Cell population and method for obtaining same

Abstract

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

Description

[0001] This application is a divisional application of a patent application with Chinese application number 202080089828.2, invention title "Cell population and method for obtaining the same", and filing date October 8, 2020. Technical Field

[0002] 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, cord blood or peripheral blood in a culture medium containing 4 or less factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, and serum. The present invention also relates to a therapeutic composition for ischemic diseases, inflammatory diseases or intractable wounds, which contains a cell population obtained by culturing mononuclear cells derived from bone marrow, cord blood or peripheral blood in a culture medium containing 4 or less factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, and serum. Background Art

[0003] In recent years, for ischemic diseases, bone marrow mononuclear cell transplantation therapy and cell transplantation therapy using endothelial progenitor cells (EPCs) based on peripheral blood stem cell collection have been started. Therefore, there is a particular need for a technique for culturing a large amount of EPCs. An in vitro amplification method for vascular endothelial progenitor cells from CD34 and / or CD133 positive cells can provide an efficient EPC culture technique (Patent Document 1). Also, by a method for dynamically analyzing vascular endothelial cell differentiation, the existence of endothelial cell-like large colony (differentiated EPC colony) forming cells and endothelial cell-like small colony (undifferentiated EPC colony) forming cells has been 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).

[0004] As shown in International Publication WO2014 / 0561154, a method for amplifying and enriching a cell population containing vascular endothelial progenitor cells or anti-inflammatory / 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 the "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).

[0005] Prior art documents

[0006] Patent documents

[0007] Patent Document 1: International Publication WO2006 / 090882

[0008] Patent Document 2: International Publication WO2006 / 090886

[0009] Patent Document 3: International Publication WO2006 / 093172

[0010] Patent Document 4: International Publication WO2014 / 051154 Summary of the invention

[0011] Technical problem to be solved by the present invention

[0012] As a result of intensive research by the present inventors, by culturing mononuclear cells derived from bone marrow, umbilical cord blood, or peripheral blood in a medium containing four or fewer factors selected from stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, and serum, a new cell population having angiogenesis ability and wound healing ability and different from the cell population obtained by the QQ-MNC method was successfully obtained, and thus the present invention was conceived.

[0013] The present invention aims to provide a cell population. The cell population of the present invention is a cell population obtained by culturing mononuclear cells derived from bone marrow, cord blood, or peripheral blood in a culture medium containing 4 or fewer factors selected from stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, and serum.

[0014] The present invention also aims to provide a method for obtaining a cell population. The method of the present invention includes: culturing mononuclear cells derived from bone marrow, cord blood, or peripheral blood in a culture medium containing 4 or fewer factors selected from stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, and serum.

[0015] The present invention also aims to provide a therapeutic composition for ischemic diseases, inflammatory diseases, or intractable wounds. The composition of the present invention includes: a cell population obtained by culturing mononuclear cells derived from bone marrow, cord blood, or peripheral blood in a culture medium containing 4 or fewer factors selected from stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, and serum.

[0016] Technical means for solving technical problems

[0017] The present invention includes but is not limited to the following modes.

[0018] [Mode 1] A cell population obtained by culturing mononuclear cells derived from bone marrow, cord blood, or peripheral blood in a culture medium containing 4 or fewer factors selected from stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, and serum.

[0019] [Mode 2] The cell population according to Mode 1, wherein the culture medium contains: 3 or 4 factors selected from stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor.

[0020] [Mode 3] The cell population according to Mode 1 or 2, wherein the serum is bovine serum or human serum.

[0021] [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.

[0022] [Mode 5] The cell population according to any one of Modes 1 to 4, wherein the total of cells that are CD206(+), CD34(+), or CD3(+) is 60% or more, and the cells that are CCR2(−) are 95% or more.

[0023] [Mode 6] The cell population according to Mode 5, wherein the cells that are CXCR4(+) among CD206(+) are 50% or more.

[0024] [Mode 7] The cell population according to Mode 5 or 6, wherein the cells that are CXCR4(+) among CD206(+) are 80% or more.

[0025] [Mode 8] The method for obtaining the cell population according to any one of Modes 1 to 7, which includes: culturing mononuclear cells derived from bone marrow, cord blood, or peripheral blood in a medium containing 4 or fewer factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, and serum.

[0026] [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 cells that are CCR2(−) are 95% or more.

[0027] [Mode 10] The method according to Mode 8 or 9, wherein in the cell population, the cells that are CXCR4(+) among CD206(+) are 50% or more.

[0028] [Mode 11] The method according to any one of Modes 8 to 10, wherein the medium contains: 3 or 4 factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor.

[0029] [Mode 12] The method according to any one of Modes 8 to 11, wherein the serum is fetal bovine serum.

[0030] [Mode 13] The method according to any one of Modes 8 to 12, wherein the serum is contained in the medium at a concentration of 0.5% by volume or more and 10% by volume or less.

[0031] [Mode 14] A therapeutic composition for treating an ischemic disease, an inflammatory disease, or a refractory wound, which contains the cell population according to any one of Modes 1 to 7.

[0032] [Mode 15] The therapeutic composition according to Mode 14, wherein the ischemic disease is limb ischemia.

[0033] [Mode 16] The therapeutic composition according to Mode 14 or 15, wherein the ischemic disease is limb ischemia accompanied by ulcers.

[0034] [Mode 17] The therapeutic composition according to any one of Modes 14 to 16, which promotes angiogenesis and / or wound healing. Description of the Drawings

[0035] Figure 1 Figure 1 Shows the results of flow cytometry (FACS)-based fractionation of the constituent cell components of RE-01 prepared from the peripheral blood of healthy individuals. As a comparative example, the FACS results of peripheral blood mononuclear cells before culture are also shown.

