Method for producing human macrophage, differentiation-inducing agent, differentiation-inducing kit, method for inducing differentiation of human macrophage, proliferation-promoting agent for human macrophage, kit for promoting proliferation of human macrophage, method for proliferating human macrophage, and human macrophage

Abstract

This method for producing human macrophages includes a culturing step for culturing human hematopoietic progenitor cells in the presence of TREM2 signal activating agent, with a culture medium substantially free of any of a macrophage colony stimulating factor and free of a granulocyte macrophage colony stimulating factor.

Description

Method for producing human macrophages, differentiation inducer, differentiation induction kit, method for inducing differentiation of human macrophages, agent for promoting proliferation of human macrophages, kit for promoting proliferation of human macrophages, method for proliferation of human macrophages, and human macrophages

[0001] The present invention relates to a method for producing human macrophages, a differentiation inducer, a differentiation induction kit, a method for inducing differentiation of human macrophages, a proliferation promoter for human macrophages, a proliferation promotion kit for human macrophages, a method for proliferating human macrophages, and human macrophages.

[0002] When inducing mature blood cells in vitro from hematopoietic progenitor cells in bone marrow, it is necessary to add colony-stimulating factors, growth factors, hematopoietic factors, cytokines such as lymphokines, support cells such as bone marrow stromal cells, cell matrix, etc. Macrophages, which are leukocytes that contribute to infection defense and maintenance of tissue homeostasis, are derived from hematopoietic stem cells in bone marrow, and their differentiation requires stimulation with macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF). A method has been established for inducing differentiation into cells with properties of monocytes and macrophages (bone marrow-derived macrophages: BMDMs) by adding macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) to human bone marrow cells or peripheral blood leukocytes and culturing them. It is also known that interleukin-34 (IL-34) acts on M-CSF receptors to promote the differentiation of monocytes into macrophages and the proliferation of macrophages (see Non-Patent Documents 1 and 2).

[0003] Another known method involves culturing induced pluripotent stem cells (iPSCs) using a culture plate coated with an extracellular matrix, and continuously culturing them in a medium supplemented with multiple cytokines and growth factors such as BMP4, VEGF, stem cell factor (SCF), bFGF, Flt3 ligand, interleukin-3 (IL-3), thrombopoietin (TPO), M-CSF, and GM-CSF to induce differentiation into monocytes, and further culturing the induced monocytes with M-CSF to induce differentiation into mature macrophages (see Non-Patent Document 3).

[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.

[0005] JP 2015-47125 A

[0006] Suwen Wei, 8 others, “Functional overlap but differential expression of CSF-1 and IL-34 in their CSF-1 receptor-mediated regulation of myeloid cells", Journal of Leukocyte Biology, 2010, 88, 495-505 Haishan Lin, and 16 others, "Discovery of a Cytokine and Its Receptor by Functional Screening of the "Extracellular Proteome", SCIENCE, 2008, 320, 807-811 Masakatsu D. Yanagimachi, 14 others, “Robust and Highly-Efficient Differentiation of Functional Monocytic Cells from Human Pluripotent “Stem Cells under Serum-and Feeder Cell-Free Conditions”, PLOS ONE, 2013, 8(4), e59243

[0007] In the above-mentioned method of inducing differentiation of hematopoietic progenitor cells into mature macrophages using M-CSF, GM-CSF, or IL-34, it is essential to add one of these cytokines to the culture medium. BMDMs obtained by this method do not have the ability to proliferate, and therefore cannot be expanded. Furthermore, Patent Document 1 does not examine the survival period or growth characteristics after differentiation of macrophages obtained using this differentiation inducer. Furthermore, as disclosed in Non-Patent Document 3, inducing differentiation of iPSCs into mature macrophages requires extracellular matrix, multiple types of cytokines, and growth factors, and the induced monocytes must be further cultured with M-CSF.

[0008] The present invention has been made in view of the above circumstances, and aims to provide a method for producing human macrophages that can be induced from hematopoietic progenitor cells without adding cytokines to the culture medium, and that can produce human macrophages that have proliferative properties after induction and can be expanded, as well as a differentiation inducer, a differentiation induction kit, a method for inducing human macrophage differentiation, a human macrophage proliferation promoter, a human macrophage proliferation promotion kit, and a method for expanding human macrophages. Another aim of the present invention is to provide human macrophages that can be induced from hematopoietic progenitor cells without adding cytokines to the culture medium, and that have proliferative properties after induction and can be expanded.

