus. The growth conditions include RPMI, D This may include culturing in media such as MEM, EMEM or customized media.

[0057] In a specific embodiment, cells may be obtained from the umbilical cord, vaginally delivered or via other means. From any completed pregnancy, full term or sub-full term, whether by caesarean section or other route Tissues may be obtained from tissue banks. Obtaining tissue from a tissue bank is also considered within the scope of this disclosure. The tissue may be rendered substantially free of blood by any means known in the art. For example, blood may be removed before or after most of the blood has been removed, for example by aspiration or drainage. After being applied, it is physically removed by washing, rinsing, dilution, etc. Other means of obtaining tissue substantially free of blood cells include enzymatic treatment or Dissociation of umbilical tissue can be accomplished by a variety of techniques known in the art, such as mechanical disruption. This can be accomplished by any of a number of techniques, for example, tissue is aseptically cut with scissors or a scalpel. Alternatively, such tissues may be prepared by cloning intact or live cells from human tissues. may be chopped, mixed, or ground in any manner suitable for collection; Alternatively, it may be homogenized.

[0058] In a specific embodiment, any isolation procedure for the cells uses an enzymatic digestion process. Many methods are used to isolate individual cells from complex tissue matrices to facilitate their growth in culture. Many enzymes are known in the art to be useful. As discussed above, cells from tissues A wide range of digestive enzymes are available to those skilled in the art for use in the isolation. The digestive enzymes in this study are weakly digestive (e.g., deoxyribonuclease and neutral proteinase). ase) to more digestive ones (e.g., papain and trypsin ), and such enzymes are commercially available. A non-exhaustive list includes mucolytic enzyme activity, metalloproteases, neutral proteases, Serine proteases (e.g., trypsin, chymotrypsin, or elastase) and Potentially useful enzymatic activities include deoxyribonuclease, metalloprotease, and ribonuclease. and the activity is selected from the group consisting of protease activity, neutral protease activity and mucolytic activity. For example, collagenase is known to be useful in the isolation of various cells from tissues. Deoxyribonuclease can digest single-stranded DNA and may cause cell aggregation during isolation. The enzymes may be used alone or in combination. Since proteases can degrade other enzymes, it is preferable to use proteases that do not use other enzymes. The temperature and time for contact with the serine protease are monitored. Serine proteases are involved in the synthesis of alpha-2 microglobulin in serum. The medium used for digestion is preferably serum-free, since the enzyme can be inhibited by EDTA and DNase are commonly used, which may improve yield or efficiency. Some embodiments include, for example, collagenase and dispase, or collagenase The method includes enzymatic treatment with collagenase, dispase and hyaluronidase. The method uses a mixture of dispase, a neutral protease, and dispase in the dissociation step. At least one of the Clostridium histolyticum of collagenase, as well as protease activity, dispase and / or thermolysin Methods using digestion in the presence of either collagenase or dispase can be used. A method using digestion with both enzymatic activities may also be used.

[0059] Digestion with hyaluronidase activity in addition to collagenase and dispase activity Those skilled in the art will recognize that many such enzymatic treatments are available from a variety of tissue sources. It will be appreciated that methods known in the art for isolating cells are known, for example, collagenase. LIBERASE™ BLENDZYM, an enzyme combination of glyceryl esterase and neutral protease The E (Roche) series are very useful and can be used in this method. The sources of such enzymes are also known, and those skilled in the art may obtain such enzymes directly from their natural sources. Those of skill in the art will appreciate that new or additional enzymes or enzyme combinations may be used in the isolation of the cells of the present disclosure. The enzyme treatment was performed at 0.5, 1, and 1. 5 or 2 hours or more. In other embodiments, the tissue is incubated for 1 hour at 4° C. for 1 hour or more. During the enzymatic treatment, the cells are incubated at approximately 37°C. Diluting the digest may also improve cell yields when the enzyme can be used. The use of mechanical separation is preferred but not required for the isolation methods provided herein. A cell-based method can be successful in isolating the cells from the umbilicus as discussed above.

[0060] After the tissue is dissociated into any medium as discussed herein above, the cells are resuspended in The cells may be suspended in a centrifuge, a method used to separate cells from tissue or other debris. The cells may be resuspended after the step. Resuspension may be by mechanical resuspension methods or by simply resuspending the cells in culture medium. By providing growth conditions, the cells can be provided with a culture medium, a nutrient solution, etc. This allows for a wide range of options regarding nutrients, atmospheric conditions and relative humidity. The incubation temperature may be approximately 37° C., but the temperature may vary depending on other culture conditions and the location of the cells or cultures. Depending on the desired application, the temperature may be in the range of about 35°C to about 39°C.

[0061] In certain embodiments, at least in some cases, growth factors are provided in the growth medium. The cells do not require exogenous growth factors, except that these are available in the form of additional serum provided to the cells. A method is used that does not require specific growth factors to expand. Methods for obtaining umbilical cells are also provided herein. These methods are similar to those described above, but The cells are then finally resuspended and grown in a medium that contains their specific growth factors (their In this sense, The method involves targeting cells that are capable of dividing in the absence of those particular growth factors. In some embodiments, the specific cells are selective for serum-free culture. The cells can be grown and expanded in a synthetic growth medium that does not require any synthetic synthesis. Cells require certain growth factors that can be added to the medium to support and sustain them. Factors that can be added to serum-free media for growth include FGF, EGF, It may include one or more of IGF and PDGF. In some embodiments, Two, three or all four of these factors were added to serum-free or chemically defined media. In a specific embodiment, leukemia suppression is used to support or improve cell growth. Factor (LIF) is added to serum-free medium.

[0062] Also provided is a method by which cells can be expanded in the presence of about 5% to about 20% oxygen in their atmosphere. The method for obtaining the cells comprises culturing the cells in the presence of L-valine. After obtaining the cells, the cells' need for L-valine may be altered by the L-isomer. The activity can be tested and confirmed by growing the plasmid p53 on medium lacking D-valine.

[0063] In some embodiments, umbilical cord blood, umbilical cord tissue, Wharton's jelly and / or epithelium. Umbilical cord-derived cells, including cells derived from the lower umbilical cord, require at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or 200 doublings The cells can maintain a normal karyotype throughout passage. 14 Cells or A method is provided for obtaining cells capable of doubling to reach about 1000 ng / ml in culture. ×10 3 ~Approx. 1×10 6 cells / cm 2 When seeded at 1000 x 100, the cells were at least 1 x 10 14 , 1 ×10 15 , 1×10 16 Or 1×10 17 enough to generate 10 or more cells Any method that obtains cells that are capable of sufficient doublings may be used. In some embodiments, the cell number The effect is achieved within 80, 70, or 60 days or less. Umbilicus-derived mesenchymal stem cells were isolated and expanded, and expressed CD9, CD10, CD13, C 29, CD44, CD73, CD90, CD105, CD146, CD141, CD16 6, SSEA4, PDGFr-alpha, HLA-A,B,C, SOX2, OCT4 and and combinations thereof. In addition, these cells express CD14, CD31, CD34, CD45, and CD79. , CD80, CD86, CD106, CD117, CD141, HLA-DR,DP,D One or more markers selected from the group consisting of Q, STRO-1, and combinations thereof. does not produce

[0064] To measure the quality of MSC cultures, flow cytometry was performed on all cultures. were analyzed for surface expression of SH-2, SH-3, and SH-4 MSC markers, as well as CD The cells were incubated in 0.05% T cells and 14% CD45 positive cells. The tissue was peeled off using liposin-EDTA, washed with DPBS + 2% bovine albumin, and then soaked in 1% paraben. After fixation with formaldehyde and blocking with 10% serum, SH-2, SH-3 and S After separate incubation with H-4 primary antibody, PE-conjugated anti-mouse IgG (H +L) incubated with antibody. 2 Confluent M in flask SCs were washed with Tyrode's salt solution and incubated in medium 199 (M199) for 60 min. The cells were then detached using 0.05% trypsin-EDTA (Gibco). Detach the cells one at a time and place the MSCs in 40 mL of M199 + 1% human serum albumin (HSA). A;American Red Cross,Washington DC,USA) MSCs collected from each set of 10 flasks were stored at 4°C for a maximum of 4 hours and then harvested. It was timed to coincide with the end of the collection. Total 2~10'10 6 MSC / kg was reconstituted to M199+1% HSA. The cells were suspended and centrifuged at 460 g for 10 min at 20° C. The cell pellet was diluted with fresh M199 + 1% HA The cells were resuspended in S medium and centrifuged three more times at 460 g for 10 min at 20°C. Total harvest time ranged from 2 to 4 hours based on MSC yield and target dose per flask. The collected MSCs were then incubated with 10% DMSO (Research Industries, Sa (Lake City, UT, USA) and 5% HSA, Cryocyte (Baxter, Deerfield, IL, USA) was incubated in a controlled freezer. The frozen units were stored in freezer bags (USA). On the day of injection, the frozen units were Thaw at bedside in a 7°C water bath, transfer to a 60 mL syringe within 5 min, and infuse 1 Patients were given 325 to 650 mg of acetaminophen intravenously over a period of 0 to 15 minutes. Premedicate orally with aminophen and 12.5 to 25 mg of diphenhydramine Blood pressure, pulse, respiratory rate, temperature and oxygen saturation were measured at the time of injection and for 3 hours thereafter. Monitor every 15 minutes, then every 2 hours for 6 hours.

[0065] In a specific embodiment, the MSCs are derived from bone marrow for use in treating patients. Bone marrow cells are generated according to the protocol previously used in. The cells express LFA-3, ICAM- 1. PECAM-1, P-selectin, L-selectin, CD49b / CD29, CD4 9c / CD29, CD49d / CD29, CD29, CD18, CD61, 6-19, Thrombomodulin, telomerase, CD10, CD13, integrin beta, CD34 , CD56, CD117, or a combination thereof. In some embodiments, the bone marrow-derived MSCs are CD10, CD2, CD3, CD5, CD14, CD16, CD19, C31, CD33, CD45, CD64 and In some embodiments, bone marrow-derived cells may not express DRII. These progenitor cells include, at least in part, STRO-1, T May express KY-1, CD146, STRO-2 and / or VCAM-1 , CBFA-1, type II collagen, PPAR.gamma.2, osteopontin, osteo Calcine, parathyroid hormone receptor, leptin, H-ALBP, aggrecan, Ki6 7, may lack expression of glycophorin A, or a combination thereof.

