Mesenchymal stem cell

Abstract

Provided are mesenchymal stem cells that express at least one gene selected from the group consisting of TNFAIP6, CYP1B1, EREG, MME, and CXCL5.

Description

Mesenchymal stem cells

[0001] The present invention relates to a mesenchymal stem cell population having a strong anti-inflammatory effect and a method for selecting said mesenchymal stem cell population. In particular, the present invention relates to a mesenchymal stem cell population having a strong ability to induce M2 macrophages and a method for evaluating and obtaining said mesenchymal stem cell population.

[0002] Mesenchymal stem cells (MSCs) are somatic stem cells that have the ability to differentiate into bone, cartilage, and fat, as well as the ability to self-renew. Because they also have anti-inflammatory properties, they are expected to be used in regenerative medicine and the treatment of inflammatory diseases.

[0003] Macrophages include M1 macrophages, which induce inflammation, and M2 macrophages, which suppress inflammation and repair tissue (Non-Patent Document 1). It has become clear that the balance between M1 and M2 macrophages (M1 / M2 macrophage balance) is closely involved in the pathogenesis of various diseases. For example, it has been reported that disruption of the M1 / M2 macrophage balance is associated with the pathogenesis of diseases such as rheumatoid arthritis, inflammatory bowel disease, wound healing, myocardial infarction, atherosclerosis, type 2 diabetes, sepsis-associated lung injury, acute liver injury, and Alzheimer's dementia (Non-Patent Documents 2-11). Promoting polarization toward M2 macrophages, which are anti-inflammatory macrophages, is thought to be effective in treating these diseases. Therefore, selecting cells with high M2-inducing activity is thought to be effective for cell therapy targeting these diseases. However, it is unknown what cells have high M2-inducing activity, and the criteria for selecting MSCs with high M2-inducing activity were also unknown.

[0004] Gordon, Nature Review Immunol. (2003) pp23-35Zheng et al., Stem Cells Int. 2015:2015:989473Cutolo et al., Front Immunol. 2022; 13: 867260Zhang et al., Cell Commun Signal. 2023; 21: 367.Hassanshahi et al., Cells. 2022 Sep 21;11(19):2953.Kim et al., Int J Mol Sci. 2021 Mar 8;22(5):2715.Clinical Cardiology 2014 Vol.4 No.4.pp46-47 Diabetes 64(2):pp106-108, 2021Wang et al., Front Immunol. 2023 Aug 24:14:1209438Wang et al., Front Immunol. 2021 Dec 14:12:803037Zuroff et al., Cell Mol Life Sci. 2017 Jun; 74(12): pp2167-2201.

[0005] In light of the above, an object of the present invention is to provide mesenchymal stem cells with high M2-inducing activity and cell populations containing the same, as well as methods for evaluating and obtaining mesenchymal stem cells with high M2-inducing activity and cell populations containing the same.

[0006] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have succeeded in obtaining and characterizing mesenchymal stem cells with high M2-inducing activity and a cell population containing the same, thereby completing the present invention. Specifically, the present invention is as follows: [1] Mesenchymal stem cells expressing at least one gene selected from the group consisting of the TNFAIP6 gene, CYP1B1 gene, EREG gene, MME gene, and CXCL5 gene. [1-1] Mesenchymal stem cells that promote induction into M2 macrophages and express at least one gene selected from the group consisting of the TNFAIP6 gene, CYP1B1 gene, EREG gene, MME gene, and CXCL5 gene. [1-2] Mesenchymal stem cells for treating M2-related diseases, expressing at least one gene selected from the group consisting of the TNFAIP6 gene, CYP1B1 gene, EREG gene, MME gene, and CXCL5 gene.

[0007] [2-1-1] Mesenchymal stem cells according to any one of [1] to [1-2], which express the TNFAIP6 gene, the CYP1B1 gene, and the EREG gene. [2-1-2] Mesenchymal stem cells according to any one of [1] to [1-2], which express the TNFAIP6 gene and the CYP1B1 gene. [2-1-3] Mesenchymal stem cells according to any one of [1] to [1-2], which express the TNFAIP6 gene and the EREG gene. [2-1-4] Mesenchymal stem cells according to any one of [1] to [1-2], which express the CYP1B1 gene and the EREG gene. [2-2-1] Mesenchymal stem cells according to any one of [1] to [1-2], which express the CYP1B1 gene, the MME gene, and the CXCL5 gene. [2-2-2] Mesenchymal stem cells according to any one of [1] to [1-2], which express the CYP1B1 gene and the MME gene. [2-2-3] Mesenchymal stem cells according to any one of [1] to [1-2], which express the CYP1B1 gene and the CXCL5 gene. [2-2-4] Mesenchymal stem cells according to any one of [1] to [1-2], which express the MME gene and the CXCL5 gene.

[0008] [3-1-1] Mesenchymal stem cells according to any one of [1] to [1-2], in which the expression of at least one gene selected from the group consisting of the TNFAIP6 gene, the CYP1B1 gene, and the EREG gene is enhanced. [3-1-2] Mesenchymal stem cells according to any one of [2-1-1] to [2-1-4], in which the expression of the TNFAIP6 gene, the CYP1B1 gene, and the EREG gene is enhanced. [3-2-1] Mesenchymal stem cells according to any one of [1] to [1-2], in which the expression of at least one gene selected from the group consisting of the CYP1B1 gene, the MME gene, and the CXCL5 gene is enhanced. [3-2-2] Mesenchymal stem cells according to any one of [2-2-1] to [2-2-4], in which the expression of the CYP1B1 gene, the MME gene, and the CXCL5 gene is enhanced.

[0009] [4] The mesenchymal stem cells according to any one of [1] to [3-2-2], wherein the mesenchymal stem cells are derived from bone marrow. [5] The mesenchymal stem cells according to any one of [1] to [4], wherein the mesenchymal stem cells are highly purified mesenchymal stem cells.

[0010] [6] A cell population comprising the mesenchymal stem cells according to any one of [1] to [5]. [6-1] A cell population comprising mesenchymal stem cells that promote induction into M2 macrophages and express at least one gene selected from the group consisting of the TNFAIP6 gene, the CYP1B1 gene, the EREG gene, the MME gene, and the CXCL5 gene. [6-1-1] The cell population according to [6-1], wherein the gene is at least one gene selected from the group consisting of the TNFAIP6 gene, the CYP1B1 gene, and the EREG gene. [6-1-2] The cell population according to [6-1-1], wherein the genes are the TNFAIP6 gene, the CYP1B1 gene, and the EREG gene. [6-1-3] The cell population according to [6-1-2], in which expression of the TNFAIP6 gene, the CYP1B1 gene, and the EREG gene is enhanced. [6-2-1] The cell population according to [6-1], wherein the gene is at least one gene selected from the group consisting of the CYP1B1 gene, the MME gene, and the CXCL5 gene. [6-2-2] The cell population according to [6-2-1], wherein the gene is the CYP1B1 gene, the MME gene, and the CXCL5 gene. [6-2-3] The cell population according to [6-2-2], wherein expression of the CYP1B1 gene, the MME gene, and the CXCL5 gene is enhanced. [6-3] The cell population according to any one of [6-1] to [6-2-3], wherein the mesenchymal stem cells are derived from bone marrow. [6-4] A cell population comprising bone marrow-derived mesenchymal stem cells that promote induction into M2 macrophages and express at least one gene selected from the group consisting of the TNFAIP6 gene, the CYP1B1 gene, the EREG gene, the MME gene, and the CXCL5 gene. [6-5] The cell population according to any one of [6-1] to [6-4], wherein the mesenchymal stem cells are highly purified mesenchymal stem cells. [6-6] A cell population comprising bone marrow-derived mesenchymal stem cells for use in treating M2-related diseases, which express at least one gene selected from the group consisting of TNFAIP6 gene, CYP1B1 gene, EREG gene, MME gene, and CXCL5 gene.

[0011] [7] A cell population described in any one of [6] to [6-6], wherein the relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.01 or more, the relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.03 or more, the relative expression level of the EREG gene to the expression level of the GAPDH gene is 0.003 or more, the relative expression level of the MME gene to the expression level of the GAPDH gene is 0.01 or more, and / or the relative expression level of the CXCL5 gene to the expression level of the GAPDH gene is 0.001 or more. [7-1] A cell population according to any one of [6] to [6-6], wherein the relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.05 or more, the relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.09 or more, the relative expression level of the EREG gene to the expression level of the GAPDH gene is 0.01 or more, the relative expression level of the MME gene to the expression level of the GAPDH gene is 0.04 or more, and / or the relative expression level of the CXCL5 gene to the expression level of the GAPDH gene is 0.005 or more. [7-1-1] A cell population comprising bone marrow-derived mesenchymal stem cells that promote induction into M2 macrophages and express the TNFAIP6 gene, CYP1B1 gene, and EREG gene, wherein the relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.01 or more, the relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.03 or more, and the relative expression level of the EREG gene to the expression level of the GAPDH gene is 0.003 or more. [7-1-2] A cell population containing bone marrow-derived mesenchymal stem cells for treating an M2-related disease, which express the TNFAIP6 gene, the CYP1B1 gene, and the EREG gene, wherein the relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.01 or more, the relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.03 or more, and the relative expression level of the EREG gene to the expression level of the GAPDH gene is 0.003 or more.