[0009] The present inventors have discovered that macrophage-like cells emerge when human hematopoietic progenitor cells are stimulated with a ligand for TREM2 (triggering receptor expressed on myeloid cells 2), a lipid-recognition receptor, and have completed the present invention.

[0010] (Method for producing human macrophages) The method for producing human macrophages described herein comprises a culture step of culturing human hematopoietic progenitor cells in a medium that is substantially free of both macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor in the presence of a TREM2 signal activator.

[0011] The medium may be substantially free of interleukin-34.

[0012] The medium may be substantially free of cytokines.

[0013] In the culturing step, the human hematopoietic progenitor cells may be cultured in the absence of supporting cells and extracellular matrix.

[0014] The TREM2 signaling activator may be a lipid.

[0015] The TREM2 signaling activator may be a brain-derived lipid or a lipid having multiple long-chain fatty acid groups.

[0016] The TREM2 signal activator may be a compound represented by formula (I), an ester thereof, or a salt thereof. (having 60 to 90 carbon atoms, R １ represents a saturated or unsaturated aliphatic hydrocarbon group, R ２ represents a saturated or unsaturated aliphatic hydrocarbon group which may have a cyclic structure or a substituent.

[0017] The TREM2 signal activator may be a phospholipid.

[0018] The phospholipid may be a lipid that constitutes a biological membrane.

[0019] The phospholipid may be cardiolipin.

[0020] The surface of a cell culture vessel for culturing the human hematopoietic progenitor cells may be coated with the TREM2 signaling activator.

[0021] The human hematopoietic progenitor cells may be human myeloid progenitor cells.

[0022] (Differentiation Inducer) The differentiation inducer of human hematopoietic progenitor cells into human macrophages described in the present specification contains a TREM2 signal activator and is substantially free of both human macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.

[0023] (Differentiation induction kit) The differentiation induction kit for human hematopoietic progenitor cells into human macrophages described in this specification comprises a cell culture vessel whose surface is coated with a TREM2 signal activator, and is used for culturing human hematopoietic progenitor cells in a medium that is substantially free of both macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.

[0024] (Method for inducing differentiation of human macrophages) The method for inducing differentiation of human macrophages described herein comprises a culture step of culturing human hematopoietic progenitor cells in a medium that is substantially free of both macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor in the presence of a TREM2 signal activator.

[0025] (Human Macrophage Proliferation Promoting Agent) The human macrophage proliferation promoting agent described herein contains a TREM2 signal activator, and is substantially free of both macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.

[0026] (Kit for promoting proliferation of human macrophages) The kit for promoting proliferation of human macrophages described in the present specification comprises a cell culture vessel whose surface is coated with a TREM2 signal activator, and is used for culturing human hematopoietic progenitor cells in a medium that is substantially free of both macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.

[0027] (Method for Proliferating Human Macrophages) The method for proliferating human macrophages described herein comprises a culture step of culturing human hematopoietic progenitor cells in a medium that is substantially free of both macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor in the presence of a TREM2 signal activator.

[0028] (Human Macrophages) The human macrophages described herein have the ability to proliferate in the presence of a TREM2 signaling activator after 10 days of subculture in a medium substantially free of both macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.

[0029] The human macrophages described herein are cells in which TREM2-dependent differentiation is induced by culturing human hematopoietic progenitor cells in a medium that is substantially free of both macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor in the presence of a TREM2 signal activator.

[0030] According to the present invention, human macrophages can be induced from hematopoietic progenitor cells without adding cytokines to the culture medium, and after induction, they have the ability to proliferate and can be expanded.

[0031] 1 is a diagram showing images of macrophage-like adherent cells cultured in the presence of various lipids in Example 1. 2 is a diagram showing images of BMDM cultured in the presence of M-CSF or GM-CSF in Comparative Example 1. 3 is a diagram showing the results of dot plot analysis of BMDM induced with M-CSF or GM-CSF and human macrophages induced with brain lipids, mycolic acid, or cardiolipin in Test Example 1. 4 is a diagram showing the results of dot plot analysis of human macrophages induced with L-3-phosphatidylcholine plasmalogen, sulfatide, palmitic acid, stearic acid, or cholesterol in Test Example 1. 5 is a diagram showing the number of human macrophages 14 days and 28 days after the start of induction in Test Example 1. 6 is a diagram showing changes in macrophage number up to 28 days after the start of induction in Test Example 1. 7 is a diagram showing the results of evaluation of phagocytic activity in Test Example 2. 8 is a diagram showing the concentration of TNF-α in the supernatant in Test Example 3. 9 is a diagram showing the concentration of IL-10 in the supernatant in Test Example 3. 1 is a graph showing the cell counts on days 9 to 57 of culture in Test Example 4. FIG. 2 is a graph showing the fold increase in cell counts in Test Example 4.

