The secretome of vMSCs and its applications

Abstract

The present invention relates to a newly identified secretome of vMSCs and its uses. The vMSCs obtained by culturing under the specific culture conditions of the present invention are novel MSCs that express the CD141 cell surface antigen, unlike conventional MSCs. The secretome structure secreted by vMSCs differs from that of conventional MSCs and significantly increases the vascular sprouting of other stem cells. Therefore, the secretome can be used to promote angiogenesis in stem cells and to prevent or treat vascular diseases or vascular dysfunction.

Description

[Technical Field]

[0001] The present invention relates to a newly identified secretome of vMSCs and uses thereof. [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 grow from pre-existing mature endothelial cells. Arteriogenesis involves the remodeling of pre-existing arteriolar connections into collateral vessels. Vasculogenesis occurs through 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 directly inserting into newly forming vessels or secreting various angiogenic and trophic factors. Therefore, endothelial progenitor cells (EPCs) are attracting attention as potential targets in the field of regenerative medicine and for therapeutic purposes through revascularization. As a result, research is underway to enhance the function of endothelial progenitor cells. For example, research has been conducted on the production of recombinant EPCs through ex vivo gene modification, and techniques have been developed to enhance the proangiogenic capacity of EPCs using vascular endothelial growth factor (VEGF) or hypoxia-inducible factor-1α. Currently, cell therapies for ischemic vascular disease are broadly divided into two types: 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 difficult to expand in vitro, have difficulty maintaining their function during expansion, are prone to immune rejection when using allogeneic or xenogeneic EPCs, and are difficult to differentiate into and function as complex blood vessels.Furthermore, conventional therapeutic drugs developed based on mesenchymal stem cells targeting ischemic diseases do not engraft and disappear, so they rely on paracrine effects rather than angiogenesis, and currently cannot demonstrate 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 individual cellular materials consisting of numerous cells capable of replacing various tissues that constitute the body through a series of processes to alter the biological properties of the cells through an ex vivo culture process, and then injecting these cells into recipients or creating human tissues for therapeutic purposes. 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 difficult. Active research is currently underway. Stem cells have great potential in cell therapy because they have the ability to differentiate into various cells from damaged tissues. However, at the current stage of development, the survival rate after transplantation is not high, and it is difficult to find examples of widespread and stable success in 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 secretome secreted by stem cells.

[0005] It is also an object of the present invention to provide a pharmaceutical composition for revascularization or angiogenesis.

[0006] Another object of the present invention is to provide stem cells with increased angiogenic potential.

[0007] Another object of the present invention is to provide a cell therapeutic composition for vascular regeneration or angiogenesis.

[0008] It is also an object of the present invention to provide a pharmaceutical composition for preventing or treating vascular diseases or vascular dysfunction.

[0009] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating diseases of the central nervous system.

[0010] Another object of the present invention is to provide a method for producing the novel stem cell-derived secretome.

[0011] Another object of the present invention is to provide uses of secretome secreted from stem cells expressing the CD141 cell surface antigen.

[0012] Another object of the present invention is to provide uses of stem cells that have been treated with secretome secreted from stem cells that express the CD141 cell surface antigen or with culture medium of stem cells that express the CD141 cell surface antigen.

[0013] It is also an object of the present invention to provide a method for treating vascular disease or vascular dysfunction.

[0014] A further object of the present invention is to provide a method for preventing or treating a neurological disease. [Means for solving the problem]

[0015] To achieve the above object, the present invention provides a novel secretome secreted by stem cells.

[0016] The present invention also provides a pharmaceutical composition for revascularization or angiogenesis, which comprises the novel stem cell culture medium or secretome.

[0017] The present invention also provides stem cells obtained by processing the secretome secreted by the novel stem cells.

[0018] The present invention also provides a cell therapy composition for vascular regeneration or angiogenesis, comprising the stem cells.

[0019] The present invention also provides a pharmaceutical composition for preventing or treating vascular diseases or vascular dysfunction, comprising the secretome or stem cells treated therewith.

[0020] The present invention also provides a pharmaceutical composition for preventing or treating a cranial nervous system disease.

[0021] The present invention also provides a method for producing the novel stem cell-derived secretome.

[0022] The present invention also provides the use of secretomes secreted from stem cells expressing the CD141 cell surface antigen for use in revascularization or angiogenesis.

[0023] The present invention also provides uses for revascularization or angiogenesis of stem cells treated with secretome secreted from stem cells expressing the CD141 cell surface antigen or culture medium of stem cells expressing the CD141 cell surface antigen.

[0024] The present invention also provides a method for treating vascular disease or vascular dysfunction.

[0025] Furthermore, the present invention provides a method for preventing or treating a cranial nervous system disease. [Effects of the Invention]

[0026] Vasculogenic multipotent stem cells (vMSCs) obtained by culturing under the specific culture conditions of the present invention share some characteristics 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. The secretome structure secreted by vMSCs differs from that of conventional MSCs and significantly increases the vascular sprouting of other stem cells. Therefore, vMSCs can be used to promote angiogenesis in stem cells and to prevent or treat vascular diseases or vascular dysfunction. [Brief explanation of the drawings]