[0012] [7-1-3] The cell population according to [7-1-1] or [7-1-2], wherein the relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.05 or more, the relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.09 or more, and the relative expression level of the EREG gene to the expression level of the GAPDH gene is 0.01 or more. [7-2-1] A cell population comprising bone marrow-derived mesenchymal stem cells that promote induction into M2 macrophages and express the CYP1B1 gene, MME gene, and CXCL5 gene, wherein the relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.03 or more, the relative expression level of the MME gene to the expression level of the GAPDH gene is 0.01 or more, and the relative expression level of the CXCL5 gene to the expression level of the GAPDH gene is 0.001 or more. [7-2-2] A cell population for treating an M2-related disease, comprising bone marrow-derived mesenchymal stem cells expressing the CYP1B1 gene, the MME gene, and the CXCL5 gene, wherein the relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.03 or more, the relative expression level of the MME gene to the expression level of the GAPDH gene is 0.01 or more, and the relative expression level of the CXCL5 gene to the expression level of the GAPDH gene is 0.001 or more. [7-2-3] The cell population according to [7-2-1] or [7-2-2], wherein the relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.09 or more, the relative expression level of the MME gene to the expression level of the GAPDH gene is 0.04 or more, and the relative expression level of the CXCL5 gene to the expression level of the GAPDH gene is 0.005 or more. [7-3-1] A cell population containing bone marrow-derived mesenchymal stem cells expressing the TNFAIP6 gene, the CYP1B1 gene, and the EREG gene for use as a therapeutic agent for an M2-related disease, wherein the relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.01 or more, the relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.03 or more, and the relative expression level of the EREG gene to the expression level of the GAPDH gene is 0.003 or more.[7-3-2] A cell population containing bone marrow-derived mesenchymal stem cells expressing the CYP1B1 gene, the MME gene, and the CXCL5 gene for use as a therapeutic agent for an M2-related disease, wherein the relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.03 or more, the relative expression level of the MME gene to the expression level of the GAPDH gene is 0.01 or more, and the relative expression level of the CXCL5 gene to the expression level of the GAPDH gene is 0.001 or more.

[0013] [7-4] The cell population according to [7-1-2], [7-2-2], [7-3-1], or [7-3-2], wherein the M2-associated disease is at least one selected from the group consisting of autoimmune diseases, arteriosclerosis, arteriosclerosis-related diseases, sepsis-associated diseases, liver diseases, and brain diseases. [7-5] The cell population according to [7-1-2], [7-2-2], [7-3-1], or [7-3-2], wherein the M2-associated disease is at least one selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease, wound healing, myocardial infarction, atherosclerosis, type 2 diabetes, lung damage associated with sepsis, acute liver damage, and Alzheimer's disease.

[0014] [8] The cell population according to any one of [6] to [7-5], wherein the mesenchymal stem cells are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and have a coefficient of variation of forward scattered light in flow cytometry of 35% or less. [8-1] The cell population according to any one of [6] to [7-5], wherein the mesenchymal stem cells are cells and their progeny obtained by the following steps: (a) obtaining a population of mesenchymal stem cells that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), (b) culturing clones of the mesenchymal stem cells in single cell culture, and (c) recovering and expanding clones that proliferate rapidly. [8-2] The cell population according to any one of [6] to [7-5], wherein the mesenchymal stem cells are produced by a production method comprising the following steps: (a) obtaining a population of mesenchymal stem cells that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90); (b) culturing the clones of the mesenchymal stem cells in single cell culture; and (c) recovering and expanding clones that proliferate rapidly.

[0015] [8-3] The cell population according to [8-2], wherein the production method can further include one or more of the following steps (d) to (f): (d) measuring the coefficient of variation of forward scattered light by flow cytometry for the cell population after expansion culture, (e) selecting a cell population having a coefficient of variation of forward scattered light measured by flow cytometry of 35% or less, and (f) further culturing the selected cell population.

[0016] [9] A composition comprising the cell population described in any one of [6] to [8-3] or a culture thereof. [9-1] A composition for promoting induction into M2 macrophages, comprising the cell population described in any one of [6] to [8-3] or a culture thereof. [9-2] A pharmaceutical composition comprising the cell population described in any one of [6] to [8-3] or a culture thereof.

[0017]

[10] The pharmaceutical composition according to [9-2], which is used as a therapeutic agent for an M2-related disease.

[11] The pharmaceutical composition according to

[10] , wherein the M2-related disease is at least one selected from the group consisting of autoimmune diseases, arteriosclerosis, arteriosclerosis-related diseases, sepsis-related diseases, liver diseases, and brain diseases. [11-1] The pharmaceutical composition according to

[10] , wherein the M2-related disease is at least one selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease, wound healing, myocardial infarction, atherosclerosis, type 2 diabetes, lung damage associated with sepsis, acute liver damage, and Alzheimer's disease.

[0018]

[12] A method for selecting cells capable of promoting induction into M2 macrophages, comprising the step of measuring the expression of at least one gene selected from the group consisting of TNFAIP6 gene, CYP1B1 gene, EREG gene, MME gene, and CXCL5 gene in a test mesenchymal stem cell.

[13] A method for evaluating the ability of a test mesenchymal stem cell to promote induction into M2 macrophages, comprising the step of measuring the expression of at least one gene selected from the group consisting of TNFAIP6 gene, CYP1B1 gene, EREG gene, MME gene, and CXCL5 gene in the test mesenchymal stem cell.

[14] A method for producing the cells, comprising the steps of culturing the test mesenchymal stem cells and collecting cells capable of promoting induction into M2 macrophages using the expression of at least one gene selected from the group consisting of TNFAIP6 gene, CYP1B1 gene, EREG gene, MME gene, and CXCL5 gene as an indicator.

[0019]

[15] A method for treating an M2-related disease, comprising administering a cell population containing mesenchymal stem cells that express at least one gene selected from the group consisting of the TNFAIP6 gene, the CYP1B1 gene, the EREG gene, the MME gene, and the CXCL5 gene. [15-1] The method for treating an M2-related disease according to

[15] , wherein the M2-related disease is at least one selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease, wound healing, myocardial infarction, atherosclerosis, type 2 diabetes, lung damage associated with sepsis, acute liver damage, and Alzheimer's disease.

[0020]

[16] Use of a cell population containing mesenchymal stem cells expressing at least one gene selected from the group consisting of the TNFAIP6 gene, the CYP1B1 gene, the EREG gene, the MME gene, and the CXCL5 gene for treating an M2-related disease. [16-1-1] Use of a cell population containing mesenchymal stem cells expressing at least one gene selected from the group consisting of the TNFAIP6 gene, the CYP1B1 gene, and the EREG gene for treating an M2-related disease, wherein the relative expression level of the TNFAIP6 gene to that of the GAPDH gene is 0.01 or more, the relative expression level of the CYP1B1 gene to that of the GAPDH gene is 0.03 or more, and the relative expression level of the EREG gene to that of the GAPDH gene is 0.003 or more.

[0021] [16-1-2] Use of a cell population containing mesenchymal stem cells that expresses at least one gene selected from the group consisting of the TNFAIP6 gene, the CYP1B1 gene, and the EREG gene for treating an M2-related disease, wherein the relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.05 or more, the relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.09 or more, and the relative expression level of the EREG gene to the expression level of the GAPDH gene is 0.01 or more. [16-2-1] Use of a cell population containing mesenchymal stem cells that expresses at least one gene selected from the group consisting of the CYP1B1 gene, the MME gene, and the CXCL5 gene for treating an M2-related disease, wherein the relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.03 or more, the relative expression level of the MME gene to the expression level of the GAPDH gene is 0.01 or more, and the relative expression level of the CXCL5 gene to the expression level of the GAPDH gene is 0.001 or more. [16-2-2] Use of a cell population containing mesenchymal stem cells expressing at least one gene selected from the group consisting of the CYP1B1 gene, the MME gene, and the CXCL5 gene for treating an M2-related disease, wherein the relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.09 or more, the relative expression level of the MME gene to the expression level of the GAPDH gene is 0.04 or more, and the relative expression level of the CXCL5 gene to the expression level of the GAPDH gene is 0.005 or more. [16-3] The use according to any one of [16-1-1] to [16-2-2], wherein the M2-related disease is at least one selected from the group consisting of autoimmune diseases, arteriosclerosis, arteriosclerosis-related diseases, sepsis-related diseases, liver diseases, and brain diseases. [16-4] The use according to any one of [16-1-1] to [16-2-2], wherein the M2-related disease is at least one selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease, wound healing, myocardial infarction, atherosclerosis, type 2 diabetes, lung damage associated with sepsis, acute liver damage, and Alzheimer's disease.

[0022] The present invention makes it possible to evaluate and obtain mesenchymal stem cells with high M2-inducing activity and cell populations containing the same. It also makes it possible to evaluate and obtain mesenchymal stem cells with strong anti-inflammatory activity and cell populations containing the same. As a result, the cells and cell populations can be used for diseases in which M2 macrophages are involved in the treatment.

[0023] Figure 1 shows the results of principal component analysis. MSC1 and 2 represent clones with high M2 shift activity (Gr1), MSC3 and 4 represent clones with low M2 shift activity (Gr2), and MSC5 and 6 represent Lonza BMMSCs (Gr3). The proximity of the plots indicates the proximity of the gene expression profiles. Figure 2 shows the results of DEG analysis. The vertical axis represents the fold change value, and the horizontal axis represents the p-value. Genes with a higher expression ratio and a greater likelihood of significant differences are expressed at the upper ends of the graph. Figure 3 shows the correlation between the relative expression level (horizontal axis) and M2 shift activity (vertical axis) of each clone for candidate genes that are particularly strongly associated with M2 shift activity. Figure 4 shows the correlation between the relative expression level (horizontal axis) and IL-6 production suppression rate (vertical axis) of each clone for candidate genes that are particularly strongly associated with M2 shift activity. Figure 5 shows the correlation between the relative expression level (horizontal axis) and IL-6 production suppression rate (vertical axis) of each clone for candidate genes that are particularly strongly associated with M2 shift activity. Figure 6 shows the relative expression level of each clone for candidate genes that are particularly strongly associated with M2 shift activity. 1 shows the protein expression levels of candidate genes that are particularly strongly associated with the M2 shift action, and FIG. 2 shows the protein expression levels of control genes.