[0036] Figure 2 Figure 2 Shows the results of flow cytometry (FACS) analysis of the constituent cell components of RE-01 prepared from the peripheral blood of other healthy individuals different from Figure 1 .

[0037] Figure 3 Figure 3 Shows 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. As a comparative example, a cell population (MNC-QQ) obtained by culturing in a serum-free medium containing five factors was used.

[0038] Figure 4 Figure 4 Shows the results of investigating the angiogenesis ability of RE-01 using human umbilical vein endothelial cells (HUVEC). As comparative examples, the cases of using MNC-QQ instead of RE-01 and using only HUVEC were investigated. Figure 4 A shows the time change of the number of formed lumens from the start of culture, Figure 4 B shows the number of formed lumens 2.75 hours after the start of culture. Figure 4 C shows the number of fluorescently labeled test cells incorporated into the lumen structure 2.75 hours after the start of culture.

[0039] Figure 5 Figure 5 Shows the results of flow cytometry (FACS)-based fractionation of the constituent cell components of RE-01 prepared from the peripheral blood of patients with critical limb ischemia. Detailed Description of the Invention

[0040] ​​​​​1. Cell population

[0041] The present invention relates to a cell population.

[0042] The cell population of the present invention is obtained by culturing mononuclear cells derived from bone marrow, cord blood or peripheral blood in a culture medium containing 4 or less factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, and serum.

[0043] The cell population is obtained by culturing mononuclear cells derived from bone marrow, cord blood or peripheral blood. The "mononuclear cells" for obtaining the cell population refer to the general term for cells with a round nucleus contained in peripheral blood, bone marrow, cord blood, etc., including lymphocytes, monocytes, macrophages, vascular endothelial progenitor cells, hematopoietic stem cells, etc. For example, bone marrow, cord blood or peripheral blood is collected from an animal, and the component is extracted by, for example, using a collecting tube for separating mononuclear cells or subjecting it to density gradient centrifugation, thereby obtaining mononuclear cells. As the 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.

[0044] There is no particular limitation on the animal species from which the bone marrow, cord blood or peripheral blood is derived. The animal species include: general mammals including humans to whom cell transplantation therapy for diseases such as ischemic diseases, inflammatory diseases or refractory wounds can be applied. From the perspective of clinical application, humans are preferred.

[0045] There is no particular limitation on the subject from which the bone marrow, cord blood or peripheral blood is derived. As one mode, for example, it can be a healthy person, a diabetic patient or a patient with severe lower limb ischemia.

[0046] Stem cell factor (SCF) is a glycoprotein composed of 248 amino acids with a molecular weight of approximately 30,000. Due to alternative splicing, there are soluble and membrane-bound forms. The SCF used to obtain the cell population can be any type of SCF as long as it is useful for culturing mononuclear cells. The soluble form is preferred. There is no particular limitation on the source of SCF, etc. Without limitation, a recombinant expected to have a stable supply is preferred, and a human recombinant is particularly preferred. There are commercially available products. The concentration of SCF in the culture medium varies depending on the type of SCF used, and there is no particular limitation as long as it is useful for culturing mononuclear cells. If it is human recombinant SCF, without limitation, for example, it is 10 - 1000 ng / mL, preferably 50 - 500 ng / mL, more preferably approximately 100 ng / mL.

[0047] Interleukin-6 (IL-6) is a glycoprotein with a molecular weight of 210,000 that was isolated as a factor inducing the final differentiation of B cells into antibody-producing cells. IL-6 is generally known to be involved in immune responses, the hematopoietic system, the proliferation and differentiation of nervous system cells, acute-phase reactions, etc. The IL-6 used to obtain the cell population is not particularly limited and can be appropriately selected. If it is used in the culture of human mononuclear cells, human IL-6 is preferred, and a recombinant with stable supply is particularly preferred. 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, without limitation, for example, it is 1 - 500 ng / mL, preferably 5 - 100 ng / mL, and more preferably about 20 ng / mL.

[0048] FMS-like tyrosine kinase 3 ligand (FL) is widely known as a ligand of a receptor-type tyrosine kinase that plays an important role in early hematopoietic control. There are several products based on alternative splicing, and it has been reported that it stimulates the proliferation of hematopoietic stem cells. 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. There are commercially available products. The concentration of FL in the culture medium varies depending on the type of FL used and is not particularly limited as long as it is useful for the culture of mononuclear cells. In the case of human recombinant Flt-3 ligand, without limitation, for example, it is 10 - 1000 ng / mL, preferably 50 - 500 ng / mL, and more preferably about 100 ng / mL.

[0049] Thrombopoietin (TPO) is a type of hematopoietic cytokine that is known to specifically act in the process of megakaryocyte formation from hematopoietic stem cells and promote the production of megakaryocytes. The source, etc. of the TPO used to obtain the cell population are not particularly limited. A recombinant with stable supply is preferred, and a human recombinant is particularly preferred. There are commercially available products. The concentration of TPO in the culture medium varies depending on the type of TPO used and is not particularly limited as long as it is useful for the culture of mononuclear cells. In the case of human recombinant TPO, without limitation, for example, it is 1 - 500 ng / mL, preferably 5 - 100 ng / mL, and more preferably about 20 ng / mL.