[0027] [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 novel markers specific to CD141 + vMSCs that distinguish them from BM-MSCs. [Figure 4] Figure 4A shows the results of analyzing angiogenesis-related cytokines in the secretome secreted by vMSCs using a Human Angiogenesis Array: Figure 4A: Analysis of the CD141+ vMSC culture medium (EGM-2-FBS + 2% hPL) alone (control group); Figure 4B: Analysis of the secretome obtained by culturing CD141+ vMSCs in culture medium; Figure 4C: Fold change values ​​of factors significantly increased in the CD141+ vMSC secretome compared to the control group; and Figure 4D: Quantitative measurement of mean pixel density of blots. [Figure 5]Figure 5A shows the results of analyzing cytokines involved in angiogenesis in the secretome secreted by BM-MSCs using a Human Angiogenesis Array. Figure 5A shows the results of analyzing BM-MSC culture medium (StemMACSTMMSC expansion media) alone (control group); Figure 5B shows the results of analyzing secretomes obtained by culturing BM-MSCs in culture medium; Figure 5C shows the difference values ​​of factors significantly increased in the BM-MSC secretome compared to the control group; and Figure 5D shows the results of quantitatively measuring the mean pixel density of the blot. [Figure 6] Figure 6 shows the results of ELISA analysis of HGF levels in the secretomes of vMSCs: Figure 6A: HGF concentrations in the secretomes of CD141+ vMSCs at passage 3 from various donors; Figure 6B: Secreted HGF concentration per cell, calculated by dividing the HGF concentrations in the secretomes of CD141+ vMSCs at passage 3 from various donors by the cell number; and Figure 6C: HGF levels in the secretomes obtained by culturing CD141+ vMSCs and BM-MSCs at passage 3 in growth factor-deficient culture conditions (MEMα + 0.2% hPL). [Figure 7] Figure 7 shows the results of ELISA analysis of VEGF levels in BM-MSC secretomes: Figure 7A: VEGF concentrations in BM-MSC secretomes at passage 3 from various donors; Figure 7B: Secreted VEGF concentration per cell, calculated by dividing VEGF concentrations in BM-MSC secretomes at passage 3 from various donors by cell number; and Figure 7C: VEGF levels in secretomes obtained by culturing CD141+ vMSCs and BM-MSCs at passage 3 in growth factor-deficient culture conditions (MEMα + 0.2% hPL). [Figure 8] FIG. 1 is a schematic diagram illustrating a method for producing spheroids using HUVEC cells and non-contact co-culture using these with vMSCs. [Figure 9] FIG. 10 is a diagram confirming the sprouting ability of HUVEC spheroids in response to HGF, a secretome component of vMSCs. [Figure 10]FIG. 1 is a schematic diagram illustrating a method for producing spheroids using vMSC cells, and the use of the spheroids for treatment with angiogenic factors or non-contact co-culture with vMSCs. [Figure 11] FIG. 10 shows the sprouting ability of CD141+ vMSCs spheroids in response to VEGF and bFGF. [Figure 12] FIG. 10 is a graph confirming the sprouting ability of CD141 + vMSCs spheroids in response to HGF. [Figure 13] FIG. 10 shows the vMSC secretome and the vMSC germination-promoting effect of HGF therein confirmed through non-contact co-culture with vMSC spheroids and vMSCs. [Figure 14] FIG. 1 is a schematic diagram illustrating a method for producing spheroids using vMSC cells and bFGF treatment or non-contact co-culture with BM-MSCs using the spheroids. [Figure 15] This figure confirms the mRNA expression levels of bFGF in vMSCs and BM-MSCs. MSCs: BM-MSCs. [Figure 16] This figure confirms the sprouting ability of vMSC spheroids after treatment with bFGF, which is secreted in large amounts by BM-MSCs, or treatment with its inhibitor. PD173074: bFGF signal transduction inhibitor. [Figure 17] This figure shows the BM-MSC secretome and the effect of bFGF in it on promoting vMSC sprouting, confirmed by non-contact co-culture with vMSC spheroids and BM-MSCs. MSCs: BM-MSC; and PD173074: bFGF signaling pathway inhibitor. BEST MODE FOR CARRYING OUT THE INVENTION

[0028] The present invention will be described in detail below by way of examples 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 well-known technologies or configurations 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 is not limited thereby. The present invention is susceptible to various modifications and applications within the scope of the claims set forth below and the scope of equivalents interpreted thereunder.

[0029] Furthermore, the terminology used in this specification is used to appropriately express 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.

[0030] 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 within the scope of the present invention. The contents of all publications referenced in this specification are incorporated herein.

[0031] 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.

[0032] In one aspect, the present invention relates to a culture medium for stem cells that express the CD141 (thrombomodulin™) cell surface antigen.

[0033] In one embodiment, the stem cells expressing the cell surface antigen CD141 may be vasculogenic multipotent stem cells (vMSCs).

[0034] In one embodiment, the stem cells expressing the cell surface antigen CD141 are CD141 cells deposited under accession number KCLRF-BP-00524. + vMSCs (CD141 + Vasculogenic Multipotent Stem Cells).

[0035] In one embodiment, the culture medium of stem cells expressing the CD141 cell surface antigen may be a culture obtained during or after culturing the cells in a medium, or a supernatant thereof. The culture medium of stem cells expressing the CD141 cell surface antigen may be a culture obtained during or after culturing the cells, or a filtrate thereof, a fraction thereof, or a concentrate of the supernatant thereof, a freeze-dried product thereof, or a supercritically dried product thereof. The concentrate may be a culture of stem cells expressing the CD141 cell surface antigen or its supernatant concentrated about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, or about 20-fold, or may be one obtained by removing apotosomes and cell debris using a 200-600 nm filter and then concentrating the concentrate about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, or about 20-fold using an ultracentrifuge.

[0036] In one embodiment, the filtrate may be obtained by filtering the culture medium to remove apotosomes (about 1000 to 600 nm in size), which are cell death-related microorganisms.

[0037] In one embodiment, the CD141 cell surface antigen-expressing stem cell culture medium may contain a secretome, which is bioactive molecules produced and secreted extracellularly through cell-cell interactions during the culture process of the cells. The secretome may include substances such as cell-derived proteins, cytokines, growth factors, exosomes, and nucleic acids (DNA and RNA), and such various bioactive molecules may be secreted from cells into membrane-bound extracellular vesicles, such as exosomes.

[0038] In one embodiment, the stem cells expressing the CD141 cell surface antigen may be derived from bone marrow, fat, blood, dental pulp, or tonsils.

[0039] In one aspect, the present invention relates to the secretome secreted from stem cells that express the CD141 cell surface antigen.

[0040] In one embodiment, the stem cells expressing the cell surface antigen CD141 are CD141 + vMSCs (CD141 + Vasculogenic Multipotent Stem Cells).

[0041] In one embodiment, the stem cells expressing the cell surface antigen CD141 are CD141 cells deposited under accession number KCLRF-BP-00524. + vMSCs (CD141 + Vasculogenic Multipotent Stem Cells).

[0042] In one embodiment, the secretome may be contained in a culture supernatant, a filtrate, or a concentrate thereof obtained by culturing stem cells expressing the CD141 cell surface antigen in a serum-free medium containing human platelet lysate.

[0043] In one embodiment, the secretome may be contained in a culture supernatant obtained by culturing stem cells expressing the CD141 cell surface antigen in a medium containing human platelet lysate, heparin, EGF, ascorbic acid, and hydrocortisone, but without FGF.

[0044] In one embodiment, the culture medium may be a culture medium in which the stem cells are cultured in a two-dimensional or three-dimensional culture method.

[0045] In one embodiment, the secretome may include Endoglin, HGF, IGFBP-2, IL-8, Pentraxin 3, TIMP-1, or uPA.

[0046] In one embodiment, the secretome may include extracellular vesicles, proteins, nucleic acids, growth factors, or cytokines secreted by the stem cells, the extracellular vesicles may be exosomes, and the exosomes may be exosomes with a size of 200 to 100 nm.

[0047] In one embodiment, when the secretome of the present invention includes exosomes secreted by the stem cells, the culture medium may be filtered through a 200-600 nm filter to remove apoptosomes (approximately 1000-600 nm in size), which are cell death-related microorganisms.

