Macrophage, method for producing macrophage, differentiation inducer, macrophage proliferation promoter, and method for proliferating macrophages

Abstract

To provide a macrophage production method capable of yielding macrophages that have a prolonged survival rate, allow for freezing preservation, and offer versatility as a tool in scientific studies.SOLUTION: A method for producing macrophages comprises a culture step for culturing hematopoietic precursor cells in the presence of a TREM2 signal activator.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for producing macrophages, a differentiation inducer, a differentiation induction kit, a method for culturing macrophages, a macrophage proliferation promoter, a macrophage proliferation promotion kit, a method for proliferating macrophages, and macrophages. [Background technology]

[0002] Macrophages are white blood cells that contribute to infection defense and tissue homeostasis. In vivo, macrophages are derived from hematopoietic stem cells in the bone marrow, and their differentiation requires stimulation with macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF). In fact, a method has been established to induce differentiation of mouse bone marrow cells into cells with the properties of monocytes and macrophages (bone marrow derived macrophages: BMDMs) by culturing them in vitro in the presence of M-CSF. BMDMs are widely used as a research tool for in vitro macrophages.

[0003] BMDMs have the following problems: i) the cost of mass culturing BMDMs is high because M-CSF used for induction is expensive, ii) they can only survive for about a week, iii) proliferation stops after differentiation, so the number of cells obtained from one culture is limited, and iv) they cannot be thawed and recultured after cryopreservation due to the above iii). In addition to BMDMs, various macrophage cell lines derived from rodents and humans are frequently used in in vitro research. These cells do not have the problems i) to iv) above, but because they are tumor cells, their use in in vivo tests such as administration to living organisms is limited.

[0004] Patent Document 1 discloses a differentiation inducer that induces differentiation of monocytes into macrophages without using M-CSF, which contains a compound that has angiotensin II receptor antagonistic activity and peroxisome proliferator-activated receptor-γ activating activity. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2015-47125 A [Non-patent literature]

[0006] [Non-Patent Document 1] Hadas Keren-Shaul, et al., "A Unique Microglia Type Associated with Restricting Development of Alzheimer's Disease", Cell, 2017, 169, 1276-1290 [Non-Patent Document 2] Diego Adhemar Jaitin, et al., “Lipid-Associated Macrophages Control Metabolic Homeostasis in a Trem2-Dependent Manner”, Cell, 2019, 178, 686-698 Summary of the Invention [Problem to be solved by the invention]

[0007] However, Patent Document 1 does not consider the survival period of macrophages obtained by the differentiation inducer, their proliferation characteristics after differentiation, and the feasibility of cryopreservation.

[0008] The present invention has been made in consideration of the above circumstances, and aims to provide a method for producing macrophages that can yield macrophages that have a long survival and proliferation period, can be cryopreserved, and are highly versatile as a research tool, a differentiation inducer, a differentiation induction kit, a method for culturing macrophages, a macrophage proliferation promoter, a macrophage proliferation promotion kit, and a method for proliferating macrophages. Another aim of the present invention is to provide macrophages that have a long survival and proliferation period, can be cryopreserved, and are highly versatile as a research tool. [Means for solving the problem]

[0009] It has been revealed that immune cell remodeling occurs at the onset of disease, and macrophage populations with special characteristics appear in tissues, playing an important role in exacerbating or suppressing the disease. For example, activated microglia (Disease-associated microglia: DAM) that show special characteristics appear in the brains of Alzheimer's disease model mice (see Non-Patent Document 1). In addition, macrophage populations (Lipid-associated macrophages: LAM) that do not exist under normal conditions appear in the adipose tissue of mice that have developed lifestyle-related diseases due to high-fat diet loading (see Non-Patent Document 2). DAM and LAM are one of the molecules that are characteristically highly expressed in TREM2 (triggering receptor expressed on myeloid cells 2), a lipid recognition receptor. Non-Patent Documents 1 and 2 show that TREM2 is deeply involved in the induction of these disease-related macrophages.

[0010] The present inventors discovered that macrophage-like cells appear when hematopoietic progenitor cells are stimulated with a TREM2 signal activator, and thus completed the present invention.

[0011] (Method for producing macrophages) The method for producing macrophages described herein includes: The method includes a culture step of culturing hematopoietic progenitor cells in the presence of a TREM2 signal activator.

[0012] The TREM2 signal activator is It may be a lipid.

[0013] The TREM2 signal activator is It may be a brain-derived lipid or a lipid having two long chain fatty groups.

[0014] The TREM2 signal activator is It may be a compound represented by formula (I), an ester thereof or a salt thereof. [ka] (The number of carbon atoms is 60 to 90, and R 1 represents a saturated or unsaturated aliphatic hydrocarbon group, R 2 represents a cyclic structure or a saturated or unsaturated aliphatic hydrocarbon group which may have a substituent.

[0015] The surface of the cell culture vessel for culturing the hematopoietic progenitor cells is The antibody may be coated with the TREM2 signal activator.

[0016] The hematopoietic progenitor cells are The cells may be myeloid progenitor cells.

[0017] The hematopoietic progenitor cells are It may be a macrophage-dendritic cell precursor cell or a common monocytic precursor cell.

[0018] (Macrophage differentiation inducer) The agent for inducing differentiation of hematopoietic progenitor cells into macrophages described herein is Contains TREM2 signal activator.

[0019] (Macrophage differentiation induction kit) The kit for inducing differentiation of hematopoietic progenitor cells into macrophages described in the present specification comprises: The device is provided with a cell culture vessel whose surface is coated with a TREM2 signal activator.

[0020] (Macrophage Cultivation Method) The method for culturing macrophages described herein comprises: The method includes a culture step of culturing hematopoietic progenitor cells in the presence of a TREM2 signal activator.

[0021] (Macrophage proliferation promoter) The macrophage proliferation promoter described herein is Contains TREM2 signal activator.

[0022] (Macrophage proliferation promotion kit) The macrophage proliferation promoting kit described herein comprises: The device is provided with a cell culture vessel whose surface is coated with a TREM2 signal activator.

[0023] (Method for Proliferating Macrophages) The method for expanding macrophages described herein includes: The method includes a culturing step of culturing macrophages in the presence of a TREM2 signal activator.

[0024] (Macrophage) The macrophages described herein are The cells have the ability to proliferate after 10 days of subculture in a medium that does not contain either macrophage colony-stimulating factor or granulocyte-macrophage colony-stimulating factor and contains a TREM2 signal activator.

[0025] The macrophages described herein are By culturing hematopoietic progenitor cells in a medium that does not contain either macrophage colony-stimulating factor or granulocyte-macrophage stimulating factor, but does contain a TREM2 signal activator, differentiation is induced in a TREM2-dependent manner.