[0050] Vascular endothelial growth factor (VEGF) is a growth factor that acts specifically on vascular endothelial progenitor cells (EPCs) and is known to be mainly produced in cells around blood vessels. Several VEGF proteins of different sizes are produced by alternative splicing. For the VEGF used to obtain the cell population, any type of VEGF can be used as long as it can form a colony of EPCs. Preferably, it is VEGF165. There are no particular limitations on the source of VEGF, etc. Preferably, it is a recombinant expected to have a stable supply, and particularly preferably, it is a human recombinant. There are commercially available products known. The concentration of VEGF in the culture medium varies depending on the type of VEGF used and is not particularly limited as long as it is useful for the culture of mononuclear cells. In the case of human recombinant VEGF165, without limitation, for example, it is about 5 - 500 ng / mL, preferably about 20 - 100 ng / mL, and more preferably about 50 ng / mL.

[0051] Regarding various factors added to the culture medium used in the culture of mononuclear cells, without limitation, they can be unified as factors derived from the same animal species as the animal from which the mononuclear cells are sourced. By thus unifying the sources of mononuclear cells and various factors, cell cultures suitable for allotransplantation such as allogeneic transplantation can be obtained. In addition, by using mononuclear cells derived from the individual intended for cell transplantation, cell cultures suitable for autologous transplantation can also be obtained.

[0052] The above-mentioned respective components can be dissolved in the culture medium at a given concentration, or concentrated solutions (stock solutions) of the respective components can be prepared in advance, and the culture medium for culturing mononuclear cells can be prepared by diluting to a given concentration with the culture medium. For example, after dissolving the necessary components in a commercially available culture medium so that it has a given concentration, it can be sterilized by filtration sterilization, etc., or a stock solution sterilized by filtration sterilization, etc. can be added to the commercially available culture medium in a sterile manner and diluted to prepare the culture medium. Filtration sterilization can be carried out according to the methods commonly practiced in the art, for example, using microporous filters (Millipore filters) of 0.22 μm, 0.45 μm, etc.

[0053] The "culture medium" used in the present invention can utilize the media commonly used in the art. For example, media well-known as media for the proliferation of hematopoietic stem cells can be used. Regarding the basal medium used as the culture medium, for example, Stemline II, DMEM, MEM, IMDM, RPMI, SCGM, EBM, etc. can be cited.

[0054] To obtain the cell population, the medium for culturing mononuclear cells contains serum in addition to 4 or fewer factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor.

[0055] 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 medium is not particularly limited. In one embodiment, the serum is contained in the medium at 0.1% by volume or more, 0.3% by volume or more, 0.5% by volume or more. The upper limit of the concentration of serum in the medium is not particularly restricted. In one embodiment, it is within 30% by volume, within 20% by volume, within 10% by volume, within 5% by volume. Without limitation, the serum is contained in the medium at a concentration of 0.1% by volume or more and 20% by volume, and at a concentration of 0.5% by volume or more and 10% by volume.

[0056] The culturing of mononuclear cells is carried out by adding a cell suspension containing mononuclear cells to a medium containing the factors and serum. As the cell suspension, the body fluid itself containing mononuclear cells (for example, bone marrow fluid, cord blood, peripheral blood) can also be used. The culturing conditions of mononuclear cells are not particularly limited and can be carried out under conditions usually practiced in this field. Without limitation, for example, the culturing is carried out in a 5% CO2 atmosphere at about 37°C. Regarding the culturing period, without limitation, for example, it is 3 days or more and within 5 days, within 6 days, within 7 days, within 10 days. For example, 3 days - 10 days, 3 days - 6 days. Regarding the concentration of mononuclear cells in the medium, as long as the culturing of mononuclear cells can be carried out, it is not particularly limited. For example, it is about 0.1 - 10×10 6 cells / ml, more preferably about 0.5 - 5×10 6 cells / ml.

[0057] In this specification, "cell population" refers to the general term for cells obtained by culturing mononuclear cells in a medium containing the factors and serum.

[0058] The culture medium used in the culturing of mononuclear cells contains 4 or fewer factors selected from the factors of stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor.

[0059] In one embodiment, the culture medium contains: two or more factors selected from stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, or three or more factors. In one embodiment, the culture medium contains: two or more and four or less factors selected from the five factors. In one embodiment, the culture medium contains: three or four factors selected from stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor.

[0060] Without limitation, the culture medium used in the culture of mononuclear cells, so the culture medium used in the culture method of the present invention, for example, contains: 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 contains: a combination of about 80-120 ng / ml of FMS-like tyrosine kinase 3 ligand, about 15-25 ng / ml of thrombopoietin, and about 40-60 ng / ml of vascular endothelial growth factor.

[0061] In one embodiment, a cell population obtained by culturing mononuclear cells derived from bone marrow, 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 growth factor, and serum contains more cells that are CD206(+), CD34(+), or CD3(+) compared to the cell population of mononuclear cells before culturing. Without limitation, by culturing, the proportion of cells that are CD206(+), CD34(+), or CD3(+) is amplified to 1.5 times or more, 2 times or more, 3 times or more. Without limitation, in the cell population, the total of cells that are CD206(+), CD34(+), or CD3(+) is 50% or more, 60% or more, 70% or more, 80% or more, 90% or more of the entire cell population. And the cell population contains more cells that are CCR2(−) compared to the cell population of mononuclear cells before culturing. Without limitation, by culturing, the proportion of cells that are CCR2(−) is amplified to 1.5 times or more, 2 times or more, 3 times or more. Without limitation, in the cell population, the cells that are CCR2(−) are 60% or more, 80% or more, 90% or more, 95% or more of the entire cell population. Without limitation, in one embodiment, the cells that are CD206(+), CD34(+), and CD3(+) are 60% or more, and the cells that are CCR2(−) are 95% or more.

