Composition and use thereof

Abstract

According to the present invention, a graft rejection reaction is reduced, and extracellular vesicles derived from cells having physiological activity against at least one disease among kidney diseases, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-obstructive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory diseases are provided. A composition according to the present disclosure is used to treat at least one disease among kidney diseases, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-obstructive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory diseases, and contains extracellular vesicles of a regenerative cell population derived from a biological sample-derived monocytes or a culture thereof.

Description

Compositions and uses thereof

[0001] The present disclosure relates to compositions and uses thereof.

[0002] Cell-based treatments have been attempted for various diseases, including inflammatory diseases, kidney diseases, peripheral arterial disease (PAD), cerebral infarction, and pulmonary hypertension (PHD). Autologous cells have been used as the cells. However, when using autologous cells, cells must be obtained from the patient, processed, and then administered to the patient, which requires time and involves problems of variability in processing. For this reason, treatments using allogeneic cells have also been attempted (Patent Document 1).

[0003] Special Publication No. 2011-529957

[0004] However, when the allogeneic cells are transplanted into the patient, an immune response (graft rejection) against the allogeneic cells is induced after the transplant, and the allogeneic cells are eliminated from the patient, resulting in a problem that a sufficient therapeutic effect cannot be obtained.

[0005] Therefore, the present disclosure aims to provide a composition comprising cell-derived extracellular vesicles that reduce transplant rejection and have physiological activity against at least one of kidney disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease.

[0006] To achieve the above-mentioned objective, the composition of the present disclosure is a composition for use in treating at least one of kidney disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease, wherein the composition comprises extracellular vesicles of a regenerative cell population derived from mononuclear cells derived from a biological sample or a culture thereof.

[0007] The present disclosure provides a composition for use in treating at least one of renal disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease, wherein the composition is a composition of the present disclosure.

[0008] The present disclosure relates to a composition for use in promoting the proliferation of vascular endothelial cells, wherein the composition is the composition of the present disclosure.

[0009] The present disclosure provides a composition for use in inducing angiogenesis, wherein the composition is a composition of the present disclosure.

[0010] The present disclosure relates to a composition for use in inhibiting fibrosis, wherein the composition is a composition of the present disclosure.

[0011] According to the present disclosure, a composition comprising extracellular vesicles that reduces transplant rejection and has physiological activity against at least one of kidney disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease can be provided.

[0012] FIG. 1 is a graph showing the particle size, production amount per cell, and average diameter of extracellular vesicles in Example 1. FIG. 2 is an electron microscope image showing the marker expression profile, the lipid bilayer structure of extracellular vesicles, and a graph showing the secretion amount of extracellular vesicles and the amount of extracellular vesicle protein in Example 1. FIG. 3 is a histogram showing marker expression in Example 1. FIG. 4 is a graph showing the proliferation of HUVEC cells in Example 1. FIG. 5 is a photograph and graph showing the growth area of ​​HUVEC cells in Example 1. FIG. 6 is a graph showing the expression level of miRNA in Example 1. FIG. 7 is a graph showing the body weight and organ weight of rats in Example 1. FIG. 8 is a graph showing the percentages of CD3-positive cells, CD4-positive cells, CD8-positive cells, regulatory T cells, and CD11b / c-positive cells in Example 1. FIG. 9 is a graph showing the expression level of miRNA in Example 1. FIG. 10 is a graph showing serum leukocyte leukemia (BUN) levels in Example 1. Figure 11 is a photograph and graph showing the degree of fibrosis in Example 1. Figure 12 is a photograph showing microvessel density in ischemic-damaged renal tissue in Example 1, and a graph showing the number of CD31-positive microvessels and the results of transcriptome analysis. Figure 13 is a photograph showing microvessel density in ischemic-damaged renal tissue on days 4 and 28 after the onset of R-IRI in Example 1, and a graph showing the number of CD31-positive microvessels. Figure 14 is a photograph and graph showing the results of IVIS measurement in Example 1. Figure 15 is a graph and diagram showing the results of elucidating the mechanism of fibrosis reduction, using various analytical methods, in which administration of RACev significantly reduces renal fibrosis 4 weeks after the onset of K-IRI in Example 1.

[0013] <Definition> In the present disclosure, the term "disease" includes at least one of renal disease, peripheral arterial disease (PAD), pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease, but is not particularly limited as long as it is a disease that can be treated with the composition of the present disclosure.

[0014] As used herein, the term "biological sample" refers to a sample derived from a living organism, from which cells constituting the living organism can be extracted or isolated.

[0015] As used herein, "mononuclear cells" refers to cells with round nuclei contained in peripheral blood, bone marrow, umbilical cord blood, etc. Examples of the mononuclear cells include lymphocytes, monocytes, macrophages, endothelial progenitor cells, hematopoietic stem cells, etc. The mononuclear cells may further include CD34 and / or CD133 positive cells.

[0016] As used herein, "extracellular vesicles" refer to membrane-containing vesicles secreted from cells. Extracellular vesicles are generally thought to be formed in endosomes of the cell of origin and then released outside the cell. Therefore, extracellular vesicles typically contain a lipid bilayer membrane and a lumen, with the lumen surrounded by the lipid bilayer membrane. Furthermore, the lipid bilayer membrane contains lipids derived from the cell membrane of the cell of origin. The lumen contains cytoplasm derived from the cell of origin. Extracellular vesicles are classified into, for example, exosomes, microvesicles (MVs), apoptotic bodies, etc., based on their size and / or surface markers.

[0017] As used herein, the term "regenerative cell population" refers to a cell population containing cells with regenerative function. The regenerative function refers to the function of repairing or improving the function of a tissue, organ, or tissue, and any one of these functions may be exhibited. Specific examples of the regenerative function include vascular endothelial cell proliferation activity and / or angiogenesis promotion activity.

[0018] As used herein, the term "cell population" refers to a cell preparation containing desired cells or a composition containing desired cells. Therefore, as used herein, the cell population can also be referred to as, for example, a cell preparation or a composition. In the cell population, the proportion of desired cells among all cells (hereinafter also referred to as "purity") can be quantified, for example, as the proportion of cells expressing one or more markers expressed by the desired cells. The purity is, for example, the proportion among live cells. The purity can be measured, for example, by methods such as flow cytometry, immunohistochemistry, and in situ hybridization.

[0019] As used herein, the purity of the cell preparation is, for example, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0020] As used herein, "culture" refers to a substance comprising cells obtained by culturing the cells of interest and / or the medium for said cells.

[0021] As used herein, the term "umbilical cord" refers to a white tubular tissue that connects a fetus to a placenta. In the present disclosure, the origin of the "umbilical cord" is not particularly limited, and examples thereof include the umbilical cords of mammals such as mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, dolphins, and sea lions. The umbilical cords are preferably from primates, and more preferably from humans.

[0022] As used herein, "umbilical cord blood" refers to blood derived from a fetus contained in the umbilical cord.

[0023] As used herein, "miRNA" refers to non-coding RNA of about 18 to about 25 bases in length. The miRNA may also be referred to as "microRNA," "miR," etc.

[0024] As used herein, "miRNA precursor" refers to a transcript originating from genomic DNA and comprising non-coding RNA that includes one or more miRNA sequences. The miRNA precursor may also be referred to as "pri-miRNA" or "pre-miRNA." The miRNA precursor typically has a hairpin structure and contains the base sequence of a mature miRNA.

[0025] As used herein, "mir-X" refers to a miRNA precursor with the number X, and "miR-X" refers to the mature form (miRNA) of the miRNA with the number X. When two miRNAs originate from opposite arms of the same miRNA precursor, the two miRNAs are indicated using the suffixes "-3p" or "-5p." Hereinafter, unless otherwise specified, when a miRNA precursor contains two miRNAs, the "miR-X" refers to both the -3p and -5p miRNAs. Furthermore, when the relative expression levels of two miRNAs are known and the expression level of one miRNA is lower than that of the other miRNA, the miRNA with the lower expression level is indicated with an asterisk after its name. Information such as the base sequences of each "mir-X" and "miR-X" can be found on miRBase (http: / / www.mirbase.org / ).

[0026] As used herein, "promotion of cell proliferation" means that the proliferation of target cells is significantly promoted. The target cells are desired cells. The promotion can also be referred to as improvement, enhancement, or increase. Specifically, the "promotion of cell proliferation" means that the proliferation of target cells is significantly promoted in a group treated with a test substance, compared to a control group not treated with the test substance or a control group treated with a substance that does not have cell proliferation-promoting activity. Therefore, as used herein, even if the cell proliferation activity of the control group is reduced compared to the start of the test, "promotion of cell proliferation" can be said to have occurred as long as the proliferation of target cells is significantly promoted compared to the control group. The cell proliferation activity can be measured, for example, as described in Example 1 below.

[0027] As used herein, "promoting angiogenesis" means that the neovascularization or proliferation of blood vessels is significantly promoted. The promotion can also be referred to as improvement, enhancement, or increase. Specifically, the "promoting angiogenesis" means that the neovascularization or proliferation of blood vessels is significantly promoted in a group treated with a test substance, compared to a control group not treated with the test substance or a control group treated with a substance without pro-angiogenic activity. Therefore, as used herein, even if the amount of neovascularization or proliferation of blood vessels in the control group is reduced compared to the control group at the start of the test, it can be said that "promoting angiogenesis" has occurred as long as the amount of neovascularization or proliferation of blood vessels is significantly promoted compared to the control group. The pro-angiogenic activity can be measured, for example, according to Example 1 described below.

[0028] As used herein, "fibrosis suppression" means that fibrosis in a subject is significantly suppressed. The suppression can also be referred to as reduction, lowering, suppression, inhibition, or prevention. Specifically, the "fibrosis suppression" means that fibrosis in a subject treated with a test substance is significantly suppressed compared to a control group not treated with the test substance or a control group treated with a substance without fibrosis suppression activity. Therefore, as used herein, even if fibrosis in the control group is suppressed compared to the control group at the start of the test, it can be said that "fibrosis suppression" has occurred as long as fibrosis in the subject is significantly suppressed compared to the control group. The fibrosis suppression activity can be measured, for example, according to Example 1 described below.

[0029] As used herein, "graft rejection" or "immune rejection" refers to a host reaction that rejects a graft caused by differences in major histocompatibility antigens in cell, tissue, organ, or organ transplants. The graft rejection reaction can also be referred to as an "allo-reaction" or "allograft rejection" when the graft is between the same species. Furthermore, the graft rejection reaction can also be referred to as a "xenograft rejection" when the graft is between different species. The "graft rejection reaction" can be measured, for example, according to Example 1 described below.

[0030] As used herein, "suppression of graft rejection" or "suppression of immune rejection" means that graft rejection or immune rejection is significantly suppressed. The suppression can also be referred to as reduction, lowering, suppression, inhibition, or prevention. The "suppression of graft rejection" means that the graft rejection is significantly suppressed in a group treated with a test substance, compared to a control group treated with a substance that induces graft rejection. As a specific example, when examining the graft rejection of the extracellular vesicles, if the graft rejection is significantly suppressed in a group treated with the extracellular vesicles, compared to a control group treated with cells from which the extracellular vesicles are derived, it can be said that the extracellular vesicles suppress graft rejection. The "graft rejection" can be measured, for example, according to Example 1 described below.

[0031] As used herein, "extraction" refers to a state in which a specific component is concentrated compared to before extraction. Therefore, the extraction can also be referred to as concentration or enrichment. The "extraction" can be carried out, for example, by obtaining at least one concentration step.

[0032] As used herein, "isolated" refers to a state in which a particular component has been identified and separated and / or recovered from its natural state. The "isolation" can be achieved, for example, by at least one purification step.

[0033] As used herein, "positive (+)" means that a higher signal is detected by an analytical method such as flow cytometry, which utilizes an antigen-antibody reaction, compared to a negative control reaction using negative control cells that do not express the antigen or an antibody that does not react with the antigen.