[0048] In one embodiment, the secretome may have increased HGF and decreased VEGF compared to secretome secreted from bone marrow-derived mesenchymal stem cells.

[0049] In one embodiment, the secretome of the present invention may be used in combination with a drug delivery vehicle, which may be a liposome, LNP, hydrogel, or biodegradable tissue engineering implant.

[0050] In one embodiment, the secretome of the present invention may be contained or encapsulated in a nanoparticle.

[0051] As used herein, the term "secretome" refers interchangeably to one or more molecules and / or biological factors secreted by the stem cells of the present invention into the extracellular space (e.g., culture medium). Secretomes can include extracellular vesicles (e.g., exosomes, microparticles, etc.), proteins, nucleic acids, growth factors, cytokines, and / or other molecules secreted by cells into the extracellular space (e.g., culture medium). Secretomes include secretory protein bodies, which refer to the sum of the protein components described above. Secretory protein bodies refer to the components released by cells into the extracellular environment after intracellular gene transcription, translation, and post-translational modification. Secretomes can be left unpurified or further processed (e.g., secretome components can be present in the culture medium or purified, isolated, and / or enriched from the culture medium or an extract, portion, or fraction thereof). The secretome can further include one or more substances that are not secreted from the cell (eg, culture medium, additives, nutrients, etc.).

[0052] The term "exosome" as used in the present invention refers to minute bodies that are excreted in various secretory forms from MVEs (multivescular endosomes), which are special organelles within cells, depending on the cellular environment. These bodies are approximately 30 to 100 nm in size, contain various growth factors, cytokines, and nucleic acids (DNA and RNA (particularly microRNA)), and have a liposome-like structure composed of a phospholipid membrane.

[0053] As used herein, the term "isolated" means material that has been removed from its original environment and has therefore been altered "by the hand of man" from its natural state.

[0054] As used herein, the term "enrichment" refers to selectively concentrating or increasing the amount of one or more components in a composition relative to one or more other components. In some instances, enrichment can involve reducing or lowering (e.g., removing or eradicating) the amount of an undesired substance, or can involve specifically selecting or isolating a desired substance from a composition.

[0055] 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 in an unlimited manner 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.

[0056] 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 capable of differentiating 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.

[0057] The term "differentiation" as used herein refers to the phenomenon in which less specialized cells develop into specific cells, and the size, shape, membrane potential, metabolic activity, and response to signals change 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.

[0058] The term "expression" as used herein 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 transcription and detoxification processes, but also post-transcriptional and post-detoxification processes that may 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-detoxification transformation of polypeptides.

[0059] As used herein, the term "overexpression" refers to a significant upregulation of the mRNA or protein expression of a specific gene through intracellular gene transcription or translation.

[0060] 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.

[0061] 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 polymerase chain reaction (RT-PCR), nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization, nucleic acid microarray, Northern blot, or DNA chip 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; or 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.

[0062] In one aspect, the present invention relates to a pharmaceutical composition for revascularization or angiogenesis, which comprises, as an active ingredient, a culture medium of stem cells that express the CD141 (thrombomodulin™) cell surface antigen.

[0063] In one embodiment, revascularization can include arterial regeneration, small arteriole regeneration, venous regeneration, capillary regeneration, cerebrovascular barrier (BBB) ​​regeneration, or retinal revascularization.

[0064] In one embodiment, the culture medium of stem cells expressing the CD141 cell surface antigen may be a culture obtained during or after culturing the cells in a medium, or a supernatant thereof. The culture medium of stem cells expressing the CD141 cell surface antigen may be a culture obtained during or after culturing the cells, or a filtrate thereof, a fraction thereof, or a concentrate of the supernatant thereof, a freeze-dried product thereof, or a supercritically dried product thereof. The concentrate may be a culture of stem cells expressing the CD141 cell surface antigen or its supernatant concentrated by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, or about 20-fold, or may be one obtained by removing apotosomes and cell debris using a filter with a MWCO of 600 nm or greater, and then concentrating the concentrate by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, or about 20-fold using an ultracentrifuge.

[0065] In one embodiment, the filtrate may be obtained by filtering the culture medium through a 200 to 600 nm filter to remove apoptosomes (size: about 1000 to 600 nm), which are cell death-related microorganisms.

[0066] In one embodiment, the CD141 cell surface antigen-expressing stem cell culture medium may contain a secretome, which is bioactive molecules produced and secreted extracellularly through cell-cell interactions during the culture process of the cells. The secretome may include substances such as cell-derived proteins, cytokines, growth factors, exosomes, and nucleic acids (DNA and RNA), and such various bioactive molecules may be secreted from cells into membrane-bound extracellular vesicles, such as exosomes.

[0067] In one embodiment, the secretome may include extracellular vesicles, proteins, nucleic acids, growth factors, or cytokines secreted by the stem cells, and the extracellular vesicles may be exosomes.

[0068] In one embodiment, the stem cells expressing the CD141 cell surface antigen (Vasculogenic Multipotent Stem Cells, vMSCs) may be the cells deposited under accession number KCLRF-BP-00524.

[0069] In one embodiment, the culture medium may be a culture medium obtained by culturing stem cells expressing the CD141 cell surface antigen in a serum-free medium.

[0070] In one embodiment, the composition may further comprise a culture medium for bone marrow-derived mesenchymal stem cells (BM-MSCs).

[0071] In one embodiment, the bone marrow-derived mesenchymal stem cell culture medium may be a culture or a supernatant obtained during or after culturing the cells in a medium. The bone marrow-derived mesenchymal stem cell culture medium may be a concentrate or lyophilized product of a culture, a fraction thereof, or a supernatant obtained during or after culturing the bone marrow-derived mesenchymal stem cells. The concentrate may be a bone marrow-derived mesenchymal stem cell culture or a supernatant thereof concentrated about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or 20-fold. The bone marrow-derived mesenchymal stem cell culture medium may contain cell-derived proteins, i.e., a secretome, that produce and secrete useful proteins extracellularly through cell-cell interactions during the culture process.

[0072] In one aspect, the present invention relates to a pharmaceutical composition for revascularization or angiogenesis comprising a secretome secreted from stem cells expressing the CD141 cell surface antigen.

[0073] In one embodiment, the secretome secreted from stem cells expressing the CD141 cell surface antigen may include Endoglin, HGF, IGFBP-2, IL-8, Pentraxin 3, TIMP-1, or uPA.

[0074] In one embodiment, the secretome secreted from stem cells expressing the CD141 cell surface antigen may have increased HGF and decreased VEGF compared to the secretome secreted from bone marrow-derived mesenchymal stem cells.

[0075] In one embodiment, the composition may further comprise a secretome secreted from bone marrow-derived mesenchymal stem cells.

