Method for producing bone marrow-derived vascular endothelial progenitor cells with enhanced angiogenesis using PDGF-BB, and method for producing a cell therapy agent for treating vascular dysplasia and related diseases using the same.

Abstract

This invention provides novel cells and compositions that can effectively improve the angiogenic capacity of vascular endothelial progenitor cells. [Solution] (i) Having the cellular phenotypes of CD31-, CD309-, CD45- and CD34-, (ii) Endothelial progenitor cells with enhanced angiogenic capacity, characterized by increased laminin β1 expression compared to endothelial progenitor cells (EPCs) that have not been treated with PDGF-BB (Platelet-derived growth factor-BB).

Description

[Technical Field]

[0001] This invention relates to a method for producing bone marrow-derived vascular endothelial progenitor cells with enhanced angiogenesis using PDGF-BB, and a method for producing a cell therapy agent for treating vascular dysplasia and related diseases using the same. [Background technology]

[0002] Endothelial progenitor cells (EPCs) were the first cells identified in peripheral blood and are cells that promote angiogenesis.

[0003] New blood vessels are generated through the processes of angiogenesis, arteriogenesis, and vasculogenesis. Neoangiogenesis involves the proliferation and migration of endothelial cells that grow from already existing mature endothelial cells; arteriogenesis is the process of remodeling existing arteriolar connections in collateral vessels; and vasculogenesis proceeds through the differentiation of endothelial progenitor cells into mature endothelial cells. Therefore, endothelial progenitor cells that leave the bone marrow and circulate migrate to the site of vascular injury and participate in new angiogenesis through direct insertion into newly formed blood vessels or through various forms of neoangiogenesis and the secretion of trophic factors.

[0004] Therefore, vascular endothelial progenitor cells (EPCs) are attracting attention as a potential target in the field of regenerative medicine and for therapeutic purposes through re-angiogenesis. Accordingly, research is underway to enhance the function of vascular endothelial progenitor cells. For example, research has been conducted on the production of recombinant EPCs by modifying ex vivo genes, and techniques have been studied to increase the pro-angiogenic capacity of EPCs using vascular endothelial growth factor (VEGF) or hypoxia-inducible factor-1α.

[0005] However, conventional methods like this have limitations when applying vascular endothelial progenitor cells to the site of vascular injury, such as low cell transplantation rates and low activation rates due to the damaged tissue environment, which prevents the desired therapeutic effect from being achieved.

[0006] Furthermore, existing therapies utilizing vascular endothelial progenitor cells have involved concentrating CD34+ cells, simply isolated from the patient's blood via leukapheresis and FACS, using minimally manipulated cell therapy, and transplanting them into myocardial infarction sites requiring angiogenesis for vascular regeneration therapy. However, such therapies exhibit significant patient-to-patient variations in the number of vascular endothelial progenitor cells (frequency of vascular endothelial progenitor cells in the blood: 0.02-0.0002%) and their activity levels. As a result, they have not been able to achieve a consistent level of increase in cardiac output after transplantation (e.g., contractility (LVEF, left ventricular ejection fraction) increasing from approximately 50% before treatment to approximately 55% after treatment), and have therefore not been utilized as a new medical technology for patients.

[0007] Therefore, there is a need to develop new technologies that can effectively enhance the angiogenic capacity of vascular endothelial progenitor cells and enable their useful use as cell therapy agents for vascular dysplasia and related diseases. [Overview of the project] [Problems that the invention aims to solve]

[0008] Therefore, the inventors analyzed the cellular characteristics of vascular endothelial progenitor cells isolated from bone marrow according to their culture passage and evaluated their vascular regeneration capacity. They confirmed that when vascular endothelial progenitor cells are treated with PDGF-BB, the angiogenesis ability, cell migration, cell proliferation, and cell viability of the vascular endothelial progenitor cells can be effectively improved. Furthermore, they confirmed that when mixed cells, a mixture of vascular endothelial progenitor cells and mesenchymal stem cells, are treated with PDGF-BB and co-cultured, an even greater angiogenesis effect is observed. Accordingly, the inventors have completed the present invention by establishing a method for producing a novel cell therapy agent that can effectively improve the angiogenesis ability of vascular endothelial progenitor cells.

[0009] Therefore, the object of the present invention is to provide a method for producing vascular endothelial progenitor cells with enhanced angiogenesis ability using PDGF-BB (Platelet-derived growth factor-BB).

[0010] Another object of the present invention is to provide an angiogenesis-promoting composition for the treatment of angioplasty-related diseases, comprising isolated endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs), and PDGF-BB (Platelet-derived growth factor-BB) as active ingredients.

[0011] Another object of the present invention is to provide a method for producing a cell therapy agent for the treatment of vascular dysplasia-related diseases, comprising the step of mixing isolated endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs), and PDGF-BB (Platelet-derived growth factor-BB).

[0012] Another object of the present invention is to provide a cell therapy agent for the treatment of angioplasty-related diseases, manufactured by the method of the present invention. [Means for solving the problem]

[0013] To achieve the aforementioned objectives of the present invention, the present invention provides a method for producing endothelial progenitor cells with enhanced angiogenic ability, comprising the step of treating isolated endothelial progenitor cells (EPCs) with PDGF-BB (Platelet-derived growth factor-BB) and culturing them.

[0014] In one embodiment of the present invention, the vascular endothelial progenitor cells can be described as human bone marrow-derived vascular endothelial progenitor cells (BM-EPC).

[0015] In one embodiment of the present invention, vascular endothelial progenitor cells treated with PDGF-BB may exhibit enhanced cell migration, increased cell viability, enhanced cell proliferation, and increased expression of the extra-matrix protein laminin β1.

[0016] Furthermore, the present invention provides an angiogenesis-promoting composition for the treatment of angioplasty-related diseases, comprising isolated endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs), and PDGF-BB (Platelet-derived growth factor-BB) as active ingredients.

[0017] In one embodiment of the present invention, the vascular endothelial progenitor cells can be described as human bone marrow-derived vascular endothelial progenitor cells (BM-EPC).