[0034] As used herein, "negative (-)" means that a signal equivalent to or less than that detected in a negative control reaction using negative control cells that do not express the antigen or an antibody that does not react with the antigen is detected.

[0035] As used herein, "treatment" means therapeutic treatment and / or prophylactic treatment. As used herein, "treatment" means treating, curing, preventing, suppressing, ameliorating, or improving a disease, pathology, or disorder, or halting, inhibiting, reducing, or delaying the progression of a disease, pathology, or disorder. As used herein, "prevention" means reducing the likelihood of developing a disease or pathology, or delaying the onset of a disease or pathology. The "treatment" may be, for example, treatment of a patient who develops a target disease, or treatment of an animal model of the target disease.

[0036] As used herein, the term "effective dose" means an amount sufficient to provide a therapeutic effect for a disease when administered to a patient with the disease.

[0037] As used herein, the term "subject" refers to an animal or a cell, tissue, or organ derived from an animal, and is used to particularly include humans. The term "animal" refers to both humans and non-human animals. Examples of non-human animals include mammals such as mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, dolphins, and sea lions.

[0038] The present disclosure will be described below using examples, but the present disclosure is not limited to the following examples and can be implemented with any modifications. Furthermore, the descriptions in this disclosure can be mutually incorporated unless otherwise specified. In this specification, when the expression "~" is used, it is used to mean including the numerical or physical values ​​before and after it. In addition, in this specification, the expression "A and / or B" includes "A only," "B only," and "both A and B."

[0039] <Composition> In one embodiment, the present disclosure provides cell-derived extracellular vesicles or a composition comprising the same, which reduce transplant rejection and have physiological activity against at least one of renal disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease. The composition of the present disclosure is a composition used for treating at least one of renal disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease, wherein the composition comprises extracellular vesicles of a regenerative cell population derived from mononuclear cells derived from a biological sample or a culture thereof. The composition of the present disclosure, which inhibits graft rejection and is derived from the regenerative cell population, exhibits physiological activities such as vascular endothelial cell proliferation-promoting activity and angiogenesis-promoting activity against at least one of kidney disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease. Therefore, the composition of the present disclosure can be suitably used for treating or improving the prognosis of at least one of kidney disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease, as described below.

[0040] The regenerative cell population is a cell population induced from monocytes derived from the biological sample. The regenerative cell population includes, for example, cells with regenerative function that can be induced from the monocytes. Examples of the cells with regenerative function include vascular endothelial progenitor cells and anti-inflammatory macrophages, and may also include Th2 cells, regulatory T cells (Tregs), and the like. The vascular endothelial progenitor cells and anti-inflammatory macrophages have, for example, angiogenesis-promoting activity and anti-inflammatory activity as their regenerative functions, and are therefore extremely useful in that they can not only promote angiogenesis but also suppress inflammation at inflamed sites such as ulcers. The regenerative cell population includes one or more types of cells with regenerative function.

[0041] The endothelial progenitor cells (EPCs) are cells capable of differentiating into vascular endothelial cells. The endothelial progenitor cells can be identified using cell surface markers such as CD34, CD133, KDR (kinase insert domain receptor, CD309), and CD105.

[0042] The endothelial progenitor cells may be further classified according to their degree of differentiation. In this case, the endothelial progenitor cells can be further classified into differentiated EPC colony cells and undifferentiated EPC colony cells. The differentiated EPC colony cells mainly contain cells with a diameter of 20 to 50 μm (CFU-large cell like EC, hereinafter also referred to as "large EPC colony"). The undifferentiated EPC colony cells mainly contain cells with a diameter of 20 μm or less (CFU-small cell like EC, hereinafter also referred to as "small EPC colony"). In the differentiation of the endothelial progenitor cells into vascular endothelial cells, small EPC colonies that appear at an early stage can be considered to be EPC colonies at an early differentiation stage with excellent proliferation potential, while large EPC colonies that appear at a later stage can be considered to be EPC colonies at a late differentiation stage with excellent vascular development (neogenesis) potential (see, for example, Masuda H. et al., Circulation Research, 109: 20-37 (2011)). The differentiated EPC colony cells are, for example, superior in regenerative functions such as angiogenesis or repair compared to the undifferentiated EPC colony cells, and therefore the vascular endothelial precursor cells are preferably differentiated EPC colony forming cells.

[0043] The anti-inflammatory macrophages are a type of macrophage that secrete anti-inflammatory cytokines (IL-10, IL-4) and the like, and are cells that contribute to angiogenesis or repair. The anti-inflammatory macrophages can be identified using cell surface markers such as CD206, CD163, and CD169. The anti-inflammatory macrophages are preferably M2 macrophages because they are excellent in regenerative functions such as angiogenesis or repair.

[0044] The proportion of cells with regenerative function among all cells in the regenerative cell population is not particularly limited, and may be, for example, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The proportion of cells can be calculated, for example, by measuring the proportion of cells positive for the cell surface marker of interest using the cell surface marker with regenerative function. The measurement can be performed, for example, using flow cytometry.

[0045] The proportion of cells with the regenerative function among all cells in the regenerative cell population is typically increased by two-fold, preferably four-fold or more, compared to the proportion of cells with the corresponding regenerative function in mononuclear cells derived from the biological sample.

[0046] When the cells with regenerative function are vascular endothelial progenitor cells and anti-inflammatory macrophages, the proportion of the cells with regenerative function among all cells in the regenerative cell population can be calculated, for example, as the sum of the proportion of CD34-positive cells and the proportion of CD206-positive cells.

[0047] In the regenerative cell population, the cells with regenerative function are derived from the monocytes. Therefore, the biological sample used to derive the regenerative cell population may be any biological sample containing the monocytes. The monocytes are abundant in blood and lymph. Therefore, the biological sample may be a tissue, organ, or tissue containing blood vessels and / or lymphatic vessels, or may be blood and / or lymph. Specific examples of the biological sample include bone marrow; blood, such as umbilical cord blood or peripheral blood; lymph; and the like.

[0048] The biological sample may be a biological sample collected from a subject to be administered, treated, or treated with the composition of the present disclosure (hereinafter collectively referred to as the "administration subject"), or may be a biological sample collected from a subject other than the administration subject. From the viewpoint of avoiding limitations during preparation, it is desirable to use a biological sample collected from a subject other than the administration subject. As shown in the Examples described below, the regenerative cell population may be cells derived from a biological sample collected from a subject other than the administration subject (allogeneic cells) or cells derived from a biological sample collected from the administration subject (autologous cells). In the present disclosure, the extracellular vesicles can be suitably prepared using a biological sample collected from a subject other than the administration subject because the graft rejection reaction is suppressed. Furthermore, it has been confirmed that the extracellular vesicles exert a therapeutic effect without being eliminated, for example, by immune rejection. Therefore, the extracellular vesicles can be prepared, for example, as an off-the-shelf formulation.

[0049] The method for preparing the biological sample is not particularly limited and can be appropriately determined depending on, for example, the type of biological sample. Specifically, when the biological sample is bone marrow, the biological sample can be obtained by, for example, performing bone marrow aspiration on the subject. When the biological sample is peripheral blood, the biological sample can be obtained by, for example, collecting venous blood. When the biological sample is umbilical cord blood, the biological sample can be obtained by, for example, isolating the umbilical cord and then collecting the blood from the umbilical cord.

[0050] The method for separating mononuclear cells from the biological sample can be carried out in accordance with a method commonly used in the art, for example, by using a cell suspension prepared from the biological sample and a mononuclear cell separation solution such as Ficoll or Percoll to separate the mononuclear cells from other components.

[0051] The method for inducing the cells with regenerative function from monocytes derived from the biological sample can be carried out by, for example, a known method depending on the type of the cells with regenerative function. Specifically, when inducing vascular endothelial progenitor cells from the monocytes, the induction method can be carried out by, for example, culturing the monocytes in the presence of SCF, IL-6, FLT3L, TPO, and / or VEGF. Furthermore, when inducing anti-inflammatory macrophages from the monocytes, the induction method can be carried out by, for example, culturing the monocytes in the presence of IL-10, IL-4, M-CSF, and / or G-CSF.

[0052] The induction method is preferably a method capable of inducing both the vascular endothelial progenitor cells and the anti-inflammatory macrophages from the monocytes. For example, the induction method described in WO 2014 / 051154 can be referred to.

[0053] Since the regenerative cell population can induce cells with the regenerative function, for example, containing extracellular vesicles having the pro-angiogenic activity and the anti-inflammatory activity, it is preferable to culture the mononuclear cells in the presence of factors such as stem cell factor (SCF), interleukin-6 (IL-6), FMS-like tyrosine kinase 3 ligand (Flt3L), thrombopoietin (TPO), and / or vascular endothelial growth factor (VEGF). The culture can be performed using a medium containing the factors.

[0054] The mononuclear cells to be cultured may be a desired cell population obtained by selecting a desired cell population from mononuclear cells derived from the biological sample, or the mononuclear cells derived from the biological sample themselves. It is preferable to use mononuclear cells obtained without selecting CD34- and / or CD133-positive cells (unsorted mononuclear cells) as the mononuclear cells to be cultured. As a result, the regenerative cell population obtained after the culture is a cell population in which the differentiated EPC colony-forming cells, the anti-inflammatory macrophages, and anti-inflammatory Th2 cells and regulatory T cells are increased (amplified), compared to when mononuclear cells selected as CD34-positive cells are used. Therefore, by using the unsorted mononuclear cells, for example, a cell population enriched in the proportion of cells having the regenerative function can be obtained as the regenerative cell population, and thereby extracellular vesicles can be obtained that have excellent physiological activity against at least one of kidney disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease, such as vascular endothelial cell proliferation-promoting activity, angiogenesis-promoting activity, fibrosis-suppressing activity, graft rejection-suppressing activity, and immunosuppressive activity.

[0055] The SCF is a glycoprotein consisting of 248 amino acids and having a molecular weight of approximately 30,000. The SCF is generated in soluble and membrane-bound forms by alternative splicing. The SCF used for the culture may be any type of SCF as long as it is useful for culturing regenerative cell populations such as EPCs. The soluble form is preferred. The origin of the SCF is not particularly limited, but recombinant SCF is preferred because of its expected stable supply, and human recombinant SCF is particularly preferred. Human recombinant SCF is commercially available. The concentration of SCF in the medium can be set depending on the type of SCF and is not particularly limited as long as it is useful for culturing regenerative cell populations such as EPCs. Specifically, when the SCF is human recombinant SCF, the concentration of SCF in the medium is, for example, 10 to 1000 ng / ml, preferably 50 to 500 ng / ml, and more preferably about 100 ng / ml.

[0056] IL-6 is a glycoprotein with a molecular weight of approximately 21,000 that was isolated as a factor that induces the terminal differentiation of B cells into antibody-producing cells. It is known to be involved in immune responses, proliferation and differentiation of hematopoietic and nervous system cells, acute phase reactions, and the like. The origin of IL-6 is not particularly limited; however, recombinant IL-6 is preferred, and human recombinant IL-6 is particularly preferred, as it is expected to be supplied stably. Human recombinant IL-6 is commercially available. The concentration of IL-6 in the medium varies depending on the type of IL-6 used, but is not particularly limited as long as it is useful for culturing regenerative cell populations such as EPCs. Specifically, when the IL-6 is human recombinant IL-6, the concentration of IL-6 in the medium is, for example, 1 to 500 ng / ml, preferably 5 to 100 ng / ml, and more preferably about 20 ng / ml.