[0076] In one embodiment, the secretome secreted from the bone marrow-derived mesenchymal stem cells may include Angiogenin, Endoglin, HGF, IGFBP-2, IGFBP-3, IL-8, Pentraxin 3, TIMP-4, uPA, or VEGF.

[0077] The term "vascular regeneration" as used in the present invention refers to repairing damage to damaged blood vessels, and refers to promoting angiogenesis, i.e., the regeneration of damaged blood vessels, using the composition of the present invention to prevent already damaged blood vessels from being further damaged by persistent external forces, etc. The term "angiogenesis" or "vasculogenesis" refers to the natural healing process of forming new blood vessels to supply blood to organs or damaged tissues, and angiogenesis plays an important role in the progression and treatment of many diseases.

[0078] In one aspect, the present invention relates to a composition for promoting neovascularization of stem cells, comprising a culture medium of stem cells expressing the CD141 cell surface antigen or a secretome secreted from stem cells expressing the CD141 cell surface antigen.

[0079] In one aspect, the present invention relates to stem cells that have been treated with a culture medium of stem cells that express the CD141 cell surface antigen or a secretome secreted from stem cells that express the CD141 cell surface antigen.

[0080] In one embodiment, the stem cells may be cardiac progenitor cells (CPCs), endothelial progenitor cells (EPCs), endothelial colony-forming cells (ECFCs), vasculogenic progenitor cells (VPCs), mesenchymal stem cells, embryonic stem cells, or myoblasts, and the mesenchymal stem cells may be umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs), umbilical cord-derived mesenchymal stem cells (UC-MSCs), adipose-derived mesenchymal stem cells (AD-MSCs), or bone marrow-derived mesenchymal stem cells (BM-MSCs).

[0081] In one embodiment, the EPCs may be cord blood-derived EPCs.

[0082] In one embodiment, the stem cells are stem cells that express the CD141 cell surface antigen, i.e., CD141 + They may be vMSCs, or bone marrow- or adipose-derived stem cells that express the CD141 cell surface antigen.

[0083] In one embodiment, the stem cells may have increased angiogenic potential due to the culture medium or secretome treatment.

[0084] In one embodiment, the stem cells may further process the culture medium of bone marrow-derived mesenchymal stem cells or secretome secreted from bone marrow-derived mesenchymal stem cells.

[0085] In one embodiment, the stem cells may have increased vascular sprouting ability, i.e., neovascularization ability, compared to stem cells not treated with the culture medium or secretome.

[0086] In one embodiment, the stem cells may be 3D cultured stem cells.

[0087] In one aspect, the present invention relates to a cell therapeutic composition for revascularization or angiogenesis, which comprises, as an active ingredient, the stem cells treated with the secretome or culture medium of the present invention.

[0088] In one embodiment, the cell therapy composition can be cryopreserved and used for revascularization.

[0089] 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. When administered orally, the cells can be encapsulated in a transplant material or the like and administered.

[0090] The term "cellular therapeutic agent" as used herein refers to a therapeutic agent used in tissue regeneration therapy by using living autologous, allogenic, or xenogenic stem cells, which are expanded and selected ex vivo and then introduced into the body to restore cellular tissue and function.

[0091] In one aspect, the present invention relates to a transplant material for vascular regeneration, which comprises, as an active ingredient, stem cells obtained by treating secretome secreted from stem cells expressing the CD141 cell surface antigen, or a cell therapy composition containing the same.

[0092] In one embodiment, the implant material may be a surgical implant, medical article, or medical device such as a film, membrane, sheet, rod, screw, anchor, pin, implant, stent, surgical suture, tissue regeneration support, bionanofiber, hydrogel, biosponge, bone plate, and bone graft.

[0093] In one embodiment, when the implant is a hydrogel, it can be administered orally and used to promote revascularization of internal organs.

[0094] In one embodiment, the implant may further include a biodegradable polymer.

[0095] 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.

[0096] In one embodiment, the transplant material may be a transplant material in which stem cells secreted from stem cells expressing the CD141 cell surface antigen of the present invention, or a cell therapy agent containing the same, are inoculated onto a composite scaffold for tissue engineering.

[0097] In one embodiment, when the implant 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.

[0098] 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.

[0099] In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating vascular diseases or vascular dysfunction, comprising as an active ingredient a secretome or culture medium secreted from stem cells expressing the CD141 cell surface antigen of the present invention, or the stem cells of the present invention treated with the secretome or culture medium.

[0100] 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.

[0101] In one embodiment, the vascular disease or vascular dysfunction may be 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.

[0102] In one embodiment, the composition may promote angiogenesis or induction of blood vessel formation, revascularization or tissue regeneration, or improve vascular function, increase tissue perfusion and new blood vessel formation.

[0103] In one aspect, the present invention relates to a cell therapeutic composition for treating cranial and nervous system diseases, which comprises as an active ingredient a secretome or culture medium secreted from stem cells expressing the CD141 cell surface antigen of the present invention, or the stem cells of the present invention treated with the secretome or culture medium.

[0104] In one embodiment, the neurological disease may be a degenerative brain disease, a psychiatric disease, or a developmental disorder.

[0105] 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).

[0106] In one embodiment, the psychiatric disorder may be bipolar disorder, autism, depression, hyperactivity, attention deficit, post-traumatic stress disorder (PTSD), anxiety disorder, sleep disorder, panic disorder, intellectual disability, memory loss, drug addiction, schizophrenia, obsessive-compulsive disorder, delusions of grandiosity, personality disorder, alcoholism, or bipolar disorder.

[0107] 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.

[0108] 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.

[0109] 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 ingredient using suitable methods in the art or methods disclosed in Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton PA, 18th, 1990).

[0110] In one embodiment, the pharmaceutical composition may be in one or more dosage forms selected from the group including oral formulations, topical formulations, suppositories, sterile injectable solutions, and sprays.

[0111] 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, formulations can be prepared as desired depending on the disease or ingredients using appropriate methods in the art or the methods disclosed in Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton PA, 18th, 1990).

[0112] The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions.

[0113] The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable additive, such as 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 of the present invention is preferably present in an amount of 0.1 to 90 parts by weight of the composition, but is not limited thereto.

[0114] The composition of the present invention can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) or orally depending on 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.

[0115] Liquid preparations for oral administration of the composition of the present invention include suspensions, liquid preparations, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, they may also contain various excipients such as wetting agents, sweeteners, flavorings, preservatives, etc. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, etc.

[0116] In one aspect, the present invention relates to a method for producing secretomes derived from vMSCs, comprising culturing stem cells expressing the CD141 cell surface antigen in a serum-free medium containing human platelet lysate.

