Differentiation and use of human microglia-like cells from pluripotent stem cells and hematopoietic primordial cells

Abstract

This invention provides a method for producing microglia cells. [Solution] A method for producing human microglia-like cells (iMGLs) from pluripotent stem cells (PSCs) is provided, comprising the steps of (i) differentiating PSCs using a culture medium supplemented with hematopoietic differentiation factors to produce induced hematopoietic primordial cells (iHPCs), and (ii) differentiating CD43+ iHPCs into human microglia-like cells (iMGLs) using a microglia differentiation medium.

Description

Technical Field

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 464,925, filed Feb. 28, 2017. The content of the above application is hereby expressly incorporated by reference in its entirety.

[0002] Description of R&D Funded by the Federal Government This invention was made with government support under Grant No. AG048099 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Embodiments of (i) human microglia-like cells (iMGL) and (ii) methods of making iMGL are described herein.

Background Art

[0004] Microglia cells are innate immune cells of the CNS and are known to play a role in the physiological development of the CNS. Furthermore, microglia cells are known to play a role in neuropathologies such as Alzheimer's disease. There are deficiencies in the techniques for obtaining microglia cells to further investigate the role played by microglia cells in CNS development and neuropathologies.

Summary of the Invention

Means for Solving the Problems

[0005] In some embodiments, a method of producing human microglia-like (iMGL) from pluripotent stem cells (PSC) is provided. In some embodiments, the method comprises (i) differentiating PSC using a medium supplemented with hematopoietic differentiation factors to produce induced hematopoietic progenitor cells (iHPC), and (ii) isolating CD43 + iHPC, and (iii) differentiating CD43 + iHPC into iMGL using a microglia differentiation medium, and (iv) maturing the iMGL.

[0006] In some embodiments, the method includes (i) differentiating PSCs using a medium supplemented with hematopoietic differentiation factors, and (ii) using a microglial differentiation medium to differentiate CD43 + This includes a step of differentiating iHPC into iMGL.

[0007] In some embodiments, a method is provided for producing human microglia-like (iMGL) cells from a first type of cell. In some embodiments, the method includes (i) differentiating the first type of cell into induced hematopoietic cells (iHPCs), and (ii) differentiating the iHPCs to produce iMGLs.

[0008] In some embodiments, the PSCs do not originate from embryoid bodies. In some embodiments, the PSCs include single-cell PSCs.

[0009] In some embodiments, PSCs include induced PSCs (iPSCs). In some embodiments, PSCs include embryonic stem cells (ESCs). In some embodiments, PSCs include mammalian PSCs. In some embodiments, PSCs are of human origin. In some embodiments, PSCs are mouse PSCs.

[0010] In some embodiments, a method is provided for producing iMGL from PSCs, comprising the steps of (i) differentiating PSCs into iHPCs and (ii) differentiating iHPCs into iMGLs.

[0011] In some embodiments, a composition of iMGL is provided that includes the expression of one or any combination of two or more of the following genes: RUNX1, SPI1, CSF1FR, CX3CR1, TGFBR1, RSG10, GAS6, MERTK, PSEN2, PROS1, P2RY12, P2RY13, GPR34, C1Q, CR3, CABLES1, BHLHE41, TREM2, TYROBP, ITGAM, APOE, SLCO2B1, SLC7A8, PPARD, TMEM119, GPR56, C9orf72, GRN, LRRK2, TARDBP, and CRYBB1.

[0012] In some embodiments, a method is provided for evaluating the secretion of chemokines, cytokines, and other inflammatory molecules, comprising the steps of (i) treating iMGL with lipopolysaccharide, IFNγ, or IL-1β, and (ii) measuring chemokines, cytokines, and other secreted factors from iMGL that may function as potential biomarkers for various inflammatory or neurodegenerative disease states. Some embodiments also provide a method for profiling the secretion of inflammatory molecules from iMGL, comprising (i) treating iMGL with lipopolysaccharide, IFNγ, TNFα, or IL-1β, and (ii) measuring inflammatory markers secreted by iMGL.

[0013] In some embodiments, a method is provided for evaluating the migration of iMGLs, comprising the steps of (i) treating iMGLs with ADP and (ii) measuring the migration of iMGLs. Some embodiments also provide a method for evaluating the migration of iMGLs, comprising (i) treating iMGLs with ADP and (ii) evaluating the motility and migration of iMGLs in response to chemical stimuli.

[0014] In some embodiments, a method is provided for inducing calcium migration in an iMGL, comprising the steps of (i) treating the iMGL with ADP and (ii) inducing calcium migration in the iMGL. In some embodiments, the method for inducing calcium migration in an iMGL comprises (i) treating the iMGL with ADP and (ii) examining the calcium flux signal in the iMGL, wherein the calcium flux signal is generated in response to an electrical, biological, or chemical stimulus.

[0015] In some embodiments, a method is provided for differentially controlling gene expression in iMGLs, comprising the steps of (i) co-culturing iMGLs with neurons or astrocytes, and (ii) differentially controlling genes in the iMGLs.

[0016] In some embodiments, a method is provided for incorporating iMGL into a CNS / brain (e.g., neuron) environment, comprising the steps of (i) co-culturing iMGL with hiPSC 3D brain organoids (BORGs), and (ii) introducing iMGL into the BORGs. Some embodiments relate to a method for incorporating iMGL into a 3D CNS environment, comprising co-culturing iMGL with hiPSC 3D brain organoids (BORGs), wherein the iMGLs migrate to the BORGs, aggregate in the BORGs, or are incorporated into the BORGs.

[0017] In some embodiments, a method is provided for differentially controlling gene expression in iMGLs, comprising the steps of (i) exposing iMGLs to any of the following compounds: Aβ, tau, fluorescently labeled Aβ, pHrodo-labeled brain-derived tau oligomers, and other brain-derived proteins associated with neurodegenerative diseases, namely synuclein, huntingtin, or prions; and (ii) differentially controlling genes in the iMGLs. Some embodiments also provide a method for establishing an iMGL gene expression profile that is similar to the in vivo state of iMGLs, comprising co-culturing the iMGLs with neurons, astrocytes, or other cells of the central nervous system, thereby replicating the in vivo state more closely with respect to the iMGLs than is present with respect to the iMGLs when the iMGLs are not co-culturified with neurons, astrocytes, or other cells of the central nervous system. Some embodiments relate to a method for testing microglial dysregulation in health and disease using iMGL, comprising (i) exposing iMGL to a compound selected from the group consisting of Aβ, tau, fluorescently labeled Aβ, pHrodo-labeled brain-derived tau oligomers, and alpha-synuclein, and (ii) profiling an iMGL omics signature selected from RNA-seq, proteomics, metabolomics, and lipidomics.

[0018] In some embodiments, a method is provided for phagocytosis of human synaptosomes (hS) in an iMGL, comprising the steps of (i) exposing the iMGL to hS, and (ii) measuring the phagocytosis of hS. In some embodiments, a method is provided for testing microglial phagocytosis of a compound, comprising (i) exposing the iMGL to a compound selected from the group consisting of Aβ, tau, fluorescently labeled Aβ, and pHrodo-labeled brain-derived tau oligomers, wherein the compound is phagocytosed, endocytosis, or ingested by the iMGL, and (ii) measuring the phagocytosis, endocytosis, or ingestion of the compound.

[0019] Some embodiments relate to a method for investigating the role of microglia in synaptic pruning and synaptic plasticity, comprising (i) exposing human synaptosomes to iMGLs and (ii) evaluating phagocytosis of human synaptosomes by iMGLs.

[0020] In some embodiments, a method is provided for determining gene regulation, comprising (i) exposing iMGL to one or more of the factors CX3CL1, CD200, and TGFβ in any combination, and (ii) evaluating one or more of the differentially regulated genes in any combination: P2ry12, EGR1, TGFβ1, ETV5, CX3CR1, APOE, BIN1, CD33, GPR84, COMT, APP, PSEN1, PSEN2, HTT, GRN, FUS, TARDP, VCP, SNCA, C9ORF72, LRRK2, and SOD1.

[0021] In some embodiments, a method is provided for evaluating the engraftment of iMGL into nerve tissue (e.g., cortex), comprising (i) implanting iMGL into nerve tissue and (ii) evaluating the engraftment of iMGL into nerve tissue.

[0022] In some embodiments, a method is provided for evaluating the interaction of iMGL with AD neuropathies, comprising (i) implanting iMGL in the hippocampus and (ii) evaluating the interaction of iMGL in the hippocampus.

[0023] In some embodiments, a method is provided for testing human microglia in a 3D neural environment, which includes implanting iMGLs into the brain of a mammal.

[0024] Some embodiments of the methods, kits, and compositions provided herein relate to a culture medium for supporting the production of human iHPCs, comprising one or more of FGF2, BMP4, activin A, and LiCl. Some embodiments of the methods and compositions provided herein relate to a culture medium for supporting the production of human iHPCs, comprising one or more of FGF2 and VEGF. Some embodiments of the methods and compositions provided herein relate to a culture medium for supporting the production of human iHPCs, comprising one or more of FGF2, VEGF, TPO, SCF, IL3, and IL6. Some embodiments relate to a kit for supporting the production of human iHPCs, comprising a culture medium comprising one or more of FGF2, BMP4, activin A, and LiCl. Some embodiments relate to a kit for supporting the production of iHPCs, comprising a culture medium comprising one or more of FGF2 and VEGF. Some embodiments relate to a kit for supporting the production of human iHPCs, comprising a culture medium comprising one or more of FGF2, VEGF, TPO, SCF, IL3, and IL6.

[0025] Some embodiments of the methods, kits, and compositions provided herein relate to a culture medium for supporting the production of human iMGLs, comprising one or more of CSF-1, IL-34, and TGFβ1. Some embodiments relate to a kit for supporting the production of human iMGLs, comprising a culture medium comprising one or more of CSF-1, IL-34, and TGFβ1.

[0026] Some embodiments of the methods, kits, and compositions provided herein relate to a culture medium for supporting the maturation or maintenance of iMGL, comprising one or more of CD200 and CX3CL1. Some embodiments relate to a kit for supporting the maturation or maintenance of iMGL, comprising a culture medium comprising one or more of CD200 and CX3CL1.

[0027] The compositions and related methods summarized above and further detailed below describe specific actions taken by a practicing physician, but it should be understood that they may also include instructions for these actions by another party. Thus, actions such as "implanting iMGL into a mammalian brain" include "instructing the implantation of iMGL into a mammalian brain." [Brief explanation of the drawing]