[0024] 1. Overview The present invention relates to mesenchymal stem cells expressing at least one gene selected from the group consisting of TNFAIP6, CYP1B1, EREG, MME, and CXCL5. The mesenchymal stem cells of the present invention can promote induction into M2 macrophages. The present invention also relates to a cell population containing the mesenchymal stem cells. The present inventors investigated the possibility of using a cell population with high M2-inducing activity as a treatment for diseases in which disruption of the M1 / M2 macrophage balance is associated with pathogenesis. The present inventors evaluated the anti-inflammatory potential of each MSC clone using two indicators: the ability to promote induction into M2 macrophages, which are anti-inflammatory macrophages, and the inhibitory effect on dextran sulfate sodium (DSS)-induced colitis. By comparing gene expression in clones with strong and weak anti-inflammatory activity, they narrowed down the number of candidate marker genes for selecting MSC clones with strong anti-inflammatory activity to 23.

[0025] Furthermore, the expression levels of candidate genes in 11 MSC clones were quantified by qPCR. From the 23 genes, three genes (TNFAIP6, CYP1B1, and EREG) correlated with the M2 macrophage induction activity of each clone, one gene (MME) with high expression levels in three clones with particularly high M2 shift activity, and one gene (CXCL5) correlated with the DSS-induced colitis inhibitory effect of each clone were identified as selection markers. This established a method for evaluating cell populations with high M2 macrophage induction activity, as well as a method for evaluating cell populations with high M2 macrophage induction activity and their functionality. The present invention successfully obtained such cells. "Evaluation" and "evaluating" refer to determining whether the tested cells have the desired function, whether they have the ability to promote M2 macrophage induction, or whether they pass or fail in terms of their ability to promote M2 macrophage induction. The process of performing this "evaluation" to identify a cell population with a high M2 macrophage-inducing activity is also referred to as "selection" or "selection" in this specification.

[0026] 2. Marker Genes for Selecting MSC Clones with Anti-inflammatory Activity In the present invention, the marker genes for selecting MSC clones with anti-inflammatory activity are the TNFAIP6 gene, CYP1B1 gene, EREG gene, MME gene, and CXCL5 gene, and one or more of these genes can be used for selection.

[0027] Mesenchymal stem cells expressing at least one of the above five genes, preferably mesenchymal stem cells in which expression of the gene is enhanced, have a high M2 macrophage induction effect or a high M1 to M2 shift effect (M2 shift effect), and can therefore be used for diseases in which M2 macrophages are involved in the treatment.

[0028] Here, "enhanced" gene expression means either that expression is higher than the steady state of the gene, or that expression is higher relative to that of housekeeping genes such as the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, β-actin gene, β2-microglobulin gene, and HPRT1 (hypoxanthine phosphoribosyltransferase 1) gene, or ribosomal RNA. For example, expression can be said to be "enhanced" when, relative to the expression level of the GAPDH gene, the relative expression level of the TNFAIP6 gene is 0.01 or more, the relative expression level of the CYP1B1 gene is 0.03 or more, the relative expression level of the EREG gene is 0.003 or more, the relative expression level of the MME gene is 0.01 or more, or the relative expression level of the CXCL5 gene is 0.001 or more (details of relative expression levels will be described later).

[0029] The terms "M2 macrophage induction-promoting action," "M2 macrophage-inducing action," "M2-inducing action," "M2-inducing activity," "M2 shift action," "M2 shift activity," and "ability to promote induction into M2 macrophages" refer to an action that promotes polarization into M2 macrophages.

[0030] The terms "highly effective in promoting M2 macrophage induction" and "strongly effective in promoting M2 macrophage induction" refer to, for example, the induction of polarization into M2 macrophages that is 3-fold or more, preferably 3.5-fold or more, more preferably 4-fold or more, and even more preferably 4.5-fold or more, compared to when M2 macrophages are induced in a medium containing only IL-4. Specifically, this can be measured according to the method described in Example 2.

[0031] 2. Mesenchymal Stem Cells and Cell Populations In the present invention, mesenchymal stem cells expressing at least one of the above five genes are (mesenchymal) somatic stem cells derived from mesodermal tissue. Mesenchymal stem cells can be obtained from various tissues, such as bone marrow, adipose tissue, placental tissue (placental chorion, etc.), dental pulp, umbilical cord tissue, umbilical cord blood, and amniotic membrane, but in the present invention, those derived from bone marrow are preferred. The purification process is, for example, as follows.

[0032] A small amount of fat fragments collected from humans or non-human mammals (e.g., cows, monkeys, cats, mice, rats, guinea pigs, hamsters, pigs, dogs, rabbits, sheep, horses, goats, etc.) are enzymatically treated to obtain a mixed population of cell types. A floating adipocyte population is separated by centrifugation, and fibroblast-like cells that settle and proliferate on the bottom surface when placed in contact with the ceiling of a culture vessel filled with culture medium are then proliferated by subculture. In addition, mesenchymal stem cells derived from iPS cells or commercially available mesenchymal stem cells can also be used in the present invention.

[0033] In a typical culture method for obtaining bone marrow-derived mesenchymal stem cells, cells extracted from bone marrow fluid (mainly bone marrow mononuclear cells) are seeded into a culture vessel, and the culture medium is changed several times until the cells adhere to the culture vessel and proliferate stably. After that, when sufficient proliferation as mesenchymal stem cells is confirmed, the mesenchymal stem cells are detached from the culture vessel and distributed among multiple culture vessels for subculture. Proliferation can be confirmed, for example, by determining whether the cultured cells reach confluence or semi-confluence.

[0034] "Confluent" refers to a state in which cultured cells cover 90% or more of the surface of the culture vessel (culture surface). "Semi-confluent" refers to a state in which cultured cells cover 70-90% of the surface of the culture vessel (culture surface). The size and type of culture equipment used can be changed appropriately depending on the cell growth rate.

[0035] In another embodiment of the present invention, it is desirable to use mesenchymal stem cells that have been further selected by flow cytometry, affinity chromatography, magnetic beads, or the like. Therefore, the present inventors previously established a method for isolating and cloning MSCs directly from human bone marrow using flow cytometry (Mabuchi Y, et al. LNGFR(+)THY-1(+)VCAM-1(hi+) cells reveal functionally distinct subpopulations in mesenchymal stem cells. Stem Cell Reports. 2013 Jul 11;1(2):152-65.). This method makes it possible to obtain homogeneous MSCs without contamination by other cells. Furthermore, they found that among the MSCs established using this method, clones with high proliferation potential and high uniformity in the size of the proliferated cells tended to maintain high differentiation potential (Mabuchi Y, et al. LNGFR(+)THY-1(+)VCAM-1(hi+) cells reveal functionally distinct subpopulations in mesenchymal stem cells. Stem Cell Reports. 2013 Jul 11;1(2):152-65.).

[0036] As one example, in a previous study, the present inventors succeeded in isolating rapidly expanding cell clones (RECs) from LNGFR (CD271)-positive mesenchymal stem cells (CD271+ cells) or LNGFR (CD271) and Thy-1 (CD90)-copositive mesenchymal stem cells (CD271+CD90+ cells) (Mabuchi Y, et al. LNGFR(+)THY-1(+)VCAM-1(hi+) cells reveal functionally distinct subpopulations in mesenchymal stem cells. Stem Cell Reports. 2013 Jul 11;1(2):152-65.). RECs differ from mesenchymal stem cell populations prepared by other methods in that they are derived from the expansion of single cells extracted from the body, and can therefore be referred to as "highly purified mesenchymal stem cells." Here, "high purity" means that RECs are derived from a single cell at the time they are extracted from a living organism, and therefore are highly uniform and pure not only in terms of genomic information but also in terms of epigenomic information.

[0037] RECs are cells that can reach semi-confluence in three weeks when seeded individually in a 96-well plate. Highly purified mesenchymal stem cells (RECs) are obtained by isolating LNGFR (CD271)-positive or LNGFR (CD271) and Thy1 (CD90)-copositive cells individually from the bone marrow mononuclear cell fraction using a cell sorter, seeding them in a 96-well plate, and selecting only fast-proliferating MSCs, resulting in an extremely homogeneous cell population.

[0038] RECs are cloned mesenchymal stem cells derived from a single mesenchymal stem cell that is positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90). These mesenchymal stem cells can be obtained, for example, according to the method described in WO2009 / 31678. The method is outlined below.

[0039] First, LNGFR (CD271)-positive (CD271+) or CD271 and CD90-copositive (CD271+CD90+) cells are isolated individually from a cell population containing human mesenchymal stem cells. If the cell population containing human mesenchymal stem cells also contains hematopoietic cells, a step of selecting CD45 and CD235a-conegative (CD45-CD235a-) cells may be added to select non-hematopoietic cells.

[0040] The material from which this cell population can be obtained is not particularly limited, but examples include bone marrow, adipose tissue, umbilical cord blood, peripheral blood, etc. Bone marrow may be from the spine, sternum, ilium, etc. ES cells and iPS cells can also be used as materials. In this case, mesenchymal stem cells derived from iPS cells or commercially available mesenchymal stem cells can also be used, and the cell population obtained by separating these mesenchymal stem cells into single cells and then expanding them is used.

[0041] When preparing a cell population, if the material is in the form of cell clumps containing mesenchymal stem cells, the material can be subjected to physical treatment such as pipetting or enzymatic treatment with trypsin, collagenase, etc., as necessary. Furthermore, if the material contains red blood cells, it is preferable to hemolyze the red blood cells beforehand. Using the cell population prepared as described above, CD271+ cells or CD271+CD90+ cells are isolated one by one.

[0042] Methods for isolating CD271+ cells or CD271+CD90+ cells one by one include, for example, methods using antibodies. The antibodies are anti-CD271 antibodies, or anti-CD271 and anti-CD90 antibodies, which can select CD271+ cells or CD271+CD90+ cells. When using flow cytometry for selection, live cells can be selected in a short period of time by using an appropriate combination of anti-CD271 antibodies labeled with different fluorescent dyes, such as FITC, PE, or APC, or anti-CD271 and anti-CD90 antibodies. In addition to flow cytometry, CD271+CD90+ cells can also be isolated one by one using a single-cell dispenser.

