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

Abstract

Provided are in vitro cell populations comprising human microglia-like cells, and in vitro methods for producing human microglia-like cells. [Solution] Provided is an in vitro cell population comprising human microglia-like cells (iMGL), wherein at least 70% of the cells in the population are iMGL that express P2RY12 and TREM2. Also provided is an in vitro method for producing human microglia-like cells (iMGL), comprising contacting human induced hematopoietic progenitor cells (iHPC) with a microglia differentiation medium containing CSF-1, IL-34, and TGFβ1 or a TGFβ mimetic to differentiate the iHPC into iMGL.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 464,925, filed February 28, 2017. The contents of the above-referenced application are expressly incorporated herein by reference in their entirety.

[0002] Declaration of Federally Funded R&D This invention was made with government support under Grant AG048099 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Described herein are embodiments of (i) human microglia-like cells (iMGLs) and (ii) methods for producing iMGLs. [Background technology]

[0004] Microglial cells are innate immune cells of the CNS and are known to play a role in the physiological development of the CNS. Furthermore, microglial cells are known to play a role in neurological disorders such as Alzheimer's disease. However, there are limitations in the techniques for obtaining microglial cells to further investigate their role in CNS development and neurological disorders. Summary of the Invention [Means for solving the problem]

[0005] In some embodiments, methods for producing human microglia-like (iMGL) cells from pluripotent stem cells (PSCs) are provided. In some embodiments, the methods include (i) differentiating PSCs using a medium supplemented with hematopoietic differentiation factors to produce induced hematopoietic progenitor cells (iHPCs); and (ii) expressing CD43 + (iii) isolating iHPCs and (iv) differentiating them using microglial differentiation medium to differentiate them into CD43 + The method includes (iv) differentiating iHPCs into iMGLs, and (iv) maturing the iMGLs.

[0006] In some embodiments, the method comprises (i) differentiating PSCs using a medium supplemented with hematopoietic differentiation factors, and (ii) differentiating PSCs using a microglial differentiation medium to differentiate CD43 + and differentiating iHPCs into iMGLs.

[0007] In some embodiments, methods are 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 an induced hematopoietic progenitor cell (iHPC), and (ii) differentiating the iHPC to produce an iMGL.

[0008] In some embodiments, the PSCs are not derived from embryoid bodies, hi some embodiments, the PSCs comprise single cell PSCs.

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

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

[0011] In some embodiments, compositions of iMGL are provided that comprise expression of any 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 for assessing the secretion of chemokines, cytokines, and other inflammatory molecules is provided, comprising (i) treating iMGL with lipopolysaccharide, IFNγ, or IL-1β, and (ii) measuring secreted factors from the iMGL that may function as potential biomarkers of inflammation or various neurodegenerative disease states. Some embodiments relate to 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 the iMGL.

[0013] In some embodiments, a method for evaluating iMGL migration is provided, comprising: (i) treating iMGL with ADP; and (ii) measuring iMGL migration. Some embodiments relate to a method for evaluating iMGL migration, comprising: (i) treating iMGL with ADP; and (ii) evaluating iMGL motility and migration in response to chemical stimulation.

[0014] In some embodiments, a method for generating a calcium transient in an iMGL is provided, the method comprising: (i) treating the iMGL with ADP; and (ii) generating a calcium transient in the iMGL. Some embodiments relate to a method for generating a calcium transient in an iMGL, the method comprising: (i) treating the iMGL with ADP; and (ii) examining a 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 for differentially regulating gene expression in iMGL is provided, comprising: (i) co-culturing iMGL with neurons or astrocytes; and (ii) differentially regulating genes in iMGL.

[0016] In some embodiments, a method for incorporating iMGL into a CNS / brain (e.g., neuron) environment is provided, comprising: (i) co-culturing iMGL with hiPSC 3D cerebral organoids (BORG); and (ii) allowing iMGL to invade into BORG. Some embodiments relate to a method for incorporating iMGL into a 3D CNS environment, comprising co-culturing iMGL with hiPSC 3D cerebral organoids (BORG), wherein iMGL migrates to, assembles into, or is incorporated into BORG.

[0017] In some embodiments, a method for differentially regulating gene expression in iMGL is provided, comprising: (i) exposing iMGL 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, i.e., synuclein, huntingtin, and prion; and (ii) differentially regulating genes in iMGL. Some embodiments relate to a method for establishing an iMGL gene expression profile that resembles the in vivo state of iMGL, comprising co-culturing iMGL with neurons, astrocytes, or other cells of the central nervous system, thereby recreating the in vivo state for iMGL more than exists for iMGL when iMGL is not co-cultured with neurons, astrocytes, or other cells of the central nervous system. Some embodiments relate to methods of using iMGL to test for microglial dysregulation in health and disease, 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 omic signature selected from RNA-seq, proteomics, metabolomics, and lipidomics.

[0018] In some embodiments, a method of phagocytosis of human synaptosomes (hS) in iMGL is provided, the method comprising: (i) exposing iMGL to hS and (ii) measuring the phagocytosis of hS. Some embodiments relate to a method of testing microglial phagocytosis of a compound, the method comprising: (i) exposing iMGL to a compound selected from the group consisting of Aβ, tau, fluorescently labeled Aβ, and pHrodo-labeled brain-derived tau oligomers, whereby the compound is phagocytosed, endocytosed, or taken up by iMGL; and (ii) measuring the phagocytosis, endocytosis, or uptake 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 iMGL to human synaptosomes; and (ii) assessing human synaptosome phagocytosis by iMGL.

[0020] In some embodiments, a method for determining gene regulation is provided, comprising: (i) exposing iMGL to any combination of one or more of the following factors: CX3CL1, CD200, and TGFβ; and (ii) evaluating any combination of any one or more of the following differentially regulated genes: 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 for assessing the engraftment of iMGL into neural tissue (e.g., cortex) is provided, comprising: (i) transplanting iMGL into neural tissue; and (ii) assessing the engraftment of iMGL into neural tissue.

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

[0023] In some embodiments, methods are provided for studying human microglia in a 3D neural environment, comprising transplanting iMGLs into the brain of a mammal.