[0018] The present invention also provides a method for producing a cell therapeutic agent for treating angiogenesis-deficiency-related diseases, which includes the step of mixing isolated endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs), and PDGF-BB (Platelet derived growth factor-BB).

[0019] In one embodiment of the present invention, the isolated endothelial progenitor cells and mesenchymal stem cells may be mixed at a cell number ratio of 1:1 to 2:1.

[0020] In one embodiment of the present invention, the endothelial progenitor cells may be human bone marrow-derived endothelial progenitor cells, and the mesenchymal stem cells may be human bone marrow-derived mesenchymal stem cells.

[0021] In one embodiment of the present invention, the angiogenesis-deficiency-related disease may be an ischemic disease, diabetic ulcer, gangrene, occlusive vascular disease, cardiovascular disease, or local anemia.

[0022] In one embodiment of the present invention, the ischemic disease may be ischemic myocardial infarction, ischemic heart disease, ischemic vascular disease, ischemic eye disease, ischemic renal failure, ischemic retinopathy, ischemic stroke, or ischemic lower limb disease.

[0023] The present invention also provides a cell therapeutic agent for treating angiogenesis-deficiency-related diseases, which is produced by the method of the present invention.

[0024] In one embodiment of the present invention, the cell therapeutic agent may be one in which human bone marrow-derived endothelial progenitor cells and human bone marrow-derived mesenchymal stem cells are mixed at a cell number ratio of 1:1 to 2:1 and have angiogenesis ability.

[0025] In one embodiment of the present invention, the cell therapeutic agent may be for intramedullary administration, intravenous administration, subcutaneous administration, intramuscular administration, or intraperitoneal administration.

[0026] In one embodiment of the present invention, the angioplasty-related disease may be ischemic disease, diabetic ulcer, gangrene, occlusive vascular disease, cardiovascular disease, or local anemia.

[0027] In one embodiment of the present invention, the ischemic disease may be ischemic myocardial infarction, ischemic heart disease, ischemic vascular disease, ischemic eye disease, ischemic renal failure, ischemic retinopathy, ischemic stroke, or ischemic lower limb disease.

[0028] The present invention also provides a method for preventing or treating vascular dysplasia-related diseases, comprising the step of administering the vascular formation-promoting composition to an individual.

[0029] The present invention also provides a method for preventing or treating angioplasty-related diseases, comprising the step of administering a cell therapy agent produced by the above-described manufacturing method to an individual. [Effects of the Invention]

[0030] The present invention has discovered that vascular endothelial progenitor cells treated with PDGF-BB have enhanced angiogenesis, cell migration, cell viability, and cell proliferation. Furthermore, by confirming that further enhanced angiogenesis is observed when PDGF-BB is treated with a mixture of vascular endothelial progenitor cells and mesenchymal stem cells, the present invention ultimately has the effect of providing a novel cell therapy agent and a method for producing the same for the effective treatment of vascular dysplasia-related diseases. [Brief explanation of the drawing]

[0031] [Figure 1a] This is the result of analyzing the characteristics of human bone marrow-derived vascular endothelial progenitor cells (BM-EPCs) according to the present invention, and confirms the ability of human BM-EPCs to form colonies before passage (P0). [Figure 1b] The results of analyzing the characteristics of human bone marrow-derived vascular endothelial progenitor cells (BM-EPCs) according to the present invention were obtained by performing double fluorescence staining and observing the expression of markers in vascular endothelial progenitor cells (FITC-UEA-I binding: green, vWF: red). [Figure 1c] This is the result of analyzing the characteristics of human bone marrow-derived vascular endothelial progenitor cells (BM-EPCs) according to the present invention, and the concentration of growth factors contained in the culture medium of BM-EPCs prepared separately for passages 2 to 4 (P2 to P4) was measured. [Figure 1d] The results of analyzing the characteristics of human bone marrow-derived vascular endothelial progenitor cells (BM-EPCs) according to the present invention are obtained by analyzing cells expressing cell surface markers in passages 1 to 3 (P1 to P3) using a parenchyma cell analyzer. [Figure 1e] This is the result of analyzing the characteristics of human bone marrow-derived vascular endothelial progenitor cells (BM-EPCs) according to the present invention, specifically the tube-forming ability of BM-EPCs at passages 3 to 6 (P3 to P6). [Figure 1f] This shows the results of analyzing the characteristics of human bone marrow-derived vascular endothelial progenitor cells (BM-EPCs) according to the present invention, specifically the results of analyzing the tube formation ability of 3-passage BM-EPCs with and without human bone marrow-derived mesenchymal stem cells (BM-MSCs). [Figure 2] This is the result of measuring the PDT (population doubling time) of human bone marrow-derived vascular endothelial progenitor cells (BM-EPCs) obtained by subculturing. [Figure 3] This is the result of analyzing the changes in inflammatory adhesion factor expression in vascular endothelial progenitor cells (BM-EPCs) induced by TNF-α treatment using Western blotting. [Figure 4] The results of analyzing the angiogenic capacity of vascular endothelial progenitor cells (BM-EPCs) at different treatment concentrations of VEGF, HGF, and PDGF-BB in vitro are shown. The left-hand figure is an image showing the angiogenic capacity of BM-EPCs 4 hours after treatment with each growth factor, while the right-hand graph shows the quantitative results of the number of meshes, junctions, segments, and total length. [Figure 5] This is the result of analyzing the angiogenic capacity of vascular endothelial progenitor cells (BM-EPCs) at different PDGF-BB treatment concentrations and time intervals. [Figure 6]This study confirmed the increased migratory capacity of vascular endothelial progenitor cells (BM-EPCs) at different PDGF-BB treatment concentrations and time intervals. The results involved scratching cross-sections of vascular endothelial progenitor cells and then examining the degree of wound healing through cell migration following PDGF-BB treatment. [Figure 7] This study confirmed the increased cell viability (cell activity) of vascular endothelial progenitor cells (BM-EPCs) after treatment with PDGF-BB. The results were obtained by treating vascular endothelial progenitor cells of different passages (P2, P3, P4) with PDGF-BB at different concentrations, and then analyzing the cell viability using a WST assay. [Figure 8] This is the result of confirming the cell proliferation activity of vascular endothelial progenitor cells (BM-EPCs) treated with PDGF-BB using the BrdU assay method. [Figure 9] This study analyzes changes in the expression of extracellular matrix proteins in vascular endothelial progenitor cells (BM-EPCs) following treatment with PDGF-BB. Specifically, it analyzes the expression level of Laminini-β1 at different PDGF-BB treatment concentrations using immunofluorescence staining and Western blotting. [Figure 10] This study confirmed the increased internalization of PDGFRβ in vascular endothelial progenitor cells (BM-EPCs) following treatment with PDGF-BB. The results were obtained by immunofluorescence staining of vascular endothelial progenitor cells 24 hours after treatment with PDGF-BB at different concentrations. [Figure 11] This study analyzed the angiogenic capacity of vascular endothelial progenitor cells and bone marrow mesenchymal stem cells treated with PDGF-BB. The images show microscopic observations of the angiogenic capacity of co-cultured vascular endothelial progenitor cells and bone marrow mesenchymal stem cells, varying in PDGF-BB treatment concentration and duration. The graph below quantitatively measures the number of meshes, junctions, segments, and total length for each experimental group. [Modes for carrying out the invention]