[0062] By culturing mononuclear cells derived from bone marrow, cord blood or peripheral blood in a medium containing 4 or fewer factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, and serum, a cell population containing more CD206(+), CD34(+), and CD3(+) can be stably obtained. Also, compared with the cell population obtained by the QQ-MNC method, the proportion of CXCR4-positive cells in CD206-positive cells containing anti-inflammatory macrophages M2 is significantly increased. Without limitation, the inventors observed the following effects through the above culture.

[0063] The increase in CD206 positivity containing anti-inflammatory macrophages M2;

[0064] The increase in CD34-positive cells and CD133-positive cells containing vascular endothelial progenitor cells (EPC);

[0065] The increase in CD3-positive cells such as T cells, particularly helper T cells and angiogenic T cells.

[0066] On the other hand, a decrease in CCR2-positive cells, B cells (CD19-positive cells), and NK cells (CD56-positive cells) as inflammatory monocytes / macrophages was observed.

[0067] Without limitation, in one approach, the cell population obtained by culturing mononuclear cells derived from bone marrow, cord blood or peripheral blood in a medium containing 4 or fewer factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, and serum, compared with the cell population obtained by the QQ-MNC method, shows a higher recovery rate. Also, the survival rate is higher. Moreover, the stability after recovery is also more excellent. These effects can bring higher therapeutic effects.

[0068] In one approach, in a cell population obtained by culturing mononuclear cells derived from bone marrow, cord blood, or peripheral blood in a culture medium containing 4 or fewer factors selected from stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, and serum, compared to the cell population of the mononuclear cells before culturing, it contains more cells that are CD206(+) and CXCR4(+). Without limitation, through culturing, the proportion of cells that are CD206(+) and CXCR4(+) is amplified to 1.5 times or more, 2 times or more, 3 times or more. Without limitation, the cells that are CD206(+) and CXCR4(+) are 50% or more, 60% or more, 70% or more, 80% or more. In one approach, the cells that are CD206(+) and CXCR4(+) are 50% or more. In one approach, the cells that are CD206(+) and CXCR4(+) are 80% or more.

[0069] The cell population of the present invention, compared to the cell population obtained by the QQ-MNC method, particularly contains more cells that are CD206(+) and CXCR4(+).

[0070] 2. Method for obtaining a cell population

[0071] The present invention also relates to a method for obtaining a cell population.

[0072] The method of the present invention includes: culturing mononuclear cells derived from bone marrow, cord blood, or peripheral blood in a culture medium containing 4 or fewer factors selected from stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, and serum.

[0073] Each of "cell population", "mononuclear cell", "factors such as stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor", "serum", "culture medium", etc. is as described in "1. Cell population" above.

[0074] The proportions of cells that are CD206(+), CD34(+), and CD3(+) in the cell population, the proportion of cells that are CCR2(−), and the proportion of cells that are CD206(+) and CXCR4(+) are also as described in "1. Cell population" above.

[0075] In one approach, in the cell population obtained by the said method, the cells that are CD206(+), CD34(+), and CD3(+) are 80% or more, and the cells that are CCR2(−) are 95% or more.

[0076] In one way, among the cell population obtained by the method, the cells that are CD206(+) and CXCR4(+) account for more than 50%.

[0077] In one way, in the method, the culture medium contains: 3 factors or 4 factors selected from stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor.

[0078] In one way, in the method, the serum is bovine serum or human serum. In one way, the serum is fetal bovine serum. In one way, the serum is contained in the culture medium at a concentration of 0.5% by volume or more and 10% by volume.

[0079] 3. Therapeutic composition

[0080] The present invention also relates to a therapeutic composition for ischemic diseases, inflammatory diseases, or intractable wounds. The therapeutic composition of the present invention contains the cell population described in "1. Cell population". The mononuclear cells as the source of the cell population can be derived from the subject (autologous) using the therapeutic composition, or from a source other than the subject using the therapeutic composition (allogeneic).

[0081] "Ischemic disease" is a disease caused by a decrease in blood volume leading to a decline in blood flow in tissues, resulting in tissue damage such as cell degeneration, atrophy, and fibrosis. Ischemia is roughly classified 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 diseases" include limb ischemia, ischemic ulcers, ischemic heart disease, cerebral infarction, etc. "Cerebral infarction" (or encephalomalacia) is caused by the obstruction or stenosis of the arteries supplying nutrition to the brain, resulting in cerebral ischemia, and the brain tissue necroses or becomes in a state close to necrosis due to oxygen or nutrient deficiency, and is classified into cerebral thrombosis and cerebral embolism. "Limb ischemia" is a disease in which the arteries supplying blood to the hands and feet are narrowed or blocked. If this obstructive arteriosclerosis becomes severe, it becomes severe limb ischemia, causing symptoms such as pain and intractable ulcers, and in the worst case, amputation may be required. Without limitation, in one way, the limbs are the lower limbs.

[0082] Without limitation, in one way, the ischemic disease is limb ischemia. The ischemic disease is limb ischemia accompanied by ulcers.

[0083] "Inflammatory disease" is a general term for diseases that cause symptoms due to tissue damage and other abnormalities caused by certain reasons. Inflammatory diseases include Crohn's disease, liver cirrhosis, hepatitis, ulcerative colitis, inflammatory bowel disease, etc.