[0066] Bone marrow was aspirated from the posterior iliac crest to obtain approximately 10–30 mL, which was then collected in a container and placed in a cryostat. The container may be a sodium heparin-containing tube. Good manufacturing practice (GMP) may be used. Individuals may be sedated or not during this procedure. Local anesthesia can be provided by administering bone marrow cells to a patient using, for example, Darbe The cells may be washed with a washing solution such as dilute phosphate buffered saline (DPBS), RPMI or PBS. The cells were plated at approximately 1–2 × 10 7 cells / m The cells can then be layered at a concentration of 100 μL. For approximately 30 minutes or for a period sufficient to separate mononuclear cells from debris and red blood cells. The cells may then be washed with a solution such as PBS and centrifuged at 175 cm 2 Culture in a suitable culture vessel such as a tissue culture flask with 10% FCS in DMEM or similar medium. Approximately 1 × 10 6 The flasks can then be plated at a density of 1000 cells / mL. A minimum of 30 million bone marrow mononuclear cells can be loaded into the 100-well plate. Allow to adhere for 4 hours. MSCs may be allowed to adhere for 72 hours or longer. C can be subjected to medium exchange every 3 to 4 days. Then, 0.05% trypsin-EDTA was used to culture the cells. 2. Harvest the adherent cells using a suitable method such as the one described above, and culture approximately 1×10 6 / 175cm 2 The bone marrow MSCs may be administered intravenously or In certain embodiments, the individual in need of intrathecal administration (at least the neurological disorders associated with radiation) The dosage can be determined by one of skill in the art and can be administered intrathecally to a patient suffering from a cellular degenerative condition, for example. Depending on various characteristics of the individual receiving the vesicles, the average dose is about 10 per kilogram of the individual's measured body weight. Cell numbers ranging from 0 to 10 million MSCs can be administered intravenously. Body measurements are performed at a cell count of approximately 2 to 5 million cells per kilogram of body weight. It is possible.

[0067] In a specific embodiment, the regenerative composition is isolated from peripheral blood, bone marrow or umbilical cord blood. The hematopoietic stem cells include hematopoietic stem cells, which may be CD34+ cells. The hematopoietic stem cells include hematopoietic stem cells that express c-kit, Sca- 1, CD34, CD133 or a combination thereof. The hematopoietic stem cells may lack expression of C38 or other lineage markers. These hematopoietic stem cells can be derived from the blood system of a mammal, including but not limited to rats. Hematopoietic stem cells can be collected by isolating them from the blood or tissue organs of a mammal. Examples of sources include peripheral blood, mobilized peripheral blood, bone marrow, umbilical cord blood, adipose stromal vascular cells, and progenitor cells. Hematopoietic stem cells include those derived from the following: The antibody can be collected from the donor by any known method. For example, see U.S. Patent Publication No. 2013-202366. No. 6,286,213 details procedures for obtaining and purifying stem cells from mammalian cadavers. Stem cells can be obtained from bone marrow collection or peripheral blood stem cell collection, both of which are well known in the art. Stem cells can be harvested from humans by conventional methods (which are well known in the art). The stem cells can be obtained from a source such as human tissue and then cultured using stem cell expansion techniques. "Methods and compositions for r the ex vivo replication of stem cells, for the optimization of hematopoietic pr ogenitor cell cultures,and for increasein g the metabolism,GM-CSF secretion and / or IL-6 secretion of human stromal cells” No. 6,326,198 to Emerson et al. "Selective expansion of targe Kraus et al. U.S. Patent No. 6,338,942; and “Method f or promoting hematopoietic and cell prol The paper is entitled "R No. 6,335,195 to Daggers et al. The entirety of which is incorporated herein by reference. In some embodiments, The resulting stem cells are cultured to expand the population of stem cells. Stem cells harvested from a donor source are not expanded using such techniques. The method can be used to cryopreserve stem cells.

[0068] In some embodiments of the present disclosure, specific types of stem cells, such as pluripotent stem cells, If there are risks associated with the stem cells, the stem cells may be covered with a membrane and capsule prior to implantation. It is contemplated that any of a number of available methods for encapsulating cells may be used. In some embodiments, the cells are individually encapsulated. In embodiments where the cells are removed after implantation, many cells are encapsulated within the same membrane. In the case of endothelial cells, structures with relatively large sizes that encapsulate many cells, such as the inside of a single membrane, are favorable for recovery. In various embodiments for microencapsulating stem cells, A wide variety of materials can be used, including, for example, polymer capsules. , alginate-poly-L-lysine-alginate microcapsules, poly-L-lysine Barium alginate capsule, Barium alginate capsule, Polyacrylonitrile / Poly Polyvinyl chloride (PAN / PVC) hollow fibers and polyethersulfone (PES) hollow fibers For microencapsulation of cells, which may be used when administering stem cells. This technique is known to those skilled in the art and is described, for example, in U.S. Pat. No. 5,639,275 (e.g., We developed a biocompatible capsule for the long-term maintenance of cells stably expressing biologically active molecules. Further methods of encapsulation are described in European Patent Publication No. 301. ,777 and U.S. Pat. Nos. 4,353,888; 4,744,933; ,749,620; Same No. 4,814,274; Same No. 5,084,350; Same No. 5, No. 089,272; No. 5,578,442; No. 5,639,275; and No. All of the above, related to the encapsulation of stem cells, are described in US Pat. No. 5,676,943. The portions are incorporated herein by reference. Certain embodiments include methods for treating stem cells with a biopolymer. or incorporated into polymers, such as synthetic polymers. Examples of biopolymers include fibronectin. tin, fibrin, fibrinogen, thrombin, collagen and proteoglycans Other factors discussed above, such as cytokines, may also be involved. In another embodiment of the present disclosure, stem cells may be incorporated into the interstices of a three-dimensional gel. Larger polymers or gels can be surgically implanted. Small enough particles Polymers or gels that can be formulated as polymers or fibers are another common and more convenient non- It may be administered by surgical route.

[0069] In some embodiments, the regenerative composition comprises at least one skeletal muscle-derived mesenchymal stem cell. The skeletal muscle-derived mesenchymal stem cells express CD13, CD34, CD56, and CD117. or a combination thereof, and may express CD10, CD2, CD5, CD14, CD19 , may lack expression of CD33, CD45 and / or DRII.

[0070] In some embodiments of the present disclosure, the mesenchymal stem cells are cultured in an immature state. and / or allow the generation of a systemically acting regenerative effect. The cells were cultured with a substance that can maintain high expression of genes / mitochondrial cells required for the In some embodiments, the systemic effect is a tissue regenerative composition treated with These substances are targeted to induce regeneration in tissues homologous to the reversin), umbilical cord blood serum, lithium, GSK-3 inhibitors, resveratrol, Pterostilbene, selenium, selenium-containing compounds, EGCG ((-)-epigallocatechin-3-gallate), valproic acid and valproates, especially valproic acid In a specific embodiment of the present disclosure, the compound is selected from the group consisting of 0 Levelsin at a concentration of 0.5 to 10 μM, preferably 1 μM, is added to the mesenchymal stem cell culture. In a specific embodiment of the present disclosure, resveratrol is added at a concentration of 10 to 100 μM, preferably Preferably, it is envisaged to use 50 μM. The present disclosure relates to selenium or selenium-containing compounds. The present invention provides a method for using the substance at a concentration of 0.05 to 0.5 μM, preferably 0.1 μM. In a specific embodiment, the umbilical cord blood serum is present at a concentration of 0.1% to 20% by volume of the tissue culture medium. In some embodiments, the present disclosure provides a method for the preparation of a composition comprising administering EGCG or a combination thereof to a subject in need thereof. It is expected to be used at a concentration of 0.001 to 0.1 μM, preferably about 0.01 μM. In certain embodiments, the present disclosure provides a method for treating valproic acid or sodium valproate. It is envisaged that concentrations of 1 to 10 μM will be used, with 5 μM being a particular embodiment. In an embodiment, mesenchymal stem cells are dedifferentiated to have higher expression of regenerative genes. Dedifferentiation is achieved by using stem cells with a high level of immaturity, such as cytoplasmic transplantation and cytoplasmic transfer. The method can be achieved by cell transfer or cell fusion, and these stem cells include pluripotent stem cells. In such a culture / co-culture procedure, the cell culture medium is active protein degradation inhibitors, optionally in combination with one or more of the substances specified above. The use of at least one protein degradation inhibitor in the cell culture medium of the present disclosure By combining the mRNA or any endogenous gene, reprogrammed proteins are produced in cells. The longer the cells remain in the cells, the greater the chance of reprogramming by transfected mRNA. Ramification is promoted in a much improved manner.

[0071] In some embodiments of the present disclosure, the regenerative composition comprises at least one pluripotent stem cell. The pluripotent stem cells are stem cells, parthenogenetic stem cells, induced pluripotent stem cells, somatic Stem cells derived from cell nuclear transfer, stem cells derived from cytoplasmic transfer, stimulation-induced acquisition of pluripotency or their The cells may be of any origin or type, including any combination of the above. Any method for this purpose may be used.

[0072] V. Inhibitors

[0073] The present disclosure, in certain embodiments, uses one or more inhibitors to inhibit, e.g., regenerative activity Generate cells with the ability to induce a regenerative abscobal effect or use the cells to Examples of inhibitors include protein degradation inhibitors (transient or (other than those listed above), protease inhibitors, proteasome inhibitors and / or lysosomal inhibitors In one embodiment, the proteosome inhibitor is MG132, Selected from the group consisting of TMC-95A, TS-341 and MG262. In the form, protease inhibitors include aprotinin, G-64, leupeptin-hemisulfuric acid, In a specific embodiment, the compound is selected from the group consisting of aryl, aryl sulfate, aryl phosphate, and combinations thereof. In this case, the lysosomal inhibitor is ammonium chloride. The present invention also includes a cell culture medium containing at least one transient inhibitor of mRNA degradation. Use similarly extends the half-life of the reprogramming factors. To allow the transfected reprogramming mRNA molecule to be translated in the cell. Suitable conditions allow for oxygen content in cell culture media between 0.5 and 21%. More details Without wishing to be bound by theory, it is believed that the use of oxygen inhibits HIF- Triggering Oct4 via 1α may lead to further induction of Oct4 or increases, and in these situations, oxygen concentrations lower than those found in the atmosphere are used, which is 0. In a specific embodiment, the condition is to regulate the mRNA expression level in the cell. The molecules may be suitable for and selected to aid in the reprogramming of cells; more particularly, These conditions are 30 to 38°C, preferably 31 to 37°C, and most preferably 32 to 36°C. The glucose content of the medium must be less than 4.6 g / L and less than 4.5 g / L. less than 4g / L, less than 3g / L, less than 2g / L, or approximately 1g / L or approximately 1 DMEM medium containing 1 g / L glucose is referred to as "DMEM low glucose Courses" include PAA, Omega Scientific, Perbio and Bi These compounds are commercially available from companies such as Osera. Although it is not our intention to do so, high glucose conditions may be detrimental to cellular senescence in vitro. support (e.g., by methylation or epigenetic changes) and thereby In a specific embodiment of the present disclosure, the cell culture medium glucose 0.1g / L~4.6g / L, 0.5g / L~4.5g / L or 1g / L~4g / L.

