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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M1/00—Apparatus for enzymology or microbiology
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

• 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.
• Macrophages which are white blood cells that contribute to infection defense and maintenance of tissue homeostasis, 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).
• M-CSF macrophage colony-stimulating factor
• GM-CSF granulocyte-macrophage colony-stimulating factor
• a method has been established to induce differentiation into cells with the 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 white blood cells and culturing them.
• M-CSF macrophage colony-stimulating factor
• GM-CSF granulocyte-macrophage colony-stimulating factor
• IL-34 acts on the M-CSF receptor to promote differentiation of monocytes into macrophages and proliferation of macrophages (see Non-Patent Documents 1 and 2).
• iPSCs induced pluripotent stem cells
• a culture plate coated with an extracellular matrix a culture plate coated with an extracellular matrix
• 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
• BMP4 vascular endothelial growth factor
• SCF stem cell factor
• bFGF Flt3 ligand
• IL-3 interleukin-3
• TPO thrombopoietin
• M-CSF GM-CSF
• GM-CSF GM-CSF
• 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- ⁇ activating activity.
• Non-Patent Document 3 when inducing differentiation of mature macrophages from iPSCs, extracellular matrix, multiple types of cytokines, and growth factors are required, and the induced monocytes must be further cultured with M-CSF.
• the present invention has been made in consideration of the above-mentioned circumstances, and aims to provide a method for producing human macrophages that can be induced from hematopoietic precursor cells without adding cytokines to the culture medium, and that can produce human macrophages that have proliferation properties after induction and can be expanded, 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, and a method for expanding human macrophages.
• Another aim of the present invention is to provide human macrophages that can be induced from hematopoietic precursor cells without adding cytokines to the culture medium, that have proliferation properties after induction, and can be expanded.
• the inventors 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 thus completed the present invention.
• the method for producing human macrophages described herein comprises: The method includes 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.
• the medium is It may be substantially free of interleukin-34.
• the medium is It may be substantially free of cytokines.
• the human hematopoietic progenitor cells may be cultured under conditions in which feeder cells and extracellular matrix are not added.
• the TREM2 signal activator is It may be a lipid.
• the TREM2 signal activator is The lipid may be brain-derived or may have multiple long-chain fatty acid groups.
• the TREM2 signal activator is It may be a compound represented by formula (I), an ester thereof or a salt thereof.
• R 1 represents a saturated or unsaturated aliphatic hydrocarbon group having 60 to 90 carbon atoms
• R 2 represents a saturated or unsaturated aliphatic hydrocarbon group which may have a cyclic structure or a substituent.
• the TREM2 signal activator is It may be a phospholipid.
• the phospholipid is The lipid may be a lipid that constitutes a biological membrane.
• the phospholipid is It may be cardiolipin.
• the surface of the cell culture vessel for culturing the human hematopoietic progenitor cells is The antibody may be coated with the TREM2 signal activator.
• the human hematopoietic progenitor cells are The cells may be human myeloid progenitor cells.
• the agent for inducing differentiation of human hematopoietic progenitor cells into human macrophages described herein is The composition comprises a TREM2 signal activator and is substantially free of both macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.
• the kit for inducing differentiation of human hematopoietic progenitor cells into human macrophages described in the present specification comprises: A cell culture vessel having a surface coated with a TREM2 signal activator is provided, It is used in the culture of human hematopoietic progenitor cells in a medium that is substantially free of both macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.
• the method for inducing differentiation of human macrophages described herein comprises the steps of: The method includes 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.
• Human macrophage proliferation promoter The agent for promoting proliferation of human macrophages described herein is It contains a TREM2 signal activator and is substantially free of both macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.
• the kit for promoting proliferation of human macrophages described herein comprises: A cell culture vessel having a surface coated with a TREM2 signal activator is provided, It is used in the culture of human hematopoietic progenitor cells in a medium that is substantially free of both macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.
• the method for expanding human macrophages described herein comprises: The method includes 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.
• human macrophages The human macrophages described herein are In the presence of a TREM2 signal activator, the cells have the ability to proliferate from day 10 onwards when subcultured in a medium substantially free of both macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.
• human macrophages described herein are In the presence of a TREM2 signal activator, human hematopoietic progenitor cells were cultured in a medium substantially free of both macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, thereby inducing differentiation in a TREM2-dependent manner.
• 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.
• FIG. 1 shows images of macrophage-like adherent cells cultured in the presence of various lipids in Example 1.
• FIG. 1 shows images of BMDM cultured in the presence of M-CSF or GM-CSF in Comparative Example 1.