[0024] Some embodiments of the methods, kits, and compositions provided herein relate to a medium for supporting the generation of human iHPCs, the medium comprising one or more of FGF2, BMP4, activin A, and LiCl. Some embodiments of the methods and compositions provided herein relate to a medium for supporting the generation of human iHPCs, the medium comprising one or more of FGF2 and VEGF. Some embodiments of the methods and compositions provided herein relate to a medium for supporting the generation of human iHPCs, the medium comprising one or more of FGF2, VEGF, TPO, SCF, IL3, and IL6. Some embodiments relate to a kit for supporting the generation of human iHPCs, the kit comprising a medium comprising one or more of FGF2, BMP4, activin A, and LiCl. Some embodiments relate to a kit for supporting the generation of iHPCs, the kit comprising a medium comprising one or more of FGF2 and VEGF. Some embodiments relate to a kit for supporting the generation of human iHPCs, the kit comprising a 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 medium for supporting the production of human iMGL, the medium comprising one or more of CSF-1, IL-34, and TGFβ 1. Some embodiments relate to a kit for supporting the production of human iMGL, the kit comprising a medium comprising one or more of CSF-1, IL-34, and TGFβ 1.

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

[0027] It should be understood that the compositions and related methods summarized above and further detailed below describe specific actions to be taken by a practitioner, but may also include direction of these actions by another party. Thus, an action such as "implanting an iMGL into the brain of a mammal" includes "directing the implantation of an iMGL into the brain of a mammal." [Brief explanation of the drawings]