[0028] [Figure 1-1]Figure 1A. Schematic diagram of a fully defined iMGL differentiation protocol. (i) Human iPSCs differentiate into CD43+ iHPCs in 10 days and are then cultured in serum-free microglia differentiation medium containing human recombinant MCSF, IL-34, and TGFβ-1. Differentiation is carried out for a further 25 days, after which iMGLs are exposed to human recombinant CD200 and CX3CL1 for 3 days. (ii) Representative image of iHPCs in cell culture on day 10. Scale bar = 100 μm. (iii) By day 14, iMGLs express PU.1 (bright spot) and TREM2 (bright spot). Scale bar = 50 μm. (iv) Representative phase-contrast image of iMGLs on day 38. Figure 1B. Schematic diagram of differentiation from iPSC to iHPC. (i) Single-cell iPSCs differentiate in known-composition media supplemented with hematopoietic differentiation factors using 5% O2 (day 4) and 20% O2 (day 6). (ii) After 10 days, CD43+ iHPCs are CD235a+ / CD41a+. Figure 1C iMGLs develop from CD45+ / CX3CR1-(A1) and CD45+ / CX3CR1+(A2) progenitor cells. Figure 1D CD45 fluorescence intensity shows that iMGLs (dark outer spots) maintain their CD45lo-int profile when compared to monocyte-derived macrophages (MD-Mφ). Figure 1E iMGL progenitor cells are CD11blo and increase their CD11b expression as they mature. In 14DIV, a small population (approximately 11%) of cells with CD11bint-hi were detected. Figure 1F CD11b fluorescence intensity demonstrates that iMGLs increase CD11b expression as they age, similar to mouse microglial progenitor cells. Figure 1G. Maygrünwald-Giemsa staining of monocytes, MD-Mφ, fetal microglia, and iMGL. Both fetal microglia and iMGL exhibit a higher nucleus-to-cytoplasmic ratio morphology compared to monocytes and MD-Mφ. Scale bar = 16 μm. Figure 1H. Differentiation results in purity exceeding 96%, assessed by the co-localization of microglia-enriched protein P2ry12 and microglia-enriched Trem2 (merge panel shows overlays of nuclear panels with overlapping expression of P2ry12, Trem2, and P2ry12 and Trem2).Figure 1I shows that iMGL also exhibits elongated protrusions and expresses Cx3Cr1 (upper left panel) and hCyto (upper right panel). The merged panel shows that hCyto expression (bright spots) localizes to the same region as Cx3Cr1 expression. [Figure 1-2]Figure 1A. Schematic diagram of a fully defined iMGL differentiation protocol. (i) Human iPSCs differentiate into CD43+ iHPCs in 10 days and are then cultured in serum-free microglia differentiation medium containing human recombinant MCSF, IL-34, and TGFβ-1. Differentiation is carried out for a further 25 days, after which iMGLs are exposed to human recombinant CD200 and CX3CL1 for 3 days. (ii) Representative image of iHPCs in cell culture on day 10. Scale bar = 100 μm. (iii) By day 14, iMGLs express PU.1 (bright spot) and TREM2 (bright spot). Scale bar = 50 μm. (iv) Representative phase-contrast image of iMGLs on day 38. Figure 1B. Schematic diagram of differentiation from iPSC to iHPC. (i) Single-cell iPSCs differentiate in known-composition media supplemented with hematopoietic differentiation factors using 5% O2 (day 4) and 20% O2 (day 6). (ii) After 10 days, CD43+ iHPCs are CD235a+ / CD41a+. Figure 1C iMGLs develop from CD45+ / CX3CR1-(A1) and CD45+ / CX3CR1+(A2) progenitor cells. Figure 1D CD45 fluorescence intensity shows that iMGLs (dark outer spots) maintain their CD45lo-int profile when compared to monocyte-derived macrophages (MD-Mφ). Figure 1E iMGL progenitor cells are CD11blo and increase their CD11b expression as they mature. In 14DIV, a small population (approximately 11%) of cells with CD11bint-hi were detected. Figure 1F CD11b fluorescence intensity demonstrates that iMGLs increase CD11b expression as they age, similar to mouse microglial progenitor cells. Figure 1G. Maygrünwald-Giemsa staining of monocytes, MD-Mφ, fetal microglia, and iMGL. Both fetal microglia and iMGL exhibit a higher nucleus-to-cytoplasmic ratio morphology compared to monocytes and MD-Mφ. Scale bar = 16 μm. Figure 1H. Differentiation results in purity exceeding 96%, assessed by the co-localization of microglia-enriched protein P2ry12 and microglia-enriched Trem2 (merge panel shows overlays of nuclear panels with overlapping expression of P2ry12, Trem2, and P2ry12 and Trem2).Figure 1I shows that iMGL also exhibits elongated protrusions and expresses Cx3Cr1 (upper left panel) and hCyto (upper right panel). The merged panel shows that hCyto expression (bright spots) localizes to the same region as Cx3Cr1 expression. [Figure 1-3]Figure 1A. Schematic diagram of a fully defined iMGL differentiation protocol. (i) Human iPSCs differentiate into CD43+ iHPCs in 10 days and are then cultured in serum-free microglia differentiation medium containing human recombinant MCSF, IL-34, and TGFβ-1. Differentiation is carried out for a further 25 days, after which iMGLs are exposed to human recombinant CD200 and CX3CL1 for 3 days. (ii) Representative image of iHPCs in cell culture on day 10. Scale bar = 100 μm. (iii) By day 14, iMGLs express PU.1 (bright spot) and TREM2 (bright spot). Scale bar = 50 μm. (iv) Representative phase-contrast image of iMGLs on day 38. Figure 1B. Schematic diagram of differentiation from iPSC to iHPC. (i) Single-cell iPSCs differentiate in known-composition media supplemented with hematopoietic differentiation factors using 5% O2 (day 4) and 20% O2 (day 6). (ii) After 10 days, CD43+ iHPCs are CD235a+ / CD41a+. Figure 1C iMGLs develop from CD45+ / CX3CR1-(A1) and CD45+ / CX3CR1+(A2) progenitor cells. Figure 1D CD45 fluorescence intensity shows that iMGLs (dark outer spots) maintain their CD45lo-int profile when compared to monocyte-derived macrophages (MD-Mφ). Figure 1E iMGL progenitor cells are CD11blo and increase their CD11b expression as they mature. In 14DIV, a small population (approximately 11%) of cells with CD11bint-hi were detected. Figure 1F CD11b fluorescence intensity demonstrates that iMGLs increase CD11b expression as they age, similar to mouse microglial progenitor cells. Figure 1G. Maygrünwald-Giemsa staining of monocytes, MD-Mφ, fetal microglia, and iMGL. Both fetal microglia and iMGL exhibit a higher nucleus-to-cytoplasmic ratio morphology compared to monocytes and MD-Mφ. Scale bar = 16 μm. Figure 1H. Differentiation results in purity exceeding 96%, assessed by the co-localization of microglia-enriched protein P2ry12 and microglia-enriched Trem2 (merge panel shows overlays of nuclear panels with overlapping expression of P2ry12, Trem2, and P2ry12 and Trem2).Figure 1I shows that iMGL also exhibits elongated protrusions and expresses Cx3Cr1 (upper left panel) and hCyto (upper right panel). The merged panel shows that hCyto expression (bright spots) localizes to the same region as Cx3Cr1 expression. [Figure 1-4]Figure 1A. Schematic diagram of a fully defined iMGL differentiation protocol. (i) Human iPSCs differentiate into CD43+ iHPCs in 10 days and are then cultured in serum-free microglia differentiation medium containing human recombinant MCSF, IL-34, and TGFβ-1. Differentiation is carried out for a further 25 days, after which iMGLs are exposed to human recombinant CD200 and CX3CL1 for 3 days. (ii) Representative image of iHPCs in cell culture on day 10. Scale bar = 100 μm. (iii) By day 14, iMGLs express PU.1 (bright spot) and TREM2 (bright spot). Scale bar = 50 μm. (iv) Representative phase-contrast image of iMGLs on day 38. Figure 1B. Schematic diagram of differentiation from iPSC to iHPC. (i) Single-cell iPSCs differentiate in known-composition media supplemented with hematopoietic differentiation factors using 5% O2 (day 4) and 20% O2 (day 6). (ii) After 10 days, CD43+ iHPCs are CD235a+ / CD41a+. Figure 1C iMGLs develop from CD45+ / CX3CR1-(A1) and CD45+ / CX3CR1+(A2) progenitor cells. Figure 1D CD45 fluorescence intensity shows that iMGLs (dark outer spots) maintain their CD45lo-int profile when compared to monocyte-derived macrophages (MD-Mφ). Figure 1E iMGL progenitor cells are CD11blo and increase their CD11b expression as they mature. In 14DIV, a small population (approximately 11%) of cells with CD11bint-hi were detected. Figure 1F CD11b fluorescence intensity demonstrates that iMGLs increase CD11b expression as they age, similar to mouse microglial progenitor cells. Figure 1G. Maygrünwald-Giemsa staining of monocytes, MD-Mφ, fetal microglia, and iMGL. Both fetal microglia and iMGL exhibit a higher nucleus-to-cytoplasmic ratio morphology compared to monocytes and MD-Mφ. Scale bar = 16 μm. Figure 1H. Differentiation results in purity exceeding 96%, assessed by the co-localization of microglia-enriched protein P2ry12 and microglia-enriched Trem2 (merge panel shows overlays of nuclear panels with overlapping expression of P2ry12, Trem2, and P2ry12 and Trem2).Figure 1I shows that iMGL also exhibits elongated protrusions and expresses Cx3Cr1 (upper left panel) and hCyto (upper right panel). The merged panel shows that hCyto expression (bright spots) localizes to the same region as Cx3Cr1 expression. [Figure 1-5]Figure 1A. Schematic diagram of a fully defined iMGL differentiation protocol. (i) Human iPSCs differentiate into CD43+ iHPCs in 10 days and are then cultured in serum-free microglia differentiation medium containing human recombinant MCSF, IL-34, and TGFβ-1. Differentiation is carried out for a further 25 days, after which iMGLs are exposed to human recombinant CD200 and CX3CL1 for 3 days. (ii) Representative image of iHPCs in cell culture on day 10. Scale bar = 100 μm. (iii) By day 14, iMGLs express PU.1 (bright spot) and TREM2 (bright spot). Scale bar = 50 μm. (iv) Representative phase-contrast image of iMGLs on day 38. Figure 1B. Schematic diagram of differentiation from iPSC to iHPC. (i) Single-cell iPSCs differentiate in known-composition media supplemented with hematopoietic differentiation factors using 5% O2 (day 4) and 20% O2 (day 6). (ii) After 10 days, CD43+ iHPCs are CD235a+ / CD41a+. Figure 1C iMGLs develop from CD45+ / CX3CR1-(A1) and CD45+ / CX3CR1+(A2) progenitor cells. Figure 1D CD45 fluorescence intensity shows that iMGLs (dark outer spots) maintain their CD45lo-int profile when compared to monocyte-derived macrophages (MD-Mφ). Figure 1E iMGL progenitor cells are CD11blo and increase their CD11b expression as they mature. In 14DIV, a small population (approximately 11%) of cells with CD11bint-hi were detected. Figure 1F CD11b fluorescence intensity demonstrates that iMGLs increase CD11b expression as they age, similar to mouse microglial progenitor cells. Figure 1G. Maygrünwald-Giemsa staining of monocytes, MD-Mφ, fetal microglia, and iMGL. Both fetal microglia and iMGL exhibit a higher nucleus-to-cytoplasmic ratio morphology compared to monocytes and MD-Mφ. Scale bar = 16 μm. Figure 1H. Differentiation results in purity exceeding 96%, assessed by the co-localization of microglia-enriched protein P2ry12 and microglia-enriched Trem2 (merge panel shows overlays of nuclear panels with overlapping expression of P2ry12, Trem2, and P2ry12 and Trem2).Figure 1I shows that iMGL also exhibits elongated protrusions and expresses Cx3Cr1 (upper left panel) and hCyto (upper right panel). The merged panel shows that hCyto expression (bright spots) localizes to the same region as Cx3Cr1 expression. [Figure 1-6]Figure 1A. Schematic diagram of a fully defined iMGL differentiation protocol. (i) Human iPSCs differentiate into CD43+ iHPCs in 10 days and are then cultured in serum-free microglia differentiation medium containing human recombinant MCSF, IL-34, and TGFβ-1. Differentiation is carried out for a further 25 days, after which iMGLs are exposed to human recombinant CD200 and CX3CL1 for 3 days. (ii) Representative image of iHPCs in cell culture on day 10. Scale bar = 100 μm. (iii) By day 14, iMGLs express PU.1 (bright spot) and TREM2 (bright spot). Scale bar = 50 μm. (iv) Representative phase-contrast image of iMGLs on day 38. Figure 1B. Schematic diagram of differentiation from iPSC to iHPC. (i) Single-cell iPSCs differentiate in known-composition media supplemented with hematopoietic differentiation factors using 5% O2 (day 4) and 20% O2 (day 6). (ii) After 10 days, CD43+ iHPCs are CD235a+ / CD41a+. Figure 1C iMGLs develop from CD45+ / CX3CR1-(A1) and CD45+ / CX3CR1+(A2) progenitor cells. Figure 1D CD45 fluorescence intensity shows that iMGLs (dark outer spots) maintain their CD45lo-int profile when compared to monocyte-derived macrophages (MD-Mφ). Figure 1E iMGL progenitor cells are CD11blo and increase their CD11b expression as they mature. In 14DIV, a small population (approximately 11%) of cells with CD11bint-hi were detected. Figure 1F CD11b fluorescence intensity demonstrates that iMGLs increase CD11b expression as they age, similar to mouse microglial progenitor cells. Figure 1G. Maygrünwald-Giemsa staining of monocytes, MD-Mφ, fetal microglia, and iMGL. Both fetal microglia and iMGL exhibit a higher nucleus-to-cytoplasmic ratio morphology compared to monocytes and MD-Mφ. Scale bar = 16 μm. Figure 1H. Differentiation results in purity exceeding 96%, assessed by the co-localization of microglia-enriched protein P2ry12 and microglia-enriched Trem2 (merge panel shows overlays of nuclear panels with overlapping expression of P2ry12, Trem2, and P2ry12 and Trem2).Figure 1I shows that iMGL also exhibits elongated protrusions and expresses Cx3Cr1 (upper left panel) and hCyto (upper right panel). The merged panel shows that hCyto expression (bright spots) localizes to the same region as Cx3Cr1 expression. [Figure 2-1]Figure 2A shows 3D principal component analysis (PCA) of iMGL, human adult microglia (adult MG), and human fetal microglia (fetal MG) (adult MG and fetal MG are located in the same circled cluster), CD14+ / CD16- monocytes (CD14 M), CD14+ / 16+ monocytes (CD16 M) (CD14 M and CD16 M are located in the same circled cluster), blood dendritic cells (blood DC), iHPC, and iPSCs (FPKM≧1, n=23,580 genes). PCA analysis reveals that iMGL clusters with adult MG and fetal MG but not with other myeloid cells. PC1 (21.3% var) reflects the time series of differentiation of iPSCs into iHPCs (arrows from iPSC cluster to iHPC cluster) and subsequent differentiation into iMGLs (arrows from iHPC cluster to iMGL cluster). PC2 (15.4% var) reflects the trajectory to blood DCs. PC3 (7.6% var) reflects the trajectory to monocytes. Figure 2B Heatmap and bi-clustering (Euclidean distance) of 300 microglia, bone marrow, and other immune-related genes (Butovsky et al., 2014, Hickman et al., 2013, Zhang et al., 2014). Pseudocounts were used for FPKM values ​​(FPKM+1), log2 converted, and each gene was standardized in its respective row (n=300). Representative profiles are shown for genes that are regulated up and down in both human microglia (fetal / adult) and iMGL. Figure 2C Bar graphs of microglia-specific enriched genes, measured as FPKM+1 followed by Log2 conversion [Log2(FPKM+1)] in iMGL, fetal MG and adult MG, blood DCs, CD14 M, and CD16 M, presented as mean ± SEM. Following analysis of data using one-way ANOVA, Tukey's modified multiple comparison post-hoc test was performed. Statistical annotations represent the maximum p-values ​​for iMGL, fetal MG, and adult MG compared to other myeloid cells: CD14 M (n=5), CD16 M (n=4), blood DC (n=3), iMGL (n=6), fetal MG (n=3), and adult MG (n=3).*p<0.05、**p<0.01、***p<0.001、****p<0.0001。 [Figure 2-2]Figure 2A shows 3D principal component analysis (PCA) of iMGL, human adult microglia (adult MG), and human fetal microglia (fetal MG) (adult MG and fetal MG are located in the same circled cluster), CD14+ / CD16- monocytes (CD14 M), CD14+ / 16+ monocytes (CD16 M) (CD14 M and CD16 M are located in the same circled cluster), blood dendritic cells (blood DC), iHPC, and iPSCs (FPKM≧1, n=23,580 genes). PCA analysis reveals that iMGL clusters with adult MG and fetal MG but not with other myeloid cells. PC1 (21.3% var) reflects the time series of differentiation of iPSCs into iHPCs (arrows from iPSC cluster to iHPC cluster) and subsequent differentiation into iMGLs (arrows from iHPC cluster to iMGL cluster). PC2 (15.4% var) reflects the trajectory to blood DCs. PC3 (7.6% var) reflects the trajectory to monocytes. Figure 2B Heatmap and bi-clustering (Euclidean distance) of 300 microglia, bone marrow, and other immune-related genes (Butovsky et al., 2014, Hickman et al., 2013, Zhang et al., 2014). Pseudocounts were used for FPKM values ​​(FPKM+1), log2 converted, and each gene was standardized in its respective row (n=300). Representative profiles are shown for genes that are regulated up and down in both human microglia (fetal / adult) and iMGL. Figure 2C Bar graphs of microglia-specific enriched genes, measured as FPKM+1 followed by Log2 conversion [Log2(FPKM+1)] in iMGL, fetal MG and adult MG, blood DCs, CD14 M, and CD16 M, presented as mean ± SEM. Following analysis of data using one-way ANOVA, Tukey's modified multiple comparison post-hoc test was performed. Statistical annotations represent the maximum p-values ​​for iMGL, fetal MG, and adult MG compared to other myeloid cells: CD14 M (n=5), CD16 M (n=4), blood DC (n=3), iMGL (n=6), fetal MG (n=3), and adult MG (n=3).*p<0.05、**p<0.01、***p<0.001、****p<0.0001。 [Figure 2-3]Figure 2A shows 3D principal component analysis (PCA) of iMGL, human adult microglia (adult MG), and human fetal microglia (fetal MG) (adult MG and fetal MG are located in the same circled cluster), CD14+ / CD16- monocytes (CD14 M), CD14+ / 16+ monocytes (CD16 M) (CD14 M and CD16 M are located in the same circled cluster), blood dendritic cells (blood DC), iHPC, and iPSCs (FPKM≧1, n=23,580 genes). PCA analysis reveals that iMGL clusters with adult MG and fetal MG but not with other myeloid cells. PC1 (21.3% var) reflects the time series of differentiation of iPSCs into iHPCs (arrows from iPSC cluster to iHPC cluster) and subsequent differentiation into iMGLs (arrows from iHPC cluster to iMGL cluster). PC2 (15.4% var) reflects the trajectory to blood DCs. PC3 (7.6% var) reflects the trajectory to monocytes. Figure 2B Heatmap and bi-clustering (Euclidean distance) of 300 microglia, bone marrow, and other immune-related genes (Butovsky et al., 2014, Hickman et al., 2013, Zhang et al., 2014). Pseudocounts were used for FPKM values ​​(FPKM+1), log2 converted, and each gene was standardized in its respective row (n=300). Representative profiles are shown for genes that are regulated up and down in both human microglia (fetal / adult) and iMGL. Figure 2C Bar graphs of microglia-specific enriched genes, measured as FPKM+1 followed by Log2 conversion [Log2(FPKM+1)] in iMGL, fetal MG and adult MG, blood DCs, CD14 M, and CD16 M, presented as mean ± SEM. Following analysis of data using one-way ANOVA, Tukey's modified multiple comparison post-hoc test was performed. Statistical annotations represent the maximum p-values ​​for iMGL, fetal MG, and adult MG compared to other myeloid cells: CD14 M (n=5), CD16 M (n=4), blood DC (n=3), iMGL (n=6), fetal MG (n=3), and adult MG (n=3).*p<0.05、**p<0.01、***p<0.001、****p<0.0001。 [Figure 2-4]Figure 2A shows 3D principal component analysis (PCA) of iMGL, human adult microglia (adult MG), and human fetal microglia (fetal MG) (adult MG and fetal MG are located in the same circled cluster), CD14+ / CD16- monocytes (CD14 M), CD14+ / 16+ monocytes (CD16 M) (CD14 M and CD16 M are located in the same circled cluster), blood dendritic cells (blood DC), iHPC, and iPSCs (FPKM≧1, n=23,580 genes). PCA analysis reveals that iMGL clusters with adult MG and fetal MG but not with other myeloid cells. PC1 (21.3% var) reflects the time series of differentiation of iPSCs into iHPCs (arrows from iPSC cluster to iHPC cluster) and subsequent differentiation into iMGLs (arrows from iHPC cluster to iMGL cluster). PC2 (15.4% var) reflects the trajectory to blood DCs. PC3 (7.6% var) reflects the trajectory to monocytes. Figure 2B Heatmap and bi-clustering (Euclidean distance) of 300 microglia, bone marrow, and other immune-related genes (Butovsky et al., 2014, Hickman et al., 2013, Zhang et al., 2014). Pseudocounts were used for FPKM values ​​(FPKM+1), log2 converted, and each gene was standardized in its respective row (n=300). Representative profiles are shown for genes that are regulated up and down in both human microglia (fetal / adult) and iMGL. Figure 2C Bar graphs of microglia-specific enriched genes, measured as FPKM+1 followed by Log2 conversion [Log2(FPKM+1)] in iMGL, fetal MG and adult MG, blood DCs, CD14 M, and CD16 M, presented as mean ± SEM. Following analysis of data using one-way ANOVA, Tukey's modified multiple comparison post-hoc test was performed. Statistical annotations represent the maximum p-values ​​for iMGL, fetal MG, and adult MG compared to other myeloid cells: CD14 M (n=5), CD16 M (n=4), blood DC (n=3), iMGL (n=6), fetal MG (n=3), and adult MG (n=3).*p<0.05、**p<0.01、***p<0.001、****p<0.0001。 [Figure 3-1]Figure 3A Flow cytometry analysis shows that iMGL (outer dark spot area in the three panels) is CD45lo-int, similar to fetal MG (innermost spot area in the three panels). Figure 3B iMGL (dark spot in the left panel) is different from CD45-hi MD-Mφ (gray spot in the left panel). The CD11b intensity histogram (left histogram) reveals that fetal MG expresses slightly more CD11b than iMGL, but less than MD-Mφ. Figure 3C iMGL secretes cytokines and chemokines when stimulated for 24 hours with either IFNγ (20 ng / ml), IL-1β (20 ng / ml), or LPS (100 ng / ml) by ELISA multiplex. Figure 3D ADP (100 μM) induces iMGL migration in a Transwell chamber (5 μm). Prior exposure to the P2ry12 antagonist PSB0739 (50 μM, 1 hour) completely inactivates ADP-induced iMGL migration (***p<0.0001). Figure 3E ADP induces calcium flux in iMGL via the P2ry12 receptor. (Left) Exposure to ADP results in increased calcium influx (I340 / I380 ratio) in the medium group (bright trace), but not in the PSB0739-treated group (dark trace). (Right) Representative images of ADP-induced calcium flux at 240 seconds in the medium (top) and PSB0739 (bottom). Figure 3F iMGL phagocytoses human brain-derived synaptosomes (hS). Representative images recorded with Amnis Imagestream show phagocytosis of hS by MD-Mφ and iMGL. Figure 3G Quantification of phagocytosis shows that iMGL internalizes hS at 50% of macrophage capacity (p<0.0001). Figure 3H Representative images of iMGL phagocytosis of hS in the presence of either the MerTK inhibitor UNC569 (top) or the anti-CD11b antibody (bottom). Figure 3I (top) iMGL phagocytosis of hS is reduced by approximately 12% (second bar from the right, p<0.05) by blocking MerTK, but by inhibiting CR3 via CD11b blockade (p<0.0001, rightmost bar).(Below) Sub-analysis of iMGL showing phagocytic events reveals similar mean amounts of internal transfer across the entire treatment group (p=0.1165). All histograms are reported as mean ± SEM. One-way ANOVA for cytokine and migration assays, followed by Dunnett's multiple comparison post-hoc test, ***p<0.0001, **p<0.001, *p<0.05, cytokine assay: n=3 wells / group. Migration assay: n=5 fields / condition. Calcium assay: medium (n=37 cells), PSB0739 treatment (n=17 cells), I340 / I380 expressed as mean ± SEM at each time point. Phagocytosis assay: MD-Mφ vs iMGL: unpaired t-test, **p<0.001, n=3 wells / group. MERTK and CR3 assays, one-way ANOVA, followed by Tukey's multiple comparison post-hoc test, ***p<0.0001; n=6, n=3 wells / group for each medium. [Figure 3-2]Figure 3A Flow cytometry analysis shows that iMGL (outer dark spot area in the three panels) is CD45lo-int, similar to fetal MG (innermost spot area in the three panels). Figure 3B iMGL (dark spot in the left panel) is different from CD45-hi MD-Mφ (gray spot in the left panel). The CD11b intensity histogram (left histogram) reveals that fetal MG expresses slightly more CD11b than iMGL, but less than MD-Mφ. Figure 3C iMGL secretes cytokines and chemokines when stimulated for 24 hours with either IFNγ (20 ng / ml), IL-1β (20 ng / ml), or LPS (100 ng / ml) by ELISA multiplex. Figure 3D ADP (100 μM) induces iMGL migration in a Transwell chamber (5 μm). Prior exposure to the P2ry12 antagonist PSB0739 (50 μM, 1 hour) completely inactivates ADP-induced iMGL migration (***p<0.0001). Figure 3E ADP induces calcium flux in iMGL via the P2ry12 receptor. (Left) Exposure to ADP results in increased calcium influx (I340 / I380 ratio) in the medium group (bright trace), but not in the PSB0739-treated group (dark trace). (Right) Representative images of ADP-induced calcium flux at 240 seconds in the medium (top) and PSB0739 (bottom). Figure 3F iMGL phagocytoses human brain-derived synaptosomes (hS). Representative images recorded with Amnis Imagestream show phagocytosis of hS by MD-Mφ and iMGL. Figure 3G Quantification of phagocytosis shows that iMGL internalizes hS at 50% of macrophage capacity (p<0.0001). Figure 3H Representative images of iMGL phagocytosis of hS in the presence of either the MerTK inhibitor UNC569 (top) or the anti-CD11b antibody (bottom). Figure 3I (top) iMGL phagocytosis of hS is reduced by approximately 12% (second bar from the right, p<0.05) by blocking MerTK, but by inhibiting CR3 via CD11b blockade (p<0.0001, rightmost bar).(Below) Sub-analysis of iMGL showing phagocytic events reveals similar mean amounts of internal transfer across the entire treatment group (p=0.1165). All histograms are reported as mean ± SEM. One-way ANOVA for cytokine and migration assays, followed by Dunnett's multiple comparison post-hoc test, ***p<0.0001, **p<0.001, *p<0.05, cytokine assay: n=3 wells / group. Migration assay: n=5 fields / condition. Calcium assay: medium (n=37 cells), PSB0739 treatment (n=17 cells), I340 / I380 expressed as mean ± SEM at each time point. Phagocytosis assay: MD-Mφ vs iMGL: unpaired t-test, **p<0.001, n=3 wells / group. MERTK and CR3 assays, one-way ANOVA, followed by Tukey's multiple comparison post-hoc test, ***p<0.0001; n=6, n=3 wells / group for each medium. [Figure 3-3]Figure 3A Flow cytometry analysis shows that iMGL (outer dark spot area in the three panels) is CD45lo-int, similar to fetal MG (innermost spot area in the three panels). Figure 3B iMGL (dark spot in the left panel) is different from CD45-hi MD-Mφ (gray spot in the left panel). The CD11b intensity histogram (left histogram) reveals that fetal MG expresses slightly more CD11b than iMGL, but less than MD-Mφ. Figure 3C iMGL secretes cytokines and chemokines when stimulated for 24 hours with either IFNγ (20 ng / ml), IL-1β (20 ng / ml), or LPS (100 ng / ml) by ELISA multiplex. Figure 3D ADP (100 μM) induces iMGL migration in a Transwell chamber (5 μm). Prior exposure to the P2ry12 antagonist PSB0739 (50 μM, 1 hour) completely inactivates ADP-induced iMGL migration (***p<0.0001). Figure 3E ADP induces calcium flux in iMGL via the P2ry12 receptor. (Left) Exposure to ADP results in increased calcium influx (I340 / I380 ratio) in the medium group (bright trace), but not in the PSB0739-treated group (dark trace). (Right) Representative images of ADP-induced calcium flux at 240 seconds in the medium (top) and PSB0739 (bottom). Figure 3F iMGL phagocytoses human brain-derived synaptosomes (hS). Representative images recorded with Amnis Imagestream show phagocytosis of hS by MD-Mφ and iMGL. Figure 3G Quantification of phagocytosis shows that iMGL internalizes hS at 50% of macrophage capacity (p<0.0001). Figure 3H Representative images of iMGL phagocytosis of hS in the presence of either the MerTK inhibitor UNC569 (top) or the anti-CD11b antibody (bottom). Figure 3I (top) iMGL phagocytosis of hS is reduced by approximately 12% (second bar from the right, p<0.05) by blocking MerTK, but by inhibiting CR3 via CD11b blockade (p<0.0001, rightmost bar).(Below) Sub-analysis of iMGL showing phagocytic events reveals similar mean amounts of internal transfer across the entire treatment group (p=0.1165). All histograms are reported as mean ± SEM. One-way ANOVA for cytokine and migration assays, followed by Dunnett's multiple comparison post-hoc test, ***p<0.0001, **p<0.001, *p<0.05, cytokine assay: n=3 wells / group. Migration assay: n=5 fields / condition. Calcium assay: medium (n=37 cells), PSB0739 treatment (n=17 cells), I340 / I380 expressed as mean ± SEM at each time point. Phagocytosis assay: MD-Mφ vs iMGL: unpaired t-test, **p<0.001, n=3 wells / group. MERTK and CR3 assays, one-way ANOVA, followed by Tukey's multiple comparison post-hoc test, ***p<0.0001; n=6, n=3 wells / group for each medium. [Figure 3-4]Figure 3A Flow cytometry analysis shows that iMGL (outer dark spot area in the three panels) is CD45lo-int, similar to fetal MG (innermost spot area in the three panels). Figure 3B iMGL (dark spot in the left panel) is different from CD45-hi MD-Mφ (gray spot in the left panel). The CD11b intensity histogram (left histogram) reveals that fetal MG expresses slightly more CD11b than iMGL, but less than MD-Mφ. Figure 3C iMGL secretes cytokines and chemokines when stimulated for 24 hours with either IFNγ (20 ng / ml), IL-1β (20 ng / ml), or LPS (100 ng / ml) by ELISA multiplex. Figure 3D ADP (100 μM) induces iMGL migration in a Transwell chamber (5 μm). Prior exposure to the P2ry12 antagonist PSB0739 (50 μM, 1 hour) completely inactivates ADP-induced iMGL migration (***p<0.0001). Figure 3E ADP induces calcium flux in iMGL via the P2ry12 receptor. (Left) Exposure to ADP results in increased calcium influx (I340 / I380 ratio) in the medium group (bright trace), but not in the PSB0739-treated group (dark trace). (Right) Representative images of ADP-induced calcium flux at 240 seconds in the medium (top) and PSB0739 (bottom). Figure 3F iMGL phagocytoses human brain-derived synaptosomes (hS). Representative images recorded with Amnis Imagestream show phagocytosis of hS by MD-Mφ and iMGL. Figure 3G Quantification of phagocytosis shows that iMGL internalizes hS at 50% of macrophage capacity (p<0.0001). Figure 3H Representative images of iMGL phagocytosis of hS in the presence of either the MerTK inhibitor UNC569 (top) or the anti-CD11b antibody (bottom). Figure 3I (top) iMGL phagocytosis of hS is reduced by approximately 12% (second bar from the right, p<0.05) by blocking MerTK, but by inhibiting CR3 via CD11b blockade (p<0.0001, rightmost bar).(Below) Sub-analysis of iMGL showing phagocytic events reveals similar mean amounts of internal transfer across the entire treatment group (p=0.1165). All histograms are reported as mean ± SEM. One-way ANOVA for cytokine and migration assays, followed by Dunnett's multiple comparison post-hoc test, ***p<0.0001, **p<0.001, *p<0.05, cytokine assay: n=3 wells / group. Migration assay: n=5 fields / condition. Calcium assay: medium (n=37 cells), PSB0739 treatment (n=17 cells), I340 / I380 expressed as mean ± SEM at each time point. Phagocytosis assay: MD-Mφ vs iMGL: unpaired t-test, **p<0.001, n=3 wells / group. MERTK and CR3 assays, one-way ANOVA, followed by Tukey's multiple comparison post-hoc test, ***p<0.0001; n=6, n=3 wells / group for each medium. [Figure 3-5]Figure 3A Flow cytometry analysis shows that iMGL (outer dark spot area in the three panels) is CD45lo-int, similar to fetal MG (innermost spot area in the three panels). Figure 3B iMGL (dark spot in the left panel) is different from CD45-hi MD-Mφ (gray spot in the left panel). The CD11b intensity histogram (left histogram) reveals that fetal MG expresses slightly more CD11b than iMGL, but less than MD-Mφ. Figure 3C iMGL secretes cytokines and chemokines when stimulated for 24 hours with either IFNγ (20 ng / ml), IL-1β (20 ng / ml), or LPS (100 ng / ml) by ELISA multiplex. Figure 3D ADP (100 μM) induces iMGL migration in a Transwell chamber (5 μm). Prior exposure to the P2ry12 antagonist PSB0739 (50 μM, 1 hour) completely inactivates ADP-induced iMGL migration (***p<0.0001). Figure 3E ADP induces calcium flux in iMGL via the P2ry12 receptor. (Left) Exposure to ADP results in increased calcium influx (I340 / I380 ratio) in the medium group (bright trace), but not in the PSB0739-treated group (dark trace). (Right) Representative images of ADP-induced calcium flux at 240 seconds in the medium (top) and PSB0739 (bottom). Figure 3F iMGL phagocytoses human brain-derived synaptosomes (hS). Representative images recorded with Amnis Imagestream show phagocytosis of hS by MD-Mφ and iMGL. Figure 3G Quantification of phagocytosis shows that iMGL internalizes hS at 50% of macrophage capacity (p<0.0001). Figure 3H Representative images of iMGL phagocytosis of hS in the presence of either the MerTK inhibitor UNC569 (top) or the anti-CD11b antibody (bottom). Figure 3I (top) iMGL phagocytosis of hS is reduced by approximately 12% (second bar from the right, p<0.05) by blocking MerTK, but by inhibiting CR3 via CD11b blockade (p<0.0001, rightmost bar).