[0084] 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. Examples of "diseases that can be treated by angiogenesis and / or wound healing" include ischemic diseases (e.g., ischemic heart diseases such as myocardial infarction and angina pectoris, lower limb ischemia such as arteriosclerosis obliterans of the lower extremities, Buerger's disease, vascular injury). In addition, it can be used to heal wounds such as skin ulcers or to fabricate artificial blood vessels. The applicable effects of the therapeutic composition can be confirmed by methods known per se. For example, in the case of a disease that can be treated by angiogenesis being a lower limb ischemic disease, the therapeutic effect after transplantation can be evaluated by investigating the blood flow in the lower limbs and the necrosis improvement rate. The measurement of the increase in blood flow can be carried out by measuring the value of laser Doppler imaging analysis. In addition, the necrosis improvement rate can be measured by visual observation in the form of a limb salvage score.

[0085] The usage mode of the therapeutic composition is not particularly limited. It can be the cell population itself as the active ingredient, or a product obtained by suspending the cell population 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 solutions, phosphate buffer solutions, physiological saline, or DMEM as a serum-free medium can be used. In addition, the liquid medium can also contain compounds that contribute to cell survival such as albumin. 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 with a cryopreservation solution for cells, etc.

[0086] The applicable subjects of the therapeutic composition are not particularly limited as long as they are subjects in need of treatment for ischemic diseases, inflammatory diseases, or intractable wounds. Without limitation, it includes humans, monkeys, chimpanzees, dogs, cats, cows, horses, mice, guinea pigs, etc.

[0087] 4. Kit

[0088] The present invention also relates to a kit for culturing mononuclear cells derived from bone marrow, cord blood, or peripheral blood and obtaining a cell population.

[0089] The kit includes: a culture medium containing 4 or fewer 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 pre-added to the culture medium or stored in separate containers and added to the culture medium during use.

[0090] 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 "1. Cell population" above.

[0091] 5. Treatment methods, etc.

[0092] The present invention also relates to a treatment method for ischemic diseases, inflammatory diseases, or intractable wounds, which comprises: applying the cell population described in "1. Cell population" to a subject in need.

[0093] The present invention also relates to the use of the cell population described in "1. Cell population" in the treatment method for ischemic diseases, inflammatory diseases, or intractable wounds, which comprises applying to a subject in need, or, the use in a therapeutic composition for ischemic diseases, inflammatory diseases, or intractable wounds.

[0094] "Ischemic diseases", "inflammatory diseases", or "intractable wounds", "application mode of cell population", "application subject", etc. are as described in "3. Therapeutic composition".

[0095] Examples

[0096] Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples. Those skilled in the art can easily modify and change the present invention according to the description of this specification, and these modifications and changes are also included in the technical scope of the present invention.

[0097] Example 1 Cultivation of RE-01 Cell Population

[0098] In this example, mononuclear cells were isolated from peripheral blood and cultured.

[0099] (1) Isolation of Mononuclear Cells

[0100] From healthy individuals or diabetic volunteers, 100 mL of peripheral blood was collected using a BD vacutainer (registered trademark) CPT (trademark, manufactured by BD) mononuclear cell separation blood collection tube. After blood collection, the blood collection tube was centrifuged as it was, transported, and refrigerated until the start of culture after arrival. The portion above the gel barrier in the CPT (trademark) blood collection tube containing mononuclear cells and plasma was recovered into a centrifuge tube, and the portion above the gel barrier in the blood collection tube such as the gel barrier was washed with a small amount of EDTA-PBS and recovered into the same centrifuge tube. For the centrifuge tube containing the recovered cells, after volume expansion with EDTA-PBS, centrifugation was performed (300×g, room temperature, 15 minutes), and the cells as a precipitate were recovered. Regarding the recovered cells, in order to remove the mixed red blood cells, they were incubated in ACK lysis buffer (15 mL / tube) (manufactured by Gibco, Thermo-Fisher) at room temperature for 5 minutes. The composition of the ACK lysis buffer was NH4Cl 8290 mg / l; KHCO3 1000 mg / l; EDTA.Na2·2H2O 37 mg / l. Then, by volume expansion with EDTA-PBS and centrifugation (200×g, room temperature, 10 minutes) or (100×g, room temperature, 15 minutes), the operation of recovering cells was repeated 2 times.

[0101] (2) Culture in serum medium

[0102] The mononuclear cells recovered in (1) were suspended in 1 mL of proliferation medium (added 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 part of it was used for cell counting using trypan blue. In the obtained cell proliferation medium, the cell concentration was adjusted to 1×10 6 / mL, and 2 mL was inoculated into each well of a 6-well culture plate. It was cultured under normal culture conditions (37 °C, 5% CO2) for 5 days. After 5 days of culture, it was recovered into a centrifuge tube, and centrifugal washing was performed 3 times at 250×g (~1000 rpm) for 7 to 10 minutes. After centrifugation, except for the samples used for cell counting, viability determination, and QC (confirmation) test, the remaining cells were suspended in PlasmaLyte A, 2.5% human albumin serum, and the concentration was 8×10 5The adjustment was made in the manner of cells / mL. Hereinafter, the obtained cell population is also referred to as "RE-01".

[0103] Example 2 Evaluation of the characteristics of the RE-01 cell population

[0104] (1) Flow cytometry analysis

[0105] In this example, in Example 1, for the cell population (referred to as "RE-01") obtained by culturing starting from the peripheral blood of a healthy person using a medium containing three factors and serum, flow cytometry analysis was performed to further clarify the characteristics of the cell population.

