Novel blood vessel-forming stem cells

Abstract

The present invention relates to newly identified stem cells, vMSCs, with angiogenic properties. The vMSCs identified by culturing under the specific culture conditions of the present invention are novel MSCs that express the CD141 cell surface antigen, unlike conventional MSCs. vMSCs have excellent cell expansion ability and direct angiogenic effects. In addition to the paracrine effects of other MSCs, they also have autocrine effects, unlike conventional stem cells. Therefore, they have higher survival and survival rates in tissues than conventional stem cell therapeutics, and can be used as a stem cell therapeutic agent for vascular regeneration that directly forms blood vessels.

Description

[Technical Field]

[0001] The present invention relates to newly identified multipotent stem cells, vMSCs, that have angiogenic properties. [Background technology]

[0002] New blood vessels are generated through the processes of angiogenesis, arteriogenesis, and vasculogenesis. Angiogenesis involves the proliferation and migration of endothelial cells that arise from pre-existing mature endothelial cells. Arteriogenesis involves the remodeling of pre-existing arteriolar connections into collateral vessels. Vasculogenesis is driven by the differentiation of endothelial progenitor cells into mature endothelial cells. Therefore, circulating endothelial progenitor cells (EPCs) migrate to sites of vascular injury and participate in new blood vessel formation by either direct insertion into newly forming vessels or by secreting various angiogenic and trophic factors. Therefore, endothelial progenitor cells (EPCs) are attracting attention as potential targets in the field of regenerative medicine and therapeutic revascularization. As a result, research is underway to improve the function of endothelial progenitor cells. For example, research is being conducted on the production of recombinant EPCs through ex vivo gene modification, and techniques are being investigated to improve the proangiogenic capacity of EPCs using vascular endothelial growth factor (VEGF) or hypoxia-inducible factor-1α. Currently, cell therapy for ischemic vascular diseases uses two main types of cells: endothelial progenitor cells (EPCs), which play a role in vascular regeneration, and mesenchymal stem cells, which secrete large amounts of growth factors that contribute to angiogenesis and indirectly contribute to angiogenesis through a paracrine effect. However, conventional EPCs are extremely difficult to expand in vitro, and it is difficult to maintain their function during expansion. In both allogeneic and xenogeneic cases, they are a cell group with many problems, such as immune rejection, and they have limitations in their differentiation and function into complex blood vessel structures.Furthermore, conventional therapeutic agents developed based on mesenchymal stem cells targeting ischemic diseases are lost without engraftment, and therefore rely on paracrine effects rather than angiogenesis as a therapeutic method, and have not yet demonstrated clear therapeutic effects in clinical settings.

[0003] Cell therapy products, including stem cell therapy products, are defined as pharmaceuticals used for therapeutic, diagnostic, and preventive purposes through a series of processes, such as expanding or selecting living autologous, allogeneic, or xenogeneic cells in vitro or otherwise altering the biological properties of cells, to restore cell and tissue function. Cell therapy transplantation using cell therapy products involves isolating and preparing cellular materials composed of numerous cells capable of substituting for various tissues that are expressed as tissues similar to normal or healthy tissues in the body. Cell materials are then cultured in an ex vivo environment to alter the biological properties of the cells, and these cells are then injected into the recipient or used to create human tissue for treatment. Stem cell therapy products specifically refer to cell therapy products that use stem cells. Current representative applications include neurological disorders, cardiac diseases, pulmonary diseases, liver diseases, and cancer, where recovery and regeneration of lost cells is essential but where regeneration is difficult. Active research is underway. Stem cells have great potential in cell therapy because they have the potential to differentiate into various cells needed in damaged tissues. However, at the current level of development, the survival rate after transplantation is not high, and it is difficult to find examples of widespread and stable success in actual clinical applications. Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a novel stem cell.

[0005] Another object of the present invention is to provide a cell therapeutic composition for vascular regeneration or angiogenesis.

[0006] Another object of the present invention is to provide a graft material for revascularization.

[0007] It is also an object of the present invention to provide a pharmaceutical composition for preventing or treating vascular diseases or vascular dysfunction.

[0008] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating diseases of the central nervous system.

[0009] Another object of the present invention is to provide a novel method for producing stem cells.

[0010] It is also an object of the present invention to provide a use for stem cells expressing the CD141 cell surface antigen for use in revascularization or angiogenesis.

[0011] It is also an object of the present invention to provide a method for revascularization or angioplasty.

[0012] It is also an object of the present invention to provide a method for treating vascular disease or vascular dysfunction.

[0013] A further object of the present invention is to provide a method for treating diseases of the central nervous system. [Means for solving the problem]

[0014] To achieve the above object, the present invention provides stem cells that express the CD141 cell surface antigen.

[0015] The present invention also provides a cell therapy composition for vascular regeneration or angiogenesis, comprising the stem cells.

[0016] The present invention also provides a graft material for revascularization.

[0017] The present invention also provides a pharmaceutical composition for preventing or treating vascular disease or vascular dysfunction.

[0018] The present invention also provides a pharmaceutical composition for preventing or treating a cranial nervous system disease.

[0019] The present invention also provides a method for producing stem cells that express the CD141 cell surface antigen.

[0020] The present invention also provides the use of stem cells expressing the CD141 cell surface antigen for use in revascularization or angiogenesis.

[0021] The present invention also provides a method of revascularization or angiogenesis.

[0022] The present invention also provides a method for treating vascular disease or vascular dysfunction.

[0023] Furthermore, the present invention provides a method for treating a neurological disease. [Effects of the Invention]

[0024] The vMSCs (multipotent stem cells) obtained by culturing under the specific culture conditions of the present invention share some properties with conventional MSCs (mesenchymal stem cells or mesenchymal stromal cells). However, unlike conventional MSCs, they are novel MSCs that express the CD141 cell surface antigen. These vMSCs have excellent cell expansion capacity and direct vascular endothelialization effects. They also exhibit the paracrine effects of vascular regeneration-related factors, as do other MSCs, and, unlike conventional stem cells, they also exhibit the autocrine effects of vascular regeneration-related factors. Therefore, they have higher survival and survival rates in tissues than conventional stem cell therapeutics, and can be used as a stem cell therapeutic agent for vascular regeneration that directly forms blood vessels. [Brief explanation of the drawings]

[0025] [Figure 1] FIG. 1 shows the colony morphology of CD141+ vMSCs formed in various extracellular matrices. [Figure 2] FIG. 1 shows the cell characteristics of CD141+ vMSCs confirmed by marker expression. [Figure 3] FIG. 1 shows the results of marker screening to identify new markers specific to CD141 + vMSCs that can be distinguished from BM-MSCs. [Figure 4] FIG. 1 shows the cell expansion capacity of CD141+ vMSCs. [Figure 5] FIG. 1 shows the evaluation of pluripotency, a stem cell characteristic of CD141+ vMSCs. [Figure 6] This figure confirms the vascular network formation ability of CD141+ vMSCs, BM-MSCs, and HUVECs using culture media (scale bar = 200 μm). [Figure 7] Figure 7 shows the measurement of autocrine HGF expression levels and analysis of c-MET receptor-mediated signaling activity in CD141+ vMSCs: Figure 7a: HGF concentration in the culture medium of CD141+ vMSCs cultured in EGM-2-FBS + 2% hPL medium; Figure 7b: HGF concentration in the culture medium of BM-MSCs cultured in StemMACS medium; Figure 7c: HGF concentration in the culture medium of CD141+ vMSCs and BM-MSCs at passage 3 when cultured under the same conditions in a medium lacking angiogenic growth factors (MEM alpha + 0.2% hPL); Figure 7d: HGF receptor (c-Met) expression levels and c-Met activity (phosphorylation) in CD141+ vMSCs and BM-MSCs at different passages; Figure 7e: Quantification of c-Met expression levels; and Figure 7f: Quantification of c-Met activity (phosphorylation). [Figure 8] FIG. 8 shows the analysis of HGF-induced vascular sprouting ability in CD141+ vMSC spheroids and BM-MSC spheroids: FIG. 8a: CD141+ vMSCs; and FIG. 8b: BM-MSCs. [Figure 9] FIG. 10 shows an analysis of the blood vessel sprouting ability of CD141+ vMSC spheroids induced by bFGF secreted by BM-MSCs. [Figure 10]These figures confirm the vascular sprouting ability of CD141+ vMSC spheroids induced by VEGF or bFGF: Figures 10a and 10c: vascular sprouting ability of vMSC spheroids induced by VEGF or bFGF; Figures 10b and 10d: vascular sprouting ability of BM-MSC spheroids induced by VEGF or bFGF; and Figure 10e: VEGFR2 mRNA expression in vMSCs and BM-MSCs (control group: HUVECs). [Figure 11] FIG. 10 is a diagram confirming that HGF secreted by vMSCs promotes the vascular sprouting ability of HUVEC spheroids. [Figure 12] FIG. 1 shows the results of a comparison of the characteristics of EPCs and CD141 + vMSCs reported in previous literature. [Figure 13] This figure compares the vascular network formation ability of CD141+ vMSCs and BM-MSCs in a normal environment, and confirms the cell composition of combined stem cells combining CD141+ vMSCs and BM-MSCs to maximize vascular regeneration potential (scale bar = 500 μm). [Figure 14] This figure confirms the composition of a composite stem cell that combines CD141+ vMSCs and BM-MSCs to maximize the vascular regeneration potential of CD141+ vMSCs in an inflammatory environment (scale bar = 500 μm). [Figure 15] FIG. 1 is a schematic diagram showing the process of isolating CD141+ vMSCs from adipose tissue. [Figure 16] FIG. 10 is a diagram comparing the cell morphology of CD141+ vMSCs and AD-MSCs at different passages. [Figure 17] FIG. 10 shows a comparison of the cumulative cell numbers of CD141+ vMSCs and AD-MSCs. [Figure 18] FIG. 10 shows a comparison of cell surface markers between CD141+ vMSCs and AD-MSCs. [Figure 19] FIG. 10 shows a comparison of the angiogenic potential of CD141+ vMSCs and AD-MSCs. [Figure 20] FIG. 1 shows an established animal model of severe hind limb ischemia. [Figure 21]FIG. 10 is a diagram evaluating the foot preservation effect in a severe hind limb ischemia model by treatment with combined stem cells that combine CD141+ vMSCs and BM-MSCs. [Figure 22] FIG. 10 shows an evaluation of the grade of necrosis in ischemic lesions in a severe limb ischemia model treated with combined stem cells that combine CD141+ vMSCs and BM-MSCs. [Figure 23] FIG. 11 is a diagram analyzing changes in blood flow in a severe lower limb ischemia model following treatment with combined stem cells that combine CD141+ vMSCs and BM-MSCs. [Figure 24] FIG. 10 is a diagram showing the foot-preserving effect of a combined stem cell formulation combining CD141+ vMSCs and BM-MSCs, evaluated to determine the minimum effective dose as a stem cell therapeutic agent. [Figure 25] FIG. 10 shows the evaluation of the grade of ischemic lesion necrosis and blood flow to determine the minimum effective dose of combined stem cells combining CD141+ vMSCs and BM-MSCs as a stem cell therapeutic agent. [Figure 26] FIG. 10 is a graph evaluating the foot-preserving effect of combined stem cells combining CD141+ vMSCs and BM-MSCs compared with administration of each cell alone. [Figure 27] FIG. 10 is a graph evaluating the effect of combined stem cells combining CD141+ vMSCs and BM-MSCs compared with administration of each cell alone, based on the grade of ischemic lesion necrosis. [Figure 28] This figure shows an evaluation of the effect of combined stem cells combining CD141+ vMSCs and BM-MSCs compared with administration of each cell alone, based on blood flow volume. [Figure 29] Macroscopic observation of angiogenesis in a combination of CD141+ vMSCs and BM-MSCs in a critical limb ischemia model: white dotted line: area where blood vessels were removed; and yellow arrow: newly formed blood vessels. [Figure 30] This figure shows the angiogenesis of combined stem cells, CD141+ vMSCs and BM-MSCs, in a severe hindlimb ischemia model, confirmed by immunofluorescence staining: White box: site of blood vessel removal (location of ischemia). [Figure 31] Figures showing in vivo angiogenesis of combined stem cells combining CD141+ vMSCs and BM-MSCs in a severe hindlimb ischemia model confirmed by immunohistochemistry: Figure 31a: Vascular structure and markers; and Figure 31b: Results of immunohistochemistry. [Figure 32] FIG. 10 is a diagram showing an analysis of in vivo angiogenesis of combined stem cells combining CD141+ vMSCs and BM-MSCs in a severe hind limb ischemia model, classified by vessel diameter. [Figure 33] This figure confirms the safety of combined stem cells that combine CD141+ vMSCs and BM-MSCs. BEST MODE FOR CARRYING OUT THE INVENTION