[0057] Flt3L is known as a ligand for receptor tyrosine kinase, which plays an important role in regulating early hematopoiesis. Several alternative splicing products of Flt3L are known, and it has been reported that Flt3L stimulates the proliferation of hematopoietic stem cells. Any type of Flt3L may be used as long as it is useful for culturing regenerative cell populations such as EPCs. The origin of Flt3L is not particularly limited, but recombinant Flt3L is preferred because of its expected stable supply, and human recombinant Flt3L is particularly preferred. Human recombinant Flt3L is commercially available. The concentration of Flt3L in the medium varies depending on the type of Flt3L used, but is not particularly limited as long as it is useful for culturing regenerative cell populations such as EPCs. As a specific example, when the Flt3L is human recombinant Flt3L, the concentration of Flt3L in the medium is, for example, 10 to 1000 ng / ml, preferably 50 to 500 ng / ml, more preferably about 100 ng / ml.

[0058] TPO is a type of hematopoietic cytokine known to act specifically on the process of megakaryocyte production from hematopoietic stem cells and promote megakaryocyte production. The origin of the TPO is not particularly limited; however, recombinant TPO is preferred, and human recombinant TPO is particularly preferred, as it is expected to be supplied stably. Human recombinant TPO is commercially available. The concentration of TPO in the medium varies depending on the type of TPO used, but is not particularly limited as long as it is useful for culturing regenerative cell populations such as EPCs. Specifically, when the TPO is human recombinant TPO, the concentration of TPO in the medium is, for example, 1 to 500 ng / ml, preferably 5 to 100 ng / ml, and more preferably about 20 ng / ml.

[0059] VEGF is a growth factor that acts specifically on EPCs and is known to be produced mainly in perivascular cells. Several types of VEGF proteins with different sizes are produced by alternative splicing. The VEGF may be any type of VEGF as long as it is capable of colony formation by EPCs contained in the regenerative cell population, and is preferably VEGF165. The origin of the VEGF is not particularly limited, but recombinant VEGF is preferred because of its expected stable supply, and human recombinant VEGF is particularly preferred. Human recombinant VEGF is commercially available. The concentration of VEGF in the medium varies depending on the type of VEGF used and is not particularly limited as long as it is useful for culturing regenerative cell populations such as EPCs. When the VEGF is human recombinant VEGF165, the concentration of VEGF in the medium is, for example, about 5 to 500 ng / ml, preferably about 20 to 100 ng / ml, and more preferably about 50 ng / ml.

[0060] The various factors added to the medium are derived from animals of the same or different species as the animal from which the monocytes are derived, but it is preferable to use factors derived from the same species. By unifying the origin of the monocytes and various factors in this way, a regenerative cell population suitable for allogeneic transplants such as allogeneic transplants and extracellular vesicles derived therefrom can be obtained. Furthermore, by using monocytes derived from the recipient, it is also possible to obtain extracellular vesicles suitable for allogeneic transplants. In this way, by culturing a regenerative cell population containing EPCs or the like in an environment that does not contain any components derived from xenogeneic animals, the resulting regenerative cell population and extracellular vesicles derived therefrom can reduce the risk of infection and rejection upon administration, etc.

[0061] The medium used for the culture may be prepared by dissolving the various factors in a basal medium to a predetermined concentration, or by preparing a concentrated solution (stock solution) containing the various factors in advance and diluting it with the desired medium to a predetermined concentration. The medium used for the culture may be prepared by dissolving the various factors required in a commercially available basal medium to a predetermined concentration and then sterilizing it by filtration sterilization or the like, or by aseptically adding and diluting a stock solution sterilized by filtration sterilization or the like to a commercially available basal medium. The filtration sterilization can be performed according to methods commonly practiced in the art, for example, using a Millipore filter with a pore size of 0.22 μm or 0.45 μm.

[0062] The basal medium can be a basal medium commonly used in the field, for example, a basal medium known as a medium for growing hematopoietic stem cells. The basal medium may be a serum-containing medium or a serum-free medium, i.e., a serum-free medium. In culturing the regenerative cell population, using a serum-free medium as the medium and / or basal medium allows for efficient induction (expansion) of cells with regenerative function during the culture (induction) of the regenerative cell population from the mononuclear cells. For this reason, the medium is preferably a serum-free medium. Examples of the medium or basal medium include DMEM, MEM, and IMDM.

[0063] The medium contains at least one factor, preferably three or more factors, and more preferably all factors selected from the group consisting of SCF, IL-6, FLT3L, TPO, and VEGF. Therefore, the medium used for the culture contains, for example, any of the following: (1) SCF, (2) IL-6, (3) FLT3L, (4) TPO, or (5) VEGF; (6) a combination of SCF and IL-6, (7) a combination of SCF and FLT3L, (8) a combination of SCF and TPO, (9) a combination of SCF and VEGF, (10) a combination of IL-6 and FLT3L, (11) a combination of IL-6 and TPO, (12) a combination of IL-6 and VEGF, (13) a combination of FLT3L and TPO, (14) a combination of FLT3L and VEGF, or (15) a combination of TPO and VEGF; (16) a combination of SCF, IL-6, and FLT3L, (17) a combination of SCF, IL-6, and TPO, (18) a combination of SCF, FLT3L, and TPO, (19) a combination of SCF, FLT3L, and VEGF, (20) a combination of IL-6, FLT3L, and TPO, or (21) a combination of IL-6, FLT3L, and VEGF; (22) a combination of SCF, IL-6, FLT3L, and TPO, (23) a combination of SCF, IL-6, FLT3L, and VEGF, or (24) a combination of IL-6, FLT3L, TPO, and VEGF; (25) a combination of SCF, IL-6, FLT3L, TPO, and VEGF.

[0064] The medium more preferably contains SCF, IL-6, FLT3L, TPO, and VEGF, and even more preferably contains about 50 ng / mL VEGF, about 100 ng / mL SCF, about 20 ng / mL IL-6, 100 ng / mL FLT3L, and about 20 ng / mL TPO.

[0065] The mononuclear cells can be cultured using a medium containing the various factors by adding a cell suspension containing the mononuclear cells to the medium containing the various factors. The cell suspension may be a body fluid containing the mononuclear cells (e.g., bone marrow fluid, umbilical cord blood, peripheral blood, etc.). The conditions for culturing the mononuclear cells are not particularly limited, and the mononuclear cells can be cultured under conditions commonly used in the art. Specific examples of the culture conditions include, for example, 5% CO ２ The mononuclear cells are cultured in a humid atmosphere at 37°C for 7 days or more (for example, 10 days or more). The concentration (seeding density) of the mononuclear cells in the medium is not particularly limited as long as it allows the culture of a regenerative cell population such as EPCs. The concentration (seeding density) of the mononuclear cells in the medium is, for example, about 0.5 to 10 x 10 ５ cells / ml, more preferably about 1-5 x 10 ５ cells / ml, more preferably about 3-4 x 10 ５ cells / ml.

[0066] The regenerative cell population culture may be a culture obtained by inducing the regenerative cell population from the mononuclear cells, or may be a culture obtained by culturing the induced regenerative cell population.

[0067] The medium and culture conditions for the regenerative cell population are not particularly limited, and the explanation of the medium and culture conditions for the mononuclear cells can be used, for example. Specifically, the medium for the regenerative cell population can be, for example, a medium obtained by adding serum, preferably human serum, to the basal medium. The origin of the serum may be the same as or different from that of the regenerative cell population, but is preferably the same. The serum may contain extracellular vesicles. Therefore, when adding serum to the basal medium, it is preferable to use serum from which extracellular vesicles have been removed. Serum from which extracellular vesicles have been removed is commercially available. The concentration of the serum is, for example, 1 to 20 (v / v)%, preferably 1 to 10 (v / v)%.

[0068] The regenerative cell population can be cultured by adding a cell suspension containing the regenerative cell population to a medium for the regenerative cell population. The culture conditions for the regenerative cell population are not particularly limited, and the regenerative cell population can be cultured under conditions commonly used in the art. Specific examples of the culture conditions include, for example, 5% CO ２ The regenerative cell population is cultured at 37°C for 1 to 10 days (for example, 1 to 2 days) under a 50% CO2 atmosphere. The concentration (seeding density) of the regenerative cell population in the medium is not particularly limited as long as it allows the regenerative cell population to be cultured. The concentration (seeding density) of the regenerative cell population in the medium is, for example, about 1 x 10 ４ ~1 x 10 ７ cells / ml, more preferably about 1 x 10 ５ ~1 x 10 ６ cells / ml, more preferably about 5 x 10 ５ cells / ml.

[0069] As described above, the extracellular vesicles are secreted from the cells from which they are derived, and therefore, the extracellular vesicles may be isolated or extracted from the regenerative cell population or a culture thereof.

[0070] When extracted or isolated from the regenerative cell population, the extracellular vesicles can be isolated or extracted by, for example, disrupting the regenerative cell population to release the extracellular vesicles from the cells of the regenerative cell population, separating a soluble fraction from the resulting suspension, and then subjecting the soluble fraction to an extracellular vesicle isolation or extraction method. The extracellular vesicles can be isolated or extracted using methods commonly used in the art, such as centrifugation, affinity purification using membrane proteins derived from the regenerative cell population or membrane proteins present in the membranes of the extracellular vesicles, or filtration using porous membranes such as filters. Examples of such filters include commercially available membrane filters, such as a 0.22 μm membrane filter (manufactured by Millipore). The membrane proteins of the extracellular vesicles are described below. Commercially available kits can be used to isolate or extract the extracellular vesicles, such as the qEv isolation kit (manufactured by Izon Sciences).

[0071] When the extracellular vesicles are isolated or extracted by centrifugation, the centrifugation conditions may be any conditions that allow the extracellular vesicles to be precipitated, and specific examples include 10,000 to 300,000 x g or 90,000 to 180,000 x g for 20 to 60 minutes. The temperature during the centrifugation is, for example, 0 to 10°C, preferably 0 to 4°C.

[0072] The culture is composed of, for example, the regenerative cell population and its medium (conditioned medium or culture supernatant). Therefore, when isolating extracellular vesicles from the culture, the extracellular vesicles may be isolated or extracted from the regenerative cell population or the conditioned medium, or from both the regenerative cell population and the conditioned medium. Separating the regenerative cell population and the conditioned medium from the culture can be performed according to methods commonly used in the art, such as filtration through a filter or centrifugation. When the culture contains the regenerative cell population, the extracellular vesicles can be isolated or extracted in the same manner as in the extraction or isolation from the regenerative cell population described above. When the conditioned medium is used, the isolation or extraction of extracellular vesicles can be performed, for example, by centrifugation, affinity purification using membrane proteins derived from the regenerative cell population present in the membrane of the extracellular vesicles, or the membrane proteins, or filtration using a porous membrane such as a filter. The isolation or extraction of the extracellular vesicles may be carried out using a commercially available kit, and as a specific example, the qEv isolation kit (manufactured by Izon Sciences) can be used.

[0073] Because the extracellular vesicles are derived from the regenerative cell population, they contain components derived from the regenerative cell population. The extracellular vesicles have, for example, a lipid bilayer membrane and a lumen, and the lumen is surrounded by the lipid bilayer membrane. In the extracellular vesicles, the lipid bilayer membrane is derived from, for example, a membrane of the regenerative cell population, specifically, a membrane of a cell of the regenerative cell population. Examples of the membrane include a cell membrane; a membrane of an intracellular organelle such as a mitochondria, an endoplasmic reticulum, an endosome, a lysosome, or a Golgi apparatus; and the like. In the extracellular vesicles, the lumen contains, for example, cytoplasm derived from the regenerative cell population, specifically, cytoplasm derived from the cells of the regenerative cell population. Therefore, the extracellular vesicles contain, for example, miRNA, DNA, and / or proteins derived from the cytoplasm of the cells of the regenerative cell population.