[0106] To the cells (1.5×10 6 cells / 300 μL - FACS buffer) suspended in FACS buffer (composition: 2 mM EDTA-PBS supplemented with 2% FBS), 10 μL of FC blocking reagent (manufactured by Miltenyi) was added, and the mixture was incubated at 4°C for 30 minutes. The incubated cell population was equally aliquoted into each tube for staining reaction (100 μL / tube × 3 tubes). 2 μL of the primary antibody was added to each sample (aliquot), and the mixture was cultured at 4°C for 20 minutes. Then, it was washed twice with 1 mL of FACS buffer, and the stained cells were suspended in FACS buffer (5×10 5 cells / 200 - 300 μL - FACS buffer). Flow cytometry measurement was performed using a BD FACSAria (trademark) III cell sorter (manufactured by BD).

[0107] 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 following commercially available products were used as antibodies against each cell surface marker.

[0108] Anti-CD206 antibody: PE / Cy7-labeled anti-human CD206 (MMR) antibody (manufactured by BioLegend);

[0109] Anti-CD34 antibody: PE-labeled anti-human CD34 antibody (manufactured by BioLegend);

[0110] Anti-CD3 antibody: Alexa Fluor700-labeled anti-CD3 antibody (manufactured by BioLegend);

[0111] Anti-CXCR4 antibody: APC-labeled anti-human CD184 (CXCR4) antibody (manufactured by BD);

[0112] Anti-CCR2 antibody: PerCP / Cy5.5-conjugated human CD192 (CCR2) antibody (manufactured by BioLegend).

[0113] The results are shown in Figure 1 . As Figure 1 shown, in the FACS-based Scatter analysis of RE-01, the viable cells were roughly divided into three groups (referred to as "Region A", "Region B", and "Region C" in this specification) according to the size of the constituent cell components (horizontal axis: FSC) and the density of cell components such as granules contained in the cells (vertical axis: SSC). In the peripheral blood mononuclear cells before culture, only Regions A and B appeared, and Region C was not observed. Region A mainly contains lymphocytes, and Region B contains monocytes. In RE-01 obtained by culturing using a proliferation medium ((2) serum medium in Example 1), in addition to the migration of vascular endothelial progenitor cells (CD34-positive cells) from Region A to Region B, a large number of Region C cells containing M2 macrophages (CD206-positive cells) also appeared.

[0114] In addition, the results of flow cytometry analysis of RE-01 obtained by culturing mononuclear cells collected from other healthy individuals using the medium containing three factors and serum described in Example 1 are shown in Figure 2 . Similarly, in the case of healthy individuals of different individuals, three regions A, B, and C appeared. In addition, the total of cells positive for CD206(+), CD34(+), or CD3(+) was 86.57%, and CCR2(+) was 0.33%, so the cells negative for CCR2 were 99.67%. And among CD206(+), the cells positive for CXCR4 were 92.97%.

[0115] Example 3 Comparison between RE-01 cell population and MNC-QQ cell population

[0116] (1) Culture of RE-01 and MNC-QQ

[0117] Mononuclear cells collected from healthy individuals and diabetic patients were cultured using a medium containing three factors and serum in the same manner as in Example 1 to obtain RE-01.

[0118] As a comparative example, after separating mononuclear cells in the same manner as RE-01 cells, stemline (registered trademark) II Hematopoietic Stem Cell Expansion Medium (manufactured by Sigma-Aldrich, CatNo. S0192) was used. Without adding serum, 100 units / mL penicillin, 100 μg / mL streptomycin, and 50 ng / mL VEGF 165 , 20 ng / mL TPO, 100 ng / mL Flt-3 ligand, 100 ng / mL SCF, and 20 ng / mL IL-6 were each added aseptically to prepare. The resulting cells were prepared at a concentration of 1×10 6 / mL, and 2 mL was inoculated into each well of a 6-well culture plate and cultured under normal culture conditions (37 °C, 5% CO2) for 7 days. After 7 days of culture, the cells were collected into a centrifuge tube and centrifuged and washed 3 times at 250×g (~1000 rpm) for 7 to 10 minutes. After centrifugation, except for the samples used for cell count determination, viability determination, and QC (confirmation) test, the remaining cells were suspended in PlasmaLyte A and 2.5% human albumin serum and prepared at a concentration of 8×10 5 cells / mL. Hereinafter, the resulting cell population will also be referred to as "MNC-QQ".

[0119] (2) Flow cytometry analysis

[0120] In Example 1, flow cytometry was performed on the cell population obtained by culturing starting from the peripheral blood of a diabetic patient using a medium containing 3 factors and serum, and the cell population (MNC-QQ) obtained by culturing using a serum-free medium containing the 5 factors described in (1) above, and the two were compared. Specifically, the expression of each cell surface marker was investigated using an antibody against each cell surface marker in the obtained cell population.

[0121] The results are as Figure 3 shown. Compared with MNC-QQ, RE-01 had a higher recovery rate and viability after culture. Also, the proportion of cells in region C of the newly formed cell population after culture was higher, especially the proportion of CD206+ cells, which is one of the cell types responsible for the effectiveness of the cell population. And surprisingly, the proportion of CXCR4+ in CD206+ was significantly higher, indicating that the cell populations of RE-01 and MNC-QQ differed in their cell composition.

[0122] (3) acLDL uptake ability

[0123] The cell population (RE-01) prepared from the peripheral blood mononuclear cells of diabetic patients in Example 1 through culture using a culture medium containing three factors and serum was used to investigate its acLDL (acetylated low-density lipoprotein) uptake ability, compared with the cell population (MNC-QQ) obtained through culture using a serum-free medium containing five factors as a comparative example.