[0026] The present invention will be described in detail below with reference to the accompanying drawings. However, the following examples are presented as examples of the present invention, and if it is determined that a detailed description of a well-known technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, such detailed description may be omitted and the present invention will not be limited thereby. The present invention is susceptible to various modifications and applications within the scope of the claims below and the scope of equivalents interpreted therefrom.

[0027] Furthermore, the terminology used in this specification is used to appropriately describe a preferred embodiment of the present invention, and may vary depending on the intention of a user or operator, or the practice of the field to which the present invention pertains. Therefore, the definition of the term should be based on the contents of this specification as a whole. Throughout the specification, when a part is said to "include" a certain element, this does not mean that other elements are excluded, but that other elements may also be included, unless otherwise specified.

[0028] Unless otherwise defined, all technical terms used in the present invention are used in the sense that they are commonly understood by those of ordinary skill in the art in the relevant field of the present invention. In addition, although preferred methods or samples are described in this specification, similar or equivalent methods or samples are also included in the scope of the present invention. The contents of all publications referenced in this specification are incorporated herein.

[0029] Throughout this specification, "%" used to indicate the concentration of a particular substance is (w / w)% for solid / solid, (w / v)% for solid / liquid, and (v / v)% for liquid / liquid, unless otherwise specified.

[0030] In one aspect, the present invention relates to stem cells that express the CD141 (thrombomodulin™) cell surface antigen.

[0031] In one embodiment, the stem cells expressing the cell surface antigen CD141 may be vasculogenic multipotent stem cells (vMSCs).

[0032] In one embodiment, the cells are CD141 cells deposited under accession number KCLRF-BP-00524. + They may also be vMSCs (Vasculogenic Multipotent Stem Cells).

[0033] In one embodiment, the vMSCs may be derived from umbilical cord blood, umbilical cord, adipose tissue, or bone marrow, and more preferably from bone marrow or adipose tissue.

[0034] In one embodiment, the vMSCs may have revascularization or angiogenic potential.

[0035] In one embodiment, the vMSCs may be multipotent and have the ability to differentiate into adipocytes, osteocytes, chondrocytes, or myocytes.

[0036] In one embodiment, the vMSCs may overexpress HGF (hepatocyte growth factor) and its receptor compared to other stem cells.

[0037] In one embodiment, the cells may have a population doubling time (PDT) of 0.5 to 1.5 days.

[0038] In one embodiment, the vMSCs can differentiate into vascular endothelium in vivo, and the vascular endothelium may be CD31 positive.

[0039] In one embodiment, the vMSCs can promote neovascularization through basic fibroblast growth factor (bFGF) or HGF.

[0040] In one embodiment, the vMSCs may express CD105, CD90, CD29, CD73, or CD44 without expressing CD31, CD309, CD34, eNOS, VE-cadherin, or VEGF receptor.

[0041] In one embodiment, the vMSCs may not form new blood vessels with VEGF.

[0042] In one embodiment, the stem cells may be adult stem cells, or may be mesenchymal stem cells (MSCs) or have the properties thereof.

[0043] In one embodiment, the mesenchymal stem cells may be umbilical cord blood-derived mesenchymal stem cells (UCB-MSC), umbilical cord-derived mesenchymal stem cells (UC-MSC), adipose-derived mesenchymal stem cells (AD-MSC), or bone marrow-derived mesenchymal stem cells (BM-MSC).

[0044] The term "stem cell" as used herein refers to an undifferentiated cell that can differentiate into various cells that make up biological tissues and can reproduce without limit to form specialized cells of tissues and organs. Stem cells are totipotent or pluripotent cells that can develop. Stem cells proliferate into mature, fully formed cells of tissues.

[0045] The term "mesenchymal stem cells" as used herein refers to undifferentiated cells with multipotency derived from mammalian, including human, adult cells, preferably human, and refers to stem cells with multipotency that can differentiate into adipocytes, bone cells, chondrocytes, muscle cells, nerve cells, and cardiomyocytes. Mesenchymal stem cells can be derived from various adult cells, such as bone marrow, blood, brain, skin, adipose (i.e., adipose tissue or adipocytes), umbilical cord blood, and Wharton's jelly from the umbilical cord.

[0046] The term "differentiation" as used herein refers to the phenomenon in which an insufficiently specialized cell develops into a specific cell, and changes in cell size, morphology, membrane potential, metabolic activity, and response to signals to a specific type of cell. Differentiation refers to the occurrence of qualitative differences between initially homogeneous parts of a biological system, or the resulting division into qualitatively distinct subsystems. In particular, stem cell differentiation refers to the phenomenon in which stem cells develop in a directional manner into cells with specific functions.

[0047] As used herein, the term "expression" generally refers to the cellular process by which a biologically active polypeptide is produced from a DNA sequence and exhibits biological activity in a cell. In this sense, gene expression not only includes the transcription and translation processes, but also post-transcriptional and post-translational processes that can affect the biological activity of a gene or gene product. Such processes include, but are not limited to, RNA synthesis, processing, and transport, as well as polypeptide synthesis, transport, and post-translational modification of polypeptides.

[0048] As used herein, the term "overexpression" refers to the significant up-regulation of the mRNA or protein expression of a particular gene through intracellular gene transcription or translation.

[0049] As used herein, the term "not expressed" means that the expression level of a particular gene into mRNA or protein is significantly "down-regulated" by intracellular gene transcription or translation, resulting in almost no expression.

[0050] In the present invention, the expression can be confirmed by measuring the expression level of a gene or mRNA by polymerase chain reaction, real-time RT-PCR, reverse transcription polymerase chain reaction, competitive RT-PCR, nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization, nucleic acid microarray, Northern blot, or DNA chip method using a nucleic acid sequence, a nucleic acid sequence complementary to the nucleic acid sequence, or a primer pair, probe, or primer pair and probe that specifically recognize the nucleic acid sequence and a fragment of the complementary sequence. The expression can also be confirmed by measuring the expression level of a gene or mRNA by Western blot, enzyme-linked immunosorbent assay (ELISA), or by using an antibody, antibody fragment, aptamer, avidity multimer, or peptidomimetics that specifically recognizes the full-length protein or a fragment thereof. Protein expression levels can be measured and confirmed by radioimmunoassay (RIA), radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry, or protein microarray.

[0051] The term "vascular regeneration" as used herein refers to the repair of damaged blood vessels. The use of the compositions of the present invention promotes angiogenesis, i.e., the regeneration of damaged blood vessels, preventing further damage to already damaged blood vessels due to persistent external forces. "Angiogenesis" or "vasculogenesis" refers to the natural healing process of forming new blood vessels to supply blood to organs or damaged tissues. Angiogenesis plays an important role in the progression and treatment of many diseases. The vMSCs of the present invention possess angiogenic potential even in a medium lacking angiogenic growth factors. They secrete high concentrations of the angiogenic growth factor HGF and overexpress its receptor, c-Met, thereby promoting vascular sprouting using HGF as an autocrine factor. Furthermore, HGF secreted by vMSCs has a paracrine effect, enhancing the angiogenic potential of surrounding vascular endothelial cells. Furthermore, bFGF secreted by BM-MSCs promotes vascular sprouting, thereby exerting a paracrine effect. Thus, the vMSCs of the present invention have revascularizing, angiogenic or vasculogenic effects through autocrine and paracrine effects.

[0052] The term "angiogenesis" as used in the present invention can be achieved through the autocrine and paracrine effects of vMSCs. vMSCs have the ability to form angiogenesis even in a medium lacking angiogenic growth factors. They secrete high concentrations of the angiogenic growth factor HGF and overexpress its receptor, c-Met, thereby promoting vascular sprouting using HGF as an autocrine factor. Furthermore, HGF secreted by vMSCs promotes vascular sprouting from mature vascular endothelial cells (HUVECs), thereby exerting a paracrine effect. Furthermore, bFGF secreted by BM-MSCs promotes vascular sprouting, thereby exerting a paracrine effect.

[0053] In one aspect, the present invention relates to a cell therapeutic composition for vascular regeneration or angiogenesis, which comprises the stem cells of the present invention as an active ingredient.

[0054] In one embodiment, the composition may further comprise bone marrow-derived mesenchymal stem cells or adipose-derived mesenchymal stem cells.

[0055] In one embodiment, the bone marrow-derived mesenchymal stem cells or adipose-derived mesenchymal stem cells may not express the CD141 cell surface antigen.

[0056] In one embodiment, the stem cells (vMSCs) of the present invention and the bone marrow-derived mesenchymal stem cells or adipose-derived mesenchymal stem cells may be contained in a ratio of 1:10 to 10:1 based on the number of cells, preferably 1:1 to 10:1, more preferably 1:1 to 3:1, and most preferably 2:1.

[0057] In one embodiment, the composition may further comprise HGF or bFGF.

[0058] In one embodiment, the revascularization may include arterial regeneration, small arteriole regeneration, venous regeneration, capillary regeneration, blood-brain barrier (BBB) ​​regeneration, or retinal revascularization.

[0059] In one embodiment, the stem cells may be 3D cultured stem cells.

[0060] In one embodiment, the cell therapy composition can be cryopreserved and used for revascularization.

[0061] The cell therapy agent can be administered to the human body via any common route as long as it can reach the target tissue, and can be administered parenterally or orally. In the case of oral administration, the cells can be encapsulated in a transplant material or the like and administered.