[0074] The lipid bilayer membrane of the extracellular vesicles contains, for example, membrane components of the cells of the regenerative cell population. Therefore, the extracellular vesicles can be defined, for example, using membrane components present in the regenerative cell population. Examples of the membrane components include membrane proteins. Examples of the membrane proteins include extracellular vesicle markers such as CD9, CD63, Alix (ALG-2-interacting protein X), and Hsp-70 (Heat Shock Protein 70); cell surface markers of the cells; and the like. The cell surface marker can be appropriately determined depending on the type of cell. When the regenerative cell population includes EPCs, examples of the cell surface marker include CD34, CD133, KDR (Kinase insert domain receptor, CD309), and CD105. When the regenerative cell population includes anti-inflammatory macrophages, examples of the cell surface marker include CD206, CD163, and CD169. The lipid bilayer membrane of the extracellular vesicles contains, for example, one or more types of membrane components. The membrane components can be measured, for example, by flow cytometry using an antibody against a molecule of interest.

[0075] The lumen of the extracellular vesicles contains, for example, the cytoplasm of cells of the regenerative cell population. Therefore, the extracellular vesicles can be defined using, for example, cytoplasmic components present in the regenerative cell population. Examples of the cytoplasmic components include miRNAs. Examples of the miRNAs include miR-15b-5p, miR-29b-3p, miR-29c-3p, miR-92a-2-5p, miR-126-3p, miR-126-5p, miR-133a-3p, miR-133b, miR-146a-3p, and miR-146b-5p. , miR-150-5p, miR-181b-3p, miR-195-3p, miR-195-5p, miR-200b-5p, miR-3 02a-5p, miR-142-3p, miR-10a-3p, miR-17-3p, miR-20b-5p, miR-21-5p, miR -24-2-5p, miR-29a-5p, miR-30b-5p, miR-30c-5p, miR-30e-3p, miR-30e-5 p, miR-32-5p, miR-92a-3p, miR-103a-2-5p, miR-144-5p, miR-148a-3p, miR Examples of miRNAs present in extracellular vesicles of the regenerative cell population include miR-199b-5p, miR-200a-3p, miR-205-5p, miR-210-3p, miR-221-5p, miR-324-5p, miR-363-3p, miR-373-3p, miR-509-3p, miR-633, and let-7c-5p. Among the miRNAs, miR-92a-2-5p, miR-133a-3p, miR-133b, miR-146a-3p, miR-200b-5p, miR-302a-5p, and miR-142-3p are particularly presumed to be specific miRNAs present in extracellular vesicles of the regenerative cell population. For this reason, the lumen of the extracellular vesicles preferably contains miR-92a-2-5p, miR-133a-3p, miR-133b, miR-146a-3p, miR-200b-5p, miR-302a-5p, and / or miR-142-3p. The lumen of the extracellular vesicles contains, for example, one or more types of cytoplasmic components. The cytoplasmic components can be measured, for example, by flow cytometry using an antibody against a molecule of interest.Furthermore, when the cytoplasmic component is a nucleic acid molecule, the cytoplasmic component can be measured using, for example, a sequencer, etc. In the case of a peripheral blood-derived regenerative cell population, the miRNA preferably includes, for example, miR-15b-5p, miR-29b-3p, miR-92a-2-5p, miR-146a-3p, miR-146b-5p, miR-150-5p, miR-195-3p, miR-195-5p, miR-200b-5p, miR-302a-5p, and / or miR-142-3p. In addition, in the case of a regenerative cell population derived from umbilical cord blood, the miRNA may be, for example, miR-10a-3p, miR-17-3p, miR-20b-5p, miR-21-5p, miR-24-2-5p, miR-29a-5p, miR-30b-5p, miR-30c-5p, miR-30e-3p, miR-30e-5p, miR-32-5p, miR-92a-3p, miR- Preferably, the miR-103a-2-5p, miR-144-5p, miR-148a-3p, miR-199b-5p, miR-200a-3p, miR-205-5p, miR-210-3p, miR-221-5p, miR-324-5p, miR-363-3p, miR-373-3p, miR-509-3p, miR-633, and / or let-7c-5p.

[0076] Among the miRNAs, miR-20b-5p, miR-30b-5p, miR-30c-5p, miR-30e-3p, miR-30e-5p, miR-32-5p, miR-92a-2-5p, miR-92a-3p, miR-199b-5p, miR-200a-3p, miR-363-3p, and miR-509-3p are generally known to have anti-apoptotic activity. Among the miRNAs, miR-17-3p, miR-20b-5p, miR-103a-2-5p, miR-150-5p, miR-181b-3p, miR-200a-3p, miR-205-5p, miR-302a-5p, miR-324-5p, miR-363-3p, and miR-633 are generally known to have cell proliferation-promoting activity. Among the miRNAs, miR-150-5p is generally known to have cell migration-promoting activity. Among the miRNAs, miR-21-5p, miR-29a-5p, miR-29b-3p, miR-29c-3p, miR-133a-3p, miR-133b, miR-200a-3p, and miR-373-3p are generally known to have anti-fibrotic activity, particularly activity to suppress the expression of TGF-β. Among the miRNAs, miR-15b-5p, miR-29c-3p, miR-126-3p, miR-126-5p, miR-146a-3p, miR-146b-5p, miR-181b-3p, miR-195-3p, miR-195-5p, miR-200b-5p, miR-210-3p, miR-221-5p, miR-633, and let-7c-5p are generally known to have angiogenesis-promoting activity. Among the miRNAs, miR-10a-3p, miR-21-5p, miR-24-2-5p, miR-142-3p, miR-144-5p, and miR-148a-3p are generally known to have anti-inflammatory activity.

[0077] The components contained in the lumen of the extracellular vesicles may be replaced with components other than the cytoplasm of the regenerative cell population. In this case, the lumen is loaded with a desired cargo. Examples of the cargo include low-molecular-weight compounds, proteins or peptides such as antibodies, and nucleic acids.

[0078] The average diameter (weighted average) of the extracellular vesicles is, for example, 1 to 500 nm, preferably 10 to 250 nm, and more preferably 30 to 150 nm. The average diameter can be measured in accordance with Example 1 described below. The average diameter of the extracellular vesicles can be adjusted, for example, by filtering a liquid containing the extracellular vesicles using a filter having a desired pore size.

[0079] The composition of the present disclosure may further contain a pharmaceutically acceptable carrier, such as a suspending agent, solubilizing agent, stabilizer, isotonicity agent, preservative, anti-adsorption agent, surfactant, diluent, vehicle, pH adjuster, soothing agent, buffer, sulfur-containing reducing agent, antioxidant, etc., for administering the extracellular vesicles, and these can be appropriately added within a range that does not impair the effects of the present disclosure.

[0080] The compositions of the present disclosure may be used, for example, in vitro or in vivo, and may be used, for example, as research reagents or pharmaceuticals.

[0081] The subject to which the composition of the present disclosure is administered is not particularly limited. When the composition of the present disclosure is used in vivo, the subject to which the composition is administered can be, for example, a human or a non-human animal other than a human. Examples of the non-human animal include mammals such as mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, dolphins, and sea lions; birds; and fish. When the composition of the present disclosure is used in vitro, the subject to which the composition is administered can be, for example, a cell, tissue, organ, or organ. Examples of the cell include cells collected from a living body, cultured cells, and the like. Examples of the tissue, organ, or organ include tissue (biological tissue) or organ, and the like collected from a living body. Examples of the cell include muscle cells, iPS cells (induced pluripotent stem cells), stem cells, and the like.

[0082] When the composition of the present disclosure is used in vivo, the subject to which the composition is administered may be, for example, a subject diagnosed with or suspected of having a kidney disease, such as acute kidney injury, transitional chronic kidney disease, chronic kidney disease, or immunosuppression after kidney transplantation.

[0083] When the composition of the present disclosure is used in vivo, the subject to which it is administered may be, for example, a subject diagnosed with or suspected of having peripheral arterial disease.

[0084] When the composition of the present disclosure is used in vivo, the subject to which it is administered may be, for example, a subject diagnosed with or suspected of having cerebral infarction.

[0085] When the composition of the present disclosure is used in vivo, the subject to which it is administered may be, for example, a subject diagnosed with or suspected of having pulmonary hypertension.

[0086] When the composition of the present disclosure is used in vivo, the subject to which it is administered may be, for example, a subject diagnosed with or suspected of having spinal cord ischemia.

[0087] When the composition of the present disclosure is used in vivo, the subject to which it is administered may be, for example, a subject diagnosed with or suspected of having non-occlusive intestinal ischemia.

[0088] When the composition of the present disclosure is used in vivo, the subject to which it is administered may be, for example, a subject diagnosed with or suspected of having ocular ischemic syndrome.

[0089] When the composition of the present disclosure is used in vivo, the subject to which the composition is administered may be, for example, a subject diagnosed with or suspected of having a fibrotic disease. Examples of the fibrotic disease include pulmonary fibrosis, myocardial fibrosis, and myelofibrosis. Examples of the inflammatory disease include autoimmune diseases, inflammatory bowel diseases, and inflammatory myopathy.

[0090] When the composition of the present disclosure is used in vivo, the subject to which the composition is administered can be, for example, a subject diagnosed with or suspected of having an inflammatory disease. Examples of the inflammatory disease include autoimmune diseases, inflammatory bowel diseases, and inflammatory myopathies. Examples of the autoimmune disease include rheumatoid arthritis, systemic lupus erythematosus, polymyositis, Sjögren's syndrome, systemic sclerosis, glomerulonephritis, and vasculitis. Examples of the inflammatory bowel disease include ulcerative colitis and Crohn's disease. Examples of the inflammatory myopathies include dermatomyositis, polymyositis, necrotizing autoimmune myositis, and (idiopathic) inclusion body myositis.

[0091] The conditions for use (administration conditions) of the composition of the present disclosure are not particularly limited, and the administration form, administration time, dosage, etc. can be appropriately set depending on the type of subject to be administered, etc.

[0092] The composition of the present disclosure can be administered, for example, orally or parenterally, such as intravenously. However, intravenous administration is preferred because it can be administered safely and stably, regardless of the skill of the administerer.

[0093] The dosage of the composition of the present disclosure is an effective dose, specifically, an amount that, when administered to a subject, can provide a therapeutic effect (therapeutic effect) for at least one of renal disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease, compared to a non-administered subject. Specifically, the dosage can be appropriately determined, for example, depending on the age, weight, symptoms, etc. of the subject. Specifically, the dosage is, for example, 10 extracellular vesicles per administration. ４ ~10 ９ pieces / kg weight, 10 ４ ~10 ８ pieces / kg weight, 10 ４ ~10 ７ Preferably, the number of the cells per kg of body weight is 10 ４ ~10 ８ pieces / kg weight, 10 ４ ~10 ７ per kg of body weight.

[0094] The composition of the present disclosure may be administered once or multiple times. The multiple times may be, for example, two, three, four, five, or more times. The number of administrations may be appropriately determined while confirming the therapeutic effect on the subject. When administering multiple times, the administration interval may be appropriately determined while confirming the therapeutic effect on the subject, and may be, for example, once a day, once a week, once every two weeks, once a month, once every three months, or once every six months.

[0095] As described above, the composition of the present disclosure inhibits graft rejection and is derived from the regenerative cell population, and therefore has physiological activity, such as angiogenesis promoting activity, against at least one of renal disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease. Therefore, the composition of the present disclosure can be suitably used, for example, as described below, for treating or improving the prognosis of at least one of renal disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease.

[0096] Use of Extracellular Vesicles or Compositions In another aspect, the present disclosure provides a composition for use in treating at least one of renal disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease, or a method using the same. In this case, the present disclosure provides a composition for use in treating at least one of renal disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease, wherein the composition is the composition of the present disclosure. Also, the present disclosure provides a method for treating a patient with at least one of renal disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease, wherein the composition of the present disclosure is administered to the patient.