[0028] [Figure 1]Figure 1A: Schematic of the fully defined iMGL differentiation protocol. (i) Human iPSCs are differentiated into CD43+ iHPCs for 10 days and then cultured in serum-free microglial differentiation medium containing human recombinant MCSF, IL-34, and TGFβ-1. Differentiation continues for an additional 25 days, after which iMGLs are exposed to human recombinant CD200 and CX3CL1 for 3 days. (ii) Representative images of iHPCs in cell culture on day 10. Scale bar = 100 μm. (iii) By day 14, iMGLs express PU.1 (bright spots) and TREM2 (bright spots). Scale bar = 50 μm. (iv) Representative phase-contrast images of iMGLs on day 38. Figure 1B: Schematic of iPSC-to-iHPC differentiation. (i) Single-cell iPSCs are differentiated in chemically defined medium 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 CD45-independent profile when compared with monocyte-derived macrophages (MD-Mφ). Figure 1E: iMGL progenitor cells are CD11blo and increase their CD11b expression as they mature. At 14 DIV, a small population (approximately 11%) of CD11bint-hi cells was detected. Figure 1F: CD11b fluorescence intensity demonstrates that CD11b expression increases as iMGLs senesce, resembling mouse microglial progenitor cells. Figure 1G. May-Grünwald-Giemsa staining of monocytes, MD-Mφ, fetal microglia, and iMGL. Both fetal microglia and iMGL exhibit morphology with a high nuclear-to-cytoplasmic ratio compared to monocytes and MD-Mφ. Scale bar = 16 µm. Figure 1H. Differentiation results in greater than 96% purity as assessed by colocalization of the microglia-enriched protein P2ry12 and microglia-enriched Trem2 (the merge panel shows an overlay of the nuclei panels, where expression of P2ry12, Trem2, and P2ry12 and Trem2 overlap).Figure 1I. iMGLs also exhibit elongated processes and express Cx3Cr1 (top left panel) and hCyto (top right panel). The merge panel shows that hCyto expression (bright spots) is localized in the same region as Cx3Cr1 expression. [Figure 2]Figure 2A: 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 within the same circled cluster), CD14+ / CD16- monocytes (CD14M), CD14+ / CD16+ monocytes (CD16M) (CD14M and CD16M are located within the same circled cluster), blood dendritic cells (blood DCs), iHPCs, 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 timeline of iPSC differentiation into iHPCs (arrow from iPSC cluster to iHPC cluster) and subsequent differentiation into iMGLs (arrow from iHPC cluster to iMGL cluster). PC2 (15.4% var) reflects a trajectory to blood DCs. PC3 (7.6% var) reflects a trajectory to monocytes. Figure 2B: Heatmap and biclustering (Euclidean distance) of 300 microglia, myeloid, and other immune-related genes (Butovsky et al., 2014, Hickman et al., 2013, Zhang et al., 2014). Pseudo-counting was used for FPKM values ​​(FPKM+1), which were log2-transformed, and each gene was normalized in each row (n=300). Representative profiles are shown for genes up- and down-regulated in both human microglia (fetal / adult) and iMGL. Figure 2C: Bar graphs of microglia-specific enriched genes measured as FPKM+1 followed by Log2-transformation [Log2(FPKM+1)] in iMGL, fetal MG, and adult MG, blood DCs, CD14 M, and CD16 M, presented as mean ± SEM. Data were analyzed using one-way ANOVA followed by Tukey's corrected multiple comparison post-hoc test. Statistical annotations represent the maximum p-values ​​for iMGL, fetal MG, and adult MG relative 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]Figure 3A: Flow cytometry analysis revealed that iMGLs (outer dark spot regions in all three panels) are CD45-intense, similar to fetal MGs (innermost spot regions in all three panels). Figure 3B: iMGLs (dark spots in the left panel) differ from CD45-hi MD-Mφs (gray spots in the left panel). Histograms of CD11b intensity (left histogram) reveal that fetal MGs express slightly more CD11b than iMGLs but less than MD-Mφs. Figure 3C: iMGLs secrete 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). Pre-exposure to the P2ry12 antagonist PSB0739 (50 μM, 1 h) completely abolished ADP-induced iMGL migration (***p<0.0001). Figure 3E. ADP induces iMGL calcium flux through the P2ry12 receptor. (Left) Exposure to ADP leads to an increase in calcium influx (I340 / I380 ratio) in the vehicle group (light trace), but not in the PSB0739-treated group (dark trace). (Right) Representative images of ADP-induced calcium flux at 240 s in vehicle (top) and PSB0739 (bottom) treatments. Figure 3F. iMGL phagocytose human brain-derived synaptosomes (hS). Representative images recorded with Amnis Imagestream show the 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 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 40% (p<0.0001, right bar) by inhibiting CR3 via CD11b blockade.(Bottom) Subanalysis of iMGL showing phagocytic events reveals similar average amounts of internalization across treatment groups (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: vehicle (n=37 cells), PSB0739-treated (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 for vehicle, n=3 wells / group. [Figure 4]Figure 4A: A heatmap of 25 immune genes with variants associated with LOAD reveals that the major risk factors APOE and TREM2 are highly expressed in iMGL, adult MG, and fetal MG. Figure 4B: iMGL internalize fluorescently labeled fAβ and pHrodo-dye BDTO. Representative images recorded with an Amnis Image StreamX Mark II. Figure 4C: iMGL were exposed to unlabeled fAβ (5 μg ml) and BDTO (5 μg / ml) for 24 h, and mRNA expression of 19 GWAS genes was assessed via qPCR array. For each gene tested, the bar corresponding to fAβ treatment is on the left, and the bar corresponding to BDTO treatment is on the right. fAβ treatment increased the expression of 10 genes by more than two-fold compared to vehicle, including MS4A6A (6.3-fold), CD33 (6.1-fold), ABCA7 (5.8-fold), TYROBP (4.98-fold), and TREM2 (4.85-fold). Meanwhile, BDTO exposure increased the expression of four genes by more than two-fold compared to vehicle. Six genes were differentially expressed in fAβ compared to BDTO. The fAβ and BDTO preparations were confirmed by dot blot analysis with conformation-specific antibodies for oligomers (A11), fibers (OC), and non-structure-specific antibodies for human Aβ (6E10) and tau oligomers (tau22). Target genes were normalized to GAPDH and compared to vehicle expression by ΔΔCt. Bars represent mean ± SEM of fold expression. Red hash bars indicate ΔΔCt = 1. Two-way ANOVA followed by Sidak'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]Figure 5A: Schematic of iMGL coculture with or without rat hippocampal neurons. Figure 5B: iMGL cocultured with neurons was collected and assessed by flow cytometry, and the transcriptome was assessed by RNA sequencing. Figure 5C: A heatmap of gene expression in iMGL and iMGL-HC highlights uniquely enriched genes. Figure 5D: Differential gene expression analysis highlights 156 upregulated and 244 downregulated genes in iMGL-HC. Figure 5E: A scatterplot of differentially expressed genes [>2Log2(FPKM+1)] highlights that TRIM14, CABLES1, MMP2, SIGLEC 11 and 12, MITF, and SLC2A5 were enriched in iMGL-HC, suggesting that iMGL responds appropriately to the neural environment. Cells cultured alone were enriched for COMT, EGR2, EGR3, and FFAR2, suggesting a primed microglial phenotype. [Figure 6]iMGLs (5 x 10 cells) were added to culture media containing a single BORG for 7 days. (Panel A) Representative brightfield images of iMGLs detected in and near BORG after 3 days. iMGLs were found at the border of the BORG media (arrows) and adhered to it, rather than floating freely in the media, suggesting complete chemotaxis of iMGLs. (Panel B) Representative images of iMGLs in the outer and inner radii of BORG. (Panel C) Embedded iMGLs exhibit macrophage-like morphology (white arrows) and elongated processes (black arrows), indicating ECM remodeling and surveillance, respectively. Simultaneous evaluation of the morphology of iMGLs embedded in undamaged (Panels D-F) and injured (Panels G-I) BORG. (Panel D) Immunohistochemical analysis of BORG revealed that iMGLs began to tile evenly throughout the BORG, protruding branched processes for environmental surveillance. BORGs are representative of the developing brain in vitro and contain neurons and astrocytes that self-organize into a cortical-like distribution, but lack microglia