(Below) Sub-analysis of iMGL showing phagocytic events reveals similar mean amounts of internal transfer across the entire treatment group (p=0.1165). All histograms are reported as mean ± SEM. One-way ANOVA for cytokine and migration assays, followed by Dunnett's multiple comparison post-hoc test, ***p<0.0001, **p<0.001, *p<0.05, cytokine assay: n=3 wells / group. Migration assay: n=5 fields / condition. Calcium assay: medium (n=37 cells), PSB0739 treatment (n=17 cells), I340 / I380 expressed as mean ± SEM at each time point. Phagocytosis assay: MD-Mφ vs iMGL: unpaired t-test, **p<0.001, n=3 wells / group. MERTK and CR3 assays, one-way ANOVA, followed by Tukey's multiple comparison post-hoc test, ***p<0.0001; n=6, n=3 wells / group for each medium. [Figure 3-6]Figure 3A Flow cytometry analysis shows that iMGL (outer dark spot area in the three panels) is CD45lo-int, similar to fetal MG (innermost spot area in the three panels). Figure 3B iMGL (dark spot in the left panel) is different from CD45-hi MD-Mφ (gray spot in the left panel). The CD11b intensity histogram (left histogram) reveals that fetal MG expresses slightly more CD11b than iMGL, but less than MD-Mφ. Figure 3C iMGL secretes cytokines and chemokines when stimulated for 24 hours with either IFNγ (20 ng / ml), IL-1β (20 ng / ml), or LPS (100 ng / ml) by ELISA multiplex. Figure 3D ADP (100 μM) induces iMGL migration in a Transwell chamber (5 μm). Prior exposure to the P2ry12 antagonist PSB0739 (50 μM, 1 hour) completely inactivates ADP-induced iMGL migration (***p<0.0001). Figure 3E ADP induces calcium flux in iMGL via the P2ry12 receptor. (Left) Exposure to ADP results in increased calcium influx (I340 / I380 ratio) in the medium group (bright trace), but not in the PSB0739-treated group (dark trace). (Right) Representative images of ADP-induced calcium flux at 240 seconds in the medium (top) and PSB0739 (bottom). Figure 3F iMGL phagocytoses human brain-derived synaptosomes (hS). Representative images recorded with Amnis Imagestream show phagocytosis of hS by MD-Mφ and iMGL. Figure 3G Quantification of phagocytosis shows that iMGL internalizes hS at 50% of macrophage capacity (p<0.0001). Figure 3H Representative images of iMGL phagocytosis of hS in the presence of either the MerTK inhibitor UNC569 (top) or the anti-CD11b antibody (bottom). Figure 3I (top) iMGL phagocytosis of hS is reduced by approximately 12% (second bar from the right, p<0.05) by blocking MerTK, but by inhibiting CR3 via CD11b blockade (p<0.0001, rightmost bar).(Below) Sub-analysis of iMGL showing phagocytic events reveals similar mean amounts of internal transfer across the entire treatment group (p=0.1165). All histograms are reported as mean ± SEM. One-way ANOVA for cytokine and migration assays, followed by Dunnett's multiple comparison post-hoc test, ***p<0.0001, **p<0.001, *p<0.05, cytokine assay: n=3 wells / group. Migration assay: n=5 fields / condition. Calcium assay: medium (n=37 cells), PSB0739 treatment (n=17 cells), I340 / I380 expressed as mean ± SEM at each time point. Phagocytosis assay: MD-Mφ vs iMGL: unpaired t-test, **p<0.001, n=3 wells / group. MERTK and CR3 assays, one-way ANOVA, followed by Tukey's multiple comparison post-hoc test, ***p<0.0001; n=6, n=3 wells / group for each medium. [Figure 3-7]Figure 3A Flow cytometry analysis shows that iMGL (outer dark spot area in the three panels) is CD45lo-int, similar to fetal MG (innermost spot area in the three panels). Figure 3B iMGL (dark spot in the left panel) is different from CD45-hi MD-Mφ (gray spot in the left panel). The CD11b intensity histogram (left histogram) reveals that fetal MG expresses slightly more CD11b than iMGL, but less than MD-Mφ. Figure 3C iMGL secretes cytokines and chemokines when stimulated for 24 hours with either IFNγ (20 ng / ml), IL-1β (20 ng / ml), or LPS (100 ng / ml) by ELISA multiplex. Figure 3D ADP (100 μM) induces iMGL migration in a Transwell chamber (5 μm). Prior exposure to the P2ry12 antagonist PSB0739 (50 μM, 1 hour) completely inactivates ADP-induced iMGL migration (***p<0.0001). Figure 3E ADP induces calcium flux in iMGL via the P2ry12 receptor. (Left) Exposure to ADP results in increased calcium influx (I340 / I380 ratio) in the medium group (bright trace), but not in the PSB0739-treated group (dark trace). (Right) Representative images of ADP-induced calcium flux at 240 seconds in the medium (top) and PSB0739 (bottom). Figure 3F iMGL phagocytoses human brain-derived synaptosomes (hS). Representative images recorded with Amnis Imagestream show phagocytosis of hS by MD-Mφ and iMGL. Figure 3G Quantification of phagocytosis shows that iMGL internalizes hS at 50% of macrophage capacity (p<0.0001). Figure 3H Representative images of iMGL phagocytosis of hS in the presence of either the MerTK inhibitor UNC569 (top) or the anti-CD11b antibody (bottom). Figure 3I (top) iMGL phagocytosis of hS is reduced by approximately 12% (second bar from the right, p<0.05) by blocking MerTK, but by inhibiting CR3 via CD11b blockade (p<0.0001, rightmost bar).(Below) Sub-analysis of iMGL showing phagocytic events reveals similar mean amounts of internal transfer across the entire treatment group (p=0.1165). All histograms are reported as mean ± SEM. One-way ANOVA for cytokine and migration assays, followed by Dunnett's multiple comparison post-hoc test, ***p<0.0001, **p<0.001, *p<0.05, cytokine assay: n=3 wells / group. Migration assay: n=5 fields / condition. Calcium assay: medium (n=37 cells), PSB0739 treatment (n=17 cells), I340 / I380 expressed as mean ± SEM at each time point. Phagocytosis assay: MD-Mφ vs iMGL: unpaired t-test, **p<0.001, n=3 wells / group. MERTK and CR3 assays, one-way ANOVA, followed by Tukey's multiple comparison post-hoc test, ***p<0.0001; n=6, n=3 wells / group for each medium. [Figure 4-1]Figure 4A Heatmaps of 25 immunogenes with LOAD-related variants reveal that the major risk factors APOE and TREM2 are highly expressed in iMGL, adult MG, and fetal MG. Figure 4B iMGL internally transports fluorescently labeled fAβ and pHrodo-dye BDTO. Representative image recorded with Amnis Image StreamX Mark II. Figure 4C iMGL was exposed to unlabeled fAβ (5 μg-ml-1) and BDTO (5 μg / ml) for 24 hours, and mRNA expression of 19 GWAS genes was evaluated via qPCR array. For each gene tested, the bar corresponding to fAβ is on the left and the bar corresponding to BDTO treatment is on the right. fAβ treatment resulted in a more than twofold increase in the expression of 10 genes, including MS4A6A (6.3x), CD33 (6.1x), ABCA7 (5.8x), TYROBP (4.98x), and TREM2 (4.85x), compared to the medium. Conversely, BDTO exposure resulted in a more than twofold increase in the expression of 4 genes compared to the medium. Six genes were differentially expressed in fAβ compared to BDTO. Preparations of fAβ and BDTO were confirmed by dot blot analysis using conformation-specific antibodies for oligomer (A11), fiber (OC), and non-structure-specific antibodies for human Aβ (6E10) and tau oligomer (tau22). Target genes were standardized against GAPDH and compared with medium-mediated expression using ΔΔCt. Bars indicate the mean ± SEM expression fold. Red hash bars represent ΔΔCt=1. Two-way ANOVA followed by Sidaq's multiple comparison post-hoc test, ***p<0.0001, **p<0.001, *p<0.05; n=6 wells / group. Data are expressed as mean ± SEM. [Figure 4-2]Figure 4A Heatmaps of 25 immunogenes with LOAD-related variants reveal that the major risk factors APOE and TREM2 are highly expressed in iMGL, adult MG, and fetal MG. Figure 4B iMGL internally transports fluorescently labeled fAβ and pHrodo-dye BDTO. Representative image recorded with Amnis Image StreamX Mark II. Figure 4C iMGL was exposed to unlabeled fAβ (5 μg-ml-1) and BDTO (5 μg / ml) for 24 hours, and mRNA expression of 19 GWAS genes was evaluated via qPCR array. For each gene tested, the bar corresponding to fAβ is on the left and the bar corresponding to BDTO treatment is on the right. fAβ treatment resulted in a more than twofold increase in the expression of 10 genes, including MS4A6A (6.3x), CD33 (6.1x), ABCA7 (5.8x), TYROBP (4.98x), and TREM2 (4.85x), compared to the medium. Conversely, BDTO exposure resulted in a more than twofold increase in the expression of 4 genes compared to the medium. Six genes were differentially expressed in fAβ compared to BDTO. Preparations of fAβ and BDTO were confirmed by dot blot analysis using conformation-specific antibodies for oligomer (A11), fiber (OC), and non-structure-specific antibodies for human Aβ (6E10) and tau oligomer (tau22). Target genes were standardized against GAPDH and compared with medium-mediated expression using ΔΔCt. Bars indicate the mean ± SEM expression fold. Red hash bars represent ΔΔCt=1. Two-way ANOVA followed by Sidaq's multiple comparison post-hoc test, ***p<0.0001, **p<0.001, *p<0.05; n=6 wells / group. Data are expressed as mean ± SEM. [Figure 4-3]Figure 4A Heatmaps of 25 immunogenes with LOAD-related variants reveal that the major risk factors APOE and TREM2 are highly expressed in iMGL, adult MG, and fetal MG. Figure 4B iMGL internally transports fluorescently labeled fAβ and pHrodo-dye BDTO. Representative image recorded with Amnis Image StreamX Mark II. Figure 4C iMGL was exposed to unlabeled fAβ (5 μg-ml-1) and BDTO (5 μg / ml) for 24 hours, and mRNA expression of 19 GWAS genes was evaluated via qPCR array. For each gene tested, the bar corresponding to fAβ is on the left and the bar corresponding to BDTO treatment is on the right. fAβ treatment resulted in a more than twofold increase in the expression of 10 genes, including MS4A6A (6.3x), CD33 (6.1x), ABCA7 (5.8x), TYROBP (4.98x), and TREM2 (4.85x), compared to the medium. Conversely, BDTO exposure resulted in a more than twofold increase in the expression of 4 genes compared to the medium. Six genes were differentially expressed in fAβ compared to BDTO. Preparations of fAβ and BDTO were confirmed by dot blot analysis using conformation-specific antibodies for oligomer (A11), fiber (OC), and non-structure-specific antibodies for human Aβ (6E10) and tau oligomer (tau22). Target genes were standardized against GAPDH and compared with medium-mediated expression using ΔΔCt. Bars indicate the mean ± SEM expression fold. Red hash bars represent ΔΔCt=1. Two-way ANOVA followed by Sidaq's multiple comparison post-hoc test, ***p<0.0001, **p<0.001, *p<0.05; n=6 wells / group. Data are expressed as mean ± SEM. [Figure 5-1]Figure 5A Schematic diagram of iMGL co-cultures with or without rat hippocampal neurons. Figure 5B iMGL co-cultured with neurons was collected, evaluated by flow cytometry, and the transcriptome was assessed by RNA sequencing. Figure 5C Heatmaps of gene expression in iMGL and iMGL-HC highlight intrinsically enriched genes. Figure 5D Differential gene expression analysis highlights 156 upregulated genes and 244 downregulated genes in iMGL-HC. Figure 5E Scatter plot of differentially expressed genes [>2Log2(FPKM+1)] highlights enrichment of TRIM14, CABLES1, MMP2, SIGLEC 11 and 12, MITF, and SLC2A5 in iMGL-HC, suggesting that iMGL responds appropriately to the neuronal environment. Cells cultured alone were enriched for COMT, EGR2, EGR3, and FFAR2, suggesting a primed microglial phenotype. [Figure 5-2] Figure 5A Schematic diagram of iMGL co-cultures with or without rat hippocampal neurons. Figure 5B iMGL co-cultured with neurons was collected, evaluated by flow cytometry, and the transcriptome was assessed by RNA sequencing. Figure 5C Heatmaps of gene expression in iMGL and iMGL-HC highlight intrinsically enriched genes. Figure 5D Differential gene expression analysis highlights 156 upregulated genes and 244 downregulated genes in iMGL-HC. Figure 5E Scatter plot of differentially expressed genes [>2Log2(FPKM+1)] highlights enrichment of TRIM14, CABLES1, MMP2, SIGLEC 11 and 12, MITF, and SLC2A5 in iMGL-HC, suggesting that iMGL responds appropriately to the neuronal environment. Cells cultured alone were enriched for COMT, EGR2, EGR3, and FFAR2, suggesting a primed microglial phenotype. [Figure 6]iMGLs (5 × 10⁵ cells) were added to a culture medium containing a single BORG for 7 days. (Panel A) Representative bright-field images of iMGLs detected in and near the BORG after 3 days. The iMGLs were found at the boundary of the culture medium (arrow) of the BORG and adhered to it, but rather than floating freely in the medium, suggesting complete chemotaxis of the iMGLs. (Panel B) Representative images of iMGLs at the outer and inner radii of the BORG. (Panel C) Embedded iMGLs exhibit macrophage-like morphology (white arrow) and elongated protrusions (black arrow), indicating ECM remodeling and surveillance, respectively. Simultaneous evaluation of the morphology of iMGLs embedded in undamaged (Panels D-F) BORGs and damaged (Panels G-I) BORGs. (Panel D) Immunohistochemical analysis of the BORG revealed that the iMGLs began to tile evenly throughout the BORG, projecting branched protrusions for environmental surveillance. BORG is a representative brain that develops in vitro and contains neurons and astrocytes that self-organize in a cortical-like distribution, but lacks microglia, iMGLs. (Panels E-F) Representative immunofluorescence images of iMGLs with elongating processes in a 3D neural environment at high magnification. (Panels G-I) Representative images of iMGL morphology observed in damaged BORGs. (Panels H-I) Spheroidal iMGLs reminiscent of amoeba-like microglia are distributed in damaged BORGs and closely resemble activated microglia, demonstrating that iMGLs respond appropriately to nerve injury. Scale bars = 50 μm in A-C, 200 μm in D and G, 80 μm in E and H, and 15 μm in panels F and I. [Figure 7]Figure 7A. Characterization of fluidity of monocytes, dendritic cells, and commercially available HPCs. Human CD14+ / CD16- monocytes and CD14+ / CD16+ inflammatory monocytes were isolated from the blood of young, healthy individuals (18–39 years) using FACs. Cells were first gated for viability (not shown), then gated for CD14 to avoid leukocyte contamination, and finally isolated by CD16 expression and collected for RNA. Figure 7B. Characterization of fluidity of monocytes, dendritic cells, and commercially available HPCs. Human bone marrow dendritic cells (blood DCs) were isolated from the blood of young, healthy individuals (18–39 years) using FACs following an untreated bone marrow DC enrichment kit. To avoid contamination by plasmacytoid DCs, DCs were stained with CD123, and bone marrow DC subtypes CD1c and CD141 were collected for RNA. Figure 7C. Characterization of fluidity of monocytes, dendritic cells, and commercially available HPCs. We identified cells from a commercially available HPC source (CD43+ / 235a+ / CD41+) and used them for comparison with HPC differentiation and subsequent iMGL differentiation in the laboratory. [Figure 8-1]Figure 8A: RNA-seq coverage maps and gene FPKM values ​​for the bone marrow-specific genes RUNX1, PU.1, and CSF1R in CD14+ / 16- monocytes (CD14 M), CD14+ / 16+ monocytes (CD16 M), and iPS-derived microglia-like cells (iMGL). Figure 8B: RNA-seq coverage maps and gene FPKM values ​​for the monocyte-specific genes IRF1, KLF4, and NR4A1 in CD14+ / 16- monocytes (CD14 M), CD14+ / 16+ monocytes (CD16 M), and iPS-derived microglia-like cells (iMGL). Figure 8C shows RNA-seq coverage maps and gene FPKM values ​​for the microglia-enriched genes P2RY12, OLFML3, and GPR34 in CD14+ / 16- monocytes (CD14 M), CD14+ / 16+ monocytes (CD16 M), and iPS-derived microglia-like cells (iMGL) in iMGL. For all RNA coverage maps (Figures 8A, 8B, and 8C), the y-axis represents reads per million (RPM) scaled according to all samples. Histogram comparisons using FPKM values ​​for all genes are shown as mean ± sem. Biological replicates for CD14 M (N=5), CD16 (N=4), and iMGL (N=6) are included in the comparisons by one-way ANOVA followed by Tukey's multiple comparison post-hoc test. **p<0.001, ***p<0.0001. Figure 8D shows representative volcano plots of differentially expressed genes (p-value < 0.001, 2x change) in iMGL (right side of the plot), CD14 M (left side of the plot), and non-significant (bright area at the bottom of the plot). Major genes are labeled. Particle change (log2) and -log10 (p-value) are shown on the x and y axes, respectively. The gray dashed vertical lines indicate a 2x change in gene expression. The Venn diagram shows the total number of differentially expressed genes for each condition. Figure 8E shows representative volcano plots of differentially expressed genes (p-value < 0.001, 2x change) in iMGL (right side of the plot), CD16 M (left side of the plot), and non-significant (bright area at the bottom of the plot). Major genes are labeled. Particle change (log2) and -log10 (p-value) are shown on the x and y axes, respectively.The gray dashed vertical lines indicate a twofold change in gene expression. The Venn diagram shows the total number of genes expressed differentially under each condition. [Figure 8-2]Figure 8A: RNA-seq coverage maps and gene FPKM values ​​for the bone marrow-specific genes RUNX1, PU.1, and CSF1R in CD14+ / 16- monocytes (CD14 M), CD14+ / 16+ monocytes (CD16 M), and iPS-derived microglia-like cells (iMGL). Figure 8B: RNA-seq coverage maps and gene FPKM values ​​for the monocyte-specific genes IRF1, KLF4, and NR4A1 in CD14+ / 16- monocytes (CD14 M), CD14+ / 16+ monocytes (CD16 M), and iPS-derived microglia-like cells (iMGL). Figure 8C shows RNA-seq coverage maps and gene FPKM values ​​for the microglia-enriched genes P2RY12, OLFML3, and GPR34 in CD14+ / 16- monocytes (CD14 M), CD14+ / 16+ monocytes (CD16 M), and iPS-derived microglia-like cells (iMGL) in iMGL. For all RNA coverage maps (Figures 8A, 8B, and 8C), the y-axis represents reads per million (RPM) scaled according to all samples. Histogram comparisons using FPKM values ​​for all genes are shown as mean ± sem. Biological replicates for CD14 M (N=5), CD16 (N=4), and iMGL (N=6) are included in the comparisons by one-way ANOVA followed by Tukey's multiple comparison post-hoc test. **p<0.001, ***p<0.0001. Figure 8D shows representative volcano plots of differentially expressed genes (p-value < 0.001, 2x change) in iMGL (right side of the plot), CD14 M (left side of the plot), and non-significant (bright area at the bottom of the plot). Major genes are labeled. Particle change (log2) and -log10 (p-value) are shown on the x and y axes, respectively. The gray dashed vertical lines indicate a 2x change in gene expression. The Venn diagram shows the total number of differentially expressed genes for each condition. Figure 8E shows representative volcano plots of differentially expressed genes (p-value < 0.001, 2x change) in iMGL (right side of the plot), CD16 M (left side of the plot), and non-significant (bright area at the bottom of the plot). Major genes are labeled. Particle change (log2) and -log10 (p-value) are shown on the x and y axes, respectively.The gray dashed vertical lines indicate a twofold change in gene expression. The Venn diagram shows the total number of genes expressed differentially under each condition. [Figure 8-3]Figure 8A: RNA-seq coverage maps and gene FPKM values ​​for the bone marrow-specific genes RUNX1, PU.1, and CSF1R in CD14+ / 16- monocytes (CD14 M), CD14+ / 16+ monocytes (CD16 M), and iPS-derived microglia-like cells (iMGL). Figure 8B: RNA-seq coverage maps and gene FPKM values ​​for the monocyte-specific genes IRF1, KLF4, and NR4A1 in CD14+ / 16- monocytes (CD14 M), CD14+ / 16+ monocytes (CD16 M), and iPS-derived microglia-like cells (iMGL). Figure 8C shows RNA-seq coverage maps and gene FPKM values ​​for the microglia-enriched genes P2RY12, OLFML3, and GPR34 in CD14+ / 16- monocytes (CD14 M), CD14+ / 16+ monocytes (CD16 M), and iPS-derived microglia-like cells (iMGL) in iMGL. For all RNA coverage maps (Figures 8A, 8B, and 8C), the y-axis represents reads per million (RPM) scaled according to all samples. Histogram comparisons using FPKM values ​​for all genes are shown as mean ± sem. Biological replicates for CD14 M (N=5), CD16 (N=4), and iMGL (N=6) are included in the comparisons by one-way ANOVA followed by Tukey's multiple comparison post-hoc test. **p<0.001, ***p<0.0001. Figure 8D shows representative volcano plots of differentially expressed genes (p-value < 0.001, 2x change) in iMGL (right side of the plot), CD14 M (left side of the plot), and non-significant (bright area at the bottom of the plot). Major genes are labeled. Particle change (log2) and -log10 (p-value) are shown on the x and y axes, respectively. The gray dashed vertical lines indicate a 2x change in gene expression. The Venn diagram shows the total number of differentially expressed genes for each condition. Figure 8E shows representative volcano plots of differentially expressed genes (p-value < 0.001, 2x change) in iMGL (right side of the plot), CD16 M (left side of the plot), and non-significant (bright area at the bottom of the plot). Major genes are labeled. Particle change (log2) and -log10 (p-value) are shown on the x and y axes, respectively.The gray dashed vertical lines indicate a twofold change in gene expression. The Venn diagram shows the total number of genes expressed differentially under each condition. [Figure 8-4]Figure 8A: RNA-seq coverage maps and gene FPKM values ​​for the bone marrow-specific genes RUNX1, PU.1, and CSF1R in CD14+ / 16- monocytes (CD14 M), CD14+ / 16+ monocytes (CD16 M), and iPS-derived microglia-like cells (iMGL). Figure 8B: RNA-seq coverage maps and gene FPKM values ​​for the monocyte-specific genes IRF1, KLF4, and NR4A1 in CD14+ / 16- monocytes (CD14 M), CD14+ / 16+ monocytes (CD16 M), and iPS-derived microglia-like cells (iMGL). Figure 8C shows RNA-seq coverage maps and gene FPKM values ​​for the microglia-enriched genes P2RY12, OLFML3, and GPR34 in CD14+ / 16- monocytes (CD14 M), CD14+ / 16+ monocytes (CD16 M), and iPS-derived microglia-like cells (iMGL) in iMGL. For all RNA coverage maps (Figures 8A, 8B, and 8C), the y-axis represents reads per million (RPM) scaled according to all samples. Histogram comparisons using FPKM values ​​for all genes are shown as mean ± sem. Biological replicates for CD14 M (N=5), CD16 (N=4), and iMGL (N=6) are included in the comparisons by one-way ANOVA followed by Tukey's multiple comparison post-hoc test. **p<0.001, ***p<0.0001. Figure 8D shows representative volcano plots of differentially expressed genes (p-value < 0.001, 2x change) in iMGL (right side of the plot), CD14 M (left side of the plot), and non-significant (bright area at the bottom of the plot). Major genes are labeled. Particle change (log2) and -log10 (p-value) are shown on the x and y axes, respectively. The gray dashed vertical lines indicate a 2x change in gene expression. The Venn diagram shows the total number of differentially expressed genes for each condition. Figure 8E shows representative volcano plots of differentially expressed genes (p-value < 0.001, 2x change) in iMGL (right side of the plot), CD16 M (left side of the plot), and non-significant (bright area at the bottom of the plot). Major genes are labeled. Particle change (log2) and -log10 (p-value) are shown on the x and y axes, respectively.The gray dashed vertical lines indicate a twofold change in gene expression. The Venn diagram shows the total number of genes expressed differentially under each condition. [Figure 8-5]Figure 8A: RNA-seq coverage maps and gene FPKM values ​​for the bone marrow-specific genes RUNX1, PU.1, and CSF1R in CD14+ / 16- monocytes (CD14 M), CD14+ / 16+ monocytes (CD16 M), and iPS-derived microglia-like cells (iMGL). Figure 8B: RNA-seq coverage maps and gene FPKM values ​​for the monocyte-specific genes IRF1, KLF4, and NR4A1 in CD14+ / 16- monocytes (CD14 M), CD14+ / 16+ monocytes (CD16 M), and iPS-derived microglia-like cells (iMGL). Figure 8C shows RNA-seq coverage maps and gene FPKM values ​​for the microglia-enriched genes P2RY12, OLFML3, and GPR34 in CD14+ / 16- monocytes (CD14 M), CD14+ / 16+ monocytes (CD16 M), and iPS-derived microglia-like cells (iMGL) in iMGL. For all RNA coverage maps (Figures 8A, 8B, and 8C), the y-axis represents reads per million (RPM) scaled according to all samples. Histogram comparisons using FPKM values ​​for all genes are shown as mean ± sem. Biological replicates for CD14 M (N=5), CD16 (N=4), and iMGL (N=6) are included in the comparisons by one-way ANOVA followed by Tukey's multiple comparison post-hoc test. **p<0.001, ***p<0.0001. Figure 8D shows representative volcano plots of differentially expressed genes (p-value < 0.001, 2x change) in iMGL (right side of the plot), CD14 M (left side of the plot), and non-significant (bright area at the bottom of the plot). Major genes are labeled. Particle change (log2) and -log10 (p-value) are shown on the x and y axes, respectively. The gray dashed vertical lines indicate a 2x change in gene expression. The Venn diagram shows the total number of differentially expressed genes for each condition. Figure 8E shows representative volcano plots of differentially expressed genes (p-value < 0.001, 2x change) in iMGL (right side of the plot), CD16 M (left side of the plot), and non-significant (bright area at the bottom of the plot). Major genes are labeled. Particle change (log2) and -log10 (p-value) are shown on the x and y axes, respectively.The gray dashed vertical lines indicate a twofold change in gene expression. The Venn diagram shows the total number of genes expressed differentially under each condition. [Figure 9-1] Figure 9A: The Spearman correlation matrix of the biological samples used in RNA sequencing highlights strong within-group correlations. iMGL correlates well with fetal and adult MG, suggesting strong similarities in gene expression between samples. Figure 9B: Histograms of major genes found across various samples. CD14 and FCGR3A (also known as CD16) were expressed in all bone marrow cells, including microglia, but were enriched in CD14 M and CD16 M, respectively. As expected, FLT3 was highly expressed in blood DCs but not in other cells and was barely detectable in all three microglia groups. The monocyte / macrophage-specific transcription factor KLF2 was enriched only in CD14 M and CD16 M. On the other hand, GATA1 and OCT4 were detected only in iHPC and iPSC, respectively. [Figure 9-2] Figure 9A: The Spearman correlation matrix of the biological samples used in RNA sequencing highlights strong within-group correlations. iMGL correlates well with fetal and adult MG, suggesting strong similarities in gene expression between samples. Figure 9B: Histograms of major genes found across various samples. CD14 and FCGR3A (also known as CD16) were expressed in all bone marrow cells, including microglia, but were enriched in CD14 M and CD16 M, respectively. As expected, FLT3 was highly expressed in blood DCs but not in other cells and was barely detectable in all three microglia groups. The monocyte / macrophage-specific transcription factor KLF2 was enriched only in CD14 M and CD16 M. On the other hand, GATA1 and OCT4 were detected only in iHPC and iPSC, respectively. [Figure 10-1]Figure 10A Representative immunofluorescence images of iMGL expressing the microglia markers CX3CR1 (left panel) and TREM2 (center and right panels). hCyto (center and left panels) is a cytoplasmic marker. The merged panel (right panel) shows co-localization of CX3CR1, hCyto, and TREM2. Figure 10B Representative immunofluorescence images of iMGL expressing the microglia markers TGFBR1 (left panel) and MERTK (left and center panels). Dapi (center and right panels) is a nuclear marker. The merged panel (right panel) shows co-localization of TGFBR1, MERTK, and the nucleus. Figure 10C Representative immunofluorescence images of iMGL expressing the microglia markers PROS1 (left panel), ITGB5 (center and left panels), and TREM2 (center and right panels). The merged panel (right panel) shows co-localization of PROS1, ITGB5, and TREM2. Figure 10D Representative bright-field and immunofluorescence images recorded by an Amnis Imagestream flow cytometer visualizing Ec phagocytosis in macrophages (top) and iMGLs (bottom). Figure 10E As expected, quantification of the percentage of phagocytic cells (top) reveals that iMGLs (right bar) phagocytose Ec nearly 10 times less than macrophages (left bar). The amount of Ec internally transported by GMFI within phatocytic cells (bottom) further indicates the greater phagocytic capacity of macrophages compared to iMGLs. [Figure 10-2]Figure 10A Representative immunofluorescence images of iMGL expressing the microglia markers CX3CR1 (left panel) and TREM2 (center and right panels). hCyto (center and left panels) is a cytoplasmic marker. The merged panel (right panel) shows co-localization of CX3CR1, hCyto, and TREM2. Figure 10B Representative immunofluorescence images of iMGL expressing the microglia markers TGFBR1 (left panel) and MERTK (left and center panels). Dapi (center and right panels) is a nuclear marker. The merged panel (right panel) shows co-localization of TGFBR1, MERTK, and the nucleus. Figure 10C Representative immunofluorescence images of iMGL expressing the microglia markers PROS1 (left panel), ITGB5 (center and left panels), and TREM2 (center and right panels). The merged panel (right panel) shows co-localization of PROS1, ITGB5, and TREM2. Figure 10D Representative bright-field and immunofluorescence images recorded by an Amnis Imagestream flow cytometer visualizing Ec phagocytosis in macrophages (top) and iMGLs (bottom). Figure 10E As expected, quantification of the percentage of phagocytic cells (top) reveals that iMGLs (right bar) phagocytose Ec nearly 10 times less than macrophages (left bar). The amount of Ec internally transported by GMFI within phatocytic cells (bottom) further indicates the greater phagocytic capacity of macrophages compared to iMGLs. [Figure 10-3]Figure 10A Representative immunofluorescence images of iMGL expressing the microglia markers CX3CR1 (left panel) and TREM2 (center and right panels). hCyto (center and left panels) is a cytoplasmic marker. The merged panel (right panel) shows co-localization of CX3CR1, hCyto, and TREM2. Figure 10B Representative immunofluorescence images of iMGL expressing the