• FIG. 1 shows 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.
• FIG. 1 shows the results of dot blot analysis of human macrophages induced with L-3-phosphatidylcholine plasmalogen, sulfatide, palmitic acid, stearic acid, or cholesterol in Test Example 1.
• FIG. 1 shows images of macrophage-like adherent cells cultured in the presence of various lipids in Example 1.
• FIG. 1 shows images of BMDM cultured in the presence of M-CSF or GM-CSF in Comparative Example 1.
• FIG. 1 shows the results of dot plot analysis of
• FIG. 1 shows the number of human macrophages 14 days and 28 days after the start of induction in Test Example 1.
• FIG. 1 shows changes in macrophage count up to 28 days after the start of induction in Test Example 1.
• FIG. 1 shows the results of evaluation of phagocytic activity in Test Example 2.
• FIG. 1 shows the concentration of TNF- ⁇ in the supernatant in Test Example 3.
• FIG. 1 shows the concentration of IL-10 in the supernatant in Test Example 3.
• FIG. 1 shows the cell counts on days 9 to 57 of culture in Test Example 4.
• FIG. 13 is a graph showing the fold increase in cell count in Test Example 4.
• the method for producing human macrophages includes a culture step of culturing human hematopoietic progenitor cells in a medium that is substantially free of both M-CSF and GM-CSF in the presence of a TREM2 signal activator.
• TREM2 is an immunoglobulin superfamily receptor that associates with the adaptor molecule DAP12 (DNAX-activating protein of 12 kDa).
• the TREM2 signal activator may be any substance that specifically binds to TREM2 and generates a signal via DAP12.
• 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, a TREM2 ligand, etc.
• 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 in this specification includes an antigen-binding fragment 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 can 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) and Christoph Stein et al., Antibodies(1):88-123 (2012).
• scFv single
• the TREM2 signal activator is, for example, a ligand for TREM2 or a lipid that functions as a TREM2 ligand.
• lipids include sphingoglycolipids, phospholipids, sulfated glycolipids, and fatty acids.
• 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.
• 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 the long-chain fatty acid group include fatty acid groups with 6 to 60 or 10 to 50 carbon atoms, and the number of carbons that constitute the carbon chain of each long-chain fatty acid group may be the same or different.
• 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 derived lipids or synthetic lipids.
• the TREM2 signal activator may be a composition in which multiple lipids are mixed, and may be a composition containing lipids obtained by extraction using an organic solvent from animal-derived tissues such as the brain, plant-derived tissues, or microorganisms such as bacteria, or may be a composition in which any two or more types of lipids are artificially mixed. Or it may be a synthetic lipid that binds to TREM2.
• One embodiment of the TREM2 signal activator includes cells, microorganisms, or lipids derived from microorganisms that have lipids on the cell surface.
• the TREM2 signal activator is a brain-derived lipid, MA, or CL, for example, represented by the following formula.
• CL is a phospholipid specific to mitochondria.
• 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.
• R 1 represents a saturated or unsaturated aliphatic hydrocarbon group.
• the number of carbon atoms in R 1 is, for example, 6 to 60, 10 to 50, 20 to 40, 22 to 30, or 22 to 26.
• R 2 represents a saturated or unsaturated aliphatic hydrocarbon group which may have a cyclic structure or a substituent.
• the number of carbon atoms in R 2 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 may be included in R 2 as a cycloalkylene group, or may be included in R 2 as a cycloalkyl group.
• R 2 may contain one to several cyclic structures.
• the substituent is not particularly limited, and examples thereof include a methyl group, a hydroxyl group, a carbonyl group, and a carboxyl group.
• the salt of the compound represented by formula (I) above is not particularly limited as long as it is a pharmacologically acceptable salt, and may be either an acid salt or a basic salt.
• 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, metathionate, tetrahydrofuran
• organic acid salts include benzenesulfonate, 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, trifluoroacetate (TFA) salt, maleate, dimaleate, and muconate.
• TFA trifluoroacetate
• the ester of the compound represented by formula (I) above is not particularly limited as long as it is a pharmacologically acceptable ester, and examples thereof include carbonate esters, phosphate esters, nitrate esters, sulfate esters, borate esters, and sulfonate esters. Note that this embodiment also includes various hydrates and solvates of the compound of formula (I) and its salts, as well as crystalline polymorphic substances.
• the TREM2 signal activator may be selected by acting the substance to be screened on cells expressing TREM2 and DAP12, and using as an indicator the expression of a reporter protein caused by the TREM2 signal, for example, the TREM2 signal, which is activated by the binding of the substance to TREM2.