[0032] The present invention is characterized by providing a method for producing vascular endothelial progenitor cells with enhanced angiogenesis, a novel cell therapy agent for treating vascular dysplasia-related diseases using these cells, and a method for producing the same.

[0033] Blood vessels are formed by the proliferation of vascular endothelial cells. Angiogenesis occurs in the early stages of development, followed by neovascularization, where new blood vessels are formed from existing capillaries. Angiogenesis is tolerated by the body only in special cases, such as when a wound is healing, and is strictly suppressed otherwise.

[0034] Furthermore, endothelial progenitor cells (EPCs) are cells derived from the bone marrow that can mature into endothelial cells, which form blood vessels. They can be identified in whole blood by detecting specific markers such as CD31, CD34, CD133, CD144, and CD309. Endothelial progenitor cells play an important role in vascular regeneration (re-endothelialization) and neovascularization.

[0035] On the other hand, while excessive angiogenesis can be a major cause of disease exacerbation, under-formation of blood vessels is also a cause of serious illness. In particular, under-formation of blood vessels, or vascular dysplasia, can induce necrosis, ulcers, and ischemia, which in turn induce dysfunction of tissues and organs. Diseases such as arteriosclerosis, myocardial infarction, and angina pectoris are also caused by poor blood supply.

[0036] Therefore, to reduce tissue damage caused by hypoxia or malnutrition due to vascular dysfunction (underformation), and to facilitate smooth tissue regeneration, the role of inducing or promoting new angiogenesis is extremely important.

[0037] In particular, angiogenesis is an essential part of the wound healing process for the regeneration of damaged skin tissue. The initial steps of wound healing involve an inflammatory response with cell necrosis and blood vessel destruction. Following this inflammatory response, a series of processes occur in which biological mediators such as kallikrein, thrombin, and plasmin are formed, along with the devascularization of blood components, platelet activation, and blood coagulation.

[0038] On the other hand, methods utilizing endogenous stem cells that regenerate damaged blood vessels and methods utilizing vascular endothelial progenitor cells or vascular endothelial progenitor cell-like cells have been applied as treatment methods for diseases caused by such vascular malformation, but significant efficacy has not yet been confirmed. Patients with underlying diseases have significantly lower numbers and activity of vascular endothelial progenitor cells compared to healthy individuals, so there is a need for technologies that can increase the number of vascular endothelial progenitor cells and enhance angiogenesis.

[0039] Therefore, the inventors investigated methods to enhance the angiogenic capacity of vascular endothelial cells as an effective cell therapy for treating vascular dysplasia-related diseases. As a result, they discovered that when vascular endothelial progenitor cells were treated with PDGF-BB (Platelet derived growth factor-BB) and cultured, not only was the angiogenic capacity of the PDGF-BB-treated vascular endothelial progenitor cells enhanced, but cell activity and cell proliferation were also significantly improved.

[0040] Therefore, the present invention provides a method for producing endothelial progenitor cells with enhanced angiogenic ability, comprising the step of treating isolated endothelial progenitor cells (EPCs) with PDGF-BB (Platelet-derived growth factor-BB) and culturing them.

[0041] In the present invention, the vascular endothelial progenitor cells may be human bone marrow-derived vascular endothelial progenitor cells (BM-EPC).

[0042] Furthermore, treating the isolated vascular endothelial progenitor cells with PDGF-BB can be carried out by adding PDGF-BB to the culture medium of the vascular endothelial progenitor cells. In this case, vascular endothelial progenitor cells that have been subcultured for 1 to 3 (P1 to P3) can be used, and preferably, vascular endothelial progenitor cells that have been subcultured for 2 to 3 (P2 to P3) can be used.

[0043] The aforementioned "subculture" is a method of cell proliferation that involves periodically transferring cells to a new culture medium every 5 to 7 days in order to continue the cell lineage. Specifically, "passage" refers to the growth of cells in a culture vessel from the initial seed culture stage to the stage where the cells are actively growing (confluence) in the same culture vessel.

[0044] In one embodiment of the present invention, vascular endothelial progenitor cells from different passages were treated with PDGF-BB, and then the cell activity (cell viability) of the vascular endothelial progenitor cells was analyzed. It was shown that cell activity increased in vascular endothelial progenitor cells from passages P1 to P3 after PDGF-BB treatment, whereas there was no significant difference in vascular endothelial progenitor cells from passage P4 after PDGF-BB treatment. Therefore, it is preferable to treat isolated vascular endothelial progenitor cells from passages P1 to P3 with PDGF-BB.

[0045] The PDGF-BB treatment can be applied to the culture medium to a concentration of 10-40 ng / ml.