[0074] In some embodiments of the present disclosure, the enhanced abscopal effect is due to the increased efficacy of the regenerative composition. or endogenous stem cells secrete regenerative compositions systemically from distant sites By administering one or more epigenetic modifiers that enhance the ability to respond to signals. In certain embodiments, the regenerative composition used is a composition that induces epigenetic changes. The epigenetic acting composition may be enhanced by the regenerative composition. The epithelium may be delivered at the same time as, or prior to, or after, administration of the regenerative composition. The genetically active composition may be administered to the same or different anatomical site as the regenerative composition (regenerated tissue). The therapeutic agent may be administered to any part of the body, potentially including the site of injury.

[0075] Examples of epigenetic modifiers include epigenetic modifiers, e.g., D NA demethylating agents, histone deacetylase (HDAC) inhibitors, histone modifiers, and cell cycle manipulation agents and pluripotency or tissue specificity promoting agents, e.g., pluripotent cell Promotes cell growth or the production of pluripotent cells, growth factors, hormones and bioactive molecules Examples of DNA demethylating agents include 5-azacytidine (5- aza), N-methyl-N'-nitro-N-nitrosoguanidine, temozolomide, procaine Examples of methylation inhibitors include, but are not limited to, demethylation inhibitors. Citabine, 5-azacytidine, hydralazine, procainamide, mitoxantrone, zeaxanthrone Branidine, 5-fluorodeoxycytidine, 5-fluorocytidine, DNA methyltransferase Antisense oligonucleotides against spherase or DNA methylation-related Other inhibitors of the enzyme include

[0076] Examples of HDAC inhibitors include hydroxamic acids, cyclic peptides, benzamides, and short-chain lipids. These include, but are not limited to, fatty acids and depudecin. Examples of hydroxamic acids and derivatives of hydroxamic acids include trichostatin A (TSA ), Suberoylanilide hydroxamic acid (SAHA), Oxamflatin (oxamfl atin), Suberic acid bishydroxamic acid (SBHA), m-carboxycinnamic acid bis Hydroxamic acid hydrochloride (CBHA) and pyroxamide Examples of cyclic peptides include, but are not limited to, trapoxin A, apicidin (a picidin) and FR901228. Examples of short chain amides include, but are not limited to, MS-27-275. Examples of fatty acids include butyrates (e.g., butyric acid, phenylbutyrate (PB) and C I-994 (acetyldinaline) is one example. Examples of histone modifiers include, but are not limited to, PARP, human endonucleases of zeste, These include, but are not limited to, hancer, valproic acid and trichostatin A. In some embodiments, RNA transfection and target cell culture containing the RNA Specific compounds used in cell culture media to facilitate dedifferentiation of cells into pluripotent cells. Modifiers include trichostatin A, valproic acid, zebularine and / or 5-aza. The target cells into which the RNA is introduced are then incubated in a RT-PCR flask until dedifferentiated (pluripotent) cells are obtained. The cells are cultured in the medium for a period of time sufficient to promote NA transfection. In some embodiments, the method includes altering the epigenetic integrity of a recipient or target cell. Other methods and procedures involved in controlling or altering the chronic state (e.g., DNA demethylating agents and and / or histone demethylating agents, histone deacetylase inhibitors and / or may be combined with exposure to histone modifiers. Thus, the present disclosure provides several Methods for altering cell fate or phenotype in morphology are described. By using the modified composition, the disclosed method can dedifferentiate or Can transdifferentiate.

[0077] VI. Exosomes

[0078] Exosomes, also called "particles," contain vesicles or flattened spheres that contain a lipid bilayer. The particles are 2-200 nm, 5-200 nm, 10-200 nm, and 15-2 00nm, 20~200nm, 30~200nm, 40~200nm, 50~200nm , 60~200nm, 70~200nm, 80~200nm, 90~200nm, 100 ~200nm, 110~200nm, 120~200nm, 130~200nm, 140 ~200nm, 150~200nm, 160~200nm, 170~200nm, 180 ~200nm, 190~200nm, 2~150nm, 5~150nm, 10~150n m, 15~150nm, 20~150nm, 30~150nm, 40~150nm, 50 ~150nm, 60~150nm, 70~150nm, 80~150nm, 90~150 nm, 100~150nm, 110~150nm, 120~150nm, 130~150 nm, 140~150nm, 2~100nm, 5~100nm, 10~100nm, 15 ~100nm, 20~100nm, 30~100nm, 40~100nm, 50~100 nm, 60~100nm, 70~100nm, 80~100nm, 90~100nm, 2 ~50nm, 5~50nm, 10~50nm, 15~50nm, 20~50nm, 30~ 50 nm, 40-50 nm, or any range therebetween. Any method that measures exosome size, including light scattering or nanoparticle tracking analysis, can be used. These particles are formed by inward budding of the endosomal membrane. The particles may have a density of about 1.13 to 1.19 g / mL. These particles are composed of cholesterol and sucrose. Gommyelin, CD9, CD63, CD81, ANXA2, ENO1, HSP90AA1, EEF1A1, YWHAE, SDCBP, PDCD6IP, ALB, YWHAZ, EEF 2, ACTG1, LDHA, HSP90AB1, ALDOA, MSN, ANXA5, PG Markers such as K1 and CFL1, and lipid raftomas such as GM1, GM3, and flotillin These particles may be enriched in the lysosome, phospholipase A, and the src protein kinase Lyn. lipids, phosphatidylserine, phosphatidylinositol, phosphatidylcholine, Sphingomyelin, ceramide, glycolipids, cerebrosides, steroids, cholesterol and and combinations thereof. These particles may contain at least one lipid raft. or any cell of the present disclosure, or mesenchymal stem cell conditioned medium (MSC-CM), or One or more proteins present in any of the disclosed media (e.g., MSC or MSC-CM) They may include RNA, e.g., miRs, proteins that are characteristic or specific for a particular gene. The particles may comprise MSCs, or any of the cells disclosed herein, or a culture of MSCs. or one or more genes found in any medium of the present disclosure. The particles may carry a gene product or a gene product secreted by the MSC or any cell of the disclosure. Combinations of such particles and any molecules contained therein may include molecules that have been (In particular, proteins or polypeptides) can be used to treat or prevent disease, for example. For the purpose of detecting MSCs or any cell of the present disclosure, These may be used to supplement or replace the activity of media conditioned by The particles bind to cytosolic proteins found in the cytoskeleton, such as tubulin, actin, and the like. annexins and actin-binding proteins, intracellular membrane fusion and transport, e.g., annexins and rab proteins, signaling proteins, e.g., protein kinases, 14-3- 3 and heterotrimeric G proteins, metabolic enzymes, e.g., peroxidase, pyruvate Kinase and lipid kinases, as well as enolase-1 and the tetraspanin family For example, CD9, CD63, CD81 and CD82. In the present invention, the particles may contain one or more tetraspanins. and / or microRNA. The particle may comprise MSCs or any of the molecules of the present disclosure. For any therapeutic purpose for which the cells, or MSC-CM, or any medium of the present disclosure may be used. It can be used.

[0079] In certain embodiments, the exosomes, i.e., particles, are derived from mesenchymal stem cells or fibroblasts. blast cells, or any of the cells of the present disclosure (including regenerative cells) The cells may be derived from foreskin, adipose tissue, placenta, ear lobe, omentum, Wharton's jelly or other tissue. The mesenchymal stem cells may be derived from any tissue origin, including combinations of these. The cells are selected from the group consisting of CD90, CD73, CD105 and combinations thereof. The culture medium may include human umbilical tissue-derived cells having a marker that is consistent with the present invention. The culture medium may include DMEM. The DMEM may be made phenol red free. phospholipid, transferrin or selenoprotein (ITS) or any combination thereof The medium may be supplemented with FGF2. The medium may be supplemented with PDGF AB. The concentration of FGF2 can be about 5 ng / mL FGF2. The concentration of PDGF AB can be about The medium may also contain glutamine-penicillin-streptomycin. or β-mercaptoethanol, or any combination thereof. Incubate for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more, e.g., 3 days. It can be done.

[0080] Conditioned medium containing exosomes can be obtained by separating cells from the medium. The conditioned medium can be centrifuged, for example at 500 g. It can also be concentrated by filtering through a membrane. The membrane may comprise a >1000 kDa membrane. The conditioned medium may be shrunk by about 50 fold or less. The conditioned medium may be subjected to liquid chromatography, such as HPLC. The conditioned medium can be separated by size exclusion. Any size exclusion medium, such as Sepharose, can be used. A matrix may be used. As an example, the TSK Guard column SWXL, 6×4 0 mm or TSK gel G4000SWXL, 7.8 x 300 mm can be used. The buffer may include any physiological medium, such as saline. It may contain 150 mM NaCl The chromatography system may include a 20 mM phosphate buffer having a pH of 7.2. Equilibration may be performed at a flow rate of 0.5 mL / min. The elution mode may be isocratic. The progress of elution may be followed using UV absorbance at 100 nm. Quasi-elastic light scattering (QELS) detection Fractions may be examined for dynamic light scattering (DLS) using a chromatograph. The fraction that is generated by the general method as described above can be retained. The fractions eluted at a retention time of 11 to 13 minutes, such as 12 minutes, were analyzed by dynamic light scattering. The particle r at this peak is found to be h is about 45 to 55 nm. Such fractions may include mesenchymal stem cell particles, such as exosomes.