[0117] In one embodiment, the method may further include collecting the culture medium after the culturing step, and centrifuging the collected culture medium to collect a culture supernatant.

[0118] In one embodiment, the method may further include filtering and concentrating the collected culture supernatant after the step of collecting the culture supernatant.

[0119] In one embodiment, the filtration may be performed by filtering through a 200 to 600 nm filter to remove apoptosomes.

[0120] In one embodiment, the apoptosomes can be removed by filtration through a filter having a MWCO of 200 nm or by centrifugation at 10,000 to 15,000 g.

[0121] In one embodiment, the method may further comprise freeze-drying or supercritical drying.

[0122] In one embodiment, the method may further comprise the step of dialyzing the collected culture supernatant after the step of collecting the culture supernatant.

[0123] In one embodiment, the concentration of HGF can be monitored as an indicator during the production process of vMSC-derived secretome.

[0124] In one aspect, the present invention relates to the use of secretomes secreted from stem cells expressing the CD141 cell surface antigen for use in revascularization or angiogenesis.

[0125] In one aspect, the present invention relates to the use of stem cells treated with secretome secreted from stem cells expressing the CD141 cell surface antigen or culture medium of stem cells expressing the CD141 cell surface antigen for revascularization or angiogenesis.

[0126] In one aspect, the present invention relates to a method for treating a vascular disease or vascular dysfunction, comprising administering to an individual having the vascular disease or vascular dysfunction a secretome secreted from stem cells expressing the CD141 cell surface antigen or a culture medium of stem cells expressing the CD141 cell surface antigen.

[0127] In one aspect, the present invention relates to a method for preventing or treating a cranial nervous system disease, comprising administering a secretome secreted from stem cells expressing the CD141 cell surface antigen or a culture medium of stem cells expressing the CD141 cell surface antigen to an individual having the cranial nervous system disease. DETAILED DESCRIPTION OF THE INVENTION

[0128] 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.

[0129] Example 1. Isolation of novel multipotent stem cells from bone marrow

[0130] 1-1. Optimal culture of vMSCs

[0131] Cryopreserved human bone marrow mononuclear cells (BM-MNCs) (STEMCELL Technologies, Vancouver, Canada; LONZA, Basel, Switzerland) were thawed at 37°C, or bone marrow aspirates collected with patient consent were subjected to density gradient centrifugation on Ficoll-paque (Cytiva, MA, USA) to obtain BM-MNCs. The resulting BM-MNCs were placed in a tissue culture-treated flask using fetal bovine serum (FBS)-deficient 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). The resulting BM-MNCs were cultured at 1.0–2.0 × 10 cells / ml. 5 cells / cm 2 The cells were then aliquoted at a density of 10 × 10 and cultured for 7–12 days. During the primary culture period, the medium was changed every 1–4 days. Colonies of vMSCs (vasculogenic multipotent stem cells), the novel multipotent stem cells of the present invention, began to be observed from 3–5 days of culture. The vMSC cells forming the colonies observed during primary culture were spindle-shaped and varied greatly in length and size. Significant colony expansion was observed between 7 and 10 days. The first subculture of vMSCs was performed when most colonies showed a density of 80–90% or higher. Following the general method for subculture of adherent cells, single cells were dissociated using trypsin or a trypsin substitute and then placed in a new flask at a density of 1.0–5.0 × 10. 3 cells / cm 2The vMSCs of the present invention are cultured in EGM-2 containing FBS, with the basic components of the EGM-2 kit being used as is, and are then subcultured 5 to 7 times.

[0132] 1-2. Confirmation of extracellular matrix adhesion properties of vMSCs

[0133] 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 2 We 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) (ThermoFisher, MA, USA), and collagen type 1 peptide (Corning). The cells constituting vMSC colonies exhibited consistent spindle-shaped morphology across all coating materials, demonstrating the feasibility of culturing on various extracellular matrices. Cultures on ECM-coated flasks yielded more colonies than standard TC-treated flasks, but the difference was not significant (Figure 1). While primary culture yields may vary depending on the presence or type of ECM coating, there was little difference in proliferation rates beyond P1.

[0134] The novel vMSCs of the present invention cannot maintain the characteristics of CD141+ vMSCs when cultured using the FBS of the existing EGM-2 bullet kit instead of human PL, and the addition of human PL further increases CD141 expression. Therefore, the culture medium must be made by adding human PL to the EGM-2 bullet kit (Lonza) and excluding FBS. However, since there are no problems with growth even if IGF and VEGF are removed, IGF and VEGF can also be excluded.

[0135] Example 2. Marker characterization of vMSCs

[0136] 2-1. Confirmation of EPC and vascular endothelial cell marker expression

[0137] 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 fluor 488-conjugated vWF antibody (Abcam, 1:500), fluorescein-conjugated UEA-1 (Vectorlabs, 3:100), and APC (allophycocyanin)-conjugated antibodies for CD29, CD31, CD34, CD44, CD45, CD73, CD90, CD105, and CD309 (Miltenyi Biotec, 1:50). Isotype IgG conjugated with APC (Miltenyi Biotec, 1:50) and Alexa fluor 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% NGS (normal goat serum) 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 fluorescence microscope.

[0138] Flow cytometry analysis confirmed 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 confirmed 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.

[0139] 2-2. Discovery of new markers specific to vMSCs

[0140] To discover specific markers for vMSCs of the present invention that distinguish them from BM-MSCs, we performed marker screening using a MACS system that can analyze 378 surface antigen markers using a flow cytometer. R The experiment was performed using Marker Screen, human, version 02 (Miltenyi Biotec) according to the manufacturer's guidelines. 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 The mixture was then dispensed at 1 / well and 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 a concentration of 200 μl / well and analyzed using a NovoCyte 3000 flow cytometer (Agilent Technologies) equipped with a NovoSampler (Agilent Technologies) and NovoExpress software.

[0141] 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 analysis of CD141, CD282, and PEAR1 (Platelet Endothelial Aggregation Receptor 1) in 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.

[0142] As can be seen from the above results, the stem cells CD141 identified in the present invention have the ability to differentiate into multiple cells and promote angiogenesis.+ Unlike EPCs, vMSCs do not express CD31, CD309, or CD34; unlike vascular endothelial cells, they do not express eNOS or VE-cadherin; and unlike mesenchymal stem cells, they express CD141. + They were named Vasculogenic Multipotent Stem Cells and deposited at the Korea Cell Line Research Foundation (KCLRF) with the accession number KCLRFBP00524.