[0124] RE-01 or MNC-QQ (1×10 5 cells) recovered into a 1.5 mL tube were suspended in 500 μL of a basal medium (Stemline II), 5 μL of acLDL labeled with Alexa Fluor 488 was added, and the mixture was incubated at 37 °C for 60 minutes. 1 mL of FACS buffer was added to the tube after incubation, and the tube was inverted and mixed. Then, a centrifugation operation was performed for 5 minutes (250×g, 4 °C), and the cells were recovered in the form of a precipitate. The recovered cells (RE-01) were suspended in 300 μL of FACS buffer and stored on ice until measurement. Just before measurement, DAPI (2 μL) was added, and the proportion of cells that had taken up acLDL and DAPI was determined through measurement using a flow cytometer.

[0125] The results are shown in Table 1 below.

[0126]

[0127] As shown in Table 1, it can be seen that overall, especially in region C, the uptake of acLDL is relatively high. This also corroborates that for the two cell populations (RE-01 and MNC-QQ), the difference in composition shown in Example 2 leads to differences in function.

[0128] (4) Angiogenic ability

[0129] In this example, a cell population (RE-01) obtained by culturing mononuclear cells collected from the blood of healthy individuals 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 five factors was used. Human umbilical vein endothelial cells (HUVEC) (obtained from Lonza, Basel, Switzerland) with the ability to form lumens were used at passages 8 - 10, and the cells of each group were co-cultured with HUVEC. Basement membrane matrix (Corning (registered trademark) Matrigel (registered trademark) (manufactured by Corning, NY, USA)) was dispensed at 50 μl / well into a 96-well plate and plated at 37°C for 30 minutes. On the other hand, to label each cell population with DiI-Ac-LDL, it was suspended in 500 μl of IMDM + 5 μl of DiI-Ac-LDL and incubated at 37°C for 1 hour.

[0130] After labeling, each cell population was resuspended in PBS at a concentration of 1×10 3 / 20 μl, and HUVEC was resuspended in PBS at a concentration of 5×10 3 / 20 μl. The cell suspensions with adjusted cell numbers were mixed so that the test cell population and HUVEC were in a 1:1 ratio, and 50 μl was added to Matrigel respectively. Culturing and photographing were carried out in an incubator of a time-lapse fluorescence microscope (manufactured by Olympus, Tokyo, Japan) at 37°C and 5% CO2 for more than 10 hours. After the culturing was completed, at a magnification of 100 times, the number of formed lumens and the number of fluorescently labeled test cells incorporated into the lumen structure were counted by visual observation.

[0131] The results are as Figure 4 shown. Figure 4 A shows the change in the number of formed lumens over time from the start of culturing, Figure 4 B shows the number of formed lumens 2.75 hours after the start of culturing. In addition Figure 4C represents the number of fluorescently labeled test cells incorporated into the luminal structure 2.75 hours after the start of culture. The ability to form lumens when HUVECs are co-cultured with RE-01 for a certain period of time is increased compared to culturing only HUVECs or co-culturing HUVECs with MNC-QQ. Also, in the case of RE-01, more cells are incorporated into the formed luminal structure. That is, it can be seen that in vitro, RE-01 has a higher in vitro angiogenesis ability compared to MNC-QQ, which was cultured in serum-free medium for a longer period (7 hours). This also corroborates that for the two cell populations (RE-01 and MNC-QQ), the difference in composition shown in Example 3 results in a difference in function.

[0132] (5) Effect based on a mouse model of hindlimb ischemia

[0133] A cell population obtained by culturing mononuclear cells collected from the blood of healthy individuals using the same method as in Example 1 was used, and a mouse model of hindlimb ischemia was used to investigate its therapeutic effect on hindlimb ischemia. As a comparative example, a cell population (MNC-QQ) obtained by culturing using a serum-free medium containing 5 factors was used.

[0134] (6) Stability test

[0135] A cell population obtained by culturing mononuclear cells collected from the blood of healthy individuals using the same method as in Example 1 was used to investigate the stability from the time of recovery to 72 hours in terms of cell number and viability. As a comparative example, a cell population (MNC-QQ) obtained by culturing using a serum-free medium containing 5 factors was used.

[0136] Example 4 Culturing and characterization of the RE-01 cell population

[0137] In this example, mononuclear cells were isolated from the peripheral blood of patients with severe hindlimb ischemia and cultured. And the resulting cell population was characterized.

[0138] (1) Isolation of mononuclear cells

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

[0140] Specifically, 100 mL of peripheral blood was collected from a patient with severe lower limb ischemia using a BD vacutainer (registered trademark) CPT (trademark, manufactured by BD) blood collection tube for mononuclear cell separation. After blood collection, the blood collection tube was centrifuged as it was, transported, and refrigerated until the start of culture after arrival. The portion above the gel barrier in the CPT (trademark) blood collection tube containing mononuclear cells and plasma was recovered into a centrifuge tube, and the portion above the gel barrier in the blood collection tube, such as the gel barrier, was washed with a small amount of EDTA-PBS and recovered into the same centrifuge tube. For the centrifuge tube containing the recovered cells, after volume expansion with EDTA-PBS, centrifugation was performed (300×g, room temperature, 15 minutes), and the cells as a precipitate were recovered. Regarding the recovered cells, in order to remove the mixed red blood cells, they were incubated in ACK lysis buffer (15 mL / tube) (manufactured by Gibco, Thermo-Fisher) at room temperature for 5 minutes. The composition of the ACK lysis buffer was NH4Cl 8290 mg / l; KHCO3 1000 mg / l; EDTA.Na2·2H2O 37 mg / l. Then, by volume expansion with EDTA-PBS and centrifugation (200×g, room temperature, 10 minutes) or (100×g, room temperature, 15 minutes), the operation of recovering the cells was repeated 2 times.

