Cell population and method for obtaining the same

Abstract

The present invention provides a cell population and a method for obtaining the same. The cell population of the present invention is obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood or peripheral blood in a culture medium containing four or less factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin and vascular endothelial cell growth factor, and serum.

Description

Technical Field

[0001] The present invention relates to a cell population and a method for obtaining the same. The cell population of the present invention is a cell population obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood or peripheral blood in a culture medium containing no more than four factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin and vascular endothelial cell growth factor, and serum. The present invention also relates to a composition for treating ischemic diseases, inflammatory diseases or refractory wounds, comprising a cell population obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood or peripheral blood in a culture medium containing no more than four factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin and vascular endothelial cell growth factor, and serum. Background Art

[0002] In recent years, bone marrow mononuclear cell transplantation therapy and cell transplantation therapy using endothelial progenitor cells (EPC) collected from peripheral blood stem cells have been started for ischemic diseases. Therefore, a large number of EPC-cultured techniques are particularly needed. The in vitro expansion method of endothelial progenitor cells from CD34 and / or CD133-positive cells can provide an efficient EPC culture technology (Patent Document 1). In addition, through the dynamic analysis method of endothelial cell differentiation, the existence of endothelial cell-like large colony-forming cells (differentiated EPC colonies) and endothelial cell-like small colony-forming cells (undifferentiated EPC colonies) is clarified, and the therapeutic effect of cell transplantation can be predicted and grasped (Patent Document 2). And a method for efficiently amplifying CD34 and / or CD133-positive cells from bone marrow mononuclear cells has been disclosed (Patent Document 3).

[0003] As shown in International Publication WO2014 / 0561154, a method for expanding a cell population enriched in endothelial progenitor cells or anti-inflammatory and immune tolerance-inducing cells from a mononuclear cell fraction derived from bone marrow, umbilical cord blood or peripheral blood is disclosed (hereinafter, also referred to as "QQ-MNC method" in this specification). This method expands a cell population containing EPCs in vitro by culturing mononuclear cells in a serum-free medium containing five factors: (1) stem cell factor (SCF), (2) interleukin-6 (IL-6), (3) FMS-like tyrosine kinase 3 ligand (FL), (4) thrombopoietin (TPO) and (5) vascular endothelial growth factor (VEGF).

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: International Publication No. WO2006 / 090882

[0007] Patent Document 2: International Publication WO2006 / 090886

[0008] Patent Document 3: International Publication No. WO2006 / 093172

[0009] Patent Document 4: International Publication No. WO2014 / 051154 Summary of the invention

[0010] Technical Problems to be Solved by the Invention

[0011] As a result of intensive research, the inventors have successfully obtained a new cell population that has angiogenesis and wound healing abilities and is different from the cell population obtained by the QQ-MNC method by culturing mononuclear cells derived from bone marrow, umbilical cord blood or peripheral blood in a culture medium containing no more than four factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin and vascular endothelial cell growth factor, and serum, thereby conceiving of the present invention.

[0012] The present invention aims to provide a cell population. The cell population of the present invention is obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood or peripheral blood in a culture medium containing four or less factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin and vascular endothelial cell growth factor, and serum.

[0013] The present invention also aims to provide a method for obtaining a cell population, which comprises culturing mononuclear cells derived from bone marrow, umbilical cord blood or peripheral blood in a culture medium containing no more than four factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin and vascular endothelial cell growth factor, and serum.

[0014] The present invention also aims to provide a composition for treating ischemic diseases, inflammatory diseases or refractory wounds. The composition of the present invention comprises: a cell population obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood or peripheral blood in a culture medium containing no more than four factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin and vascular endothelial cell growth factor, and serum.

[0015] Technical means to solve technical problems

[0016] The present invention includes but is not limited to the following aspects.

[0017] [Mode 1] A cell population obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood or peripheral blood in a culture medium containing no more than four factors selected from the group consisting of stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin and vascular endothelial cell growth factor, and serum.

[0018] [Mode 2] The cell population according to Mode 1, wherein the culture medium contains: three factors or four factors selected from the group consisting of stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor.

[0019] [Mode 3] The cell population according to Mode 1 or 2, wherein the serum is bovine serum or human serum.

[0020] [Mode 4] The cell population according to any one of Modes 1 to 3, wherein the serum is contained in the culture medium at a concentration of 0.5% by volume or more and 10% by volume or less.

[0021] [Mode 5] The cell population according to any one of Modes 1 to 4, wherein the total of cells expressing CD206(+), CD34(+) or CD3(+) is 60% or more, and the total of cells expressing CCR2(-) is 95% or more.

[0022] [Mode 6] The cell population according to Mode 5, wherein 50% or more of the CD206(+) cells are CXCR4(+).

[0023] [Mode 7] The cell population according to Mode 5 or 6, wherein 80% or more of the CD206(+) cells are CXCR4(+).

[0024] [Method 8] A method for obtaining a cell population according to any one of Methods 1 to 7, comprising: culturing mononuclear cells derived from bone marrow, umbilical cord blood or peripheral blood in a culture medium containing no more than four factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin and vascular endothelial growth factor, and serum.

[0025] [Mode 9] The method according to Mode 8, wherein, in the cell population, the total of cells that are CD206(+), CD34(+) or CD3(+) is 60% or more, and the total of cells that are CCR2(-) is 95% or more.

[0026] [Mode 10] The method according to Mode 8 or 9, wherein in the cell population, 50% or more of the cells are CD206(+) and CXCR4(+).

[0027] [Mode 11] The method according to any one of Modes 8 to 10, wherein the culture medium contains three factors or four factors selected from the group consisting of stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor.

[0028] [Mode 12] The method according to any one of Modes 8 to 11, wherein the serum is fetal bovine serum.

[0029] [Aspect 13] The method according to any one of aspects 8 to 12, wherein the serum is contained in the culture medium at a concentration of 0.5% by volume or more and 10% by volume or less.

[0030] [Embodiment 14] A composition for treating an ischemic disease, an inflammatory disease or a refractory wound, comprising the cell population according to any one of Embodiments 1 to 7.

[0031] [Mode 15] The therapeutic composition according to Mode 14, wherein the ischemic disease is limb ischemia.