• TREM2 and DAP12 can be expressed in cells by known methods, for example, by introducing a gene expression plasmid 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 genes controlled by NFAT (nuclear factor of activated T cells)-type transcription factors are expressed by the TREM2 signal.
• 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 to activate the TREM2 signal.
• the same method can also be used to confirm whether or not any lipid functions as a TREM2 ligand.
• a medium substantially free of both M-CSF and GM-CSF refers to a medium in which at least one of M-CSF and GM-CSF is contained, if at all, only in trace amounts, and to which M-CSF and GM-CSF have not been intentionally added.
• a medium substantially free of both M-CSF and GM-CSF refers to a medium that does not contain either M-CSF or 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.
• cases in which at least one of M-CSF and GM-CSF is contained in a trace amount in the medium include cases in which the cultured human hematopoietic progenitor cells secrete at least one of M-CSF and GM-CSF, or cases in which at least one of M-CSF and GM-CSF is contained in the additives described below.
• the medium may be a known medium that can be used 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.
• human macrophages can be differentiated from human hematopoietic progenitor cells without using cytokines such as stem cell factor (SCF), thrombopoietin (TPO), Flt3 ligand (FL), IL-6, IL-34, and granulocyte colony-stimulating factor (G-CSF) in addition to M-CSF and GM-CSF.
• SCF stem cell factor
• TPO thrombopoietin
• FL Flt3 ligand
• IL-6 IL-6
• IL-34 granulocyte colony-stimulating factor
• G-CSF granulocyte colony-stimulating factor
• the "medium substantially free of M-CSF and GM-CSF” does not substantially contain IL-34 or cytokines.
• substantially free of IL-34 or cytokines refers to a medium in which, if IL-34 or cytokines are contained, they are in trace amounts, and to which IL-34 or cytokines are not intentionally added, as in the above-mentioned "medium substantially free of both M-CSF and GM-CSF.”
• the "medium substantially free of M-CSF and GM-CSF” does not substantially contain any agonist (antibody, compound, etc.) that activates one or more cytokine receptors.
• the "medium substantially free of M-CSF and GM-CSF” does not substantially contain 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.
• the "medium substantially free of M-CSF and GM-CSF" does not substantially contain any agonists that activate M-CSF receptors, agonists that activate GM-CSF receptors, or agonists that activate IL-34 receptors, and may, for example, not substantially contain any agonists that activate any cytokine receptors.
• 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.
• the concentration of human hematopoietic progenitor cells is not particularly limited, but for example, 1 ⁇ 10 3 to 1 ⁇ 10 7 cells or 1 ⁇ 10 4 to 1 ⁇ 10 6 cells are seeded per unit culture area (cm 2 ).
• 1 ⁇ 10 5 human hematopoietic progenitor cells are seeded per unit culture area (cm 2 ).
• human macrophages can be differentiated from human hematopoietic progenitor cells without using supporting cells and extracellular matrix. Therefore, preferably, in the culturing step, human hematopoietic progenitor cells are cultured under conditions without adding supporting cells and extracellular matrix.
• human hematopoietic progenitor cells may be cultured in a cell culture vessel holding a TREM2 signal activator.
• the cell culture vessel include a cell culture plate, a cell culture flask, and a cell culture dish.
• the TREM2 signal activator is insoluble in water and insoluble in a liquid medium
• human hematopoietic progenitor cells may be cultured in a cell culture vessel whose surface is coated with the TREM2 signal activator.
• the mass of the lipid coating the surface of the cell culture vessel is, for example, 1.0 ⁇ g/cm 2 or more, 1.5 to 20 ⁇ g/cm 2 , 2 to 18 ⁇ g/cm 2 , 3 to 16 ⁇ g/cm 2 , and preferably 3 to 12 ⁇ g/cm 2 for 1 ⁇ 10 4 to 1 ⁇ 10 6 human hematopoietic progenitor cells.
• 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.
• the cells may be cultured by a cell culture method using a known medium.
• the culture conditions are, for example, 37°C and 5% CO2 concentration.
• it is preferable to replace 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.
• the human macrophages obtained by the manufacturing method according to this embodiment can be induced from hematopoietic precursor cells without adding cytokines to the culture medium. Such macrophages are called lipid induced macrophages (LIM). Furthermore, the human macrophages have proliferation properties after induction and can be expanded. The number of days (survival period) that the human macrophages can be cultured is at least 28 days or more.