[0062] In one aspect, the present invention relates to a cell therapeutic composition for treating brain and nervous system diseases, which comprises the stem cells of the present invention as an active ingredient.

[0063] In one embodiment, the neurological disease may be a degenerative brain disease, a psychiatric disorder, or a developmental disorder.

[0064] In one embodiment, the degenerative brain disease may be cognitive impairment, Alzheimer's disease, dementia with Lewy bodies, frontotemporal dementia, Parkinson's disease, Creutzfeldt-Jakob disease (CJD), Huntington's disease, multiple sclerosis, or Guillain-Barré syndrome (GBS).

[0065] In one embodiment, the mental illness may be bipolar disorder, autism, depression, hyperactivity, attention deficit disorder, autism, post-traumatic stress disorder (PTSD), anxiety disorder, sleep disorder, panic disorder, intellectual disability, memory loss, drug addiction, schizophrenia, obsessive-compulsive disorder, delusions of grandeur, personality disorder, alcoholism, or bipolar disorder.

[0066] In one embodiment, the developmental disorder may be epilepsy, cerebral palsy, developmental language disorder, sensory disturbance, learning disorder, attention deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), intellectual disability, cognitive impairment, or impulse control disorder.

[0067] The term "stem cell therapeutic agent" as used in the present invention refers to a therapeutic agent used for tissue regeneration therapy by using living autologous, allogenic, or xenogenic stem cells that are expanded and selected in vitro and then introduced into the body to restore cellular tissue and function.

[0068] In one aspect, the present invention relates to a transplant material for revascularization, which comprises the stem cells of the present invention or the cell therapy composition of the present invention as an active ingredient.

[0069] In one embodiment, the graft material may be a surgical implant, medical product, or medical device such as a film, membrane, sheet, rod, screw, anchor, pin, implant, stent, surgical suture, tissue regeneration scaffold, bionanofiber, hydrogel, biosponge, bone plate, and bone graft.

[0070] In one embodiment, when the implant is a hydrogel, it can be administered orally and used to promote revascularization of internal organs.

[0071] In one embodiment, the implant may further include a biodegradable polymer.

[0072] In one embodiment, the transplant material may be a composite scaffold for tissue engineering, which may comprise a scaffold prepared by molding a biodegradable polymer and containing the stem cells of the present invention or the cell therapeutic composition of the present invention.

[0073] In one embodiment, the transplant material may be a transplant material in which the stem cells or cell therapeutic agent of the present invention are seeded onto a composite scaffold for tissue engineering.

[0074] In one embodiment, when the implant material is a hydrogel, the polymer forming the hydrogel may be polyethylene glycol (PEG), polyethylene oxide (PEO), polyhydroxyethyl methacrylate (PHEMA), polyacrylic acid (PAA), polyvinyl alcohol (PVA), poly(N-isopropylacrylamide) (PNIPAM), polyvinylpyrrolidone (PVP), polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), gelatin, hyaluronic acid, alginate, carrageenan, chitosan, hydroxyalkyl cellulose, alkyl cellulose, silicone, rubber, agar, carboxyvinyl copolymer, polydioxolane, polyacrylacetate, polyvinyl chloride, or maleic anhydride / vinyl ether.

[0075] The term "biodegradable polymer" as used herein refers to a polymer that spontaneously decomposes gradually in vivo after a certain period of time and has one or more of the following properties: biocompatibility, blood affinity, anti-calcification properties, cell nutrition, and intercellular matrix formation. While the type of biodegradable polymer is not particularly limited in the present invention, representative examples include fibrin, collagen, gelatin, chitosan, alginate, hyaluronic acid, dextran, polylactic acid, poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly-ε-(caprolactone), polyanhydride, polyorthoester, polyvinyl alcohol, polyethylene glycol, polyurethane, polyacrylic acid, poly-N-isopropylacrylamide, poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) copolymer, copolymers thereof, and mixtures thereof. The composite support can be manufactured by molding a biodegradable polymer using a conventional method, such as a solvent-casting and particle-leaching technique, a gas-forming technique, a fiber extrusion and fabric-forming process, a thermally induced phase separation technique, an emulsion freeze-drying method, or a high-pressure gas expansion method.

[0076] In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating vascular diseases or vascular dysfunction, comprising the stem cells of the present invention or the cell therapy composition of the present invention as an active ingredient.

[0077] In one embodiment, the vascular disease may be traumatic vascular injury, peripheral vascular disease, cardiovascular disease, cerebrovascular disease, or ischemic disease, and the ischemic disease may be ischemic myocardial infarction, ischemic heart disease, ischemic vascular disease, ischemic enteritis, ischemic eye disease, ischemic glaucoma, ischemic renal failure, ischemic retinopathy, ischemic stroke, or ischemic lower limb disease.

[0078] In one embodiment, the vascular disease or vascular dysfunction may be critical limb ischemia, diabetic neovascularization disorder, vascular dementia, diabetic organ damage, arteriosclerosis, angina pectoris, peripheral cardiovascular disease, hypertension, cerebral infarction, sequelae of brain injury, heart failure, peripheral circulatory disorder, myocardial infarction, arterial occlusive disease, stroke, spinal cord injury, sequelae of spinal nerves, degenerative disease, sequelae of cerebral infarction, peripheral neuropathy, diabetic ulcer, presbyopia, age-related hearing loss, sequelae of brain surgery, ischemic stroke, or subarachnoid hemorrhage.

[0079] In one embodiment, the composition may induce angiogenesis or vasculogenesis, promote vascular regeneration or tissue regeneration, or improve vascular function, increase tissue perfusion and new blood vessel formation.

[0080] In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating a brain and nervous system disease, comprising the stem cell of the present invention or the cell therapy composition of the present invention as an active ingredient.

[0081] In one embodiment, the neurological disease may be a degenerative brain disease, a psychiatric disorder, or a developmental disorder.

[0082] In one embodiment, the degenerative brain disease may be cognitive impairment, Alzheimer's disease, dementia with Lewy bodies, frontotemporal dementia, Parkinson's disease, Creutzfeldt-Jakob disease (CJD), Huntington's disease, multiple sclerosis, or Guillain-Barré syndrome (GBS).

[0083] In one embodiment, the mental illness may be bipolar disorder, autism, depression, hyperactivity, attention deficit disorder, autism, post-traumatic stress disorder (PTSD), anxiety disorder, sleep disorder, panic disorder, intellectual disability, memory loss, drug addiction, schizophrenia, obsessive-compulsive disorder, delusions of grandeur, personality disorder, alcoholism, or bipolar disorder.

[0084] In one embodiment, the developmental disorder may be epilepsy, cerebral palsy, developmental language disorder, sensory disturbance, learning disorder, attention deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), intellectual disability, cognitive impairment, or impulse control disorder.

[0085] The pharmaceutical compositions of the present invention are administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment, without causing adverse effects. The effective dose level can be determined based on factors including the individual's health status, the type and severity of vascular disease or vascular dysfunction, the activity of the drug, sensitivity to the drug, the method, time, route and excretion rate of administration, duration of treatment, coadministered or concurrently used drugs, and other factors well known in the medical field. The compositions of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, in single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can achieve maximum effect at the minimum dose without adverse effects, which can be easily determined by one skilled in the art.

[0086] The pharmaceutical compositions of the present invention may contain carriers, diluents, excipients, or combinations of two or more thereof commonly used in biological preparations. The term "pharmaceutically acceptable" as used herein means that the composition is non-toxic to cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition. For example, compounds listed in the Merck Index, 13th ed., Merck & Co. Inc., saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture of one or more of these components may be used. Other common additives, such as antioxidants, buffers, and bacteriostatic agents, may be added as needed. Furthermore, the compositions may be formulated into commonly used dosage forms, such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets, by adding diluents, dispersants, surfactants, binders, and lubricants. Furthermore, formulations can be prepared as desired depending on the disease or component using methods suitable in the art or methods disclosed in Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton PA, 18th, 1990).

[0087] In one embodiment, the pharmaceutical composition may be in one or more dosage forms selected from the group including oral preparations, topical preparations, suppositories, sterile injectable solutions, and sprays.

[0088] The compositions of the present invention may also contain carriers, diluents, excipients, or combinations of two or more thereof commonly used in biological preparations. Pharmaceutically acceptable carriers are not particularly limited as long as they are suitable for in vivo delivery of the composition. For example, compounds listed in the Merck Index, 13th ed., Merck & Co. Inc., physiological saline, sterile water, Ringer's solution, buffered physiological saline, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture of one or more of these components may be used. Other common additives, such as antioxidants, buffers, and bacteriostatic agents, may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the compositions into commonly used dosage forms, such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets. Furthermore, the composition may be formulated according to a suitable method in the art or Remington's Pharmaceutical Sciences. (Mack Publishing Company, Easton PA, 18th, 1990) can be used to prepare formulations suitable for each disease or component.

[0089] The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions.

[0090] The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable additive, and in this case, examples of the pharmaceutically acceptable additive include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicone dioxide, calcium hydrogen phosphate, lactose, mannitol, candy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive according to the present invention may include: The content of the additive in the composition is preferably 0.1 to 90 parts by weight, but is not limited to this.

[0091] The composition of the present invention can be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally, or topically) according to the intended method, and the dosage range varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, severity of disease, etc. The daily dosage of the composition of the present invention is 0.0001 to 10 mg / ml, preferably 0.0001 to 5 mg / ml, and more preferably administered once or in divided doses per day.

[0092] In one aspect, the present invention relates to a method for producing stem cells that express the CD141 cell surface antigen, comprising culturing bone marrow-derived human bone marrow mononuclear cells (cryopreserved, BM-MNCs) or adipose-derived stroma vascular fraction (SVF) in a serum-free medium containing human platelet lysate.

[0093] In one aspect, the present invention relates to the use of stem cells expressing the CD141 cell surface antigen for use in revascularization or angiogenesis.

[0094] In one aspect, the invention relates to a method of revascularization or angiogenesis comprising administering to an individual stem cells that express the CD141 cell surface antigen.

[0095] In one aspect, the present invention relates to a method for treating a vascular disease or vascular dysfunction comprising administering stem cells expressing the CD141 cell surface antigen to an individual suffering from the vascular disease or vascular dysfunction.

[0096] In one aspect, the present invention relates to a method for treating a cranial nervous system disease, comprising administering stem cells expressing the CD141 cell surface antigen to an individual suffering from the cranial nervous system disease. DETAILED DESCRIPTION OF THE INVENTION

[0097] The present invention will be described in more detail with reference to the following examples, which are intended to illustrate the content of the present invention and are not intended to limit the present invention.