[0032] [Embodiment 16] The therapeutic composition according to Embodiment 14 or 15, wherein the ischemic disease is limb ischemia accompanied by ulcer.

[0033] [Aspect 17] The therapeutic composition according to any one of Aspects 14 to 16, which promotes angiogenesis and / or wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] [ Figure 1 ] Figure 1 The results of flow cytometry (FACS) fractionation of constituent cell components of RE-01 prepared from healthy human peripheral blood are shown. As a comparative example, the results of FACS of peripheral blood mononuclear cells before culture are also shown.

[0035] [ Figure 2 ] Figure 2 Expressing about Figure 1 The results of flow cytometry (FACS) analysis of the constituent cell components of RE-01 prepared from peripheral blood of other healthy subjects.

[0036] [ Figure 3 ] Figure 3 The results of flow cytometry (FACS) analysis of the constituent cell components of RE-01 prepared from the peripheral blood of a 53-year-old female diabetic patient are shown. As a comparative example, a cell population (MNC-QQ) cultured in a serum-free medium containing five factors was used.

[0037] [ Figure 4 ] Figure 4 The results of investigating the angiogenesis ability of RE-01 using human umbilical vein endothelial cells (HUVEC) are shown. As comparative examples, the case where MNC-QQ was used instead of RE-01 and the case where only HUVEC was used were investigated. Figure 4 A represents the time change of the number of formed lumens from the start of culture, Figure 4 B shows the number of lumens formed 2.75 hours after the start of culture. Figure 4 C shows the number of fluorescently labeled test cells enclosed in the tubular structure 2.75 hours after the start of culture.

[0038] [ Figure 5 ] Figure 5 The present invention shows the results of flow cytometry (FACS) fractionation of the constituent cell components of RE-01 prepared from the peripheral blood of patients with critical limb ischemia. DETAILED DESCRIPTION

[0039] 1. Cell Population

[0040] The present invention relates to a cell population.

[0041] The cell population of the present invention is obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood or peripheral blood in a culture medium containing four or less factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin and vascular endothelial cell growth factor, and serum.

[0042] The cell population is obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood or peripheral blood. "Mononuclear cells" for obtaining the cell population refer to the general term for cells with round nuclei contained in peripheral blood, bone marrow or umbilical cord blood, etc., including lymphocytes, monocytes, macrophages, endothelial progenitor cells, hematopoietic stem cells, etc. For example, bone marrow, umbilical cord blood or peripheral blood is collected from an animal, and the component is extracted, for example, by using a blood collection tube for separating mononuclear cells, or by supplying it to a density gradient centrifugation method, thereby obtaining mononuclear cells. As a density gradient centrifugation method, there is no particular limitation as long as a mononuclear cell component is formed. For example, Histopaque-1077 (Sigma-Aldrich) can be used.

[0043] There is no particular limitation on the animal species used as the source of bone marrow, umbilical cord blood or peripheral blood. The animal species include general mammals including humans that can be used for cell transplantation therapy for diseases such as ischemic diseases, inflammatory diseases or refractory trauma. From the perspective of clinical application, humans are preferred.

[0044] The subjects of bone marrow, umbilical cord blood or peripheral blood are not particularly limited. In one embodiment, for example, the subjects may be healthy subjects, diabetic patients or patients with severe lower limb ischemia.

[0045] Stem cell factor (SCF) is a glycoprotein with a molecular weight of about 30,000 and composed of 248 amino acids. Due to selective splicing, there are soluble and membrane-bound types. The SCF used to obtain the cell population can be any type of SCF as long as it is useful for the culture of mononuclear cells. It is preferably a soluble type. There is no particular limitation on the source of SCF. Without limitation, it is preferably a recombinant that is expected to have a stable supply, and human recombinants are particularly preferred. Commercially available products are known. The concentration of SCF in the culture medium varies depending on the type of SCF used, and is not particularly limited as long as it is useful for the culture of mononuclear cells. If it is a human recombinant SCF, it is not limited, for example, 10 to 1000 ng / mL, preferably 50 to 500 ng / mL, and more preferably about 100 ng / mL.

[0046] Interleukin 6 (IL-6) is a glycoprotein with a molecular weight of 210,000 that was isolated as a factor that induces the final differentiation of B cells into antibody-producing cells. It is known that IL-6 is generally related to immune response, hematopoietic system, proliferation and differentiation of neural cells, acute phase response, etc. The IL-6 used to obtain the cell population is not particularly limited and can be appropriately selected. If used in the culture of human mononuclear cells, human IL-6 is preferred, and recombinant forms that are expected to have a stable supply are particularly preferred. It is known that there are commercially available products. The concentration of IL-6 in the culture medium varies depending on the type of IL-6 used, and is not particularly limited as long as it is useful for the culture of mononuclear cells. In the case of human recombinant IL-6, it is not limited, for example, 1 to 500 ng / mL, preferably 5 to 100 ng / mL, and more preferably about 20 ng / mL.

[0047] FMS-like tyrosine kinase 3 ligand (FL) is widely known as a ligand of a receptor tyrosine kinase that plays an important role in the control of early hematopoiesis. Several products based on alternative splicing are known, and it is reported that they stimulate the proliferation of hematopoietic stem cells. As for the FL used to obtain the cell population, any type of FL can be used as long as it is useful for the culture of mononuclear cells. It is known that there are commercially available products. The concentration of FL in the culture medium varies depending on the type of FL used, and there is no particular limitation as long as it is useful for the culture of mononuclear cells. In the case of human recombinant Flt-3 ligand, it is not limited, for example, 10 to 1000 ng / mL, preferably 50 to 500 ng / mL, and more preferably about 100 ng / mL.

[0048] Thrombopoietin (TPO) is a type of hematopoietic cytokine, and it is known that it specifically plays a role in the process of forming megakaryocytes from hematopoietic stem cells, promoting the production of megakaryocytes. The source of TPO used to obtain the cell population is not particularly limited. It is preferably a recombinant that is expected to have a stable supply, and a human recombinant is particularly preferred. It is known that there are commercially available products. The concentration of TPO in the culture medium varies depending on the type of TPO used. As long as it is useful for the culture of mononuclear cells, it is not particularly limited. In the case of human recombinant TPO, it is not limited, for example, 1 to 500 ng / mL, preferably 5 to 100 ng / mL, and more preferably about 20 ng / mL.