[0097] Examples of the diseases include kidney disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory diseases. Examples of the kidney disease include acute kidney injury, transitional chronic kidney disease, chronic kidney disease, and immunosuppression after kidney transplantation. Examples of the fibrosis include pulmonary fibrosis, myocardial fibrosis, and myelofibrosis. Examples of the inflammatory disease include autoimmune diseases, inflammatory bowel diseases, and inflammatory myopathy. Examples of the autoimmune disease include rheumatoid arthritis, systemic lupus erythematosus, polymyositis, Sjögren's syndrome, systemic sclerosis, glomerulonephritis, and vasculitis. Examples of the inflammatory bowel disease include ulcerative colitis and Crohn's disease. Examples of the inflammatory muscle diseases include dermatomyositis, polymyositis, necrotizing autoimmune myositis, and (idiopathic) inclusion body myositis.

[0098] In another aspect, the present disclosure provides a composition for use in promoting the proliferation of vascular endothelial cells or a method using the same. In this case, the present disclosure provides a composition for use in promoting the proliferation of vascular endothelial cells, wherein the composition is the composition of the present disclosure. The present disclosure also provides a method for promoting the proliferation of vascular endothelial cells, comprising the step of promoting the proliferation of the vascular endothelial cells by contacting the vascular endothelial cells with a composition, wherein the composition comprises the composition of the present disclosure.

[0099] In another aspect, the present disclosure provides a composition for use in inducing angiogenesis or a method using the same. In this case, the present disclosure provides a composition for use in inducing angiogenesis, wherein the composition is a composition of the present disclosure. Also, the present disclosure provides a method for inducing angiogenesis, wherein the composition of the present disclosure is used.

[0100] The method for inducing angiogenesis of the present disclosure involves, for example, administering the extracellular vesicles or compositions to a subject in whom angiogenesis is to be induced. Examples of subjects in whom angiogenesis is to be induced include patients with kidney disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), and the like. In this case, the induction of angiogenesis can also be referred to as the induction of angiogenesis in the patient's kidney, brain, lung, bone marrow, intestine, eye, ischemic muscle, or a site where ischemia-reperfusion injury has occurred (e.g., central or peripheral blood vessels).

[0101] In another aspect, the present disclosure provides a composition for use in inhibiting fibrosis or a method using the same. In this case, the present disclosure provides a composition for use in inhibiting fibrosis, wherein the composition is the composition of the present disclosure. Also, the present disclosure provides a method for inhibiting fibrosis, wherein the composition of the present disclosure is used.

[0102] In the method for inhibiting fibrosis of the present disclosure, for example, the extracellular vesicles or composition are administered to a subject in which fibrosis is to be inhibited. Examples of subjects in which fibrosis is to be inhibited include patients with kidney disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), and the like. In this case, the induction of angiogenesis can also be considered as the inhibition of fibrosis in the patient's kidney, brain, lung, bone marrow, intestine, eye, ischemic muscle, or site of ischemia-reperfusion injury (e.g., central or peripheral blood vessels).

[0103] <Use of extracellular vesicles or compositions> The present disclosure relates to a composition for use in treating at least one of kidney disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease. The present disclosure also relates to a composition for use in promoting the proliferation of vascular endothelial cells. The present disclosure also relates to a composition for use in promoting angiogenesis. The present disclosure also relates to a composition for use in inhibiting fibrosis.

[0104] The present disclosure will be described in detail below using examples, but the present disclosure is not limited to the embodiments described in the examples. Unless otherwise specified, commercially available reagents, kits, etc. were used according to their protocols.

[0105] [Example 1] It was confirmed that the composition of the present disclosure has vascular endothelial cell proliferation activity, reduces graft rejection, restores renal function, has anti-fibrosis activity, and has pro-angiogenic activity. Furthermore, the therapeutic effect of RAC-derived extracellular vesicles (RACev) on renal ischemia-reperfusion injury (IRI) was evaluated in comparison with a vehicle-administered group.

[0106] (1) Cell Culture Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy human volunteers after obtaining written informed consent in accordance with the ethical committee (CS-00201623). Peripheral blood (30 mL) was collected using a vacutainer (BD).

[0107] Peripheral blood mononuclear cells (PBMNCs) were isolated from peripheral blood by density gradient centrifugation using Histopaque-1077 (Sigma-Aldrich, #10771) according to the methods described in Asahara et al., Science, 275: 964-7 (1997) and Amankeldi et al., PLoSONE, (2019), 14(3):e0205477. Isolated PBMNCs were washed with phosphate buffered saline (PBS)-ethylenediaminetetraacetic acid (EDTA) and then suspended in serum-free medium (QQ medium) to prepare a cell suspension. The CD34 positivity rate of isolated PBMNCs was 0.23±0.03%, and the CD133 positivity rate was 0.20±0.07%. The number of CD34 positive cells per 100 ml of peripheral blood was (27.8±4.5) x 104, and the number of CD133 positive cells was (23.2±6.9) x 104.

[0108] The serum-free medium (hereinafter referred to as "QQ medium" or "QQcm") used for culturing (inducing) the regenerative cell population was prepared by aseptically adding the following factors to stemline™ II Hematopoietic Stem Cell Expansion Medium (Sigma-Aldrich, Cat No. S0192) to achieve the specified concentrations.

[0109] (Concentration of each factor in QQ medium) rhSCF (Peprotech, Inc., Cat. No. #300-07): 100 ng / ml rhFlt3L (Peprotech, Inc., Cat. No. #300-19): 100 ng / ml rhTPO (Peprotech, Inc., Cat. No. #300-18): 20 ng / ml rhVEGF (Peprotech, Inc., Cat. No. #100-02): 50 ng / ml rhIL-6 (Peprotech, Inc., Cat. No. #200-06): 20 ng / ml Penicillin / Streptomycin (Gibco), Cat. No. 15140122: 100 U / 100 μg / ml) r: recombinant h: human origin

[0110] The PBMNC was placed in a 10 cm dish (manufactured by Sumitomo Bakelite Co., Ltd.) at 10 × 10 ７ After seeding, cells were cultured at 10 ml per dish at 37°C and 5% CO ２ The cells were cultured for 7 days under conditions of 37°C and 5% CO. Thus, a regenerative cell population (RAC) was prepared. Unless otherwise specified below, the culture conditions were 37°C, 5% CO. ２ , was carried out under humid environmental conditions.

[0111] (2) Preparation of Extracellular Vesicles After the culture, the RAC was collected and centrifuged. The resulting pellet was suspended in an extracellular vesicle production medium to prepare a cell suspension. The composition of the extracellular vesicle production medium was X-vivo 15 medium (Lonza, Cat. No. BE02-054Q) supplemented with 5% (v / v) of exosome-removed fetal bovine serum. The resulting cell suspension was placed in a 10 cm dish at a concentration of 2.5 × 10 ５ cells / ml / dish (250k), or 5 x 10 ５ After seeding at a density of 500 kJ / ml per dish, the cells were cultured for 48 hours.

[0112] After the incubation, the culture supernatant (conditioned medium) was collected and centrifuged at 300 × g and 4°C for 10 minutes to remove cells. The resulting supernatant fraction was then centrifuged at 2,000 × g and 12°C for 20 minutes, and the supernatant fraction was collected to remove apoptotic bodies and microvesicles. The supernatant fraction was filtered through a 0.2 μm filter (Millipore Merck, Cat. No. SLGS033SS) to remove extracellular vesicles with a particle size greater than 200 nm. The resulting filtrate was then centrifuged at 174,000 × g and 4°C for 110 minutes to precipitate and recover extracellular vesicles (exosomes). The recovered precipitate was suspended in the medium or buffer solution (maximum 3 × 50 μl) used in the experiments described below. The number and size of EVs were then measured by nanoparticle tracking analysis using a NanoSight 500 (Malvern Panalytical) according to the manufacturer's manual.

[0113] For labeling of extracellular vesicles, CM-Dil dye (C7000, Thermo Fisher Scientific) was added to the filtrate at 2 μmol / L after filtration through a 0.2 μm filter. The resulting reaction solution was then centrifuged at 174,000 × g and 4°C for 110 minutes. The extracellular vesicles were then washed with PBS and centrifuged again under the same conditions. The same washing and centrifuging procedures were repeated. The recovered precipitate was then suspended in the medium or buffer solution (maximum 3 × 50 μl) used in the experiments described below.

[0114] Labeled extracellular vesicles and control solution (physiological saline) were injected into the tail vein using a 24G angiocatheter one day after the onset of R-IRI. Three hours after extracellular vesicle injection, the animals were euthanized, and organs such as the kidneys, spleen, lungs, and heart were removed for further extracellular vesicle tracking analysis. Extracellular vesicle trapping at the injury site was analyzed using an IVIS Lumina III (Perkin Elmer). The excitation wavelength filter for the DiI dye was set at 520 nm, and the emission wavelength filter was set at 570 nm.

[0115] (3) Animals Used in Experiments All animal experiments were conducted in accordance with AAALAC International standards at Shonan Health Innovation Park (approval number AU-00030960). Male Lewis rats (6-10 weeks old) weighing 150-250 g were purchased from Charles River Laboratories through Oriental Yeast Co., Ltd. Animals used in experiments were maintained under standard conditions (20±2°C, 50-60% relative humidity, 12-hour / 12-hour light / dark cycle). Animals used in experiments had free access to water and food.

[0116] (4) Characterization of Extracellular Vesicles. The extracellular vesicles prepared in the above section "(2) Preparation of Extracellular Vesicles" were analyzed for their mean diameter, production volume per cell, marker expression, and lipid membrane and lumen structures. Specifically, mean diameter, production volume per cell, and marker expression were measured by flow cytometry. A DxFlex Flow Cytometry (Beckman Coulter) flow cytometer equipped with three lasers (405 nm, 488 nm, and 638 nm) was used with the following configuration changes: the 405 / 10 VSSC filter was moved to the V450 channel in the WDM. The detector configuration in the software (CytExpert software) was updated and assigned to the VSSC channel in the WDM. Before each data acquisition, the flow path of the flow cytometer was washed with a cleaning solution (FlowClean) and then passed through water filtered through a 10 μm filter.

[0117] In addition, the flow cytometer (DxFlex FACS machine, Beckman Coulter) was calibrated using latex beads of different sizes (100 nm or 200 nm), and then extracellular vesicles were measured. Furthermore, the particle diameter of each extracellular vesicle was calculated by comparing the side-scattered light (SSC) intensity of the latex beads with that of each extracellular vesicle. The mean diameter (weighted mean) was then calculated based on the obtained particle diameter. Furthermore, the particle diameter of each extracellular vesicle was calculated in the same manner, except that a nanoparticle tracking analysis (NTA) device (ZetaView®, DKSH) was used instead of the flow cytometer, and the mean diameter (weighted mean) was calculated. It was confirmed that the mean diameter obtained by the flow cytometer and the mean diameter obtained by the NTA device were approximately the same values.

[0118] The expression of the markers was measured using the flow cytometer after staining the extracellular vesicles with an anti-CD9 antibody (PE-labeled, Biolegend, Cat. No. #312106) and an anti-CD63 antibody (PE-labeled, Biolegend, Cat. No. #353004). Controls were performed in the same manner, except that control antibodies were used instead of the various antibodies.

[0119] The yield per cell was 2.5 x 10 based on the count and measured sample volume in the flow cytometer. ５ The amount of extracellular vesicles produced per cell (number of extracellular vesicles) was calculated.

[0120] The structures of the lipid membrane and lumen were observed using a scanning electron microscope (JEM-1400, manufactured by JEOL Ltd.).

[0121] A reference example was carried out in the same manner, except that human mesenchymal stem cells (MSCs, manufactured by Lonza, Cat. No. BE02-054Q) were used instead of the RAC. The results are shown in Figures 1 to 4. Statistical analysis in the graphs was performed using the Mann-Whitney test.