[0143] Example 3. Secretome analysis of vMSCs

[0144] 3-1.Human Angiogenesis Array

[0145] vMSCs (CD141 + To analyze the secretome secreted by vMSCs or BM-MSCs, bone marrow-derived vMSCs (CD141 + Human angiogenesis arrays were performed using secretomes of cultured vMSCs and bone marrow-derived MSCs (BM-MSCs). Specifically, 1.0 × 10 vMSCs and BM-MSCs at passage 3 were placed in a T-75 flask. 5 Each aliquot was placed in EGM2-2% FBS + 2% hPL medium and StemMACS TM After culturing in MSCS expansion medium, cell culture supernatant was collected on day 4 of culture. Then, Human Angiogenesis Array (R&D Systems, ARY007, Minneapolis, USA) was performed according to the manufacturer's instructions. As a control, BM-MSCs were cultured in the respective culture medium (BM-MSC culture medium: StemMACS TM We used MSC expansion media and vMSC culture medium (EGM2-2% FBS + 2% hPL) to identify factors that were significantly increased in vMSC or BM-MSC culture supernatants compared to the control group. Blots containing the results were quantitatively analyzed using the protein array analyzer in the ImageJ program.

[0146] As a result, vMSCs (CD141 + In the culture supernatants of vMSCs, the levels of Endoglin, HGF, IGFBP-2, IL-8, Pentraxin 3, TIMP-1, and uPA were significantly higher than those of the control culture medium. Specifically, HGF was 6.5-fold higher, IL-8 was 80.3-fold higher, Pentraxin 3 was 27.1-fold higher, and uPA was 15-fold higher (Figure 4). In the culture supernatants of BM-MSCs, the levels of Angiogenin, Endoglin, HGF, IGFBP-2, IGFBP-3, IL-8, Pentraxin 3, TIMP-4, uPA, and VEGF were significantly higher than those of the control culture medium. Specifically, Endoglin was 4.6-fold higher, IGFBP-2 and IL-8 were 3.9-fold higher, uPA was 39.7-fold higher, and VEGF was 6.5-fold higher (Figure 5).

[0147] 3-2.ELISA analysis

[0148] vMSC (CD141 + To confirm the levels of HGF and VEGF, among factors involved in angiogenesis, in the secretome secreted by vMSCs (1.3 × 10 5 ) and BM-MSCs (2 × 10 5 ) were cultured, and the cell culture supernatant was collected on the fourth or fifth day of culture, and the cells were counted to reflect the cell number. To confirm the secretion levels of HGF and VEGF when vMSCs and BM-MSCs were cultured under similar growth factor-deficient conditions, 9 × 10 vMSCs and BM-MSCs at the third passage were cultured in a 6-well plate. 4The cells were plated at 1 / well and cultured in MEMα (Gibco, NY, USA) containing 0.2% hPL (PL BioScience, Aachen, Germany) as a growth factor-deficient medium. After replacing the medium with fresh medium on day 1, cell culture supernatants were collected on day 4. HGF and VEGF levels were then analyzed by ELISA using Quantikine ELISA (R&D Systems, Minneapolis, USA) according to the manufacturer's instructions, and absorbance was measured at 450 nm using a microplate reader (Molecular Device, SpectraMax ABS).

[0149] HGF ELISA analysis revealed that HGF concentrations in the vMSC culture supernatant ranged from 5,400 pg / mL to 53,600 pg / mL (average 25,200 pg / mL) (Figure 6A). The HGF concentration was divided by the number of cells to determine the amount of HGF secreted per cell. The vMSCs secreted 10 4 The HGF secretion levels ranged from 340 pg to 5,600 pg per cell (average 1,700 pg) (Figure 6B). When vMSCs and BM-MSCs were cultured under the same conditions using growth factor-deficient medium, the HGF level in the vMSC culture supernatant was measured at 10,600 pg / mL, while that in the BM-MSC culture supernatant was measured at 1,100 pg / mL, approximately 9-fold lower than that in the vMSC culture supernatant (Figure 6C).

[0150] Furthermore, VEGF ELISA analysis revealed that VEGF levels in the culture supernatant of BM-MSCs ranged from 200 pg / mL to 1,600 pg / mL (average 820 pg / mL) (Fig. 7A). 4 The cells secreted VEGF at levels ranging from 55 pg to 160 pg per cell (average 100 pg) (Figure 7B). When vMSCs and BM-MSCs were cultured under the same conditions using a growth factor-deficient medium, VEGF levels were measured in the culture supernatant of vMSCs at 330 pg / mL, while those of BM-MSCs were measured at 620 pg / mL, approximately two-fold higher than those of vMSCs (Figure 7C).

[0151] These results confirmed that vMSCs (CD141-positive cells) secrete high levels of HGF, whereas BM-MSCs secrete high levels of VEGF. Furthermore, vMSCs secrete not only HGF but also IL-8, Pentraxin 3, and uPA, which are involved in angiogenesis, at very high levels, and BM-MSCs secrete not only VEGF but also uPA at very high levels.

[0152] Example 4. Effect of vMSC secretome on promoting cell sprouting of vascular endothelial cells

[0153] To confirm the effect of vMSC secretome on promoting angiogenesis in vascular endothelial cells, HUVEC cells were cultured as spheroids using the hanging drop method, co-cultured with vMSCs, and then a spheroid sprouting assay was performed. Specifically, HUVECs (1.33 × 10) were cultured in M199 medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% FBS and 0.2% methylcellulose. 4HUVEC cells were suspended at 1000x1000 cells / mL and dispensed into 30 μL aliquots onto the lids of Petri dishes. Sterile distilled water was added to the bottom of the Petri dishes to maintain humidity. The Petri dish lids were inverted 180 degrees and placed on the bottom of the Petri dishes containing sterile water. The cells were then cultured at 37°C and 5% CO2 for 24 hours to form HUVEC spheroids (Hanging drop in Figure 8). The HUVEC spheroids were then suspended in M199 medium containing 20% ​​FBS and 0.95% methylcellulose. The spheroids were then mixed 1:1 (v / v) with a Type I collagen solution containing 30 mg / mL type IA collagen gelatin, 10x Medium (Sigma-Aldrich), and 10x reconstitution buffer (0.05 N NaOH, 0.261 M NaHCO3, and 0.2 M HEPES) at an 8:1:1 (v / v) ratio. This mixture was dispensed in 0.7 mL aliquots into 24-well plates and polymerized for 30 minutes at 37°C in 5% CO. 1 × 10 vMSCs diluted in 100 μL of M199 medium were placed on the polymerized collagen gel containing HUVEC spheroids. 5 The vMSCs were then cultured for 24 hours. To neutralize HGF in the vMSC secretome, 500 ng / mL of an HGF-neutralizing antibody (MAB294, R&D System; Anti-HGF Ab) was mixed into M199 medium and applied in 100 μl aliquots onto the polymerized collagen gel containing HUVEC spheroids. The gel was then cultured for 24 hours. Mouse IgG (IgG) (500 ng / mL) was used as a control (Figure 8, Sprouting Assay). Images of spheroid sprouting were captured using a Nikon microscope and quantitatively analyzed using ImageJ software (NIH) for the number of sprouts, cumulative sprout length (the sum of all sprout lengths from a single spheroid), and average sprout length (the average length of all sprouts from all spheroids).