[0043] Before using these methods, dead cells may be removed by reacting the cell population with a fluorescent dye (e.g., PI) that stains dead cells and then removing the fluorescently stained cells. Next, LNGFR (CD271)-positive cells or LNGFR and Thy1 (CD90)-copositive cells isolated individually are cultured as single cells (clones), and the most rapidly proliferating lot is selected to obtain RECs with excellent proliferation, differentiation, and migration abilities.

[0044] Here, "fast proliferation" and "high-speed proliferation" mean that when cells are seeded one cell per well of a 96-well culture plate and cultured, the culture plate has a proliferation rate such that the culture plate becomes confluent or semi-confluent three weeks or earlier after the start of culture (doubling time is approximately 24±4 hours).

[0045] Moderately / Slowly Expanding Cells, i.e., cells that do not reach semi-confluence or confluence after three weeks of single cell culture, are discarded. RECs collected from each well selected as RECs are transferred to a 6-well plate and cultured, then transferred to a T75 culture flask and cultured until they reach semi-confluence (expansion culture). The expanded cells are then collected separately. RECs from one well constitute one lot.

[0046] RECs are obtained by clonal sorting, in which one cell is seeded per well, and therefore the genetic traits of the proliferated cells are all identical.

[0047] Alternatively, the REC to be used for selection can be evaluated in advance using the REC marker (anti-Ror2). For example, after the expansion culture, adherent and proliferated cells are collected from all the lots, and a portion of each lot (1-3 × 10 5A small number of cells (approximately 1000 cells) are selected and single-stained with an anti-Ror2 monoclonal antibody. A technique for single-staining with an anti-Ror2 monoclonal antibody is known (WO2016 / 17795). In summary, the percentage of REC marker-positive cells among the collected cells is determined by flow cytometry analysis using the REC marker. The percentage can be determined by quantitative PCR to quantify Ror2 mRNA expression, or by manually determining the percentage using a microscope. Lots (cell populations) with a positive percentage above a certain value (e.g., 65%) are considered acceptable and can be used for selection.

[0048] In one embodiment of the present invention, highly purified mesenchymal stem cells REC are a population of Thy-1 (CD90)-positive, rapidly proliferating mesenchymal stem cell clones, and preferably satisfy at least one of the following characteristics (a) and (b): (a) the coefficient of variation of forward scattered light in flow cytometry is 35% or less, and (b) the average cell diameter is 20 μm or less.

[0049] In yet another embodiment, the highly purified mesenchymal stem cells REC are cells and their progeny obtained by the steps of: (a) obtaining a population of mesenchymal stem cells that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), (b) culturing clones of the mesenchymal stem cells in single cell culture, and (c) recovering and expanding clones that proliferate rapidly. Preferably, the highly purified mesenchymal stem cells REC have a coefficient of variation of forward scattered light in flow cytometry of 35% or less.

[0050] In one embodiment of the present invention, a method for producing highly purified mesenchymal stem cells (REC) comprises the following steps: (a) obtaining a population of mesenchymal stem cells that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), (b) culturing clones of the mesenchymal stem cells in single cell culture, and (c) recovering rapidly proliferating clones and expanding them. The production method can further comprise one or more of the following steps: (d) measuring the coefficient of variation of forward scattered light by flow cytometry for the cell population after the expansion culture, (e) selecting a cell population having a coefficient of variation of forward scattered light measured by flow cytometry of 35% or less, and (f) further culturing the selected cell population.

[0051] By examining the proliferation potential, adipocyte differentiation potential, mesenchymal stem cell marker expression levels, and cell size uniformity of individual REC clones and analyzing the correlations among these, it became possible to select highly pure, uniform RECs with higher proliferation and differentiation potential. The selection can be performed using the coefficient of variation (CV) of forward scattered light in flow cytometry and the average cell size as indicators. Forward scattered light in flow cytometry refers to light scattered at a small angle forward relative to the axis of the laser beam. Forward scattered light consists of scattered light, diffracted light, and refracted light of the laser beam generated on the cell surface and is proportional to the surface area or size of the cell. Therefore, forward scattered light can provide information about the size of the sample.

[0052] The CV value is the standard deviation divided by the mean value, and is a value used to relatively evaluate the variability of data in different units, or the relationship between the data and the variability relative to the mean value. For example, RECs with high proliferation and differentiation potential, whose CV value of the forward scattered light intensity is 35% or less, can be selected. RECs with a CV value of 35% or less in forward scattered light intensity are cell populations composed of cells of uniform size. Preferably, the CV value is 30% or less, 25% or less, or 20% or less. Furthermore, the average cell size in a population of REC cells with high proliferation and differentiation potential is 20 μm or less. Preferably, the average cell size is 18 μm or less, for example, in the range of 10 μm to 18 μm, 12 μm to 18 μm, or 14 μm to 18 μm.

[0053] Examples of methods for evaluating cell size variation using the CV value of forward scattered light intensity as an index include, but are not limited to, the following: (a) PI staining is performed on REC for each lot, and a gate is set on the PI-negative live cell population to exclude dead cells from the analysis. (b) The PI-negative live cell population is developed on an FSC / SSC cytogram, and a gate (P1) is set on the main cell population to exclude debris and noise from the analysis. (c) The cell population within the P1 gate is developed on an FSC histogram, a marker (M1) is set, and the CV value is measured.

[0054] 3. Gene Expression in Mesenchymal Stem Cells In the present invention, gene expression in mesenchymal stem cells can be defined as the relative expression level compared to an endogenous control gene. Endogenous control genes are often genes that are expressed at similar levels in all tissues, and the expression levels of GAPDH, β-actin, β2-microglobulin, HPRT1, ribosomal RNA, etc. are used. These relative expression levels can be calculated by correcting the expression level of the target gene with the expression level of the endogenous control gene. Specifically, the value (Ct1-Ct2) obtained by subtracting the Ct value (Ct2) of the endogenous control gene from the Ct value (Ct1) of the target gene is called the ΔCt value, and the relative expression level is calculated by 2- ΔCt is calculated as the value of

[0055] In the present invention, gene expression in mesenchymal stem cells is exemplified as follows, using the GAPDH gene as an endogenous control gene: (1-1) The relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.01 or more. (1-2) The relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.05 or more. (2-1) The relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.03 or more. (2-2) The relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.09 or more.

[0056] (3-1) The relative expression level of the EREG gene to the expression level of the GAPDH gene is 0.003 or more. (3-2) The relative expression level of the EREG gene to the expression level of the GAPDH gene is 0.01 or more. (4-1) The relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.01 or more, and the relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.03 or more. (4-2) The relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.01 or more, and the relative expression level of the CYP1B1 gene is 0.09 or more.

[0057] (5-1) The relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.01 or more, and the relative expression level of the EREG gene is 0.003 or more. (5-2) The relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.01 or more, and the relative expression level of the EREG gene is 0.01 or more. (6-1) The relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.03 or more, and the relative expression level of the EREG gene is 0.003 or more. (6-2) The relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.03 or more, and the relative expression level of the EREG gene is 0.01 or more.

[0058] (7-1) The relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.01 or more, the relative expression level of the CYP1B1 gene is 0.03 or more, and the relative expression level of the EREG gene is 0.003 or more. (7-2) The relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.01 or more, the relative expression level of the CYP1B1 gene is 0.03 or more, and the relative expression level of the EREG gene is 0.01 or more. (7-3) The relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.01 or more, the relative expression level of the CYP1B1 gene is 0.09 or more, and the relative expression level of the EREG gene is 0.003 or more. (7-4) The relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.01 or more, the relative expression level of the CYP1B1 gene is 0.09 or more, and the relative expression level of the EREG gene is 0.01 or more.

[0059] (7-5) The relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.05 or more, the relative expression level of the CYP1B1 gene is 0.03 or more, and the relative expression level of the EREG gene is 0.003 or more. (7-6) The relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.05 or more, the relative expression level of the CYP1B1 gene is 0.03 or more, and the relative expression level of the EREG gene is 0.01 or more. (7-7) The relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.05 or more, the relative expression level of the CYP1B1 gene is 0.09 or more, and the relative expression level of the EREG gene is 0.003 or more. (7-8) The relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.05 or more, the relative expression level of the CYP1B1 gene is 0.09 or more, and the relative expression level of the EREG gene is 0.01 or more.

[0060] (8-1) The relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.05 or more, and the relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.03 or more. (8-2) The relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.05 or more, and the relative expression level of the CYP1B1 gene is 0.09 or more.

[0061] (9-1) The relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.05 or more, and the relative expression level of the EREG gene is 0.003 or more. (9-2) The relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.05 or more, and the relative expression level of the EREG gene is 0.01 or more. (10-1) The relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.09 or more, and the relative expression level of the EREG gene is 0.003 or more. (10-2) The relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.09 or more, and the relative expression level of the EREG gene is 0.01 or more.

[0062] (11-1) The relative expression level of the MME gene to the expression level of the GAPDH gene is 0.01 or more. (11-2) The relative expression level of the MME gene to the expression level of the GAPDH gene is 0.04 or more.

[0063] (12-1) The relative expression level of the CXCL5 gene to the expression level of the GAPDH gene is 0.001 or more. (12-2) The relative expression level of the CXCL5 gene to the expression level of the GAPDH gene is 0.005 or more. (13-1) The relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.03 or more, and the relative expression level of the MME gene is 0.01 or more. (13-2) The relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.03 or more, and the relative expression level of the MME gene is 0.04 or more.

[0064] (14-1) The relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.03 or more, and the relative expression level of the CXCL5 gene is 0.001 or more. (14-2) The relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.03 or more, and the relative expression level of the CXCL5 gene is 0.005 or more. (15-1) The relative expression level of the MME gene to the expression level of the GAPDH gene is 0.01 or more, and the relative expression level of the CXCL5 gene is 0.001 or more. (15-2) The relative expression level of the MME gene to the expression level of the GAPDH gene is 0.01 or more, and the relative expression level of the CXCL5 gene is 0.005 or more.