[0026] The macrophages described herein are After cryopreservation, the cells have the ability to proliferate in a medium that does not contain either macrophage colony-stimulating factor or granulocyte-macrophage colony-stimulating factor, but does contain a TREM2 signal activator.

[0027] The macrophages described herein are After 24 hours of culture in a medium containing lipopolysaccharide, the concentrations of TNF-α, IL-6 and nitric oxide in the culture supernatant are 30% or less compared to those of bone marrow-derived macrophages induced from bone marrow cells with macrophage colony-stimulating factor.

[0028] The macrophages described herein are Among the genes shown in Table 1 below, expression of one or more genes is higher than that of at least one selected from alveolar macrophages, BMDM, M1-induced BMDM, M2-induced BMDM, Kupffer cells, microglia, osteoclasts, and peritoneal exudate macrophages. [Table 1]

[0029] The macrophages described herein are Among the genes shown in Table 2 below, expression of one or more is lower compared to at least one selected from alveolar macrophages, BMDM, M1-induced BMDM, M2-induced BMDM, Kupffer cells, microglia, osteoclasts, and peritoneal exudate macrophages. [Table 2] Effect of the Invention

[0030] According to the present invention, macrophages that have a long survival and proliferation period, can be cryopreserved, and are highly versatile as research tools can be obtained. [Brief description of the drawings]

[0031] [Figure 1]Figure 1 shows images of macrophages induced to differentiate from mouse bone marrow cells (A) shows an image of macrophages induced to differentiate with brain lipids (B) shows an image of macrophages induced to differentiate with M-CSF (C). [Diagram 2] FIG. 1 shows dot plots obtained by flow cytometry analysis in Test Example 2. [Diagram 3] FIG. 13 is a diagram showing a univariate histogram of surface antigens obtained by flow cytometry analysis in Test Example 2. [Figure 4] FIG. 13 is a graph showing the number of cells relative to the brain lipid mass per unit culture area in Test Example 3. [Diagram 5] Fig. 1 shows the change in cell count over time in Test Example 4. The upper row of (A) shows the cell count after 0 to 21 days of culture. The lower row of (A) shows the increase fold of the cell count after 0 to 21 days of culture, with the cell count on day 0 taken as 1. The upper row of (B) shows the cell count after 0 to 544 days of culture. The lower row of (B) shows the increase fold of the cell count after 0 to 544 days of culture, with the cell count on day 0 taken as 1. (C) shows the cell count of macrophages cultured in the presence or absence of brain lipids. [Figure 6] FIG. 13 is a graph showing the change over time in the number of macrophages thawed after cryopreservation in Test Example 5. [Figure 7] 1 shows the number of macrophages after stimulation with brain lipids in Test Example 6. (A) shows the number of macrophages differentiated from mixed-cultured Lin-positive cells and Lin-negative cells. (B) shows the number of macrophages differentiated from singly-cultured Lin-positive cells and Lin-negative cells. [Figure 8] FIG. 13 is a graph showing the viable cell count after differentiation induction of each mouse bone marrow cell in Test Example 7. [Figure 9] FIG. 13 shows ligand activity using TREM2 reporter cells according to Test Example 8. [Figure 10] FIG. 13 is a graph showing the number of viable cells after differentiation of mouse bone marrow cells was induced with a test substance in Test Example 8. [Figure 11] FIG. 13 is a graph showing the phagocytic ability of macrophages in Test Example 9. [Figure 12]FIG. 1 shows the cytokine production response of macrophages to lipopolysaccharide (LPS) stimulation in Test Example 10. (A), (B), (C), (D), and (E) show the concentrations of monocyte chemoattractant protein-1 (MCP-1), TNF-α, IL-6, IL-10, and nitric oxide (NO), respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described with reference to the accompanying drawings, in which: FIG.

[0033] The method for producing macrophages according to the present embodiment includes a culturing step of culturing hematopoietic progenitor cells in the presence of a TREM2 signaling activator. TREM2 is an immunoglobulin superfamily receptor that associates with DAP12 (DNAX-activating protein of 12 kDa), an adaptor molecule. The TREM2 signaling activator may be any substance that specifically binds to TREM2 and generates a signal via DAP12.

[0034] The TREM2 signal activator is, for example, an antibody or an antigen-binding fragment thereof or a compound having TREM2 agonist activity or DAP12 agonist activity, and a TREM2 ligand. The antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may also be a human chimeric antibody, a humanized antibody, or a human antibody. In this specification, the antibody includes the antigen binding of the antibody. The antigen-binding fragment is a protein or peptide comprising a portion (partial fragment) of an antibody, which retains the functional binding ability of the antibody to an antigen, and may be, for example, F(ab')2, Fab', Fab, Fab3, single-chain Fv (scFv), (tandem) bispecific single-chain Fv (sc(Fv)2), single-chain triple body, nanobody, divalent VHH, pentavalent VHH, minibody, (two-chain) diabody, tandem diabody, bispecific tribody, bispecific bibody, dual affinity retargeting molecule (DART), triabody (or tribody), tetrabody (or [sc(Fv)2]2), or disulfide-linked Fv (dsFv), or a polymer thereof (Nature Biotechnology, 29(1):5-6 (2011); Maneesh Jain et al., TRENDS in Biotechnology, 25(7)(2007):307-316, and Christoph Stein et al., Antibodies(1):88-123(2012).

[0035] The TREM2 signal activator is, for example, a TREM2 ligand or lipid. A lipid is an organic compound that is insoluble in water and easily soluble in an organic solvent. There is no particular limitation on the lipid, and it may be liquid or solid at room temperature. Examples of the lipid include sphingoglycolipids, phospholipids, sulfated glycolipids, and fatty acids. Preferably, the lipid is a lipid having two long-chain fatty acid groups. Examples of the long-chain fatty acid group include fatty acid groups having 6 to 60 or 10 to 50 carbon atoms, and the number of carbon atoms constituting the carbon chain of each long-chain fatty acid group may be the same or different.

[0036] The fatty acid may be a saturated fatty acid or an unsaturated fatty acid. Preferred examples of lipids include 2-tetradecylhexadecanoic acid (THA), β-glucosylceramide (β-GluCer), α-galactosylceramide (α-GalCer), phosphatidylcholine (PC), lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE), sulfatide (Sulf), and mycolic acid (MA). The TREM2 signal activator may be a mixture of multiple types of lipids, such as total lipids extracted from animal-derived tissues such as the brain, plant-derived tissues, and microorganisms such as bacteria using an organic solvent, or synthetic lipids that bind to TREM2. One embodiment of the TREM2 signal activator includes cells or microorganisms having lipids on the cell surface. Preferably, the TREM2 signal activator is a brain-derived lipid or MA.