[0049] Vascular endothelial cell growth factor (VEGF) is a growth factor that exerts a specific effect on endothelial progenitor cells (EPC), and is known to be mainly produced in cells around blood vessels. Several VEGF proteins of different sizes are produced by selective splicing. As for the VEGF used to obtain the cell population, any type of VEGF can be used as long as the EPC colony can be formed. VEGF165 is preferred. There is no particular limitation on the source of VEGF. It is preferably a recombinant that is expected to have a stable supply, and a human recombinant is particularly preferred. It is known that there are commercially available products. The concentration of VEGF in the culture medium varies depending on the type of VEGF used, and there is no particular limitation as long as it is useful for the culture of mononuclear cells. In the case of human recombinant VEGF165, it is not limited, for example, about 5 to 500 ng / mL, preferably about 20 to 100 ng / mL, and more preferably about 50 ng / mL.

[0050] The various factors added to the culture medium used in the culture of mononuclear cells can be unified, without limitation, as factors derived from an animal of the same species as the animal from which the mononuclear cells are derived. By unifying the sources of mononuclear cells and various factors in this way, a cell culture suitable for allogeneic transplantation such as allogeneic transplantation can be obtained. In addition, by using mononuclear cells derived from an individual intended for cell transplantation, a cell culture suitable for allogeneic transplantation can also be obtained.

[0051] The components described above can be dissolved in a culture medium at a given concentration, or a concentrated solution (stock solution) of each component can be prepared in advance, and the culture medium for culturing mononuclear cells can be prepared by diluting the culture medium to a given concentration. For example, the culture medium can be prepared by dissolving the necessary components in a commercially available culture medium in a manner such that the components are at a given concentration, sterilizing the solution by filtration sterilization, etc., or adding the stock solution sterilized by filtration sterilization, etc. to a commercially available culture medium in an aseptic manner and diluting the solution. Filtration sterilization can be performed according to the methods commonly used in the field, for example, using a 0.22 μm, 0.45 μm millipore filter, etc.

[0052] The "culture medium" used in the present invention can utilize a culture medium commonly used in the field, for example, a culture medium widely known as a culture medium for the proliferation of hematopoietic stem cells can be used. As for the basal culture medium used as the culture medium, for example, Stemline II, DMEM, MEM, IMDM, RPMI, SCGM, EBM, etc. can be cited.

[0053] In order to obtain the cell population, the medium for culturing mononuclear cells contains serum in addition to no more than four factors selected from the group consisting of stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin and vascular endothelial growth factor.

[0054] The type of serum is not particularly limited. Without limitation, in one embodiment, the serum is bovine serum or human serum. In one embodiment, the serum is fetal bovine serum and / or human serum albumin. The concentration of serum in the culture medium is not particularly limited. In one embodiment, the serum is contained in the culture medium at 0.1% by volume or more, 0.3% by volume or more, or 0.5% by volume or more. The upper limit of the concentration of serum in the culture medium is not particularly limited. In one embodiment, it is within 30% by volume, within 20% by volume, within 10% by volume, or within 5% by volume. Without limitation, the serum is contained in the culture medium at a concentration of 0.1% by volume or more and 20% by volume, and is contained in the culture medium at a concentration of 0.5% by volume or more and 10% by volume.

[0055] The mononuclear cell culture is performed by adding a cell suspension containing the mononuclear cells to a culture medium containing the factors and serum. As the cell suspension, a body fluid containing the mononuclear cells itself (e.g., bone marrow fluid, umbilical cord blood, peripheral blood) may also be used. The culture conditions of the mononuclear cells are not particularly limited, and may be carried out under conditions commonly practiced in this field. Without limitation, for example, culture is performed at about 37°C in a 5% CO2 atmosphere. With respect to the culture period, without limitation, for example, it may be more than 3 days, within 5 days, within 6 days, within 7 days, or within 10 days. For example, 3 days to 10 days, 3 days to 6 days. With respect to the concentration in the culture medium of the mononuclear cells, as long as the culture of the mononuclear cells can be performed, it is not particularly limited, and for example, it is about 0.1 to 10×10 6 cells / ml, more preferably about 0.5 to 5×10 6 cells / ml.

[0056] In the present specification, the "cell population" refers to a general term for cells obtained by culturing mononuclear cells in a medium containing the above-mentioned factors and serum.

[0057] The culture medium used for culturing mononuclear cells contains four or less factors selected from the group consisting of stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial cell growth factor.

[0058] In one embodiment, the culture medium comprises: two or more factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial cell growth factor, or three or more factors. In one embodiment, the culture medium comprises: two or more factors selected from the five factors but less than four factors. In one embodiment, the culture medium comprises: three factors or four factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial cell growth factor.

[0059] Without limitation, the culture medium used in the culture of mononuclear cells, and therefore the culture medium used in the culture method of the present invention, for example, comprises: i) a combination of FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor; or ii) a combination of stem cell factor, FMS-like tyrosine kinase 3 ligand, and vascular endothelial growth factor. The culture medium more preferably comprises: a combination of about 80 to 120 ng / ml of FMS-like tyrosine kinase 3 ligand, about 15 to 25 ng / ml of thrombopoietin, and about 40 to 60 ng / ml of vascular endothelial growth factor.

[0060] In one embodiment, a cell population obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood or peripheral blood in a medium containing no more than four factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin and vascular endothelial cell growth factor, and serum, contains more CD206(+), CD34(+) or CD3(+) cells than the cell population of mononuclear cells before culture. Without limitation, by culture, the proportion of cells that are CD206(+), CD34(+) or CD3(+) is expanded to 1.5 times or more, 2 times or more, or 3 times or more. Without limitation, in the cell population, the total number of cells that are CD206(+), CD34(+) or CD3(+) is 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the entire cell population. And the cell population contains more CCR2(-) cells than the cell population of mononuclear cells before culture. Without limitation, by culturing, the proportion of CCR2(-) cells is expanded to 1.5 times or more, 2 times or more, or 3 times or more. Without limitation, in the cell population, CCR2(-) cells account for 60% or more, 80% or more, 90% or more, or 95% or more of the entire cell population. Without limitation, in one embodiment, cells that are CD206(+), CD34(+), and CD3(+) account for 60% or more, and cells that are CCR2(-) account for 95% or more.