[0065] (16-1) The relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.03 or more, the relative expression level of the MME gene is 0.01 or more, and the relative expression level of the CXCL5 gene is 0.001 or more. (16-2) The relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.03 or more, the relative expression level of the MME gene is 0.01 or more, and the relative expression level of the CXCL5 gene is 0.005 or more. (16-3) The relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.03 or more, the relative expression level of the MME gene is 0.04 or more, and the relative expression level of the CXCL5 gene is 0.001 or more. (16-4) The relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.03 or more, the relative expression level of the MME gene is 0.04 or more, and the relative expression level of the CXCL5 gene is 0.005 or more.

[0066] (16-5) The relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.09 or more, the relative expression level of the MME gene is 0.01 or more, and the relative expression level of the CXCL5 gene is 0.001 or more. (16-6) The relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.09 or more, the relative expression level of the MME gene is 0.01 or more, and the relative expression level of the CXCL5 gene is 0.005 or more. (16-7) The relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.09 or more, the relative expression level of the MME gene is 0.04 or more, and the relative expression level of the CXCL5 gene is 0.001 or more. (16-8) The relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.09 or more, the relative expression level of the MME gene is 0.04 or more, and the relative expression level of the CXCL5 gene is 0.005 or more.

[0067] (17-1) The relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.09 or more, and the relative expression level of the MME gene is 0.01 or more. (17-2) The relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.09 or more, and the relative expression level of the MME gene is 0.04 or more.

[0068] (18-1) The relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.09 or more, and the relative expression level of the CXCL5 gene is 0.001 or more. (18-2) The relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.09 or more, and the relative expression level of the CXCL5 gene is 0.005 or more. (19-1) The relative expression level of the MME gene to the expression level of the GAPDH gene is 0.04 or more, and the relative expression level of the CXCL5 gene is 0.001 or more. (19-2) The relative expression level of the MME gene to the expression level of the GAPDH gene is 0.04 or more, and the relative expression level of the CXCL5 gene is 0.005 or more.

[0069] (20-1) The relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.01 or more, the relative expression level of the CYP1B1 gene is 0.03 or more, the relative expression level of the EREG gene is 0.003 or more, the relative expression level of the MME gene is 0.01 or more, and the relative expression level of the CXCL5 gene is 0.001 or more. (20-2) The relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.01 or more, the relative expression level of the CYP1B1 gene is 0.09 or more, the relative expression level of the EREG gene is 0.003 or more, the relative expression level of the MME gene is 0.04 or more, and the relative expression level of the CXCL5 gene is 0.005 or more. (20-3) The relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.05 or more, the relative expression level of the CYP1B1 gene is 0.09 or more, the relative expression level of the EREG gene is 0.01 or more, the relative expression level of the MME gene is 0.01 or more, and the relative expression level of the CXCL5 gene is 0.001 or more. (20-4) The relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.05 or more, the relative expression level of the CYP1B1 gene is 0.09 or more, the relative expression level of the EREG gene is 0.01 or more, the relative expression level of the MME gene is 0.04 or more, and the relative expression level of the CXCL5 gene is 0.005 or more.

[0070] 4. Composition and Pharmaceutical Composition for Promoting Induction of M2 Macrophages In the present invention, the composition and pharmaceutical composition for promoting induction of M2 macrophages comprise the mesenchymal stem cells, a cell population thereof, or a culture thereof. In another aspect of the present invention, the composition and pharmaceutical composition for promoting induction of M2 macrophages comprise the highly purified mesenchymal stem cells, a cell population thereof, or a culture thereof.

[0071] The compositions of the present invention include mesenchymal stem cells themselves as well as cultures of the mesenchymal stem cells. The term "culture" refers to any of the culture supernatant, the cultured cells themselves, and processed products of the culture supernatant and the cultured cells. Examples of media used for culture include media commonly used for culturing animal cells, such as DMEM and RPMI-1640. The media may also contain additives such as serum, cytokines, antibiotics, growth factors, and buffers.

[0072] In the case of culture supernatant, the "processed product" includes a product from which unnecessary solids have been removed by centrifugation, filtration, or the like, or a frozen or lyophilized product thereof, and in the case of cultured cells, the "processed product" includes a product from which unnecessary solids have been removed by disrupting the cells, or a frozen or lyophilized product thereof. Note that the culture supernatant can also be diluted 2-fold, 3-fold, 5-fold, 10-fold, or the like with fresh medium or the like before use.

[0073] In the present invention, mesenchymal stem cells are administered in the form of a pharmaceutical composition. In one embodiment, such a composition contains a pharmaceutically acceptable carrier and / or excipient. The administration route is not particularly limited, but includes, for example, subcutaneous injection, intradermal injection, intramuscular injection, intralymph node injection, intravenous injection, intraarterial injection, intraperitoneal injection, intrathoracic injection, direct injection into a local area, direct application, or direct implantation into a local area. According to one embodiment of the present invention, the injectable solution is filled into a syringe and administered via a needle or catheter intravenously, intraarterially, intramyocardially, intra-articularly, intrahepatic artery, intramuscularly, epidurally, gingivally, intraventricularly, subcutaneously, intradermally, intraperitoneally, or into the portal vein, but is not limited thereto. Particularly preferred is injection, such as intravenous injection or intravenous drip infusion. The terms "carrier" and "excipient" refer to compositions commonly used in the art to facilitate cell storage, administration, and / or biological activity.

[0074] Carriers used in the compositions of the present invention include, for example, physiological saline, aqueous dextrose, lactose, Ringer's solution, buffer solutions, etc. Excipients include starch, cellulose, glucose, lactose, etc. The compositions containing mesenchymal stem cells of the present invention are prepared as appropriate liquid suspensions, for example, in buffer solutions or culture media. Suspensions for injection may contain sodium carboxymethylcellulose, sorbitol, dextran, etc.

[0075] In the present invention, target diseases requiring promotion of induction into M2 macrophages (also referred to as M2-related diseases) or diseases to which a pharmaceutical composition is administered include, for example, autoimmune diseases, arteriosclerosis and its related diseases, sepsis-related diseases, liver diseases, and brain diseases. Specific examples include rheumatoid arthritis, inflammatory bowel disease, wound healing, myocardial infarction, atherosclerosis, type 2 diabetes, lung damage associated with sepsis, acute liver damage, and Alzheimer's dementia.

[0076] The dosage and administration of the pharmaceutical composition when administered to a subject having the above-mentioned disease is, for example, at least 1 x 10 cells. ６ cell / ml concentration (e.g., 1x10 ６ cell / ml, 5x10６ cell / ml, 1x10 ７ The dosage and method of use of the cultured product can be determined appropriately depending on the purpose of use.

[0077] 5. Method for Evaluating the Ability to Promote Induction into M2 Macrophages In the present invention, the method for evaluating the ability to promote induction into M2 macrophages comprises the step of measuring the expression of at least one gene selected from the group consisting of the TNFAIP6 gene, the CYP1B1 gene, the EREG gene, the MME gene, and the CXCL5 gene in a test mesenchymal stem cell.

[0078] Gene expression can be measured by extracting total RNA from the cells and performing quantitative real-time PCR using cDNA synthesized from the total RNA as a template. If the measurement results show that the gene expression in the mesenchymal stem cells satisfies any of the relative expression levels described in (1-1) to (20-4) of Section 3 (3. Gene Expression in Mesenchymal Stem Cells), the test mesenchymal stem cells can be determined to have the ability to promote induction into M2 macrophages or the activity of promoting induction into M2 macrophages.

[0079] 6. Method for producing cells capable of promoting induction into M2 macrophages In the present invention, cells capable of promoting induction into M2 macrophages may be produced by collecting cells determined to have the ability to promote induction into M2 macrophages or the activity of promoting induction into M2 macrophages in Section 5 above (5. Method for evaluating the ability to promote induction into M2 macrophages).

[0080] The present invention will be explained in more detail below with reference to examples, although the scope of the present invention is not limited to these examples.

[0081] Example 1: Preparation of Highly Purified Mesenchymal Stem Cells Multiple lots of RECs were prepared according to a known method (WO2016 / 17795). More specifically, bone marrow aspirate was collected from healthy donors, and mononuclear cells were prepared by density gradient centrifugation. Bone marrow mononuclear cells were stained with anti-LNGFR and anti-Thy1. LNGFR-positive and Thy1-positive cells were clonally sorted and cultured in 96-well culture plates using flow cytometry (FCM, cell sorter). After three weeks, the culture plates were photographed under a microscope, and cells in semiconfluent wells were designated as RECs. RECs in one well were collected as one lot. The collected RECs were seeded separately into 6-well plates and cultured, then transferred to T75 culture flasks and further cultured until semiconfluent.

[0082] 1x10 cells were used to evaluate the cell proliferation potential of each lot. 5 REC cells were seeded onto a 100 mm culture dish and cultured at 37°C in a 5% CO environment for 5 days. After that, the cell number and average cell size were measured using a cell counter. The culture medium used was DMEM medium (Fujifilm Wako Pure Chemical Industries) supplemented with FBS, basic FGF, Hepes, and penicillin-streptomycin.

[0083] In addition, to evaluate the fat differentiation potential of each lot, 5x10 4 REC cells were seeded onto a 24-well plate and cultured at 37°C in a 5% CO2 environment for 2 days. The medium was then replaced with adipogenesis-inducing medium and cultured for an additional 14 days. After 14 days of culture, Oil Red 0 staining was performed, and the lipid droplet area was calculated by image analysis. The adipogenesis-inducing medium used was the above-mentioned culture medium supplemented with dexamethasone, indomethacin, and IBMX. In this example, a lot of REC with high cell proliferation and adipogenesis potential (hereinafter referred to as REC clone) was used.