[0046] The aforementioned culture can be carried out using cell culture media commonly used in this industry. In one embodiment of the present invention, culture was performed using a-MEM medium supplemented with 0.2% FBS.

[0047] According to one embodiment of the present invention, when the culture medium of human bone marrow-derived vascular endothelial progenitor cells was treated with the neovascularization-promoting factors VEGF, HGF, and PDGF-BB, respectively, the structure in relation to the ability to form a vascular network was observed. It was shown that VEGF and HGF did not have a sufficient effect on vascular network formation, while angiogenesis was significantly increased only in the group specifically treated with PDGF-BB.

[0048] Through this, the inventors confirmed that PDGF-BB can be used as a method for producing vascular endothelial progenitor cells with improved angiogenesis ability.

[0049] In this invention, experiments have confirmed that vascular endothelial progenitor cells treated with PDGF-BB not only exhibit increased vascular network formation, but also possess enhanced cell migration, increased cell viability, increased cell proliferation, and increased expression of the extra-matrix protein laminin β1.

[0050] Therefore, the present invention can provide a method for producing vascular endothelial progenitor cells with enhanced angiogenic ability, comprising the step of treating isolated vascular endothelial progenitor cells with PDGF-BB and culturing them, and can also provide a method for increasing the cell number of vascular endothelial progenitor cells, comprising the step of treating isolated vascular endothelial progenitor cells with PDGF-BB and culturing them.

[0051] Furthermore, the present invention also has the characteristic of being able to provide a composition for promoting angiogenesis that contains endothelial progenitor cells (EPCs) and PDGF-BB (Platelet-derived growth factor-BB) as active ingredients.

[0052] The angiogenesis-promoting composition of the present invention contains both vascular endothelial progenitor cells and PDGF-BB as active ingredients, and as previously described, it is characterized by promoting angiogenesis through the action of promoting cell migration, cell viability, and cell proliferation by vascular endothelial progenitor cells whose angiogenesis ability has been enhanced by PDGF-BB.

[0053] Therefore, since the angiogenesis-promoting composition according to the present invention has the effect of preventing or treating diseases associated with angioplasty, the present invention can provide a pharmaceutical composition for the prevention or treatment of angioplasty-related diseases containing the angiogenesis-promoting composition as an active ingredient.

[0054] Angioplasty-related diseases that can be prevented or treated by the angioplasty-promoting composition according to the present invention may include, but are not limited to, ischemic diseases, diabetic ulcers, gangrene, occlusive vascular diseases, cardiovascular diseases, and local anemia, and the ischemic diseases may include, but are not limited to, ischemic myocardial infarction, ischemic heart disease, ischemic vascular disease, ischemic eye disease, ischemic renal failure, ischemic retinopathy, ischemic stroke, and ischemic lower limb disease.

[0055] The angiogenesis-promoting composition or pharmaceutical composition for the prevention or treatment of angiogenesis-related diseases according to the present invention may contain a pharmaceutically effective amount of an active ingredient alone as a physiologically active ingredient, or may contain one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0056] In the foregoing, "pharmaceutically effective amount" means an amount sufficient to exhibit the desired physiological or pharmacological activity when administered to an animal or human. However, the pharmaceutically effective amount can be appropriately varied depending on the age, weight, health condition, sex, route of administration, and duration of treatment of the recipient.

[0057] Furthermore, in the foregoing, "pharmaceutically acceptable" means that it is physiologically acceptable and does not typically cause gastrointestinal disorders, allergic reactions such as dizziness, or similar reactions when administered to humans. Examples of the carrier, excipients, and diluents may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. They may also further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives.

[0058] Furthermore, the compositions of the present invention can be formulated using methods known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to a mammal, and can be formulated in a variety of forms for parenteral administration.

[0059] Typical parenteral dosage forms are injectable forms, preferably isotonic aqueous solutions or suspensions. Injectable dosage forms can be manufactured by art known to the art using suitable dispersants or wetting agents and suspending agents. For example, each component can be dissolved in saline or buffer solution to form an injectable dosage form.

[0060] The compositions according to the present invention can be administered through various routes, including transdermal, subcutaneous, intravenous, or intramuscular, and the dosage of the active ingredient can be appropriately selected depending on various factors such as the route of administration, the patient's age, sex, weight, and the severity of the patient's condition. Furthermore, the compositions according to the present invention can be administered in parallel with known compounds that can enhance the desired effect.

[0061] The present invention can also provide a method for producing a cell therapy agent for the treatment of vascular dysplasia-related diseases, the method comprising the step of mixing isolated endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs), and PDGF-BB (Platelet-derived growth factor-BB).

[0062] In the method for producing the cell therapy agent of the present invention, the isolated vascular endothelial progenitor cells and mesenchymal stem cells may be a mixed cell obtained by mixing them in a cell number ratio of 1:1 to 2:1, and preferably, vascular endothelial progenitor cells and mesenchymal stem cells can be used mixed in a cell number ratio of 2:1.

[0063] The aforementioned "mesenchymal stem cells (MSCs)" refer to multipotent stem cells that can differentiate into a variety of mesodermal cells, including bone, cartilage, fat, and muscle cells. These mesenchymal stem cells are found in umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and chorionic villi. These may be derived from membranes, decidua, or placenta, and preferably from human bone marrow-derived mesenchymal stem cells (BM-MSCs). Mesenchymal stem cells have the ability to migrate directly to the site of bodily injury and regenerate damaged tissues and cells, can proliferate easily in vitro, and are pluripotent cells capable of differentiating into various cell morphologies, making them useful targets in gene therapy and cell therapy.

[0064] The vascular endothelial progenitor cells used in the present invention may be human bone marrow-derived vascular endothelial progenitor cells.

[0065] Furthermore, in the production of the cell therapy agent according to the present invention, perivascular cells may be used instead of mesenchymal stem cells.

[0066] The aforementioned "pericytes" refer to vascular wall cells located within the basement membrane of the microvessel system, forming specific local contact with the vascular endothelium. These are connective tissue cells surrounding small blood vessels, also called Rouget cells, adventitial cells, or mural cells, and are known to surround 10% to 50% of the area outside the vascular endothelium. They are elongated, contractile cells that surround the pre-capillary arterioles outside the basement membrane.