and iMGLs. (Panels E-F) Representative immunofluorescence images of iMGLs with extending processes within a 3D neural environment at high magnification. (Panels G-I) Representative images of iMGL morphology observed in injured BORGs. (Panels H-I) Spherical iMGLs reminiscent of amoeboid microglia are distributed in injured BORGs and closely resemble activated microglia, demonstrating that iMGLs respond appropriately to neural 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 the mobility 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 humans (18–39 years old) by 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 analysis. Figure 7B. Characterization of the mobility of monocytes, dendritic cells, and commercially available HPCs. Human myeloid dendritic cells (blood DCs) were isolated from the blood of young, healthy humans (18–39 years old) using a crude myeloid DC enrichment kit followed by FACs. To avoid contamination with plasmacytoid DCs, DCs were stained with CD123, and myeloid DC subtypes CD1c and CD141 were collected for RNA analysis. Figure 7C. Characterization of the mobility of monocytes, dendritic cells, and commercially available HPCs. A commercially available source of HPCs (CD43+ / 235a+ / CD41+) cells was identified and used for comparison with in-lab HPC differentiation and further iMGL differentiation. [Figure 8]Figure 8A: RNA-seq coverage maps and gene FPKM values ​​for the myeloid-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. RNA-seq coverage maps and gene FPKM values ​​for CD14+ / 16- monocytes (CD14 M), CD14+ / 16+ monocytes (CD16 M), and iPS-derived microglia-like cells (iMGL) for the microglia-enriched genes P2RY12, OLFML3, and GPR34 in iMGL. For all RNA coverage maps (Figures 8A, 8B, and 8C), the y-axis represents reads per million (RPM) scaled for all samples. Histogram comparisons using FPKM values ​​for all genes are shown as mean ± s.e.m. Biological replicates for CD14 M (N = 5), CD16 (N = 4), and iMGL (N = 6) were included for comparison by one-way ANOVA followed by Tukey's multiple comparison post-hoc test. **p<0.001, ***p<0.0001. Figure 8D. Representative volcano plots of differentially expressed genes (p-value < 0.001, 2-fold change) in iMGL (right part of the plot), CD14 M (left part of the plot), and non-significant (lower light part of the plot). Key genes are labeled. Fold change (log2) and -log10 (p-value) indicate the x- and y-axes, respectively. Gray dashed vertical lines indicate 2-fold changes in gene expression. Venn diagrams show the total number of differentially expressed genes per condition. Figure 8E. Representative volcano plots of differentially expressed genes (p-value < 0.001, 2-fold change) in iMGL (right part of the plot), CD16 M (left part of the plot), and non-significant (lower light part of the plot). Key genes are labeled. Fold change (log2) and -log10 (p-value) indicate the x- and y-axes, respectively.Gray dashed vertical lines indicate a 2-fold change in gene expression. Venn diagrams show the total number of differentially expressed genes per condition. [Figure 9] Figure 9A: Spearman correlation matrix of biological samples used in RNA sequencing highlights strong intragroup correlations. iMGL correlates well with fetal and adult MG, suggesting strong gene expression similarities between samples. Figure 9B: Histogram of key genes found across the various samples. CD14 and FCGR3A (also known as CD16) were expressed in all myeloid cells, including microglia, but were enriched in CD14M and CD16M, respectively. As expected, FLT3 was highly expressed by blood DCs but not other cells, and was barely detectable in all three microglial groups. The monocyte / macrophage-specific transcription factor KLF2 was enriched only in CD14M and CD16M. On the other hand, GATA1 and OCT4 were only detected in iHPCs and iPSCs, respectively. [Figure 10]Figure 10A: Representative immunofluorescence images of iMGLs expressing microglial markers CX3CR1 (left panel) and TREM2 (center, right panels). hCyto (center, left panel) is a cytoplasmic marker. The merge panel (right panel) shows the colocalization of CX3CR1, hCyto, and TREM2. Figure 10B: Representative immunofluorescence images of iMGLs expressing microglial markers TGFBR1 (left panel) and MERTK (left, center panels). Dapi (center, right panel) is a nuclear marker. The merge panel (right panel) shows the colocalization of TGFBR1, MERTK, and the nucleus. Figure 10C: Representative immunofluorescence images of iMGLs expressing microglial markers PROS1 (left panel), ITGB5 (center, left panel), and TREM2 (center, right panel). The merge panel (right panel) shows the colocalization of PROS1, ITGB5, and TREM2. Figure 10D: Representative bright-field and immunofluorescence images recorded by an Amnis Imagestream flow cytometer visualizing Ec phagocytosis within macrophages (top) and iMGLs (bottom). Figure 10E: As expected, quantification of the percentage of phagocytic cells (top) reveals that iMGLs (right bar) phagocytose nearly 10-fold less Ec than macrophages (left bar). The amount of Ec internalized by GMFI within phagocytic cells (bottom) further demonstrates the greater phagocytic capacity of macrophages compared to iMGLs. [Figure 11]iMGLs express genes associated with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Parkinson's disease (PD), and dementia with Lewy bodies (DLB), which contribute to microglial dysfunction. A bar graph of genes involved in neurodegenerative diseases detected in iMGLs, similar to fetal and adult MG, is presented as FPKM+1 followed by Log2 transformation [Log2(FPKM+1)] and presented as mean ± SEM. Similar to isolated human primary microglia, iMGLs express valosin-containing protein (VCP), FUS, C9ORF72, proganulin (GRN), TDP-43 (TARDBP), LRRK2, superoxide dismutase (SOD), and synuclein (SNCA). Recent literature implicates microglial dysfunction in mutations or loss of function of these genes involved in the pathogenesis of ALS (C9ORF72, SOD1, TARDBP, FUS), FTD (VCP, C9ORF72, GRN, TARDBP), PD (LRRK2, SNCA), and DLB (SNCA), suggesting the utility of iMGL in examining the mechanisms underlying these genes in these neurological disorders. [Figure 12] GO terms from differential gene expression analysis of iMGL cocultured with hippocampal neurons. The gene expression profile of iMGL cocultured with rat hippocampal neurons (iMGL-HC) was enriched by soluble and insoluble factors present in 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 complimentary gene profile with 20 statistically significant GO biological modules (iMGL histogram), including terms characteristic of cholesterol homeostasis, characteristic TNFα signaling via NF-κB, leukocyte differentiation, and regulation of IL-1β secretion. [Figure 13] Figures 13A-13P. iMGLs transplanted into the brains of either wild-type or AD transplant-competent mice resemble cerebral microglia. In the brains of xenotransplant-competent mice, transplanted iMGLs ramify and interact with the neural environment. (A-L) After 2 months in vivo, iMGLs transplanted into mice exhibit long-term survival with advanced dendrites similar to endogenous microglia found in the brain. (A) Transplanted iMGLs labeled with P2ry12 (HPA HPA014518, Sigma) and human nuclei (ku80) exhibit long-term survival in mice. (B-D) At higher magnification, P2ry12 is highly expressed in iMGL dendrites, both of which suggest homeostatic microglia monitoring the brain environment. (E-H) Branched iMGLs also express microglia-enriched Tmem119, recognized by a human-specific Tmem119 antibody (ab185333, Abcam, identified and validated in [Bennet et al., PNAS 2016]), as well as the human cytoplasmic marker SC121 (hCyto). (I-L) At higher magnification, 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) Transplanted iMGLs extend protrusions and migrate toward plaques. iMGLs completely envelop amyloid plaques (O) and begin phagocytosing amyloid (P). Scale bars: (A, E, N) = 30 μm, (B-D, F-H, I-L, O, P) = 10 μm, (M) = 300 μm. n = 3 animals per study. [Figure 14]Figures 14A-14F. Genomic stability of iPSCs and iMGLs. (Figure 14A) Top: Representative fluorescence images of iPSCs expressing the 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. (Figure 14B-C) Karyotype and Pluritest scores show that all iPS lines generated using Sendai virus and used in this study were karyotypically normal