[0037] The TREM2 signal activator may be a compound represented by formula (I), an ester thereof, or a salt thereof. The number of carbon atoms in one molecule of the compound is, for example, 60 to 90 or 70 to 80. 1 R represents a saturated or unsaturated aliphatic hydrocarbon group. 1 The number of carbon atoms in R is, for example, 6 to 60, 10 to 50, 20 to 40, 22 to 30, or 22 to 26. 2 R represents a cyclic structure or a saturated or unsaturated aliphatic hydrocarbon group which may have a substituent. 2 The number of carbon atoms constituting the cyclic structure is, for example, 6 to 60, 10 to 50, 20 to 40, 22 to 30, or 22 to 26. The number of carbon atoms constituting the cyclic structure is, for example, 3 to 6. The cyclic structure is a cycloalkylene group represented by R 2 The cycloalkyl group may be included in R 2 R 2 The substituent is not particularly limited and may be a methyl group, a hydroxy group, a carbonyl group, a carboxyl group, or the like.

[0038] [ka]

[0039] The salt of the compound represented by the above formula (I) is not particularly limited as long as it is a pharmacologically acceptable salt, and may be either an acid salt or a basic salt. Examples of the salt include alkali metal salts such as lithium salt, sodium salt, and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, and phosphate, as well as formate, acetate, oxalate, propionate, hexanoate, cyclopentanepropionate, glycolate, pyruvate, lactate, malonate, succinate, malate, fumarate, tartrate, dibenzoyltartrate, ditoluoyltartrate, citrate, benzoate, o-(4-hydroxybenzoyl)benzoate, cinnamate, mandelate, methionine, methyl ... and organic acid salts such as ethanesulfonate, ethanesulfonate, 1,2-ethanedisulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-chlorobenzenesulfonate, 2-naphthalenesulfonate, p-toluenesulfonate, aspartate, camphorsulfonate, glucoheptanoate, 3-phenylpropionate, trimethylacetate, tert-butylacetate, lauryl sulfate, gluconate, glutamate, hydroxynaphthoate, salicylate, stearate, trifluoroacetate (TFA) salt, maleate, dimaleate, and muconate.

[0040] The ester of the compound represented by the above formula (I) is not particularly limited as long as it is a pharmacologically acceptable ester, and examples thereof include carbonate ester, phosphate ester, nitrate ester, sulfate ester, borate ester, sulfonate ester, etc. Note that this embodiment also includes various hydrates and solvates of the compound of formula (I) and its salts, and crystalline polymorphic substances.

[0041] The TREM2 signal activator may be selected using cells in which TREM2 and DAP12 are expressed and a reporter protein is expressed by the signal of TREM2 due to the binding of a ligand. TREM2 and DAP12 can be expressed in cells by a known method, for example, by introducing a gene expression plasmid or the like into the cells. Any reporter protein can be used, and examples of the reporter protein include green fluorescent protein (GFP) and the luminescent protein luciferase. It is known that a gene controlled by a NFAT (nuclear factor of activated T cells) type transcription factor is expressed by a TREM2 signal. Therefore, preferably, cells having a NFAT type transcription factor may be used as cells in which TREM2 and DAP12 are expressed. Specifically, cells in which a gene encoding a reporter protein is introduced as a gene whose expression is controlled by a NFAT type transcription factor and in which TREM2 and DAP12 are expressed may be used for evaluating the ligand.

[0042] Hematopoietic progenitor cells are cells contained in bone marrow or umbilical cord blood. Hematopoietic progenitor cells are, for example, myeloid progenitor cells. As hematopoietic progenitor cells, bone marrow cells collected from bone marrow or mononuclear cells collected from umbilical cord blood may be used. As hematopoietic progenitor cells, macrophage and dendritic cell progenitors (MDP) and common monocyte progenitors (cMoP) are particularly preferred.

[0043] In the culture step, the concentration of hematopoietic progenitor cells is not particularly limited, but may be, for example, 2 ) per 1×10 3 ~1×10 7 cells or 1 x 10 4 ~1×10 6 The cells are seeded. Preferably, the cells are seeded in a unit culture area (cm 2 ) per 1×10 5 Hematopoietic progenitor cells are seeded.

[0044] In the culturing step, hematopoietic progenitor cells may be cultured in a cell culture vessel holding a TREM2 signal activator. Examples of cell culture vessels include cell culture plates, cell culture flasks, and cell culture dishes. When the TREM2 signal activator is insoluble in water and insoluble in liquid medium, hematopoietic progenitor cells may be cultured in a cell culture vessel whose surface is coated with the TREM2 signal activator. When brain-derived lipids are used as the TREM2 signal activator, the mass of lipids coating the surface of the cell culture vessel may be, for example, 1×10 4 ~1×10 6 1.0 μg / cm for each hematopoietic progenitor cell 2 Above 1.5 to 20 μg / cm 2 , 2-18μg / cm 2 , 3-16μg / cm 2 , preferably 3 to 12 μg / cm 2 To coat the surface of a cell culture vessel with a TREM2 signal activator, the ligand is dissolved (suspended) in an organic solvent or the like, added to the cell culture vessel, and the organic solvent is then dried.

[0045] In the culture step, the cells may be cultured using a known medium by a cell culture method. The culture conditions are, for example, 37° C., CO 2 The concentration is 5%. During the culture, it is preferable to change the medium, for example, every 3 to 4 days. Macrophage-like adherent cells can be obtained about 7 to 10 days after the start of culture.

[0046] The macrophages obtained by the production method according to the present embodiment maintain their proliferation as long as they are stimulated with a TREM2 signal activator, and the number of days (survival period) that they can be cultured is at least 540 days or more. Furthermore, when the macrophages are cryopreserved, thawed and cultured, they maintain their proliferation, so that the macrophages can be cryopreserved. Furthermore, since the proliferation of the macrophages stops when cultured in the absence of a TREM2 signal activator, they are not tumor cells, and their use in vivo is not limited, making them highly versatile as a research tool. Furthermore, the macrophages can also be used in cell therapy for diseases such as cancer.

[0047] In another embodiment, a macrophage is provided, and differentiation of the macrophage is induced in a TREM2-dependent manner by culturing hematopoietic progenitor cells in a medium that does not contain either M-CSF or GM-CSF and contains a TREM2 signal activator.