[0081] In some embodiments of the present disclosure, the systemically acting regenerative effect is achieved by the use of regenerative cell-derived These regenerative cells are generated by administering a cell lysate of, for example, mesenchymal stem cells. In some embodiments, the cells may be mesenchymal stem cells, or any of the cells of the present disclosure. is derived from the umbilical cord but may be of any tissue type. Umbilical Cord / Wharton's Cells for Clinical Applications The induction of mesenchymal stem cells from jelly has been reported in the art and is incorporated by reference. In some embodiments of the present disclosure, xeno-free medium is used to mesenchymal stem cells or any of the cells of the present disclosure are grown and sensitized with components such as fetal bovine serum. In some embodiments of the present disclosure, In the present invention, the mesenchymal stem cells are pretreated using a method for enhancing regenerative activity, the method including: Histone deacetylase inhibitors such as valproic acid and GSK-3 inhibitors such as lithium These include treatment with acetic acid [74-79], incubation under hypoxia, and treatment with carbon monoxide

[80] . In some embodiments, fibroblasts are utilized as an alternative to stem cell administration. can be.

[0082] In some embodiments, for example, mesenchymal stem cells are cultured in the presence of aphidicolin. to arrest them in S phase, and the cells are then Repeat centrifugation and resuspension in phosphate-buffered saline (PBS) three times as described. The cells can then be synchronized in G2 by washing the cells. For example, the doubling time may be approximately 24 hours. Cells with chromatin deficiency can be allowed to enter the G2 phase by incubating for 6 to 12 hours. For cells with shorter or longer doubling times, the appropriate In some embodiments of the present disclosure, the mesenchymal stem cells are cultured in a culture medium containing ... The cells were incubated in 0.5 μg / mL nocodazole for 17–20 h. The mitotic cells can be synchronized in mitosis by vigorous shaking. The detached G1 doublets can be discarded or the detached cells can be The exfoliated cells collected can be left behind with the majority (>80%) of mitotic cells. The cells are then centrifuged at 500 g for 10 minutes at 4°C in a 10 mL conical tube. Synchronized or unsynchronized cells can be harvested by standard methods and placed in a 10 mL Conica The cells can be washed by centrifugation at 500 g for 10 minutes at 4° C. in a 100% aqueous solution. Discard the supernatant and resuspend the cell pellet in a total volume of 50 mL of cold PBS. Keep the cells at 4 °C. Centrifuge at 500g for 10 minutes. Repeat this washing step to separate the cell pellet into approximately 20 volumes. of ice-cold interphase cell lysis buffer (20 mM Hepes, pH 8.2, 5 mM MgCl 2. 1 mM DTT, 10 pM aprotinin, 10 pM leupeptin, 10 pM pepstatin 10 pM trypsin A, 10 pM soybean trypsin inhibitor, 100 pM PMSF and 2 The cells were then resuspended in 100 μg / mL cytochalasin B (10 μg / mL cytochalasin B). Discard the supernatant and aliquot the cell pellet in 1 volume or less. Carefully resuspend the interphase cells in lysis buffer. Incubate the cells on ice for 1 hour. The cells are then sonicated using a tip sonicator. Dissolve the mixture by pulverization or Dounce homogenization using a glass mortar and pestle. Cell lysis is carried out until at least 90% of the cells and nuclei are lysed. Lysis of at least 90% of the cells and nuclei can be assessed using phase contrast microscopy. The duration and power of sonication required to obtain a desired extract depends on the type of microorganisms used to prepare the extract. This may vary depending on the type of cell.

[0083] In some embodiments, the cell lysate is placed in a 1.5 mL centrifuge tube and centrifugeed using a tabletop centrifuge. Centrifuge the tube at 10,000-15,000 g for 15 minutes at 4°C. Remove from the centrifuge and immediately place on ice. Remove the supernatant using a 200 μL pipette tip. Carefully collect and pool the supernatant from several tubes and place on ice. This cell extract was diluted to a volume of 20 pL per tube. Aliquot on ice, immediately flash-frozen in liquid nitrogen and can be stored at -80°C until use. Alternatively, place the cell extract in an ultracentrifuge tube on ice (e.g., SW55 TiLo). (Appropriate for Beckman). If necessary, add mineral oil to the top of the tube. The extract was centrifuged at 200,000 g for 3 hours at 4°C to determine the amount of cytoplasmic At the end of the centrifugation, discard the oil. Carefully collect the supernatant and discard. Pool if necessary and place in a cold 1.5 mL tube on ice.

[0084] In certain embodiments, the mesenchymal stem cell or any cell of the present disclosure, the lysate comprises a cell Generated by rinsing 3-4 times with PBS and then alpha-MEM or DMEM / F1 Culture medium such as 2 (Gibco) is added without additives or serum. 12-24 hours Afterwards, the cells were washed twice with PBS and collected by scraping with a rubber policeman. The mixture was then collected in 100 mL Falcon tubes (Becton Dickinson). The cells were washed and lysed in ice-cold cell lysis buffer (20 mM HEPES, pH 8.2, 50 mM M NaCl, 5 mM MgCl.sub.2, 1 mM dithiothreitol and protein Resuspend in 1 volume of lysis buffer (ATPase inhibitor cocktail), pellet at 400 g, and resuspend in 1 volume of cell lysis buffer. Resuspend until all cells and nuclei are lysed (as determined by phase contrast microscopy). The cells were then sonicated in 200 μL aliquots using a 2 mm diameter probe. Sonicate the lysate on ice using a sonicator. Sonicate the lysate at 10,000 to 14,000 x g for 30 min at 4°C. Centrifuge at 1000g for 15-30 minutes to remove crude material and any soluble material that may have remained. Pellet undissociated cells. Aliquot the supernatant and freeze and store in liquid nitrogen or Protein concentrations of the extracts were determined by Bradford assay. Analyze and adjust pH to approximately 7.0 + / - 0.4 before use and dilute with water if necessary. Adjust the osmolality to -300 mOsm by using a method such as

[0085] In addition to cell lysates, conditioned medium of the cells may also be used. , may be administered intranasally, via aerosolized means, or orally, intravenously, subcutaneously, The compound may be administered intravenously, rectally, intramuscularly, intraarticularly or sublingually.

[0086] Conditioned media can be generated to enrich for secreted factors or as a source of exosomes. In some embodiments, the exosomes are purified by ultracentrifugation. , using chromatography or based on adhesion to a substrate.

[0087] VI. Fibroblasts

[0088] Embodiments of the present disclosure include fibroblasts derived from autologous, allogeneic, syngeneic and / or xenogeneic sources. The disclosed methods and compositions are useful in treating inflammatory conditions. The present invention relates to a method for treating cancer, autoimmune conditions, or other degenerative conditions, comprising administering to the patient a therapeutically effective amount of certain engineered cells. In particular, the cells include at least fibroblasts of any kind. A means of manipulating blastocytes, as well as actively inhibiting degenerative processes, lines of different tissue origin In one embodiment of the present disclosure, fibroblasts capable of inhibiting immune responses are disclosed. fibroblasts to harness the blastogenic potential and to prevent and / or treat degenerative conditions; In at least one embodiment, the present invention uses fibroblasts as a cell therapy for treatment. by treating fibroblasts with one or more specific agents and / or conditions. In certain embodiments, the degenerative process can be treated directly or indirectly. The agents include interferon gamma and / or platelet-rich plasma, and some In some cases, at least interferon gamma and / or platelet-rich plasma (and / or platelet-rich lysate) demonstrated that fibroblasts actively suppress immune responses directly or indirectly. Fibroblasts cultured under these conditions may confer the ability to inhibit autoimmune disorders or The compound is administered to an individual suffering from or at risk of an inflammatory or other degenerative disorder. The route of administration, dosage and frequency will depend on the disease process and stage of the disease. may be determined and optimized according to usual practice in medicine.

[0089] In some embodiments of the present disclosure, the regenerative composition comprises at least one fibroblast. The fibroblasts may be of any origin, for example, foreskin, fat, placenta, ear lobe, from any tissue type, including omentum, Wharton's jelly, or a combination thereof. Methods for isolating fibroblasts are well known in the art, and any such method may include: In certain embodiments, fibroblasts can be obtained by, for example, , NANOG, OCT-4, SSEA-4, CD90, CD105, CD73, stem cell factors express at least one marker, which may be a marker, a receptor, or a combination thereof The fibroblasts may be cultured before being used in the method for regeneration. Any suitable method and medium for culturing fibroblasts, including methods and media. Nutrient solutions may be used. In certain embodiments, fibroblasts produce exosomes. The exosomes may be used to generate exosomes for use in certain methods disclosed herein. In a specific embodiment, the fibroblasts are used as a live composition. and the apoptotic vesicles can be used as a regenerative composition. In a specific embodiment, fibroblasts are cultured to obtain or induce miRNA. and the miRNA derived or obtained from the fibroblasts may be used in a regenerative composition. It can be used as:

[0090] In certain embodiments, fibroblasts (whether allogeneic, autologous, or xenogeneic) In a non-manipulated manner (e.g., interferon gamma, etc.), The antibody is administered to an individual without prior exposure to any one or more specific agents, but has no immunomodulatory activity. Gender-specific natural sources (e.g. placental fibroblasts or adipose tissue-associated fibroblasts) In another embodiment of the present disclosure, the immature phenotype of any fibroblast is selected from the group consisting of To achieve this, the fibroblasts are cultured under conditions that can induce retrodifferentiation, Here, the immature phenotype correlates with high anti-inflammatory and / or immunomodulatory potential. For example, fibroblasts have been shown to be resistant to valproic acid (Moon et al., 2008; Huang et al., 2010). In the presence of one or more histone deacetylase inhibitors, such as In addition to HDAC inhibitors, other methods for inducing dedifferentiation of fibroblasts have been stage, e.g., 8-Br-cAMP (Wang et al., 2011); M-CSF treatment treatment (Li et al., 2016); exposure to levolecin (Li et al., 20 16); and / or exposure to stem cell extracts (Xiong et al., 2014) may also be used in the context of the present disclosure. , extracellular markers such as VEGFR-2, CD34 and / or CD133, and To assess intracellular markers such as SOX-2, NANOG and / or OCT-4 This can be done by

[0091] In certain embodiments, dedifferentiated fibroblasts are used. Such fibroblasts include: The cells are induced to dedifferentiate, and the dedifferentiated cells are then engineered to produce certain factors. In a specific embodiment, the induction of dedifferentiation of fibroblasts is achieved by dedifferentiating fibroblasts from By culturing with the cytoplasm of cells with a more undifferentiated phenotype compared to fibroblasts, In some cases, dedifferentiation of fibroblasts is achieved by converting fibroblasts into more This is done by culturing cells with a more undifferentiated phenotype. The typed cells can be any kind of stem cell, such as a pluripotent stem cell.