[0098] Example 1. Isolation of novel multipotent stem cells from bone marrow

[0099] 1-1. Optimal culture of vMSCs

[0100] Cryopreserved human bone marrow mononuclear cells (BM-MNCs) (STEMCELL Technologies, Vancouver, Canada; LONZA, Basel, Switzerland) were thawed at 37°C, or bone marrow aspirates obtained with patient consent were subjected to density gradient centrifugation using Ficoll-paque (Cytiva, MA, USA) to obtain BM-MNCs. The obtained BM-MNCs were placed in a tissue culture-treated flask using fetal bovine serum (FBS)-depleted EGM-2 bullet kit medium (Lonza; Basel, Switzerland) supplemented with 2% human platelet lysate (PL bioscience, GmbH, Germany) and 2 units / mL heparin (Sigma-Aldrich, St. Louis, MO, USA) (EGM-2-FBS + 2% hPL). 5 cells / cm 2The cells were then aliquoted at a density of 1.0 × 10 to 5.0 × 10 for primary culture for 7 to 12 days. During the primary culture period, the medium was changed every 1 to 4 days. Colonies of vMSCs (Vasculogenic Multipotent Stem Cells), the novel multipotent stem cells of the present invention, began to be observed from the 3rd to 5th day of culture. The vMSC cells forming the colonies observed during primary culture were spindle-shaped and varied greatly in length and size, and the colonies were observed to expand significantly between the 7th and 10th days. The first subculture of vMSCs was performed when the majority of colonies showed a density of 80 to 90% or higher. Similar to the general subculture method for adherent cells, the cells were separated into single cells using trypsin or a trypsin substitute and then placed in a new flask at a density of 1.0 to 5.0 × 10. 3 cells / cm 2 The vMSCs of the present invention are cultured using the basic components of the EGM-2 kit without any modification, and subcultured at a density of 70-90%, allowing for 5-7 passages. Unlike EPCs (endothelial progenitor cells), which are cultured in EGM-2 containing FBS, the vMSCs of the present invention are cultured by excluding FBS from EGM-2 and adding human platelet lysate (PL).

[0101] 1-2. Confirmation of extracellular matrix adhesion properties of vMSCs

[0102] The ECM complex was composed of human fibroblast-derived HumaTein (100 μg / mL, ROKIT Healthcare, Seoul, Republic of Korea), human type I collagen (0.2 μg / cm 2 ) (Corning, NY, USA), fibronectin (1 μg / cm 2We cultured vMSCs in culture flasks coated with various extracellular matrices (ECMs), including porcine collagen type 1-P (Cellmatrix Type IP) (Nitta Gelatin, Osaka, Japan), collagen type I from rat tail (50–825 μg / mL) (ThremoFisher, MA, USA), and collagen type 1 peptide (Corning). The cells constituting vMSC colonies appeared spindle-shaped across all coating materials, demonstrating their viability. Cultures on ECM-coated flasks yielded more colonies than standard TC-treated flasks, but the difference was not significant (Figure 1). While the recovery rate of primary cultures may vary depending on the presence or type of ECM coating, there was little difference in proliferation rates beyond P1.

[0103] When the novel vMSCs of the present invention are cultured using the FBS of the conventional EGM-2 bullet kit instead of human PL, they are unable to maintain the characteristics of CD141+ vMSCs, and the addition of human PL further increases CD141 expression. Therefore, it is essential to add human PL to the EGM-2 bullet kit (Lonza) and exclude FBS from the culture medium. Since there is no problem with growth even if IGF and VEGF are removed, IGF and VEGF can also be excluded.

[0104] Example 2. Analysis of marker characteristics of vMSCs

[0105] 2-1. Confirmation of EPC and vascular endothelial cell marker expression

[0106] The expression of conventional EPC and vascular endothelial cell markers in the vMSCs of the present invention was confirmed by flow cytometry, and the expression of eNOS and VE-cadherin, which are expressed in mature vascular endothelial cells, was confirmed by immunofluorescence staining analysis and compared with that of bone marrow mononuclear cells (BM-MSCs) and human umbilical cord endothelial cells (HUVECs), respectively. Specifically, for flow cytometry, vMSCs and BM-MSCs were resuspended in PEB buffer containing 2 mM EDTA and 0.5% bovine serum albumin (BSA) (Sigma-Aldrich), and then blocked with human FcR blocking reagent (Miltynyi Biotec) for 10 minutes at 4°C. Cells were then stained with Alexa flour 488-conjugated vWF antibody (Abcam, 1:500), fluorescein-labeled UEA-1 (Vectorlabs, 3:100), and APC (allophycocyanin)-conjugated antibodies against CD29, CD31, CD34, CD44, CD45, CD73, CD90, CD105, and CD309 (Miltenyl Biotec, 1:50). Isotype IgG conjugated with APC (Miltenyl Biotec, 1:50) and Alexa flour 488 (Abcam, 1:500) served as controls. After antibody staining, cells were washed with PEB buffer and centrifuged at 1,000 × g and 4°C for 5 minutes. Finally, cells were resuspended in PEB buffer and analyzed using a NovoCyte 3000 flow cytometer (Agilent Technologies, Santa Clara, CA, USA) and NovoExpress software. For immunofluorescence analysis, vMSCs and HUVECs were cultured on coverslips, fixed with 3.7% formaldehyde (Sigma-Aldrich), and then permeabilized with 0.2% Triton X-100.For blocking, the sections were incubated with 20% normal goat serum (NGS) at room temperature for 1 hour, followed by incubation overnight at 4°C with primary antibodies e-NOS (Cell Signaling Technology, MA, USA, 1:400) and VE-cadherin (Cell Signaling Technology, 1:400) diluted in 20% NGS. The next day, the sections were incubated with Alexa Fluor 488-conjugated anti-rabbit secondary antibody (Invitrogen, MA, USA, 1:1000) diluted in 20% NGS for 1 hour at room temperature. For sample observation, the sections were mounted with DAPI-containing mounting solution (Vector Laboratories, CA, USA) and images were captured using a Leica microscope.

[0107] Flow cytometry analysis showed that the vMSCs of the present invention express all known MSC markers, including CD105, CD90, CD29, CD73, and CD44, but do not express CD31, CD309, or CD34, which are commonly expressed by previously reported EPCs (Figure 2a). Furthermore, immunofluorescence staining analysis showed that the vMSCs of the present invention, unlike the positive control HUVECs, do not express eNOS or VE-cadherin (Figure 2c). This indicates that vMSCs share more markers with MSCs than with EPCs.

[0108] 2-2. Discovery of new markers specific to vMSCs

[0109] To discover specific markers for vMSCs of the present invention that distinguish them from BM-MSCs, we performed marker screening using MACS, which can analyze 378 surface antigen markers by flow cytometry. R The experiment was performed using Marker Screen, human, version 02 (Miltenyi Biotec) according to the manufacturer's instructions. Specifically, vMSCs (5.7 × 10) from the same donor at passage 3 were cultured. 7 ) and BM-MSCs (5.7 × 10 7) were resuspended in PEB buffer and blocked with human FcR blocking reagent (Miltenyi Biotec) for 10 minutes at 4°C. Then, 1.2 × 10 cells were added to four 96-well plates containing six isotype controls and 378 antibodies. 5 After dispensing at 1 / well, the mixture was incubated at 4°C for 20–30 minutes. After washing with PEB buffer and centrifuging at 300 × g for 5 minutes, the mixture was resuspended in PEB buffer at 200 μl / well and analyzed using a NovoCyte 3000 flow cytometer (Agilent Technologies) equipped with a NovoSampler (Agilent Technologies) and NovoExpress software.

[0110] Marker screening identified approximately 21 markers that were differentially expressed between vMSCs and BM-MSCs (Figure 3a). To validate these markers, we performed flow cytometry for CD141, CD282, and PEAR1 (Platelet Endothelial Aggregation Receptor 1) on vMSCs and BM-MSCs derived from more than 10 donors using APC-conjugated CD141, CD282, and PEAR1 (Miltenyi Biotec, 1:50). The results showed that CD141 was consistently expressed in 70–99% of vMSCs but not in BM-MSCs (Figure 3b). Furthermore, CD282 was not expressed in BM-MSCs, but was expressed in vMSCs at low levels (30%) and high levels (90%), demonstrating significant donor-to-donor variability (Figure 3c). Furthermore, PEAR1 expression was found to be 70-99% in vMSCs, but in BM-MSCs, expression ranged from as low as 20% to as high as 90%, demonstrating significant donor-to-donor variability (Figure 3d).This demonstrates that CD141 is a clear marker for distinguishing vMSCs from BM-MSCs.

[0111] Example 3. Analysis of cell expansion capacity of vMSCs

[0112] CD141+ To evaluate the cell expansion capacity of vMSCs in comparison with BM-MSCs, CD141 cells were cultured in more than 20 donors using the method described in Example 1. + vMSCs were cultured up to passage 3 and transfected with CD141 + BM-MNCs were cultured simultaneously with primary vMSC culture using StemMACS TM expansion media, 4.0~7.0×10 in XF (Miltenyi Biotec, Bergisch Gladbach, Germany) (StemMACS) medium 4 cells / cm 2 After dispensing, CD141 + The primary culture was performed for the same period as vMSCs, and the subculture was performed using CD141 + This was carried out at the same time as vMSC. + PDT (population doubling time) for each passage of vMSCs and BM-MSCs and the number of primary cultured MNCs were 1 × 10 7 The cells were fixed in a centrifuge tube and the cumulative cell number was measured up to the third passage.

[0113] As a result, the average PDT from P1 to P3 was CD141 + For vMSCs, it was 0.84 days (20 hours), and for BM-MSCs, it was 24 days (30 hours). + The vMSCs were shown to divide approximately 1.5 times faster (Figures 4a and 4b). + For vMSCs, 3.0 × 10 cells were cultured within the third passage (days 19–22). 10 In the case of BM-MSCs, approximately 1.8 x 10 cells were obtained. 9 It was shown that CD141 cells were obtained (Fig. 4C and d). + We were able to obtain 16 to 17 times more cells from vMSCs than from BM-MSCs, demonstrating that these cells have excellent cell expansion capacity (Figure 4e).

[0114] Example 4. Analysis of multipotency of vMSCs

[0115] 4-1.Adipose differentiation characteristics

[0116] CD141 + To confirm the multipotency of vMSCs, we cultured them in a 3rd passage CD141 + vMSCs and BM-MSCs were cultured in adipogenesis-inducing medium. Specifically, CD141 + vMSCs were grown in EGM-2-FBS + 2% hPL medium, and BM-MSCs were grown in StemMACS medium at 1.0x10 3 / cm 2 Once the cells reached nearly 100% density, they were cultured in StemPro, an adipogenesis-inducing medium. TM The medium was replaced with Adipogenesis Differentiation Kit (Gibco) medium, and cultured for 14 days with fresh medium every 3–4 days. After fixing each cell type with 3.7% formaldehyde (Sigma-Aldrich), the cells were incubated with 60% isopropanol (Daejung, Sihueng, Republic of Korea) for 5 minutes at room temperature. They were then stained with 0.3% Oil Red O (Sigma-Aldrich) solution containing 60% isopropanol for 20 minutes at room temperature. After washing with distilled water, the cells were imaged under a microscope (Nikon, Tokyo, Japan). The CD141 + vMSCs were shown to differentiate into fat, similar to MSCs (Figure 5).