[0061] By culturing mononuclear cells derived from bone marrow, cord blood or peripheral blood in a medium containing less than 4 factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin and vascular endothelial cell growth factor, and serum, a cell population containing more CD206(+), CD34(+) and CD3(+) can be stably obtained. And compared with the cell population obtained by the QQ-MNC method, the proportion of CXCR4-positive cells in CD206-positive anti-inflammatory macrophages M2 is significantly increased. Without limitation, the inventors observed the following effects through the culture.

[0062] Increased CD206 positivity, including anti-inflammatory macrophages M2;

[0063] CD34-positive cells and CD133-positive cells including endothelial progenitor cells (EPC) increased;

[0064] T cells, especially CD3-positive cells such as helper T cells and angiogenic T cells, increase.

[0065] On the other hand, a decrease in CCR2-positive cells, B cells (CD19-positive cells), and NK cells (CD56-positive cells), which are inflammatory monocytes / macrophages, was observed.

[0066] Without limitation, in one embodiment, a cell population obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood or peripheral blood in a medium containing no more than four factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin and vascular endothelial cell growth factor, and serum shows a higher recovery rate than a cell population obtained by the QQ-MNC method. In addition, the survival rate is higher. In addition, the stability after recovery is also more excellent. These effects can bring about a higher therapeutic effect.

[0067] In one embodiment, a cell population obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood or peripheral blood in a medium containing no more than four factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin and vascular endothelial cell growth factor, and serum, contains more cells with CXCR4(+) among CD206(+) cells than a cell population of mononuclear cells before culturing. Without limitation, by culturing, the proportion of cells with CXCR4(+) among CD206(+) cells is expanded to 1.5 times or more, 2 times or more, or 3 times or more. Without limitation, the proportion of cells with CXCR4(+) among CD206(+) cells is 50% or more, 60% or more, 70% or more, or 80% or more. In one embodiment, the proportion of cells with CXCR4(+) among CD206(+) cells is 50% or more. In one embodiment, the proportion of cells with CXCR4(+) among CD206(+) cells is 80% or more.

[0068] The cell population of the present invention contains more CXCR4(+) cells among the CD206(+) cells, compared with the cell population obtained by the QQ-MNC method.

[0069] 2. Method for obtaining cell population

[0070] The present invention also relates to a method for obtaining a cell population.

[0071] The method of the present invention comprises: culturing mononuclear cells derived from bone marrow, umbilical cord blood or peripheral blood in a culture medium containing four or less factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin and vascular endothelial cell growth factor, and serum.

[0072] The factors of "cell population", "mononuclear cells", "stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin and vascular endothelial growth factor", "serum", "culture medium" etc. are as described in the above "1. Cell population".

[0073] The proportions of CD206(+), CD34(+) and CD3(+) cells in the cell population, the proportion of CCR2(-) cells, and the proportion of CXCR4(+) cells in CD206(+) cells are also as described in the above-mentioned "1. Cell population".

[0074] In one embodiment, in the cell population obtained by the method, 80% or more of the cells are CD206(+), CD34(+), and CD3(+), and 95% or more of the cells are CCR2(-).

[0075] In one embodiment, in the cell population obtained by the method, 50% or more of the CD206(+) cells are CXCR4(+).

[0076] In one embodiment, in the method, the culture medium contains three factors or four factors selected from the group consisting of stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor.

[0077] In one embodiment, in the method, the serum is bovine serum or human serum. In one embodiment, the serum is fetal bovine serum. In one embodiment, the serum is contained in the culture medium at a concentration of 0.5 volume % to 10 volume %.

[0078] 3. Therapeutic compositions

[0079] The present invention also relates to a therapeutic composition for ischemic diseases, inflammatory diseases or refractory wounds. The therapeutic composition of the present invention comprises the cell population described in "1. Cell population". The mononuclear cells as the source of the cell population may be derived from the subject to which the therapeutic composition is used (autologous) or from a source other than the subject to which the therapeutic composition is used (allogeneic).

[0080] "Ischemic disease" is a disease caused by a decrease in blood volume, which leads to a decrease in blood flow in the tissue, resulting in tissue damage such as cell degeneration, atrophy, and fibrosis. Ischemia is roughly divided into obstructive ischemia, compressive ischemia, spastic ischemia, and compensatory ischemia according to its cause. If ischemia persists, cell degeneration, atrophy, and fibrosis will occur. "Ischemic disease" includes limb ischemia, ischemic ulcers, ischemic heart disease, cerebral infarction, etc. "Cerebral infarction" (or encephalomalacia) is caused by blockage or stenosis of the arteries that supply nutrients to the brain, resulting in cerebral ischemia, brain tissue necrosis due to lack of oxygen or nutrition, or a state close to necrosis, and is classified into cerebral thrombosis and cerebral embolism. "Limb ischemia" is a disease caused by stenosis or blockage of the arteries that supply blood to the hands and feet. If the obstructive arteriosclerosis becomes severe, it will become severe limb ischemia, resulting in symptoms such as pain and intractable ulcers. In the worst case, amputation may be required. Without limitation, in one embodiment, the limbs are the lower limbs.

[0081] Without limitation, in one embodiment, the ischemic disease is limb ischemia. The ischemic disease is limb ischemia accompanied by ulcer.

[0082] "Inflammatory diseases" is a general term for diseases that cause symptoms due to abnormalities such as tissue damage. Inflammatory diseases include Crohn's disease, cirrhosis, hepatitis, ulcerative colitis, inflammatory bowel disease, etc.

[0083] The therapeutic composition promotes angiogenesis and / or wound healing. Therefore, it can be applied to diseases that can be treated by angiogenesis and / or wound healing. As "diseases that can be treated by angiogenesis and / or wound healing", for example, ischemic diseases (for example, ischemic heart diseases such as myocardial infarction and angina, lower limb ischemia such as lower limb ischemic arteriosclerosis, Buerger's disease, vascular damage. In addition, it can be used to heal wounds such as skin ulcers, or to make artificial blood vessels. The applicable effect of the therapeutic composition can be confirmed by a method known per se. For example, in the case where the disease that can be treated by angiogenesis is lower limb ischemic disease, for example, the therapeutic effect after transplantation can be evaluated by investigating the lower limb blood flow and the necrosis improvement rate. The increase in blood flow can be measured by measuring the value of laser Doppler imaging analysis. In addition, the necrosis improvement rate can be measured by naked eye observation in the form of a limb salvage score.