[0032] Embodiments of the present invention will be described with reference to the drawings. Note that the present invention is not limited to the following embodiments and drawings. Note that in the following embodiments, the expressions "have," "include," or "contain" also include the meaning of "consisting of" or "consisting of."

[0033] The method for producing human macrophages according to this embodiment includes a culturing step of culturing human hematopoietic progenitor cells in a medium substantially free of M-CSF and GM-CSF in the presence of a TREM2 signaling activator. TREM2 is an immunoglobulin superfamily receptor that associates with the adaptor molecule DAP12 (DNAX-activating protein of 12 kDa). 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, a TREM2 ligand, or the like, that has TREM2 agonist activity or DAP12 agonist activity. 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. The antibody herein includes an antigen-binding fragment of an antibody. An antigen-binding fragment is a protein or peptide that contains a portion (partial fragment) of an antibody, and retains the functional binding ability of the antibody to the antigen, such as F(ab') ２ ,Fab',Fab,Fab ３ , single-chain Fv (scFv), (tandem) bispecific single-chain Fv (sc(Fv) ２ ), single-chain triplebodies, nanobodies, divalent VHHs, pentavalent VHHs, minibodies, (two-chain) diabodies, tandem diabodies, bispecific tribodies, bispecific bibodies, dual affinity retargeting molecules (DARTs), triabodies (or tribodies), tetrabodies (or [sc(Fv) ２ ] ２), or disulfide-linked Fv (dsFv), or a polymer thereof (see Nature Biotechnology, 29(1):5-6(2011) and Christoph Stein et al., Antibodies(1):88-123(2012)).

[0035] The TREM2 signal activator is, for example, a ligand for TREM2 or a lipid that functions as a TREM2 ligand. Examples of lipids include glycosphingolipids, phospholipids, sulfated glycolipids, and fatty acids. For example, the phospholipid may be a lipid that constitutes a biological membrane such as a cell membrane or a mitochondrial membrane. The fatty acid may be a saturated fatty acid or an unsaturated fatty acid. Preferably, the lipid that functions as a TREM2 ligand is a lipid having multiple long-chain fatty acid groups, more preferably 2 to 6 or 2 to 4 long-chain fatty acid groups. Examples of long-chain fatty acid groups 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] Preferred examples of lipids that function as TREM2 ligands include mycolic acid (hereinafter also referred to simply as "MA"), cardiolipin (hereinafter also referred to simply as "CL"), L-3-phosphatidylcholine plasmalogen (hereinafter also referred to simply as "PC"), sulfatide (hereinafter also referred to simply as "Sulf"), palmitic acid (hereinafter also referred to simply as "Palm"), stearic acid (hereinafter also referred to simply as "Stea"), and cholesterol (hereinafter also referred to simply as "Chol"). Such lipids may be naturally occurring lipids or synthetic lipids. Furthermore, the TREM2 signaling activator may be a composition containing a mixture of multiple lipids, such as a composition containing lipids obtained by extraction from animal-derived tissues such as the brain, plant-derived tissues, or microorganisms such as bacteria using an organic solvent, or a composition containing an artificial mixture of any two or more types of lipids. Alternatively, the TREM2 signaling activator may be a synthetic lipid that binds to TREM2. One embodiment of the TREM2 signaling activator includes cells having lipids on their cell surfaces, microorganisms, and lipids derived from microorganisms. Preferably, the TREM2 signal activator is a brain-derived lipid, MA, or CL, illustratively represented by the following formula: CL is a phospholipid specific to mitochondria.

[0037]

[0038] 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 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 represents a saturated or unsaturated aliphatic hydrocarbon group which may have a cyclic structure or a substituent. ２ The number of carbon atoms in 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 formed by ２ The cycloalkyl group may be included in R ２ may be included in R２ The substituent is not particularly limited and may be a methyl group, a hydroxy group, a carbonyl group, a carboxyl group, or the like.

[0039]

[0040] The salt of the compound represented by 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, methyl methacrylate ... 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, trimethyl acetate, tert-butyl acetate, lauryl sulfate, gluconate, glutamate, hydroxynaphthoate, salicylate, stearate, trifluoroacetic acid (TFA) salt, maleate, dimaleate, and muconate.