[0117] 4-2.Osteodifferentiation characteristics

[0118] CD141 + To confirm the multipotency of vMSCs, we cultured them in a 3rd passage CD141 + vMSCs and BM-MSCs were cultured in osteogenic differentiation medium. Specifically, CD141 + vMSCs were grown in EGM-2-FBS + 2% hPL medium, and BM-MSCs were grown in StemMACS medium at 1.0x10 3 / cm 2 Once the cells reached a density of 80-90%, they were cultured in StemPro, a bone differentiation-inducing medium. TMThe medium was replaced with fresh medium from the Osteogenesis Differentiation Kit (Gibco) and cultured for 21 days, with a fresh medium change every 3–4 days. After fixing each cell type with 3.7% formaldehyde (Sigma-Aldrich), the fixed cells were stained with 2% Alizarin Red S (Sigma-Aldrich) solution at room temperature for 20 minutes. After washing with distilled water, the cells were imaged under a microscope (Nikon, Tokyo, Japan). The CD141 + vMSCs were shown to undergo osteogenic differentiation, similar to MSCs (Figure 5).

[0119] 4-3. Cartilage differentiation characteristics

[0120] CD141 + To confirm the multipotency of vMSCs, we cultured them in a 3rd passage CD141 + vMSCs and BM-MSCs were cultured in chondrogenic differentiation medium. + vMSCs were grown in EGM-2-FBS + 2% hPL medium, and BM-MSCs were grown in StemMACS medium at 1.0x10 3 / cm 2 Once the cells reached nearly 100% density, they were cultured in StemPro, a chondrogenic differentiation medium. TM The medium was replaced with fresh medium from the Chobdrogenesis Differentiation Kit (Gibco) and cultured for 21 days, with a fresh medium change every 3-4 days. After fixing each cell type with 3.7% formaldehyde (Sigma-Aldrich), the fixed cells were incubated with 3% acetic acid (Sigma-Aldrich) at room temperature for 3 minutes, and then stained with 1% alcian blue at room temperature for 30 minutes. After washing with distilled water, the cells were imaged under a microscope (Nikon, Tokyo, Japan). The CD141 + vMSCs were shown to differentiate into cartilage, similar to MSCs (Figure 5).

[0121] As a result, the novel CD141 of the present invention +It was confirmed that vMSCs possess multipotency, a property not previously reported for EPCs, and that, like MSCs, they can differentiate into fat, bone, and cartilage.

[0122] Example 5. Analysis of the revascularization potential of vMSCs

[0123] CD141 prepared in the above example + To compare the vascular regeneration potential of vMSCs with that of BM-MSCs and mature endothelial cells (HUVECs), a Matrigel tube formation assay was performed. Specifically, Matrigel (Corning Inc., NY, USA) was applied to μ-slides (ibidi, Grafelfing, Germany) at 10 μl per well and CD141 was added. + vMSCs, BM-MSCs, and HUVECs were suspended in growth factor-deficient α-MEM (GIBCO, NY, USA) containing 0.2% human phospholipase A (hPL) or EGM2 medium containing proangiogenic growth factors such as VEGF and FGF, respectively, and plated onto Matrigel at 6,000 cells / well. They were then cultured overnight (more than 16 hours) and imaged using a microscope (Leica, Wetzlar, Germany).

[0124] As a result, BM-MSCs were unable to form a vascular network structure in either medium. HUVECs, which are vascular endothelial cells, formed a vascular network structure very well in EGM2 medium containing angiogenic growth factors, but were unable to maintain a vascular network structure in MEMα + 0.2% hPL medium, which does not contain angiogenic growth factors. + In the case of vMSCs, it was shown that they formed very large and thick vascular networks in EGM2 medium, while they formed small and numerous vascular networks in MEMα + 0.2% hPL medium (Figure 6). + vMSCs have been shown to have angiogenic potential even in a medium lacking angiogenic growth factors.

[0125] Example 6. Analysis of the ability of vMSCs to secrete angiogenic growth factors

[0126] 6-1. Analysis of HGF secretion ability of vMSCs

[0127] In order to confirm not only the wound healing but also the secretion ability of HGF (Hepatocyte growth factor), a potent angiogenic growth factor, ELISA (enzyme-linked immunosorbent assay) analysis was performed. Specifically, as in the previous example, CD141 + vMSCs and BM-MSCs were cultured in their respective culture media (EGM-2-FBS + 2% hPL and StemMACS) in a 6-well plate, 9 × 10 4 The cells were aliquoted at a concentration of 1000 cells / well. The next day, the medium was replaced with growth factor-free MEMα + 0.2% hPL to eliminate differences due to medium, and the cells were cultured for 48 hours. The culture medium was then collected and analyzed for HGF secretion by measuring absorbance at 450 nm using a microplate reader (Molecular Device, SpectraMax ABS) according to the manufacturer's instructions using Quantikine HGF ELISA (R&D Systems, Minneapolis, USA). The absorbance was then divided by the cell number to determine the amount of HGF secretion per cell. The cells were cultured in this manner until the fourth passage, and the culture supernatant was collected immediately before subculture to analyze the level of HGF secretion.

[0128] CD141 of the same passage + Comparison of vMSCs and BM-MSCs revealed that CD141 + vMSCs were shown to secrete 14-130 times more HGF than BM-MSCs (Fig. 7a and b), and under the same conditions, CD141 + It was shown that vMSCs secreted approximately 9 times more HGF than BM-MSCs (Fig. 7c).

[0129] 6-2. Analysis of HGF receptor expression in vMSCs

[0130] As in the previous example, CD141 +vMSCs and BM-MSCs were cultured and lysed at various passages using 2 mM PMSF (phenylmethylsulphonyl fluoride, Sigma-Aldrich) and lysis buffer (Cell Signaling Tech.). The cells were centrifuged at 14,000 × g for 10 minutes at 4°C, and the supernatant was collected to obtain protein lysates. Protein lysates were adjusted to the same concentration as determined by BCA analysis (Thermo Fisher Scientific) and separated by SDS-PAGE and transferred to a nitrocellulose membrane. After blocking with 5% skim milk in TBS-T for 1 hour at room temperature, the membranes were incubated overnight at 4°C with antibodies against c-Met (Cell Signaling Tech., 1:1000), phosphor-c-Met (Cell Signaling, 1:1000), and α-tubulin (Sigma-Aldrich, 1:4000). The next day, the membrane was reacted with an HRP-conjugated secondary antibody at room temperature for 1 hour. After the antibody reaction was completed, the membrane was treated with EZ-western Lumi pico (Dogen, Seoul, Korea) to detect light. The signal was then detected using a Chemiluminator and quantified using ImageJ software to confirm the expression level and activation (phosphorylation) of the HGF receptor c-Met at each passage.

[0131] As a result, CD141 + vMSC cells (CD141+ vMSCs) were shown to express significantly more HGF receptor (c-Met) than BM-MSC cells (Figures 7d and 7e), and no difference in activity was observed between the two cells (Figures 7d and 7f). + vMSCs not only secrete large amounts of HGF themselves, but also express large amounts of the HGF receptor (c-Met), confirming that they can use HGF as an autocrine factor.

[0132] 6-3. Confirmation of angiogenic mechanism

[0133] 6-3-1. Confirmation of neovascularization ability by autocrine factors

[0134] CD141 + To confirm the mechanism of angiogenic action of vMSCs, we analyzed their angiogenic sprouting capacity by HGF in comparison with BM-MSCs. + vMSC spheroids and BM-MSC spheroids were generated using the hanging drop method, respectively, and then treated with HGF or an HGF neutralizing antibody to inhibit HGF, followed by an in vitro spheroid sprouting assay. + vMSCs and BM-MSCs 2x10 4 CD141 cells / mL were suspended in M199 medium containing 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA), 1% P / S, and 0.2% methylcellulose (Sigma-Aldrich, St. Louis, MO, USA). The cells were inoculated in droplets onto the lid of a Petri dish at a volume of 30 μL. The lid of the inoculated Petri dish was then inverted and incubated at 37°C in a 5% CO2 incubator for 24 hours to allow spheroid formation. CD141 cells were formed after 24 hours of incubation. +vMSC and BM-MSC spheroids were mixed with 3 mg / mL collagen type IA (final concentration: 1.44 mg / mL; Nitta Gelatin) and methylcellulose (final concentration: 0.48%; Sigma-Aldrich) (collagen gel mixture), and 0.7 mL of each mixture was dispensed into a 24-well plate. The collagen gel mixture containing the spheroids was polymerized for 30 minutes in a 37°C, 5% CO2 incubator. For the HGF neutralizing antibody-treated group, 500 ng / mL of HGF neutralizing antibody (MAB294, R&D Systems; Anti-HGF Ab) was added to the collagen gel mixture containing the spheroids and then polymerized (3 hours before HGF treatment). After polymerization, HGF (Peprotech, New Jersey, USA) and / or HGF neutralizing antibody (control: 500 ng / mL mouse IgG, IgG) were diluted in M199 (Sigma-Aldrich) medium and applied in 100 μL portions onto the collagen gel mixture. After 48 hours of culture, spheroid sprouts were photographed under a Nikon microscope. The number of spheroids, average sprout length, and total sprout length were measured using ImageJ software (NIH).

[0135] Quantitative analysis of angiogenesis showed that HGF treatment significantly increased vascular sprouting in the vMSC spheroids of the present invention, whereas simultaneous treatment with HGF and an HGF neutralizing antibody reduced vascular sprouting (Figure 8a).On the other hand, treatment with HGF or its neutralizing antibody did not affect vascular sprouting in the BM-MSC (MSCs) spheroids (Figure 8b).

[0136] In other words, unlike BM-MSCs, which have HGF-independent sprouting ability, CD141 + vMSCs have HGF-dependent sprouting ability, secrete large amounts of HGF themselves, and express large amounts of c-Met, indicating that they can germinate using HGF as an autocrine factor.

[0137] 6-3-2. Comparison of angiogenesis by BM-MSC-secreted bFGF

[0138] CD141+ To confirm the mechanism of angiogenesis of vMSCs, BM-MSCs and / or bFGF receptor inhibitor PD173074 (Tocris, Bristol, UK) were diluted in M199 medium (Sigma-Aldrich) during in vitro spheroid sprouting assay as in Example 6-3-1. 100 μl of each solution was applied to the collagen gel mixture and cultured for 48 hours. + The effect of bFGF secreted by BM-MSCs on vMSC spheroid sprouting was examined.

[0139] As a result, CD141 + Compared with the untreated group (NT), vMSC spheroids were significantly more sensitive to CD141 + When vMSC spheroids were cultured with BM-MSCs, CD141 + The blood vessel sprouting ability of vMSCs was improved (Figure 9). Treatment with PD173074, a bFGF receptor inhibitor, improved CD141 expression by BM-MSCs. + It was shown that the vascular sprouting ability of vMSCs was reduced (Figure 9).

[0140] That is, the bFGF receptor inhibitor PD173074 suppressed the paracrine effect of BM-MSCs on the vascular sprouting of vMSCs, and the CD141 + It was speculated that a synergistic effect on angiogenesis would occur when vMSCs were used in combination with HGF, bFGF, or BM-MSCs.