[0084] The use of the therapeutic composition is not particularly limited. It can be the cell group itself as an active ingredient, or it can be a product obtained by suspending the cell group in a liquid medium. The liquid medium is not particularly limited as long as it is a liquid that can be injected into the human body. For example, isotonic electrolyte infusion, phosphate buffer, physiological saline or DMEM as a serum-free culture medium can be used. In addition, the liquid medium can also contain compounds such as albumin that help cell survival. As a compound containing albumin, serum derived from a patient can be preferably cited. In addition, in the case of cryopreservation, it can also be a product suspended in a frozen cell preservation solution.

[0085] The therapeutic composition is applicable to any subject who needs treatment for ischemic diseases, inflammatory diseases or refractory wounds, and is not particularly limited to humans, monkeys, chimpanzees, dogs, cats, cows, horses, mice, guinea pigs, etc.

[0086] 4. Test kit

[0087] The present invention also relates to a kit for culturing mononuclear cells derived from bone marrow, umbilical cord blood or peripheral blood and obtaining cell populations.

[0088] The kit comprises: a culture medium comprising no more than four factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin and vascular endothelial growth factor, and serum. Each factor and serum can be added to the culture medium in advance, or stored in respective containers and added to the culture medium when used.

[0089] "Cell population", "mononuclear cells", various factors including "stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin and vascular endothelial growth factor", "serum, "culture medium" etc. are as described in the above "1. Cell population".

[0090] 5. Treatment methods, etc.

[0091] The present invention also relates to a method for treating ischemic diseases, inflammatory diseases or refractory wounds, comprising: applying the cell population described in "1. Cell population" to a subject in need.

[0092] The present invention also relates to the use of the cell population described in "1. Cell population" in a method for treating ischemic diseases, inflammatory diseases or intractable wounds, which includes applying it to a subject in need, or using it in a composition for treating ischemic diseases, inflammatory diseases or intractable wounds.

[0093] "Ischemic disease", "inflammatory disease" or "refractory wound", "application mode of cell population", "application target" and the like are as described in "3. Therapeutic composition".

[0094] Example

[0095] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples. A person skilled in the art can easily modify and change the present invention based on the description of this specification, and these modifications and changes are also included in the technical scope of the present invention.

[0096] Example 1 Cultivation of RE-01 Cell Population

[0097] In this example, mononuclear cells were isolated from peripheral blood and cultured.

[0098] (1) Isolation of mononuclear cells

[0099] 100 mL of peripheral blood was collected from healthy volunteers or diabetic patients using a BD vacutainer (registered trademark) CPT (trademark) BD company) mononuclear cell separation blood collection tube. After blood collection, the blood collection tube was centrifuged and transported, and refrigerated until the start of culture. The part above the gel barrier in the CPT (trademark) blood collection tube containing mononuclear cells and plasma was recovered in a centrifuge tube, and the part above the gel barrier and other blood collection tubes were washed with a small amount of EDTA-PBS and recovered in the same centrifuge tube. For the centrifuge tube in which the cells were recovered, the volume was increased with EDTA-PBS, and then centrifuged (300×g, room temperature, 15 minutes) to recover the cells as precipitates. For the recovered cells, in order to remove the mixed red blood cells, they were incubated in ACK hemolysis buffer (15 mL / tube) (Gibco, Thermo-Fisher company) at room temperature for 5 minutes. The composition of ACK hemolysis buffer was NH4Cl 8290 mg / l; KHCO3 1000 mg / l; EDTA.Na2·2H2O 37 mg / l. Then, the volume was increased with EDTA-PBS and centrifuged (200×g, room temperature, 10 minutes) or (100×g, room temperature, 15 minutes) twice to recover the cells.

[0100] (2) Cultivation in serum medium

[0101] The mononuclear cells recovered in (1) were suspended in 1 mL of proliferation medium (supplemented with 50 ng / mL VEGF 165 , 20 ng / mL TPO, 100 ng / mL Flt-3 ligand, 100 units / mL penicillin, 100 μg / mL streptomycin, 0.5% FBS stemline (registered trademark) II Hematopoietic Stem Cell Expansion Medium (manufactured by Sigma-Aldrich, Cat No. S0192)), and a portion thereof was used to count the number of cells using trypan blue. The cell concentration in the obtained cell proliferation medium was 1×10 6 / mL, and 2mL was inoculated into each well of a 6-well culture plate. Culture was carried out for 5 days under normal culture conditions (37°C, 5% CO2). After 5 days of culture, the cells were collected in a centrifuge tube and washed by centrifugation at 250×g (~1000rpm) for 7 to 10 minutes three times. After centrifugation, except for samples used for cell number determination, survival rate determination, and QC (confirmation) tests, the remaining cells were suspended in PlasmaLyte A, 2.5% human albumin serum, and the concentration was 8×10 5The obtained cell population is hereinafter also referred to as "RE-01".

[0102] Example 2 Evaluation of characteristics of RE-01 cell population

[0103] (1) Flow cytometry analysis

[0104] In this example, in Example 1, flow cytometry analysis was performed on a cell population (called "RE-01") obtained from the peripheral blood of a healthy person by culturing in a medium containing three factors and serum in order to further clarify the characteristics of the cell population.

[0105] Cells (1.5×10 6 10 μL of FC blocking reagent (Miltenyi) was added to each aliquot (100 μL / tube × 3 tubes) and incubated at 4°C for 30 minutes. The incubated cell population was equally distributed to each staining reaction tube (100 μL / tube × 3 tubes). 2 μL of primary antibody was added to each aliquot and incubated at 4°C for 20 minutes. Then, the cells were washed twice with 1 mL of FACS buffer and the stained cells were suspended in FACS buffer (5 × 10 5 Cells / 200 to 300 μL-FACS buffer). Flow cytometry was performed using BD FACSAria (trademark) III cell sorter (manufactured by BD).

[0106] Specifically, the expression of each cell surface marker in the obtained cell population was investigated using antibodies against each cell surface marker. It should be noted that the antibodies against each cell surface marker used were the following commercially available products.