[0067] The aforementioned perivascular cells are relatively undifferentiated pluripotent cells that support blood vessels and can differentiate into fibroblasts, smooth muscle cells, or macrophages as needed. Furthermore, these perivascular cells play an important role not only in angiogenesis but also in the stability of the blood-brain barrier and can regulate blood flow in the microvessel system through their adhesion to endovascular cells.

[0068] Therefore, the production of the cell therapy agent according to the present invention can enhance the angiogenic ability of vascular endothelial progenitor cells by treatment with PDGF-BB, and can further enhance angiogenesis, vascular regeneration, and vascular barrier stability through co-culture with mesenchymal stem cells or perivascular cells.

[0069] Therefore, the present invention can provide not only a method for producing a cell therapy agent for the treatment of angioplasty-related diseases, but also a cell therapy agent for the treatment of angioplasty-related diseases produced by the method described above.

[0070] The cell therapy agent of the present invention may further include a support for containing the cells, preferably a biodegradable support. The biodegradable support may be, but is not limited to, a hydrogel such as fibrin glue, hyaluronic acid, gelatin, collagen, alginic acid, cellulose, pectin, chitin, polyglycolic acid, or polylactic acid.

[0071] Furthermore, the cell therapy agent may further contain a pharmaceutically acceptable carrier, the pharmaceutically acceptable carrier can be a mixture of saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components, and other common additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. The cell therapy agent can be further formulated into injectable dosage forms such as aqueous solutions, suspensions, or emulsions by adding diluents, dispersants, surfactants, binders, and lubricants, and can be used for intramedullary administration, intravenous administration, subcutaneous administration, intramuscular administration, or intraperitoneal administration.

[0072] Furthermore, the vascular dysplasia-related diseases that the cell therapy agent according to the present invention aims to treat may be selected from the group consisting of ischemic diseases, diabetic ulcers, gangrene, occlusive vascular diseases, cardiovascular diseases, and local anemia.

[0073] The aforementioned ischemic diseases may be selected from the group consisting of ischemic myocardial infarction, ischemic heart disease, ischemic vascular disease, ischemic eye disease, ischemic renal failure, ischemic retinopathy, ischemic stroke, and ischemic lower limb disease, but are not limited to these.

[0074] The present invention also provides a method for preventing or treating vascular dysplasia-related diseases, comprising the step of administering the vascular formation-promoting composition to an individual.

[0075] The present invention also provides a method for preventing or treating angioplasty-related diseases, comprising the step of administering a cell therapy agent produced by the above-described manufacturing method to an individual.

[0076] The components and technical features of the present invention will be described in more detail below through the following examples. However, the following experimental examples are merely illustrative of the content of the present invention, and the scope of the invention is not limited by these examples. [Examples]

[0077] <Experimental Example 1> Experimental Method 1-1. Cell culture and EPC-specific analysis Human BM mononuclear cells (MNCs) were purchased from STEMCELL Technologies Inc. (Vancouver, Canada). For EPC culture, BM-MNCs were cultured in endothelial growth medium-2 (EGM-2, Lonza, Basel, Switzerland) in 100 mm dishes coated with type I collagen (CellMatrix, VA, USA). The medium was changed daily for the first 7 days, and then every 3 days thereafter. EPCs were subcultured at 70-80% confluence. For MSC culture, BM-MNCs were cultured in 100 mm dishes in mesenchymal stem cell growth medium (MSCGM, Lonza). The medium was changed every 3 days, and subcultured at 70-80% confluence.

[0078] For immunofluorescence (IF) staining, BM-EPC (1.0 × 10) 4 The cells were cultured on collagen type I coated coverslips, fixed with 3.7% formaldehyde, permeabilized with 0.2% Triton x-100 buffer for 10 minutes, and blocked using 20% ​​normal goat serum. Ulex europaeus agglutinin (UEA)-I lectin binding was performed using fluorescein (FITC)-labeled UEA-I lectin (Sigma Aldrich, St. Louis, MO). Subsequently, Von Willebrand factor (vWF, abcam, Cambridge, England), PDGFRβ (abcam), VEGFR2 (abcam), and Laminin (Santa Cruz, California, USA) were used as primary antibodies, and Alexa fluor 488 or 568-conjugated secondary antibodies were used as secondary antibodies. The coverslips were mounted using mounting medium containing DAPI (VECTOR, Burlingame, CA). The images were acquired using fluorescence microscopes (Leica, Wetzlar, Germany).

[0079] Expression analysis of surface markers UEA-I (VECTOR), vWF (abcam), CD31, CD309, CD105, CD45, and CD34 was performed using Novocyte (Agilent, CA, USA) and analyzed with Novosoftware (Agilent).

[0080] For the reaction analysis of BM-EPC with TNF-α, BM-EPC (1.0 × 10) 5 The cells were cultured in a 6-well plate coated with type I collagen, and after adding 10 ng / ml TNF-α (PeproTech Inc, Rocky Hill, USA) and culturing for 24 hours, the expression levels of ICAM-1 (abcam) and VCAM-1 (Santa Cruz) were analyzed by Western blotting.

[0081] 1-2.ELISA analysis EPC (2.0 x 10 5 Cells were cultured in 100 mm culture dishes, and conditioned medium (EPC) was collected on day 4. Subsequently, levels of HGF, TGF-β1, VEGF, and PDGF-BB were measured according to the guidelines of the Quantikine ELISA manufacturer (R&D Systems, Minneapolis, USA). Absorbance was measured at 450 nm using a microplate reader (Biotek, VT, USA).

[0082] 1-3. Tube Formation Assay Matrigel (Corning Inc., NY, USA) was applied to μ-slides (Ibidi, Grafelfing, Germary) at a rate of 10 μl per well. EPC (4.0 × 10 3 ) or EPC (4.0 × 10 3 ) and MSC(2.0×10 3Mixed cells were suspended in α-MEM (GIBCO, NY, USA) medium containing 0.2% FBS, seeded onto Matrigel, and then cultured under treatment with VEGF, HGF, and PDGF-BB (PeproTech) at concentrations of 0-40 ng / ml. Images were subsequently acquired using a microscope (Leica).