and pluripotent. Pluritest is a microarray-based assessment of pluripotency based on iPS whole-transcriptome analysis referenced to a library of functionally validated iPSCs (Muller, FJ et al. 2011). (Figure 14D-E) Maintenance of genomic stability over the course of iMGL differentiation using pluripotent iPS or commercially available hematopoietic progenitor cells. CNV assessment of differentiated iMGL revealed that genomic stability was maintained over the course of differentiation. (Figure 14D) Representative Nanostring nCounterKaryotype results demonstrate that microglia derived from ADRC iPS line 22 do not inherit extrachromosomal DNA over the course of differentiation. (Figure 14E) Quantification of 338 probe sets across all 24 chromosomes did not reveal any chromosomal abnormalities (n = 6). (Figure 14F) Representative analysis of iMGL derived from iPSCs showed a strong correlation of CNVs (r = 0.929), demonstrating the assay's sensitivity and genomic stability in derived iMGL. [Figure 15] Figure 15A-B. Assessment of iMGL purity by P2RY12 / TREM2 colocalization and flow cytometric characterization of monocytes, dendritic cells, and commercially available iHPCs. (Figure 15A) Specificity assessment of rabbit anti-human P2ry12 (HPA014518, also recently validated) and goat anti-human Trem2 (R&D, AF1828) in human monocytes and iMGLs. Scale bar = 20 μm. (Figure 15B) Representative immunofluorescence images (from five representative lines) of iMGL purity by P2ry12 / Trem2 / DAPI colocalization. Scale bar = 100 μm. [Figure 16] Figures 16A-E. TGFβ-1, CX3CL1, CD200, and their effects on key microglial genes are associated with neuronal function and environmental regulation. (Figures 16A-B) TGFβ1 maintains core microglial genes. Withdrawal of TGFβ1 for 24 hours strongly impacts the microglial transcriptome. Consistent with in vivo mouse studies, TGFβ removal reduces expression of key microglial genes, including surface receptors P2RY12, TGFβR1, and CX3CR1, while also reducing expression of microglial transcription factors EGR1 and ETV5. AD-related pathway genes, such as BIN1, CD33, and APOE, are also affected by the absence of TGFβ. Removal of CX3CL1 and CD200 does not alter core microglial identity but influences state by affecting homeostatic gene expression, such as COMT and APOE (Figure 16B). (Figure 16C) Differential gene expression analysis reveals that the presence of TGFβ increases the expression of 1,262 genes in iMGL, while the absence of TGFβ decreases the expression of 1,517 genes, further supporting previous studies highlighting the role of TGFβ in microglial development, gene signatures, and function. (Figure 16D) KEGG pathway analysis highlights that TGFβ-elevated microglial core genes regulate pathways for CNS diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. (Figure 16E) Fold changes in AD GWAS loci genes relative to iMGL by TGFβ. Statistics reflect one-way ANOVA followed by Dunnett's multiple comparison post-hoc test. *p<0.05, **p<0.001, ***p<0.0001. [Figure 17]Figure 17A-C Microglial AD-GWAS and other CNS disease-related genes can be tested using iMGL. (Figure 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 involved in regulating microglial inflammation and function in AD, such as CD33, ABCA7, TYROBP, and TREM2. Stimulation of iMGL without 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 and in survival and homeostasis. (Figure 17C) Key neurodegeneration-related genes, APP (AD), SCNA (PD), and HTT (HD), are expressed in iMGL and primary cultured microglia. iMGLs also express genes associated with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and dementia with Lewy bodies (DLB), supporting previous research on microglial dysfunction. A bar graph of genes involved in neurodegenerative diseases detected in iMGLs, similar to fetal and adult MG, is expressed as Log2(FPKM+1) and presented as mean ± SEM. Similar to isolated human primary cultured microglia, iMGLs express valosin-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), suggesting the utility of iMGL in examining the mechanisms underlying 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 microglial cells engraft and phagocytose Aβ, similar to human fetal microglia. (A-D) Human fetal microglia (hCyto) were transplanted into an immunodeficient AD mouse model, Rag5×fAD, and responded to beta-amyloid plaques. Fetal microglia were observed surrounding plaques (C) and phagocytosing Aβ (C-D). (E-H) Similar to fetal microglia, iMGL (hCyto) cells surround and phagocytose beta-amyloid plaques. Scale bars (A, B, E, F) = 20 μm, (C, D, H, G) = 5 μm. [Figure 19] Assessment of the percentage of (i) P2ry12-expressing cells (left bar), (ii) TREM2-expressing cells (middle bar), and (iii) P2ry12-, TREM2-, and DAPI-expressing cells (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 panels). However, TREM1 expression (center column of panels) is highly enriched in macrophages, distinguishing macrophage ontogeny from iMGLs, exemplified by the low TREM1 expression typical of microglia within the CNS. [Figure 21] iMGLs are highly motile in vitro. Time-lapse phase-contrast imaging (over 24 h) of iMGL motility in culture reveals that iMGLs (enclosed in boxes) are highly motile, monitor their environment by protrusion, and demonstrate the ability to migrate in response to, for example, injury or stress. [Figure 22] Co-culture of iMGLs with iPSC-derived astrocytes results in branched iMGLs. When co-cultured with astrocytes differentiated by GFAP expression (left panel), iMGLs (Iba-1, middle panel) branched and extended protrusions in 2D in vitro culture, further demonstrating that cues derived from the CNS environment can further educate iMGLs 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, the resident macrophages of the CNS, demonstrating their potential to outcompete endogenous mouse microglia in MITRG mice. (A) Confocal microscopy reveals successful engraftment of iHPCs detected by a human nucleus-specific antibody (top panel of 23A) and expressing the myeloid marker Iba-1 (middle panel of 23A). (B) Transplanted iHPCs differentiate into microglia expressing P2ry12 (top right panel of 23B), demonstrating that human CSF1 expression is mediated by MITRG humanization, allowing human cells to outcompete endogenous mouse cells. [Figure 24] Microglia-specific expression of P2ry12 is detectable in iHPCs after only two weeks of engraftment. Two weeks after iHPC transplantation, cells that survive transplantation and are labeled with a human nucleus-specific antibody (left column of panels) begin to express Iba-1 (middle left column of panels), indicating cells of myeloid origin. Furthermore, P2ry12, a microglia-specific gene highly expressed in homeostatic microglia, can be detected (middle right column of panels; arrow). [Figure 25] Transplanted hematopoietic progenitor cells differentiate into microglia in the mouse brain and express TMEM119. After two months, engrafted HPCs express TMEM119 (middle left panel), a microglial marker (Bennet et al., 2016) that is prominently expressed within the highly branched processes of maturing microglia. The human specificity of the TMEM119 antibody is demonstrated by colocalization of TMEM119 with an antibody specific for human nuclei (middle right panel), but not all nuclei (DAPI, left panel). [Figure 26]Transplanted human cells express the homeostatic microglial marker, P2ry12. Engrafted iMGLs, distinguished by the human-specific nuclear marker (left center column of panels), are distinguishable from endogenous mouse cell nuclei, which are stained only with the nonspecific nuclear stain DAPI (left center column of panels). Engrafted iMGLs express the homeostatic microglial marker P2ry12 (center column of panels), highlighting the elongated branching processes typical of in vivo microglia. This contrasts with the commonly used microglial marker Iba-1, which exhibits a cytoplasmic distribution in microglia (right center column of panels). [Figure 27] iMGLs can be used to examine astrocyte-microglia crosstalk in vivo. Transplanted iMGLs (Iba-1, upper left panel) are observed to interact with endogenous mouse astrocytes (GFAP, lower left panel) in vivo. Recent studies (Liddelow et al., 2017) have implicated astrocyte-microglia crosstalk in influencing immune responses in the CNS. DETAILED DESCRIPTION OF THE INVENTION