[0092] In some embodiments of the present disclosure, dedifferentiated fibroblasts are useful in the disclosed methods. The cells may be used to express telomerase, NANOG, Sox2, beta-III-thiol, UBLIN, NF-M, MAP2, APP, GLUT, NCAM, NeuroD, Nur r1, GFAP, NG2, Olig1, alkaline phosphatase, vimentin, osteo Nectin, osteoprotegerin, osterix, adipsin, erythropoietin , SM22-alpha, HGF, c-MET, alpha-1-anthryltrypsin, Luroplasmin, AFP, PEPCK1, BDNF, NT-4 / 5, TrkA, BMP2 , BMP4, FGF2, FGF4, PDGF, PGF, TGF alpha, TGF beta, expressing one or more markers selected from the group consisting of VEGF and combinations thereof do.

[0093] In certain embodiments, the undifferentiated cells (and / or the cytoplasm of the undifferentiated cells) Fibroblast cultures were treated with: a) valproic acid; b) trichostatin A; c) phenylbutyrate. ;d) vorinostat;e) belinostat;f) LAQ824;g) panobinostat ;h) entinostat;i) CI994;j) mocetinostat;k) sulforaphane and l) a histone deacetylase inhibitor selected from the group consisting of: The method is carried out under conditions including the presence of one or more histone deacetylase inhibitors, such as a histone cytotoxic agent. In a specific case, the undifferentiated cells (and / or the cytoplasm of the undifferentiated cells) are Exposure to these histone deacetylase inhibitors resulted in dedifferentiated fibroblasts. The individual's ability to regenerate one or more discs is enhanced.

[0094] In certain embodiments, the undifferentiated cells (and / or the cytoplasm of the undifferentiated cells) Fibroblasts were cultured under conditions that included the presence of one or more DNA methyltransferase inhibitors. The DNA methyltransferase inhibitors are: a) decitabine ;b) 5-azacytidine;c) Zebularine;d) RG-108;e) procaine hydrochloride; f) procainamide hydrochloride; g) hydralazine hydrochloride; h) epigallocatechin gallate i) chlorogenic acid; j) caffeic acid; and h) combinations thereof. In a specific case, undifferentiated cells (and / or the cytoplasm of undifferentiated cells) Dedifferentiated cells were then exposed to one or more DNA methyltransferase inhibitors. The ability of fibroblasts to regenerate one or more of the individual's discs is increased.

[0095] In certain cases, a medium that allows the growth of dedifferentiated fibroblasts may be used to differentiate dedifferentiated fibroblasts. Contains one or more factors known to be mitogenic for fibroblasts, For example, a) FGF-1; b) FGF-2; c) FGF-5; d) EGF; e) CN TF;f)KGF-1;g)PDGF;h)Platelet-rich plasma;i)TGF-alpha;j) and k) one or more factors selected from the group consisting of HGF-1; and combinations thereof. In a specific case, undifferentiated cells (and / or the cytoplasm of undifferentiated cells) are dedifferentiated. One or more compounds known to be mitogenic for cultured fibroblasts By exposure to the above factors, the dedifferentiated fibroblasts can repopulate one or more of the individual's discs. Increased ability to survive.

[0096] In a specific embodiment, the dedifferentiated fibroblasts are cultured to obtain the conditioned medium. In the case of , dedifferentiated fibroblasts are cultured, exosomes are generated from the dedifferentiated cells, Obtain exosomes from the conditioned medium. In certain cases, exosomes are derived from dedifferentiated cells. Exosomes are harvested from fibroblasts while they are in a proliferative state. The transformed fibroblasts were grown in a medium that did not contain growth factors or had significantly lower levels of proliferation-inducing factors. It can be recovered from the dedifferentiated fibroblasts while they are cultured in a medium containing growth factors. Exosomes grow in a medium containing a certain level of oxygen for a certain period of time. For example, exosomes can be collected from dedifferentiated fibroblasts cultured in a culture medium such as 2%~8%, 2%~7%, 2%~6%, 2%~5%, 2%~4%, 2%~3%, 3% ~8%, 3%~7%, 3%~6%, 3%~5%, 3%~4%, 4%~8%, 4%~7%, 4%~6%, 4%~5%, 5%~8%, 5%~7%, 5%~6%, 6%~8%, 6%~7 Fibroblasts were cultured in a medium containing 7% or 8% oxygen. Exosomes are obtained by culturing dedifferentiated fibroblasts in culture for a certain period of time. The cells may be harvested for a period of time that may or may not include the oxygen levels described above. Exosomes may not be present. Dedifferentiated fibroblasts cultured in medium for 0, 11, 12, 13, 14, or 15 days The cells can be collected from, for example, 1-15, 1-14, 1-13, 1-12, 1-11, , 1~10, 1~9, 1~8, 1~7, 1~6, 1~5, 1~4, 1~3, 1~2, 2~ 15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2~6, 2~5, 2~4, 2~3, 3~15, 3~14, 3~13, 3~12, 3~11 , 3~10, 3~9, 3~8, 3~7, 3~6, ​​3~5, 3~4, 4~15, 4~14, 4~13, 4~12, 4~11, 4~10, 4~9, 4~8, 4~7, 4~6, 4~5, 5~15, 5~14, 5~13, 5~12, 5~11, 5~10, 5~9, 5~8, 5~ 7, 5~6, 6~15, 6~14, 6~13, 6~12, 6~11, 6~10, 6~9, 6~8, 6~7, 7~15, 7~14, 7~13, 7~12, 7~11, 7~10, 7~ 9, 7~8, 8~15, 8~14, 8~13, 8~12, 8~11, 8~10, 8~9, 9~15, 9~14, 9~13, 9~12, 9~11, 9~10, 10~15, 10~1 4, 10~13, 10~12, 10~11, 11~15, 11~14, 11~13, 11 ~12, 12~15, 12~14, 12~13, 13~15, 13~14 or 14~1 The cells may be cultured for 5 days.

[0097] In one embodiment of the present disclosure, the fibroblasts used for immunomodulation are, e.g. and / or b) expressing one or more immune modulating proteins. These engineered cells are then used to induce immune tolerance. At least in some cases, individual and disease characteristics will determine which genes are linear. In these cases, the autoantigen may be a polynucleotide. The fibroblasts can be transfected with the peptide in the form of a nucleotide, which can regulate immune function. They are cultured to regulate immune responses or transfected with genes that allow immune regulation. Genes of particular interest for transfection to induce immune regulation include These include at least the following: Fas ligand, TGF-beta, IL-4, IL- 10. HLA-G, indoleamine 2,3 deoxygenase, galectin family members members, galectin 3, arginase and / or IL-20 (de Jesus e t al.,2016;Wang et al.,2011;Zhao et al., 2010;Min et al.,2001;Cancedda et al.,200 1) Any of the genes described herein or active portions thereof may be expressed in a CMV promoter. romote (Artuc et al., Exp. Dermatol. 1995, 4:3 17-21) containing a promoter sequence that allows expression in fibroblasts of mammalian Examples of suitable constructs include pcDNA3, pc DNA3.1(+ / -), pGL3, PzeoSV2(+ / -), pDisplay, p EF / myc / cyto, pCMV / myc / cyto (each of which is similar to Inv (commercially available from suppliers such as Serogen) or in human foreskin cells. The pSH expression vector (Ventura avenae) allows for the controlled expression of polynucleotides. nd Villa,1993,Biochem.Biophys.Commun.192 :867-9), but are not limited to these. Retrotransfection allows cloning and transgenes are transcribed from the CMV promoter. Retroviral vectors and packages, including the -X vectors pLNCX and pLXSN An example of a gestation system is Clontech, San Diego, Calif., US Vectors derived from Mo-MuLV such as pBabe are commercially available from A. Vectors are also included, in which the transgene is transcribed from the 5'LTR promoter. After transfection of the smid is complete, allow detachment from the tissue culture plate. Fibroblasts are harvested by any suitable means, such as trypsinization, and placed in a suitable container or medium for growth. Approximately 3 days after transfection, the cell culture medium is transferred to the modified fibroblast The medium is then replaced with one suitable for the proliferation and expansion of the cells. One example is Neurobasal-A (Inv iRogen), 1% D-glucose (Sigma Aldrich), 1% penicillin 2 containing retinoic acid / streptomycin / glutamine (Invitrogen) % B27 supplement (Invitrogen), 0.2% EGF (Peprotech ,USA), 0.08% FGF-2 (Peprotech), 0.2% heparin (Sig Aldrich, USA) and valproic acid (Sigma A) at concentrations up to 1 μM. The medium is Neurobasal A (NBA) growth medium containing ldrich. The medium was changed, for example, three times a week, and reprogrammed until extensive colony formation was achieved. Cells should be periodically re-plated (e.g., Re-plate up to 2-8 times, once a week, and gradually increase the number of times as colonies begin to emerge. Various quality control measures are in place for practitioners of the present disclosure to administer cells clinically. Examples of criteria for cell qualification include those known in the art, such as flow cytometry. By which means, marker identification, viability, endotoxin content, and microbial and Evaluation of the presence of mycoplasma contamination is one example.

[0098] In certain embodiments of the present disclosure, the fibroblasts are cultured to enhance the viability and proliferation of the fibroblasts. The cells are cultured ex vivo using means known in the art for preserving the cells. The present invention provides a modification of known culture procedures to reduce recognition of fibroblasts by the patient's immune system. In one embodiment, the fibroblasts are supplemented with a xenogeneic component, such as fetal bovine serum. The xenogeneic components are cultured in conditions lacking the ability to induce antibody and T cell responses. It is known that inflammatory bowel disease triggers a number of immunological responses (Selvaggi et al. .,1997;Mackensen et al.,2000;Kadri et al. .,2007;Forni et al.,1976;Lauer et al.,19 83) Many individuals have autoimmune responses to IgM isotypes related to fetal bovine serum. Since natural antibodies exist (Irie et al., 1974), in the presence of fetal bovine serum After administration of the grown cells, rejection, inflammation or anaphylaxis may occur ( Macy et al., 1989). In a specific embodiment, the present disclosure relates to fibroblasts. Further features to reduce the immunogenicity of the cells include human platelet-rich plasma, platelet lysates, and , cord blood serum, autologous serum and / or fetal bovine serum with a defined cytokine mixture. The procedure for culturing tissue in xeno-free medium includes substitution of the tissue of interest for other cell types. This technique is known in the art and is incorporated by reference (Riordan et al., 2015).