[0084] Example 2: Effect of REC cells on promoting M2 macrophage induction 2-1. Collection of REC culture supernatants Eleven REC clones (1708AC#1, 1807#1, 1807#10, 1706AC(1)#11, 1706AC(1)#15, 1807#5, 1606#15, 1707(3)#2, 1807#29, 1706AC(1)#5, 1611#9) were suspended in DMEM low glucose medium (containing 20% ​​FBS, 100 units / mL penicillin-streptomycin (P / S), 0.01 mol / L HEPES, and 20 ng / mL bFGF) and collected at 2 × 10 5 Cells were seeded onto 10-cm dishes at a cell density of 100 cells / dish and cultured in a CO2 incubator (37°C, 5% CO2). After confirming that each cell line reached approximately 70-80% confluency, the culture supernatant was aspirated, RPMI 1640 medium was added, and culture was resumed in a CO2 incubator (37°C, 5% CO2). Two days after initiating culture in RPMI 1640 medium, the culture supernatant was collected. The collected culture supernatant was centrifuged (1500 rpm, 5 minutes, 4°C), and the supernatant was collected again and stored in a refrigerator until use.

[0085] 2-2. Measurement of M2 shift activity in each REC clone. Bone marrow was isolated from the femur and tibia of male C57BL / 6j mice, and bone marrow cells were collected from the obtained bone marrow in RPMI1640 medium (containing 10% FBS, 100 units / mL P / S, and 0.01 mol / mL HEPES). The collected bone marrow cell suspension was filter-sterilized and then centrifuged (1500 rpm, 5 minutes, 4°C). After centrifugation, the number of cells was counted, and the cell suspension was placed in a 10-cm dish in RPMI1640 medium containing 20 ng / mL mouse M-CSF, at a concentration of 1 × 10 7 The bone marrow cells were seeded at a cell density of 1000 cells / dish and cultured for 7 days in a CO2 incubator (37°C, 5% CO2). After 7 days of culture, the culture supernatant was removed with PBS using an aspirator, and the cells were washed with PBS. The washed cells were then detached using accutase and collected.

[0086] The collected cell suspension was centrifuged (1500 rpm, 5 minutes, 4°C) and the cells were counted. Cells were suspended and seeded in RPMI1640 medium (M0 group), cells were seeded in RPMI1640 medium containing 20 ng / mL mouse IL-4 (M2 group), and cells were suspended and seeded in RPMI1640 medium containing 20 ng / mL mouse IL-4 (+ 50% v / v REC culture supernatant) (M2 + REC group). A negative control group, in which no antibody staining was performed during flow cytometry, was prepared by seeding cells in RPMI1640 medium containing 20 ng / mL mouse IL-4, as in the M2 group. The prepared cell suspension was placed in a 6-well plate at 1 × 10 6 The cells were seeded at a cell density of 100 cells / well and cultured for 2 days in a CO2 incubator (37°C, CO2 concentration 5%).

[0087] After two days of culture, cells were harvested from each well and resuspended in 100 μL of HBSS. Each cell suspension was stained with APC-conjugated anti-mouse CD206 antibody (CD206-APC) (Biolegend) for 30 minutes in the dark on ice. 30 minutes after the start of staining, 500 μL of HBSS was added to each cell suspension, followed by centrifugation (3000 rpm, 5 minutes, 4°C) and removal of the supernatant. Each cell was suspended in 400 μL of HBSS containing 2 μg / mL propidium iodide solution (PI), and the resulting cell suspension was filtered through a 40 μm mesh to form a single-cell suspension. The mean fluorescence intensity (MFI) of CD206-APC in live cells in each single-cell suspension was measured using a CytoFLEX Flow Cytometer (Beckman Coulter). The M2 shift activity of each REC clone was expressed as the MFI value of CD206-APC in the M2+REC group, with the MFI of CD206-APC in the M2 group set at 1.

[0088] The M2 shift activity of each clone is shown in Table 1. Although the M2 shift activity differed depending on the clone, it was revealed that the shift activity was 1.55 to 4.63 times higher than that of the control.

[0089]

[0090] Example 3: Promotion of M2 macrophage induction by human bone marrow-derived mesenchymal stem cells (human BMMSCs) 3-1. Collection of human BMMSC culture supernatant Two types of human BMMSCs (Lonza) 20TL293908 and 21TL076024 were suspended in DMEM low glucose medium (containing 20% ​​FBS, 100 units / mL P / S, 0.01 mol / mL HEPES, and 20 ng / mL bFGF) to obtain a cell suspension of 2 × 10 5 Cells were seeded onto 10-cm dishes at a cell density of 100 cells / dish and cultured in a CO2 incubator (37°C, 5% CO2). After confirming that each cell line reached approximately 70-80% confluency, the culture supernatant was aspirated, RPMI 1640 medium was added, and culture was resumed in a CO2 incubator (37°C, 5% CO2). Two days after initiating culture in RPMI 1640 medium, the culture supernatant was collected. The collected culture supernatant was centrifuged (1500 rpm, 5 minutes, 4°C), and the supernatant was collected again and stored in a refrigerator until use.

[0091] 3-2. Examination of M2 shift activity in BMMSCs. Bone marrow was isolated from the femurs and tibias of male C57BL / 6j mice, and bone marrow cells were collected from the obtained bone marrow in RPMI1640 medium (containing 10% FBS, 100 units / mL P / S, and 0.01 mol / mL HEPES). The collected bone marrow cell suspension was filter-sterilized and then centrifuged (1500 rpm, 5 minutes, 4°C). After centrifugation, the number of cells was counted, and the cell suspension was placed in a 10-cm dish in RPMI1640 medium containing 20 ng / mL mouse M-CSF, at a concentration of 1 × 10 7 The bone marrow cells were seeded at a cell density of 1000 cells / dish and cultured for 7 days in a CO2 incubator (37°C, 5% CO2). After 7 days of culture, the culture supernatant was removed with PBS using an aspirator, and the cells were washed with PBS. The washed cells were then detached using accutase and collected.

[0092] The collected cell suspension was centrifuged (1500 rpm, 5 min, 4°C) and the cells were counted. Cells were suspended and seeded in RPMI 1640 medium (M0 group), cells were seeded in RPMI 1640 medium containing 20 ng / mL mouse IL-4 (M2 group), and cells were seeded in RPMI 1640 medium containing 20 ng / mL mouse IL-4 (+ 50% v / v BMMSC culture supernatant) (M2 + BMMSC group). A negative control group (M2 group) was used, in which cells were not stained with any antibodies during flow cytometry analysis, but were seeded in RPMI 1640 medium containing 20 ng / mL mouse IL-4.

[0093] The prepared cell suspension was added to a 6-well plate at 1 × 10 6 Cells were seeded at a density of 100 cells / well and cultured for 2 days in a CO2 incubator (37°C, 5% CO2). After 2 days of culture, cells were harvested from each well and resuspended in 100 μL of HBSS. Each cell suspension was stained with APC-conjugated anti-mouse CD206 antibody (CD206-APC) (Biolegend) for 30 minutes on ice in the dark. 30 minutes after the start of staining, 500 μL of HBSS was added to each cell suspension, followed by centrifugation (3000 rpm, 5 minutes, 4°C) and removal of the supernatant. Each cell was suspended in 400 μL of HBSS containing 2 μg / mL propidium iodide solution (PI). The resulting cell suspension was passed through a 40 μm mesh to obtain a single-cell suspension.

[0094] The mean fluorescence intensity (MFI) of CD206-APC in live cells in each single-cell suspension was measured using a CytoFLEX Flow Cytometer (Beckman Coulter). The M2 shift activity of BMMSCs was expressed as the MFI of CD206-APC in the M2+BMMSC group, with the MFI of CD206-APC in the M2 group set at 1. The M2 shift activity of each lot is shown in Table 2.

[0095]

[0096] Example 4 Cell Selection Using a DSS Model 4-1. Preparation of a DSS-Induced Colitis Model Mouse Female C57BL / 6j mice were administered a 3% DSS solution, prepared by dissolving dextran sulfate sodium (DSS) (MP BIO) in tap water, in their drinking water for 3 days to prepare a DSS-induced colitis model mouse. The control group received tap water as drinking water.

[0097] 4-2. Preparation and administration of cells for administration A cell suspension of each REC clone in DMEM low glucose medium (containing 20% ​​FBS, 100 Units / mL L / S, 0.01 mol / mL HEPES, and 20 ng / mL bFGF) was placed in a 75 cm 2 1×10 in flask 5 Cells were seeded at a density of 100 cells / flask and cultured in a CO2 incubator (37°C, 5% CO2). On the day of cell administration, after confirming that each cell line had reached approximately 70-80% confluency, the culture supernatant was aspirated and each cell line was washed with PBS. After washing with PBS, the cells were detached by trypsinization and collected.

[0098] The collected cell suspension was centrifuged (200 g, 5 minutes, room temperature), and the supernatant was removed by aspirating. The obtained cell pellet was resuspended in PBS(-), and the cell number was counted. 6 A cell suspension was prepared at a concentration of 1 × 10 cells / mL. The prepared cell suspension was stored on ice until administration. Before starting administration of 3% DSS aqueous solution in drinking water, 1 × 10 cells were added to mice under isoflurane anesthesia. 6 The cell suspension was slowly administered via the orbital venous plexus at a dose of 200 μL / mouse.

[0099] 4-3. Serum Collection and Measurement of Serum IL-6 Concentration Three days after the start of administration of 3% DSS aqueous solution in drinking water, whole blood samples were collected from each mouse and allowed to stand at room temperature for at least 60 minutes. Then, each sample was centrifuged (15,000 rpm, 10 minutes, 4°C). The supernatant was collected and centrifuged again (15,000 rpm, 10 minutes, 4°C). The supernatant was dispensed into 1.5 mL tubes and stored in a freezer until use. IL-6 concentration in each serum sample was assessed using ELISA Max Standard Set Mouse IL-6 (Biolegend). Each procedure followed the protocol for ELISA Max Standard Set Mouse IL-6 (Biolegend). Absorbance was measured using GloMax® Discover (Promega).

[0100] The IL-6 lowering effect of each clone is shown in Table 3.