[0041] The ester of the compound represented by 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 encompasses various hydrates and solvates of the compound of formula (I) and its salts, as well as crystalline polymorphic substances.

[0042] TREM2 signal activators may be selected by allowing a substance to be screened to act on cells expressing TREM2 and DAP12, and using the TREM2 signal activated by the binding of the substance to TREM2, for example, expression of a reporter protein caused by the TREM2 signal, as an indicator. TREM2 and DAP12 can be expressed in cells by known methods, for example, by introducing a gene expression plasmid or the like into the cells. Any reporter protein can be used, and examples of reporter proteins include green fluorescent protein (GFP) and the luminescent protein luciferase. It is known that TREM2 signaling causes the expression of genes controlled by NFAT (nuclear factor of activated T cells)-type transcription factors. Therefore, preferably, cells having NFAT-type transcription factors are used as cells for expressing TREM2 and DAP12. Specifically, TREM2- and DAP12-expressing cells into which a gene encoding a reporter protein has been introduced as a gene whose expression is controlled by an NFAT-type transcription factor can be used to evaluate the ability of the cell to activate TREM2 signals. A similar method can also be used to confirm whether any lipid functions as a TREM2 ligand.

[0043] A "medium substantially free of both M-CSF and GM-CSF" refers to a medium that contains at least one of M-CSF and GM-CSF in trace amounts, if any, and to which M-CSF and GM-CSF have not been intentionally added. In other words, a "medium substantially free of both M-CSF and GM-CSF" refers to a medium that contains neither M-CSF nor GM-CSF, or in some cases, contains at least one of M-CSF and GM-CSF in an amount insufficient to differentiate human hematopoietic progenitor cells. Examples of medium containing at least one of M-CSF and GM-CSF in trace amounts include when the cultured human hematopoietic progenitor cells secrete at least one of M-CSF and GM-CSF, or when at least one of M-CSF and GM-CSF is contained in the additives described below.

[0044] The medium may be a known medium suitable for culturing human hematopoietic progenitor cells, such as RPMI medium. If necessary, fetal bovine serum (FBS) or the like may be added to the medium. The method for producing human macrophages according to this embodiment allows differentiation of human hematopoietic progenitor cells into human macrophages without the use of cytokines such as stem cell factor (SCF), thrombopoietin (TPO), Flt3 ligand (FL), IL-6, IL-34, or granulocyte colony-stimulating factor (G-CSF) in addition to M-CSF and GM-CSF. Therefore, preferably, a "medium substantially free of M-CSF and GM-CSF" is substantially free of IL-34 or cytokines. Note that "substantially free of IL-34 or cytokines" refers to a medium to which IL-34 or cytokines are not intentionally added, and in which IL-34 or cytokines are present in trace amounts, similar to the above-described "medium substantially free of both M-CSF and GM-CSF."

[0045] Preferably, a "medium substantially free of M-CSF and GM-CSF" is substantially free of agonists (antibodies, compounds, etc.) that activate one or more cytokine receptors. For example, a "medium substantially free of M-CSF and GM-CSF" is substantially free of any one or more selected from agonists that activate M-CSF receptors, agonists that activate GM-CSF receptors, and agonists that activate IL-34 receptors. More preferably, a "medium substantially free of M-CSF and GM-CSF" is substantially free of any agonists that activate M-CSF receptors, agonists that activate GM-CSF receptors, and agonists that activate IL-34 receptors; for example, it may be substantially free of any agonists that activate any cytokine receptors.

[0046] Human hematopoietic progenitor cells are cells contained in human bone marrow or umbilical cord blood. Human hematopoietic progenitor cells are, for example, human myeloid progenitor cells. As human hematopoietic progenitor cells, bone marrow cells collected from human bone marrow or mononuclear cells collected from umbilical cord blood may be used.

[0047] In the culture step, the concentration of the human hematopoietic progenitor cells is not particularly limited, but may be, for example, ２ ) per 1 x 10 ３ ~1 x 10 ７ cells or 1 x 10 ４ ~1 x 10 ６ The cells are seeded. Preferably, the unit culture area (cm ２ ) per 1 x 10 ５ In the method for producing human macrophages according to this embodiment, human hematopoietic progenitor cells can be differentiated into human macrophages without using feeder cells or extracellular matrix. Therefore, in the culturing step, the human hematopoietic progenitor cells are preferably cultured under conditions in which feeder cells and extracellular matrix are not added.