microglia markers TGFBR1 (left panel) and MERTK (left and center panels). Dapi (center and right panels) is a nuclear marker. The merged panel (right panel) shows co-localization of TGFBR1, MERTK, and the nucleus. Figure 10C Representative immunofluorescence images of iMGL expressing the microglia markers PROS1 (left panel), ITGB5 (center and left panels), and TREM2 (center and right panels). The merged panel (right panel) shows co-localization of PROS1, ITGB5, and TREM2. Figure 10D Representative bright-field and immunofluorescence images recorded by an Amnis Imagestream flow cytometer visualizing Ec phagocytosis in macrophages (top) and iMGLs (bottom). Figure 10E As expected, quantification of the percentage of phagocytic cells (top) reveals that iMGLs (right bar) phagocytose Ec nearly 10 times less than macrophages (left bar). The amount of Ec internally transported by GMFI within phatocytic cells (bottom) further indicates the greater phagocytic capacity of macrophages compared to iMGLs. [Figure 11-1]iMGL expresses genes associated with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Parkinson's disease (PD), and Lewy body dementia (DLB), leading to microglial dysfunction. A bar graph of genes involved in neurodegenerative diseases, presented as mean ± SEM and detected in iMGL as in fetal MG and adult MG, is shown, represented as FPKM+1 followed by Log2 conversion [Log2(FPKM+1)]. Similar to isolated human primary microglia, iMGL expresses balossin-containing protein (VCP), (FUS), C9ORF72, proganulin (GRN), TDP-43 (TARDBP), LRRK2, superoxide dismutase (SOD), and synuclein (SNCA). Recent literature suggests that microglial dysfunction is associated with mutations or loss of function in these genes involved in the pathogenesis of ALS (C9ORF72, SOD1, TARDBP, FUS), FTD (VCP, C9ORF72, GRN, TARDBP), PD (LRRK2, SNCA), and DLB (SNCA), indicating the usefulness of iMGL in investigating the underlying mechanisms of these genes in these neurological disorders. [Figure 11-2]iMGL expresses genes associated with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Parkinson's disease (PD), and Lewy body dementia (DLB), leading to microglial dysfunction. A bar graph of genes involved in neurodegenerative diseases, presented as mean ± SEM and detected in iMGL as in fetal MG and adult MG, is shown, represented as FPKM+1 followed by Log2 conversion [Log2(FPKM+1)]. Similar to isolated human primary microglia, iMGL expresses balossin-containing protein (VCP), (FUS), C9ORF72, proganulin (GRN), TDP-43 (TARDBP), LRRK2, superoxide dismutase (SOD), and synuclein (SNCA). Recent literature suggests that microglial dysfunction is associated with mutations or loss of function in these genes involved in the pathogenesis of ALS (C9ORF72, SOD1, TARDBP, FUS), FTD (VCP, C9ORF72, GRN, TARDBP), PD (LRRK2, SNCA), and DLB (SNCA), indicating the usefulness of iMGL in investigating the underlying mechanisms of these genes in these neurological disorders. [Figure 12-1] GO terms from differential gene expression analysis of iMGL cultured with hippocampal neurons. The gene expression profile of iMGL (iMGL-HC) co-cultured with rat hippocampal neurons was enhanced by soluble and insoluble factors present in the neurons. Genes upregulated in iMGL-HC were associated with 20 statistically significant GO biological modules (iMGL-HC histogram), including positive cholesterol efflux, lipid transport, positive regulation of immune response, negative regulation of leukocyte differentiation, and cell adhesion molecules. Cells cultured in the absence of neurons had a complementary gene profile with 20 statistically significant GO biological modules (iMGL histogram), which included characteristic cholesterol homeostasis, characteristic TNFα signaling by NF-κB, leukocyte differentiation, and regulation of IL-1β secretion. [Figure 12-2] GO terms from differential gene expression analysis of iMGL cultured with hippocampal neurons. The gene expression profile of iMGL (iMGL-HC) co-cultured with rat hippocampal neurons was enhanced by soluble and insoluble factors present in the neurons. Genes upregulated in iMGL-HC were associated with 20 statistically significant GO biological modules (iMGL-HC histogram), including positive cholesterol efflux, lipid transport, positive regulation of immune response, negative regulation of leukocyte differentiation, and cell adhesion molecules. Cells cultured in the absence of neurons had a complementary gene profile with 20 statistically significant GO biological modules (iMGL histogram), which included characteristic cholesterol homeostasis, characteristic TNFα signaling by NF-κB, leukocyte differentiation, and regulation of IL-1β secretion. [Figure 13]Figures 13A-13P show that iMGLs transplanted into the brains of either wild-type or AD transplant-competent mice resemble brain microglia. In the brains of mice adapted to xenotransplantation, transplanted iMGLs branch and interact with the neural environment. (A-L) Two months in vivo, transplanted iMGLs in mice exhibit high dendrites similar to endogenous microglia found in the brain and show long-term survival rates. (A) Transplanted iMGLs labeled with P2ry12 (HPA HPA014518, Sigma) and human nucleus (ku80) show long-term survival rates in mice. (B-D) At high magnification, P2ry12 is highly expressed in iMGL dendrites, both suggesting homeostatic microglia that monitor the brain environment. (E~H) Branched iMGLs express microglia-enriched Tmem119, which is recognized by the human-specific Tmem119 antibody (ab185333, Abcam, identified and validated in [Bennet et al, PNAS 2016]), as well as the human cytoplasmic marker (maker) SC121 (hCyto). (I~L) At higher magnifications, representative iMGLs express P2ry12, hCyto, and Iba1 (ab5076, Abcam). (M~P) Human iMGLs (hCyto) transplanted into AD immunodeficient mice (Marsh et al, PNAS 2016) interact with and phagocytose amyloid plaques. (I~J) The transplanted iMGLs extend their projections and migrate to the plaques. The iMGLs completely enclose the amyloid plaques (O) and begin to phagocytose the amyloid (P). Scale bar; (A, E, N) = 30 μm, (B~D, F~H, I~L, O, P) = 10 μm, (M) = 300 μm. n = 3 animals per test. [Figure 14-1]Figures 14A-14F: Genomic stability of iPSCs and iMGLs. (Figure 14A) Top: Representative fluorescence images of iPSCs expressing pluripotency markers OCT4 and SOX2. Scale bar = 300 μm. Bottom: Functional validation of iPSC pluripotency. Representative fluorescence images of iPSCs differentiated into endoderm, mesoderm, and ectoderm, and stained for Sox17, T(Brachyury), and Otx2, respectively, to validate differentiation potential. Scale bar = 200 μm. (Figures 14B-C) Karyotype and Pluritest score were generated using Sendai virus, indicating that all iPS strains used in this test are karyotype normal and pluripotent. Pluritest is a microarray-based pluripotency assessment based on whole iPS transcriptome analysis referencing a library of functionally validated iPSCs (Muller, FJ et al. 2011). (Figure 14D-E) Maintenance of genomic stability throughout the differentiation process of iMGL using pluripotent iPS or commercially available hematopoietic progenitor cells. CNV evaluation of differentiated iMGL revealed that genomic stability was maintained throughout the differentiation process. (Figure D) Representative Nanostring nCounterKaryotype results demonstrate that microglia derived from ADRC iPS strain 22 do not inherit extrachromosomal DNA throughout the differentiation process. (Figure 14E) Quantification of a 338 probe set across all 24 chromosomes revealed no chromosomal abnormalities whatsoever (n=6). (Figure 14F) Representative analysis of iMGL derived from that iPSC showed a strong correlation of CNV (r²=0.929), demonstrating the sensitivity and genomic stability of the assay for the derived iMGL. [Figure 14-2]Figures 14A-14F: Genomic stability of iPSCs and iMGLs. (Figure 14A) Top: Representative fluorescence images of iPSCs expressing pluripotency markers OCT4 and SOX2. Scale bar = 300 μm. Bottom: Functional validation of iPSC pluripotency. Representative fluorescence images of iPSCs differentiated into endoderm, mesoderm, and ectoderm, and stained for Sox17, T(Brachyury), and Otx2, respectively, to validate differentiation potential. Scale bar = 200 μm. (Figures 14B-C) Karyotype and Pluritest score were generated using Sendai virus, indicating that all iPS strains used in this test are karyotype normal and pluripotent. Pluritest is a microarray-based pluripotency assessment based on whole iPS transcriptome analysis referencing a library of functionally validated iPSCs (Muller, FJ et al. 2011). (Figure 14D-E) Maintenance of genomic stability throughout the differentiation process of iMGL using pluripotent iPS or commercially available hematopoietic progenitor cells. CNV evaluation of differentiated iMGL revealed that genomic stability was maintained throughout the differentiation process. (Figure D) Representative Nanostring nCounterKaryotype results demonstrate that microglia derived from ADRC iPS strain 22 do not inherit extrachromosomal DNA throughout the differentiation process. (Figure 14E) Quantification of a 338 probe set across all 24 chromosomes revealed no chromosomal abnormalities whatsoever (n=6). (Figure 14F) Representative analysis of iMGL derived from that iPSC showed a strong correlation of CNV (r²=0.929), demonstrating the sensitivity and genomic stability of the assay for the derived iMGL. [Figure 14-3]Figures 14A-14F: Genomic stability of iPSCs and iMGLs. (Figure 14A) Top: Representative fluorescence images of iPSCs expressing pluripotency markers OCT4 and SOX2. Scale bar = 300 μm. Bottom: Functional validation of iPSC pluripotency. Representative fluorescence images of iPSCs differentiated into endoderm, mesoderm, and ectoderm, and stained for Sox17, T(Brachyury), and Otx2, respectively, to validate differentiation potential. Scale bar = 200 μm. (Figures 14B-C) Karyotype and Pluritest score were generated using Sendai virus, indicating that all iPS strains used in this test are karyotype normal and pluripotent. Pluritest is a microarray-based pluripotency assessment based on whole iPS transcriptome analysis referencing a library of functionally validated iPSCs (Muller, FJ et al. 2011). (Figure 14D-E) Maintenance of genomic stability throughout the differentiation process of iMGL using pluripotent iPS or commercially available hematopoietic progenitor cells. CNV evaluation of differentiated iMGL revealed that genomic stability was maintained throughout the differentiation process. (Figure D) Representative Nanostring nCounterKaryotype results demonstrate that microglia derived from ADRC iPS strain 22 do not inherit extrachromosomal DNA throughout the differentiation process. (Figure 14E) Quantification of a 338 probe set across all 24 chromosomes revealed no chromosomal abnormalities whatsoever (n=6). (Figure 14F) Representative analysis of iMGL derived from that iPSC showed a strong correlation of CNV (r²=0.929), demonstrating the sensitivity and genomic stability of the assay for the derived iMGL. [Figure 14-4]Figures 14A-14F: Genomic stability of iPSCs and iMGLs. (Figure 14A) Top: Representative fluorescence images of iPSCs expressing pluripotency markers OCT4 and SOX2. Scale bar = 300 μm. Bottom: Functional validation of iPSC pluripotency. Representative fluorescence images of iPSCs differentiated into endoderm, mesoderm, and ectoderm, and stained for Sox17, T(Brachyury), and Otx2, respectively, to validate differentiation potential. Scale bar = 200 μm. (Figures 14B-C) Karyotype and Pluritest score were generated using Sendai virus, indicating that all iPS strains used in this test are karyotype normal and pluripotent. Pluritest is a microarray-based pluripotency assessment based on whole iPS transcriptome analysis referencing a library of functionally validated iPSCs (Muller, FJ et al. 2011). (Figure 14D-E) Maintenance of genomic stability throughout the differentiation process of iMGL using pluripotent iPS or commercially available hematopoietic progenitor cells. CNV evaluation of differentiated iMGL revealed that genomic stability was maintained throughout the differentiation process. (Figure D) Representative Nanostring nCounterKaryotype results demonstrate that microglia derived from ADRC iPS strain 22 do not inherit extrachromosomal DNA throughout the differentiation process. (Figure 14E) Quantification of a 338 probe set across all 24 chromosomes revealed no chromosomal abnormalities whatsoever (n=6). (Figure 14F) Representative analysis of iMGL derived from that iPSC showed a strong correlation of CNV (r²=0.929), demonstrating the sensitivity and genomic stability of the assay for the derived iMGL. [Figure 15-1] Figures 15A-B: Evaluation of iMGL purity by P2RY12 / TREM2 colocalization and characterization of monocytes, dendritic cells, and commercially available iHPCs by flow cytometry. (Figure 15A) Specificity evaluation of rabbit anti-human P2ry12 (HPA014518, similarly recently validated) and goat anti-human Trem2 (R&D, AF1828) in human monocytes and iMGL. Scale bar = 20 μm. (Figure 15B) Representative immunofluorescence images of iMGL purity by P2ry12 / Trem2 / DAPI colocalization (from 5 representative strains). Scale bar = 100 μm. [Figure 15-2] Figures 15A-B: Evaluation of iMGL purity by P2RY12 / TREM2 colocalization and characterization of monocytes, dendritic cells, and commercially available iHPCs by flow cytometry. (Figure 15A) Specificity evaluation of rabbit anti-human P2ry12 (HPA014518, similarly recently validated) and goat anti-human Trem2 (R&D, AF1828) in human monocytes and iMGL. Scale bar = 20 μm. (Figure 15B) Representative immunofluorescence images of iMGL purity by P2ry12 / Trem2 / DAPI colocalization (from 5 representative strains). Scale bar = 100 μm. [Figure 16-1]Figures 16A-E show that TGFβ-1, CX3CL1, CD200, and their effects on major microglial genes are linked to the regulation of neuronal function and the environment. (Figures 16A-B) TGFβ1 maintains core microglial genes. 24-hour removal of TGFβ1 strongly affects the microglial transcriptome. Consistent with in vivo mouse studies, TGFβ removal reduces the expression of major microglial genes, including surface receptors P2RY12, TGFβR1, and CX3CR1, while also reducing the expression of microglial transcription factors EGR1 and ETV5. AD-related pathway genes such as BIN1, CD33, and APOE are also affected by TGFβ deficiency. Removal of CX3CL1 and CD200 does not alter core microglial identity but affects state by influencing the expression of homeostatic genes such as COMT and APOE (Figure 16B). (Figure 16C) Differential gene expression analysis reveals that the presence of TGFβ increases the expression of 1262 genes in iMGL, while the absence of TGFβ decreases the expression of 1517 genes, further supporting previous studies highlighting the role of TGFβ in microglial development, gene signature, and function. (Figure 16D) KEGG pathway analysis highlights that microglial core genes elevated by TGFβ modulate pathways in CNS diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. (Figure 16E) TGFβ-induced multiplicative changes in AD GWAS locus genes in iMGL. Statistics reflect one-way ANOVA followed by Dunnett's multiple comparison post-hoc tests. *p<0.05, **p<0.001, ***p<0.0001. [Figure 16-2]Figures 16A-E show that TGFβ-1, CX3CL1, CD200, and their effects on major microglial genes are linked to the regulation of neuronal function and the environment. (Figures 16A-B) TGFβ1 maintains core microglial genes. 24-hour removal of TGFβ1 strongly affects the microglial transcriptome. Consistent with in vivo mouse studies, TGFβ removal reduces the expression of major microglial genes, including surface receptors P2RY12, TGFβR1, and CX3CR1, while also reducing the expression of microglial transcription factors EGR1 and ETV5. AD-related pathway genes such as BIN1, CD33, and APOE are also affected by TGFβ deficiency. Removal of CX3CL1 and CD200 does not alter core microglial identity but affects state by influencing the expression of homeostatic genes such as COMT and APOE (Figure 16B). (Figure 16C) Differential gene expression analysis reveals that the presence of TGFβ increases the expression of 1262 genes in iMGL, while the absence of TGFβ decreases the expression of 1517 genes, further supporting previous studies highlighting the role of TGFβ in microglial development, gene signature, and function. (Figure 16D) KEGG pathway analysis highlights that microglial core genes elevated by TGFβ modulate pathways in CNS diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. (Figure 16E) TGFβ-induced multiplicative changes in AD GWAS locus genes in iMGL. Statistics reflect one-way ANOVA followed by Dunnett's multiple comparison post-hoc tests. *p<0.05, **p<0.001, ***p<0.0001. [Figure 16-3]Figures 16A-E show that TGFβ-1, CX3CL1, CD200, and their effects on major microglial genes are linked to the regulation of neuronal function and the environment. (Figures 16A-B) TGFβ1 maintains core microglial genes. 24-hour removal of TGFβ1 strongly affects the microglial transcriptome. Consistent with in vivo mouse studies, TGFβ removal reduces the expression of major microglial genes, including surface receptors P2RY12, TGFβR1, and CX3CR1, while also reducing the expression of microglial transcription factors EGR1 and ETV5. AD-related pathway genes such as BIN1, CD33, and APOE are also affected by TGFβ deficiency. Removal of CX3CL1 and CD200 does not alter core microglial identity but affects state by influencing the expression of homeostatic genes such as COMT and APOE (Figure 16B). (Figure 16C) Differential gene expression analysis reveals that the presence of TGFβ increases the expression of 1262 genes in iMGL, while the absence of TGFβ decreases the expression of 1517 genes, further supporting previous studies highlighting the role of TGFβ in microglial development, gene signature, and function. (Figure 16D) KEGG pathway analysis highlights that microglial core genes elevated by TGFβ modulate pathways in CNS diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. (Figure 16E) TGFβ-induced multiplicative changes in AD GWAS locus genes in iMGL. Statistics reflect one-way ANOVA followed by Dunnett's multiple comparison post-hoc tests. *p<0.05, **p<0.001, ***p<0.0001. [Figure 16-4]Figures 16A-E show that TGFβ-1, CX3CL1, CD200, and their effects on major microglial genes are linked to the regulation of neuronal function and the environment. (Figures 16A-B) TGFβ1 maintains core microglial genes. 24-hour removal of TGFβ1 strongly affects the microglial transcriptome. Consistent with in vivo mouse studies, TGFβ removal reduces the expression of major microglial genes, including surface receptors P2RY12, TGFβR1, and CX3CR1, while also reducing the expression of microglial transcription factors EGR1 and ETV5. AD-related pathway genes such as BIN1, CD33, and APOE are also affected by TGFβ deficiency. Removal of CX3CL1 and CD200 does not alter core microglial identity but affects state by influencing the expression of homeostatic genes such as COMT and APOE (Figure 16B). (Figure 16C) Differential gene expression analysis reveals that the presence of TGFβ increases the expression of 1262 genes in iMGL, while the absence of TGFβ decreases the expression of 1517 genes, further supporting previous studies highlighting the role of TGFβ in microglial development, gene signature, and function. (Figure 16D) KEGG pathway analysis highlights that microglial core genes elevated by TGFβ modulate pathways in CNS diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. (Figure 16E) TGFβ-induced multiplicative changes in AD GWAS locus genes in iMGL. Statistics reflect one-way ANOVA followed by Dunnett's multiple comparison post-hoc tests. *p<0.05, **p<0.001, ***p<0.0001. [Figure 16-5]Figures 16A-E show that TGFβ-1, CX3CL1, CD200, and their effects on major microglial genes are linked to the regulation of neuronal function and the environment. (Figures 16A-B) TGFβ1 maintains core microglial genes. 24-hour removal of TGFβ1 strongly affects the microglial transcriptome. Consistent with in vivo mouse studies, TGFβ removal reduces the expression of major microglial genes, including surface receptors P2RY12, TGFβR1, and CX3CR1, while also reducing the expression of microglial transcription factors EGR1 and ETV5. AD-related pathway genes such as BIN1, CD33, and APOE are also affected by TGFβ deficiency. Removal of CX3CL1 and CD200 does not alter core microglial identity but affects state by influencing the expression of homeostatic genes such as COMT and APOE (Figure 16B). (Figure 16C) Differential gene expression analysis reveals that the presence of TGFβ increases the expression of 1262 genes in iMGL, while the absence of TGFβ decreases the expression of 1517 genes, further supporting previous studies highlighting the role of TGFβ in microglial development, gene signature, and function. (Figure 16D) KEGG pathway analysis highlights that microglial core genes elevated by TGFβ modulate pathways in CNS diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. (Figure 16E) TGFβ-induced multiplicative changes in AD GWAS locus genes in iMGL. Statistics reflect one-way ANOVA followed by Dunnett's multiple comparison post-hoc tests. *p<0.05, **p<0.001, ***p<0.0001. [Figure 17-1]Figures 17A-C show that microglia AD-GWAS and other CNS disease-related genes can be tested using iMGL. (Figures 17A-B) iMGL AD-related GWAS genes respond differentially to fAβ when primed with or without CD200 and CX3CL1. Exposure of iMGL to CNS factors, CD200 and CX3CL1, "primes" the response to fAβ by increasing the expression of genes that play a role in regulating microglial inflammation and function in AD, such as CD33, ABCA7, TYROBP, and TREM2. Stimulation of iMGL unexposed to CD200 or CX3CL1 with fAβ increases the expression of AD GWAS-related genes CLU and APOE, which are involved in the response to misfolded proteins as well as survival and homeostasis. (Figure 17C) Major neurodegeneration-related genes, APP (AD), SCNA (PD), and HTT (HD), are expressed in iMGL and primary cultured microglia. iMGL also expresses genes associated with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Lewy body dementia (DLB), supporting previous research on microglial dysfunction. A bar graph of genes involved in neurodegenerative diseases, detected in iMGL as well as in fetal MG and adult MG, expressed as Log2(FPKM+1) and presented as mean ± SEM. Similar to isolated human primary cultured microglia, iMGL expresses balossin-containing protein (VCP), FUS-binding protein (FUS), proganulin (GRN), TDP-43 (TARDBP), LRRK2, and superoxide dismutase (SOD). Recent literature suggests that microglial dysfunction is associated with mutations or loss of function in these genes involved in the pathogenesis of ALS (SOD1, TARDBP, FUS), FTD (VCP, GRN, TARDBP), PD (LRRK2, SNCA), and DLB (SNCA), indicating the usefulness of iMGL in testing the underlying mechanisms of these genes in these neurological disorders. Statistics reflect one-way ANOVA followed by Tukey's multiple comparison post-hoc test. *p<0.05, **p<0.001, ***p<0.0001. [Figure 17-2] Figures 17A-C show that microglia AD-GWAS and other CNS disease-related genes can be tested using iMGL. (Figures 17A-B) iMGL AD-related GWAS genes respond differentially to fAβ when primed with or without CD200 and CX3CL1. Exposure of iMGL to CNS factors, CD200 and CX3CL1, "primes" the response to fAβ by increasing the expression of genes that play a role in regulating microglial inflammation and function in AD, such as CD33, ABCA7, TYROBP, and TREM2. Stimulation of iMGL unexposed to CD200 or CX3CL1 with fAβ increases the expression of AD GWAS-related genes CLU and APOE, which are involved in the response to misfolded proteins as well as survival and homeostasis. (Figure 17C) Major neurodegeneration-related genes, APP (AD), SCNA (PD), and HTT (HD), are expressed in iMGL and primary cultured microglia. iMGL also expresses genes associated with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Lewy body dementia (DLB), supporting previous research on microglial dysfunction. A bar graph of genes involved in neurodegenerative diseases, detected in iMGL as well as in fetal MG and adult MG, expressed as Log2(FPKM+1) and presented as mean ± SEM. Similar to isolated human primary cultured microglia, iMGL expresses balossin-containing protein (VCP), FUS-binding protein (FUS), proganulin (GRN), TDP-43 (TARDBP), LRRK2, and superoxide dismutase (SOD). Recent literature suggests that microglial dysfunction is associated with mutations or loss of function in these genes involved in the pathogenesis of ALS (SOD1, TARDBP, FUS), FTD (VCP, GRN, TARDBP), PD (LRRK2, SNCA), and DLB (SNCA), indicating the usefulness of iMGL in testing the underlying mechanisms of these genes in these neurological disorders. Statistics reflect one-way ANOVA followed by Tukey's multiple comparison post-hoc test. *p<0.05, **p<0.001, ***p<0.0001. [Figure 17-3]Figures 17A-C show that microglia AD-GWAS and other CNS disease-related genes can be tested using iMGL. (Figures 17A-B) iMGL AD-related GWAS genes respond differentially to fAβ when primed with or without CD200 and CX3CL1. Exposure of iMGL to CNS factors, CD200 and CX3CL1, "primes" the response to fAβ by increasing the expression of genes that play a role in regulating microglial inflammation and function in AD, such as CD33, ABCA7, TYROBP, and TREM2. Stimulation of iMGL unexposed to CD200 or CX3CL1 with fAβ increases the expression of AD GWAS-related genes CLU and APOE, which are involved in the response to misfolded proteins as well as survival and homeostasis. (Figure 17C) Major neurodegeneration-related genes, APP (AD), SCNA (PD), and HTT (HD), are expressed in iMGL and primary cultured microglia. iMGL also expresses genes associated with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Lewy body dementia (DLB), supporting previous research on microglial dysfunction. A bar graph of genes involved in neurodegenerative diseases, detected in iMGL as well as in fetal MG and adult MG, expressed as Log2(FPKM+1) and presented as mean ± SEM. Similar to isolated human primary cultured microglia, iMGL expresses balossin-containing protein (VCP), FUS-binding protein (FUS), proganulin (GRN), TDP-43 (TARDBP), LRRK2, and superoxide dismutase (SOD). Recent literature suggests that microglial dysfunction is associated with mutations or loss of function in these genes involved in the pathogenesis of ALS (SOD1, TARDBP, FUS), FTD (VCP, GRN, TARDBP), PD (LRRK2, SNCA), and DLB (SNCA), indicating the usefulness of iMGL in testing the underlying mechanisms of these genes in these neurological disorders. Statistics reflect one-way ANOVA followed by Tukey's multiple comparison post-hoc test. *p<0.05, **p<0.001, ***p<0.0001. [Figure 18] iPS-derived microglia cells engraft and phagocytose Aβ, similar to human fetal microglia. (A-D) Human fetal microglia (hCyto) are transplanted into the immunodeficient AD mouse model, Rag5×fAD, and respond to beta-amyloid plaques. Fetal microglia are observed around the plaques (C) and phagocytose Aβ (C-D). (E-H) Similar to fetal microglia, iMGL(hCyto) are located around beta-amyloid plaques and phagocytose them. Scale bars (A, B, E, F) = 20 μm, (C, D, H, G) = 5 μm. [Figure 19] Evaluation of the percentages of (i) cells expressing P2ry12 (left bar), (ii) cells expressing TREM2 (center bar), and (iii) cells expressing P2ry12, TREM2, and DAPI (right bar) from Figure 1H. [Figure 20] Expression of the peripheral macrophage marker TREM1 is low in iMGLs. Both iMGLs and macrophages express the myeloid protein Iba-1 (left column of the panel). However, TREM1 expression (center column of the panel) is highly enriched in macrophages, distinguishing macrophage ontogeny from iMGLS, which is exemplified by the low TREM1 expression typical of microglia in the CNS. [Figure 21] iMGLs are highly motile in vitro. Time-lapse phase-contrast images of iMGL motility in culture (over 24 hours) reveal that iMGLs (enclosed in a box) are highly motile, monitoring their environment by protruding, and demonstrating the ability of iMGLs to migrate in response to, for example, injury or stress. [Figure 22] Co-culturing iMGLs with iPSC-derived astrocytes results in branched iMGLs. When co-culturing with astrocytes distinguished by GFAP expression (left panel), iMGLs (Iba-1, center panel) branch and extend their protrusions in 2D in vitro culture, further demonstrating that cues derived from the CNS environment can further educate iMGLs, allowing them to adopt the in vivo phenotype of microglia found in the brain. [Figure 23]Figures 23A-23B Humanization of mouse brains using human hematopoietic progenitor cells. iHPCs differentiate into microglia, which are commensal macrophages of the CNS, and demonstrate the potential to overcome endogenous mouse microglia in MITRG mice. (A) Confocal microscopy reveals successful engraftment of iHPCs expressing Iba-1 (center panel of 23A), a bone marrow marker, which can be detected by human nucleus-specific antibodies (upper panel of 23A). (B) The transplanted iHPCs differentiate into microglia expressing P2ry12 (upper right panel of 23B), and humanization of MITRG expressing human CSF1 allows human cells to overcome endogenous mouse cells. [Figure 24] Microglia-specific expression of P2ry12 can be detected in iHPCs as early as two weeks after engraftment. Two weeks after iHPC transplantation, cells that survived the transplant and were labeled with human nucleus-specific antibodies (left column of the panel) begin expressing Iba-1 (left-center column of the panel), indicating bone marrow origin. Furthermore, P2ry12, a microglia-specific gene highly expressed in homeostatic microglia, can be detected (right-center column of the panel; arrow). [Figure 25] Transplanted hematopoietic primordial cells differentiate into microglia in the mouse brain and express TMEM119. After two months, HPCs engrafted in the mouse brain express TMEM119 (left-center column of the panel), a microglia marker (Bennet et al., 2016) that is prominently expressed in the highly branched processes of maturing microglia. The human specificity of the TMEM119 antibody is demonstrated by co-localization of TMEM119 with an antibody specific to the human nucleus (right-center column of the panel), but not in all nuclei (DAPI, left column of the panel). [Figure 26]The transplanted human cells express the homeostatic microglia marker, P2ry12. Engrafted iMGLs, distinguishable by human-specific nuclear markers (left-center column of the panel), can be differentiated from endogenous mouse cell nuclei (left column of the panel), which are stained only with the nonspecific nuclear stain DAPI. Engrafted iMGLs express the homeostatic microglia marker P2ry12 (center column of the panel), which highlights the elongated branching processes typical of in vivo microglia. This shows a cytoplasmic cell distribution in microglia, in contrast to the commonly used microglia marker Iba-1 (right-center column of the panel). [Figure 27] iMGLs can be used to study astrocyte-microglia crosstalk in vivo. Transplanted iMGLs (Iba-1, upper left panel) are observed to interact with endogenous mouse astrocytes in vivo (GFAP, lower left panel). Recent studies (Liddelow et al., 2017) suggest astrocyte-microglia crosstalk influencing immune responses in the CNS. [Modes for carrying out the invention]