[0083] 1-4. Cell viability analysis and BrdU (5-Bromo-2'-deoxyuridine) cell labeling For cell viability analysis, EPC(1.0 × 10) 4 The samples were seeded into 96-well plates coated with collagen type I, starved for 18 hours in EBM-2 containing 0.2% FBS, and then treated with 0, 10, 20, and 40 ng / ml of PDGF-BB for 24 hours, respectively. WST analysis was performed according to the manufacturer's guidelines (Roche, Basel, Switzerland). Absorbance was measured at 450 nm using a multi-plate reader (Biotek).

[0084] For BrdU cell labeling, EPC (1.0 × 10 4 The cells were cultured in collagen type I coated coverslips in fresh medium, then treated with 20 ng / ml PDGF-BB for 20 hours, followed by the addition of 10 μM BrdU (Sigma Aldrich) and cultured for 4 hours. The cells were then immobilized using methanol for 10 minutes. The coverslips were then treated with 2N HCl at 37°C for 1 hour, neutralized with 0.1 M borate buffer, treated with primary antibody to detect BrdU (Roche), and subsequently treated with FITC-conjugated secondary antibody (Invitrogen, California, USA). The cells were cultured, stained with PI (Sigma Aldrich) as a control, and images were obtained by fluorescence microscopy (Leica).

[0085] 1-5. Western blot The BM-EPC lysate was obtained with lysis buffer (Cell signaling, Danvers, MA) supplemented with 2 mM PMSF (Sigma Aldrich). Protein concentration was measured by BCA assay (Thermo Scientific, Waltham, MA). The EPC lysate and conditioned medium were subjected to SDS-PAGE electrophoresis and transferred to a nitrocellulose membrane. Subsequently, primary antibodies for detecting VCAM-1 (Santa Cruz), ICAM-1 (abcam), laminin-β1 (Santa Cruz) and α-tubulin (Sigma Aldrich) were used, and an HRP-conjugated secondary antibody (BD, San Jose, CA, USA) was added for reaction. Subsequently, the membrane after the antibody reaction was developed using EZ-Western Lumi Pico (Dogen, Seoul, Korea), signals were detected using a chemilluminator, and then quantified with ImageJ software.

[0086] 1-6. Wound healing assay (scratch assay) EPC cell monolayers on 6-well plates coated with type I collagen were maintained in starvation with 0.2% FBS + EBM-2 for 18 hours. Subsequently, the cells were scratched with a 200 μl pipette tip, washed with 1xDPBS, and then cultured in 0.2% FBS + α-MEM medium containing 0, 10, 20 ng / ml of PDGF-BB. The wound healing process was observed with a microscope (Leica).

[0087] 1-7. Statistical analysis All data were presented as mean ± standard deviation and considered significant when the P value was less than 0.05 ( * p < 0.05, ** p < 0.01, *** p < 0.001).

Example

[0088] <Experimental Example 2> Phenotypic and characteristic analysis of endothelial progenitor cells (EPCs) derived from human bone marrow mononuclear cells (MNCs). Human bone marrow-derived vascular endothelial progenitor cells (BM-EPCs) exhibiting colony formation were isolated from human bone marrow mononuclear cells, and it was shown that BM-EPC colonies had a morphology closer to spindle morphology than cobblestone morphology (Figure 1a). Immunofluorescence staining, ELISA, parenchymal cell analysis, and Matrigel tube formation analysis were performed to confirm the phenotype of the isolated vascular endothelial progenitor cells. Expression of UEA-1 lectin binding and vWF markers, which are common markers for endothelial cells, was confirmed by double fluorescence staining (Figure 1b).

[0089] Subsequently, major angiogenic factors such as HGF, TGF-β1, VEGF, and PDGF-BB were measured using ELISA in BM-EPC condition media for each passage. The results showed that human bone marrow-derived vascular endothelial progenitor cells (BM-EPCs) secrete HGF, TGF-β1, and VEGF, and that their expression levels decreased with passage. Furthermore, PDGF-BB was not secreted in all 2-4 passage cultures (Figure 1c).

[0090] Furthermore, the expression levels of cell surface markers were measured through parenchyma cell analysis. Human BM-EPCs cultured for 1-3 passages showed low expression of CD31 (<2%), CD309 (<3%), CD45 (<2%), and CD34 (<1%), but high expression of CD105 (>95%) (Figure 1d). In addition, BM-EPCs showed high UEA-I binding (>99%) and vWF (>99%) expression, as confirmed by fluorescence staining after 1-3 passages. Matrigel tube formation analysis showed that BM-EPCs had very low tube formation ability up to 3 passages, but showed tube formation ability after 4-6 passages (Figure 1e).

[0091] Furthermore, while BM-EPCs showed very low tube-forming ability after three passages, a well-formed vascular network could be observed when BM-EPCs and BM-MSCs were mixed in a 2:1 ratio (Figure 1f).

[0092] Therefore, through these results, the inventors confirmed that human bone marrow mononuclear cell-derived vascular endothelial progenitor cells (BM-EPCs) possess colony-forming ability and are a very rapidly dividing cell population, with the time to double the cell number up to three passages (P3) being approximately 1.5 days (Figure 2). Furthermore, BM-EPCs were shown to divide very rapidly in the initial passage, secrete angiogenic factors such as VEGF, HGF, and TGF-β1, and further enhance vascular network formation when cultured with BM-MSC cells. Thus, these results indicate that mixed cells obtained by co-culturing BM-EPCs and BM-MSCs can be usefully used as cell therapy agents for the treatment of various ischemic and chronic vascular diseases. [Examples]

[0093] <Experimental Example 3> Confirmation of increased expression of inflammatory adhesion factors in vascular endothelial progenitor cells by TNF-α treatment. TNF-α is known to upregulate the expression of pro-inflammatory adhesion factors such as ICAM-1 and VCAM-1 in vascular endothelial cells. Therefore, the inventors analyzed, through Western blot analysis, whether the human bone marrow-derived vascular endothelial progenitor cells (BM-EPCs) of the present invention can induce the expression of pro-inflammatory factors upon TNF-α treatment.