[0048] Furthermore, as shown in Test Example 4 below, BMDM induced from bone marrow cells with M-CSF lost proliferation ability after 5 days when passaged in a medium not containing M-CSF, whereas the macrophages had proliferation ability after 10 days of passaged in the presence of a TREM2 signaling activator, even in the absence of M-CSF in the medium. Thus, in another embodiment, macrophages are provided, which have proliferation ability after 10 days of passaged in a medium not containing either M-CSF or GM-CSF and containing a TREM2 signaling activator, preferably after 15 days, 20 days, 50 days, or 100 days, more preferably after 200 days, 300 days, 400 days, and even more preferably after 500 days, after the start of induction or after cryopreservation.

[0049] As shown in the following Test Example 5, BMDM thawed after cryopreservation did not proliferate even in the presence of M-CSF, whereas the thawed macrophages proliferated in the presence of a TREM2 signal activator. Therefore, in another embodiment, macrophages capable of proliferating in a medium containing neither M-CSF nor GM-CSF but a TREM2 signal activator after cryopreservation are provided. The cryopreservation temperature is not particularly limited as long as it is a temperature normally used for the cryopreservation of cells, for example, −80° C. The period of cryopreservation is not particularly limited, and may be immediately after confirmation of freezing, or may be 5, 10, 20, or 30 days after freezing, or may be several months or several years.

[0050] Furthermore, as shown in the following Test Example 10, the macrophages were cultured in a medium containing LPS (e.g., 10 ng / ml) for 24 hours, and the concentrations of MCP-1, TNF-α, IL-6, and NO in the culture supernatant were lower than those of BMDM cultured in a medium containing LPS for 24 hours. Thus, in another embodiment, macrophages are provided, and the concentrations of TNF-α, IL-6, and nitric oxide in the culture supernatant of the macrophages after 24 hours of culture in a medium containing LPS are 30% or less compared to BMDM induced from bone marrow cells with M-CSF. The concentration of TNF-α in the culture supernatant may be 25% or less or 20% or less compared to BMDM. The concentration of IL-6 in the culture supernatant may be 25% or less, 20% or less, or 15% or less compared to BMDM. The concentration of NO in the culture supernatant may be 25% or less compared to BMDM.

[0051] Furthermore, the macrophages of this embodiment may have higher expression of one or more, two or more, three or more, four or more, five or more, ten or more, twenty or more, thirty or more, forty or more, fifty or more, sixty or more, seventy or more, eighty or more, or eighty-eight of the genes listed in Table 3, compared to at least one, preferably two or more, three or more, four or more, five or more, six or more, seven or more, or all eight of these genes selected from alveolar macrophages, BMDM, M1-induced BMDM, M2-induced BMDM, Kupffer cells, microglia, osteoclasts, and peritoneal exudate macrophages.

[0052] [Table 3]

[0053] Independently of, or in addition to, the expression of the genes in Table 3, the macrophages express 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, or more than 100 of the genes listed in Table 4. or 80 or more, or 87 of the above may have lower expression than at least one, preferably two or more, three or more, four or more, five or more, six or more, seven or more, or all eight of the above selected from alveolar macrophages, BMDM, M1-induced BMDM, M2-induced BMDM, Kupffer cells, microglia, osteoclasts, and peritoneal exudate macrophages.

[0054] [Table 4]

[0055] Preferably, the expression of the gene is the expression level of RNA. Throughout this specification, the term "level" refers to an index of abundance expressed in a numerical value, such as concentration, amount, or an index that can be used instead of concentration. Thus, the level may be a measured value or a value converted into concentration. The level may also be an absolute value such as abundance and abundance per unit area, or a relative value compared to a control set as necessary.

[0056] The macrophages of this embodiment may have one, two or more, three or more, four or more, five or more, or all of the characteristics arbitrarily selected from the characteristics described above.

[0057] In another embodiment, there is provided a method for preparing macrophages, a method for producing macrophages, a method for inducing macrophages, a method for culturing macrophages, or a method for proliferating macrophages, which includes the above-mentioned culturing step. The method for culturing macrophages allows macrophages to be maintained for a long period of time. Furthermore, the method for proliferating macrophages causes macrophages to proliferate. Preferably, the method for producing macrophages, the method for preparing macrophages, the method for producing macrophages, the method for inducing macrophages, the method for culturing macrophages, and the method for proliferating macrophages do not use either M-CSF or GM-CSF.

[0058] In another embodiment, a differentiation inducer containing a TREM2 signal activator is provided. The differentiation inducer induces differentiation from hematopoietic progenitor cells to macrophages. The differentiation inducer may contain, in addition to the TREM2 signal activator, a solvent, water, ethanol, polyhydric alcohol, oil, surfactant, thickener, preservative, pH adjuster, and the like. The differentiation inducer may be, for example, in liquid, gel, cream, or solid form. Preferably, the differentiation inducer is added to a cell culture vessel or applied to the surface of the cell culture vessel to coat the surface.

[0059] The differentiation inducer makes it possible to obtain macrophages from hematopoietic progenitor cells that have a long survival time and can be cryopreserved. The macrophages are highly versatile as a research tool and can be easily used in various experiments.

[0060] In another embodiment, there is provided a macrophage proliferation promoter comprising a TREM2 signaling activator. The proliferation promoter promotes the proliferation of macrophages, particularly macrophages obtained by the above-mentioned production method. The proliferation promoter may contain other components of the TREM2 signaling activator, as with the differentiation inducer.

[0061] In another embodiment, a differentiation induction kit is provided, comprising a cell culture vessel whose surface is coated with a TREM2 signal activator. The differentiation induction kit is used to induce differentiation from hematopoietic progenitor cells to macrophages. The differentiation induction kit may further comprise a medium such as a basal medium, a serum-reduced medium, or a serum-free medium, serum necessary for cell culture such as fetal bovine serum (FBS), and other known additives added to the medium.

[0062] According to the cell culture vessel, the surface of which has been pre-coated with a TREM2 signal activator, macrophages that have a long survival time and can be cryopreserved can be easily obtained simply by seeding hematopoietic progenitor cells in the cell culture vessel and culturing them. The differentiation induction kit can also be used as a kit for promoting macrophage proliferation.

[0063] Preferably, the differentiation inducer and macrophage proliferation promoter do not contain either M-CSF or GM-CSF. Also, the macrophage differentiation induction kit and macrophage proliferation promotion kit do not contain either M-CSF or GM-CSF. EXAMPLES

[0064] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0065] Example 1: Preparation of lipid-induced macrophages (LIM) [Extraction of mouse brain lipids] Mouse brains (approximately 400 mg / mouse) were submerged in 1 ml of methanol in a polypropylene tube and cut into pieces with scissors. The entire sample was transferred to a glass test tube, and 1 ml of methanol and 4 ml of chloroform were added to make the volume ratio of chloroform to methanol (MeOH) 2:1 (CM 2:1). The sample was mixed at room temperature for 3 hours using a shaker (200 rpm). The sample was centrifuged at 2000 rpm for 15 minutes, and the supernatant was filtered and collected in a glass bottle as extract 1.