[0029] Microglia are innate immune cells of the CNS, playing crucial roles in synaptic plasticity, neurogenesis, homeostasis, and immune activity. Microglia also play a vital role in neurological disorders, including Alzheimer's disease (AD), highlighting the need to improve our understanding of their function in both health and disease. Currently, testing human microglia remains challenging due to the rarity and difficulty of obtaining primary cultured cells from human fetal or adult CNS tissue. Therefore, the development of renewable sources of human microglia, including pluripotent stem cells (PSCs), induced pluripotent stem cells (iPSCs), and embryonic stem cells (ESCs), is urgently needed.

[0030] The challenge in generating microglia from iPSCs stems from their unique developmental origin. Clear lineage tracing studies have shown that microglia originate from yolk sac primordial erythrocytes (EMPs) generated during primordial hematopoiesis. EMPs further develop into early primordial macrophages, migrate into the developing neural tube, and become primordial microglial cells. Subsequently, primordial microglial cells mature and develop branching processes used to monitor their environment, promote CNS development, regulate synaptic plasticity, and respond to CNS injury and pathology.

[0031] The generation of patient-derived iPSCs has facilitated new opportunities to investigate the relationship between genetic risk factors and disease. Recently, genome-wide association studies (GWAS) have identified several genes expressed by microglia that are associated with the risk of developing late-onset Alzheimer's disease (LOAD). While the function of microglia and the role of these genes in AD are only just beginning to be investigated in mouse models, the generation of human microglia-like cells, as described herein, makes it possible to investigate human-specific genes that cannot be modeled in mice.

[0032] In Alzheimer's disease (AD), microglia clusters surrounding beta-amyloid plaques highlight their inefficiency in beta-amyloid clearance. Microglia are also involved in the neuroinflammatory components of AD pathogenesis, including cytokine / chemokine secretion that exacerbates disease pathology. Furthermore, AD GWAS genes such as TREM2 and CD33 may be affected by AD pathology and involved in AD progression. Microglia are also key regulators of brain development, neuronal homeostasis, and many neurological disorders. Therefore, a deeper understanding of both pathological and disease-associated gene effects on human microglia and microglial function is urgently needed.

[0033] Some embodiments described herein provide methods for the effective and robust generation of human iPSC microglia-like cells (iMGLs) that are similar to fetal and adult microglia. These methods produce iMGLs useful for investigating neurological diseases such as Alzheimer's disease (AD). In some embodiments described herein, microglia-like cells (iMGLs) are differentiated from iPSCs and their function in neurological diseases such as Alzheimer's disease (AD) is tested.

[0034] The iMGLs described herein develop in vitro, similar to in vivo microglia. Analysis of the entire transcriptome shows they are remarkably similar to adult and fetal human microglia. Functional evaluation of these iMGLs reveals that they secrete cytokines in response to inflammatory stimuli, migrate, undergo calcium translocation, and robustly phagocytose CNS substrates, similar to adult / fetal microglia.

[0035] These iMGLs can be used to (i) examine the effects of fibrillary Aβ and brain-derived tau oligomers on AD-related gene expression, and (ii) identify mechanisms involved in synaptic pruning. Furthermore, iMGLs can be used in high-throughput testing of microglial function, providing important new insights into human neurological disorders.

[0036] The following sections provide various embodiments of methods for producing iMGL, and various embodiments of the structure and function of iMGL. Furthermore, methods for using iMGL are also provided. In addition, a non-limiting detailed description of the methods is also provided. How to create an iMGL:

[0037] In some embodiments, a method is provided for producing human microglia-like cells (iMGLs) from pluripotent stem cells (PSCs). In some embodiments, the method comprises (i) differentiating PSCs using a medium supplemented with hematopoietic differentiation factors to produce induced hematopoietic primordial cells (iHPCs), and (ii) CD43 +The step of isolating iHPC, and (iii) using a microglial differentiation medium to differentiate CD43 + The step of differentiating iHPC into human microglia-like cells (iMGL), and (iv) the step of maturing iMGL. In some embodiments, the HPC generation technique enables the collection of a medium in which (iii) is performed without isolating iHPC. +

[0038] In some embodiments, the method includes the step of differentiating PSC using a medium supplemented with a hematopoietic differentiation factor, and (ii) differentiating CD43 + The step of differentiating iHPC into iMGL using a microglial differentiation medium.

[0039] Some embodiments of the methods and compositions provided herein relate to a method of producing human iMGL from a first type of cell, the method comprising (i) differentiating the first type of cell into iHPC, and (ii) differentiating iHPC to produce iMGL. In some embodiments, the first type of cell is neither a PSC nor an ESC.

[0040] In some embodiments, the PSC is not derived from embryoid bodies. In some embodiments, the PSC comprises single cell PSC. In some embodiments, the PSC is not CD43 + before differentiation. In some embodiments, the PSC is not CD34 + before differentiation. In some embodiments, the PSC is not CD31 + before differentiation. In some embodiments, the PSC is not CD45 + before differentiation.

[0041] In some embodiments, the PSC is an induced PSC (iPSC) or includes it. In some embodiments, the PSC is an embryonic stem cell (ESC) or includes it. In some embodiments, the PSC is a mammalian PSC. In some embodiments, the PSC is a human PSC. In some embodiments, the PSC is a mouse PSC. ​

[0042] Differentiating single-cell PSCs In some embodiments, differentiating PSCs to produce iHPCs involves an incubation period of 5 to 15 days. For example, the incubation period is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days. In some embodiments, the incubation period is 10 days. In some embodiments, the percentage of oxygen to which the PSCs are exposed varies over the 10-day period. In some embodiments, during the incubation period, the iPSCs are incubated in a hypoxic or normal oxygen environment. In some embodiments, the PSCs are incubated in a hypoxic or normal oxygen environment for days 1 to 10 of the incubation period. In some embodiments, during the first part of the 10-day period, the PSCs are exposed to an oxygen environment of 3% to 7%. In some embodiments, the first part of the 10-day period is 4 days (days 1 to 4), and the oxygen environment is 5%. In some embodiments, during the second part of a 10-day period, the PSCs are exposed to an oxygen environment between 15% and 25%. In some embodiments, the second part of a 10-day period is 6 days (days 5-10), and the oxygen environment to which the PSCs are exposed is 20%. In some embodiments, differentiating the PSCs to produce iHPCs includes an incubation period between 3 and 21 days. In some embodiments, the incubation period is up to 28 days. In some embodiments, the incubation period exceeds 28 days. In some embodiments, the incubation period is less than 3 days.

[0043] In some embodiments, the hematopoietic differentiation factors used to differentiate PSCs include FGF2, BMP4, activin A, LiCl, VEGF, TPO, SCF, IL3, and IL6. The culture medium contains one or more of these factors in any combination. In some embodiments, PSCs are incubated in different media throughout the incubation period of the PSC differentiation process. In some embodiments, a 10-day incubation period is provided in which, on day 1, the medium contains FGF2, BMP4, activin A, and LiCl; on days 3 and 4, the medium contains FGF2 and VEGF; and on days 5-10, the medium contains FGF2, VEGF, TPO, SCF, IL3, and IL6. Some embodiments relate to a culture medium containing one or a combination of the factors FGF2, BMP4, activin A, LiCl, VEGF, TPO, SCF, IL3, and IL6. Some embodiments relate to a culture medium containing one or a combination of factors FGF2, BMP4, activin A, and LiCl. Some embodiments relate to a culture medium containing one or a combination of factors FGF2 and VEGF. Some embodiments relate to a culture medium containing one or a combination of factors FGF2, VEGF, TPO, SCF, IL3, and IL6.

[0044] In some embodiments, the concentration of each factor in the culture medium is between 5 ng / ml and 100 ng / ml. In some embodiments, the concentration of each factor in the culture medium is between 30 ng / ml and 70 ng / ml or between 40 ng / ml and 60 ng / ml. In some embodiments, the concentration of each factor in the culture medium is 50 ng / ml. In some embodiments, the concentration of activin A in the culture medium is between 9 ng / ml and 16 ng / ml or between 11 ng / ml and 14 ng / ml. In some embodiments, the concentration of activin A in the culture medium is 12.5 ng / ml. In some embodiments, the concentration of LiCl in the culture medium is between 1 nM and 3 nM. In some embodiments, the concentration of LiCl in the culture medium is between 1 mM and 3 mM. In some embodiments, the concentration of LiCl in the culture medium is 2 mM.

[0045] Isolation of iHPC Using any method known in the art, iHPC or CD43 + Isolate iHPC. In some embodiments, iHPC or CD43 + The method used to isolate iHPC is FACS. In some embodiments, the isolation step is CD43 + This includes selecting a marker. In some embodiments, CD43 + HPCs are isolated using markers other than CD34. In some embodiments, the isolation step is performed using CD34 + This includes selecting cells. In some embodiments, the isolation step involves CD31 + Cells or CD45 + This includes selecting cells. In some embodiments, the isolation step includes selecting another marker known to identify iHPCs.

[0046] In some embodiments, by isolating iHPC, iHPC with a purity of over 80%, for example, over 90%, is isolated. In some embodiments, CD43 + By isolating iHPC, CD43 with a purity exceeding 80%, for example, exceeding 90%, can be obtained. + iHPC is isolated.

[0047] Differentiate iHPC into iMGL iMGLs can be matured using any method known in the art for maturing microglial cells.

[0048] In some embodiments, CD43 + Differentiating iHPC into iMGL involves an incubation period of 20 to 30 days. In some embodiments, the incubation period is 25 days.

[0049] In some embodiments, the culture medium used to differentiate iHPCs into iMGLs contains one or a combination of factors CSF-1, IL-34, and TGFβ1. In some embodiments, the culture medium contains all of factors CSF-1, IL-34, and TGFβ1. In some embodiments, the concentration of CSF-1 in the culture medium is between 5 ng / ml and 50 ng / ml. In some embodiments, the concentration of CSF-1 in the culture medium is between 15 ng / ml and 35 ng / ml or between 20 ng / ml and 30 ng / ml. In some embodiments, the concentration of CSF-1 in the culture medium is 25 ng / ml. In some embodiments, the concentration of IL-34 in the culture medium is between 25 ng / ml and 125 ng / ml. In some embodiments, the concentration of IL-34 in the culture medium is between 80 ng / ml and 120 ng / ml or between 90 ng / ml and 110 ng / ml. In some embodiments, the concentration of IL-34 in the culture medium is 100 ng / ml. In some embodiments, the concentration of TFGβ-1 in the culture medium is between 2.5 ng / ml and 100 ng / ml. In some embodiments, the concentration of TFGβ-1 in the culture medium is between 30 ng / ml and 70 ng / ml or between 40 ng / ml and 60 ng / ml. In some embodiments, the concentration of TGFβ-1 in the culture medium is 50 ng / ml. Some embodiments relate to a culture medium containing one or a combination of factors CSF-1, IL-34, and TGFβ1.

[0050] In some embodiments, the culture medium used to differentiate iHPCs into iMGLs contains TFGβ-2. In some embodiments, the concentration of TFGβ-2 in the medium is between 2.5 ng / ml and 100 ng / ml. In some embodiments, the concentration of TFGβ-2 in the medium is between 30 ng / ml and 70 ng / ml or between 40 ng / ml and 60 ng / ml. In some embodiments, the concentration of TFGβ-2 in the medium is 50 ng / ml.

[0051] In some embodiments, the culture medium used to differentiate iHPCs into iMGLs contains a TFGβ mimetic. Examples of TGFβ mimetics include IDE-1 and IDE-2. In some embodiments, the TFGβ mimetic has one or more off-target effects and / or affects the SOX signaling pathway. In some embodiments, the concentration of the TFGβ mimetic in the culture medium is between 2.5 ng / ml and 100 ng / ml. In some embodiments, the concentration of the TFGβ mimetic in the culture medium is between 30 ng / ml and 70 ng / ml or between 40 ng / ml and 60 ng / ml. In some embodiments, the TGFβ mimetic activates the TGFβ signaling pathway.

[0052] In some embodiments, the culture medium used to differentiate iHPCs into iMGLs is serum-free medium.

[0053] iMGL's maturity In some embodiments, maturing the iMGL involves an incubation period of 1 to 5 days. In some embodiments, the incubation period for maturing the iMGL is 3 days.

[0054] In some embodiments, the maturation step includes incubating iMGL in a culture medium containing either or both CD200 and CX3CL1. In some embodiments, CD200 is human recombinant CD200 and CX3CL1 is human recombinant CX3CL1.

[0055] In some embodiments, the concentrations of CD200 and CX3CL1 in the culture medium are between 1 ng / ml and 1 μg / ml. In some embodiments, the concentrations of CD200 and CX3CL1 in the culture medium are between 80 ng / ml and 120 ng / ml or between 90 ng / ml and 110 ng / ml. In some embodiments, the concentration of CD200 and CX3CL1 is 100 ng / ml.

[0056] Characteristics of the produced iMGL In some embodiments, iMGL produced using the methods described herein yields a pure population of iMGL with a purity between 70% and 100%. In some embodiments, iMGL produced using the methods described herein yields a pure population of iMGL with a purity between 80% and 100%. For example, the population of iMGL may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 96%, 97%, 98%, 99%, 99%, or 100%. In some embodiments, the population of iMGL produced exceeds 96%.

[0057] The purity of iMGL is assessed by utilizing any method known in the art for determining the purity of microglial cells. In some embodiments, the purity level is assessed by the expression and / or co-localization of factors P2RY12 and TREM12. In some embodiments, the purity level is assessed by the expression and / or co-localization of Trem2, Iba1, and / or Pu1.

[0058] iMGL produced by any of the methods described herein will express any factor or any combination of factors expressed by typical microglial cells. In some embodiments, the produced iMGL will express c-kit - / CD45 + In some embodiments, c-kit - / CD45 + iMGLs are detected using flow cytometry, immunofluorescence microscopy, qPCR, RNA-seq, or proteomics. In some embodiments, other cell types are detected using flow cytometry, immunofluorescence microscopy, qPCR, RNA-seq, or proteomics. In some embodiments, the produced iMGLs are a population of two distinct iMGLs: (1) CD45 + / CX3CR1 - and (2) CD45+ / CX3CR1 + Includes. In some embodiments, the iMGL produced is CD43 + CD235a + , or CD41 + In some embodiments, the iMGL produced is CD43 + / CD235a + / CD41 + That is the case.

[0059] Any of the methods for producing iMGL described herein involves the cellular constraint on the microglial lineage of CD43 in the early stages of the differentiation process. + This brings about a differentiation process in iHPC. In some embodiments, c-kit - / CD45 + iMGL is CD43 + iHPCs are detected on day 14 of the incubation period used to differentiate iMGLs. Determination of whether or not a cell is constrained to the iMGL lineage is performed by testing the expression of any factor known to be a marker of cells constrained to the microglial fate. In some embodiments, whether or not a cell is constrained to the iMGL lineage is determined by evaluating the expression of the transcription factor PU.1 and / or the microglia-enriched protein Trem2. In some embodiments, cell markers are detected using flow cytometry, immunofluorescence microscopy, qPCR, RNA-seq, or proteomics.

[0060] In some embodiments, a method is provided for producing iMGL from induced PSCs, comprising the steps of (i) differentiating PSCs into induced hematopoietic primordial cells (iHPCs) and (ii) differentiating iHPCs to produce iMGL. In some embodiments, the method further comprises the step (iii) of maturing the iMGL produced in step (ii). In some embodiments, the PSCs include induced PSCs (iPSCs) or embryonic stem cells (ESCs). In some embodiments, the PSCs are mammalian PSCs from humans or mice, for example.

[0061] In some embodiments, the expression of mRNA and / or proteins of TRIM14, CABLES1, MMP2, SIGLEC 11 and 12, MITF, and / or SLC2A5 is enriched in the produced iMGL. In some embodiments, the expression of mRNA and / or proteins of COMT, EGR2, EGR3, and / or FFAR2 is enriched in the produced iMGL.

[0062] iMGL composition iMGL gene expression In some embodiments, iMGLs expressing specific gene profiles are provided. Any of the iMGLs described herein include a gene expression profile similar to that of microglia cells. In some embodiments, any of the iMGL compositions described herein include the expression of any of the following genes: RUNX1, PU.1, CSF1FR, CX3CR1, TGFBR1, RSG10, GAS6, PROS1, P2RY12, GPR34, C1Q, CR3, CABLES1, BHLHE41, TREM2, ITAM, APOE, SLCO2B1, SLC7A8, PPARD, C9orf72, GRN, LRRK2, TARDBP, and CRYBB1. Any of the iMGLs disclosed herein include the expression of any combination of these genes.

[0063] RUNX1, SPI1, CSF1FR, CX3CR1, TGFBR1, RSG10, GAS6, MERTK, PSEN2, PROS1, P2RY12, P2RY13, GPR34, C1Q, CR3, CABLES1, BH LHE41, TREM2, TYROBP, ITGAM, APOE, SLCO2B1, SLC7A8, PPARD, TMEM119, GPR56, C9orf72, GRN, LRRK2, TARDBP, and CRYBB1.

[0064] In some embodiments, TREM2 and P2RY12 are co-expressed in any of the iMGL compositions described herein. In some embodiments, none of the iMGL compositions described herein express one or more of the genes KLF2, TREM1, MPT, ITGAL, and ADGRE5.

[0065] iMGL's Functional Characteristics and How to Use iMGL Chemokine secretion Any iMGL described herein will stimulate chemokines within microglial cells to secrete a chemokine profile similar to that of microglial cells in response to any stimulus known in the art. In some embodiments, the secreted chemokines are any combination of one or more of TNFα, CCL2, CCL4, and CXCL10, and are secreted in response to stimulation with lipopolysaccharide, IFNγ, or IL-1β.

[0066] In some embodiments, a method is provided for stimulating chemokine secretion from iMGLs. This method comprises (i) treating iMGLs with any factor known in the art to stimulate cytokine secretion in microglial cells, and (ii) causing chemokines to be secreted from the microglial cells. In some embodiments, the factor used to treat iMGLs is lipopolysaccharide, INFγ, or IL-1β. The secreted chemokines may include any chemokines known to be secreted by microglial cells. In some embodiments, the secreted chemokines include any one or more of TNFα, CCL2, CCL4, and CXCL10.

[0067] Migration and calcium transfer Any of the iMGLs described herein migrate in response to ADP treatment, and / or ADP treatment induces calcium translocation. In some embodiments, inhibition of P2ry12 disables ADP-mediated iMGL migration and / or ADP-mediated calcium translocation. In some embodiments, inhibition of P2ry12 occurs via the inhibitor PSB0739.

[0068] In some embodiments, a method for causing iMGL to migrate is provided. This method includes (i) treating iMGL with ADP and (ii) causing iMGL to migrate. In some embodiments, a method for inducing calcium migration is provided. This method includes (i) treating iMGL with ADP and (ii) inducing calcium migration in iMGL.

[0069] phagocytosis using iMGL Any of the iMGLs described herein are capable of phagocytosis. Any of the iMGLs described herein can phagocytose any factor known in the art that can be phagocytosed by microglia. In some embodiments, the factors phagocytosed by the iMGL include one or more of Aβ, fluorescently labeled Aβ, tau, and pHrodo-labeled brain-derived tau oligomers.