[0141] 6-3-3. Confirmation of the ability of VEGF to form new blood vessels

[0142] CD141 + To confirm the mechanism of angiogenic action of vMSCs, we investigated the effects of CD141 on VEGF, an important angiogenic factor. +We confirmed whether the angiogenic potential of vMSC spheroids and BM-MSC spheroids differed by in vitro spheroid sprouting assay as described above, and compared VEGFR2 mRNA expression in BM-MSCs and vMSCs with that in HUVEC cells, a type of EPC.

[0143] As a result, CD141 + vMSC spheroids and BM-MSC spheroids did not respond (Fig. 10a-d), which is consistent with the results of BM-MSC and CD141 + Consistent with the results that vMSCs do not express VEGF receptors (Fig. 10e), bFGF treatment also increased the expression of CD141 + It was shown that vMSC spheroids had improved angiogenesis, but BM-MSC spheroids did not (Figures 10a-d).

[0144] This suggests that VEGF promotes sprouting of EPCs and endothelial cells, but CD141 + It was found that vMSCs do not express VEGFR2 (VEGF receptor 2) and do not promote vascular sprouting by VEGF. + This is clearly a characteristic of vMSCs.

[0145] 6-3-4. Confirmation of the paracrine angiogenic effect of vMSC-secreted HGF

[0146] To confirm the paracrine effect of vMSCs in promoting angiogenesis in vascular endothelial cells, HUVEC cells were cultured as spheroids using the hanging drop method and co-cultured with vMSCs in a non-contact manner, as in Example 6-3-1. To neutralize HGF, a protein secreted by vMSCs, a spheroid sprouting assay was performed. To neutralize HGF, a protein secreted by vMSCs, 500 ng / mL of an HGF neutralizing antibody (MAB294, R&D Systems; Anti-HGF Ab) was mixed with M199 medium. 100 μL of this mixture was applied to the collagen gel containing polymerized HUVEC spheroids and cultured for 24 hours. Mouse IgG (IgG) (500 ng / mL) was used as a control. Images of spheroid sprouting were quantitatively analyzed using ImageJ software (NIH) for the number of sprouts, cumulative sprout length (the sum of all sprout lengths formed in one spheroid), and average sprout length (the average of all sprout lengths formed in all spheroids).

[0147] As a result, CD141 + Non-contact co-culture with vMSCs showed a sprouting-promoting effect on HUVEC spheroids, and treatment with an HGF neutralizing antibody inhibited the HUVEC sprouting effect (Figure 11). This confirmed that HGF, a secreted protein of vMSCs, has a cell sprouting effect on vascular endothelial cells (HUVECs), i.e., it is a substance that promotes angiogenesis.

[0148] Therefore, bone marrow-derived vMSCs secrete excessive amounts of HGF, which is associated with angiogenesis, and this has a paracrine effect of promoting new blood vessel sprouting in HUVECs.

[0149] Example 7. Comparison of vMSCs and EPCs

[0150] The bone marrow-derived CD141 of the present invention was confirmed by the above results. + As a result of comparing vMSCs with peripheral blood- or umbilical cord blood-derived EPCs, the CD141 +Although vMSCs and EPCs have similar functional characteristics, they are completely different in culture conditions, cell morphology, and cell surface marker expression. In particular, CD309 and CD31, which are expressed in all types of EPCs, are different from CD141 of the present invention. + In addition, unlike EPCs, whose sprouting is promoted by VEGF, the CD141 gene of the present invention was not expressed in vMSCs. + vMSCs were not stimulated to germinate by VEGF and did not express VEGF receptors. + vMSCs possess multipotency, which has not been reported in conventional EPCs. + Although vMSC cells are functionally similar to commonly known EPCs, they are a completely different type of cell from EPCs (Figure 12).

[0151] According to the above examples, the novel stem cell CD141 identified in the present invention + Unlike EPCs, vMSCs do not express CD31, CD309, or CD34; unlike vascular endothelial cells, they do not express eNOS or VE-cadherin; unlike mesenchymal stem cells, they express CD141; compared to mesenchymal stem cells, they overexpress its receptor cMET, and have pluripotency and angiogenesis-promoting effects. Therefore, we have identified vMSCs as CD141. + The cells were named vasculogenic mesenchymal stem cells and deposited at the Korea Cell Line Research Foundation (KCLRF) with the accession number KCLRFBP00524.

[0152] Example 8. Combination of bone marrow-derived vMSCs and BM-MSCs

[0153] 8-1. Search for combinations that maximize vascular regeneration potential in normal environments

[0154] CD141 +To maximize the vascular regeneration potential of vMSCs, BM-MSCs were added. To confirm the optimal mixture ratio of the two cell types, we performed a Matrigel vascular network formation assay. Specifically, Matrigel was dispensed onto a µ-slide at 10 µl per well and allowed to gel at 37 °C for 30 minutes. CD141 + vMSCs and BM-MSCs were mixed in α-MEM containing 0.2% human pluripotent phospholipids (MEMα + 0.2% human pluripotent phospholipids) at the following cell number ratios: 1:0 (6,000 + 0), 9:1 (5,400 + 600), 2:1 (4,000 + 2,000), 1:1 (3,000 + 3,000), 1:2 (2,000 + 4,000), 1:9 (600 + 5,400), and 0:1 (0 + 6,000). Then, the mixture was plated on top of Matrigel and cultured for 16 hours. Images of each group were taken under a microscope (Nikon), and the total length, number of meshes, number of master segments, total master segment length, and number of isolated segments were used as evaluation indices using the Angiogenesis analyzer in ImageJ software.

[0155] Microscopic observations showed that there were differences in vascular network (Tube) formation depending on the cell composition ratio. Specifically, BM-MSCs alone could not form vascular networks, and the ratio of BM-MSCs containing CD141 + The higher the density of vMSCs, the more likely they were to aggregate (Fig. 13a). +Although vMSC cells alone exhibited excellent in vitro vascular network formation ability, a coarse structure was observed. However, the addition of BM-MSCs to these cells resulted in the formation of a tighter and more mature vascular network (Figure 13b). Analysis of the evaluation indices revealed no significant differences in total length, number of meshes, number of master segments, or total master segment length. However, the number of isolated segments, which indicates segments that could not be connected anywhere and is an indicator of immature blood vessels, was lowest in the 2:1 group among the three groups, with no differences in the remaining indices (Figure 13c).

[0156] That is, CD141 in normal environments + Although vMSCs alone have excellent in vitro angiogenesis ability, we confirmed that mixing them with BM-MSCs at an appropriate ratio resulted in the formation of a tighter and more mature vascular network.

[0157] 8-2. Search for combinations that maximize revascularization potential in inflammatory environments

[0158] Considering that stem cell therapeutic agents are administered in an inflammatory environment, we performed a Matrigel vascular network formation analysis in an inflammatory environment. To mimic the inflammatory environment, we used a medium supplemented with 500 pg / mL of TNF-α. CD141 + vMSCs and BM-MSCs were mixed and suspended in MEMα + 0.2% hPL supplemented with TNF-α 500 pg / mL at the following ratios: 1:0 (6,000 + 0 cells), 9:1 (5,400 + 600 cells), 2:1 (4,000 + 2,000 cells), 1:1 (3,000 + 3,000 cells), 1:2 (2,000 + 4,000 cells), 1:9 (600 + 5,400 cells), and 0:1 (0 + 6,000 cells). + A total of nine groups were compared, including vMSCs and BM-MSCs suspended in MEMα+0.2% hPL without 500 pg / mL TNF-α.

[0159] Microscopic observations showed that there were differences in vascular network formation depending on the cell composition ratio.+ vMSCs formed good vascular networks even when used alone in a normal environment, but their ability to form vascular networks was shown to be reduced in an inflammatory environment. However, when BM-MSCs were added, they were shown to form good vascular networks, and as in the normal environment, aggregation was observed when the ratio of BM-MSCs increased (Figure 14a). Furthermore, in an inflammatory environment, CD141 + When vMSC cells were present alone, the total length was shortened, and the number of master segments and mesh formation, which are indicators of mature blood vessels, decreased most rapidly, while the number of isolated segments, which are indicators of immature blood vessels, increased dramatically (Figure 14b). + When vMSCs and BM-MSCs were mixed at a 2:1 ratio, the length of blood vessels and mesh formation were maintained at a high level even under inflammatory conditions, and the number and length of master segments, in particular, were significantly increased compared to those under normal conditions. + The level was maintained at a level comparable to that of the vMSC alone group, and the isolated segment length was significantly longer than that of normal CD141 + This was observed at a lower level than in the vMSC alone group (Fig. 14b).

[0160] In other words, CD141 is essential for tight vascular formation in an inflammatory environment. + The combined use of vMSCs and BM-MSCs was found to be advantageous.

[0161] Example 9. Adipose-derived vMSCs

[0162] 9-1. Isolation of adipose tissue-derived vMSCs

[0163] Stem cells from mesodermal tissue in adipose tissue can generate bone, cartilage, muscle, and adipose tissue. Adipose tissue-derived stem cells (ASCs) have the characteristics of bone marrow mesenchymal stem cells (e.g., cell morphology, expression markers, secreted cytokines), and are currently used as an alternative source to bone marrow due to their advantages of higher initial tissue yield compared to bone marrow and the ability to transplant cells at earlier passages. Therefore, adipose-derived vMSCs were isolated as described in Example 1. Specifically, adipose tissue (approximately 1.5 g) was collected from a patient, chopped with scissors, and then dissociated into single cells using collagenase I. These were then centrifuged to obtain stromal vascular fraction (SVF). These were cultured on Humatein (Rokit Healthcare)-coated culture dishes in FBS (fetal bovine serum)-deficient EGM2 (LONZA) + 2% PL (Platelet lysate, BioScience GmbH) medium to produce adipose-derived CD141 cells. + vMSCs were obtained (Figure 15). For comparison, adipose-derived MSCs (AD-MSCs) were also obtained by culturing single cells isolated from adipose tissue in HUMA medium (Rokit Healthcare).

[0164] 9-2. Analysis of cell morphology of adipose-derived vMSCs

[0165] Adipose-derived CD141 obtained in Example 9-1 + Observation of the cell morphology of vMSCs and AD-MSCs at different passages revealed that both types of cells exhibited the spindle shape typical of MSCs, but the CD141 + It was shown that vMSCs have a longer and sharper tip (Figure 16).

[0166] 9-3. Analysis of cell expansion capacity of adipose-derived vMSCs

[0167] Adipose-derived CD141 + When vMSCs and AD-MSCs were passaged from the first to third passages (2.16 × 10 6The cumulative number of cells obtained by dispensing was confirmed. 9 Adipose-derived CD141 + vMSCs were obtained (Figure 17).

[0168] 9-4. Analysis of cell surface markers of adipose-derived vMSCs

[0169] Adipose-derived CD141 + Flow cytometry analysis of cell surface markers in vMSCs and AD-MSCs revealed that both types of cells expressed CD34 - , CD45 - , CD29 + , CD44 + , CD73 + and CD90 + The expression of CD141 and PEAR1 is characteristic of CD141 + It was shown that the expression of these two markers was high in vMSCs (EGM2-FBS+2% hPL) and low in AD-MSCs (HUMA) (Figure 18). + We confirmed that this can be used as an important marker to distinguish between vMSCs and AD-MSCs.