[0066] The brain residue remaining in the glass test tube was mixed with 2 ml of methanol and 4 ml of chloroform at room temperature for 16 hours using a shaker (200 rpm). The filtered supernatant was collected in the glass bottle containing Extraction Solution 1 and mixed well to make Extraction Solution 2. Extraction Solution 2 was stored at -30°C until use.

[0067] 1.8 ml of Extract 2 was dispensed into a weighed glass bottle, and the solvent was dried by blowing nitrogen while heating at 37°C, and the dry weight of lipids was measured. The lipids were dissolved in CM 2:1 to a concentration of 5 mg / ml to prepare a brain lipid extract.

[0068] [Coating cell culture plates with brain lipids] Brain lipid extracts were diluted with methanol to 50 μg / ml and plated on cell culture plates at 3 μg / cm 2 The solvent was dried in a clean bench.

[0069] [Induction of lipid-induced macrophages] The collected mouse bone marrow cells were diluted to 4 × 10 5 The bone marrow cell suspension was then added to the brain lipid-coated cell culture plate at 1 × 10 5 cells / 0.25ml / cm 2 The bone marrow cells were incubated at 37°C and 5% CO 2 The cells were cultured in an incubator. After 3 days of culture, half the amount of fresh medium was added to the culture plate. After that, the medium was changed every 3 to 4 days.

[0070] (result) Around 7 to 10 days after the start of culture, macrophage-like adherent cells began to appear. Figure 1(A) shows an image of LIM 14 days after the start of culture.

[0071] Comparative Example 1: Preparation of BMDM The collected mouse bone marrow cells were diluted to 4 × 10 5The bone marrow cell suspension was suspended in 10% FBS / RPMI at 2 × 10 cells / ml. 25 ng / ml M-CSF was added to the cell suspension. 2 × 10 bone marrow cell suspension was added to each well of a 24-well culture plate. 5 The bone marrow cells were added at a concentration of 0.5 ml per cell. The cells were incubated at 37°C, 5% CO 2 The cells were cultured in an incubator. After 3 days of culture, half the amount (0.25ml / well) of 25ng / ml M-CSF / 10%FBS / RPMI was added. After 5 days of culture, the supernatant was aspirated, 0.5ml / well of 10%FBS / RPMI was added, and the cells were detached with a scraper. The cells were diluted 1 / 4 with 10%FBS / RPMI, dispensed at 0.5ml / well, and 25ng / ml M-CSF was added. After that, half the amount of 25ng / ml M-CSF / 10%FBS / RPMI was replaced every 3-4 days.

[0072] (result) Five days after the start of culture, the number of cells was 4.0 to 6.5 × 10 5 Figure 1(B) shows images of M-CSF-induced BMDMs after 5 days in culture.

[0073] Test Example 1: Identification of cell types [Total RNA extraction from LIM] Total RNA was extracted from LIM according to the instructions for Sepasol (trademark)-RNA I Super G (Nacalai Tesque) as follows. 61 ml of Sepasol™-RNA I Super G was added to each sample (cells) and homogenized by pipetting. The sample was stirred with a vortex mixer and left to stand at room temperature for 5 minutes. 200 μl of chloroform was added to the sample, mixed by inversion, and left to stand at room temperature for 3 minutes. The sample was centrifuged at 12000G and 4°C for 15 minutes, and the upper aqueous phase was transferred to a 1.5 ml tube. 500 μl of isopropanol was added to the aqueous phase, and the mixed sample was left to stand at room temperature for 10 minutes. The sample was centrifuged at 12000G and 4°C for 10 minutes, and the supernatant was removed. 1 ml of 75% ethanol was added to the precipitate to wash the precipitate. It was further centrifuged at 12000G and 4°C for 5 minutes, and the supernatant was removed. The tube was opened, left to stand at room temperature for 10 minutes, and the remaining liquid was dried to obtain total RNA. 40 μl of nuclease-free water was added to the total RNA to dissolve the total RNA.

[0074] [DNase I treatment of total RNA] According to the instruction manual of Recombinant DNase I (Takara Bio), total RNA was treated with DNase I as follows. 5 μl of 10×DNase buffer, 2 μl (10 U) of Recombinant DNase I (RNase-free), 1 μl (20 U) of RNase inhibitor, and 4 μl of DEPC-treated water were added to 38 μl of total RNA. The mixture was reacted at 37° C. for 20 minutes, and 150 μl of DEPC-treated water was added to make a total volume of 200 μl of sample. 200 μl of phenol / chloroform / isoamyl alcohol (volume ratio of 25:24:1) was added to the sample and mixed. The sample was centrifuged at 12000 G at room temperature for 5 minutes. Approximately 200 μl of the aqueous phase was collected in a 1.5 ml tube, and 20 μl of 3M sodium acetate and 500 μl of ethanol were added to the tube, and the mixed sample was left at room temperature for 15 minutes.

[0075] The sample was centrifuged at 20,000G for 20 minutes at 4°C, and the supernatant was removed. 1 ml of 70% ethanol was added to wash the precipitate. The sample was centrifuged at 20,000G for 10 minutes at 4°C, and the supernatant was removed. The tube was opened and left to stand at room temperature for 10 minutes to dry the remaining liquid. 40 μl of TE buffer was added to dissolve the RNA, and the concentration was measured using a Nanodrop. The RNA was adjusted to 150 ng / μl and stored at -80°C.

[0076] [RNA-seq analysis] We used the next-generation sequencing analysis service provided by Veritas Genetics (service name: mRNA-seq (150 bp PE), data volume: 3G, number of reads: 10 million reads) to obtain fastq-formatted data.

[0077] [RNA-seq data analysis] The acquired data was compressed into fastq.gz format using the pigz command. Adapter sequences were trimmed and quality checks were performed using the fastp command. Mapping to the reference genome (mouse: GRCm38_genome) was performed using the hisat2 command. The mapping results were sorted using the samtools command and converted into a BAM file. The reads for each gene were counted using the featurecounts command, and expression levels were quantified. The obtained read count data was used to identify cell types by referencing the ImmGen database (https: / / www.immgen.org / ) using a Single R pipeline.

[0078] (result) RNA-seq data analysis identified the cell type of LIM as "macrophage."