[0048] In the culturing step, human hematopoietic progenitor cells may be cultured in a cell culture vessel holding a TREM2 signaling activator. Examples of cell culture vessels include cell culture plates, cell culture flasks, and cell culture dishes. When the TREM2 signaling activator is insoluble in water and insoluble in liquid medium, human hematopoietic progenitor cells may be cultured in a cell culture vessel whose surface is coated with the TREM2 signaling activator. When brain-derived lipids are used as the TREM2 signaling activator, the mass of lipids coating the surface of the cell culture vessel may be, for example, 1 x 10 ４ ~1 x 10 ６ 1.0 μg / cm for human hematopoietic progenitor cells ２ Above 1.5 to 20 μg / cm ２ , 2 to 18 μg / cm ２ , 3 to 16 μg / cm ２ , preferably 3 to 12 μg / cm ２ To coat the surface of a cell culture vessel with a TREM2 signaling activator, the ligand may be dissolved (suspended) in an organic solvent or the like, added to the cell culture vessel, and the organic solvent may then be dried.

[0049] In the culturing step, cells may be cultured by a cell culture method using a known medium. The culture conditions are, for example, 37°C, CO ２The 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.

[0050] The human macrophages obtained by the production method according to this embodiment can be induced from hematopoietic progenitor cells without adding cytokines to the culture medium. Such macrophages are called lipid-induced macrophages (LIM). Furthermore, these human macrophages have proliferative properties after induction and can be expanded. The number of days (survival period) that these human macrophages can be cultured is at least 28 days.

[0051] In another embodiment, human macrophages (lipid-induced macrophages) are provided, and differentiation of the human macrophages has been induced in a TREM2-dependent manner by culturing human hematopoietic progenitor cells in a medium substantially free of M-CSF and GM-CSF in the presence of a TREM2 signaling activator, preferably in the absence of feeder cells and extracellular matrix.

[0052] Furthermore, as shown in Test Example 1 below, human macrophages according to this embodiment retained the ability to proliferate in the presence of a TREM2 signaling activator, at least after 28 days of subculture, under conditions in which the medium did not contain either M-CSF or GM-CSF and did not contain feeder cells or extracellular matrix. Therefore, in another embodiment, human macrophages are provided, which, after the start of induction, retain the ability to proliferate in a medium that does not contain either M-CSF or GM-CSF and contains a TREM2 signaling activator, preferably after 14 days of subculture, preferably after 20 days or after 28 days, under conditions in which the medium did not contain either M-CSF or GM-CSF and contained a TREM2 signaling activator, did not contain feeder cells or extracellular matrix.

[0053] The human macrophages according to this embodiment may have one, two or more, three or more, four or more, five or more, or all of the above-mentioned characteristics arbitrarily selected from the above-mentioned characteristics.

[0054] In another embodiment, there is provided a method for preparing human macrophages, a method for generating human macrophages, a method for inducing differentiation of human macrophages, a method for culturing human macrophages, or a method for expanding human macrophages, which includes the above-mentioned culturing step. According to the human macrophage culturing method, etc., human macrophages can be maintained over a long period of time. Furthermore, according to the human macrophage expansion method, human macrophages are expanded. Since the human macrophages can be expanded in a medium substantially free of M-CSF and GM-CSF in the presence of a TREM2 signaling activator, another embodiment provides a method for expanding human macrophages, which includes a culturing step of culturing human macrophages in a medium substantially free of M-CSF and GM-CSF in the presence of a TREM2 signaling activator.

[0055] In another embodiment, a differentiation inducer is provided that contains a TREM2 signaling activator and is substantially free of M-CSF and GM-CSF. The differentiation inducer induces differentiation from human hematopoietic progenitor cells to human macrophages. In addition to the TREM2 signaling activator, the differentiation inducer may contain solvents, water, ethanol, polyhydric alcohols, oils, surfactants, thickeners, preservatives, pH adjusters, and the like. The differentiation inducer may be in the form of, for example, a liquid, gel, cream, or solid. 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. This differentiation inducer enables the generation of long-lived human macrophages from human hematopoietic progenitor cells.

[0056] In another embodiment, there is provided a proliferation promoter for human macrophages, which contains a TREM2 signaling activator and is substantially free of both M-CSF and GM-CSF. The proliferation promoter promotes the proliferation of human macrophages, particularly human macrophages obtained by the above-described production method. Like the differentiation inducer, the proliferation promoter may contain components other than the TREM2 signaling activator.