[0122] (5) Proliferation Promotion of Vascular Endothelial Cells The proliferation promotion activity of RACev on vascular endothelial cells was examined using human fetal vascular endothelial cells (HUVECs, Lonza, Cat. No. C2517A, Batch #18TL061650). Specifically, HUVECs were collected after 3-4 passages, and then labeled by adding Cytopainter dye (Abcam, Cat. No. #ab176735) and incubating at 37°C for 10-30 minutes. After labeling, the cells were centrifuged and washed twice. The resulting HUVEC cells were plated in a 3.5 cm dish at a concentration of 5 x 10 ４The HUVEC cells were seeded at 2 ml / dish. EGM-2 (Lonza) was used as the medium for passage of the HUVEC cells, and EBM-2 (Lonza) was used for measuring the proliferation activity of the HUVEC cells. Next, 250 kJ or 500 kJ of extracellular vesicles isolated from RAC or MSC (RACev or MSCev) were added to each dish. The cells were then cultured for 2 days under the above culture conditions. After the culture, each dish was observed using a phase-contrast microscope (6-well dish, Falcon). The number of cells per field of view (HPF) was counted in the obtained phase-contrast images, and the percentage of the area occupied by HUVEC cells relative to the total area was calculated as the growth area.

[0123] After the culture, HUVEC cells were collected from the dishes and their cell division was measured using a flow cytometer (FACS Verse, BD Biosciences) with the label as an indicator. A control was performed in the same manner, except that RACev and MSCev were not added.

[0124] (6) Library Preparation, Sequencing, and Bioinformatics Analysis. Furthermore, miRNA analysis was performed on the RACev and MSCev cultures added during HUVEC cell culture. Specifically, miRNA was purified from the RACev and MSCev cultures using miRNA purification kits (miRNeasy Mini Kit and RNeasy MinElute Cleanup Kit, QIAGEN). A sequencing library was prepared using the resulting miRNA and a library construction kit (QIAseq™). The quality of the library was inspected using the Agilent Bioanalyze and a DNA chip (High Sensitivity DNA chip, Agilent Technologies). The library was sequenced using a next-generation sequencer (NextSeq 500, Illumina) to generate 76-bp single-end reads. The expression levels of each miRNA (miR-181b-3p, miR-150-5p, miR-302a-5p, miR-92a-2-5p) were calculated from the obtained sequence data. The relative expression levels were calculated based on the expression levels of each miRNA in HUVEC cells without extracellular vesicles.

[0125] The control was performed in the same manner except that extracellular vesicles were not added. Statistical analysis in the graphs was performed using two-way ANOVA followed by Tukey's multiple comparison test.

[0126] (7) Induction of Renal Ischemia-Reperfusion Injury Rats were anesthetized with 3-4% sevoflurane (Maruishi Pharmaceutical). Through a midline abdominal incision, the two renal pedicles were clamped together and then clamped for 45 minutes using a microaneurysm clamp (FST). During the ischemic period, the rats were placed on a 37°C heating pad to maintain body temperature. After clamp removal, the renal blood flow was checked for recovery for 1 minute. Once the kidneys had returned to their original color, the abdomen was closed. Sham-operated rats underwent the same surgical procedure, except that the microaneurysm clamp was not applied.

[0127] (8) Confirmation of graft rejection One day after the onset of renal ischemia-reperfusion injury (K-IRI), RACev (1 × 10 ６ In one group, extracellular vesicles derived from regeneration-related cells were transplanted, and in the control group, PBS was transplanted. The transplantation was performed via the tail vein as described above. After each experiment, the animals were euthanized by overdosing with 5% sevoflurane. After confirming euthanasia, vital organs such as the kidneys, lungs, spleen, and liver were removed.

[0128] Specifically, 500 kJ of extracellular vesicles isolated from RACs or MSCs (RACev or MSCev) were diluted with PBS, and the resulting dilutions were administered via the tail vein at predetermined times (30 minutes, 1 day, and 2 days) after the induction of ischemia-reperfusion injury. Body weights were measured initially before the induction of ischemia-reperfusion injury (baseline) and then weekly for up to 4 weeks after the induction of injury. Kidneys were collected and weighed 4 days and 4 weeks after the induction. Peripheral blood and kidneys were collected 4 weeks after the induction. The percentages of CD3+ cells, CD4+ cells, CD8+ cells, regulatory T cells, and CD11b / c+ cells in the peripheral blood were measured using a flow cytometer. Controls were administered in the same manner, except that RACev and MSCev were not administered. The relative expression levels of each miRNA (miR-142-3p) were calculated for the administered RACev and MSCev. Statistical analysis in the graphs was performed using Dunn's multiple comparison test.

[0129] (9) Immunohistochemical Analysis. Systemic perfusion was performed with heparinized PBS (1000 U / 500 mL). Kidneys were fixed overnight in 4% paraformaldehyde (Cat. #163-20145, Fujifilm), embedded in paraffin, and cut into 2.5-3 mm thick sections. The sections were stained with Periodic Acid-Schiff (PAS) and Masson's Trichrome to determine the extent of fibrosis. To analyze microvascular density, sections were incubated with anti-CD31 (1:100) antibody and DAB. All histological photographs were taken at 200x magnification using an all-in-one fluorescence microscope, BZ-X800 (Keyence). Histological data were evaluated using a Keyence Analyzer (Keyence) with three to five high-power fields per section.

[0130] The original FASTQ files generated by NextSeq were uploaded to the Qiagen GeneGlobe Data Analysis Center (https: / / geneglobe.qiagen.com) and aligned to miRBase v21 (http: / / www.mirbase.org). All reads assigned to a specific miR were counted, associated UMIs were tabulated, and unique molecules were counted. The miR UMI count matrix was subjected to downstream analysis using StrandNGS 3.4 software (Agilent Technologies). UMI counts were quantified using the trimmed mean M-value (TMM) method. To determine target genes of differentially expressed miRs, information from PITA miRBase version 18, microRNA.org miRBase version 18, and TargetScan version 6.0 was integrated.

[0131] Next, gene ontology (GO) analysis and pathway statistical analysis were performed to determine the biological functions of the target genes. Pathway statistical analysis was performed using the PathVisio tool on the pathway collection in the Wiki Pathways database to determine pathways taking into account the number of genes included in the pathway and the number of target genes.

[0132] Kidney tissues were collected 4–6 days after the onset of R-IRI, and total RNA was isolated using a nucleic acid extraction system according to the manufacturer's instructions. RNA integrity was confirmed using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

[0133] (10) Statistical Analysis. All values ​​are expressed as mean ± SEM. The Mann-Holney U test was used for two and three nonparametric groups, followed by Dunn's multiple comparison test. One-way analysis of variance was applied to three parametric groups, followed by Dunn's multiple comparison test or Tukey's multiple comparison test. For multiple comparisons between groups at different time points, two-way analysis of variance was applied, followed by Tukey's post-hoc test. Differentially expressed miRs were determined using an absolute threshold of fold change ≥ 2. All statistical analyses were performed using GraphPad Prism 9.1 (GraphPad Prism Software). Statistical significance was set at P < 0.05.

[0134] [Results] (1) Characterization of Extracellular Vesicles Figure 1 is a graph showing the particle size, production volume per cell, and average diameter of extracellular vesicles. In Figure 1, A shows the particle size results, B shows the production volume results, and C shows the average diameter results. In Figure 1A, the horizontal axis shows the particle size (logarithm), and the vertical axis shows the relative number. In Figure 1B, the horizontal axis shows the type of sample, and the vertical axis shows the number of extracellular vesicles produced. In Figure 1C, the horizontal axis shows the type of sample, and the vertical axis shows the average diameter. As shown in Figures 1A and 1C, RAC-derived extracellular vesicles (RACev) were slightly larger than MSC-derived extracellular vesicles (MSCev), with an average diameter of 145 nm for RACev and 130 nm for MSCev. Furthermore, as shown in Figure 1B, RAC produced significantly more extracellular vesicles per cell than MSCs.

[0135] Figure 2 shows marker expression profiles, electron microscope images showing the lipid bilayer structure of extracellular vesicles, and graphs showing the amount of extracellular vesicles secreted and the amount of extracellular vesicle protein. Figure 2A shows the results of the EV biomarker expression profile of anti-CD9 and anti-CD63 in RACev. Figure 2B shows a transmission electron microscope photograph showing the lipid bilayer structure of extracellular vesicles. Figure 2C shows the results of quantification of the amount of extracellular vesicles secreted per million RACs. Figure 2D shows the number of all proteins in RACev derived from one million RACs. In Figure 2A, the horizontal axis shows the expression level of anti-CD63- or anti-CD9-positive RACev by flow cytometry, and the vertical axis shows the size of the IgG control and stained RACev. In Figure 2C, the horizontal axis shows the type of sample, and the vertical axis shows the amount of extracellular vesicles secreted. In Figure 2D, the horizontal axis indicates the type of sample, and the vertical axis indicates the number of all proteins in RACev derived from RACs. As shown in Figure 2A, the presence of extracellular vesicle-specific markers (CD9, CD63) was confirmed. Furthermore, as shown in Figure 2B, a lipid bilayer structure was confirmed. Furthermore, as shown in Figures 2C and 2D, the average number of extracellular vesicles (RACev) obtained from 1 million RACs and the number of all proteins in RACev were 2.89 x 10, respectively. ９ ±4.7 × 10 ８and 128±21.5. The size of RACev was 154.5±6.7 nm.

[0136] Next, Figure 3 shows histograms showing marker expression. Figure 3A shows the results for RACev, and Figure 3B shows the results for MSCev. In Figures 3A and 3B, the horizontal axis represents SSC intensity or the fluorescence intensity of each marker, and the vertical axis represents counts. As shown in the left panel of Figure 3A, it was found that the particle size of extracellular vesicles can be measured using a flow cytometer by using latex beads with known particle sizes. Furthermore, as shown in the center and right panels of Figure 3A, expression of CD63 and CD9 was confirmed in RACev. Furthermore, as shown in Figures 3A and 3B, RACev had higher CD63 expression levels than MSCev.

[0137] These results indicate that RACev is an extracellular vesicle because it matches MISEV2018 (Thery C et.al., 2018, J.Extracell Vesicles 7:1535750).

[0138] (2) Proliferation-Promoting Activity of Vascular Endothelial Cells Figure 4 is a graph showing the proliferation of HUVEC cells. In Figure 4, the horizontal axis indicates the type of sample, and the vertical axis indicates the number of cells per HPF. As shown in Figure 4, RACev and MSCev promoted the proliferation of vascular endothelial cells compared to the control. Also, as shown in Figure 4, RACev significantly promoted the proliferation of vascular endothelial cells compared to MSCev (MSCev-250K vs. RACev-250K: P > 0.001, MSCev-500K vs. RACev-500K: P > 0.0001).

[0139] Next, Figure 5 shows photographs and graphs illustrating the growth area of ​​HUVEC cells. Figure 5A shows a phase-contrast image, and Figure 5B shows a graph of the growth area. In Figure 5B, the horizontal axis indicates the type of sample, and the vertical axis indicates the growth area. As shown in Figures 5A and 5B, RACev and MSCev showed an increased growth area of ​​vascular endothelial cells compared to the control. Also, as shown in Figures 5A and 5B, RACev showed a significantly increased growth area of ​​vascular endothelial cells compared to MSCev (MSCev-500K vs. RACev-500K: P >0.0007). Furthermore, although not shown, we observed HUVEC cell proliferation over time using labeled RACev, and observed that when HUVEC cells took up RACev, they progressed to S phase. Furthermore, we confirmed that the proliferation-promoting activity of RACev on vascular endothelial cells was maintained up to the eighth generation, i.e., for eight cell divisions.