[0101] Example 5: Examination of gene expression profiles in REC clones and BMMSCs 5-1. Recovery of each cell type. Among the REC clones, clones with high M2 shift activity (hereinafter referred to as Gr1: 1708AC#1, 1807#1) and clones with low M2 shift activity (hereinafter referred to as Gr2: 1706AC(1)#5, 1611#9) were selected, and differences in gene expression profiles between these clones were examined. Furthermore, differences in gene expression profiles between the selected REC clones and human bone marrow-derived MSCs (BMMSCs) (Lonza) (hereinafter referred to as Gr3: 20TL293908, 21TL076024) were compared. Each cell was suspended in DMEM low glucose medium (containing 20% ​​FBS, 100 units / mL P / S, 0.01 mol / mL HEPES, and 20 ng / mL bFGF) and plated at 2 x 10 cells / cm in a 10-cm dish. 5 The cells were seeded at a cell density of 100 cells / dish and cultured in a CO2 incubator (37°C, CO2 concentration 5%).

[0102] After confirming that the confluency of each cell type reached approximately 70-80%, the culture supernatant was aspirated and each cell type was washed with PBS. After washing with PBS, the cells were detached by trypsin treatment and collected. The collected cell suspension was centrifuged (200 g, 5 minutes, room temperature), and the supernatant was aspirated and removed. The cells were resuspended in DMEM low glucose medium (containing 20% ​​FBS, 100 units / mL P / S, 0.01 mol / mL HEPES, and 20 ng / mL bFGF), and the cell number was counted and set at 1 x 10 6 A cell suspension with a concentration of 1000 cells / mL was prepared. 1 mL of the prepared cell suspension was added to each 1.5 mL tube, and the cells were precipitated to the bottom of the tube by centrifugation (16,000 g, 15 minutes, 4°C). The supernatant was then completely removed using a Pipetman.

[0103] 5-2. Total RNA Extraction Total RNA was extracted from each cell line using the Maxwell RSC simply RNA Cell Kit (Promega). The concentration of the resulting total RNA was measured using a Nano Drop ONE (Thermo Fisher Scientific). To confirm the purity of the total RNA, the RNA Integrity Number equivalent (RINe) was measured using a Tapestation 4200 High Sensitivity RNA (Agilent Technologies).

[0104] Poly(A) RNA extraction and fragmentation Poly(A) RNA was extracted and fragmented from 100 ng of the total RNA obtained using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England BioLabs) and the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs). The protocol followed the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina Instruction Manual.

[0105] mRNA was isolated using Oligo dT Beads, and the isolated poly(A) RNA was added to the NEBNext First Strand Synthesis Reaction Buffer and NEBNext Random Primers from the NEBNext Ultra II RNA Library Prep Kit for Illumina and incubated at 94°C for 15 minutes to fragment the poly(A) RNA.

[0106] 5-4. Reverse transcription and adapter ligation. The fragmented poly(A) RNA was reverse transcribed using the NEBNext First Strand Synthesis Enzyme Mix (New England BioLabs) from the NEBNext Ultra II RNA Library Prep Kit for Illumina to synthesize single-stranded cDNA, followed by synthesis of double-stranded cDNA using the NEBNext Second Strand Synthesis Enzyme Mix (New England BioLabs). The cDNA was purified using AMPureXP (Beckman Coulter). An adapter was then added using NEBNext Adaptor (New England BioLabs), and the cDNA from the adapter-ligated product was purified using AMPureXP.

[0107] 5-5. cDNA Amplification and Barcode Sequence Addition The cDNA was amplified by PCR (c1000 Touch thermal cycler, BioRad Laboratories) under the conditions in Table 4 to create a library. To identify the samples, a barcode sequence was added using NEBNext Multiplex Oligos for Illumina (New England BioLabs). The library was then purified using AMPureXP. The concentration of the library was measured using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific). The library length distribution was also confirmed by analysis using a Tapestation 4200 D1000 (Agilent Technologies).

[0108]

[0109] 5-6. Illumina NovaSeq 6000 analysis Analysis conditions: The 50 bp cDNA region and index sequence were sequenced using NovaSeq 6000 SP Reagent Kit V1.5.

[0110] 5-7. RNA-Seq Analysis Data analysis was performed using CLC Genomics Workbench 23.0.2 (hereafter referred to as GWB). BCL files output from the Illumina NovaSeq 6000 were converted to fastq files using the bcl2fastq v2.20.0.402 tool. The resulting fastq files were imported into GWB, and the reads were trimmed using the GWB Trim reads 2.8 tool. The trimmed reads were mapped to the reference sequence and tag counted using the GWB RNA-Seq Analysis 2.7 tool. The reference sequence and genome annotation were performed using Homo sapiens GRCh38 (hg38) release-110 downloaded from Ensembl using the GWB function. Using the RNA-Seq Analysis tool in CLC Genomics Workbench, we counted the number of reads for each gene ID and transcript ID, and calculated the counts for each gene (total gene reads, unique gene reads), and values ​​normalized by gene length and read amount (RPKM) and (TPM). Expression values ​​were calculated as TPM values.

[0111] 5-8. Principal component analysis All TPM values ​​were converted to log2 by adding 1, and then principal component analysis was performed using JMP Pro 15.

[0112] 5-9. DEG analysis: Using the software featureCounts, we counted the mapped fragments and calculated FPKM, FPKM-UQ, and TPM values. Furthermore, using the software DESeq2, we performed RLE normalization based on the read count values, calculated the difference in expression level between samples [log2(Fold Change)], and extracted differentially expressed genes (DEGs).

[0113] The results of the principal component analysis are shown in Figure 1. The clones with high immunoregulatory ability (Gr1: MSC1, 2), the clones with low immunoregulatory ability (Gr2: MSC3, 4), and Lonza BMMSCs (Gr3: MSC5, 6) showed relatively similar gene expression profiles within each group.

[0114] The results of the DEG analysis (Volcano plot) are shown in Figure 2. The vertical axis of the Volcano plot represents the fold change value, and the horizontal axis represents the p-value. In the Volcano plot, genes with a larger expression ratio (i.e., a greater likelihood of significant differences) are expressed at the top ends of the graph. The red plot (the right plot, with the origin (0) as the boundary) represents genes whose expression levels were increased in Gr1, a clone with high M2 shift activity, compared to Gr2, a clone with low M2 shift activity. The blue plot (the left plot, with the origin (0) as the boundary) represents genes whose expression levels were decreased in Gr1, a clone with high M2 shift activity, compared to Gr2, a clone with low M2 shift activity. Among the genes whose names are indicated in the figure, TGM2, PTGFRN, and EPB41L3 were significantly different in expression levels in BMMSCs (Gr3: MSC5, 6) compared to Gr2. In the figure, gene names other than TGM2, PTGFRN, and EPB41L3 (gene names shown in blue) are genes that were particularly noted for their increased expression levels in Gr1 compared to BMMSCs (Gr3: MSC5, 6). The gray area extending from the origin to the bottom (near the horizontal axis) is a non-DEG.

[0115] Example 6: Genes differentially expressed in REC clones and human BMMSCs Among the differentially expressed genes extracted by the DEG analysis in Example 5, 23 genes (CHI3L1, PTGFRN, TNFAIP6, CXCL8, PTGES, CXCL6, CXCL5, POSTN, TGFA, TGM2, CYP1B1, LACC1, LAMA4, AKR1C1, PID1, SLC16A6, EREG, HOXA5, PAPPA, MME, RAB27B, HCP5, FAM20A) were selected, and the relative expression levels of the 23 genes were measured in nine REC clones (1708AC#1, 1807#1, 1807#10, 1706AC(1)#11, 1807#5, 1707(3)#2, 1807#29, 1706AC(1)#5, 1611#9) and two human BMMSCs (20TL293908, 21TL076024).

[0116] Each cell was suspended in DMEM low glucose medium (containing 20% ​​FBS, 100 units / mL P / S, 0.01 mol / mL HEPES, and 20 ng / mL bFGF) and plated in a 10 cm 2 2 x 10 on a dish 5 Cells were seeded at a cell density of 1000 cells / dish and cultured in a CO2 incubator (37°C, 5% CO2). After confirming that each cell type reached approximately 70-80% confluency, the culture supernatant was aspirated and each cell type was washed with ice-cold PBS. After washing, total RNA was extracted from each cell lysate and each resulting cell type using an RNeasy® Mini Kit (QIAGEN). Total RNA was extracted according to the RNeasy® Mini Kit protocol.

[0117] The concentration of total RNA derived from each cell line was measured using a Qubit 2.0 Fluorometer (Invitrogen). cDNA was synthesized using total RNA derived from each cell line adjusted to 100 μg / mL. cDNA synthesis was performed using SuperScriptIV VILO master mix (Invitrogen) according to the manufacturer's protocol. For gene expression analysis, the obtained cDNA was used as a template and analyzed using TaqMan Gene Expression Assays (Applied Biosystems), TaqMan TM Each sample was prepared using Fast Advanced Master Mix (Applied Biosystems). The TaqMan Gene Expression Assays used are listed in Table 5.

[0118]

[0119] The Ct values ​​of 23 genes in each sample were calculated using the 7500 Fast Real-time PCR system (Applied Biosystems). GAPDH was used as a reference gene, and the relative expression level of each gene was calculated using a 2- ΔCt The relative expression level of each clone (2- ΔΔCt The correlation between the M2 shift activity and the M2 shift activity in each cell type obtained in Example 2 was evaluated by plotting the M2 shift activity (value of hEREG, hCYP1B1, and hTNFAIP6) on the horizontal axis and the M2 shift activity in each cell type obtained in Example 2 on the vertical axis ( FIG. 3 ). Candidate factors that were particularly strongly associated with the M2 shift effect were found to be hEREG, hCYP1B1, and hTNFAIP6.