[0079] Test Example 2: Expression analysis of cell surface markers of BMDM and LIM LIM 14 days after the start of induction in Example 1 and BMDM 5 days after the start of induction in Comparative Example 1 were each detached from the medium using a cell scraper and collected, and the cells were counted using a hemocytometer. The cells were centrifuged at 500 G and 4° C. for 5 minutes, and the supernatant was removed. 7 1% FBS / HBSS was added to the cells at 100 cells / ml, and the cells were suspended in the HBSS. Anti-mouse CD16 / 32 antibody (BioLegend) was added to the cell suspension at 10 μg / ml, and the cells were allowed to stand on ice for 10 minutes to block the Fcγ receptor.

[0080] Dispense 10 μl of the blocked cell suspension into a 96-well V-bottom plate at each well, and add PE anti-mouse TREM2 antibody (R&D), PE anti-mouse Dectin-1 antibody, FITC anti-mouse CD9 antibody, APC anti-mouse CD11c antibody, PE anti-mouse Siglec-F antibody, PE anti-mouse CD206 antibody, APC anti-mouse P2RY12 antibody, APC anti-mouse CX3CR1 antibody, APC anti-mouse MHC class II antibody, APC / Cyanine7 anti-mouse CD115 antibody, PE / Cyanine7 anti-mouse CD115 antibody, FITC anti-mouse F4 / 80 antibody, PE / Cyanine7 anti-mouse Ly-6C antibody, FITC anti-mouse Ly-6G antibody, APC anti-mouse CD45 antibody, or ... Anti-mouse CD11b antibody (both manufactured by BioLegend) was added to make a total volume of 20 μl / well, and the plate was left to stand on ice for 15 minutes for reaction. 1% FBS / HBSS was added at 180 μl / well for washing.

[0081] The samples were centrifuged at 500G for 5 minutes at 4°C, and the supernatant was removed. 180 μl / well of 1% FBS / HBSS was added to wash the samples. The samples were centrifuged at 500G for 5 minutes at 4°C, and the supernatant was removed. 50 μl / well of 10 μg / ml PI / 1% FBS / HBSS was added to suspend the samples. The obtained samples were analyzed by flow cytometry, and Ly6G-negative and CD11b-positive cells were extracted as macrophage cells and each surface antigen was analyzed in the univariate histogram analysis of the surface antigen. For the detection of CD115, APC / Cyanine7 anti-mouse CD115 antibody was used in the dot plot analysis by flow cytometry, and PE / Cyanine7 anti-mouse CD115 antibody was used in the univariate histogram analysis of the surface antigen.

[0082] (result) The results of dot plot analysis are shown in Figure 2. The distribution of signals for forward scatter (FSC), an index of cell size, and side scatter (SSC), an index of cell disorder (abundance of granules), was similar between LIM and BMDM. LIM showed high expression of the Pan leukocyte marker CD45 and the monocyte marker CD115, as well as high expression of the macrophage marker F4 / 80 and the granulocyte and macrophage marker CD11b.

[0083] The results of univariate histogram analysis of surface antigens are shown in Figure 3. Compared with macrophages in mouse in vivo, such as peritoneal macrophages (MΦ), splenic macrophages, Kupffer cells, and microglia, the expression patterns of various surface antigens in BMDM and LIM were very similar. The expression levels of TREM2, CD206, P2RY12, and CX3CR1 were particularly high in LIM. The results of Test Example 1 and Test Example 2 indicated that LIM is a macrophage.

[0084] Test Example 3: Examination of lipid concentration for LIM induction [Brain lipid coating] A brain lipid extract prepared in the same manner as in Example 1 was coated on a 96-well cell culture plate at 4, 2, 1, 0.5, 0.25 or 0.125 μg / well. 5 The bone marrow cell suspension was dispensed at 100 μl per well and incubated at 37°C, 5% CO 2 The cells were cultured in an incubator. After 3 days of culture, half the amount of fresh medium was added. After that, the medium was changed every 3 to 4 days. The number of viable cells was measured by WST-8 assay 7 and 14 days after the start of culture.

[0085] [WST-8 assay] A 1 / 10 volume of WST-8 (07553-15 Viable Cell Count Reagent SF, Nacalai Tesque) was added to fresh 10% FBS / RPMI medium to prepare a viable cell count measurement medium. The old medium was removed by suction, and 100 μl of viable cell count measurement medium was dispensed into each well. The medium was incubated at 37°C, 5% CO 2 The cells were cultured in an incubator for 1 hour, and the absorbance at 450 nm was measured using a microplate reader.

[0086] (result) Figure 4 shows the number of cells versus the mass of brain lipid coated per unit culture area. 2 The above is suitable for induction of LIM, especially 3 to 12 μg / cm 2 promoted the proliferation of LIM.

[0087] Test Example 4: Subculture of LIM When using a 24-well plate, the cells become 90-100% confluent around 14 days after the start of induction. Therefore, LIM or BMDM were subcultured every 14 days as follows. The culture supernatant was aspirated, 0.5 ml / well of 1 mM EDTA / PBS was added, and the cells were incubated at 37°C for 5 minutes. The 1 mM EDTA / PBS was aspirated and removed, and 0.5 ml / well of 10% FBS / RPMI was added, and the cells were suspended by pipetting. The cell number was measured using a hemocytometer. The cell suspension was diluted 1 / 4 with 10% FBS / RPMI and dispensed into a new brain lipid-coated plate at 0.5 ml / well. After that, half of the medium was replaced with 10% FBS / RPMI every 3-4 days, and the cells were subcultured again after 14 days.

[0088] In addition, LIM cultured for 586 days were plated on 24-well plates with (BL+) or without (BL-) brain lipid coating (coated with brain lipid 3 μg / cm 2 ) were seeded at 20,000 cells / well and cultured.

[0089] (result) The upper row of Fig. 5(A) shows the cell counts after 0 to 21 days of culture, and the lower row of Fig. 5(A) shows the fold increase in cell counts after 0 to 21 days of culture, with the number of cells on day 0 taken as 1. The upper row of Fig. 5(B) shows the cell counts after 0 to 544 days of culture, and the lower row of Fig. 5(B) shows the fold increase in cell counts after 0 to 544 days of culture, with the number of cells on day 0 taken as 1. BMDMs hardly increased after being serially diluted. On the other hand, LIMs survived and proliferated even after at least 544 days of culture.

[0090] Figure 5(C) shows the number of LIM cells cultured in BL+ and BL-. LIM cells did not grow or survive in BL-. Since LIM cells do not grow or survive without stimulation by brain lipids, it was shown that they are not tumorigenic cells.