[0099] In one embodiment of the present disclosure, fibroblasts are injected (e.g., in hypoxic regions) or otherwise The compound may be administered to the subject by any suitable route, including, for example, intramuscular injection. Possible routes of administration include intravenous, subcutaneous, intrathecal, oral, rectal, intrathecal, and intraretinal. These include intramental, intraventricular (inside the heart), liver, and kidney.

[0100] In certain embodiments, fibroblasts are derived from cells of the skin, heart, blood vessels, bone marrow, skeletal muscle, liver, etc. The cells may be derived from tissues including the pancreas, brain, adipose tissue, foreskin, placenta and / or umbilical cord. In certain embodiments, the fibroblasts are placental, fetal, neonatal or adult fibroblasts or is a mixture of them.

[0101] The frequency of administration of cells to an individual will depend, at least in part, on the factors described herein. The amount of the agonist may be optimized using routine methods in the art. In a specific embodiment, a single dose is required. In other embodiments, multiple cell administrations are required. The system is time and condition sensitive. The specific needs of the individual, which may vary depending on the circumstances, the loss of cells or the loss of activity of individual cells. It should be recognized that the results will depend on variables such as the rate of loss of cell viability. It is therefore expected that each individual may be monitored for appropriate dosage, and such Monitoring of such individuals is routinely practiced in the art.

[0102] Embodiments of the present disclosure provide methods for reducing the immunogenicity of certain types of fibroblasts. Fibroblasts are derived from various tissues available by biopsy (if required) or at autopsy. tissues or organs (e.g., skin, heart, blood vessels, bone marrow, skeletal muscle, liver, pancreas, brain and / or In some embodiments, the cells may be derived from fetal, neonatal, adult The present invention includes fibroblasts which may be derived from any of the following sources or a combination thereof.

[0103] In some embodiments of the present disclosure, fibroblasts are used to reduce the immunogenicity of the fibroblasts. One method involves genetic modification of blast cells, which are poorly immunogenic, such as immature dendritic cells. In another embodiment, the genetic modification includes cytoplasmic transfer using cells. Disease-causing genes, or CD40, CD80, CD86, TNF-alpha, HMGB-1 , IFN-gamma, IL-1 beta, IL-17, FAP, IL-18, IL-33, Gene editing is being used to selectively cut out costimulatory molecules, or combinations of them. can be.

[0104] In certain embodiments of the present disclosure, one or more immunomodulatory agents are operable in eukaryotic cells. The gene is expressed in universal donor fibroblasts via a recombinant expression vector capable of expressing the gene. Expression of the immunomodulatory agent can be driven by a constitutive promoter, an inducible promoter, or a tissue-specific promoter. In a specific embodiment, the vector is a retroviral vector. virus, lentivirus, adenovirus, adeno-associated virus or herpes simplex virus The vector may be a viral vector such as a DNA vector, or may be naked DNA or a plasmid vector. Non-viral vectors such as NA or minicircle DNA. Non-viral vectors such as endothelial lysates may also be used. Polynucleotides of particular interest for this application include at least the following: as ligand, TGF-beta, IL-4, IL-10, HLA-G, indoleamine 2,3-deoxygenase (IDO), galectin family member, galectin-3, a Ruginase, IL-20, HGF, PDGF-BB, EGF, IGF, GDF-5, GD F-11, angiopoietin, FGF-1, FGF-2, FGF-5, FGF-15 or or combinations thereof. In certain cases, as recombinantly expressed angiogenic agents is FAS ligand, IL-2, IL-4, IL-10, IL-20, IL-35, HL AG, I-309, IDO, iNOS, CD200, galectin 3, arginase, P GE-2, TGF-beta, CTLA-4, PD-L1, IFN-gamma or their Combinations may be included.

[0105] In certain embodiments, a therapeutically effective amount of the modified cells is administered to an individual to treat one or more immune disorders. Exemplary immunomodulatory agents include FAS ligand, IL-2R, IL-1Ra, IL-2, IL-4, IL-8, IL-10, IL-20, IL-35, HLA-G, PD-L1, I-309, IDO, iNOS, CD200, Galectin 3, sCR1, arginase, PGE-2, aspirin, atorvastatin, fluvastatin , lovastatin, pravastatin, rosuvastatin, simvastatin, pitavastatin, n-acetylcysteine, rapamycin, IVIG, naltrexone, TGF-beta, VEGF, PDGF, CTLA-4, anti-CD45RB antibody, hydroxychloroquine, Lev Lunomide, auranofin, dicyanogold, sulfasalazine, methotrexate, carbohydrate co Ruticoids, etanercept, adalimumab, abatacept, anakinra, certolisma , etanercept-szzs, golimumab, infliximab, rituximab, tosiri izumab, cyclosporine, IFN-gamma, everolimus, rapamycin or their Combinations may be included.

[0106] In certain embodiments, at least in some cases, growth factors are provided in the growth medium. The cells do not require exogenous growth factors, except that these are available in the form of additional serum provided to the cells. A method is used that does not require specific growth factors to expand. Methods for obtaining umbilical cells are also provided herein. These methods are similar to those described above, but The cells are then finally resuspended and grown in a medium that contains their specific growth factors (their In this sense, The method involves targeting cells that are capable of dividing in the absence of those particular growth factors. In some embodiments, the specific cells are selective for serum-free culture. The cells can be grown and expanded in a synthetic growth medium that does not require any synthetic synthesis. Cells require certain growth factors that can be added to the medium to support and sustain them. Factors that can be added to serum-free media for growth include FGF, EGF, It may include one or more of IGF and PDGF. In some embodiments, Two, three or all four of these factors were added to serum-free or chemically defined media. In a specific embodiment, leukemia suppression is used to support or improve cell growth. Factor (LIF) is added to serum-free medium. EXAMPLES

[0107] VII. Examples

[0108] Example 1: Stimulation of regeneration in discs distal to discs injected with human dermal fibroblasts

[0109] Five patients with degenerative disc disease were given 10 million human skin samples (e.g., CybroCe ll) Fibroblast cells are injected intradiscally. The cells are then delivered to the nucleus pulposus via fluoroscopically guided injection. Six months later, the injected discs and the discs proximal and distal to the injection area were Regeneration is observed in the discs.

[0110] Example 2: Effective treatment of bilateral osteoarthritis by administration of fibroblasts

[0111] Ten patients with osteoarthritis received 10 million CybroCell cells in each knee. Fibroblast cells were administered intra-articularly. Three months after administration, the injected knees showed signs of cartilage regeneration. Furthermore, the contralateral, uninjected knee also shows signs of cartilage regeneration. References

[0112] All publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications herein are incorporated by reference in their entirety as if each individual publication were incorporated by reference in its entirety. Each of these disclosures is incorporated by reference into the present specification to the same extent as if each respective disclosure was specifically and individually indicated to be incorporated by reference.

[0113] US Pat. No. 4,353,888

[0114] US Pat. No. 4,744,933

[0115] US Pat. No. 4,749,620

[0116] US Pat. No. 4,814,274

[0117] U.S. Pat. No. 5,084,350

[0118] U.S. Pat. No. 5,089,272

[0119] U.S. Pat. No. 5,578,442

[0120] U.S. Pat. No. 5,639,275

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[0130] U.S. Pub. 2013 / 0149286

[0131] European Patent Publication No. 301,777

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Claims

1. 1. A method of stimulating regeneration at a first tissue site in an individual, the method comprising administering fibroblasts to a first tissue site in an individual. and / or at least one comprising dedifferentiated fibroblasts and, optionally, stem cells. to a second tissue site, said second tissue site being in contact with said individual. wherein the first tissue site comprises the same tissue type as the first tissue site of.

2. Administration of the composition comprises systemic injection, local injection, systemic delivery and / or local delivery; The method of claim 1.

3. The administration of the composition may be at least 1 administration or 2, 3, 4, 5, 6, 7, 8, 9, 3. The method of claim 1 or 2, comprising 10 or more administrations.

4. 4. The method according to claim 1, wherein the first tissue has a partial or complete loss of function. The method according to claim 5.

5. The loss of function may result in cell death, tissue necrosis, atrophy, fibrosis, inflammation, fat deposition, degeneration, or other abnormalities in the tissue. Production of sex molecules, loss of elasticity, neurodegeneration, autoimmunity, complement activation, loss of cartilage, ligament rupture, muscle 5. The method of claim 4, comprising a tear, loss of connective tissue, a neoplasm, or a combination thereof. 。

6. The tissue is muscle tissue, connective tissue, epithelial tissue, endothelial tissue, nervous tissue, adipose tissue, skin tissue, , lung tissue, liver tissue, bladder tissue, kidney tissue, heart tissue, stomach tissue, intestinal tissue, spinal cord tissue, eye tissue, Any of claims 1 to 5, comprising fibrous tissue, omentum, lymphatic tissue, bone marrow or a combination thereof. The method according to any one of claims 1 to 5.