[0029] Microglia are innate immune cells of the CNS and play critical roles in synaptic plasticity, neurogenesis, homeostatic function, and immune activity. Microglia also play a key role in neurological disorders, including AD, highlighting the need for improved understanding of their function in both health and disease. Yet, the study of human microglia remains challenging due to the rarity and difficulty of obtaining primary cultures from human fetal or adult CNS tissue. Therefore, there is an urgent need to develop renewable sources of human microglia, such as from pluripotent stem cells (PSCs), including induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs).

[0030] The challenge in generating microglia from iPSCs stems from their unique developmental origin. Elucidated lineage tracing studies have shown that microglia originate from yolk sac erythroid progenitor cells (EMPs), generated during primitive hematopoiesis. EMPs further develop into early primitive macrophages, which migrate into the developing neural tube and become microglial progenitor cells. Microglial progenitor cells then mature and develop branched processes that are 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 examine 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 AD (LOAD). While the function of microglia and the role of these genes in AD are only beginning to be examined in mouse models, the generation of human microglia-like cells, as described herein, will enable the investigation of human-specific genes that cannot be modeled in mice.

[0032] In AD, microglial clusters surrounding beta-amyloid plaques highlight their inefficiency in beta-amyloid clearance. Microglia are also involved in the neuroinflammatory component of AD pathogenesis, including cytokine / chemokine secretion, which exacerbates disease pathology. Furthermore, AD GWAS genes, such as TREM2 and CD33, are affected by AD pathology and may be involved in AD progression. Microglia are also key regulators of brain development, neuronal homeostasis, and many neurological disorders. Therefore, there is an urgent need to further our understanding of the influence of both pathology and disease-related genes on human microglia and microglial function.

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

[0034] The iMGLs described herein develop in vitro similarly to in vivo microglia. Transcriptome-wide analysis shows that they closely resemble adult and fetal human microglia. Functional assessment of these iMGLs reveals that they respond to inflammatory stimuli by secreting cytokines, migrating, undergoing calcium transients, and robustly phagocytosing CNS substrates similar to adult and fetal microglia.

[0035] These iMGLs can be used, among other uses, to (i) examine the effects of fibrillar 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 studies of microglial function, providing important new insights into human neurological diseases.

[0036] In the following sections, various embodiments of methods for producing iMGL and various embodiments of the structure and function of iMGL are provided. Methods for using iMGL are also provided. Non-limiting detailed descriptions of the methods are also provided. How to make iMGL:

[0037] In some embodiments, methods for producing human microglia-like cells (iMGLs) from pluripotent stem cells (PSCs) are provided. In some embodiments, the methods include (i) differentiating PSCs to produce induced hematopoietic progenitor cells (iHPCs) using a medium supplemented with hematopoietic differentiation factors; and (ii) expressing CD43 +(iii) isolating iHPCs and (iv) differentiating them using microglial differentiation medium to differentiate them into CD43 + (iv) differentiating the iHPCs into human microglia-like cells (iMGLs); and (iv) maturing the iMGLs. In some embodiments, the HPC generation techniques are enriched for progenitor cells and express CD43 + (iii) allows collection of the medium to be performed without isolating iHPCs.

[0038] In some embodiments, the method comprises (i) differentiating PSCs using a medium supplemented with hematopoietic differentiation factors, and (ii) differentiating PSCs using a microglial differentiation medium to differentiate CD43 + and differentiating iHPCs into iMGLs.

[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 an iHPC, and (ii) differentiating the iHPC to produce iMGL. In some embodiments, the first type of cell is not a PSC or an ESC.

[0040] In some embodiments, the PSCs are not derived from embryoid bodies. In some embodiments, the PSCs comprise single cell PSCs. In some embodiments, the PSCs are CD43 + In some embodiments, the PSCs are not CD34 + In some embodiments, the PSCs are not CD31 + In some embodiments, the PSCs are not CD45 + isn't it.

[0041] In some embodiments, the PSCs are or comprise induced PSCs (iPSCs). In some embodiments, the PSCs are or comprise embryonic stem cells (ESCs). In some embodiments, the PSCs are mammalian PSCs. In some embodiments, the PSCs are human PSCs. In some embodiments, the PSCs are mouse PSCs.

[0042] Differentiating single-cell PSCs In some embodiments, differentiating PSCs to produce iHPCs involves an incubation period of between 5 and 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 normoxic environment. In some embodiments, on days 1-10 of the incubation period, the PSCs are incubated in a hypoxic or normoxic environment. In some embodiments, during the first portion of the 10-day period, the PSCs are exposed to a 3%-7% oxygen environment. In some embodiments, the first portion of the 10-day period is 4 days (days 1-4), and the oxygen environment is 5%. In some embodiments, during the second portion of the 10-day period, the PSCs are exposed to an oxygen environment between 15% and 25%. In some embodiments, the second portion of the 10-day period is 6 days (days 5-10), and the PSCs are exposed to a 20% oxygen environment. In some embodiments, differentiating the PSCs to produce iHPCs involves an incubation period of between 3 and 21 days. In some embodiments, the incubation period is up to 28 days. In some embodiments, the incubation period is greater than 28 days. In some embodiments, the incubation period is less than 3 days.