[0107] Anti-CD206 antibody: PE / Cy7-labeled anti-human CD206 (MMR) antibody (BioLegend);

[0108] Anti-CD34 antibody: PE-labeled anti-human CD34 antibody (manufactured by Bio Legend);

[0109] Anti-CD3 antibody: Alexa Fluor 700-labeled anti-CD3 antibody (manufactured by Bio Legend);

[0110] Anti-CXCR4 antibody: APC-labeled anti-human CD184 (CXCR4) antibody (manufactured by BD Biosciences);

[0111] Anti-CCR2 antibody: PerCP / Cy5.5-labeled human CD192 (CCR2) antibody (manufactured by BioLegend).

[0112] The results are shown in Figure 1 .like Figure 1 As shown, RE-01 is roughly divided into three living cell groups (referred to as "Area A", "Area B", and "Area C" in this specification) according to the size of the constituent cell components (horizontal axis: FSC) and the density of the cell constituent components such as particles contained in the cells (vertical axis: SSC) in the FACS-based Scatter analysis. In the peripheral blood mononuclear cells before culture, only the A and B regions appeared, and the C region was not observed. The A region mainly contains lymphocytes, and the B region contains monocytes. In the RE-01 obtained by culturing using a proliferation medium ((2) serum medium of Example 1), in addition to the migration of endothelial progenitor cells (CD34-positive cells) from the A region to the B region, a large number of M2 macrophages (CD206-positive cells) appeared in the C region.

[0113] In addition, the results of flow cytometric analysis of RE-01 obtained by culturing mononuclear cells collected from other healthy subjects using a medium containing three factors and serum as described in Example 1 are shown in FIG. Figure 2 . In the case of healthy individuals, three regions A, B, and C also appeared. In addition, the total number of cells that were CD206(+), CD34(+), or CD3(+) was 86.57%, and CCR2(+) was 0.33%, so the number of cells that were CCR2(-) was 99.67%. In addition, the number of cells that were CXCR4(+) among CD206(+) was 92.97%.

[0114] Example 3 Comparison of RE-01 cell population and MNC-QQ cell population

[0115] (1) Cultivation of RE-01 and MNC-QQ

[0116] Mononuclear cells collected from healthy subjects and diabetic patients were cultured in a medium containing three factors and serum in the same manner as in Example 1 to obtain RE-01.

[0117] As a comparative example, mononuclear cells were isolated in the same manner as RE-01 cells, and then stemline (registered trademark) II Hematopoietic Stem Cell Expansion Medium (manufactured by Sigma-Aldrich, Cat No. S0192) was used. No serum was added, but 100 units / mL penicillin, 100 μg / mL streptomycin, and 50 ng / mL VEGF were added. 165 , 20 ng / mL TPO, 100 ng / mL Flt-3 ligand, 100 ng / mL SCF, and 20 ng / mL IL-6 were added in a sterile manner to prepare the cells. 6 / mL method, 2mL was inoculated into each well of a 6-well culture plate, and cultured for 7 days under normal culture conditions (37°C, 5% CO2). After 7 days of culture, the cells were collected in a centrifuge tube and washed by centrifugation at 250×g (~1000rpm) for 7 to 10 minutes three times. After centrifugation, except for samples used for cell number determination, survival rate determination, and QC (confirmation) tests, the remaining cells were suspended in PlasmaLyteA, 2.5% human albumin serum, and the concentration was 8×10 5 The obtained cell population is hereinafter referred to as "MNC-QQ".

[0118] (2) Flow cytometry analysis

[0119] In Example 1, flow cytometry was performed to compare a cell population obtained by culturing in a medium containing three factors and serum from the peripheral blood of a diabetic patient and a cell population (MNC-QQ) obtained by culturing in a serum-free medium containing the five factors described in (1). Specifically, the expression of each cell surface marker in the obtained cell population was investigated using antibodies against each cell surface marker.

[0120] The results are as follows Figure 3 As shown. RE-01 has higher recovery rate and survival rate after culture than MNC-QQ. In addition, the proportion of cells in the C region, which is a newly formed cell group after culture, is higher, especially the proportion of CD206+ cells, which is one of the cell types responsible for the effectiveness of the cell group. In addition, surprisingly, the proportion of CXCR4+ in CD206+ is significantly higher, which shows that the cell groups of RE-01 and MNC-QQ are different in their cell composition.

[0121] (3) acLDL uptake capacity

[0122] The acLDL (acetylated low-density lipoprotein) uptake capacity of the cell population (RE-01) prepared from peripheral blood mononuclear cells of diabetic patients in Example 1 and cultured in a medium containing three factors and serum was investigated, as well as that of the cell population (MNC-QQ) obtained as a comparison example by culture in a serum-free medium containing five factors.

[0123] RE-01 or MNC-QQ (1×10 5 ) were suspended in 500 μL of basal culture medium (Stemline II), 5 μL of acLDL labeled with Alexa Fluor 488 was added, and incubated at 37°C for 60 minutes. 1 mL of FACS buffer was added to the tube after the incubation, and the mixture was mixed by inversion. Then, a 5-minute centrifugation operation (250×g, 4°C) was performed to recover the cells in the form of a precipitate. The recovered cells (RE-01) were suspended in 300 μL of FACS buffer, cooled in ice and stored until the measurement. DAPI (2 μL) was added just before the measurement, and the proportion of cells that had taken up acLDL and DAPI was determined by measurement using a flow cytometer.

[0124] The results are shown in Table 1 below.

[0125] [Table 1]

[0126]

[0127] As shown in Table 1, it can be seen that the uptake of acLDL is high overall, especially in the C region. This also proves that for the two cell populations (RE-01 and MNC-QQ), the different compositions shown in Example 2 lead to different functions.

[0128] (4) Angiogenesis ability

[0129] In this example, a cell population (RE-01) obtained by culturing mononuclear cells collected from the blood of healthy people using a serum medium in the same manner as in Example 1 was used to investigate its angiogenesis ability. As a comparative example, a cell population (MNC-QQ) obtained by culturing using a serum-free medium containing 5 factors was used. Human umbilical vein endothelial cells (HUVEC) (obtained from Lonza, Basel, Switzerland) with lumen formation ability were used in the form of 8 to 10 passages, and the cells of each population were co-cultured with HUVEC. Basement membrane matrix (Corning (registered trademark) Matrigel (registered trademark) (manufactured by Corning, NY, USA)) 50 μl / well was dispensed into a 96-well plate and plated at 37°C for 30 minutes. On the other hand, in order to label each cell population with DiI-Ac-LDL, it was suspended in IMDM 500 μl + DiI-Ac-LDL 5 μl and incubated at 37°C for 1 hour.