7. The tissue may be an endothelial cell, an epithelial cell, a skin cell, an endodermal cell, a mesodermal cell, a fibroblast, Bone cells, chondrocytes, natural killer cells, dendritic cells, liver cells, pancreatic cells, stromal cells, Salivary gland mucus cells, salivary gland serous cells, von Ebner gland cells, mammary gland cells, lacrimal gland cells, auditory canal glands Cells, eccrine dark cells, eccrine clear cells, apocrine cells, molar cells, skin Sebaceous gland cells, Bowman's gland cells, Brunner's gland cells, seminal vesicle cells, prostate cells, urethral gland cells, Bartholin's gland cells, Little gland cells, endometrial cells, isolated goblet cells, mucosal cells of the stomach wall, stomach Glandular zymogen cells, gastric gland acid-secreting cells, pancreatic acinar cells, Paneth cells, type II alveolar cells, Clara cells cells, somatotropes, lactotropes, thyrotropes, Gonadotropes, Corticotropes, Pituitary Intermediate Cells, Large Cells Neurosecretory cells, intestinal cells, airway cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, epithelial microglia Chief somatic cells, eosinophilic cells, adrenal cells, chromaffin cells, Leydig cells, theca interna cells, Luteal cells, granulosa luteal cells, theca luteal cells, juxtaglomerular cells, macula densa cells, perivascular pole cells , mesangial cells, vascular endothelial fenestrated cells and lymphatic endothelial fenestrated cells, vascular endothelial continuous cells and lymphatic endothelial continuous cells, vascular endothelial splenocytes and lymphatic endothelial splenocytes, synovial cells, and serous cells Membrane cells (lining the peritoneal, pleural and pericardial cavities), squamous epithelial cells, columnar cells, dark cells, vestibular The membrane cells (lining the endolymphatic space of the ear), basal cells of the stria vascularis, and peripheral cells of the stria vascularis (lining the endolymphatic space of the ear). Inner wall), Claudius cells, Boettcher cells, choroid plexus cells, pia arachnoid squamous epithelial cells, Pigmented ciliary epithelial cells, non-pigmented ciliary epithelial cells, corneal endothelial cells, PEG cells, airway ciliary cells Follicles, oviductal ciliated cells, endometrial ciliated cells, reticulum testis ciliated cells, efferent testicular ciliated cells, ciliated epithelium keratinocytes, epidermal keratinocytes, epidermal basal cells, fingernail and toenail keratinocytes, nail bed base Cells, medullary hair stem cells, cortical hair stem cells, cuticle hair stem cells, cuticle hair root sheath cells, hair follicle Root sheath cells of the layer of Henle, outer root sheath cells, hair matrix cells, surface of stratified squamous epithelium Face epithelial cells, Basal epithelial cells, Urinary epithelial cells, Inner hair cells of the organ of Corti of the ear, Corti of the ear outer hair cells of the olfactory organ, basal cells of the olfactory epithelium, cold-sensitive primary sensory neurons, heat-sensitive primary sensory neurons Neurons, Merkel cells of the epidermis, olfactory receptor neurons, pain-sensitive primary sensory neurons, photoreceptors Rod cells, photoreceptors blue-sensitive cone cells, photoreceptors green-sensitive cone cells, photoreceptors red Sensitive pyramidal cells, proprioceptive primary sensory neurons, tactile sensitive primary sensory neurons, type I Carotid body cells, type II carotid body cells (blood pH sensor), type I ciliary cells of the vestibular apparatus of the ear Vestibular nerve (acceleration and gravity), type II hair cells of the vestibular apparatus of the ear, type I taste bud cells, cholinergic nerves cells, adrenergic neurons, peptidergic neurons, inner column cells of the organ of Corti, Outer column cells of the organ of Corti, inner supporting cells of the organ of Corti, outer supporting cells of the organ of Corti, border cells of the organ of Corti , Hensen's cells of the organ of Corti, supporting cells of the vestibular apparatus, supporting cells of the taste buds, supporting cells of the olfactory epithelium, Schwann Cells, satellite cells, enteric glial cells, astrocytes, neurons, oligodendrocytes, spindle-shaped Neurons, prelental epithelial cells, crystallin-containing lens fiber cells, hepatocytes, adipocytes, white White adipocytes, brown adipocytes, liver lipid cells, renal glomerular wall cells, renal glomerular podocytes, renal juxtacellular Brush border cells of distal tubule, cells of the thin part of the loop of Henle, cells of distal tubule, cells of renal collecting duct, Type I pneumocytes, pancreatic duct cells, smooth muscle duct cells, duct cells, intestinal brush border cells, exocrine gland striated duct cells alveolus, gallbladder epithelial cells, non-ciliated cells of the efferent duct of the testis, epididymis principal cells, epididymis basal cells, ename lunar epithelial cells, semilunar epithelial cells, interdental epithelial cells of the organ of Corti, loose connective tissue fibroblasts, Corneal keratocytes, tendon fibroblasts, bone marrow reticular fibroblasts, non-epithelial fibroblasts, pericytes, Nucleus pulposus cells, cementoblasts / cementocytes, odontoblasts, odontocytes, soft tissue of hyaline cartilage Osteocytes, chondrocytes of fibrocartilage, chondrocytes of elastic cartilage, osteoblasts, osteocytes, osteoclasts, osteoporotic cells Alveolar progenitor cells, vitreous cells, stellate cells (ear), hepatic stellate cells (Ito cells), pancreatic stellate cells, red skeletal muscle Cells, white skeletal muscle cells, intermediate skeletal muscle cells, muscle spindle nuclear bag cells, muscle spindle nuclear chain cells, satellite cells cells, normal cardiomyocytes, nodal cardiomyocytes, Purkinje fiber cells, smooth muscle cells, myoepithelial cells of the iris alveolar cysts, myoepithelial cells of exocrine glands, reticulocytes, megakaryocytes, monocytes, connective tissue macrophages. Epithelium Langerhans cells, dendritic cells, microglial cells, neutrophils, eosinophils, basophils, mast cells cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells Follicles, B cells, natural killer cells, melanocytes, retinal pigment epithelial cells, oogonia / oocytes Cells, spermatids, spermatocytes, spermatogonia, spermatozoa, follicular cells, Sertoli cells, thymic epithelial cells, The present invention relates to a method for treating kidney disease comprising administering to a patient a therapeutically effective amount of a renal cell comprising administering to the patient a therapeutically effective amount of a renal cell. The method according to any one of claims 1 to 6.

8. The regenerative composition according to any one of claims 1 to 7, wherein the regenerative composition comprises at least one growth factor. The method described.

9. At least one growth factor is selected from AM, Ang, BMP, BDNF, EGF, Epo, F GF, GNDF, G-CSF, GM-CSF, GDF-9, HGF, HDGF, IGF, Migration stimulating factor, GDF-8, GDF-11, GDF-15, MGF, NGF, PlGF, PDGF, Tpo, TGF-alpha, TGF-beta, TNF-alpha, VEGF, Wnt proteins, interleukins, IL-1 alpha, IL-1 beta, IL-1F 1, IL-1F2, IL-1F3, IL-1F4, IL-1F5, IL-1F6, IL- 1F7, IL-1F8, IL-1F9, IL-2, IL-3, IL-4, IL-5, IL -6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, 35kD a alpha subunit, IL-12, 40 kDa beta subunit, IL-13, I L-14, IL-15, IL-16, IL-17A, IL-17B, IL-17C, IL -17D, IL-17E, IL-17F isoform 1, IL-17F isoform 2, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23 p1 9 subunit, IL-23 p40 subunit, IL-24, IL-25, IL-2 6, IL-27B, IL-27-p28, IL-28A, IL-28B, IL-29, I L-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36 Soluble receptors for IL-36 alpha, IL-36 beta, and IL-36 gamma. IFN, IFN-alpha, IFN-beta, IFN-gamma, IFN-lambda IFN-lambda 1, IFN-lambda 2, IFN-lambda 3, IFN-K, IFN-epsilon, IFN -kappa, IFN-tau, IFN-delta, IFN-zeta, IFN-omega, IFN -soluble receptor for insulin, proinsulin, insulin receptor eptin (LEP), leptin (LEP), and combinations thereof. Item 9. The method according to item 8.

10. The method of any one of claims 1 to 9, wherein the regenerative composition comprises platelet-rich plasma.

11. The method of claim 10, wherein the platelet-rich plasma comprises a platelet lysate.

12. The method of claim 10, wherein the platelet-rich plasma is derived from peripheral blood.

13. The method of claim 10, wherein the platelet-rich plasma is derived from umbilical cord blood.

14. The regenerative composition comprises one or more exosomes derived from at least one regenerative cell. The method according to any one of claims 1 to 13, comprising:

15. 15. The method of claim 14, wherein the regenerative cells are stem cells and / or fibroblasts. 。

16. The fibroblasts are derived from foreskin, adipose tissue, placenta, ear lobe, omentum, Wharton's jelly and their 16. The method of claim 15, wherein the tissue source is selected from the group consisting of:

17. The method according to any one of claims 14 to 16, wherein the exosome has a size of 2 nm to 200 nm. The method according to any one of claims 1 to 5.

18. Any of claims 14 to 17, wherein the exosome has a size of 30 to 150 nm.

2. The method according to claim 1.

19. The exosomes contain phospholipids, phosphatidylserine, phosphatidylinositol , phosphatidylcholine, sphingomyelin, ceramide, glycolipid, cerebroside, steatohepatitis at least one selected from the group consisting of steroids, cholesterol and combinations thereof; The method of any one of claims 14 to 18, comprising one lipid.

20. 20. The method according to claim 14, wherein the exosome comprises at least one lipid raft.

2. The method according to claim 1.

21. The exosomes are selected from the group consisting of CD9, CD63, CD81, ANXA2, ENO1, and HSP9. 0AA1, EEF1A1, YWHAE, SDCBP, PDCD6IP, ALB, YWHA Z, EEF2, ACTG1, LDHA, HSP90AB1, ALDOA, MSN, ANX A5, PGK1, CFL1, and combinations thereof. The method according to any one of claims 14 to 20, further comprising administering to the patient an antigen marker on the surface of the exosome. The method described.

22. The method according to any one of claims 1 to 21, wherein the regenerative composition comprises one or more fibroblasts. Method of posting.

23. The fibroblasts are derived from foreskin, adipose tissue, placenta, ear lobe, adipose tissue, omentum, Wharton's jelly and 23. The method of claim 22, which is derived from a tissue source selected from the group consisting of: How to.

24. The fibroblasts express NANOG, OCT-4, SSEA-4, stem cell factor receptor, and and combinations thereof. The method according to claim 22 or 23.

25. The regenerative composition of any one of claims 1 to 24, wherein the regenerative composition comprises one or more stem cells. method.

26. 26. The method of 25, wherein the stem cells comprise pluripotent stem cells.

27. The pluripotent stem cells may be embryonic stem cells, parthenogenetic stem cells, induced pluripotent stem cells, somatic stem cells, or the like. Stem cells derived from nuclear transfer, stem cells derived from cytoplasmic transfer, stimuli-induced acquisition of pluripotency and their 27. The method of claim 26, wherein the compound is selected from the group consisting of a combination.

28. 26. The method of 25, wherein the stem cells comprise hematopoietic stem cells.