[0140] Next, Figure 6 is a graph showing the expression levels of miRNAs. In Figure 6, the horizontal axis indicates the type of miRNA, and the vertical axis indicates the relative expression level. As shown in Figure 6, RACev had significantly higher expression levels of anti-apoptotic miRNAs (miR-92a-2-5p), mitogenic miRNAs (miR-181b-3p, miR-150-5p, miR-302a-5p), and miRNAs promoting cell migration (miR-150-5p) compared to MSCev. These results demonstrate that RACev promotes vascular endothelial cell proliferation by delivering anti-apoptotic miRNAs and mitogenic miRNAs.

[0141] (3) Suppression of Graft Rejection Figure 7 is a graph showing changes in body weight and organ weight (kidney weight) of subject animals after 4 weeks. Figure 7A is a graph showing changes in body weight of subject animals after 4 weeks, and Figure 7B is a graph comparing kidney weights of subject animals after 4 weeks. In Figure 7A, the horizontal axis represents time, and the vertical axis represents body weight (g). In Figure 7B, the horizontal axis represents sample type, and the vertical axis represents kidney weight (μg). Figure 7A shows that the weight gain after 4 weeks was significantly greater in the RACev-treated group than in the control group. Figure 7B shows that kidney weight after 4 weeks was increased in the control group and the RACev-treated group compared to the sham group, and was approximately equivalent. As shown in Figures 7A and 7B, no graft rejection was observed following RACev transplantation. Statistical significance was determined by two-way analysis of variance with Tukey's multiple comparison test for body weight and one-way analysis of variance with Tukey's multiple comparison test for kidney weight. Results are shown as mean ± SEM.

[0142] Next, Figure 8 is a graph showing the proportions of CD3-positive cells, CD4-positive cells, CD8-positive cells, regulatory T cells, and CD11b / c-positive cells in peripheral blood. In Figure 8, the horizontal axis indicates the type of sample, and the vertical axis indicates the proportion of each cell. In Figure 9, the horizontal axis indicates the type of sample, and the vertical axis indicates the relative expression level.

[0143] As shown in Figure 8, there was no difference between RACev, MSCev and the control in both peripheral blood and kidney, and no graft rejection was observed.

[0144] Furthermore, as shown in Figure 9, RACev had significantly higher expression levels of miRNA (miR-142-3p) with immunosuppressive (anti-inflammatory) activity compared to MSCev. miR-142-3p is known to be contained in exosomes released by regulatory T cells (Tregs). These results suggest that RACev suppresses graft rejection by delivering miRNA with immunosuppressive activity.

[0145] As shown in Figures 7 to 9, no graft rejection was observed, demonstrating that the composition of the present disclosure has an immunosuppressive effect. Furthermore, the characteristics of immune cells in peripheral blood and kidney were clarified.

[0146] (4) Improvement of Renal Function by RACev Transplantation Figure 10 is a graph showing serum creatinine (Cr) values ​​and serum BUN values. Figure 10A is a graph comparing changes in serum creatinine (Cr) values ​​over time, and Figure 10B is a graph comparing changes in serum BUN values ​​over time. In Figure 10A, the horizontal axis represents elapsed time, and the vertical axis represents serum creatinine (Cr) values ​​(mg / dL). In Figure 10B, the horizontal axis represents elapsed time, and the vertical axis represents serum BUN values ​​(mg / dL).

[0147] On the first day after the onset of acute liver dysfunction (R-AKI), 1 × 10 ６ Cell-derived EVs were injected via the tail vein, and the treatment time was adjusted based on clinical treatment guidelines. (Figure 10A) Serum creatinine (Cr) levels on day 3 after the onset of K-IRI were significantly reduced in the EV-treated group compared to the control group (P<0.007, treatment group vs. control group). (Figure 10B) Serum BUN levels were also significantly reduced in the RACev group compared to the control group (P<0.007, treatment group vs. control group). As shown in Figures 10A and 10B, RACev transplantation significantly improved renal function. Statistical significance was determined by two-way analysis of variance with Tukey's multiple comparison test. Results are shown as mean ± SEM.

[0148] Body weight (BW) of rats was measured before and every week after the onset of symptoms. Four weeks after the onset of symptoms, BW in the RACev-implanted group was increased compared with the control group (RACev: 333±6 g, control: 315±5.5 g, P<0.0053).

[0149] (5) Improvement of fibrosis Figure 11 shows photographs and graphs showing the degree of fibrosis. Figure 11A shows fluorescent micrographs showing the degree of fibrosis in each area, Figure 11B shows a graph comparing the fibrosis area in the cortex, Figure 11C shows a graph comparing the fibrosis area in the medulla, Figure 11D shows the expression level of miRNA with anti-fibrotic activity, Figure 11E shows the expression level of miRNA with anti-inflammatory activity, and Figure 11F shows the expression level of pro-fibrotic genes. In Figure 11B, the horizontal axis indicates the type of sample, and the vertical axis indicates the fibrosis area (%). In Figures 11C and 11D, the horizontal axis indicates the type of RNA, and the vertical axis indicates the relative expression level. In Figure 11F, the horizontal axis indicates the type of gene, and the vertical axis indicates the Log ratio (RACev vs. Control). As shown in Figures 11A-11C, Masson's trichrome staining demonstrated that renal fibrosis levels were preserved in the treatment group compared with the control group in both the cortex and medulla (P<0.04 for the cortex, treated group vs. control; P<0.01 for the medulla, treated group vs. control). Furthermore, as shown in Figures 11D and 11E, this anti-fibrotic effect of RACev, coupled with the delivery of miRNAs with anti-fibrotic and anti-inflammatory activities to ischemic tissue, provided beneficial protection from excessive inflammation and scar formation. Furthermore, as shown in Figure 11F, gene expression analysis revealed that the expression of most pro-fibrotic genes (e.g., NGFR, FGF2, CSFLR, COL4A1, COL8A1, and TGFb1) was significantly reduced in the RACev-treated group compared with the control group at day 6 after R-IRI. As described above, RACev transplantation increased renal function parameters by delivering miRNAs with anti-fibrotic and anti-inflammatory activities. Statistical significance was determined by one-way analysis of variance with Dunn's multiple comparison test. Results are presented as mean ± SEM (n = 8-10 per group).

[0150] (6) Angiogenesis-promoting activity of extracellular vesicles Figure 12 shows photographs showing microvessel density in ischemic-damaged kidney tissue and a graph showing the number of CD31-positive microvessels. Figure 12A shows blood vessels in ischemic-damaged kidney tissue, Figure 12B is a graph comparing blood vessel density in the cortex, Figure 12C is a graph comparing blood vessel density in the medulla, and Figure 12D is a graph showing the expression level of miRNA. In Figures 12B and 12C, the horizontal axis indicates the type of sample, and the vertical axis indicates microvessel density ( / mm ２ ) are shown. In Figure 12D, the horizontal axis indicates the type of miRNA, and the vertical axis indicates the relative expression level. Figures 12A to 12C show that the RACev-treated group enhanced microvascular density in ischemic-injured kidney tissue (P<0.003, treated group vs. control group), demonstrating the strong angiogenic properties of RACev. In Figure 12D, transcriptome analysis revealed that key angiogenic miRNAs involved in angiogenesis, so-called angiomiRs, were more highly upregulated in RACev. Statistical significance was determined by one-way analysis of variance with Dunn's multiple comparison test. Results are shown as mean ± SEM (n=8-10 per group).

[0151] Figure 13 shows photographs showing the microvessel density in ischemic-damaged renal tissue on days 6 and 28 after the onset of R-IRI, and a graph showing the number of CD31-positive microvessels. Figure 13A shows blood vessels in ischemic-damaged renal tissue, Figure 13B is a graph comparing blood vessel density in the cortex, Figure 13C is a graph comparing blood vessel density in the medulla, and Figure 13D is a graph showing genes related to angiogenesis. In Figures 13B and 13C, the horizontal axis indicates the type of sample, and the vertical axis indicates microvessel density ( / mm ２) are shown. In Figure 13D, the horizontal axis indicates gene type, and the vertical axis indicates the Log ratio (RACev vs. control). Capillary density was assessed at the acute phase on days 6 and 28 after R-IRI onset to verify its protective and angiogenic effects. Figures 13A and 13B show that microvessel density in ischemic kidney tissue was beneficially enhanced in the RACev-transplanted group compared with the control group (P < 0.005 at day 6, P < 0.003 at day 28 for both the RACev and control groups), demonstrating the potent vasculoprotective and angiogenic properties of RACev. Furthermore, transcriptome analysis revealed that RACev therapy significantly upregulated angiogenesis-related genes in ischemic kidney tissue compared with the control group (Figure 13D). Taken together, these findings demonstrate that RACev delivers angiogenic enzymes and vasoprotective miRs to promote angiogenesis and protect against hypoxia-induced renal vascular network damage at the site of ischemic injury.

[0152] (7) Selective accumulation of RACev in damaged kidneys Figure 14 is a photograph showing the results of IVIS measurement. IVIS measurement results confirmed that labeled RACev accumulated in the damaged kidneys of the transplant group. The above EV tracking analysis demonstrated that RACev selectively accumulates in the liver.

[0153] Furthermore, cell type annotation analysis was performed using the 500 most highly expressed genes to identify which parts / cells of the nephron were highly expressed. The top 10 nephron regions with the highest expression levels were proximal tubule epithelial cell segments 1 and 2, distal tubule cell types 1 and 2, and renal tubule epithelial cells. This result confirms the IVIS results, which showed that most of the labeled RACev accumulated between the cortex and medulla. Meanwhile, some of the transplanted RACev was also captured in the kidney. These EV tracking analyses demonstrated that RACev accumulated in ischemia-induced renal tissue.

[0154] Under angiogenic conditions, inflammatory cell subsets in peripheral blood mononuclear cells (PBMCs) undergo phenotypic transformation into pro-regenerative cells, including angiogenic endothelial progenitor cells, M2 macrophages, and regulatory T cells. Notably, systemic injection of RACev significantly reduced serum creatinine and blood urinary nitrogen levels on day 3 compared with the control group. Histologically, the treatment group showed less fibrosis in the cortical and medullary regions than the control group (P<0.04 and P<0.01). CD31 staining confirmed increased capillary density in the treatment group compared with the control group (P<0.003). These beneficial effects were associated with significant expression of angiogenic, anti-fibrotic, anti-inflammatory, and anti-apoptotic miRs. In vivo bioluminescence analysis demonstrated preferential accumulation of RACev in the IR-injured kidney. In conclusion, systemic transplantation of RACev improves renal function through protection from tissue fibrosis, anti-inflammation, angiogenesis, and delivery of anti-apoptotic miRs to ischemic tissues.

[0155] (8) Regulatory Role of RACev on Epithelial-Mesenchymal Transition, Inflammation, and Hypoxia After Ischemic Injury. Figure 15 shows graphs and diagrams illustrating the results of various analytical methods used to elucidate the mechanism of fibrosis reduction, in which administration of RACev significantly reduced renal fibrosis 4 weeks after the onset of K-IRI. Figures 15A and 15B show the expression levels of miRNAs, Figure 15C shows the results of cluster analysis of the most highly correlated pathway regulation in the control group, Figure 15D shows the results of network analysis of the relationships between genes in downstream mechanisms in the control group, and Figure 15E shows the results of network analysis of the relationships between genes in downstream mechanisms in the treatment group. In Figures 15A and 15B, the horizontal axis indicates the type of RNA, and the vertical axis indicates relative expression levels. First, small non-codding RNA sequencing and data analysis revealed that RACev contains several important anti-inflammatory miRs, such as miR-10a-3p, miR-21-5p, miR-24-2-5p, and miR24-3p, which regulate excessive inflammation and initiate regeneration (Fig. 15A). Furthermore, RACev contains anti-apoptotic and cell proliferation miRs (Fig. 15B). In contrast, pathway analysis in the control group revealed that renal tissue fibrosis was initiated by the hypoxia pathway (P<2.19e-02) (Fig. 15C, D). Upregulation of redundant inflammatory pathways, such as IL-6-JAK2-STAT3 (P<1.43e-03), NF-kB-mediated TNFα signaling (P<1.51e-03), and INF-γ response (P<2.19e-02), initiated the early epithelial-mesenchymal transition pathway (P<3.75e-11) (Fig. 15C, D). In the treated group, pathway analysis indicated that RACev enhanced regeneration-related pathways (Fig. 15E).