[0094] The results showed that BM-EPC does not express ICAM-1, but ICAM-1 expression significantly increases upon TNF-α treatment (Figure 3A). Furthermore, BM-EPC significantly regulates VCAM-1 expression upward upon TNF-α treatment (Figures 3B-C).

[0095] Through these results, the inventors have concluded that BM-EPC can induce an inflammatory response when treated with TNF-α, and that the BM-EPC of the present invention possesses the characteristics of vascular endothelial cells. [Examples]

[0096] <Experimental Example 4> Analysis of the effect of treatment with angiogenesis factors on enhancing the angiogenesis capacity of human bone marrow-derived vascular endothelial progenitor cells (BM-EPCs) To determine whether VEGF, HGF, and PDGF-BB, known as neovascularization-promoting factors in vascular endothelial cells, affect the vascular network formation ability of vascular endothelial progenitor cells, we conducted a study on vascular endothelial progenitor cells (4.0 × 10⁻⁶). 3 Cells (per well) were spread in α-MEM medium containing 0.2% FBS, and VEGF, HGF, and PDGF-BB were added at different concentrations (0, 10, 20, and 40 ng / ml), respectively, and dispensed onto μ-slide Matrigel. The ability to form a vascular network was then observed in terms of the number of meshes, the number of junctions, the number of segments, and the total length between two junctions.

[0097] As a result, as shown in Figure 4, VEGF did not show a significant difference in the angiogenic capacity of vascular endothelial progenitor cells, but PDGF-BB significantly increased the angiogenic capacity of vascular endothelial progenitor cells in a concentration-dependent manner. Furthermore, while the angiogenic capacity of vascular endothelial progenitor cells was also increased in the HGF-treated group, the increase in angiogenic capacity was not as significant as that of the PDGF-BB-treated group.

[0098] Therefore, the present inventors have found that treatment with PDGF-BB is extremely useful as a cell activation method that can effectively enhance the angiogenic capacity of vascular endothelial progenitor cells.

[0099] Furthermore, prior examples confirmed that BM-EPC does not secrete PDGF-BB, but that it is present in abundant amounts under wound conditions. Therefore, the inventors treated BM-EPC that had been subcultured three times with PDGF-BB at different concentrations (0, 10, 20, and 40 ng / ml), and then continuously monitored it for 18 hours to observe its tube-forming ability.

[0100] As a result, as shown in Figure 5, the number of meshes, junctions, segments, and total length between two junctions of human BM-EPC increased in a treatment concentration-dependent manner with PDGF-BB, confirming that vascular network formation was enhanced. [Examples]

[0101] <Experimental Example 5> Cellular response analysis of bone marrow-derived vascular endothelial progenitor cells (BM-EPCs) treated with PDGF-BB 5-1. Effects on the migration of vascular endothelial progenitor cells To analyze the changes in the migratory ability of vascular endothelial progenitor cells (1.0 × 10⁶) induced by PDGF-BB treatment, the gap-filling rate was measured after scratch injury to vascular endothelial progenitor cells. Specifically, in 6 wells, vascular endothelial progenitor cells (1.0 × 10⁶) were measured. 5 After culturing cells (cells / well), a starvation period of 18 hours was initiated. Then, cell cross-sections of vascular endothelial progenitor cells were scraped with a 200 μl tip and washed twice with PBS. Subsequently, α-MEM medium containing 0.2% FBS was treated with PDGF-BB at different concentrations (0, 10, 20 ng / ml), and the wound closure process was measured.

[0102] As a result, as shown in Figure 6, cell migration of vascular endothelial progenitor cells was enhanced in the group treated with PDGF-BB compared to the group not treated with PDGF-BB.

[0103] 5-2. Effects on the cell viability of vascular endothelial progenitor cells We analyzed the changes in the survival rate of vascular endothelial progenitor cells by passage after PDGF-BB treatment. For this purpose, we used vascular endothelial progenitor cells (1.0 × 10⁶) in 96 wells. 4 After culturing cells (cells / well), a starvation state was maintained for 18 hours, followed by treatment with PDGF-BB at different concentrations (0, 10, 20, 40 ng / ml) for 24 hours. After treating vascular endothelial progenitor cells with WST-1 solution, cell viability was analyzed by measuring absorbance at 450 nm.

[0104] As shown in Figure 7, cell viability increased in P2 and P3 passaged vascular endothelial progenitor cells in proportion to the PDGF-BB treatment concentration, but no significant difference was observed in P4 passages due to PDGF-BB treatment.

[0105] 5-3. Effects on the proliferation of vascular endothelial progenitor cells We analyzed the changes in cell proliferation of vascular endothelial progenitor cells induced by PDGF-BB treatment. For this purpose, we placed vascular endothelial progenitor cells (1.0 × 10⁶) in 24-well coverslips. 4 After culturing the cells, they were kept in a starvation state for 18 hours, and then treated with PDGF-BB at different concentrations (0, 20 ng / ml) for 24 hours. Then, 20 hours after PDGF-BB treatment, they were treated with 10 μM BrdU for 4 hours, and the number of cells entering the S-phase was measured. In addition, vascular endothelial cells were fixed with 3.7% formaldehyde and permeabilized, then treated sequentially with primary and secondary antibodies, and nuclear staining was performed with PI staining. Cell proliferation was analyzed by counting the number of BrdU-positive cells among PI-positive cells using a fluorescence microscope.

[0106] As a result, as shown in Figure 8, we confirmed that the number of vascular endothelial progenitor cells increased and cell proliferation occurred in the group treated with PDGF-BB compared to the group that was not treated with PDGF-BB.