29. 28. The hematopoietic stem cells are capable of multilineage reconstitution in an immunocompromised host. The method described above.

30. The hematopoietic stem cells are characterized in that they express c-kit, Sca-1, CD34, CD133 and combinations thereof. and a combination thereof.

29. The method according to item 28.

31. Any one of claims 28 to 30, wherein the hematopoietic stem cells express Sca-1 protein. The method according to claim 5.

32. The method according to any one of claims 28 to 31, wherein the hematopoietic stem cells express CD34. Law.

33. The hematopoietic stem cells according to any one of claims 28 to 32, wherein the hematopoietic stem cells express CD133. method.

34. 30. The method of claim 28, wherein the hematopoietic stem cells lack expression of one or more lineage markers.

35. The hematopoietic stem cells are characterized by the expression of CD38, CD14, CD16, CD56, or a combination thereof.

29. The method of claim 28, wherein the gene lacks expression of the gene.

36. the hematopoietic stem cells are positive for c-kit expression and positive for Sca-1 expression; and / or substantially lacks expression of lineage markers.

37. The hematopoietic stem cells are derived from peripheral blood, mobilized peripheral blood, bone marrow, umbilical cord blood, adipose stromal vascular cells, progenitor cells, or derived from a source selected from the group consisting of: derived from a cell; and combinations thereof.

29. The method of claim 28.

38. 33. The method of claim 32, wherein the hematopoietic progenitor cells are pluripotent stem cells.

39. 26. The method of claim 25, wherein the stem cells comprise mesenchymal stem cells.

40. 40. The method of claim 39, wherein the mesenchymal stem cells are plastic-adherent.

41. The mesenchymal stem cells are selected from the group consisting of CD73, CD90, CD105 and combinations thereof.

41. The method of claim 39 or 40, wherein the cell expresses a marker selected from the group consisting of:

42. The mesenchymal stem cells are composed of CD14, CD45, CD34 and combinations thereof.

42. The method of claim 39, 40 or 41, wherein the gene lacks expression of a marker selected from the group consisting of:

43. The mesenchymal stem cells are derived from bone marrow, peripheral blood, adipose tissue, mobilized peripheral blood, umbilical cord blood, Wharton's jelly, etc. , umbilical cord tissue, skeletal muscle tissue, subepithelial umbilical cord, endometrial tissue, menstrual blood, fallopian tube tissue and their 40. The method of claim 39, wherein the tissue is derived from a tissue selected from the group consisting of:

44. The mesenchymal stem cells derived from umbilical cord tissue express oxidized low density lipoprotein receptor 1, Mokine receptor ligand 3, granulocyte chemotactic protein and combinations thereof 44. The method of claim 43, wherein the cell expresses a marker selected from the group consisting of:

45. The mesenchymal stem cells derived from umbilical cord tissue are characterized by the expression of CD117, CD31, CD34, CD45 and and combinations thereof.

44. The method according to claim 44.

46. The mesenchymal stem cells derived from umbilical cord tissue express higher levels of interferon compared to human fibroblasts.

46. ​​The method according to claim 43, wherein the vector expresses kin8 and / or reticulon 1. The method described.

47. The mesenchymal stem cells derived from umbilical cord tissue are characterized by the presence of CD10, CD13, CD44, CD73, 90, and combinations thereof.

47. The method according to any one of claims 3 to 46.

48. The umbilical cord tissue-derived cells express MCP-1, MIP1 beta, IL-6, IL-8, GC P-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, RANTES, T 4. The method of claim 3, wherein the cell secretes a factor selected from the group consisting of IMP1 and combinations thereof.

48. The method according to any one of claims 3 to 47.

49. The umbilical cord tissue-derived cells are 4. Expressing a marker selected from the group consisting of NANOG and combinations thereof The method according to any one of claims 43 to 48.

50. The umbilical cord tissue-derived mesenchymal stem cells are capable of self-renewal and expansion in culture, and are blood-stimulating 43 to 45. The method of claim 42, wherein the isolated umbilical cord tissue cells are isolated from umbilical cord tissue that is qualitatively free of umbilical cord tissue.

49. The method of any one of claims 49 to 50.

51. 43 to 45. The cells derived from the umbilical cord tissue stain positive for alkaline phosphatase.

10. The method according to any one of claims 1 to 0.

52. The mesenchymal stem cells derived from the cord tissue can undergo at least 20 doublings in culture. The method of claim 43,

53. 50. The method of claim 49, wherein the cord tissue derived mesenchymal stem cells maintain a normal karyotype upon passaging. How to.

54. The umbilical cord tissue-derived mesenchymal stem cells are 90, PDGFr-alpha, PD-L2, HLA-A, B, C, and combinations thereof The method according to any one of claims 43 to 53, wherein the gene expresses a marker selected from the group consisting of Method of posting.

55. The mesenchymal stem cells derived from the cord tissue are characterized by the expression of CD31, CD34, CD45, CD80, and CD8 6, CD117, CD141, CD178, B7-H2, HLA-G, HLA-DR, D Expressing one or more markers selected from the group consisting of P, DQ, and combinations thereof. The method according to any one of claims 43 to 51, wherein the

56. The bone marrow-derived mesenchymal stem cells are Lectin, L-selectin, CD49b / CD29, CD49c / CD29, CD49d / CD29, CD29, CD18, CD61, 6-19, thrombomodulin, telomerase , CD10, CD13, CD34, CD56, CD117, integrin beta and and combinations thereof. How to.

57. 57. Any one of claims 43 or 56, wherein the bone marrow mesenchymal stem cells do not express CD10. The method according to claim 5.

58. The bone marrow mesenchymal stem cells are CD2, CD5, CD14, CD19, CD33, CD45 and / or DRII, 57. The method according to any one of claims 57.

59. The bone marrow mesenchymal stem cells are characterized by the expression of CD13, CD34, CD56, CD90, CD117 and and / or nestin.

58. The method of any one of claims 58.

60. The bone marrow-derived mesenchymal stem cells according to any one of claims 43 to 59, wherein the bone marrow-derived mesenchymal stem cells are precursor cells of mesenchymal stem cells.

13. The method according to any one of claims 1 to 12.

61. The mesenchymal progenitor cells are selected from bone marrow mesenchymal stem cells enriched in cells expressing STRO-1. The method of claim 60, comprising a population of cells.

62. 7. The mesenchymal precursor cells express both STRO-1 and VCAM-1.

62. The method according to any one of claims 0 or 61.

63. The cells expressing STRO-1 are CBFA-1, type II collagen, PPAR gamma 2, osteopontin, osteocalcin, parathyroid hormone receptor, rep Chin, H-ALBP, aggrecan, Ki67, glycophorin A, and combinations thereof 63. The method according to claim 60, wherein at least one marker selected from the group consisting of:

13. The method according to any one of claims 1 to 12.

64. The bone marrow mesenchymal stem cells are characterized by the expression of at least one of CD14, CD34 and / or CD45.

61. The method of claim 60, wherein both of said genes lack expression.

65. The cells expressing STRO-1 are VCAM-1, TKY-1, CD146, S markers selected from the group consisting of TRO-2 and combinations thereof are positive; 65. The method according to any one of claims 61 to 64.

66. The skeletal muscle stem cells are characterized by the expression of CD13, CD34, CD56, CD117 and combinations thereof.

44. The method of claim 43, wherein the gene expresses a marker selected from the group consisting of:

67. 67. Any of claims 43 or 66, wherein the skeletal muscle mesenchymal stem cells do not express CD10.

2. The method according to claim 1.

68. The skeletal muscle mesenchymal stem cells are CD2, CD5, CD14, CD19, CD33, CD4 5 and / or DRII.

68. The method according to any one of claims 67 to 67.

69. The subepithelial umbilical cord-derived mesenchymal stem cells are characterized by the expression of CD29, CD73, CD90, CD166, From SSEA4, CD9, CD44, CD146, CD105 and combinations thereof 44. The method of claim 43, having a marker selected from the group consisting of:

70. The subepithelial umbilical cord-derived mesenchymal stem cells are D106, CD86, CD80, CD19, CD117, Stro-1, HLA-DR and and combinations thereof.

70. The method according to any one of claims 69 to 70.

71. The subepithelial umbilical cord-derived mesenchymal stem cells are characterized by the expression of CD29, CD73, CD90, CD166, At least one of SSEA4, CD9, CD44, CD146 and / or CD105 71. The method of any one of claims 43, 69 or 70, further comprising expressing at least one of the following:

72. The subepithelial umbilical cord-derived mesenchymal stem cells are D106, CD86, CD80, CD19, CD117, Stro-1 and / or H 72. The method of claim 43 or any one of claims 69 to 71, wherein the mouse does not express at least one of LA-DR.

2. The method according to claim 1.

73. The mesenchymal stem cells derived from the subepithelial umbilical cord are SOX2 positive.

72. The method of any one of claims 72 to 71.

74. The mesenchymal stem cells derived from the subepithelial umbilical cord are OCT4 positive.

73. The method according to any one of claims 73 to 73.

75. The subepithelial umbilical cord-derived mesenchymal stem cells are OCT4-positive and SOX2-positive. 43 or 69-74. The method according to any one of claims 43 to 69.

76. The regenerative composition comprises one or more fibroblast-derived apoptotic vesicles.

75. The method according to any one of claims 75 to 75.

77. Any one of claims 1 to 76, wherein the regenerative composition comprises fibroblast-derived miRNA. The method according to claim 5.

78. The method of claim 77, wherein the fibroblast-derived miRNA is contained in an exosome. How to.

79. 78. The method of claim 77, wherein the fibroblast-derived miRNA is contained in an apoptotic body. The method described.

80. 78. The method of claim 77, wherein the fibroblast-derived miRNA is circulating in plasma. 。

81. The enhancement of one or more distant regenerative effects is achieved by systemic administration of one or more epigenetic acting compositions. The method of any one of claims 1 to 80, which is achieved by administration.

82. 9. The method of claim 8, wherein the systemic administration is at the site of administration of the regenerative composition or at a different site.

2. The method according to claim 1.

83. The epigenetic acting composition comprises one or more histone deacetylase inhibitors.

83. The method of claim 81 or 82, comprising:

84. The epigenetic acting composition comprises one or more DNA methyltransferases 84. The method of any one of claims 81 to 83, comprising an inhibitor.

85. The method of claim 4 , wherein the first tissue is degenerating.