[0070] In some embodiments, a method for phagocytosis of iMGL is provided. This method comprises (i) exposing iMGL to one or more of the compounds: Aβ, fluorescently labeled Aβ, tau, and pHrodo-labeled brain-derived tau oligomers, and (ii) phagocytosing the compounds.

[0071] Any of the iMGLs provided herein can phagocytose human synaptosomes (hS). In some embodiments, a method is provided for the iMGL to phagocytose hS. This method comprises (i) exposing the iMGL to hS and (ii) phagocytosing the hS. In some embodiments, the hS is fluorescently labeled.

[0072] The usefulness of iMGL in Alzheimer's disease trials Any of the iMGLs described herein can regulate gene expression in response to different stimuli. In some embodiments, the stimuli include neurons, such as rat hippocampal neurons. In some embodiments, any of the iMGLs described herein can differentially regulate one or more of the genes: CABLES, TRIM4, MITF, MMP2, and SLCA25. In some embodiments, the iMGL upregulates one or more of the genes: TYROPB, CD33, and PICALM.

[0073] In some embodiments, methods for controlling gene expression in iMGLs are provided. One such method comprises (i) co-culturing iMGLs with neurons and (ii) differentially controlling genes in the iMGLs. The neurons co-culturing with iMGLs include any neurons of any species. In some embodiments, the neurons are rat hippocampal neurons.

[0074] Another method comprises (i) exposing iMGL to one or more of the compounds: Aβ, fluorescently labeled Aβ, tau, and pHrodo-labeled brain-derived tau oligomers, and (ii) differentially regulating genes. Differentially regulated genes include any combination of genes that are differentially regulated in microglia in response to Aβ, fluorescently labeled Aβ, tau, and pHrodo-labeled brain-derived tau oligomers. In some embodiments, the differentially regulated genes are upregulated genes that include any combination of one or more of the compounds: CD33, TYROPB, and PICALM.

[0075] How to use iMGL In some embodiments, a method is provided for evaluating gene expression in iMGL in response to neuronal cues. According to some embodiments, the method comprises (i) exposing iMGL to one or more of the factors CX3CL1, CD200, and TGFβ in any combination, and (ii) evaluating one or more of the differentially regulated genes in any combination: P2ry12, EGR1, TGFβ1, ETV5, CX3CR1, APOE, BIN1, CD33, GPR84, COMT, APP, PSEN1, PSEN2, HTT, GRN, FUS, TARDP, VCP, SNCA, C9ORF72, LRRK2, and SOD1.

[0076] In some embodiments, a method is provided for evaluating the engraftment of iMGL into the cortex. According to some embodiments, this method includes (i) implanting iMGL into the cortex and (ii) evaluating the engraftment of iMGL into the cortex. In some embodiments, step (ii) occurs at least two weeks after step (i), for example, at least three weeks after step (i), at least four weeks after step (i), at least five weeks after step (i), at least six weeks after step (i), at least seven weeks after step (i), at least eight weeks after step (i), at least nine weeks after step (i), at least ten weeks after step (i), at least eleven weeks after step (i), at least twelve weeks after step (i), at least thirteen weeks after step (i), at least fourteen weeks after step (i), at least fifteen weeks after step (i), at least sixteen weeks after step (i), at least seventeen weeks after step (i), at least eighteen weeks after step (i), at least nineteen weeks after step (i), or at least twenty weeks after step (i). In some embodiments, step (ii) occurs two months after step (i). In some embodiments, the method further includes implanting iMGL into the cortex of a mouse. In some embodiments, the mouse is an MITRG mouse.

[0077] In some embodiments, methods are provided for evaluating the interaction between AD neuropathy and iMGL. In some embodiments, the method includes (i) implanting iMGL in the hippocampus and (ii) evaluating the interaction of iMGL in the hippocampus. In some embodiments, the method includes evaluating the migration of iMGL to plaques. In some embodiments, the method includes evaluating the phagocytosis of fibrous Aβ by iMGL.

[0078] In some embodiments, a method is provided for testing human microglia in a 3D neural environment, comprising implanting iMGL into a mammalian brain. In some embodiments, the mammalian brain is a mouse brain. In some embodiments, the iMGL is implanted in the hippocampus of the mouse brain. In some embodiments, the mouse is a wild-type mouse. In some embodiments, the mouse is an AD mouse strain. [Examples]

[0079] Some aspects of the embodiments described above are disclosed in further detail in the following embodiments, which are not intended in any way to limit the scope of this disclosure. [Examples]

[0080] Production of human microglia-like (iMGL) cells from induced pluripotent stem cells (iPSCs) A two-step, fully defined protocol was developed to successfully generate microglia-like cells (iMGLs) from iPSCs in just five weeks (Figure 1A). This and other examples of methods and protocols can be used in a similar manner to generate iMGLs from other PSCs, including ESCs. Using this approach, iMGLs were successfully generated from more than 10 independent iPSC lines. Firstly, this replicates the ontogeny of microglia, as iPSCs differentiate into hematopoietic primordial cells (iHPCs), which represent early primordial hematopoiesis derived from the yolk sac, giving rise to microglia during development. This protocol (Figure 1Bi) successfully generated iMGLs from CD43 cells after 10 days. + / CD235a + / CD41 + This brought about the primitive iHPC. CD43 + Cell FACS sorting revealed that this approach produced iHPC with a purity of over 90% (Figure 1Bii).

[0081] Secondly, CD43 + iHPCs were grown in serum-free differentiation medium (laboratory-formulated) containing CSF-1, IL-34, and TGFβ1. By day 14, cells expressed the bone marrow-associated transcription factor PU.1 and the microglia-enriched protein TREM2 (Figure 1A iii), demonstrating early constraint on microglial fate. This protocol resulted in a large number of iMGLs, and following their development, iMGLs were characterized in vitro every 4 days by flow cytometry. Early iMGLs on day 14 showed c-kit- / CD45 + This (Figure 1C) suggests a restriction to the myeloid lineage. Furthermore, the cells were CD45 + / CX3CR1 - (A1) and CD45 + / CX3CR1 + The population was further subdivided into (A2). CD45 expression was consistently monitored in developing iMGLs and compared to monocyte-derived macrophages (MD-Mφ). CD45 expression increased with maturation but never reached macrophage levels (Figure 1D), consistent with mouse development. A small population of iMGLs (approximately 10%) also expressed intermediate CD11b levels by day 14, which also increased with cell maturation but similarly never reached macrophage levels (Figures 1E and 1F).

[0082] By day 38, iMGLs showed high purity, as assessed by co-localization and quantification (>96%) of purine receptors P2RY12 and TREM2 (Figure 1H). One million iPSCs produced 30-40 million iMGLs using this protocol, suggesting that this approach can be easily scaled up for high-content screening. The resulting iMGLs resembled human microglia, but not monocytes or macrophages, according to cytospin / Giemsa staining (Figure 1G) and protein expression (Figure 1I). Similar to the development of mouse microglia in vivo, the in vitro developed iMGLs showed high purity of PU.1, TREM2, and CD11b. int / CD45 low It expresses and resembles fetal microglia. As iMGL matures in vitro, it becomes more branched, similar to in vivo microglia (Figure 1A iv). [Examples]

[0083] iMGL Transcriptome Analysis The transcriptome of iMGL was profiled compared to that of primary human embryonic microglia (fetal MG) and adult microglia (adult MG). CD14 + / CD16 - Monocyte (CD14 M), CD14 + / CD16 + Inflammatory monocytes (CD16 M), bone marrow dendritic cells (hematopoietic DCs), iHPCs, and iPSCs were also examined for comparison with stem cells and other bone marrow molecular signatures. Correlation analysis and principal component analysis (PCA) revealed significant similarities between iMGL and fetal MG and adult MG (fetal MG and adult MG are located within the same circled cluster in Figure 2A; see also Figure 9A). Furthermore, the first principal component PC1 (21.3% variance, arrow in Figure 2A) defined the time series of differentiation from iPSCs to iMGL cells via iHPCs, while PC2 and PC3 defined the dendritic and monocyte trajectories, respectively.

[0084] Bi-clustering analysis using 300 microglia, macrophages, and other immune-related genes adapted from previous studies identified similarities between groups and highlighted common gene clusters. This analysis again demonstrated that iMGL clusters with microglia but differs from other myeloid cells, iHPCs, and iPSCs (Figure 2B). Importantly, iMGL, fetal MG, and adult MG expressed standard microglial genes such as P2RY12, GPR34, C1Q, CABLES1, BHLHE41, TREM2, ITAM PROS1, APOE, SLCO2B1, SLC7A8, PPARD, and CRYBB1 (Figure 2C; Table 1). When compared to monocytes, iMGL expressed the myeloid genes RUNX1, PU.1, and CSF1R (Figure 8A), but did not express the monocyte-specific transcription factors IRF1, KLF4, and NR4A1 (Figure 8B). Differential analyses of iMGL, CD14 M, and CD16 M (Figures 8D and 8E) further highlighted that iMGL primarily expressed microglia genes including CX3CR1, TGFBR1, RGS10, and GAS6 (more than 2-fold change and p<0.001), but did not express monocyte and macrophage genes KLF2, TREM1, MPO, ITGAL, and ADGRE5. At the protein level, like primary cultured microglia, iMGL expressed CD45 hi Compared to MD-Mφ, CD45 lo The cells expressed the microglial surface proteins CX3CR1, TGFBR1, and PROS1 (Figures 10A, 10B, and 10C). Table 2 shows the enriched upper GO pathways in adult MG compared to fetal MG and iMGL. Table 3 shows the enriched GO pathways in fetal MG compared to adult MG and iMGL. Table 4 shows the enriched GO pathways in iMGL compared to fetal MG and adult MG. Collectively, unbiased whole transcriptome analysis strongly established iMGL as a highly similar cell model to primary human microglia that can be used to test the physiology and function of microglia in human health and disease.

[0085] [Table 1]

[0086] [Table 2]

[0087] [Table 3]

[0088] [Table 4] [Examples]

[0089] iMGL Functional Analysis iMGLs were validated as a microglia substitute using both functional and physiological assays. Cytokine / chemokine secretion by iMGLs stimulated by lipopolysaccharide (LPS) and by IL-1β and IFNγ (two cytokines elevated in AD patients and mouse models) was measured. The results showed that iMGLs secreted 10 tested cytokines at low but detectable levels (Table 5). However, in response to IFNγ or IL-1β, iMGLs secreted eight different chemokines, including TNFα, CCL2, CCL4, and CXCL10. As expected, iMGLs responded robustly to LPS by induction of all measured cytokines except CCL3 (see Table 5 for values). In summary, these data indicate that iMGL differentially releases cytokines / chemokines based on cell surface receptor stimulation, a finding closely consistent with the response observed in acutely isolated primary cultured microglia (Rustenhoven et al., 2016).

[0090] iMGLs can sense extracellular nucleotides leaked from degenerated neurons and express the microglial enriched purine receptor P2ry12, which has been shown to be important for microglial homeostasis (Figures 1H and 2C). Therefore, ADP-P2ry12-mediated chemotaxis and calcium translocation were evaluated. It was determined that iMGLs migrate robustly in response to ADP, and that ADP induces calcium translocation (Figures 3D and 3E), which can be neutralized by the P2ry12-specific inhibitor PSB0739. These physiological findings further clarify that iMGLs express a functional surface receptor, enabling quantitative analysis of microglial physiology.

[0091] Microglia, along with astrocytes, play a crucial role in synaptic pruning. Since in vitro synaptosome phagocytosis assays are an established alternative for testing pruning, we quantitatively evaluated the ability of iMGLs to phagocytose human synaptosomes (hS). Compared to MD-Mφ, phagocytosis of pHrodo-labeled hS by iMGLs was less robust (Figure 3F 3G). However, iMGLs preferentially internalized hS compared to E. coli particles, and when standardized against MD-Mφ (Figures 10D and 10E), this supports the idea that iMGLs and microglia are more biased towards homeostatic functions than MD-Mφ.

[0092] Since microglia (and iMGLs) express both C1q and CR3 (CD11b / CD18 dimer), iMGLs were used to evaluate whether synaptic pruning in human microglia is primarily involved in this pathway. Using CD11b antibody without additives significantly reduced iMGL-mediated phagocytosis of hS (-40.0%, p<0.0001) (Figures 3H and 3I). In contrast, an inhibitor of MERTK (UNC569), which is also involved in synaptic pruning, only slightly reduced iMGL-mediated hS phagocytosis (-12.6%, p<0.05) (Figures 3H and 3I). Similar to the mouse knockout study, the data indicate that MERTK plays a small role in human microglia-mediated synaptic pruning and demonstrate that C1q / CR3 is essential for human microglia-mediated synaptic pruning.

[0093] [Table 5] [Examples]

[0094] To validate the usefulness of using iMGL to test for Alzheimer's disease. Previous reports have shown that impaired microglial clearance of beta-amyloid (Aβ) is involved in the pathophysiology of Alzheimer's disease (AD). Therefore, we examined iMGLs to determine whether they could phagocytose Aβ or tau, two characteristic AD pathologies. Similar to primary cultured microglia, iMGLs internalized fluorescently labeled fibrous Aβ (Figure 4B, bottom). iMGLs also recognized and internalized pHrodo-labeled brain-derived tau oligomers (BDTO) (Figure 4B, top). The emitted fluorescence indicated the transport of pHrodo-conjugated BDTO into the acidic lysosomal compartment, demonstrating that iMGLs can actively ingest extracellular tau that may be released during neuronal cell death. These data support recent findings that microglia may play a role in tau propagation in AD and other taupathies. Taken together, these findings suggest that iMGLs can be utilized in high-throughput drug screening assays to identify compounds that enhance Aβ degradation or block exosome-mediated tau release.

[0095] Microglia genes are involved in late-onset Alzheimer's disease (AD), but little is known about how they modify disease risk. Therefore, we investigated iMGLs to determine how these genes may influence microglial function and AD risk. Hierarchical clustering using only these 25 AD-GWAS genes demonstrated that iMGLs are similar to microglia and not to peripheral bone marrow cells (Figure 4A). In the basal state investigated, both iMGLs and microglia expressed many AD-GWAS-related genes that lack mouse orthologues, namely CD33, MS4A4A, and CR1. Therefore, iMGLs can be used to test how changes in the expression of these genes affect the microglial phenotype in ways that cannot be replicated in transgenic mice. Next, we investigated fAβ or BDTO treatment to determine how it affects AD-GWAS gene expression in microglia. Following fAβ exposure, iMGL increased the expression of 10 genes (Table 6), including ABCA7 (5.79±0.44), CD33 (6.02±0.41), TREM2 (4.86±0.50), and APOE (2.52±0.19), which are involved in Aβ clearance / degradation. BDTO increased the expression of 4 genes, including CD2AP (4.62±0.45), which has been previously associated with tau-mediated toxicity. Furthermore, compared to BDTO, six genes in fAβ showed differential increases (Table 6). Furthermore, the genes CD33, TYROBP, and PICALM, which were more enriched in other myeloid cells at baseline, were upregulated by fAβ and BDTO, suggesting that proteinopathy may alter the microglial phenotype to resemble invasive peripheral myeloid cells (Stalder et al., 2005, Prinz et al., 2011, Chan et al., 2007). In addition to the AD-GWAS genes, iMGL expresses C9orf72, GRN, LRRK2, and TARDBP, and can be used to test other neurological diseases such as ALS, FTD, and DLB in which microglia play a significant role in pathogenesis (Figure 11).

[0096] [Table 6] [Examples]

[0097] iMGL interaction and function in the neural environment In the brain, neurons and glia interact with microglia, influencing function and gene expression. Therefore, iMGLs were cultured with rat hippocampal neurons (21 div) to evaluate how iMGLs respond to neuronal cues (Figure 5A). Rat hippocampal neurons were used because they readily form synapses in culture and can be generated with limited variability. iMGLs were then isolated from neurons by FACs with human-specific CD45 and CD11b antibodies and profiled at the transcriptome level (Figure 5B). Differential gene expression analysis revealed that co-culturing neurons upregulated 156 iMGL genes and downregulated 244 iMGL genes (Figures 5C and 5D). FFAR2 and COL26A1 are two genes differentially expressed in iMGLs cultured with only defined factors, indicating a developmentally primed microglial profile. In contrast, co-culturing microglia with neurons increased the expression of Siglec11 and 12, human-specific sialic acid-binding proteins that interact with the neuronal glycocalyx. Furthermore, increased expression of microglial genes CABLES1, TRIM14, MITF, MMP2, and SLCA25 is associated with both neuronal surface cues and soluble factors in microglial maturation (Figures 5E and 12).

[0098] A fundamental characteristic of microglia is their surveillance of the CNS environment through highly branched processes. To investigate how iMGLs may interact in the CNS environment, iMGLs were cultured with human iPSC (hiPSC) 3D brain organoids (BORGs). BORGs contain neurons and astrocytes that self-organize into cortical-like networks, but lack microglia (Figure 6, Panel B). To test whether iMGLs invade BORGs in a similar way to how microglia enter the developing neural tube, iMGLs were added to the BORG culture. By day 3, iMGLs were embedded in the BORGs and no longer detectable in the culture medium, suggesting rapid chemotaxis of iMGLs to neuronal queues (Figure 6, Panel A). iMGLs also tiled and extended varying degrees of branching processes in the 3D organoid environment (Figure 6, Panel B). iMGL protrusions were observed in the majority of cells and exhibited morphology similar to in vivo microglia (Figure 6, Panel B). IMARIS 3D image reconstruction of some iMGLs highlights the development of branched iMGLs in BORG. To determine whether iMGLs respond to neuronal injury, 25-gauge needles were inserted into BORG (long white arrows, Figure 6, panel C). After injury, iMGLs clustered near the injury site and at the edges of BORG (Figure 6, panel C), adopting a more amoeboid morphology, similar to "activated" microglia found in injured or diseased brains (Figure 6, panel C). Taken together, these data indicate that iMGLs can integrate into the 3D brain environment, mature, branch, and respond to injury, similar to brain microglia. [Examples]

[0099] Interaction of iMGL with neurons, astrocytes, and endothelial cells in the brain Neurons, astrocytes, and endothelial cells in the brain interact with microglia to influence gene expression and function. The differentiation protocol attempted to replicate the CNS cues present in the brain by including signals derived from these other cell types, including CX3CL1, CD200, and TGFβ. Whole transcriptome RNA-seq analysis confirmed the importance of these factors for establishing microglia in vitro (Figures 16A-E and 17A-E). TGFβ, a glial cytokine, is required for in vivo development of mouse microglia and the maintenance of microglia-specific transcriptome signatures. Differential gene expression analysis confirmed the role of TGFβ in maintaining human microglia transcriptome signatures; 1262 genes were differentially expressed in iMGLs by TGFβ, while 1517 genes were differentially expressed in iMGLs after TGFβ removal (24 hours). Many of the differentially expressed genes have been identified as core microglial signature targets, including P2RY12, TGFβR1, and CD33, transcription factors EGR1 and ETV5, and APOE (Figure 16A-C). Examination of the gene ontology highlighted neurodegenerative disease pathways, including TGFβ-dependent AD, Parkinson's disease, and Huntington's disease (Figure 16D). Furthermore, removal of TGFβ resulted in significant changes in many human microglial homeostasis targets, including TREM2, APOE, ABCA7, SPI1 (CELF1 locus), PILRA (ZCWPW1 locus), and AD GWAS locus genes (Karch et al., 2016), including the HLA-DR and MS4A gene clusters. This suggests that many identified AD GWAS genes function in maintaining microglial homeostasis (Figure 16E), clearly demonstrating the usefulness of iMGL for investigating AD GWAS gene function. [Examples]

[0100] Effects of CX3CL1 and CD200 on iMGL phenotypes CX3CL1 and CD200 are neuronal and endothelial-derived cues that can further train iMGL toward an endogenous microglia phenotype. We tested CX3CL1 and CD200 to determine how the inclusion or exclusion of these factors modulates the iMGL phenotype. Addition of CD200 and CX3CL1 to iMGL increased the expression of selected genes, including COMT (Figure 16B), CD52 (a cell surface receptor that binds to Siglec-10 and interacts with DAP12 as part of microglial cenosomes), and HLADRB5 (a member of the MHC II complex involved in AD), while maintaining similar expression levels of core microglia genes (e.g., P2RY12, TYROBP, OLFML3) and AD risk genes (Figure 17A). The results indicated that CD200 and CX3CL1 modulate the iMGL response to CNS stimuli such as fAβ. In the absence of CD200 and CX3CL1, fAβ stimulated the expression of AD-GWAS genes involved in interactions with misfolded or folded proteins, surface receptors, or anti-apoptotic events such as CLU(APOJ). Cells exposed to these two factors responded differentially to fAβ but increased the expression of genes involved in cell surface recognition of neuronal motifs or phagocytosis of CNS substrates, including MS4A, TREM2, TYROBP, CD33, and ABCA7 (Figure 178B). These tests further support the concept that the CD200-CD200R1 and / or CX3CL1-CX3CR1 axis can modulate microglia to respond to neurodegenerative states. Therefore, exposure to soluble CNS factors such as CD200 and CX3CL1 may enable access to microglia-specific transcriptional regulatory elements. [Examples]

[0101] This study investigates the effect of direct contact between iMGL and the CNS environment on iMGL maturation. Next, we investigated whether iMGL maturation could be achieved by direct contact with the CNS environment. iMGLs were cultured with rat hippocampal neurons (21 DIV) to evaluate how iMGLs responded to cues on the neuronal surface (Figure 5A). Rat hippocampal neurons were used because they readily form synapses in culture and can be generated with limited variability. Subsequently, iMGLs were isolated from neurons by FACs with human-specific CD45 and CD11b antibodies and profiled at the transcriptome level (Figure 5B). Differential gene expression analysis revealed that co-culturing neurons upregulated 156 iMGL genes and downregulated 244 iMGL genes (Figures 5C and D). FFAR2 and COL26A1 are two genes that are differentially expressed in iMGL cultured with only defined factors, indicating a developmentally primed microglia profile. In contrast, co-culturing microglia with neurons increased the expression of Siglec11 and 12, human-specific sialic acid-binding proteins that interact with neuronal glycocalyxes, function in neuroprotection, suppress pro-inflammatory signaling, and thereby maintain microglial homeostasis. Furthermore, increased expression of microglial genes CABLES1, TRIM14, MITF, MMP2, and SLC2A5 was observed. Overall, these results suggest both soluble and surface CNS cues as factors in microglial maturation (Figure 5A-F). [Examples]

[0102] iMGL interaction in the human brain environment A fundamental characteristic of microglia is their surveillance of the CNS environment through highly branched processes. To investigate how iMGLs may interact in the human brain environment, iMGLs were cultured with hiPSC 3D brain organoids (BORGs). BORGs contain neurons, astrocytes, and oligodendrocytes that self-organize into cortical-like networks, but lack microglia (Figure 6). To test whether iMGLs invade BORGs in a similar way to how microglia enter the developing neural tube, iMGLs were added to the BORG culture. By day 3, iMGLs were embedded in the BORGs and no longer detectable in the culture medium, suggesting rapid chemotaxis of iMGLs to CNS cues (Figure 6, panels A-C). Also, by day 7, iMGLs tiled and extended varying degrees of branching processes in the 3D organoid environment (Figure 6, panels D-F). To determine whether iMGLs respond to neuronal injury, BORGs were punctured with a 25-gauge needle. After injury, iMGLs cluster near the injury site, adopt a more amoeboid morphology, and resemble "activated" microglia found in injured or diseased brains (Figure 6, panels G-I). In summary, these data indicate that iMGLs are integrated into the in vitro 3D brain and CNS environment, where they can mature, branch, and respond to injury in a similar manner to brain microglia. [Examples]

[0103] Investigation of iMGL in the context of the in vivo CNS environment iMGL was tested in a vivo CNS environment. iMGL (day 38) consisted of Rag2-deficient and IL2rγ-deficient mice, and four knock-in cytokines (M-CSF) were present. h ;IL-3 / GM-CSF h TPO hHumanized forms of ) were also expressed and transplanted into the cortex of MITRG mice, enabling xenotransplantation and survival of bone marrow and other leukocytes (Figure 13). Two months after transplantation, the degree of engraftment of MITRG cortical microglia was evaluated by immunohistochemistry. Human iMGLs were distinguished from endogenous microglia using human-specific nuclear or cytoplasmic markers, ku80 (hNuclei) and SC121 (hCyto), respectively. Homeostasis and identity of transplanted microglia were assessed using P2ry12 and human-specific Tmem119 antibodies. Transplanted human iMGLs co-expressing both ku80 and P2ry12 were abundant in the MITRG brain, suggesting potential for long-term engraftment (Figure 13 panels A-D). Higher magnification images showed P2ry12 expression in highly branched iMGLs, similar to resting cortical microglia, with a membrane distribution that emphasized finer elongation processes (Figure 13 panels B-D). Tmem119 also has hCyto + It was expressed in both the cell body and the highly dendritic iMGL protrusions (Figure 13, panels E-H). hCyto + High-magnification images of the cells show that Tmem119 is primarily bound to the membrane, consistent with published studies. In summary, these findings suggest that long-term survival and engraftment of iMGLs lead to the development of highly branched microglia-like cells that express Iba1, P2ry12, and Tmem119 (Figure 13 panels I-L) and resemble endogenous quiescent microglia. Furthermore, the morphology and high expression of the homeostatic P2ry12 receptor suggest that transplanted iMGLs actively monitor the neuronal environment, which translates to potential use in testing human microglia in mouse CNS disease models. [Examples]

[0104] We will implant iMGLs into the hippocampus and determine how iMGLs interact with AD neuropathology. iMGLs were transplanted into the hippocampus of previously generated and characterized xenograft-compatible AD mice to examine how iMGLs interact with AD neuropathology in vivo (Figure 13 panels M-P and Figure 18). Transplanted iMGLs engrafted and migrated along the white matter tract, similar to developing microglia (Figure 13 panel M). In many cases, iMGLs migrated toward Aβ plaques, extended their processes, and began to surround them with walls (Figure 13 panels N-P). Numerous iMGLs also began to phagocytose fibrous Aβ (Figure 13 panels N-P, Figure 18 panels E-H). Similarly, human fetal microglia, when transplanted into the same AD transgenic model, migrated toward Aβ, extended their processes, and phagocytosed Aβ (Figure 18 panels A-D).