[0057] In another embodiment, a differentiation induction kit is provided, comprising a cell culture vessel whose surface is coated with a TREM2 signaling activator. The differentiation induction kit is used for culturing human hematopoietic progenitor cells in a medium substantially free of both M-CSF and GM-CSF, and induces differentiation of the human hematopoietic progenitor cells into human macrophages. The differentiation induction kit may further comprise a medium such as a basal medium, a serum-reduced medium, or a serum-free medium, a serum necessary for cell culture such as FBS, and other known additives added to the medium.

[0058] This differentiation induction kit includes a cell culture vessel whose surface has been pre-coated with a TREM2 signaling activator, and therefore human macrophages with long survival times can be easily obtained simply by seeding human hematopoietic progenitor cells in the cell culture vessel and culturing them. This differentiation induction kit can also be used as a kit for promoting the proliferation of human macrophages.

[0059] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0060] Example 1: Preparation of human lipid-induced macrophages (LIM) [Coating of cell culture plate with lipid] The test substance, mouse brain lipid (hereinafter also referred to simply as "BL"), MA, CL, PC, sulf, palm, steato- or chol, was diluted with methanol to 50 μg / ml. 100 μl (5 μg) of the test substance was dispensed into a 24-well cell culture plate and allowed to stand in a clean bench to dry the methanol. PC and Sulf are known to be ligands for human TREM2 (Yaming Wang and 12 others, "TREM2 Lipid Sensing Sustains the Microglial Response in an Alzheimer's Disease Model", Cell, 2015, 160, 1061-1071).

[0061] BL was prepared as follows. Mouse brains (approximately 400 mg per mouse) were submerged in 1 ml of methanol in a polypropylene tube and minced 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 a chloroform to methanol (MeOH) volume ratio of 2:1 (C-M 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 Solution 1. The brain residue remaining in the glass test tube was mixed with 2 ml of methanol and 4 ml of chloroform, and the resulting sample was mixed at room temperature for 16 hours using a shaker (200 rpm). The filtered supernatant was collected in the glass bottle containing Extract Solution 1 and mixed well to make Extract Solution 2. Extract Solution 2 was stored at -30°C until use. 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 lipid was measured. It was dissolved in C-M 2:1 to make 5 mg / ml and used as BL extract.

[0062] [Induction of LIM] Collected human bone marrow cells were cultured at 4 × 10 ５ The bone marrow cell suspension was then suspended in 10% FBS / RPMI to a concentration of 1 x 10 cells / ml. ５ cells / 0.25ml / cm ２ The bone marrow cells were added at 37°C and 5% CO ２ The cells were cultured in an incubator. After 3 days of culture, half of the fresh medium was added to the culture plate. Thereafter, the medium was changed every 3 to 4 days.

[0063] (Results) Macrophage-like adherent cells began to appear around 7 to 10 days after the start of culture. Figure 1 shows an image of LIM 14 days after the start of culture.

[0064] Comparative Example 1: Preparation of BMDM Collected human bone marrow cells were cultured at 4 × 10 ５ The bone marrow cell suspension was suspended in 10% FBS / RPMI to a concentration of 2 × 10 cells / ml. 100 ng / ml M-CSF or GM-CSF was added to the cell suspension. 2 × 10 bone marrow cell suspension was added to each well of a 24-well culture plate. ５The 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 volume (0.25 ml / well) of 100 ng / ml M-CSF or GM-CSF / 10% FBS / RPMI was added. After 5 days of culture, the supernatant was aspirated, 0.5 ml / well of 10% FBS / RPMI was added, and the cells were detached with a scraper. The cells were diluted to 1 / 4 with 10% FBS / RPMI and dispensed at 0.5 ml / well, followed by the addition of 100 ng / ml M-CSF or GM-CSF. Thereafter, half the volume of the 100 ng / ml M-CSF / 10% FBS / RPMI medium was replaced every 3 to 4 days.

[0065] (Results) 14 days after the start of culture, 2.1 to 8.7 x 10 ４ Figure 2 shows images of BMDMs after 14 days in culture.

[0066] Test Example 1: Analysis of expression of cell surface markers in BMDM and LIM LIM 14 and 28 days after the start of induction in Example 1 and BMDM 14 and 28 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 for 5 minutes at 4°C, and the supernatant was removed. ７ 1% FBS / HBSS was added to the cell suspension to give a concentration of 100 cells / ml, and the cells were suspended in the suspension. 1 / 20 volume of Human TruStain FcX (BioLegend) was added to the cell suspension, and the mixture was left on ice for 10 minutes to block the Fcγ receptor.