[0091] Test Example 5: Cryopreservation test of LIM and BMDM LIM and BMDM that were 80-90% confluent were detached from the culture plate and centrifuged at 500G for 5 minutes, and the supernatant was removed. 5 ~5×106 The cells were suspended in 1 ml of Cell Banker 1 (Nihon Zenyaku Kogyo Co., Ltd.), transferred to a cryotube, and stored at -80°C. After 30 days, the cells were thawed and seeded onto 24-well plates with no stimulation (None), 25 ng / ml M-CSF (M-CSF), or brain lipid coating (BL). The number of viable cells was measured using a hemocytometer 6 or 14 days after seeding.

[0092] (result) As shown in Figure 6, BMDMs thawed after cryopreservation did not proliferate even in the presence of M-CSF, whereas thawed LIM proliferated in the presence of brain lipid stimuli, demonstrating that LIM can be cryopreserved.

[0093] Test Example 6: Magnetic cell sorting and cell isolation using a cell sorter 1 x 10 bone marrow cells from CD45.1-positive or CD45.2-positive mice 7 100 μl of 10 μg / ml anti-Fcγ receptor (CD16 / 32) antibody / 0.5% BSA / PBS was added per cell, and the cells were suspended. The cells were left on ice for 5 minutes to block the Fcγ receptor. The following fluorescently labeled antibodies against the mature cell marker (Lineage marker: Lin) were added to the bone marrow cell suspension from each mouse and reacted. Anti-CD3-FITC (2.5μg / ml) Anti-CD19-FITC (2.5μg / ml) Anti-NK1.1-FITC (2.5μg / ml) Anti-Ly6G-FITC (2.5μg / ml) Anti-TER-119-FITC (2.5μg / ml) Anti-CD11b-FITC (2.5μg / ml)

[0094] The bone marrow cell suspension was left on ice for 15 minutes, and thereafter, the procedure was carried out according to the instructions for use of Miltenyi Biotec's Anti-FITC MicroBeads and Anti-APC MicroBeads. 7The cells were washed by adding 1 ml of MACS™ buffer (0.5% BSA / 2 mM EDTA / PBS) per cell sample, centrifuged at 300G and 4°C for 10 minutes, and the supernatant was removed. The precipitate was suspended in 80 μl of MACS™ buffer, and 20 μl of anti-FITC antibody-labeled magnetic beads were added. The mixture was left to stand at 4°C for 15 minutes, and 1 ml of MACS™ buffer was added to the sample for washing. The sample was centrifuged at 300G and 4°C for 10 minutes, and the supernatant was removed. The precipitate was suspended in 0.5 ml of MACS™ buffer. The sample was added to an LS column set in a MACS™ separator and previously washed with 3 ml of MACS™ buffer, and the flow-through liquid was collected as a Lin-negative fraction.

[0095] The LS column was washed three times with 3 ml of MACS™ buffer. The LS column was removed from the MACS™ separator and placed in a 15 ml tube. 5 ml of MACS™ buffer was added to the LS column, which was then pushed out with a plunger to recover the Lin-positive fraction. The Lin-negative fraction derived from CD45.2-positive mice and the Lin-positive fraction derived from CD45.1-positive mice were each centrifuged at 500 G for 10 minutes at 4°C, and the supernatant was removed. The resulting cells were collected at 5 x 10 6 The cells were suspended in 10% FBS / RPMI to a concentration of 100 cells / ml, and then the Lin-negative and Lin-positive fractions were strictly isolated using a cell sorter. The isolated cell sample was centrifuged at 500 G for 10 minutes at 4°C, and the supernatant was removed. The resulting cells were diluted to 4 × 10 5 The Lin-negative fraction from CD45.2-positive mice and the Lin-positive fraction from CD45.1-positive mice were mixed at a ratio of 1:9, which is almost the same as the ratio in bone marrow, and seeded on a brain lipid-coated culture plate. Each fraction was also seeded alone in the same manner. Whether or not Lin-positive cells or Lin-negative cells were induced to differentiate into LIM by brain lipid stimulation was evaluated by counting the number of CD11b-positive F4 / 80-positive macrophages using flow cytometry 5, 9, and 14 days after the start of culture.

[0096] (result) Figures 7(A) and 7(B) show the number of macrophages differentiated from Lin-positive cells derived from CD45.1-positive mice and Lin-negative cells derived from CD45.2-positive mice in the mixed culture, and the change in the number of macrophages when each fraction was cultured alone. Although the ratio of Lin-negative cells to Lin-positive cells at the start of the culture was 1:9, the majority (66-83%) of the cells induced to differentiate into LIM were Lin-negative cells. Furthermore, when Lin-negative cells were cultured alone, the induction of differentiation of a large number of LIM was observed early (on the 5th day). These results suggest that LIM mainly differentiate from myeloid precursor cells. In addition, since a certain number of macrophages were induced from Lin-positive cells, it was suggested that they may also differentiate from CD11b-positive monocytes.

[0097] Test Example 7: Induction of bone marrow cells derived from each mouse into BMDM and LIM Bone marrow cells derived from wild-type (WT) mice (C57BL / 6), TREM2 knockout mice (KO), and mice carrying the TREM2 R47H mutation were used to induce LIM and BMDM in the same manner as in Example 1 and Comparative Example 1. The TREM2 R47H mutation reduces ligand binding ability compared to the wild-type. For LIM, a WST-8 assay was performed 14 days after the start of induction.

[0098] (result) BMDMs were observed under a microscope 5 days after the start of induction, and it was confirmed that BMDMs could be induced from either mouse. Figure 8 shows the number of viable cells evaluated by the WST-8 assay. Bone marrow cells from wild-type mice were induced to differentiate into LIM cells by stimulation with brain lipids, whereas bone marrow cells from TREM2 KO and TREM2 R47H mice were not induced to differentiate into LIM cells even when stimulated with brain lipids.

[0099] Test Example 8: Evaluation of ligand activity using TREM2 reporter cells We introduced TREM2 and DAP12 cDNA into 2B4-NFAT-GFP reporter cells (obtained from the RIKEN Institute) using a retroviral vector to create reporter cells in which GFP expression is induced when a ligand binds to TREM2 expressed on the cell surface (Iizasa et al., Nature Communications, 2021, vol. 12(1), p. 2299-16).

[0100] The test substances, β-GluCer, α-GalCer, palmitic acid, stearic acid, cholesterol, PC, LPC, PE, Sulf, or MA, were diluted with methanol to 50 μg / ml. The test substances were dispensed into 96-well cell culture plates at 20 μl (1 μg) each, and the plates were placed in a clean bench to dry the methanol.

[0101] 5 × 10 reporter cells were placed on a culture plate coated with the test substance. 4 Cells were seeded at 100 μl per well. 2 The cells were cultured in an incubator for 16 hours. The culture solution was thoroughly suspended and transferred to a V-bottom 96-well plate. The samples were centrifuged at 500G for 5 minutes at 4°C, and the supernatant was removed. 10 μg / ml PI / 1% FBS / HBSS was added at 50 μl / well and suspended. GFP-positive cells in the samples were analyzed by flow cytometry.