[0154] The results showed that non-contact co-culture with CD141+ vMSCs promoted HUVEC spheroid sprouting, and that treatment with an HGF neutralizing antibody inhibited HUVEC sprouting (Figure 9). This confirms that HGF in the vMSC secretome promotes HUVEC cell sprouting, i.e., promotes angiogenesis. It can be concluded that HGF is not sufficient for promoting HUVEC sprouting, but is required for vMSC-induced HUVEC sprouting.

[0155] Therefore, bone marrow-derived vMSCs secrete excessive amounts of HGF, which is associated with angiogenesis, and their secretome promotes the sprouting of new blood vessels in HUVECs. Therefore, secretomes isolated from vMSCs cultured in serum-free culture medium can be used to promote vascular regeneration.

[0156] Example 5. Autocrine effects of the vMSC secretome

[0157] To confirm the angiogenesis-promoting effect of vMSC secretome, i.e., the autocrine effect, vMSC cells were cultured as spheroids using the hanging drop method, and then non-contact co-cultured with vMSCs and spheroid sprouting assays were performed. Specifically, vMSCs were cultured at 2 × 10 in M199 medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% FBS and 0.2% methylcellulose. 4The vMSCs were suspended at 1000kJ / mL and dispensed into 30 μL aliquots onto the lids of Petri dishes. Sterile distilled water was added to the bottom of the Petri dishes to maintain humidity. The Petri dish lids were inverted 180 degrees and placed on the bottom of the Petri dishes containing sterile water. The vMSCs were then cultured at 37°C and 5% CO2 for 24 hours to form spheroids (Hanging drop in Figure 10). The vMSC spheroids were then suspended in M199 medium containing 0.95% methylcellulose and mixed 1:1 with a Type I collagen solution (30 mg / mL type I collagen gelatin, 10x Medium (Sigma-Aldrich), and 10x reconstitution buffer (0.05 N NaOH, 0.261 M NaHCO3, and 0.2 M HEPES) at an 8:1:1 ratio. This mixture was dispensed into 24-well plates in 0.7 mL aliquots and polymerized for 30 minutes at 37°C in 5% CO2. The gel containing vMSC spheroids was treated with HGF or VEGF as angiogenic factors diluted in 100 μL of M199 medium and cultured for 48 hours. Alternatively, vMSCs were dispensed instead of angiogenic factors and co-cultured with vMSC spheroids in a non-contact manner. To neutralize HGF, a secretome component of vMSCs, 500 ng / mL of an HGF neutralizing antibody (MAB294, R&D Systems; Anti-HGF Ab) was added to M199 medium and treated (sprouting assay in Figure 10). Images of spheroid sprouting were taken using a microscope (Nikon), and the number of sprouts, cumulative sprout length (the total length of all sprouts formed from one spheroid), and average sprout length (the average length of all sprouts formed from all spheroids) were quantitatively analyzed using ImageJ software (NIH).

[0158] The results showed that vMSC spheroids responded to bFGF but not VEGF (Figure 11), and sprouting was promoted at concentrations of 20 ng / mL and 50 ng / mL HGF (Figure 12). Furthermore, when vMSC spheroids were co-cultured with vMSCs and exposed to the HGF-rich vMSC secretome, sprouting of vMSC spheroids was promoted, while sprouting increased with an HGF-neutralizing antibody (Figure 13). This suggests that the sprouting ability of vMSC spheroids promoted by the vMSC secretome is due to HGF in the secretome, demonstrating that HGF is a growth factor that exerts an autocrine effect on vMSCs.

[0159] Therefore, bone marrow-derived CD141 + vMSCs secrete excessive HGF, which is associated with angiogenesis, and their secretome expresses CD141 + CD141 to promote vMSC sprouting + Secretomes isolated from vMSCs cultured in serum-free medium were used to treat various stem cell therapeutic agents for vascular regeneration (CD141 + It can be used as an adjuvant for vMSCs or EPCs.

[0160] Example 6. Paracrine effects of the BM-MSC secretome

[0161] To confirm the effect of BM-MSC secretome on promoting angiogenesis in vMSCs, i.e., the paracrine effect, vMSC cells were cultured as spheroids using the hanging drop method (Hanging drop in Figure 14) as in Example 5, and then non-contact co-cultured with BM-MSCs, followed by a spheroid sprouting assay (Sprouting assay in Figure 14). Specifically, 3 x 10 BM-MSCs were added to a gel containing vMSC spheroids. 4100 μl of M199 medium containing BM-MSCs was dispensed and co-cultured with vMSC spheroids for 48 hours without contact or treated with bFGF (25 ng / mL). To inhibit bFGF, a component of the BM-MSC secretome, PD173074 (Tocris, Bristol, UK) (10 nM), a bFGF signaling inhibitor, was added 3 hours prior to treatment. Images of spheroid sprouts were captured using a Nikon microscope, and the number of sprouts, cumulative sprout length (the total length of all sprouts formed from a single spheroid), and average sprout length (the average length of all sprouts formed from all spheroids) were quantitatively analyzed using ImageJ software (NIH). To compare bFGF mRNA expression levels in vMSCs and BM-MSCs, total RNA was isolated from each cell line using TRIzol reagent (Invitrogen), cDNA was synthesized using SuperScript III Reverse Transcriptase (Invitrogen), and RT-PCR analysis was performed using SYBR Green reagent (Invitrogen). Human RPS9 (ribosomal protein S9 gene) was used as an endogenous control.

[0162] Comparison of bFGF mRNA levels between vMSCs (vMSCs) and BM-MSCs (MSCs) revealed significantly higher bFGF expression in BM-MSCs compared with vMSCs (Figure 15). Furthermore, bFGF promoted sprouting in vMSC spheroids, which was inhibited by pretreatment with PD173074 for 3 hours (Figure 16). Furthermore, when vMSC spheroids and BM-MSCs (MSCs) were co-cultured without contact, the secretome of BM-MSCs promoted vMSC spheroid sprouting, and pretreatment with an inhibitor of bFGF inhibited vMSC spheroid sprouting (Figure 17).

[0163] This resulted in the secretome of BM-MSCs (bone marrow-derived mesenchymal stem cells) expressing CD141 in non-contact co-culture. +The sprouting of vMSC spheroids was promoted, and the sprouting-promoting effect was suppressed by inhibition of the bFGF signaling pathway, indicating that bFGF secreted by BM-MSCs plays a role in the angiogenic effect of vMSCs. Therefore, sprouting of vMSC spheroids is promoted by bFGF, but not VEGF, and it was confirmed that the bFGF-secreting BM-MSC secretome provides a paracrine angiogenic effect to vMSCs.