[0105] Experimental methods and materials reagent Unless otherwise stated, all cell culture flasks, reagents, supplements, cytokines, and general reagents were purchased from ThermoFisher (Carlsbad, CA).

[0106] Maintenance and culture of human pluripotent stem cells (hPSCs) All stem cell studies were conducted with the approval of the UC Irvine Human Stem Cell Research Monitoring Organization (hSCRO) and the IBC Committee. The use of human tissue was subject to the compliance and approval of the Institutional Review Board (IRB). Human iPSC cell lines ADRC F5 and ADRC F14 (control subjects) were generated from UCI ADRC-induced pluripotent stem cell cores using non-integrated Sendai virus (Cytotune). iPSCs were confirmed to have normal karyotype by G-banding, be sterile, and be pluripotent by Pluritest (UCLA) analysis. iPSCs were maintained without feeders on Matrigel (MTG) in complete TeSR-E8 medium (Stemcell Technologies) in a humidified incubator (5% CO2, 37°C).

[0107] Differentiation of iPSCs into hematopoietic primordial cells (iHPCs) Human iPSC-derived hematopoietic progenitor cells were generated using defined conditions with several modifications to previously published protocols (Kennedy et al., 2007, Sturgeon et al., 2014). Briefly, iPSCs were pulverized to produce single-cell suspensions, which were then incubated in E8 medium + Y-27632 ROCK inhibitor (10 μM; R&D Systems) at a rate of 1–6 × 10⁶ cells per well. 5 Cells were seeded in a 6-well plate. In some embodiments, Y-27632 is replaced with thiazovibin (R&D systems). After culturing cells under normal oxygen (20% O2) conditions for 24 hours, the E8 medium was replaced with a differentiation medium consisting of a base medium and cytokines: IMDM / F12 (50:50), insulin (0.02 mg / ml), holotransferrin (0.011 mg / ml), sodium selenite (0.0134 mg / ml), magnesium L-ascorbic acid 2-phosphate (64 μg / ml; Sigma), monothioglycerol (400 μM), PVA (5 mg / ml; Sigma), L-alanyl-L-glutamine (2 mM), lipid concentrate of known composition (1x), non-essential amino acids (NEAA; 1x), FGF2 (50 ng / ml), BMP4 (50 ng / ml), activin-A (12.5 ng / ml), and LiCl (2 mM) under hypoxic conditions (5% O2). On day 2, the culture medium was changed to a base medium supplemented with FGF2 (50 ng / ml) and VEGF (50 ng / ml). On day 4, the culture medium was changed to a medium containing FGF2 (50 ng / ml), VEGF (50 ng / ml), TPO (50 ng / ml), SCF (50 ng / ml), IL-6 (50 ng / ml), and IL-3 (50 ng / ml). On day 6, the culture medium was supplemented with the above-mentioned medium. After culturing the cells for a further 4 days (total 10 days), CD43 was identified by FACS for iMGL differentiation. + Cells were isolated. Furthermore, iPSC-derived HPC (Cellular Dynamics) was found to be CD43 + It was identified as a commercial source of progenitor cells.

[0108] Generation of microglia-like cells from iHPC CD43 +iHPC, 1-2 x 10 per well 5 Cells were seeded at cell density in Matrigel-coated 6-well plates (BD Biosciences) containing serum-free complete differentiation medium. The differentiation medium consisted of M-CSF (25 ng / ml), IL-34 (100 ng / ml; Peprotech), and TGFβ-1 (50 ng / ml; Militenyi) added to a base medium (phenol-free DMEM / F12 (1:1), insulin (0.2 mg / ml), holotransferrin (0.011 mg / ml), sodium selenite (0.0134 mg / ml), penicillin / streptomycin (1% v / v), B27 (1% v / v), N2 (0.5%, v / v), monothioglycerol (200 μM), and additional insulin (4 μg / ml) added immediately before addition to cells). Complete differentiation medium was replenished to the cells every two days. On day 12, early iMGLs were collected (300×g for 5 minutes at 25°C), and 50% of the culture medium was changed. After 25 days of microglial differentiation (35 days from iPSC), the iMGLs were cultured for a further 3 days in fully differentiated medium supplemented with CD200 (100 ng / ml, Novoprotein) and CX3CL1 (100 ng / ml; Peprotech), and cultured with hippocampal neurons or with human brain organoids.

[0109] Isolation of PBMCs from human blood Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors using Ficoll-paque (GE Healthcare) gradient separation. Briefly, blood was placed on top of a Ficoll-Paque and centrifuged in a swing bucket rotator without braking (400×g, 40 minutes, 18°C). After centrifugation, the plasma and upper layer were removed, and the PBMCs were isolated from the interlayer. The cells were then washed once with ice-cold PBS and used immediately.

[0110] Isolation of monocytes from PBMCs CD14 and CD16 monocytes were isolated from PBMCs by negative selection using the EasySep® Monocyte Concentration Kit (Stemcell Technologies) according to the manufacturer's instructions. Isolated cells were washed three times with PBS and sorted by FACs for RNA sequencing analysis or used for further macrophage differentiation.

[0111] Monocyte-derived macrophages Isolated monocytes were incubated at 37°C in a 5% CO2 incubator in RPMI-1640 medium, 2 × 10⁶ cells per minute. 6 Cells were seeded at a concentration of cells / ml into 6 wells for tissue culture. After 2 hours, the medium was aspirated and discarded, and the adhered monocytes were washed three times with DPBS and replaced with complete medium consisting of RPMI-1640, FBS (10% v / v), penicillin / streptomycin (1% v / v), and L-alanyl-L-glutamine (2 mM). To generate MD-Mφ, M-CSF (25 ng / ml) was added to the wells, and the cells were differentiated for 5 days.

[0112] RNA-seq library construction Cells were collected, washed three times with DPBS, and stored in RNAlater and RNA preservation solution. RNA was extracted from all cell types using the RNeasy Mini Kit (Qiagen) according to the manufacturer's guidelines. RNA integrity (RIN) was measured for all samples using the Bioanalzyer Agilent 2100 series. All analyzed sequencing libraries were generated from RNA samples with a measured RIN score of 9 or higher. Poly(A) mRNA was obtained from all samples using the Illumina TruSeq mRNA stranded protocol. RNA-seq libraries were constructed using 200 ng of isolated mRNA. The libraries were quantified and standardized using the Kapa Biosystems Library Quantification Kit and sequenced as paired-end 100 bp reads on the Illumina HiSeq 2500 platform.

[0113] RNA-seq analysis RNA-seq reads were mapped to the hg38 reference genome using a STAR aligner and then mapped to Gencode version 24 gene annotations using RSEM. Genes with expression (<1 FPKM) across all samples were filtered out of all subsequent analyses. Differential gene expression analysis was performed against standardized TMM counts using EdgeR (Robinson et al., 2010). Multiple biological copies were used for all comparative analyses. Genes that were significantly differentially expressed for each comparison were determined using p-values ​​≤ 0.001 and a twofold change in expression. PCA analysis was performed using the R package rgl and plotted using plot3d. Clustering was performed using R hclust2 and visualized using Java Tree View 3.0.

[0114] ADP migration and calcium imaging assay The Transwell migration assay to ADP was performed as previously described (De Simone et al., 2010; Moore et al., 2015). iMGL (5.5 × 10⁶) 4Cells (per well) were cultured for 1 hour in serum-free basal medium without cytokines. Next, iMGL was pre-exposed to DMSO or PSB0739 (50 μM, Tocris) for 1 hour in a 5% CO2 cell culture incubator at 37°C. Then, the cells were washed three times with basal medium and seeded in a transwell migration chamber (24 wells with 5 μm polycarbonate inserts, Corning) containing adenosine 5'-phosphate (ADP, 100 μM, Sigma) in the bottom chamber at 37°C in 5% CO2. After 4 hours, the cells were washed three times and fixed in PFA (4%) at room temperature for 15 minutes. Cells were stained with Hoechst stain for 10 minutes to visualize the cell nuclei. Blinded observers counted the total number of cells per slide, then scraped the cells off the surface, washed with PBS, and recounted to record the migrating cells. Migration was reported as the number of migrating cells relative to the total number of cells per well. Cell fluorescence images were recorded using an Olympus IX71 inverted microscope.

[0115] For calcium imaging, iMGL was seeded onto poly-L-lysine coated coverslips and incubated for 1 hour with Fura-2-AM (Molecular Probes) calcium dye diluted in Ringer's solution pH=7.4 containing (mM) NaCl 140, KCl 4.5, CaCl22, MgCl21, HEPES 10, glucose 10, sucrose 5. After 1 hour incubation, the dye was washed three times with Ringer's solution and treated for 1 hour with the P2RY12 inhibitor PSB0739 (50 μM, Tocris) or a medium (DMSO) before use in the experiment. Baseline Ca 2+ Signal (I 340 / I 380 The measurement was taken for over 100 seconds, and after baseline measurement, ADP (10 μM) was introduced under steady flow. 2+Recording was performed using a Zeiss (Axiovert 35)-based imaging setup, and data acquisition was performed using Metafluor software (Molecular Devices). Data analysis was performed using Metafluor, Origin Pro, and Prism 6.0.

[0116] Immunocytochemistry and immunohistochemistry The cells were washed with cold PBS, fixed with cold PFA (4%) at 25°C for 20 minutes, and then washed three times with PBS. The cells were blocked at 25°C for 1 hour with PBS containing either 0.05% goat serum or donkey serum and Triton X100 (0.01%). Primary antibody (1:500) was added to the blocking solution overnight at 4°C. The cells were then washed three times with PBS and stained with 1:400 AlexaFluor® conjugate secondary antibody at 25°C for 1 hour. After secondary staining, the cells were washed three times and subsequently covered with DAPI-counterstain mounting media (Fluoromount, southern Biotech). Primary antibodies used in immunocytochemical analysis include: β-3 tubulin (Biolegend), GFAP (Abcam), Iba1 (Wako), ITGB5 (Abcam), MMP-9 (Novus), MerTK (Biolegend), P2RY12 (Sigma), PROS1 (Abcam), PU.1 (Cell Signaling Technology), hCytoplasm (SC121; Takara Bio Inc.), TREM2 (R&D Systems), and TGFβR1 (Abcam).

[0117] For ICC, cells were washed three times with DPBS (1x), fixed with cold PFA (4% w / v) for 20 minutes at room temperature, and then washed three times with PBS (1x). Cells were blocked at room temperature for 1 hour with blocking solution (1x PBS, 5% goat or donkey serum, 0.2% Triton X-100). ICC primary antibody was added to the blocking solution at each dilution (see below) and allowed to stand overnight at 4°C. The following day, cells were washed three times with PBS for 5 minutes and stained with 1:400 AlexaFluor® conjugate secondary antibody in the dark at room temperature for 1 hour.

[0118] After secondary staining, cells were washed three times with PBS and coversliped on DAPI-counter-stain mounting media (Fluoromount, Southern Biotech). For BORG IHC, tissue was collected, fixed dropwise in PFA (4% w / v) at room temperature for 30 minutes, and washed three times with PBS. BORG was then incubated overnight in sucrose solution (30% w / v) before being embedded in OCT (Tissue-Tek). The embedded tissue was sectioned to 20 μm using a cryostat, and the mounted slides were stored at -20°C until staining. For BORG staining, the mounted tissue was removed from storage and allowed to stand at room temperature for 30 minutes before warming. The tissue was rehydrated and washed three times with PBS (1x) at room temperature for 5 minutes each.

[0119] Heat-mediated antigen recovery was performed at 97°C for 20 minutes using citrate buffer (10 mM citrate, 0.05% Tween 20, pH=6.0), followed by cooling to room temperature. After antigen recovery, the slides were washed three times with PBS. The slides were then washed once for 15 minutes with PBS-A solution (1×PBS containing 0.1% Triton X-100). The tissue was blocked at room temperature for 1 hour with PBS-B solution (PBS-A, 0.2% BSA, and 1.5% goat or donkey serum). After blocking, the primary antibody was added to PBS-B solution (250-350 μl / slide) at an appropriate dilution (see below) and incubated overnight at room temperature. The following day, the slides were washed three times with PBS-A solution for 5 minutes each. The tissue was blocked at room temperature for 1 hour with PBS-B solution. After blocking, the slides were incubated in PBS-B (250-300 μl / slide) with AlexaFluor® conjugate secondary antibody (all 1:500) and Hoechst stain (1x) at room temperature in the dark for 2 hours.

[0120] After secondary staining, the slides were washed five times with PBS for 5 minutes each. The slides were coverslipped using fluoromounts (Southern Biotech). For mouse brain IHC, the brains were collected, fixed, and processed as described above. Suspended sections were blocked in a blocking solution (1×PBS, 0.2% Triton X-100, and 10% goat serum) at room temperature for 1 hour with gentle shaking. For human TMEM119 staining, heat-mediated antigen recovery was performed before blocking, as previously done (Bennett et al., 2016). Recovery of suspended tissue antigens was performed by placing the suspended sections in a 1.5 ml microcentrifuge tube containing 1 ml of citrate buffer and placing it on a preheated temperature block set to 100°C. After heating the tissue at 100°C for 10 minutes, it was removed and allowed to return to room temperature for 20 minutes, then washed three times with PBS for 5 minutes each, and then proceeded to the blocking step. For amyloid plaque staining of AD mouse brain, suspension sections were left standing in 1×Amylo-Glo®RTD®(Biosensis) staining solution at room temperature for 10 minutes without shaking.

[0121] After staining, each section was washed three times with PBS for 5 minutes, briefly rinsed with MiliQ DI water, returned to PBS, and blocked. The primary antibody was added to the staining solution (1× PBS, 0.2% Triton X-100, 1% goat serum) at the appropriate dilution (see below) and incubated overnight at 4°C with gentle shaking. The following day, the sections were washed three times with PBS and stained with 1:400 AlexaFluor® conjugate secondary antibody in the dark at room temperature for 1 hour with gentle shaking. After secondary staining, the sections were washed three times with PBS for 5 minutes and mounted on glass slides. After mounting, the slides were coverslipped using DAPI counterstain mounting media (Fluoromount, southern Biotech). Primary antibody: Rabbit anti-amyloid fibrils (OC) (1:1,000, EMD Millipore, AB2286) Rabbit anti-amyloid oligomer (A11) (1:1,000, EMD Millipore, AB9234) Mouse anti-amyloid 1-16aa(6e10)(1:1,000, Biolegend, 803001) Mouse anti-β3 tubulin (1:500; Biolegend, 801201) Mouse anti-human cytoplasm (SC121, 1:100; Takara Bio Inc., Y40410), Mouse anti-human nucleus (ku80, 1:100; Abcam, ab79220) Chicken anti-GFAP (1:500; Abcam, ab4674) Rabbit anti-Iba1 (1:500; Wako; 019-19741) Goat anti-Iba1 (1:100; Abcam ab5076) is recommended for use only with Alexa Fluor 488 or 555 secondary antibodies. Mouse anti-ITGB5 (1:500; Abcam, ab177004) Mouse anti-MMP-9 (1:500; EMD Millipore, AB19016) Mouse anti-human Mertk (1:500; Biolegend, 367602) Rabbit anti-P2ry12 (1:125; Sigma; HPA014518) Rabbit antipros(1:500; Abcam, ab97387) Rabbit-resistant PU.1 (1:500; Cell Signaling Technology, 2266S) Rabbit anti-human Tmem119 (1:100; Abcam, ab185333) Goat anti-human Trem2 (1:100; R&D Systems, AF1828) Rabbit anti-Tgfbr1 (1:500; Abcam, ab31013).

[0122] Confocal microscopy and bright-field imaging Immunofluorescence sections were visualized and images recorded using an Olympus FX1200 confocal microscope. To avoid nonspecific bleed-through, each laser line was excited and detected independently. All images shown represent a single confocal z-slice or z-stack. Bright-field images of cell cultures were recorded using an Evos XL Cell Imaging microscope.

[0123] Flow cytometry analysis Cells were suspended in FACs buffer (DPBS, 2% BSA, and 0.05 mM EDTA) and incubated in human Fc blocks (BD Bioscience) at 4°C for 15 minutes. For the detection of microglial surface markers, cells were stained with anti-CD11b-FITC clone ICRF44, anti-CD45-APC / Cy7 clone HI30, anti-CX3CR1-APC clone 2A9-1, anti-CD115-PE clone 9-4D, and anti-CD117-PerCP-Cy5.5 clone 104D2. Live / dead cells were all gated using ZombieViolet® live / dead stain from Biolegend (San Diego, CA). Cells were run in FACs Aria II (BD Biosciences) and analyzed with FlowJo software (FlowJo).

[0124] Sitespin and Meigrunwald Giemsa staining 1 x 10 5 Cells were suspended in 100 μl of FACs buffer and added to Shandon glass slides (Biomedical Polymers), then assembled in a cytology funnel apparatus. The assembled slides containing the cells were loaded into a cytospin instrument and centrifuged (500 rpm, 5 min). The slides were air-dried for 2 minutes and immediately stained with 100% Maygrünwald stain (Sigma) for 5 minutes. Next, the slides were washed with PBS for 1.5 minutes and immediately immersed in 4% Giemsa stain (Sigma) at room temperature for 20 minutes. The slides were washed six times with double-distilled H2O and air-dried for 10 minutes. The slides were stored using glass coverslips and permounts (Sigma).

[0125] RNA isolation and qPCR analysis Cells were stored in RNAlater stabilization reagent, and RNA was isolated using the Qiagen RNeasy Mini Kit (Valencia, CA) according to the manufacturer's guidelines. qPCR analysis was performed using the ViiA®7 Real-Time PCR System and Taqman qPCR primers. For AD-GWAS gene analysis, a custom Taqman low-density array card was used with the primers described below.

[0126] Isolation of rat cortical and hippocampal neurons All procedures were performed under IUCAC-approved protocols. Primary cultures of cortical and hippocampal neurons were derived from embryonic rats (E18). Briefly, dissected tissues were dissociated with trypsin, pulverized, and seeded in 6-well plates coated with poly-L-lysine in serum-free Neurobasal (NB medium) supplemented with B27 (1% v / v). Cells were placed in 5 × 10⁶ wells. 6 The cells were seeded at a density of cells / ml and maintained in culture until use.

[0127] Co-culture of iMGL with rat neurons Rat hippocampal or cortical neurons were cultured for 21 days with 50% medium change every 3-4 days. iMGL was added to 50% iMGL and 50% NB medium in a 1:5 ratio to the neurons (1 × 10⁶). 6 iMGL vs 5x10 6 They were cultured together with neurons. After 3 days, iMGLs were collected for RNA isolation.

[0128] Mesoscale multiplex cytokine and chemokine assays Prior to 24-hour stimulation with IFNγ (20 ng / ml), IL1β (20 ng / ml), and LPS (100 ng / ml), the iMGL culture medium was replaced with basal medium for 2 hours, after which cells were collected for RNA, and the culture supernatant was evaluated for cytokine secretion. To simultaneously evaluate multiple cytokine and chemokine analytes from the iMGL culture supernatant, the culture supernatant from each treatment group was processed and analyzed using the V-PLEX Human Cytokine 30 Plex Kit (Mesoscale) according to the manufacturer's protocol.

[0129] 3D brain organoid cell culture Human 3D brain organoids were generated with several modifications as previously described (Lancaster et al., 2013), the modifications of which are detailed in the supplementary information.

[0130] Preparation of fibrous Aβ. Fibrous fluorescent amyloid beta (fAβ) 1-42 The following was produced: In short, the fluorescently labeled Aβ peptide (Anaspec; Fremont, CA) was first dissolved in 0.1% NH4OH to a concentration of 1 mg / ml, then further diluted with endotoxin-free sterile water and incubated at 37°C for 7 days. The fAβ was thoroughly mixed before cell exposure.

[0131] Preparation of BDTO Tau oligomers were isolated by immunoprecipitation with T22 antibody using a PBS-soluble fraction of homogenate prepared from AD brain. These were then purified by high-performance protein liquid chromatography (FPLC) using PBS (pH 7.4). Additional analyses included Western blotting to detect the presence of monomeric tau or large tau aggregates (tau-5, typically appearing at the top of the stacking gel), and identification of nonspecific bands using mouse anti-IgG. Subsequently, BDTO was conjugated with pHrodo-Red according to the manufacturer's protocol.

[0132] Human synaptosomes Human tissue samples were obtained by autopsy, chopped, slowly frozen in 0.32 M sucrose containing 10% DMSO, and stored at -80°C. To obtain the crude synaptosome fraction, the tissue was thawed in a 37°C water bath and homogenized in 10 mm Tris buffer (pH 7.4) containing a proteinase inhibitor (Roche) and a phosphatase inhibitor (Sigma-Aldrich) using a glass / Teflon homogenizer (clearance 0.1-0.15 mm). The homogenate was centrifuged at 1000 g for 10 minutes at 4°C, the supernatant was removed, and the mixture was centrifuged again at 10000 g for 20 minutes at 4°C. The resulting pellet was resuspended in sucrose / Tris solution and stored at -80°C. Synaptosomes were conjugated to pHrodo-Red according to the manufacturer's protocol.

[0133] Phagocytosis assay iMGL and MD-Mφ were incubated with mouse anti-CD16 / 32Fc receptor block (2 mg / ml; BD Biosciences) at 4°C for 15 minutes. The cells were then stained with anti-CD45-APC clones (mouse cells; Tonbo Biosciences; San Diego, CA) in 1:200 flow cytometer buffer. Next, Amnis Imagestreamer was used. x Samples were analyzed using a Mark II Imaging Flow Cytometer (Millipore). Phagocytosis of E. coli, human synaptosomes, fAβ, and BDTO was analyzed using IDEAS software with the Internalization Wizard algorithm. Untreated anti-CD11b antibody (Biolegend) was used for CD11b blockade.

[0134] statistical analysis Statistical analyses were performed using Graphpad Prism 6 software. For comparisons involving more than two groups, using one-way ANOVA followed by Tukey's post-hoc test, p-values ​​adjusted for multiple comparisons were reported. For comparisons between two groups, a two-tailed Student's t-test was used. All differences were considered statistically significant if p < 0.05. Statistical analyses of RNA sequencing are detailed above, and all other statistical analyses are reported in the legend of the figures.

[0135] ADP migration and calcium imaging assay iMGL (5.5×10 4 Cells (per well) were cultured for 1 hour in serum-free basal medium without cytokines. Next, iMGL was pre-exposed to DMSO or PSB0739 (50 μM, Tocris) for 1 hour in a 5% CO2 cell culture incubator at 37°C. Then, the cells were washed three times with basal medium and seeded in a transwell migration chamber (24 wells with 5 μm polycarbonate inserts, Corning) containing adenosine 5'-phosphate (ADP, 100 μM, Sigma) in the bottom chamber at 37°C in 5% CO2. After 4 hours, the cells were washed three times and fixed in PFA (4%) at room temperature for 15 minutes. Cells were stained with Hoechst stain for 10 minutes to visualize the cell nuclei. Blinded observers counted the total number of cells per slide, then scraped the cells off the surface, washed with PBS, and recounted to record the migrating cells. Migration was reported as the number of migrating cells relative to the total number of cells per well. Cell fluorescence images were recorded using an Olympus IX71 inverted microscope.