[0067] The blocked cell suspension was dispensed into a 96-well V-bottom plate at 10 μl / well, and PE anti-mouse / human TREM2 antibody (manufactured by R&D), FITC anti-mouse / human CD11b antibody, APC anti-human CD163 antibody, APC / Cyanine7 anti-human CD45 antibody, and PE / Cyanine7 anti-human CD115 antibody (all manufactured by BioLegend) diluted in 1% FBS / HBSS to the recommended concentrations described in the instruction manual were added to a total volume of 20 μl / well, and the plate was left to react on ice for 15 minutes. The plate was then washed with 180 μl / well of 1% FBS / HBSS.

[0068] The samples were centrifuged at 500 G for 5 minutes at 4°C, and the supernatant was removed. 180 μl of 1% FBS / HBSS was added per well to wash the samples. The samples were centrifuged at 500 G for 5 minutes at 4°C, and the supernatant was removed. 50 μl of 1% FBS / HBSS was added per well to suspend the samples. The obtained samples were analyzed by flow cytometry, and CD45-positive, CD11b-positive, CD115-positive, and CD163-positive cells were extracted as macrophage cells, analyzed, and counted.

[0069] (Results) The results of dot plot analysis of BMDM induced with M-CSF or GM-CSF, and LIM induced with BL, MA, or CL are shown in Figure 3. The results of dot plot analysis of LIM induced with PC, Sulf, Palm, Tea, or Chol are shown in Figure 4. The distribution of forward scattered light (FSC), an index of cell size, and side scattered light (SSC), an index of cellular disorder (abundance of granules), was similar between LIM and BMDM. LIM showed high expression of the pan-leukocyte marker CD45 and the granulocyte and macrophage marker CD11b, as well as high expression of the monocyte marker CD115 and the macrophage marker CD163. These results indicated that LIM were macrophages.

[0070] The number of macrophages 14 and 28 days after the start of induction is shown in Figure 5. It was shown that LIM was induced by CL, PC, and Sulf, which have been reported to be TREM2 ligands, as well as BL and MA.

[0071] The change in macrophage number up to 28 days after the start of induction is shown in Figure 6. BMDM induced with M-CSF or GM-CSF was reduced compared to day 14, but LIM induced with BL or MA continued to proliferate.

[0072] Test Example 2: Evaluation of phagocytic activity BMDM induced with M-CSF or LIM induced with BL was cultured at 1 × 10 ４The cells were seeded onto a 96-well plate at 100 μl / well, and 5 μg of pHrodo Green E. coli BioParticles (Thermo Fisher Scientific) was added, followed by incubation for 3 hours at 37° C. After incubation, the cells were collected, and the phagocytic activity was evaluated by measuring the fluorescence intensity of pHrodo Green by flow cytometry.

[0073] (Results) The mean fluorescence intensity (MFI) of phagocytosed pHrodo Green E. coli BioParticles is shown in Figure 7. It was demonstrated that LIM has phagocytic ability comparable to that of BMDM.

[0074] Test Example 3: Investigation of cytokine production response to lipopolysaccharide (LPS) stimulation. BMDM induced with M-CSF or LIM induced with BL were cultured at 2 × 10 ４ The cells were seeded at 100 μl / well in a 96-well plate, 10 ng / ml LPS was added, and the cells were incubated for 24 hours at 37° C. After incubation, the culture supernatant was collected, and the concentrations of TNF-α and IL-10 in the supernatant were measured using ELISA kits (ELISA MAX Standard Set Human TNF-α and ELISA MAX Standard Set Human IL-10, manufactured by BioLegend).

[0075] (Results) Figures 8A and 8B show the concentrations of TNF-α and IL-10 in the supernatant, respectively, demonstrating that LIM has cytokine production capacity comparable to that of BMDM.

[0076] Test Example 4: Subculture of LIM When using a 24-well plate, macrophages appeared approximately 9 days after the start of induction. Therefore, LIM induced with M-CSF or GM-CSF (BMDM) or BL or CL were subcultured every 14 to 18 days as follows: The culture supernatant was aspirated, and 0.1 ml of 1 mM ethylenediaminetetraacetic acid (EDTA) / phosphate buffered saline (PBS) was added and incubated at 37°C for 5 minutes. 0.4 ml of 10% FBS / RPMI was added, and cells detached by pipetting and a cell scraper were suspended. The cell number was measured using a hemocytometer. The cell suspension was diluted 1 / 4 with 10% FBS / RPMI and dispensed at 0.5 ml per well onto a new lipid-coated plate. Half of the 10% FBS / RPMI medium was then replaced every 3 to 4 days, and the cells were subcultured again after 14 to 18 days.