[0120] In addition, the relative expression level of each clone (2- ΔCt The genes showing high relative expression levels in the three clones 1708AC#01, 1807#1, and 1807#10, which were particularly high in M2 shift activity among the cells obtained in Example 2, were evaluated using the vertical axis (value of ). hMME was identified as a candidate factor that is particularly strongly associated with the M2 shift activity.

[0121] In addition, the relative expression level of each clone (2- ΔΔCt The correlation between the IL-6 production suppression rate by each cell type obtained in Example 4 was evaluated by plotting the IL-6 production suppression rate (value of IL-6 suppression rate) on the horizontal axis and the IL-6 production suppression rate by each cell type obtained in Example 4 on the vertical axis ( FIG. 5 ). The ratio of serum IL-6 in the colitis-induced mice administered each cell type relative to the serum IL-6 concentration in the colitis-induced mice administered PBS was used as a control.

[0122] As a result, hCXCL5 was identified as a candidate factor that is particularly strongly associated with the M2 shift activity. High relative expression levels of hCXCL5 were observed in 1708AC#01, 1807#1, and 1807#10, the three clones with particularly high M2 shift activity among the cells obtained in Example 2 (Figure 6).

[0123]

[0124] Example 7 Protein expression levels of differentially expressed genes in each REC clone The protein expression levels of the candidate factor MME and the control CD73 were measured in each of 11 REC clones (1708AC#1, 1807#1, 1807#10, 1706AC(1)#11, 1807#5, 1606#15, 1706AC(1)#15, 1707(3)#2, 1807#29, 1706AC(1)#5, and 1611#9).

[0125] Each of the 11 REC clones (1708AC#1, 1807#1, 1807#10, 1706AC(1)#11, 1706AC(1)#15, 1807#5, 1606#15, 1707(3)#2, 1807#29, 1706AC(1)#5, and 1611#9) was suspended in DMEM low glucose medium (containing 20% ​​FBS, 100 units / mL penicillin-streptomycin (P / S), 0.01 mol / L HEPES, and 20 ng / mL bFGF) at a cell suspension concentration of 2 × 10 5Cells were seeded onto 10-cm dishes at a cell density of 100 cells / dish and cultured in a CO2 incubator (37°C, 5% CO2). After confirming that each cell type reached approximately 70-80% confluency, the culture supernatant was aspirated and each cell was washed with PBS. After washing with PBS, the cells were detached by trypsin treatment and collected. The collected cell suspension was centrifuged (200 g, 5 minutes, room temperature), and the supernatant was aspirated and removed. After resuspending in HBSS (containing 2% FBS, 100 units / mL P / S, and 0.01 mol / mL HEPES), the cells were counted and collected at a concentration of 2 x 10 cells in 100 μL of HBSS. 5 Three 1.5 mL tubes containing cell suspensions prepared to yield single cells were prepared for each REC clone. APC-labeled anti-human MME antibody (MME-APC) (Biolegend), APC-labeled anti-human CD73 antibody (CD73-APC) (Biolegend), or APC-labeled mouse IgG1 antibody (isotype control: iso-APC) (R&D Systems) was added to each cell suspension, and staining was performed for 30 minutes on ice in the dark. After 30 minutes, 500 μL of HBSS was added to each cell suspension, followed by centrifugation (3000 rpm, 5 minutes, 4°C) and removal of the supernatant. Each cell was suspended in 400 μL of HBSS containing 2 μg / mL propidium iodide solution (PI), and the resulting cell suspension was filtered through a 40 μm mesh to obtain a single-cell suspension. The mean fluorescence intensity (MFI) of MME-APC, CD73-APC, and Iso-APC in live cells in each single-cell suspension was measured using a CytoFLEX Flow Cytometer (Beckman Coulter). The protein expression levels of MME and CD73 were expressed as MFI values ​​for MME-APC and CD73-APC, with Iso-APC set to 1. The protein expression levels of MME and CD73 in each REC clone are shown in Table 8.

[0126] hMME showed high protein expression levels in 1708AC#01, 1807#1, and 1807#10, three clones with particularly high M2 shift activity among the cells obtained in Example 2 (Table 8, Figure 7).

[0127]

[0128] Example 8: Method for producing a cell population: After preparing MSCs, cell populations are selected by comparing the expression levels of five genes. (Step 1) Preparation of MSCs: Mesenchymal stem cells are prepared from human bone marrow (or may be isolated from dental pulp, fat, placental chorion, umbilical cord, umbilical cord blood, or amniotic membrane). Alternatively, commercially available mesenchymal stem cells are obtained and passaged until suitable for the experiment.

[0129] (Step 2) Gene Expression Analysis Gene expression analysis is performed for the following three genes according to the procedure described in Example 6. A cell population that satisfies any one of the following conditions (a) to (e) is selected: (a) the relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.01 or more; (b) the relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.03 or more; (c) the relative expression level of the EREG gene to the expression level of the GAPDH gene is 0.003 or more; (d) the relative expression level of the MME gene to the expression level of the GAPDH gene is 0.01 or more; (e) the relative expression level of the CXCL5 gene to the expression level of the GAPDH gene is 0.001 or more.

[0130] (Step 3) Measurement of M2 shift activity The selected cell population is measured for its ability to promote M2 macrophage induction according to the procedure described in Example 2. According to the present invention, a cell population exhibiting a high ability to promote M2 macrophage induction can be obtained by using the above conditions (a), (b), (c), (d), and / or (e) as indicators.

[0131] Example 9: Method for evaluating cell populations by comparing the expression levels of five genes (standard test) Any obtained mesenchymal stem cell population is subjected to gene expression analysis for the following three genes according to the procedure described in Example 6. The cell population is evaluated using as an index whether it satisfies any of the following conditions (a) to (e):

[0132] (a) the relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.01 or more; (b) the relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.03 or more; (c) the relative expression level of the EREG gene to the expression level of the GAPDH gene is 0.003 or more; (d) the relative expression level of the MME gene to the expression level of the GAPDH gene is 0.01 or more; and (e) the relative expression level of the CXCL5 gene to the expression level of the GAPDH gene is 0.001 or more. According to the present invention, a method for evaluating a cell population that exhibits a high M2 macrophage induction-promoting activity can be provided by using the above conditions (a), (b), (c), (d) and / or (e) as indicators.

[0133] Example 10 Confirmation of Therapeutic Effect (10-1) Therapeutic Effect of Selected Cells Using a Mouse Model of DSS-Induced Colitis Cells selected by the method of the present invention can be evaluated using a mouse model of DSS-induced colitis. For example, a colitis model using DSS drinking water is created according to the method described in the section "Example 4: Selection of Cells Using a DSS Model." In this example, REC cells were administered before DSS drinking water to observe the therapeutic effect in the early stages of onset. However, REC cells can also be administered after the start of DSS drinking water or around the end of the DSS drinking water period to evaluate the therapeutic effect after onset, or the effect on resolving inflammation and accelerating recovery during the repair phase.

[0134] (10-2) Therapeutic Effect of Selected Cells Using SKG Mice (Rheumatoid Arthritis Model) Cells selected by the method of the present invention can be evaluated using SKG mice, a mouse model of rheumatoid arthritis. For example, the therapeutic effect on rheumatoid arthritis can be evaluated according to the methods described in Ueyama et al., Sci Rep. 2020 Feb 20; 10(1): 3076 and Yamamoto et al., Stem Cells Transl Med. 12(3) pp169-182.

Claims

1. A cell population comprising mesenchymal stem cells that promote induction into M2 macrophages and express at least one gene selected from the group consisting of the TNFAIP6 gene, the CYP1B1 gene, the EREG gene, the MME gene, and the CXCL5 gene.

2. The cell population according to claim 1, wherein the gene is at least one gene selected from the group consisting of the TNFAIP6 gene, the CYP1B1 gene, and the EREG gene.

3. The cell population according to claim 2, wherein the genes are the TNFAIP6 gene, the CYP1B1 gene, and the EREG gene.

4. The cell population according to claim 1, wherein the gene is at least one gene selected from the group consisting of the CYP1B1 gene, the MME gene, and the CXCL5 gene.

5. The cell population of claim 4, wherein the genes are the CYP1B1 gene, the MME gene, and the CXCL5 gene.

6. The cell population of claim 1, wherein the mesenchymal stem cells are derived from bone marrow.

7. The cell population according to claim 1, wherein the mesenchymal stem cells are highly purified mesenchymal stem cells.

8. The cell population described in claim 1, wherein the relative expression level of the TNFAIP6 gene to the expression level of the GAPDH gene is 0.01 or more, the relative expression level of the CYP1B1 gene to the expression level of the GAPDH gene is 0.03 or more, the relative expression level of the EREG gene to the expression level of the GAPDH gene is 0.003 or more, the relative expression level of the MME gene to the expression level of the GAPDH gene is 0.01 or more, and / or the relative expression level of the CXCL5 gene to the expression level of the GAPDH gene is 0.001 or more.

9. The cell population described in claim 6, wherein the mesenchymal stem cells are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and have a coefficient of variation of forward scattered light in flow cytometry of 35% or less.

10. A composition comprising the cell population or a culture thereof according to any one of claims 1 to 9.

11. A composition for promoting induction of M2 macrophages, comprising the cell population or a culture thereof according to any one of claims 1 to 9.

12. A pharmaceutical composition comprising the cell population or a culture thereof according to any one of claims 1 to 9.

13. The pharmaceutical composition according to claim 12, which is used as a therapeutic agent for an M2-related disease.

14. A method for evaluating the ability of a test mesenchymal stem cell to promote induction into M2 macrophages, comprising a step of measuring the expression of at least one gene selected from the group consisting of the TNFAIP6 gene, the CYP1B1 gene, the EREG gene, the MME gene, and the CXCL5 gene in the test mesenchymal stem cell.

15. A method for producing cells, comprising the steps of culturing test mesenchymal stem cells and collecting cells capable of promoting induction into M2 macrophages using the expression of at least one gene selected from the group consisting of the TNFAIP6 gene, the CYP1B1 gene, the EREG gene, the MME gene, and the CXCL5 gene as an indicator.

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