[0107] 5-4. Effects on the expression of laminin β1, VEGFR2, and PDGFRβ The expression level of laminin β1, an extra-matrix protein in vascular endothelial progenitor cells treated with PDGF-BB, was analyzed by fluorescence staining and Western blotting. For this purpose, vascular endothelial progenitor cells (1.0 × 10⁶) were placed in coverslips in 24 wells. 4 After culturing the cells, they were kept in a starvation state for 18 hours, and then treated with PDGF-BB at different concentrations (0, 10, 20 ng / ml) for 24 hours. Vascular endothelial cells were fixed with 3.7% formaldehyde, permeabilized, treated with a primary antibody against laminin β1, then treated with a secondary antibody, stained with DAPI, and the degree of laminin β1 expression was analyzed.

[0108] As a result, as shown in 9, laminin β1 expression in vascular endothelial progenitor cells was increased in proportion to the PDGF-BB treatment concentration. These results were also confirmed by Western blotting of the culture medium of vascular endothelial progenitor cells.

[0109] Furthermore, in order to analyze the changes in VEGFR2 and PDGFRβ expression in vascular endothelial progenitor cells induced by PDGF-BB treatment, we performed the analysis using antibodies against VEGFR2 and PDGFRβ as described above. As a result, as shown in Figure 10, no VEGFR2 expression was observed in vascular endothelial progenitor cells, indicating that PDGF-BB treatment does not affect VEGFR2 expression.

[0110] On the other hand, it was shown that PDGFRβ internalization increased in proportion to the PDGF-BB treatment concentration. This is expected to be related to increased cell migration, increased cell viability, increased cell proliferation, and increased expression of the extra-substrate protein laminin β1, as previously confirmed. [Examples]

[0111] <Experimental Example 6> Confirmation of effective vascular network increase by co-culture of bone marrow-derived vascular endothelial progenitor cells (BM-EPCs) and mesenchymal stem cells and PDGF-BB treatment. Through the experiments in the above-mentioned examples, the inventors confirmed that treating BM-EPC with PDGF-BB increases the activity of the vascular network. Therefore, as a method to further enhance the vascular network formation ability of BM-EPC, the inventors conducted experiments to confirm whether co-culturing BM-EPC with mesenchymal stem cells and treating them with PDGF-BB could effectively enhance the vascular network formation ability.

[0112] For this reason, vascular endothelial progenitor cells (4.0 × 10) 3 cells / well and mesenchymal stem cells (2.0 × 10⁻¹⁰ 3 Cells per well were suspended in α-MEM medium containing 0.2% FBS in a 2:1 ratio (cell number ratio), and treated with PDGF-BB at different concentrations (0, 10, 20, 40 ng / ml). These cell cultures were then dispensed onto Matrigel μ-slides, and the degree of vascular network formation was observed.

[0113] As a result, as shown in Figure 11, the group in which vascular endothelial progenitor cells and mesenchymal stem cells were co-cultured showed increased vascular network formation ability compared to the group in which vascular endothelial progenitor cells were cultured alone. Furthermore, when the group in which vascular endothelial progenitor cells and mesenchymal stem cells were co-cultured was treated with PDGF-BB, the angiogenesis ability increased further in proportion to the treatment concentration.

[0114] Therefore, through these results, the inventors have found that mixed cells containing vascular endothelial progenitor cells and mesenchymal stem cells can be used as a composite stem cell therapeutic agent that can enhance vascular network formation and vascular regeneration capacity, and in particular, that treating these mixed cells with PDGF-BB can further enhance vascular network formation capacity.

[0115] Therefore, the mixed cells of vascular endothelial progenitor cells and mesenchymal stem cells treated with PDGF-BB as devised in the present invention can be used as a novel cell therapy agent for the treatment of ischemic diseases and other conditions caused by a decrease in vascular network formation ability.

[0116] Up to this point, the present invention has been examined primarily in terms of preferred embodiments. Those with ordinary skill in the art to which the present invention pertains should understand that the present invention can be embodied in modified forms that do not deviate from its essential characteristics. Therefore, the disclosed embodiments should be considered in an explanatory rather than restrictive manner. The scope of the present invention is defined in the claims, not in the foregoing description, and all differences within an equivalent scope should be considered as being included within the scope of the present invention.

[0117] This invention was carried out with support from the Ministry of Health and Welfare's Advanced Medical Technology Development (R&D) Research Project (Development of a combined stem cell therapy agent of autologous bone marrow mesenchymal stem cells and vascular endothelial progenitor cells, Project No.: 1465032804, Project No.: HI18C1492010021).

Claims

1. (i) Having the cellular phenotypes CD31-, CD309-, CD45- and CD34-, (ii) Vascular endothelial progenitor cells with enhanced angiogenesis ability, characterized by increased laminin β1 expression compared to vascular endothelial progenitor cells (EPCs) that have not been treated with PDGF-BB (Platelet-derived growth factor-BB).

2. The vascular endothelial progenitor cells according to claim 1, characterized in that, compared to vascular endothelial progenitor cells not treated with PDGF-BB, at least one of the following is increased: cell motility, cell viability, and cell proliferation.

3. The vascular endothelial progenitor cell according to claim 1, characterized in that it is a human bone marrow-derived vascular endothelial progenitor cell (BM-EPC).

4. A composition for the prevention or treatment of vascular dysplasia-related diseases, comprising vascular endothelial progenitor cells according to any one of claims 1 to 3.

5. The composition according to claim 4, further characterized by containing mesenchymal stem cells (MSCs).

6. The composition according to claim 5, characterized in that the ratio of vascular endothelial progenitor cells to mesenchymal stem cells is 1:1 to 2:

1.

7. The composition according to any one of claims 4 to 6, characterized in that the vascular dysplasia-related disease is ischemic disease, diabetic ulcer, gangrene, occlusive vascular disease, cardiovascular disease, or local anemia.

8. The composition according to claim 7, characterized in that the ischemic disease is ischemic myocardial infarction, ischemic heart disease, ischemic vascular disease, ischemic eye disease, ischemic renal failure, ischemic retinopathy, ischemic stroke, or ischemic lower limb disease.

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