[0164] According to the above examples, the secretome of BM-MSCs and the secretome of vMSCs can be used alone or in combination to promote vascular regeneration and various stem cell therapeutic agents for vascular regeneration (e.g., CD141 + It can be used as an additive to vMSCs or EPCs.

Claims

1. Secretome secreted from stem cells expressing the CD141 (thrombomodulin™) cell surface antigen.

2. The secretome of claim 1, wherein the stem cells expressing the CD141 cell surface antigen are vasculogenic multipotent stem cells (vMSCs) deposited under accession number KCLRF-BF-00524.

3. The secretome of claim 1 , comprising a cell-derived protein, a cytokine, a growth factor, an exosome, or a nucleic acid.

4. The secretome of claim 1, comprising Endoglin, HGF, IGFBP-2, IL-8, Pentraxin 3, TIMP-1, or uPA.

5. The secretome according to claim 1, wherein HGF is increased compared to secretome secreted from bone marrow-derived mesenchymal stem cells (BM-MSCs).

6. The secretome of claim 1 , wherein VEGF is reduced compared to secretome secreted from bone marrow-derived mesenchymal stem cells.

7. A pharmaceutical composition for revascularization or angiogenesis, comprising as an active ingredient a culture medium of stem cells expressing the CD141 cell surface antigen.

8. 8. The pharmaceutical composition for vascular regeneration or angiogenesis according to claim 7, wherein the stem cells expressing the CD141 cell surface antigen are angiogenic pluripotent stem cells deposited under accession number KCLRF-BP-00524.

9. 8. The pharmaceutical composition for vascular regeneration or angiogenesis according to claim 7, wherein the culture medium is a culture medium obtained by culturing stem cells expressing the CD141 cell surface antigen in a serum-free medium.

10. The pharmaceutical composition for vascular regeneration or angiogenesis according to claim 7, further comprising a culture medium for bone marrow-derived mesenchymal stem cells.

11. A pharmaceutical composition for revascularization or angiogenesis, comprising the secretome of claim 1.

12. 12. The pharmaceutical composition for revascularization or angiogenesis according to claim 11, comprising Endoglin, HGF, IGFBP-2, IL-8, Pentraxin 3, TIMP-1 or uPA.

13. The pharmaceutical composition for revascularization or angiogenesis according to claim 11, comprising a cell-derived protein, a cytokine, a growth factor, an exosome or a nucleic acid.

14. The pharmaceutical composition for revascularization or angiogenesis according to claim 11, further comprising a secretome secreted from bone marrow-derived mesenchymal stem cells.

15. Stem cells treated with the secretome of claim 1 or a culture medium of stem cells expressing the CD141 cell surface antigen.

16. The stem cell according to claim 15, wherein the stem cell is a cardiac progenitor cell (CPC), an EPC (Endothelial Progenitor Cell), an ECFC (Endothelial Colony Forming Cell), a VPC (Vasculogenic Progenitor Cell), a mesenchymal stem cell, an embryonic stem cell, or a myoblast.

17. The stem cell according to claim 16, wherein the mesenchymal stem cells are umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs), umbilical cord-derived mesenchymal stem cells (UC-MSCs), adipose-derived mesenchymal stem cells (AD-MSCs), or bone marrow-derived mesenchymal stem cells (BM-MSCs).

18. The stem cell according to claim 15, wherein the stem cell expresses the CD141 cell surface antigen.

19. The stem cell according to claim 18, wherein the stem cell expressing the CD141 cell surface antigen is derived from bone marrow, fat, blood, dental pulp or tonsil.

20. The stem cell according to claim 15, wherein the angiogenic potential is increased by treatment with the secretome or culture medium.

21. The stem cells according to claim 15, wherein the culture medium of the bone marrow-derived mesenchymal stem cells or the secretome secreted from the bone marrow-derived mesenchymal stem cells has been further processed.

22. A cell therapeutic composition for vascular regeneration or angiogenesis, comprising the stem cells according to claim 15 as an active ingredient.

23. A pharmaceutical composition for preventing or treating vascular diseases or vascular dysfunction, comprising the secretome of claim 1 or the stem cell of claim 15 as an active ingredient.

24. 24. The pharmaceutical composition for preventing or treating vascular disease or vascular dysfunction according to claim 23, wherein the vascular disease is traumatic vascular injury, peripheral vascular disease, cardiovascular disease, cerebrovascular disease or ischemic disease.

25. A pharmaceutical composition for preventing or treating a neurological disease, comprising the secretome of claim 1 or the stem cell of claim 15 as an active ingredient.

26. The pharmaceutical composition for preventing or treating a cranial nervous system disease according to claim 25, wherein the cranial nervous system disease is a degenerative brain disease, a psychiatric disease, or a developmental disorder.

27. 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).

27. The pharmaceutical composition for preventing or treating a cranial nervous system disease according to claim 26, wherein the cranial nervous system disease is GBS.

28. 27. The pharmaceutical composition for the prevention or treatment of a nervous system disease according to claim 26, wherein the psychiatric disease is bipolar disorder, autism, depression, hyperactivity, attention deficit, 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 bipolar disorder.

29. The developmental disorder is 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. The pharmaceutical composition for preventing or treating a cranial nervous system disease according to claim 26, wherein the compound is a compound selected from the group consisting of cerebrospinal fluid (CSF) and cerebrospinal fluid (CSF).

30. A method for producing a secretome derived from vMSCs, comprising culturing stem cells expressing the CD141 cell surface antigen in a serum-free medium containing human platelet lysate.

31. The method for producing a secretome derived from vMSCs according to claim 30, further comprising a step of filtering the culture medium.

32. Use of secretomes secreted from stem cells expressing the CD141 cell surface antigen for use in revascularization or angiogenesis.

33. Use of secretome secreted from stem cells expressing the CD141 cell surface antigen or stem cells treated with a culture medium of stem cells expressing the CD141 cell surface antigen for revascularization or angiogenesis.

34. A method for treating a vascular disease or vascular dysfunction, comprising administering a secretome secreted from stem cells expressing the CD141 cell surface antigen or a culture medium of stem cells expressing the CD141 cell surface antigen to an individual having the vascular disease or vascular dysfunction.

35. A method for preventing or treating a cranial nervous system disease, comprising administering a secretome secreted from stem cells expressing the CD141 cell surface antigen or a culture medium of stem cells expressing the CD141 cell surface antigen to an individual having the cranial nervous system disease.

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