[0043] In some embodiments, hematopoietic differentiation factors used to differentiate PSCs include FGF2, BMP4, activin A, LiCl, VEGF, TPO, SCF, IL3, and IL6. The culture medium includes any 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 includes FGF2, BMP4, activin A, and LiCl; on days 3 and 4, the medium includes FGF2 and VEGF; and on days 5-10, the medium includes FGF2, VEGF, TPO, SCF, IL3, and IL6. Some embodiments relate to culture media containing any one or combination of the factors FGF2, BMP4, activin A, LiCl, VEGF, TPO, SCF, IL3, and IL6. Some embodiments relate to a medium comprising any one or a combination of the factors FGF2, BMP4, activin A, and LiCl. Some embodiments relate to a medium comprising any one or a combination of the factors FGF2 and VEGF. Some embodiments relate to a medium comprising any one or a combination of the factors FGF2, VEGF, TPO, SCF, IL3, and IL6.

[0044] In some embodiments, the concentration of each of the factors FGF2, BMP4, VEGF, TPO, SCF, IL-3, and IL6 in the medium is between 5 ng / ml and 100 ng / ml. In some embodiments, the concentration of each of the factors FGF2, BMP4, VEGF, TPO, SCF, IL-3, and IL6 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 each of the factors FGF2, BMP4, VEGF, TPO, SCF, IL-3, and IL6 in the medium is 50 ng / ml. In some embodiments, the concentration of activin A in the 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 medium is 12.5 ng / ml. In some embodiments, the concentration of LiCl in the medium is between 1 nM and 3 nM. In some embodiments, the concentration of LiCl in the medium is between 1 mM and 3 mM, hi some embodiments, the concentration of LiCl in the medium is 2 mM.

[0045] Isolation of iHPCs iHPCs or CD43 may be isolated using any method known in the art. + In some embodiments, iHPCs or CD43 + The method used to isolate iHPCs is FACS. In some embodiments, the isolation step involves detecting CD43 + In some embodiments, the method further comprises selecting a marker for CD43. + In some embodiments, the isolation step uses a marker other than CD34 to isolate HPCs. + In some embodiments, the isolating step comprises selecting cells that are CD31 + cells or CD45 + In some embodiments, the isolating step comprises selecting for another marker known to identify iHPCs.

[0046] In some embodiments, isolating iHPCs results in isolation of iHPCs that are greater than 80% pure, for example, greater than 90% pure. + By isolating iHPCs, it is possible to obtain CD43 + iHPCs are isolated.

[0047] Differentiation of iHPCs into iMGLs Any method known in the art for maturing microglial cells can be used to mature the iMGLs.

[0048] In some embodiments, CD43 + Differentiation of iHPCs into iMGLs involves an incubation period of between 20 and 30 days. In some embodiments, the incubation period is 25 days.

[0049] In some embodiments, the medium used to differentiate iHPCs into iMGLs comprises any one or a combination of the factors CSF-1, IL-34, and TGFβ1. In some embodiments, the medium comprises all of the factors CSF-1, IL-34, and TGFβ1. In some embodiments, the concentration of CSF-1 in the medium is between 5 ng / ml and 50 ng / ml. In some embodiments, the concentration of CSF-1 in the 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 medium is 25 ng / ml. In some embodiments, the concentration of IL-34 in the medium is between 25 ng / ml and 125 ng / ml. In some embodiments, the concentration of IL-34 in the 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 medium is 100 ng / ml. In some embodiments, the concentration of TFGβ-1 in the medium is between 2.5 ng / ml and 100 ng / ml. In some embodiments, the concentration of TFGβ-1 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 TGFβ-1 in the medium is 50 ng / ml. Some embodiments relate to a medium comprising any one or combination of the factors CSF-1, IL-34, and TGFβ1.

[0050] In some embodiments, the medium used to differentiate iHPCs into iMGLs contains TGFβ-2. In some embodiments, the concentration of TGFβ-2 in the medium is between 2.5 ng / ml and 100 ng / ml. In some embodiments, the concentration of TGFβ-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 TGFβ-2 in the medium is 50 ng / ml.

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

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

[0053] Maturity of iMGL In some embodiments, maturing the iMGL comprises an incubation period of between 1 and 5 days, hi some embodiments, the incubation period for maturing the iMGL is 3 days.

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

[0055] In some embodiments, the concentration of each of CD200 and CX3CL1 in the medium is between 1 ng / ml and 1 μg / ml. In some embodiments, the concentration of each of CD200 and CX3CL1 in the 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 each of CD200 and CX3CL1 is 100 ng / ml.

[0056] Properties of the produced iMGL In some embodiments, the iMGL produced using the methods described herein results in a pure population of iMGL between 70% and 100% purity. In some embodiments, the iMGL produced using the methods described herein results in a pure population of iMGL between 80% and 100% purity. For example, the iMGL population will 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% pure. In some embodiments, the iMGL population produced is greater than 96%.

[0057] The purity of iMGL can be assessed by 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] The iMGL produced by any of the methods described herein will express any factor or any combination of factors that typical microglial cells express. In some embodiments, the iMGL produced will express c-kit - / CD45 + In some embodiments, the c-kit - / CD45 + iMGL is 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 iMGL produced is classified into two distinct iMGL populations: (1) CD45 + / CX3CR1 - and (2) CD45+ / CX3CR1 + In some embodiments, the iMGL produced comprises CD43 + , CD235a + , or CD41 + In some embodiments, the iMGL produced is CD43 + / CD235a + / CD41 + is.