[0130] After labeling, each cell population was 1×10 3 / 20μl, and 5×10 HUVEC 3 / 20μl, resuspended in PBS respectively. The suspension adjusted with the number of cells was mixed in a 1:1 ratio between the test cell population and HUVEC, and 50μl was added to Matrigel respectively. At 37°C, 5% CO2, for more than 10 hours, the cells were cultured in a time-lapse fluorescence microscope (manufactured by Olympus, Tokyo, Japan) and photographed. After the culture was completed, the number of lumens formed and the number of fluorescently labeled test cells enclosed in the lumens were counted by naked eye observation at a magnification of 100 times.

[0131] The results are as follows Figure 4 shown. Figure 4 A represents the time change of the number of formed lumens from the start of culture, Figure 4 B represents the number of lumens formed 2.75 hours after the start of culture. Figure 4C represents the number of fluorescently labeled test cells enclosed in the tubular structure 2.75 hours after the start of culture. The ability to form a lumen when HUVEC and RE-01 are co-cultured for a certain period of time is improved compared to when HUVEC is cultured alone or HUVEC is co-cultured with MNC-QQ. Moreover, for RE-01, more cells are enclosed in the formed tubular structure. That is, it can be seen that in vitro, RE-01 has a higher in vitro angiogenesis ability than MNC-QQ cultured for a longer period of time (7 hours) in serum-free medium. This also proves that for the two cell populations (RE-01 and MNC-QQ), the different compositions shown in Example 3 will lead to different functions.

[0132] (5) Effects based on mouse hind limb ischemia model

[0133] The therapeutic effect on limb ischemia was investigated using a mouse limb ischemia model using a cell population obtained by culturing mononuclear cells collected from healthy human blood in the same manner as in Example 1. As a comparative example, a cell population (MNC-QQ) obtained by culturing in a serum-free medium containing five factors was used.

[0134] (6) Stability test

[0135] The stability up to 72 hours after recovery was investigated by cell number and survival rate using a cell population obtained by culturing mononuclear cells collected from the blood of a healthy person in the same manner as in Example 1. As a comparative example, a cell population (MNC-QQ) obtained by culturing in a serum-free medium containing five factors was used.

[0136] Example 4 Cultivation and Characteristic Evaluation of RE-01 Cell Population

[0137] In this example, mononuclear cells were isolated from the peripheral blood of patients with severe lower limb ischemia and cultured, and the characteristics of the obtained cell population were evaluated.

[0138] (1) Isolation of mononuclear cells

[0139] Mononuclear cells were isolated from the peripheral blood of patients with severe lower limb ischemia. The isolation was performed in the same manner as in Example 1.

[0140] Specifically, 100 mL of peripheral blood was collected from patients with severe lower limb ischemia using a BD vacutainer (registered trademark) CPT (trademark) BD company) mononuclear cell separation blood collection tube. After blood collection, the blood collection tube was centrifuged and transported, and refrigerated until the start of culture after arrival. The part above the gel barrier in the CPT (trademark) blood collection tube containing mononuclear cells and plasma was recovered in a centrifuge tube, and the part above the gel barrier and other blood collection tubes were washed with a small amount of EDTA-PBS and recovered in the same centrifuge tube. For the centrifuge tube in which the cells were recovered, the volume was increased with EDTA-PBS, and then centrifuged (300×g, room temperature, 15 minutes) to recover the cells as precipitates. For the recovered cells, in order to remove the mixed red blood cells, they were incubated in ACK hemolysis buffer (15 mL / tube) (Gibco, Thermo-Fisher company) at room temperature for 5 minutes. The composition of ACK hemolysis buffer was NH4Cl 8290 mg / l; KHCO3 1000 mg / l; EDTA.Na2·2H2O 37 mg / l. Then, the volume was increased with EDTA-PBS and centrifuged (200×g, room temperature, 10 minutes) or (100×g, room temperature, 15 minutes) twice to recover the cells.

[0141] (2) Cultivation in serum medium

[0142] The isolated mononuclear cells were cultured in a serum medium in the same manner as in Example 1. The difference from Example 1 was that Iscove's Modified Dulbecco's Medium (IMDM) was used as the basic medium instead of stemline (registered trademark), and human albumin serum was added to FBS as the serum.

[0143] Specifically, the mononuclear cells collected in (1) were suspended in 1 mL of proliferation medium (supplemented with 50 ng / mL VEGF). 165 , 20ng / mL TPO, 100ng / mL Flt-3 ligand, 100 units / mL penicillin, 100μg / mL streptomycin, 0.5% FBS, 0.5% human albumin serum Iscove's Modified Dulbecco's Medium (IMDM), a portion of which was used to count the number of cells using trypan blue. In the obtained cell proliferation medium, the cell concentration was 1×10 6 / mL, and 2mL was inoculated into each well of a 6-well culture plate. Culture was carried out for 5 days under normal culture conditions (37°C, 5% CO2). After 5 days of culture, the cells were collected in a centrifuge tube and washed by centrifugation at 250×g (~1000rpm) for 7 to 10 minutes three times. After centrifugation, except for samples used for cell number determination, survival rate determination, and QC (confirmation) tests, the remaining cells were suspended in PlasmaLyte A, 2.5% human albumin serum, and the concentration was 8×10 5 The cells / mL method was adjusted.

[0144] (3) Flow cytometry analysis

[0145] In this example, for (1) and (2), the cell population obtained by culturing mononuclear cells isolated from the peripheral blood of patients with severe lower limb ischemia using a culture medium containing three factors and serum (referred to as "RE-01" in the same manner as in Example 1) was subjected to flow cytometry analysis in order to further clarify the characteristics of the cell population.