• LIM lipid induced macrophages
• human macrophages lipid-induced macrophages
• differentiation of the human macrophages is 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 signal activator, preferably in the absence of supporting cells and extracellular matrix.
• human macrophages had the ability to proliferate in the presence of a TREM2 signaling activator, under conditions in which the medium did not contain either M-CSF or GM-CSF, and no supporting cells or extracellular matrix was added, at least after 28 days of subculture. Therefore, in another embodiment, human macrophages are provided, which, after the start of induction, have 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 no supporting cells or extracellular matrix was added.
• the human macrophages of this embodiment may have one, two or more, three or more, four or more, five or more, or all of the characteristics described above.
• a method for preparing human macrophages a method for producing human macrophages, a method for inducing differentiation of human macrophages, a method for culturing human macrophages, or a method for proliferating human macrophages, including the above-mentioned culturing step.
• human macrophages can be maintained for a long period of time.
• human macrophages are proliferated.
• a method for proliferating human macrophages including a culturing step of culturing human macrophages in a medium substantially free of both M-CSF and GM-CSF in the presence of a TREM2 signal activator.
• a differentiation inducer in another embodiment, contains a TREM2 signal activator and is substantially free of both M-CSF and GM-CSF.
• the differentiation inducer induces differentiation from human hematopoietic progenitor cells to human macrophages.
• the differentiation inducer may contain a solvent, water, ethanol, polyhydric alcohol, oil, surfactant, thickener, preservative, pH adjuster, etc.
• the differentiation inducer may be in the form of, for example, a liquid, gel, cream, or solid.
• the differentiation inducer is added to a cell culture vessel or applied to the surface of the cell culture vessel to coat the surface. With the differentiation inducer, human macrophages with a long survival period can be obtained from human hematopoietic progenitor cells.
• a proliferation promoter for human macrophages contains a TREM2 signal activator and is substantially free of both M-CSF and GM-CSF.
• the proliferation promoter promotes proliferation of human macrophages, particularly human macrophages obtained by the above-mentioned production method.
• the proliferation promoter may contain components other than the TREM2 signal activator, similar to the differentiation inducer.
• a differentiation induction kit in another embodiment, includes a cell culture vessel whose surface is coated with a TREM2 signal activator.
• the differentiation induction kit is used for culturing human hematopoietic progenitor cells in a medium that is substantially free of both M-CSF and GM-CSF, and induces differentiation of human hematopoietic progenitor cells into human macrophages.
• the differentiation induction kit may further include a medium such as a basal medium, a serum-reduced medium, or a serum-free medium, serum necessary for cell culture such as FBS, and other known additives that are added to the medium.
• the differentiation induction kit includes a cell culture vessel whose surface has been pre-coated with a TREM2 signal activator, so long-lived human macrophages can be easily obtained simply by seeding and culturing human hematopoietic progenitor cells in the cell culture vessel.
• the differentiation induction kit can also be used as a proliferation promotion kit for human macrophages.
• Example 1 Preparation of human lipid-induced macrophages (LIM) [Coating of cell culture plates with lipids]
• the test substances mouse brain lipids (hereinafter, also simply referred to as "BL"), MA, CL, PC, Sulf, Palm, Stea, or Chol, were diluted with methanol to 50 ⁇ g/ml.
• the test substances were dispensed into 24-well cell culture plates in 100 ⁇ l (5 ⁇ g) portions, and the plates were left to stand in a clean bench to dry the methanol.
• PC and Sulf are known to be ligands for human TREM2 (Yaming Wang, et al., "TREM2 Lipid Sensing Sustains the Microglial Response in an Alzheimer's Disease Model", Cell, 2015, 160, 1061-1071).
• BL was prepared as follows. 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 (C-M 2:1). The sample was mixed at room temperature for 3 hours (200 rpm). The sample was centrifuged at 2000 rpm for 15 minutes, and the supernatant was filtered and collected in a glass bottle as Extraction 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 sample was mixed at room temperature for 16 hours (200 rpm) using a shaker.
• 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.
• 1.8 ml of extract 2 was dispensed into a weighed glass bottle, heated to 37°C and sparged with nitrogen to dry off the solvent, and the dry weight of lipids was measured. It was dissolved in C-M 2:1 to give a concentration of 5 mg/ml, and used as the BL extract.
• the collected human bone marrow cells were suspended in 10% FBS/RPMI to a concentration of 4 x 105 cells/ml.
• the bone marrow cell suspension was added to a cell culture plate coated with the test substance to a concentration of 1 x 105 cells/0.25 ml/ cm2 .