[0170] 9-5. Analysis of the angiogenic potential of adipose-derived vMSCs

[0171] Adipose-derived CD141 in an inflammatory environment to mimic the post-transplant environment + To confirm the angiogenic potential of vMSCs, adipose-derived CD141 + vMSCs and AD-MSCs were mixed alone (6,000 cells) or at a 2:1 ratio (4,000 vMSCs + 2,000 AD-MSCs), and then added to Matrigel with 1% human pluripotent lumen (hPL) and 500 pg / ml TNF-α. The vascular morphology that emerged after 24 hours was compared. The results showed that AD-MSCs alone had low angiogenic potential and the cells aggregated, whereas adipose-derived CD141 + vMSC alone or CD141 + When vMSCs and AD-MSCs were mixed at a 2:1 ratio, the angiogenic potential was enhanced compared to when AD-MSCs were cultured alone, and in particular, CD141+ A 2:1 mixture of vMSCs and AD-MSCs demonstrated the best angiogenic potential (Figure 19).

[0172] Example 10. Establishment of an animal model of severe limb ischemia

[0173] Bone marrow-derived CD141 + To confirm the efficacy of a combined stem cell therapy using vMSCs and BM-MSCs in treating critical limb ischemia, we created an animal model of critical limb ischemia. Model 1 (Figure 20) involved ligating the common and upper superficial femoral arteries with Silk. While blood flow recovered over time in most mice, this model was deemed inappropriate for observing angiogenesis and evaluating efficacy. Therefore, Model 2 (critical limb ischemia, CLI) was created by ligating both the common and superficial femoral arteries at three locations and then removing the superficial femoral artery. Most mice showed severe necrosis and tissue loss, as well as a decrease in blood flow over time (Figure 20).

[0174] Example 11: Confirmation of the effect of combined stem cells on an animal model of severe limb ischemia

[0175] 11-1. Evaluation of foot preservation rate

[0176] CD141 in the severe hindlimb ischemia mouse model (Model 2) prepared in Example 10 + The efficacy of vMSC + BM-MSC as a combined stem cell therapy was evaluated, and the effects were observed for up to 24 weeks. + The vMSC + BM-MSC (cell number 2:1) administration group (G1) was established, and immediately after the severe limb ischemia model was created, 1.2 × 10 5 The insulin was administered once into the muscle at the site of ischemia (the site of blood vessel removal) using an insulin syringe.

[0177] As a result, the final condition of the feet was observed 24 weeks after model creation. The foot preservation rate was 0% in the control group, and most individuals lost their feet / lower limbs, while the G1 group showed limb salvage of approximately 63% or more (Figure 21).

[0178] 11-2. Evaluation of ischemic lesion necrosis grade

[0179] In a mouse model of severe lower limb ischemia, the compound was administered in the same manner as in Example 11-1, and then the degree of ischemic necrosis at the site of ischemia was evaluated on a 7-point scale.

[0180] As a result, after model creation, a statistically significant difference was observed in the degree of ischemic lesion necrosis in the G1 group compared to the G0 group on days 1 and 2 after administration, with a significant difference beginning to appear at week 1 and continuing through week 24 (Figure 22) (week 1, G0: 4.3±0.8 vs. G1: 1.5±0.8, p<0.05; and week 24, G0: 4.9±0.4 vs. G1: 1.9±0.9, p<0.05).

[0181] 11-3.Analysis of changes in blood flow

[0182] In a mouse model of severe hindlimb ischemia, the blood flow rate was compared between the contralateral side and the ischemic side after administration as described in Example 11-1. The blood flow rates after the creation of the mouse model of severe hindlimb ischemia were expressed as a ratio (%). The blood flow rates were G0: 52.4±4.9 and G1: 47.3±1.4, confirming a rapid decrease in blood flow in both groups. However, from day 3, the G1 group began to show a significant improvement in blood flow compared with the G0 group, and this statistically significant improvement was maintained up to 24 weeks (Figure 23) (3 days, G0: 37.2±4.9 vs. G1: 70.8±14.3, p<0.05; and 24 weeks, G0: 11.9±6.3 vs. G1: 69±15.9, p<0.01). This suggests that CD141 +The combination of vMSCs and BM-MSCs was shown to have an effective effect on severe limb ischemia, and the blood flow recovered from day 3 to week 1 was maintained at the same level up to week 24.

[0183] 11-4. Determining the minimum effective dose

[0184] CD141 for critical limb ischemia + To determine the effective dose of the combined stem cell therapy agent consisting of vMSCs and BM-MSCs and to determine the minimal effective dose, a control group (G0), a high-dose group (1.2 × 10 6 cells / 50μL, 24,000 / μL) (G1), medium dose group (1.2×10 5 cells / 50 μL, 2,400 / μL (G2) and the low-dose group (1.2 × 10 4 The rats were divided into two groups: G1 and G2 (G3) and administered 1000 mg / kg of 1000 cells (G3). Four weeks after model creation (D0), the final foot condition was observed. The control group had a 0% foot preservation rate and almost all foot / leg loss was observed, whereas the G1 and G2 groups showed high foot preservation rates of 42.9% and 75%, respectively (Figure 24). The G3 group showed a relatively favorable foot preservation rate of 14.3% and phalangeal loss of 28.6% (Figure 24). Furthermore, the ischemic necrosis grading and blood flow measurement, which are efficacy evaluation indicators, showed statistically significant effects in the high- and medium-dose groups compared to the control group (Figure 25). While these beneficial effects were not dose-dependent, the high dose demonstrated sufficient efficacy. Therefore, the effective doses were confirmed to be the high and medium doses, and the minimum effective dose was determined to be 1.2 × 10 5 It was determined to be cells.

[0185] Example 12. Comparison of the effects of administering combined stem cells to administering single stem cells

[0186] 12-1. Evaluation of foot preservation rate

[0187] CD141 in a mouse model of critical limb ischemia+ Administration of combined vMSC and BM-MSC stem cells significantly increased the expression of each CD141 + To confirm whether vMSCs and BM-MSCs are superior to single stem cell administration, a control group (G0), CD141 + vMSC+BM-MSC group (G1), CD141 + The vMSC-only group (G2) and the BM-MSC group (G3) were set at 1.2 × 10 5 Cells were administered at a dose of 100 mg / kg / day.

[0188] The final condition of the feet was observed 4 weeks after model creation (DAY 28). In the control group, the foot preservation rate was 0%, and most individuals lost their feet / lower limbs. In the G1 group, the foot preservation rate was approximately 87.5% or more, and in the G2 group, the foot preservation rate was 25% and the phalange loss was 25%, showing relatively good results. In the G3 group, the foot preservation rate was 0% and the phalange loss was 25% (Figure 26).

[0189] 12-2. Evaluation of ischemic lesion necrosis grade

[0190] Each group in Example 12-1 was evaluated on a 7-point scale according to the severity of ischemic necrosis at the site of ischemia. No statistically significant differences were observed between the groups at model creation (time 0) or 1 day after administration (time 1). However, from day 2, a statistically significant difference began to appear in the G1 group compared to the G0 group, and this significant difference was maintained until day 28 (Figure 27) (2 days after administration, G0: 3.5±0.2 vs. G1: 1.0±0, P<0.001; and 28 days, G0: 4.8±0.3 vs. G1: 0.4±0.4, P<0.001). Furthermore, when comparing the combined and single administrations, the G1 group showed statistically significant results compared with the G3 group from days 3 to 28 (day 3, G1 vs. G3: 2.9 ± 0.4, P < 0.001; and day 28, G1 vs. G3: 4.3 ± 0.5, P < 0.001). When compared with the G2 group, statistically significant differences were observed on days 3, 14, and 28 (Figure 27) (day 3, G1 vs. EPC G2: 2.2 ± 0.3, P < 0.001; and day 28, G1 vs. G2: 2.7 ± 0.6, P < 0.01).

[0191] 12-3.Analysis of changes in blood flow

[0192] In each group in Example 12-1, blood flow was compared between the contralateral side and the ischemic side, and the results were expressed as a ratio (%). It was confirmed that blood flow decreased rapidly after the CLI model was established. Meanwhile, from day 7, the G1 group showed a statistically significant improvement in blood flow compared to the G0 group, and this statistically significant improvement was maintained until day 28 (Figure 28) (day 7, G1: 79.4±6.8 vs. G0: 11.8±1.9, p<0.001; and day 28, G1: 104.6±5.5 vs. G0: 19.4±4.0, p<0.001). Furthermore, when comparing combined and single administration, the G1 group showed statistically significant improvement from days 7 to 28 compared to the G3 group, and a statistically significant difference was observed on day 28 when compared to the G2 group (Figure 28) (day 7, G1: 79.4±6.8 VS G3: 28.8±11.0, p<0.05; day 28, G1: 104.6±5.5 VS G3: 33.6±10.1, p<0.001, G2: 53.9±9.3, p<0.001).

[0193] This allows CD141 + We confirmed that administration of a combined stem cell system consisting of vMSCs and BM-MSCs has a superior therapeutic effect on severe limb ischemia compared to administration of either cell type alone.

[0194] Example 13. Analysis of combined stem cell angiogenesis in vivo

[0195] 13-1. Confirmation of angiogenesis (naked eye)

[0196] CD141 expression at the ablated site of ischemia in a mouse model of severe hindlimb ischemia + vMSCs and BM-MSCs combined (CD141 + Four weeks after administration of vMSCs + BM-MSCs, the presence or absence of angiogenesis in vivo was observed.

[0197] The results showed that in normal mice without severe limb ischemia, the femoral arteries were clearly visible and the muscle tissue was well preserved, while in control mice with severe limb ischemia, severe inflammation and damage to the surrounding tissue were observed near the removed blood vessels. + The group administered a combination of vMSCs and BM-MSCs (CD141 + In the vMSC+BM-MSC group, new blood vessels were observed to form near the removed blood vessels (yellow arrows), and the surrounding tissue was also very well preserved (Figure 29).

[0198] 13-2. Confirmation of angiogenesis (immunofluorescence staining)

[0199] In each group of mice in Example 13-1, 3D immunofluorescence staining was used to measure CD31 in the whole muscle. + Blood vessel staining was performed to observe the blood vessel density.

[0200] As a result, in the normal group (Normal) mice, small arteries were extending from the femoral artery, while in the control group (Control) mice, the arteries appeared to have been severed ( * The blood vessel density in the ischemic lesions was very low. + The group administered a combination of vMSCs and BM-MSCs (CD141 + In the normal group (vMSC+BM-MSC), it was confirmed that small arteries were formed at a high density (Figure 30). When the vascular staining image was enlarged, a large artery (femoral artery) with an inner diameter of 321.7±31.9 μM was observed in the normal group (Normal), and CD141 + In the vMSC+BM-MSC group, small arteries with an inner diameter of 88.1 ± 5.5 μM were observed extending to the site of blood vessel removal, while in the control group, severed blood vessels were observed ( * ), and the vascular density around it was very low, with only a few thin blood vessels being observed (Figure 30).