[0156] Although the present disclosure has been described above with reference to the embodiments and examples, the present disclosure is not limited to the above-described embodiments and examples. Various modifications that can be understood by a person skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0157] This application claims priority based on Japanese Patent Application No. 2021-181301, filed on November 11, 2022, the disclosure of which is incorporated herein in its entirety.

[0158] <Supplementary Notes> Some or all of the above embodiments and examples can be described as, but are not limited to, the following supplementary notes: <Composition> (Supplementary Note 1) A composition for use in treating at least one of renal disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease, the composition comprising extracellular vesicles of a regenerative cell population induced from mononuclear cells derived from a biological sample or a culture thereof. (Appendix 2) The composition of Appendix 1, wherein the extracellular vesicles comprise miR-15b-5p, miR-29b-3p, miR-29c-3p, miR-92a-2-5p, miR-126-3p, miR-126-5p, miR-133a-3p, miR-133b, miR-146a-3p, miR-146b-5p, miR-150-5p, miR-181b-3p, miR-195-3p, miR-195-5p, miR-200b-5p, miR-302a-5p, and / or miR-142-3p. (Appendix 3) Furthermore, the extracellular vesicles may contain miR-10a-3p, miR-17-3p, miR-20b-5p, miR-21-5p, miR-24-2-5p, miR-29a-5p, miR-30b-5p, miR-30c-5p, miR-30e-3p, miR-30e-5p, miR-32-5p, miR-92a-3p, miR-103a-2-5p, miR- The composition according to any one of Appendixes 1 to 2, comprising miR-144-5p, miR-148a-3p, miR-199b-5p, miR-200a-3p, miR-205-5p, miR-210-3p, miR-221-5p, miR-324-5p, miR-363-3p, miR-373-3p, miR-509-3p, miR-633, and / or let-7c-5p. (Appendix 4) The composition according to any one of Appendixes 1 to 3, wherein the extracellular vesicles are CD9 and / or CD63 positive. (Appendix 5) The composition according to any one of Appendixes 1 to 4, wherein the average diameter of the extracellular vesicles is 10 to 500 nm. (Appendix 6) The composition according to any one of Appendixes 1 to 5, wherein the composition has the activity of promoting the proliferation of vascular endothelial cells. (Appendix 7) The composition according to any one of Appendices 1 to 6, which has angiogenesis promoting activity.(Appendix 8) The composition according to any one of Appendices 1 to 7, which has anti-fibrosis activity. (Appendix 9) The composition according to any one of Appendices 1 to 8, which suppresses graft rejection. (Appendix 10) The composition according to any one of Appendices 1 to 9, wherein the extracellular vesicles are extracellular vesicles extracted or isolated from the regenerative cell population or a culture thereof. (Appendix 11) The composition according to any one of Appendices 1 to 10, wherein the regenerative cell population is induced by culturing the mononuclear cells in the presence of stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and / or vascular endothelial growth factor. (Appendix 12) The composition according to any one of Appendices 1 to 11, wherein the regenerative cell population is induced without selection of CD34 and / or CD133 positive cells. (Appendix 13) The composition according to any one of Appendices 1 to 12, wherein the regenerative cell population comprises endothelial progenitor cells, anti-inflammatory macrophages, and regulatory T cells. (Appendix 14) The composition according to Appendices 13, wherein the endothelial progenitor cells are differentiated EPC colony-forming cells. (Appendix 15) The composition according to Appendices 13 or 14, wherein the anti-inflammatory macrophages are M2 macrophages. (Appendix 16) The composition according to any one of Appendices 1 to 15, wherein the biological sample is at least one of blood and bone marrow. (Appendix 17) The composition according to any one of Appendices 1 to 16, wherein the biological sample is at least one of peripheral blood and umbilical cord blood. <Composition for treating at least one of renal disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease> (Appendix 18) A composition for use in the therapeutic or prophylactic treatment of at least one of renal disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease, the composition being a composition described in any of Appendices 1 to 17. (Appendix 19) The composition described in Appendices 18, wherein the renal disease is acute kidney injury, transitional chronic kidney disease, chronic kidney disease, or immunosuppression after kidney transplantation.(Appendix 20) A pharmaceutical product for renal disease used for therapeutic or preventive treatment of at least one of renal disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease, comprising the composition described in any one of Appendices 1 to 17. <Composition for promoting the proliferation of vascular endothelial cells> (Appendix 21) A composition for use in promoting the proliferation of vascular endothelial cells, wherein the composition is a composition described in any one of Appendices 1 to 17. <Composition for inducing angiogenesis> (Appendix 22) A composition for use in inducing angiogenesis, wherein the composition is a composition described in any one of Appendices 1 to 17. <Composition for inhibiting fibrosis> (Appendix 23) A composition for use in inhibiting fibrosis, wherein the composition is a composition described in any one of Appendices 1 to 17. <Method for treating kidney disease> (Appendix 24) A method for treating a patient suffering from at least one of kidney disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease, comprising administering to the patient a composition described in any of Appendices 1 to 17. (Appendix 25) The method for treating the kidney disease described in Appendices 24, wherein the kidney disease is acute kidney injury, transitional chronic kidney disease, chronic kidney disease, or immunosuppression after kidney transplantation. <Method for promoting proliferation of vascular endothelial cells> (Appendix 26) A method for promoting proliferation of vascular endothelial cells, comprising a step of promoting proliferation of the vascular endothelial cells by contacting the vascular endothelial cells with a composition, wherein the composition is a composition described in any of Appendices 1 to 17. <Method for inducing angiogenesis> (Appendix 27) A method for inducing angiogenesis, comprising using a composition described in any of Appendices 1 to 17. (Appendix 28) The method of appendix 27, wherein the composition is administered to a subject in whom angiogenesis is to be induced.(Appendix 29) The method according to Appendix 28 or 29, wherein the subject in which angiogenesis is induced is a patient with at least one of renal disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease, and the induction of angiogenesis is induction of angiogenesis in the kidney, brain, lung, bone marrow, intestine, eye, ischemic muscle, or a site where ischemia-reperfusion injury has occurred in the patient with the disease. <Method for inhibiting fibrosis> (Appendix 30) A method for inhibiting fibrosis, comprising using a composition according to any one of Appendices 1 to 17. (Appendix 31) The method according to Appendix 30, wherein the composition is administered to a subject in which fibrosis is to be inhibited. (Appendix 32) The method of Appendices 30 and 31, wherein the subject for inhibiting fibrosis is a patient with at least one of renal disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease, and the inhibition of fibrosis is inhibition of fibrosis in the kidney, brain, lung, bone marrow, intestine, eye, ischemic muscle, or a site where ischemia-reperfusion injury has occurred in the patient. <Use> (Appendix 33) A composition for use in therapeutic or preventive treatment of at least one of renal disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease, wherein the composition is a composition described in any of Appendices 1 to 17. (Appendix 34) A composition for use in promoting the proliferation of vascular endothelial cells, wherein the composition is a composition described in any of Appendices 1 to 17. (Appendix 35) A composition for use in inducing angiogenesis, the composition being the composition described in any one of Appendices 1 to 17. (Appendix 36) A composition for use in inhibiting fibrosis, the composition being the composition described in any one of Appendices 1 to 17.

[0159] As described above, the composition of the present disclosure inhibits graft rejection and is derived from the regenerative cell population, thereby exhibiting physiological activities such as vascular endothelial cell proliferation-promoting activity and angiogenesis-promoting activity. Therefore, the present disclosure can be suitably used for treating or improving the prognosis of at least one of renal disease, peripheral arterial disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-occlusive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease. Therefore, the present disclosure is extremely useful, for example, in the pharmaceutical field.

Claims

1. A composition for use in the treatment of at least one of the following diseases: renal disease, peripheral artery disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-obstructive bowel ischemia, ocular ischemic syndrome, fibrosis, and inflammatory diseases, The composition comprises extracellular vesicles of a regenerative cell population derived from mononuclear cells derived from a biological sample or a culture thereof.

2. The composition according to claim 1, wherein the extracellular vesicles include miR-15b-5p, miR-29b-3p, miR-29c-3p, miR-92a-2-5p, miR-126-3p, miR-126-5p, miR-133a-3p, miR-133b, miR-146a-3p, miR-146b-5p, miR-150-5p, miR-181b-3p, miR-195-3p, miR-195-5p, miR-200b-5p, miR-302a-5p, and / or miR-142-3p.

3. Furthermore, the extracellular vesicles are miR-10a-3p, miR-17-3p, miR-20b-5p, miR-21-5p, miR-24-2-5p, miR-29a-5p, miR-30b-5p, miR-30c-5p, miR-30e-3p, miR-30e-5p, miR-32-5p, miR-92a-3p, miR-103a-2-5p, mi The composition according to claim 1, comprising R-144-5p, miR-148a-3p, miR-199b-5p, miR-200a-3p, miR-205-5p, miR-210-3p, miR-221-5p, miR-324-5p, miR-363-3p, miR-373-3p, miR-509-3p, miR-633, and / or let-7c-5p.

4. The composition according to claim 1, wherein the extracellular vesicles are CD9 and / or CD63 positive.

5. The composition according to claim 1, wherein the average diameter of the extracellular vesicles is 10 to 500 nm.

6. The composition according to claim 1, which has activity to promote the proliferation of vascular endothelial cells.

7. The composition according to claim 1, having angiogenesis-promoting activity.

8. The composition according to claim 1, having fibrosis inhibitory activity.

9. The composition according to claim 1, wherein graft rejection is suppressed.

10. The composition according to claim 1, wherein the extracellular vesicles are extracellular vesicles extracted or isolated from the regenerative cell population or its culture.

11. The composition according to claim 1, wherein the regenerative cell population is induced by culturing the mononuclear cells in the presence of a stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and / or vascular endothelial growth factor.

12. The composition according to claim 1, wherein the regenerative cell population is induced without sorting of CD34 and / or CD133-positive cells.

13. The composition according to claim 1, wherein the regenerative cell population comprises vascular endothelial progenitor cells, anti-inflammatory macrophages, and regulatory T cells.

14. The composition according to claim 13, wherein the vascular endothelial progenitor cells are differentiated EPC colony-forming cells.

15. The composition according to claim 13, wherein the anti-inflammatory macrophage is an M2 macrophage.

16. The composition according to claim 1, wherein the biological sample is at least one of blood and bone marrow.

17. The composition according to claim 1, wherein the biological sample is at least one of peripheral blood and umbilical cord blood.

18. A composition for use in the therapeutic or prophylactic treatment of at least one of the following diseases: renal disease, peripheral artery disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-obstructive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory diseases, The composition is the composition described in any one of claims 1 to 17.

19. The composition according to claim 18, wherein the kidney disease is acute kidney injury, transitional chronic kidney disease, chronic kidney disease, or immunosuppression after kidney transplantation.

20. A renal disease drug comprising the composition described in any one of claims 1 to 17, for use in the therapeutic or prophylactic treatment of at least one of the following diseases: renal disease, peripheral artery disease (PAD), cerebral infarction, pulmonary hypertension (PHD), spinal cord ischemia, non-obstructive intestinal ischemia, ocular ischemic syndrome, fibrosis, and inflammatory disease.

21. A composition for use in promoting the proliferation of vascular endothelial cells, The composition is the composition described in any one of claims 1 to 17.

22. A composition for use in inducing angiogenesis, The composition is the composition described in any one of claims 1 to 17.

23. A composition for use in suppressing fibrosis, The composition is the composition described in any one of claims 1 to 17.