• the bone marrow cells were cultured at 37°C in a 5% CO2 incubator. After 3 days of culture, half the amount of fresh medium was added to the culture plate. Thereafter, the medium was replaced every 3 to 4 days.
• Figure 1 shows an image of the LIM after 14 days from the start of culture.
• Comparative Example 1 Preparation of BMDM Collected human bone marrow cells were suspended in 10% FBS/RPMI to 4 x 105 cells/ml. 100 ng/ml M-CSF or GM-CSF was added to the cell suspension. The bone marrow cell suspension was added to each well of a 24-well culture plate to 2 x 105 cells/0.5 ml. The bone marrow cells were cultured at 37°C in a 5% CO2 incubator. After 3 days of culture, half the amount (0.25 ml/well) of 100 ng/ml M-CSF or GM-CSF/10% FBS/RPMI was added.
• the supernatant was aspirated, 0.5 ml/well of 10% FBS/RPMI was added, and the cells were peeled off with a scraper.
• the medium was diluted to 1/4 with 10% FBS/RPMI, dispensed at 0.5 ml per well, and 100 ng/ml M-CSF or GM-CSF was added. After that, half of the medium was replaced with 100 ng/ml M-CSF/10% FBS/RPMI every 3 to 4 days.
• Figure 2 shows images of BMDM after 14 days from the start of culture.
• Test Example 1 Analysis of expression of cell surface markers of BMDM and LIM LIM 14 days and 28 days after the start of induction in Example 1 and BMDM 14 days 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 500G and 4°C for 5 minutes and the supernatant was removed. 1% FBS/HBSS was added to the cell suspension to give a concentration of 1 x 107 cells/ml and suspended. 1/20 volume of Human TruStain FcX (manufactured by BioLegend) was added to the cell suspension and left on ice for 10 minutes to block Fc ⁇ receptors.
• Human TruStain FcX manufactured by BioLegend
• the samples were centrifuged at 500G for 5 minutes at 4°C, and the supernatant was removed. 180 ⁇ l/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 ⁇ l/well of 1% FBS/HBSS was added 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.
• BMDMs induced with M-CSF or GM-CSF had decreased compared to 14 days later, but LIMs induced with BL or MA continued to proliferate.
• Test Example 2 Evaluation of phagocytic activity BMDM induced with M-CSF or LIM induced with BL were seeded on a 96-well plate at 1 x 104 cells/100 ⁇ l/well, 5 ⁇ g of pHrodo Green E. coli BioParticles (Thermo Fisher Scientific) was added, and the cells were incubated 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.
• Test Example 3 Study of cytokine production response to lipopolysaccharide (LPS) stimulation BMDM induced with M-CSF or LIM induced with BL were seeded in a 96-well plate at 2 x 104 cells/100 ⁇ l/well, and 10 ng/ml LPS was added and incubated for 24 hours at 37° C. After incubation, the culture supernatant was collected, and the concentrations of TNF- ⁇ and IL-10 in the supernatant were measured using ELISA kits (ELISA MAX Standard Set Human TNF- ⁇ and ELISA MAX Standard Set Human IL-10, BioLegend).
• LPS lipopolysaccharide
• (result) 8A and 8B show the concentrations of TNF- ⁇ and IL-10 in the supernatant, respectively, demonstrating that LIM has a cytokine production ability comparable to that of BMDM.
• Test Example 4 Subculture of LIM When a 24-well plate is used, macrophages appear about 9 days after the start of induction. Therefore, BMDM induced with M-CSF or GM-CSF, or LIM induced with 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 buffer (PBS) was added and incubated at 37°C for 5 minutes. 0.4 ml of 10% FBS/RPMI was added, and the cells detached by pipetting and a cell scraper were suspended. The number of cells was measured using a hemocytometer.
• EDTA ethylenediaminetetraacetic acid
• PBS phosphate buffer
• the cell suspension was diluted to 1/4 with 10% FBS/RPMI and dispensed into a new lipid-coated plate at 0.5 ml/well. Thereafter, half of the 10% FBS/RPMI medium was replaced every 3 to 4 days, and the medium was subcultured again after 14 to 18 days.
• Figure 9A shows the cell numbers from days 9 to 57 of culture
• Figure 9B shows the fold increase in cell numbers from days 9 to 57 of culture, with the number of cells on day 9 of culture taken as 1.
• BMDM induced with M-CSF or GM-CSF hardly increased after serial dilution.
• LIM induced with BL or CL survived and proliferated even after at least 57 days of culture.
• the present invention is useful for obtaining human macrophages.

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