[0136] 3D brain organoid cell culture iPSCs were cultured and maintained in Vitronectin XF (Stem Cell Technologies) on 6-well tissue culture processed plates (BD Falcon) and maintained daily at 37°C with 5% CO2 in TeSR-E8 medium (Stem Cell Technologies). At approximately 80% confluence, iPSCs were separated from the Vitronectin XF substrate using the standard ReLeSR protocol (Stem Cell Technologies), centrifuged, pelletized, and suspended in embryoid body (EB) medium consisting of KO DMEM / F12 (Invitrogen), KOSR (20% v / v) (v / v), L-alanyl-L-glutamine (2 mM), NEAA (1x), 2-mercaptoethanol (0.1 mM), rhubFGF (4 μg / ml), HSA (0.1% v / v), and ROCK inhibitor (50 μM) to form embryoid bodies (EBs). To prevent EB from adhering to the 96-well plate, approximately 1 × 10⁻¹⁶ wells per well of a standard V-bottom 96-well plate coated with Lipidure (1% v / v; AMSBio) 4Nine cells were seeded. After 4 days in EB medium containing bFGF (4 ng / ml) and ROCK inhibitor (50 μM), both bFGF and ROCK inhibitor were discontinued, and the brain organoids were left in basic EB medium for a further 3 days (total of 7 days). After the EB medium stage, the EB medium was replaced with neuroepithelial (NE) medium consisting of DMEM / F12, N2 supplement (0.1% v / v), L-alanyl-L-glutamine (2 mM), MEM-NEAA (0.1% v / v), and heparin solution (0.2 mg / ml; Sigma), filtered using a 0.22 μm PES filter (EMD Milipore). The brain organoids were transferred to ultra-low adhesion 24-well plates (Corning) using a cut P200 pipette tip in 1 ml of NE medium with 1-2 EB cells per well. EBs were neurogenicated in NE medium for 5 days, then transferred to Matrigel (Corning) using molds made from siloconized Parafilm and sterile empty P200 boxes. Brain organoids were maintained in 6 cm suspension petri dishes with differentiation medium consisting of KO DMEM / F12 (50%), basal neuronal medium (50%), N2 supplement (0.1% v / v), B27 without vitamin A supplement (0.1% v / v), insulin solution (0.1% v / v; Sigma), 2-mercaptoethanol (0.1 mM), L-alanyl-L-glutamine (2 mM), MEM-NEAA (1x), and penicillin / streptomycin (0.1% v / v). After exposure to differentiation medium containing vitamin B27 without vitamin A for 5 days, the differentiation medium was replaced with the same preparation except that the vitamin A-free B27 was replaced with vitamin A-containing B27. At this point, the brain organoids were also transferred to a 125 ml rotating flask bioreactor (Corning) treated with Sigmacote (Sigma), where they were fed differentiation medium containing vitamin A weekly for 8 weeks. After 12 weeks, the organoids were used in a co-culture test with iMGL.

[0137] Isolation of human adult microglia and fetal microglia In short, seemingly normal cortical tissue was excised from a pharmacologically refractory, non-malignant case of temporal lobe epilepsy. The tissue was extensively washed and mechanically dissected. After gentle enzymatic digestion with trypsin and DNAse before passing through a nylon mesh filter, a single-cell suspension was produced. The single-cell suspension underwent a fickle ultracentrifugation step to remove myelin. The dissociated cells were centrifuged, counted, and 2 × 10⁶ cells were added to MEM supplemented with 5% FBS, 0.1% P / S, and 0.1% glutamine. 6 Cells were seeded at a concentration of cells / mL. Microglia were allowed to grow for 3 days, then collected and measured at 1 × 10⁶. 5 Cells were seeded at 1 / mL and maintained in culture for 6 days, during which time the cells received two TGFβ (20 ng / mL) treatments on days 3 and 5. Human fetal brain tissue was obtained from the Fetal Tissue Storage Facility (Albert Einstein College of Medicine, Bronx, NY). Total RNA was isolated using a standard Trizol (Invitrogen) protocol and stored at -80°C. In some embodiments, suspensions of small cell clumps were produced and used in a similar manner to single-cell suspensions.

[0138] iMGL transplantation in MITRG and Rag5×fAD brains All animal procedures were performed according to the IAUC protocol (IAUC#2011-3004) approved by NIH and University of California guidelines. MITRG mice were purchased from Jax (The Jackson Laboratory, #017711) and previously characterized (Rongvaux et al., 2014). MITRG mice are designed to enable xenografting and support human bone marrow engraftment. iMGLs were harvested on day 38 and suspended in 1×HBSS containing infusion buffer: M-CSF (10 ng / ml), IL-34 (50 ng / ml), and TGFβ-1 (25 ng / ml). iMGLs were delivered using the following coordinates with previously described stereotactic surgery (Blurton-Jones, et al, 2009): AP: -0.6, ML: ±2.0, DV: -1.65. Brains were collected from mice 60 days post-transplant according to an established protocol (Blurton-Jones, et al, 2009). Rag5×fAD mice were generated in this laboratory and previously characterized (Marsh et al., 2016). Rag5×fAD mice exhibit robust beta-amyloid pathology, enabling xenotransplantation of human cells. iMGL was transplanted into the hippocampus using the following coordinates: AP: -2.06, ML: ±1.75, DV: -1.95. Post-transplant, mice were killed and brains were collected using a previously established protocol. Briefly, mice were anesthetized with sodium barbiturate and perfused to the left ventricle with cold 1×HBSS for 4 minutes. Perfused mice were decapitated, the brains were extracted and fixed dropwise with PFA (4% w / v) at 4°C for 48 hours. Next, the brain was washed three times with PBS and immersed in sucrose (30% w / v) solution for 48 hours before coronal sections (40 μm) were prepared using a microtome (Leica). The suspended sections were stored in PBS sodium azide (0.05%) solution at 4°C until IHC was performed.

[0139] dot blot A serial dilution of the protein (2 μl) was blotted onto pre-moistened nitrocellulose paper and dried. After drying, the blot was blocked with 5% BSA in 1× Tris-buffered saline containing Tween 20 (TBST) at room temperature for 1 hour with gentle shaking. Next, the blot was incubated with the primary antibody (see below) at room temperature for 1 hour. Then, the blot was washed three times with TBST for 5 minutes each. Next, the blot was incubated with HRP-conjugated secondary antibody (Santa Cruz) at a ratio of 1:10,000 at room temperature for 1 hour with gentle shaking. After 1 hour, the blot was washed three times with TBST for 5 minutes each. After washing, the blot was dried on filter paper and incubated with Pierce ECL Western blotting substrate (Thermo Fisher) in the dark for 10 minutes. The blot was imaged using a ChemiDoc XRS+ imaging system (BioRad).

[0140] AD-GWAS qPCR primers The following validated and available Taqman primers were used: APOE Hs00171168_m1, CR1 Hs00559342_m1, CD33 Hs01076281_m1, ABCA7 Hs01105117_m1, TREM2 Hs00219132_m1, TREML2 Hs01077557_m1, TYROBP(DAP12) Hs00182426_m1, PICALM Hs00200318_m1, CLU Hs00156548_m1, MS4A6A Hs01556747_m1, BIN1 Hs00184913_m1, CD2AP Hs00961451_m1, CASS4 Hs00220503_m1, MEF2C Hs00231149_m1, DSG2 Hs00170071_m1, MS4A4A Hs01106863_m1, ZCWPW1 Hs00215881_m1, INPP5D Hs00183290_m1, and PTK2B Hs00169444_m1.

[0141] Various combinations or subcombinations of specific features and aspects of the embodiments disclosed above can be created, but are intended to remain within the scope of one or more of the present invention. Furthermore, any specific features, aspects, methods, properties, characteristics, qualities, attributes, elements, etc., disclosed herein relating to the embodiments can be used in all other embodiments described herein. Therefore, it should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for each other to form various modes of the disclosed invention. Accordingly, it is intended that the scope of the present invention disclosed herein should not be limited by the specific disclosed embodiments described above. Furthermore, various modifications and alternative forms of the present invention are possible, specific examples of which are shown in the drawings and described in detail herein. However, it should be understood that the present invention is not limited to any specific form or method disclosed, but rather encompasses all modifications, equivalents, and alternatives that fall within the spirit and scope of the various embodiments described and the appended claims. Any method disclosed herein does not need to be performed in the order mentioned. The methods disclosed herein include specific actions taken by a practicing physician, but may also include, explicitly or implicitly, instructions from any third party to take such actions. For example, an action such as “administering a population of proliferating NK cells” may include “instructions to administer a population of proliferating NK cells.” Furthermore, if any feature or aspect of the disclosure is described in terms of the Markush group, a person skilled in the art will understand that the disclosure is also described in terms of any individual member of the Markush group, or a subgroup of members of the Markush group.

[0142] The scope disclosed herein also includes any and all overlaps, subscopes, and combinations thereof. Words such as “at most,” “at least,” “greater than,” “less than,” and “between” include the numbers mentioned. Numbers preceded by terms such as “about” or “approximately” include the numbers mentioned. For example, “about 10 nanometers” includes “10 nanometers.” [Examples]

[0143] iHPC transplantation allows for the testing of human microglia development in a complete brain environment. Normal development and aging of human microglia in a complete CNS environment can be tested by transplanting iHPCs into mouse brains (see Figures 24 and 25). References [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12] [Table 13] [Table 14] [Table 15]

Claims

1. A method for producing human microglia-like cells (iMGLs) from pluripotent stem cells (PSCs), (i) A step of differentiating PSCs using a culture medium supplemented with hematopoietic differentiation factors to produce induced hematopoietic progenitor cells (iHPCs), (ii) Using microglia differentiation medium CD43 ＋ The process of differentiating iHPC into human microglia-like cells (iMGLs) and A method that includes this.

2. The method according to claim 1, wherein step (i) includes an incubation period of 3 to 21 days.

3. The method according to claim 2, wherein the incubation period of step (i) is 10 days.

4. The method according to claim 3, wherein the PSCs are incubated in a hypoxic or normal oxygen environment during the 1st to 10th day of the incubation period.

5. On days 1 and 2 of the incubation period, the culture medium contains hematopoietic differentiation factors FGF2, BMP4, activin A, LiCl, and VEGF; On the third and fourth days of the incubation period, the culture medium contains the hematopoietic differentiation factors FGF2 and VEGF; During the 5th to 10th day of the incubation period, the culture medium contains hematopoietic differentiation factors FGF2, VEGF, TPO, SCF, IL3, and IL6. The method according to claim 3 or 4.

6. The concentrations of FGF2, BMP4, VEGF, TPO, SCF, IL-3, and IL-6 in the culture medium are between 5 ng / ml and 100 ng / ml. The concentration of activin A in the culture medium is between 0.1 ng / ml and 30 ng / ml. The method according to claim 5.

7. The concentrations of FGF2, BMP4, VEGF, TPO, SCF, IL-3, and IL-6 in the culture medium are approximately 50 ng / ml. The concentration of activin A in the culture medium is approximately 12.5 ng / ml. The concentration of LiCl is approximately 2 mM. The method according to claim 6.

8. CD43 using fluorescence-activated cell sorting (FACS) ＋ The method according to claim 1, further comprising isolating the iHPC.

9. CD43 ＋ Isolating iHPC is CD43 ＋ The method according to claim 8, which is performed by selecting a marker.

10. The method according to claim 9, wherein the isolated iHPC has a purity of more than 80%.

11. The method according to claim 10, wherein the isolated iHPC has a purity of more than 90%.

12. The method according to claim 1, wherein step (ii) further comprises an incubation period of 20 to 30 days.

13. The method according to claim 12, wherein step (ii) further includes an incubation period of about 25 days.

14. The method according to claim 1, wherein the microglia differentiation medium in step (ii) comprises factors CSF-1, IL-34, and TGFβ1.

15. The method according to claim 14, wherein the microglia differentiation medium is serum-free.

16. The concentration of CSF-1 in the culture medium is between 5 ng / ml and 50 ng / ml. The concentration of IL-34 in the culture medium is between 25 ng / ml and 125 ng / ml. The concentration of TGFβ1 in the culture medium is between 2.5 ng / ml and 100 ng / ml. The method according to claim 14 or 15.

17. The concentration of CSF-1 is approximately 25 ng / ml. The concentration of IL-34 is approximately 100 ng / ml. The concentration of TGFβ1 is approximately 50 ng / ml. The method according to claim 16.

18. The method according to claim 1, wherein step (ii) includes maturing the iMGL during an incubation period of 1 to 5 days.

19. The method according to claim 18, wherein step (ii) includes maturing the iMGL for an incubation period of about three days.

20. The method according to claim 19, wherein step (ii) comprises maturing the iMGL by incubating the iMGL in a culture medium containing CD200 and CX3CL1.

21. The method according to claim 20, wherein CD200 is human recombinant CD200 and CX3CL1 is human recombinant CX3CL1.

22. The method according to claim 20 or 21, wherein the concentrations of CD200 and CX3CL1 in the culture medium are between 1 ng / ml and 1 μg / ml.

23. The method according to claim 22, wherein the concentration of CD200 and CX3CL1, respectively, is 100 ng / ml.

24. The iMGL produced is c-kit － / CD45 ＋ The method according to claim 1.

25. The c-kit － / CD45 ＋ The method according to claim 24, wherein iMGL can be detected on the 14th day of a 25-day incubation period.

26. The method according to claim 25, further comprising testing for the expression of a factor that is a known marker of constraint on the fate of microglia.

27. The method according to claim 26, wherein the expression of the factor being tested includes PU. 1 and TREM2.

28. where the iMGL is two separate populations of iMGL: (1) CD45 ＋ / CX3CR1 － and (2) CD45 ＋ / CX3CR1 ＋ The method according to claim 1, comprising the above.

29. The method according to claim 1, wherein the iMGL produced has a purity of at least 70%.

30. The method according to claim 29, wherein the iMGL produced has a purity of at least 96%.

31. The method according to claim 29 or 30, wherein the purity level is evaluated using the expression of a purine receptor, P2ry12, Trem2, Iba1, or Pu1.

32. The aforementioned CD43 ＋ iHPC is CD235a ＋ / CD41a ＋ The method according to claim 1.

33. A method for producing human microglia-like cells (iMGLs) from pluripotent stem cells (PSCs), (i) A process of differentiating PSCs into induced hematopoietic progenitor cells (iHPCs), (ii) A step of differentiating the iHPC to produce iMGL A method that includes this.

34. The method according to claim 33, further comprising the step (iii) of maturing the iMGL by incubating the iMGL in a culture medium containing CD200 and CX3CL1.

35. The iHPC is CD43 ＋ / CD235a ＋ / CD41 ＋ The method according to claim 33.

36. The method according to claim 33, wherein the iHPC is differentiated into the iMGL by placing the iHPC in a serum-free differentiation medium in step (ii).

37. The method according to claim 36, wherein the serum-free differentiation medium comprises MCSF, IL-34, and TGFβ1.

38. The iMGL produced above is c-kit － / CD45 ＋ The method according to claim 33.

39. The iMGL is a group of two distinct iMGLs: (1) CD45 ＋ / CX3R1 － and (2) CD45 ＋ / CX3R1 ＋ The method according to claim 33, including the method described in claim 33.

40. The method according to claim 33, wherein step (i) includes an incubation period of 5 to 15 days, and step (ii) includes an incubation period of 20 to 30 days.

41. The method according to claim 40, wherein step (i) includes a 10-day incubation period and step (ii) includes a 25-day incubation period.

42. The method according to claim 34, wherein step (iii) includes an incubation period of 1 to 5 days.

43. The method according to claim 42, wherein step (iii) includes a 3-day incubation period.

44. A composition of iMGL comprising the expression of any combination of the following genes: RUNX1, PU. 1, CSF1FR, CX3CR1, TGFBR1, RSG10, GAS6, PROS1, P2RY12, GPR34, C1Q, CR3, CABLES1, BHLHE41, TREM2, ITAM, APOE, SLCO2B1, SLC7A8, PPARD, C9orf72, GRN, LRRK2, TARDBP, and CRYBB1.

45. The composition according to claim 44, wherein TREM2 and P2RY12 are colocalized.

46. The composition according to claim 44 or 45, wherein the genes KLF2, TREM1, MPT, ITGAL, and ADGRE5 are not expressed.

47. A composition of iMGL comprising the expression of CD33, MS4A4A, and CR1 when iMGL is in the ground state.

48. The composition according to any one of claims 44 to 47, wherein the iMGL secretes any combination of chemokines: TNFα, CCL2, CCL4, and CXCL10 in response to stimulation by lipopolysaccharide, IFGγ, or IL-1β.

49. The composition according to any one of claims 44 to 47, wherein the iMGL migrates in response to the ADP.

50. The composition according to any one of claims 44 to 47, wherein ADP stimulation results in the generation of calcium transfer in the iMGL.

51. The composition according to any one of claims 44 to 47, wherein the iMGL can phagocytose human synaptosomes.

52. The composition according to any one of claims 44 to 47, wherein the iMGL can perform synaptic pruning.

53. The composition according to claim 52, wherein the synaptic pruning is mediated by the C1q / CR3 pathway.

54. The composition according to any one of claims 44 to 47, wherein the iMGL can phagocytose Aβ and tau.

55. The composition according to any one of claims 44 to 47, wherein the iMGL can phagocytose fluorescently labeled fibrous Aβ and pHo-labeled brain-derived tau oligomers.

56. A method for profiling the secretion of inflammatory molecules from iMGL, (i) Treating the iMGL with lipopolysaccharide, IFNγ, TNFα, or IL-1β, (ii) Measuring the inflammatory markers secreted by the iMGL and A method that includes this.

57. The method according to claim 56, wherein the inflammatory marker secreted by iMGL includes an inflammatory marker selected from CCL2, CCL4, and CXCL10.

58. A method for evaluating the movement of iMGL, (i) Processing the iMGL by ADP, (ii) To evaluate the motility and migration of iMGL in response to chemical stimuli. A method that includes this.

59. A method for inducing calcium migration in iMGL, (i) Processing the iMGL by ADP, (ii) Investigating the calcium flux signal in the iMGL and A method comprising the following, wherein the calcium flux signal is generated in response to an electrical, biological, or chemical stimulus.

60. A method for testing the microglial phagocytosis of compounds, (i) Exposing iMGL to a compound selected from the group consisting of Aβ, tau, fluorescently labeled Aβ, and pHo-labeled brain-derived tau oligomers, wherein the compound is phagocytosed, endocytose, or ingested by the iMGL, (ii) Measuring the phagocytosis, endocytosis, or uptake of the compound A method that includes this.

61. A method for establishing an iMGL gene expression profile similar to that of iMGL in vivo, Co-culturing iMGL with neurons, astrocytes, or other cells of the central nervous system to reproduce the in vivo state of the iMGL more accurately than when the iMGL is not co-culturing with the neurons, astrocytes, or other cells of the central nervous system. A method that includes this.

62. The method according to claim 61, wherein the neuron is a rat hippocampal neuron.

63. The method according to claim 61 or 62, wherein the differentially controlled genes are CABLES, TRIM4, MITF, MMP2, and SLCA25, and the differentially controlled genes are upregulated in iMGL.

64. A method for integrating iMGL into a 3D CNS environment, A method comprising co-culturing iMGL with hiPSC 3D brain organoids (BORG), wherein the iMGL migrates to the BORG, aggregates in the BORG, or is incorporated into the BORG.

65. A method for testing microglial dysregulation in health and disease using iMGL, (i) Exposing iMGL to a compound selected from the group consisting of Aβ, tau, fluorescently labeled Aβ, pHdo-labeled brain-derived tau oligomer, and alpha-synuclein, (ii) Profiling iMGL omics signatures selected from RNA-seq, proteomics, metabolomics, and lipidomics. A method that includes this.

66. The method according to claim 65, wherein the differentially controlled genes are CD33, TYROPB, and PICALM, and the differentially controlled genes are upregulated in iMGL.

67. A method for investigating the role of microglia in synaptic pruning and synaptic plasticity, comprising (i) exposing human synaptosomes to iMGL and (ii) evaluating the phagocytosis of human synaptosomes by the iMGL.

68. A method for evaluating gene regulation in iMGL in response to neuronal cues, (i) Exposing iMGL to a factor present in the central nervous system, selected from the group consisting of CX3CL1, CD200, and TGFβ, (ii) To evaluate differentially regulated genes, including genes selected from the group consisting of P2RY12, EGR1, TGFβR1, ETV5, CX3CR1, APOE, BIN1, CD33, GPR84, COMT, APP, PSEN1, PSEN2, HTT, GRN, FUS, TARDP, VCP, SNCA, C9ORRF72, LRRK2, and SOD1. A method that includes this.

69. A method for evaluating the engraftment of iMGL into the cortex, (i) Transplanting iMGL into the cortex, (ii) To evaluate the engraftment of the iMGL into the cortex and A method that includes this.

70. The method according to claim 69, wherein step (ii) occurs two months after step (i).

71. The method according to claim 69 or 70, further comprising implanting iMGL into the cortex of a mouse.

72. The method according to claim 71, wherein the mouse is an MITRG mouse.

73. A method for evaluating the interaction between AD neuropathy and iMGL, (i) Transplanting iMGL into the brain of a mouse, (ii) Evaluating iMGL that interacts with the mouse brain and A method that includes this.

74. The method according to claim 73, wherein step (ii) includes evaluating the migration of iMGL to a patch.

75. The method according to claim 73, wherein step (ii) includes evaluating the phagocytosis of fibrous Aβ by iMGL.

76. A method for testing human microglia in a 3D neural environment, comprising implanting iMGLs into the brain of a mammal.

77. The method according to claim 76, wherein the brain of the mammal is that of a mouse.

78. The method according to claim 77, wherein the iMGL is implanted in the hippocampus of the mouse.

79. The method according to claim 76 or 77, wherein the mouse is a wild-type mouse.

80. The method according to claim 76 or 77, wherein the mouse is an AD mouse strain.

81. The method according to claim 3, wherein the PSC is incubated in a 5% oxygen environment on days 1 to 4 of the incubation period, and the PSC is incubated in a 20% oxygen environment on days 5 to 10 of the incubation period.

82. The concentrations of FGF2, BMP4, VEGF, TPO, SCF, IL-3, and IL-6 in the culture medium are between 30 ng / ml and 70 ng / ml. The concentration of activin A in the culture medium is between 11 ng / ml and 14 ng / ml. The concentration of LiCl is between 1 mM and 3 mM. The method according to claim 5.

83. The concentration of CSF-1 in the culture medium is between 15 ng / ml and 35 ng / ml. The concentration of IL-34 in the culture medium is between 80 ng / ml and 120 ng / ml. The concentration of TGFβ1 in the culture medium is between 30 ng / ml and 70 ng / ml. The method according to claim 14 or 15.

84. The method according to claim 1, wherein the microglia differentiation medium in step (iii) comprises factors CSF-1, IL-34, and TGFβ2.

85. The concentration of CSF-1 in the culture medium is between 5 ng / ml and 50 ng / ml. The concentration of IL-34 in the culture medium is between 25 ng / ml and 125 ng / ml. The concentration of TGFβ1 in the culture medium is between 2.5 ng / ml and 100 ng / ml. The method according to claim 84.

86. The method according to claim 1, wherein the microglial differentiation medium in step (iii) comprises factors CSF-1, IL-34, and a TGFβ mimetic.

87. The method according to claim 86, wherein the TGFβ mimetic activates the TGFβ signaling pathway.

88. The method according to claim 1, wherein the PSC comprises a single-cell PSC or a small cluster of PSCs.

89. The method according to claim 1, further comprising maturing the iMGL.

90. The method according to claim 1 or 33, wherein the PSC does not originate from an embryoid body.

91. The c-kit － / CD45 ＋ The method according to claim 24, wherein iMGL is detected using flow cytometry, immunofluorescence microscopy, qPCR, RNA-seq, or proteomics.

92. The method according to claim 1 or 33, wherein the PSC includes an induced PSC (iPSC).

93. The method according to claim 1 or 33, wherein the PSC includes embryonic stem cells (ESCs).

94. The method according to any one of claims 1, 33, 92, or 93, wherein the PSC is a mammalian PSC.

95. The method according to any one of claims 1, 33, 92, or 93, wherein the PSC is a human PSC.

96. The method according to any one of claims 1, 33, 92, or 93, wherein the PSC is a mouse PSC.

97. The iHPC is CD34 ＋ CD31 ＋ or CD45 ＋ The method according to claim 35.

98. The aforementioned PSC, before differentiation, CD43 ＋ CD235a ＋ CD41 ＋ CD34 ＋ CD31 ＋ , and CD45 ＋ The method according to claim 1 or 33, which is neither of the above.

99. A method for producing human microglia-like cells (iMGLs) from a first type of cell, (i) A step of differentiating type 1 cells into induced hematopoietic primordial cells (iHPCs), (ii) A step of differentiating the iHPC to produce iMGL A method that includes this.

100. A culture medium for supporting the generation of human microglia-like cells (iHPCs), comprising FGF2, BMP4, activin A, and LiCl.

101. A culture medium for supporting the generation of human microglia-like cells (iHPCs), comprising FGF2 and VEGF.

102. A culture medium for supporting the generation of human microglia-like cells (iHPCs), comprising FGF2, VEGF, TPO, SCF, IL3, and IL6.

103. A kit containing a culture medium to support the generation of human microglia-like cells (iHPCs), including FGF2, BMP4, activin A, and LiCl.

104. A kit containing a culture medium to support the generation of human microglia-like cells (iHPCs) containing FGF2 and VEGF.

105. A kit containing a culture medium to support the generation of human microglia-like cells (iHPCs) containing FGF2, VEGF, TPO, SCF, IL3, and IL6.

106. A culture medium for supporting the generation of human microglia-like cells (iMGLs), comprising CSF-1, IL-34, and TGFβ1.

107. A kit containing a culture medium to support the generation of human microglia-like cells (iMGLs) containing CSF-1, IL-34, and TGFβ1.

108. A culture medium for supporting the maturation and maintenance of human microglia-like cells (iMGLs), comprising CD200 and CX3CL1.

109. A kit containing a culture medium to support the maturation and maintenance of human microglia-like cells (iMGLs), including CD200 and CX3CL1.

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