[0077] (Results) Figure 9A shows the cell counts from days 9 to 57 of culture, and Figure 9B shows the fold increase in cell counts from days 9 to 57 of culture, with the cell count on day 9 of culture defined as 1. BMDMs induced with M-CSF or GM-CSF showed almost no increase after serial dilution. On the other hand, LIMs induced with BL or CL survived and proliferated even after at least 57 days of culture.

[0078] The present invention allows various embodiments and modifications without departing from the broad spirit and scope of the present invention. Furthermore, the above-described embodiments are intended to illustrate the present invention and do not limit the scope of the present invention. That is, the scope of the present invention is defined by the claims, not the embodiments. Various modifications made within the scope of the claims and the meaning of the invention equivalent thereto are considered to be within the scope of the present invention.

[0079] This application is based on Japanese Patent Application No. 2023-101497, filed on June 21, 2023. The entire specification, claims, and drawings of Japanese Patent Application No. 2023-101497 are incorporated herein by reference.

[0080] The present invention is useful for obtaining human macrophages.

Claims

1. A method for producing human macrophages, comprising a culturing step of culturing human hematopoietic progenitor cells in a medium substantially free of both macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor in the presence of a TREM2 signal activator.

2. The method for producing human macrophages according to claim 1, wherein the medium is substantially free of interleukin-34.

3. The method for producing human macrophages according to claim 1 or 2, wherein the medium is substantially free of cytokines.

4. The method for producing human macrophages according to claim 1 or 2, wherein in the culturing step, the human hematopoietic progenitor cells are cultured under conditions in which supporting cells and extracellular matrix are not added.

5. The method for producing human macrophages according to claim 1 or 2, wherein the TREM2 signal activator is a lipid.

6. The method for producing human macrophages according to claim 1 or 2, wherein the TREM2 signal activator is a brain-derived lipid or a lipid having multiple long-chain fatty acid groups.

7. The method for producing human macrophages according to claim 1 or 2, wherein the TREM2 signal activator is a compound represented by formula (I), an ester thereof, or a salt thereof. (having 60 to 90 carbon atoms, 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.

8. The method for producing human macrophages according to claim 1 or 2, wherein the TREM2 signal activator is a phospholipid.

9. The method for producing human macrophages according to claim 8, wherein the phospholipid is a lipid that constitutes a biological membrane.

10. The method for producing human macrophages according to claim 8, wherein the phospholipid is cardiolipin.

11. The method for producing human macrophages according to claim 1 or 2, wherein the surface of a cell culture vessel for culturing the human hematopoietic progenitor cells is coated with the TREM2 signal activator.

12. The method for producing human macrophages according to claim 1 or 2, wherein the human hematopoietic progenitor cells are human myeloid progenitor cells.

13. An agent for inducing differentiation of human hematopoietic progenitor cells into human macrophages, comprising a TREM2 signal activator and substantially free of both macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.

14. A kit for inducing differentiation of human hematopoietic progenitor cells into human macrophages, comprising a cell culture vessel whose surface is coated with a TREM2 signal activator, and used for culturing human hematopoietic progenitor cells in a medium that is substantially free of both macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.

15. A method for inducing differentiation of human macrophages, comprising a culturing step of culturing human hematopoietic progenitor cells in a medium substantially free of both macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor in the presence of a TREM2 signal activator.

16. A proliferation-promoting agent for human macrophages, comprising a TREM2 signal activator and substantially free of both macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.

17. A kit for promoting the proliferation of human macrophages, comprising a cell culture vessel whose surface is coated with a TREM2 signal activator, and used for culturing human hematopoietic progenitor cells in a medium that is substantially free of both macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.

18. A method for proliferating human macrophages, comprising a culturing step of culturing human hematopoietic progenitor cells in a medium substantially free of both macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor in the presence of a TREM2 signal activator.

19. A human macrophage having the ability to proliferate in the presence of a TREM2 signal activator after 10 days of subculture in a medium substantially free of both macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.

20. A human macrophage in which differentiation is induced in a TREM2-dependent manner by culturing human hematopoietic progenitor cells in a medium substantially free of both macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor in the presence of a TREM2 signal activator.

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