[0141] (2) Culture in serum medium

[0142] The isolated mononuclear cells were cultured in 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 basal medium instead of stemline (registered trademark), and human albumin serum was added in addition to FBS as the serum.

[0143] Specifically, the mononuclear cells recovered in (1) were suspended in 1 mL of proliferation medium (Iscove’s Modified Dulbecco’s Medium (IMDM) 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, 0.5% human albumin serum), and a portion of it was used for cell counting using trypan blue. In the obtained cell proliferation medium, the cell concentration was 1×10 6It was prepared in the manner of / mL, and 2 mL was inoculated into each well of a 6-well culture plate. It was cultured for 5 days under normal culture conditions (37 °C, 5% CO2). After 5 days of culture, it was recovered into a centrifuge tube, and centrifugal washing was carried out 3 times for 7 to 10 minutes at 250×g (~1000 rpm). After centrifugation, except for the samples used for cell count determination, viability determination, and QC (confirmation) test, the remaining cells were suspended in PlasmaLyte A, 2.5% human albumin serum, and adjusted to a concentration of 8×10 5 cells / mL.

[0144] (3) Flow cytometry analysis

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

[0146] To the cells suspended in FACS buffer (composition: 2 mM EDTA-PBS supplemented with 2% FBS) (1.5×10 6 cells / 300 μL-FACS buffer), 10 μL of FC blocking reagent (manufactured by Miltenyi) was added, and it was incubated at 4 °C for 30 minutes. The incubated cell population was equally aliquoted into each tube for staining reaction (100 μL / tube × 3 tubes). 2 μL of primary antibody was added to each specimen (aliquot) respectively, and it was cultured at 4 °C for 20 minutes. Then it was washed twice with 1 mL of FACS buffer, and the stained cells were suspended in FACS buffer (5×10 5 cells / 200 - 300 μL-FACS buffer). Flow cytometry measurement was performed using a BD FACSAria (trademark) III cell sorter (manufactured by BD).

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

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

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

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

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

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

[0153] The results are shown in Figure 5 . As Figure 5 shown, even when culturing a cell population obtained by culturing mononuclear cells isolated from the peripheral blood of a patient with severe lower limb ischemia, three regions A, B, and C also appeared in the same manner as in Example 2. In addition, the total of cells that are CD206(+), CD34(+), or CD3(+) is 83.72%, and CCR2(+) is 1.63%, so the cells that are CCR2(−) are 98.37%. Also, 83.72% of the CD206(+) cells are CXCR4(+).

[0154] In summary, it can be seen that even for a patient with severe lower limb ischemia, similar to the healthy subjects or diabetic patients in Example 1, an RE-01 cell population can be obtained by culturing using a medium containing three factors and serum. In particular, an RE-01 cell population in which the total of cells that are CD206(+), CD34(+), or CD3(+) is 80% or more, the cells that are CCR2(−) are 95% or more, and 80% or more of the CD206(+) cells are CXCR4(+).

[0155] Industrial Applicability

[0156] By the present invention, a novel cell population having 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 agent for patients with refractory limb ischemia diseases. Thereby, for example, amputation and the like can be avoided, bringing about life innovations such as improvement of the patient's QOL and reduction of the nursing burden, and having great social significance. In addition, the provision of a novel cell population brings about the construction of a new treatment strategy and may also bring about new discoveries regarding vascular lesions as a whole. And, surprisingly, based on high effectiveness, compared with the existing QQ-MNC method, this cell population also stably achieved a high recovery rate and survival rate within a shorter culture period and was more stable after manufacture, so it also has very great advantages industrially.

Claims

1. A cell population obtained by culturing mononuclear cells derived from bone marrow, cord blood or peripheral blood in a culture medium, said culture medium containing 4 or fewer factors selected from stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, and serum.

2. The cell population according to claim 1, wherein the culture medium contains: 3 or 4 factors selected from stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor.

3. The cell population according to claim 1 or 2, wherein the serum is bovine serum or human serum.

4. The cell population according to any one of claims 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.

5. The cell population according to any one of claims 1 to 4, wherein the total of cells that are CD206(+), CD34(+) or CD3(+) is 60% or more, and the cells that are CCR2(−) are 95% or more.

6. The cell population according to claim 5, wherein the cells that are CXCR4(+) among CD206(+) are 50% or more.

7. The cell population according to claim 5 or 6, wherein the cells that are CXCR4(+) among CD206(+) are 80% or more.

8. A method for obtaining a cell population according to any one of claims 1 to 7, comprising: culturing mononuclear cells derived from bone marrow, cord blood or peripheral blood in a culture medium containing 4 or fewer factors selected from stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor, and serum.

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

10. The method according to claim 8 or 9, wherein in the cell population, the cells that are CXCR4(+) among CD206(+) are 50% or more.

11. The method according to any one of claims 8 to 10, wherein the culture medium contains: 3 or 4 factors selected from stem cell factor, interleukin-6, FMS-like tyrosine kinase 3 ligand, thrombopoietin, and vascular endothelial growth factor.

12. The method according to any one of claims 8 to 11, wherein the serum is fetal bovine serum.

13. The method according to any one of claims 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.

14. A therapeutic composition for treating ischemic diseases, inflammatory diseases or intractable wounds, comprising the cell population according to any one of claims 1 to 7.

15. The therapeutic composition according to claim 14, wherein the ischemic disease is limb ischemia.

16. The therapeutic composition according to claim 14 or 15, wherein the ischemic disease is limb ischemia accompanied by ulcers.

17. The therapeutic composition according to any one of claims 14 to 16, which promotes angiogenesis and / or wound healing.

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