[0102] In the same manner as above, each test substance was coated on a 96-well cell culture plate, and mouse bone marrow cells were cultured at 4 × 10 4 100 μl of cells were seeded into the plate per well. 2 The cells were cultured in an incubator, and the number of viable cells was measured by WST-8 assay 5, 10, 14, and 20 days after the start of culture.

[0103] (result) As shown in Figure 9, β-GluCer, α-GalCer, PC, LPC, PE, Sulf, and MA were found to have ligand activity for TREM2. As shown in Figure 10, β-GluCer, α-galactosylceramide, PC, LPC, PE, Sulf, and MA induced the differentiation of bone marrow cells into LIM.

[0104] Test Example 9: Evaluation of phagocytic activity 1 × 10 BMDM or LIM 5 Seed 1 x 10 cells / well in a 96-well plate at 200 μl / well. 6 Cfu of FITC-labeled Bacille Calmette-Guerin (BCG) was added and incubated for 4 hours at 37°C. After 4 hours, the cells were harvested and the phagocytic ability of FITC-labeled BCG (BCG-FITC) was compared by measuring the fluorescence intensity of FITC by flow cytometry.

[0105] (result) The mean fluorescence intensity (MFI) of FITC-labeled BCG is shown in Figure 11. It was shown that LIM has higher phagocytic activity than BMDM.

[0106] Test Example 10: Examination of cytokine production response to LPS stimulation 1 × 10 BMDM or LIM 5 The cells were seeded in a 96-well plate at 200 μl / well, and 10 ng / ml LPS was added and incubated for 24 hours at 37° C. After 24 hours, the culture supernatant was collected and the concentrations of MCP-1, TNF-α, IL-6, IL-10 and NO in the supernatant were measured.

[0107] (result) 12(A), (B), (C), (D), and (E) show the concentrations of MCP-1, TNF-α, IL-6, IL-10, and NO, respectively. LIM produced less inflammatory cytokines, such as IL-6 and TNF-α, as well as NO, compared to BMDM.

[0108] Test Example 11: Examination of marker molecules for LIM RNA-seq data (SRA files) of various cells were obtained from the Gene Expression Omnibus (GEO) database (https: / / www.ncbi.nlm.nih.gov / geo / ). The reference sources (GEO accession numbers) of various cells are as follows: Alveolar macrophages: GSM4476109, GSM4476110, GSM4476111 BMDM: GSM4552500, GSM4552501, GSM4552502 BMDM(M1): GSM4844180, GSM4844181, GSM4844182 BMDM(M2): GSM4844183, GSM4844184, GSM4844185 Kupffer cells: GSM4119149, GSM4119150, GSM4119151 Microglia: GSM4065894, GSM4065895, GSM4065896 Osteoclasts: GSM3179449, GSM3179450, GSM3179451 Peritoneal exudate macrophages: GSM2859803, GSM2859804, GSM2859805

[0109] BMDM(M1) cells were cultured for 24 hours with 100 ng / ml LPS and 50 ng / ml mIFNγ, and BMDM(M2) cells were cultured for 24 hours with 20 ng / ml IL-4 and 20 ng / ml IL-13.

[0110] The SRA files of various cells were converted to fastq format data using the fasterq-dump command. Read count data was obtained according to the method for analyzing RNA-seq data in Test Example 1 above. Differentially expressed genes (DEGs) were extracted using DESeq2 using the read count data of LIM and these cells. Furthermore, from each list of DEGs, DEGs (88 genes) that were commonly highly expressed in LIM when comparing LIM with all other macrophages and DEGs (87 genes) that were commonly low expressed in LIM were extracted as marker molecules. Calculate and draw custom Venn diagrams (http: / / bioinformatics.psb.ugent.be / webtools / Venn / ) were used for extraction.

[0111] (result) The number of DEGs is shown in Table 5. The extracted DEGs with high expression in LIM and DEGs with low expression in LIM are shown in Tables 6 and 7, respectively.

[0112] [Table 5]

[0113] [Table 6]

[0114] [Table 7]

[0115] The above-described embodiment is for explaining the present invention, and does not limit the scope of the present invention. That is, the scope of the present invention is indicated by the claims, not by the embodiment. Various modifications made within the scope of the claims and within the scope of the meaning of the invention equivalent thereto are considered to be within the scope of the present invention. [Industrial Applicability]

[0116] The present invention is useful for obtaining macrophages.

Claims

1. After cryopreservation, the cells have the ability to proliferate in a medium that does not contain either macrophage colony-stimulating factor or granulocyte-macrophage colony-stimulating factor and that contains a TREM2 signal activator. Macrophages.

2. A method comprising culturing hematopoietic progenitor cells in the presence of the TREM2 signal activator. The method for producing the macrophages according to claim 1 .

3. A compound comprising the TREM2 signal activator. An agent for inducing differentiation of hematopoietic progenitor cells into the macrophages according to claim 1.

4. A compound comprising the TREM2 signal activator. The macrophage proliferation promoter according to claim 1.

5. A culture step of culturing macrophages in the presence of the TREM2 signal activator. The method for expanding macrophages according to claim 1.

6. The cells have proliferation ability after 10 days of subculture in the medium containing neither macrophage colony-stimulating factor nor granulocyte-macrophage colony-stimulating factor and containing a TREM2 signal activator. The macrophage of claim 1.

7. By culturing hematopoietic progenitor cells in the medium containing neither macrophage colony-stimulating factor nor granulocyte-macrophage colony-stimulating factor but containing a TREM2 signal activator, differentiation is induced in a TREM2-dependent manner. The macrophage of claim 1.

8. the concentrations of TNF-α, IL-6 and nitric oxide in the culture supernatant after 24 hours of culture in a medium containing lipopolysaccharide are 30% or less compared to those of bone marrow-derived macrophages induced from bone marrow cells with macrophage colony-stimulating factor; The macrophage of claim 1.

9. The expression of one or more of the genes shown in Table 1 below is higher than that of at least one selected from alveolar macrophages, BMDM, M1-induced BMDM, M2-induced BMDM, Kupffer cells, microglia, osteoclasts, and peritoneal exudate macrophages; The macrophage of claim 1. Table 1

10. The expression of one or more of the genes shown in Table 2 below is lower than that of at least one selected from alveolar macrophages, BMDM, M1-induced BMDM, M2-induced BMDM, Kupffer cells, microglia, osteoclasts, and peritoneal exudate macrophages. The macrophage of claim 1. Table 2

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