[0201] 13-3. Confirmation of angiogenesis markers

[0202] The structure of blood vessels is composed of endothelial cells and pericytes (smooth muscle cells) that surround the endothelial cells (Fig. 31a). + After injecting vMSCs and BM-MSCs (2:1) into the ischemic muscles of a mouse model of severe hindlimb ischemia, immunofluorescence staining and immunohistochemistry were performed using an anti-human CD31 antibody against CD31, a marker for vascular endothelial cells, and an anti-human alpha-smooth muscle actin (A-SMA) antibody against A-SMA, a marker for perivascular cells.

[0203] As a result, CD141 + Human CD31 and human A-SMA were shown to form blood vessels in the muscle where vMSCs and BM-MSCs were administered (Fig. 31b). + We confirmed that vMSCs + BM-MSCs can form blood vessels in vivo.

[0204] 13-4. Classification by vessel diameter

[0205] CD141 + To confirm the angiogenic potential of the combined administration of vMSCs and BM-MSCs, CD141 + After administering a combination of vMSCs and BM-MSCs to the ischemic muscle of a mouse model of severe hindlimb ischemia, the ischemic muscle was stained by immunofluorescence staining using an antibody against TAGLN (Transgelin), a marker for smooth muscle cells that make up blood vessels. + The blood vessels were analyzed.

[0206] As a result, CD141 + The group administered with vMSCs and BM-MSCs showed significantly higher TAGLN than the control group. +The number of blood vessels was statistically significantly increased (Fig. 32) (Control: 31.6±3.0 vs. Combined stem cell: 57±6.8, p<0.01). Furthermore, the measurement of the vascular diameter also showed a significant increase in the number of blood vessels. + The size of the vessel inner diameter in the vMSC+BM-MSC administration group was significantly larger than that in the control group (Figure 32) (Control: 15.9±1.9μm vs. Combined stem cell: 36.8±8.4μm, p<0.5). Based on the above results, the vessels were classified by vessel diameter and the number of vessels was counted. When the diameter was 20μm or less and 20μm to 50μm or less, the number of vessels was significantly higher than that in the control group (Figure 32). + The number of blood vessels in the vMSC+BM-MSC group was not statistically significant, but the CD141 + The number of cells was confirmed to be higher in the vMSC+BM-MSC group. Furthermore, when the size was 50 μm or larger, CD141 + The number of blood vessels in the vMSC+BM-MSC administration group was significantly higher than that in the control group (Figure 32) (Control: 0 VS CD141 + vMSC+BM-MSC: 7±2, p<0.05).

[0207] As a result, CD141 + It was confirmed that combined administration of vMSCs and BM-MSCs not only increased the number of blood vessels but also increased blood flow in the ischemic area by forming blood vessels with an inner diameter of 50 μm or more.

[0208] Example 14. Confirmation of in vivo safety of combined stem cells

[0209] CD141 in normal BALB / c nude mice + To confirm the safety of combined vMSC and BM-MSC administration, biodistribution was assessed by immunohistochemistry (IHC) for CD31. + Vascular and Alpha-SMA + We confirmed the formation of blood vessels. We also performed CD31 transplants at 1, 4, 13, and 26 weeks to confirm the engraftment status. + Blood vessels and Alpha-SMA +The number of blood vessels was confirmed. Biodistribution studies were also performed using qPCR in blood and tissues / organs at 6 hours, 1 day, 1 week, 4 weeks, 13 weeks, and 26 weeks after a single dose. Furthermore, 1.2 × 10 5 cells, 6 × 10 5 cells, or 1.2 × 10 6 The toxicity was confirmed for 26 weeks after a single administration of 1 × 10 cells. 7 A single subcutaneous administration of 2.4 × 10 cells 6 Tumor formation and potential were observed for up to 26 weeks after a single intramuscular administration of 1000 cells.

[0210] IHC analysis revealed that CD141 + Combined administration of vMSCs and BM-MSCs increased the expression of CD31 + Vascular and Alpha-SMA + It was shown that significantly more blood vessels were formed compared to the control group (G0) (Figure 33). Furthermore, when the number of blood vessels was examined over time after administration, it was found to be highest at 1 week, decreased until 4 weeks, and then maintained at a similar level until 26 weeks (total number of blood vessels in Figure 33). Biodistribution tests using qPCR showed that composite stem cells were detected only in the muscles at the administration site at all observation points, and not in other organs or tissues. The administered cells were detected in all individuals up to 4 weeks, but were also detected in most individuals at 13 and 26 weeks (Pg / 100ng host DNA in Figure 33). This indicates that CD141 + The combined administration of vMSCs and BM-MSCs remained and engrafted for a long time only in the muscle at the injection site, and differentiated into vascular morphology, maintaining the therapeutic effect of critical limb ischemia. Furthermore, no toxic reactions were observed for 26 weeks at any dose (data not shown), with a non-toxic dose of approximately 1.2 × 10 6 Furthermore, as a result of observing tumor formation and potential, no tumors related to the test substance were formed, confirming the absence of tumor formation and potential. +It was confirmed that the combined administration of vMSCs and BM-MSCs did not result in toxicity or tumor formation, confirming that there are no safety issues when used as a treatment for severe lower limb ischemia.

Claims

1. Stem cells that express the CD141 (thrombomodulin™) cell surface antigen.

2. The stem cells according to claim 1, which are vasculogenic multipotent stem cells (vMSCs).

3. The stem cell according to claim 2, deposited under accession number KCLRF-BP-00524.

4. The stem cells according to claim 2, which are derived from umbilical cord blood, umbilical cord, adipose, or bone marrow.

5. The stem cell according to claim 2, which has revascularization or angiogenesis ability.

6. The stem cell according to claim 2, which has pluripotency.

7. The stem cell according to claim 2, which overexpresses HGF (hepatocyte growth factor) and its receptor compared to other stem cells.

8. The stem cells according to claim 2, which have a population doubling time (PDT) of 0.5 to 3 days.

9. The stem cell according to claim 2, wherein neovascularization is promoted by bFGF (basic fibroblast growth factor) or HGF.

10. The stem cell according to claim 2, which does not express CD31, CD309, CD34, eNOS, VE-cadherin or VEGF receptor.

11. A cell therapy composition for vascular regeneration or angiogenesis, comprising the stem cells according to claim 1 as an active ingredient.

12. The cell therapeutic composition for vascular regeneration or angiogenesis according to claim 11, further comprising bone marrow-derived mesenchymal stem cells or adipose-derived mesenchymal stem cells.

13. The cell therapeutic composition for vascular regeneration or angiogenesis according to claim 12, wherein the bone marrow-derived mesenchymal stem cells or adipose-derived mesenchymal stem cells do not express the CD141 cell surface antigen.

14. The cell therapeutic composition for vascular regeneration or angiogenesis according to claim 12, comprising the stem cells according to claim 1 and bone marrow-derived mesenchymal stem cells or adipose-derived mesenchymal stem cells in a ratio of 1:10 to 10:1 based on the number of cells.

15. The cell therapeutic composition for revascularization or angiogenesis according to claim 11, further comprising HGF or bFGF.

16. A transplant material for vascular regeneration, comprising the stem cells according to claim 1 or the cell therapy composition according to claim 11 as an active ingredient.

17. A pharmaceutical composition for preventing or treating vascular diseases or vascular dysfunction, comprising the stem cells according to claim 1 or the cell therapy composition according to claim 11 as an active ingredient.

18. The pharmaceutical composition for preventing or treating vascular disease or vascular dysfunction according to claim 17, wherein the vascular disease is traumatic vascular injury, peripheral vascular disease, cardiovascular disease, cerebrovascular disease or ischemic disease.

19. 19. The pharmaceutical composition for preventing or treating vascular disease or vascular dysfunction according to claim 18, wherein the ischemic disease is ischemic myocardial infarction, ischemic heart disease, ischemic vascular disease, ischemic enteritis, ischemic eye disease, ischemic glaucoma, ischemic renal failure, ischemic retinopathy, ischemic stroke, or ischemic lower limb disease.

20. 18. The pharmaceutical composition for the prevention or treatment of vascular disease or vascular dysfunction according to claim 17, wherein the vascular disease or vascular dysfunction is critical limb ischemia, diabetic angiogenic disorder, vascular dementia, organ damage associated with diabetes, arteriosclerosis, angina pectoris, peripheral cardiovascular disease, hypertension, cerebral infarction, sequelae of brain injury, heart failure, peripheral circulatory disorder, myocardial infarction, arterial occlusive disease, stroke, spinal cord injury, sequelae of spinal nerves, degenerative disease, sequelae of cerebral infarction, peripheral neuropathy, diabetic ulcer, presbyopia, age-related hearing loss, sequelae of brain surgery, ischemic stroke, or subarachnoid hemorrhage.

21. A pharmaceutical composition for preventing or treating a neurological disease, comprising the stem cell according to claim 1 or the cell therapy composition according to claim 11 as an active ingredient.

22. The pharmaceutical composition for preventing or treating a cranial nervous system disease according to claim 21, wherein the cranial nervous system disease is a degenerative brain disease, a psychiatric disease, or a developmental disorder.

23. Degenerative brain diseases include cognitive impairment, Alzheimer's disease, disease), dementia with Lewy bodies, frontotemporal dementia, Parkinson's disease , Creutzfeldt-Jakob disease (CJD), Huntington's disease e), multiple sclerosis, or Guillain-Barré syndrome (GBS).

24. 23. The pharmaceutical composition for the prevention or treatment of a cranial nervous system disease according to claim 22, wherein the psychiatric disease is bipolar disorder, autism, depression, hyperactivity, attention deficit disorder, autism, post-traumatic stress disorder (PTSD), anxiety disorder, sleep disorder, panic disorder, intellectual disability, memory decline, drug addiction, schizophrenia, obsessive-compulsive disorder, delusions of grandeur, personality disorder, alcoholism, or manic-depressive disorder.

25. Developmental disorders include epilepsy, cerebral palsy, developmental language disorder, sensory disorder, learning disorder, attention deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), intellectual disability, cognitive impairment, or impulse control disorder.

23. The pharmaceutical composition for preventing or treating a cranial nervous system disease according to claim 22, wherein the compound is a compound selected from the group consisting of cerebrospinal fluid, ...

26. A method for producing stem cells that express the CD141 cell surface antigen, comprising culturing bone marrow-derived human bone marrow mononuclear cells (cryopreserved, BM-MNCs) or adipose-derived stroma vascular fraction (SVF) in a serum-free medium containing human platelet lysate.

27. Use of stem cells expressing the CD141 cell surface antigen for use in revascularization or angiogenesis.

28. A method of revascularization or angiogenesis comprising administering to an individual stem cells that express the CD141 cell surface antigen.

29. A method for treating a vascular disease or vascular dysfunction, comprising administering stem cells that express the CD141 cell surface antigen to an individual suffering from the vascular disease or vascular dysfunction.

30. A method for treating a cranial nervous system disease, comprising administering stem cells expressing the CD141 cell surface antigen to an individual suffering from the cranial nervous system disease.

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