[0146] Cells (1.5×10 6 10 μL of FC blocking reagent (Miltenyi) was added to each aliquot (100 μL / tube × 3 tubes) and incubated at 4°C for 30 minutes. The incubated cell population was equally distributed to each staining reaction tube (100 μL / tube × 3 tubes). 2 μL of primary antibody was added to each aliquot and incubated at 4°C for 20 minutes. Then, the cells were washed twice with 1 mL of FACS buffer and the stained cells were suspended in FACS buffer (5 × 10 5 Cells / 200 to 300 μL-FACS buffer). Flow cytometry was performed using BD FACSAria (trademark) III cell sorter (manufactured by BD).

[0147] Specifically, the expression of each cell surface marker in the obtained cell population was investigated using antibodies against each cell surface marker. It should be noted that the antibodies against each cell surface marker used were the following commercially available products.

[0148] Anti-CD206 antibody: PE / Cy7-labeled anti-human CD206 (MMR) antibody (BioLegend);

[0149] Anti-CD34 antibody: PE-labeled anti-human CD34 antibody (manufactured by Bio Legend);

[0150] Anti-CD3 antibody: Alexa Fluor 700-labeled anti-CD3 antibody (manufactured by Bio Legend);

[0151] Anti-CXCR4 antibody: APC-labeled anti-human CD184 (CXCR4) antibody (manufactured by BD Biosciences);

[0152] Anti-CCR2 antibody: PerCP / Cy5.5-labeled human CD192 (CCR2) antibody (manufactured by BioLegend).

[0153] The results are shown in Figure 5 .like Figure 5 As shown, even in the cell population obtained by culturing mononuclear cells isolated from the peripheral blood of patients with severe lower limb ischemia, three regions of A, B, and C appeared as in Example 2. In addition, the total number of cells with CD206(+), CD34(+), or CD3(+) was 83.72%, and CCR2(+) was 1.63%, so the number of cells with CCR2(-) was 98.37%. In addition, the number of cells with CXCR4(+) among CD206(+) was 83.72%.

[0154] In summary, it can be seen that even if the cells are from patients with severe lower limb ischemia, as in the healthy people or diabetic patients in Example 1, RE-01 cell populations can be obtained by culturing using a culture medium containing three factors and serum, in particular, RE-01 cell populations in which the total number of cells that are CD206(+), CD34(+) or CD3(+) is more than 80%, the number of cells that are CCR2(-) is more than 95%, and the number of cells that are CXCR4(+) among CD206(+) is more than 80%.

[0155] Industrial Applicability

[0156] Through the present invention, a new cell population with high angiogenesis ability and wound healing ability can be provided. The cell population of the present invention can be used, for example, as a therapeutic drug for patients with refractory limb ischemic diseases. As a result, for example, amputation can be avoided, which can lead to life innovations such as improved patient QOL and reduced care burden, and has great social significance. In addition, the provision of a new cell population may not only bring about the construction of a new treatment strategy, but also bring new discoveries about vascular lesions as a whole. Moreover, it is shocking that, based on high effectiveness, the cell population has stably achieved a higher recovery rate and survival rate in a shorter culture period compared to the existing QQ-MNC method, and is more stable after manufacturing, so it also has a very large advantage in industry.

Claims

1. A cell population obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood or peripheral blood in a culture medium, wherein the culture medium contains serum and factors, wherein the factors are selected from the group consisting of: 10 to 1000 ng / mL of stem cell factor, 1 to 500 ng / mL of interleukin 6, 10 to 1000 ng / mL of FMS-like tyrosine kinase 3 ligand, 1 to 500 ng / mL of thrombopoietin and 5 to 500 ng / mL of vascular endothelial growth factor, The total of cells expressing CD206(+), CD34(+) or CD3(+) is 60% or more, and the total of cells expressing CCR2(-) is 95% or more. The serum is contained in the culture medium at a concentration of 0.5% by volume or more and 10% by volume or less.

2. The cell population according to claim 1, wherein The factors are: i) a combination of FMS-like tyrosine kinase 3 ligand, thrombopoietin and vascular endothelial growth factor; or ii) A combination of stem cell factor, FMS-like tyrosine kinase 3 ligand and vascular endothelial growth factor.

3. The cell population according to claim 1, wherein The culture medium does not contain both stem cell factor and interleukin 6 as factors.

4. The cell population according to claim 1, wherein The serum is bovine serum or human serum.

5. The cell population according to claim 1, wherein More than 50% of CD206(+) cells were CXCR4(+).

6. The cell population according to claim 1, wherein More than 80% of CD206(+) cells were CXCR4(+).

7. A method for obtaining a cell population according to any one of claims 1 to 6, comprising: Cultivating mononuclear cells derived from bone marrow, umbilical cord blood or peripheral blood in a medium containing serum and factors, wherein the factors are selected from the group consisting of 10 to 1000 ng / mL of stem cell factor, 1 to 500 ng / mL of interleukin 6, 10 to 1000 ng / mL of FMS-like tyrosine kinase 3 ligand, 1 to 500 ng / mL of thrombopoietin and 5 to 500 ng / mL of vascular endothelial cell growth factor, In the cell population, the total of cells belonging to CD206(+), CD34(+) or CD3(+) accounts for 60% or more, and the total of cells belonging to CCR2(-) accounts for 95% or more, The serum is contained in the culture medium at a concentration of 0.5% by volume or more and 10% by volume or less.

8. The method according to claim 7, wherein: Among the cell population, CXCR4(+) cells accounted for more than 50% of CD206(+) cells.

9. The method according to claim 7 or 8, wherein: The factors are: i) a combination of FMS-like tyrosine kinase 3 ligand, thrombopoietin and vascular endothelial growth factor; or ii) A combination of stem cell factor, FMS-like tyrosine kinase 3 ligand and vascular endothelial growth factor.

10. The method according to claim 7 or 8, wherein: The culture medium does not contain both stem cell factor and interleukin 6 as factors.

11. The method according to claim 7 or 8, wherein: The serum is fetal bovine serum. 12 . A composition for treating ischemic diseases, inflammatory diseases or refractory wounds, comprising the cell population according to claim 1 .

13. Use of the cell population according to any one of claims 1 to 6 for preparing a composition for treating ischemic diseases, inflammatory diseases or refractory wounds.

14. The use according to claim 13, wherein Ischemic disease refers to ischemia of the limbs.

15. The use according to claim 13 or 14, wherein Ischemic disease is ischemia of the limbs with ulcers.

16. The use according to claim 13 or 14, which promotes angiogenesis and / or wound healing.

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