[0059] Any of the methods for producing iMGL described herein may involve the expression of CD43, which commits cells to the microglial lineage early in the differentiation process. + In some embodiments, the c-kit - / CD45 + iMGL, which is CD43 + The iMGL lineage is detected on day 14 of the incubation period used to differentiate iHPCs into iMGLs. The determination of iMGL lineage commitment is made by examining the expression of any factor known to be a marker of cells committed to a microglial fate. In some embodiments, the commitment of cells to the iMGL lineage is determined by assessing the expression of the transcription factor PU.1 and / or the microglia-enriched protein Trem2. In some embodiments, the cell markers are detected using flow cytometry, immunofluorescence microscopy, qPCR, RNA-seq, or proteomics.

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

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

[0062] Composition of iMGL Gene expression of iMGL In some embodiments, iMGLs are provided that express a specific gene profile. Any of the iMGLs described herein comprise a gene expression profile similar to that of microglial cells. In some embodiments, any of the compositions of iMGLs described herein comprise 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 comprise the expression of any of these genes in any combination.

[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, in any of the iMGL compositions described herein, TREM2 and P2RY12 are co-expressed. In some embodiments, none of the iMGL compositions described herein express any 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 of the iMGLs described herein will secrete a chemokine profile similar to that of microglial cells in response to any stimulus known in the art to stimulate chemokines in microglial cells. In some embodiments, the secreted chemokines are any combination of one or more of TNFα, CCL2, CCL4, and CXCL10, secreted in response to stimulation with lipopolysaccharide, IFNγ, or IL-1β.

[0066] In some embodiments, a method for stimulating chemokine secretion from iMGL is provided. The method includes (i) treating iMGL with any agent known in the art to stimulate cytokine secretion in microglial cells, and (ii) secreting chemokines from microglial cells. In some embodiments, the agent used to treat iMGL 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 translocation Any of the iMGLs described herein migrate in response to ADP treatment, and / or ADP treatment induces calcium mobilization.In some embodiments, inhibiting P2ry12 abolishes ADP-mediated iMGL migration and / or ADP-mediated calcium mobilization.In some embodiments, inhibiting P2ry12 occurs via inhibitor PSB0739.

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

[0069] Phagocytosis by iMGL Any of the iMGLs described herein can phagocytose. Any of the iMGLs described herein can phagocytose any factor known in the art that microglia can phagocytose. In some embodiments, the factor that iMGL phagocytose includes any one or more of Aβ, fluorescently labeled Aβ, tau, and pHrodo-labeled brain-derived tau oligomers.

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

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

[0072] The utility 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 any one or more of the following genes: CABLES, TRIM4, MITF, MMP2, and SLCA25. In some embodiments, the iMGLs upregulate any one or more of the following genes: TYROPB, CD33, and PICALM.

[0073] In some embodiments, provided is a method for controlling gene expression in iMGL.One of the methods comprises: (i) co-culturing iMGL with neuron; and (ii) differentially controlling gene in iMGL.The neuron that is co-cultured with iMGL comprises any neuron of any species.In some embodiments, the neuron is rat hippocampal neuron.

[0074] Another method includes (i) exposing iMGL to any one or more of the following 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, including any one or more of CD33, TYROPB, and PICALM, in any combination.

[0075] How to use iMGL In some embodiments, provide the method for evaluating gene expression in iMGL in response to neuronal cue.According to some embodiments, this method includes: (i) exposing iMGL to any combination of one or more of the following factors: CX3CL1, CD200 and TGFβ; and (ii) evaluating any combination of one or more of the following differentially regulated genes: 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, provide a method for evaluating the engraftment of iMGL into the cortex.According to some embodiments, this method includes: (i) transplanting iMGL into the cortex; and (ii) evaluating the engraftment of iMGL into the cortex. In some embodiments, step (ii) occurs at least 2 weeks after step (i), e.g., at least 3 weeks after step (i), at least 4 weeks after step (i), at least 5 weeks after step (i), at least 6 weeks after step (i), at least 7 weeks after step (i), at least 8 weeks after step (i), at least 9 weeks after step (i), at least 10 weeks after step (i), at least 11 weeks after step (i), at least 12 weeks after step (i), at least 13 weeks after step (i), at least 14 weeks after step (i), at least 15 weeks after step (i), at least 16 weeks after step (i), at least 17 weeks after step (i), at least 18 weeks after step (i), at least 19 weeks after step (i), or at least 20 weeks after step (i). In some embodiments, step (ii) occurs 2 months after step (i). In some embodiments, the method further comprises transplanting the iMGL into the cortex of a mouse. In some embodiments, the mouse is a MITRG mouse.

[0077] In some embodiments, a method for evaluating the interaction between AD neuropathy and iMGL is provided.In some embodiments, this method comprises: (i) transplanting iMGL into hippocampus; and (ii) evaluating the interaction of iMGL in hippocampus.In some embodiments, this method comprises evaluating the migration of iMGL to plaque.In some embodiments, this method comprises evaluating the phagocytosis of fibrillar Aβ by iMGL.

[0078] In some embodiments, a method for testing human microglia in a 3D neural environment is provided, comprising transplanting iMGL into a mammalian brain. In some embodiments, the mammalian brain is a mouse brain. In some embodiments, the iMGL is transplanted into 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. [Example]

[0079] Certain aspects of the above-described embodiments are disclosed in further detail in the following examples, which are not intended to limit the scope of the disclosure in any way. [Example]

[0080] Generation 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 5 weeks (Figure 1A). This and other example methods and protocols can be used in a similar manner to generate iMGLs from other PSCs, including ESCs. Utilizing this approach, we have successfully generated iMGLs from over 10 independent iPSC lines. First, iPSCs differentiate into hematopoietic progenitor cells (iHPCs), which recapitulate the ontogeny of microglia, as iHPCs represent early primitive hematopoietic cells derived from the yolk sac that give rise to microglia during development. This protocol (Figure 1Bi) allows for the differentiation of iMGLs after 10 days, resulting in the differentiation of CD43 + / CD235a + / CD41 + CD43 + FACS sorting of the cells revealed that this approach produced iHPCs with greater than 90% purity (Fig. 1Bii).

[0081] Second, CD43 + iHPCs were grown in serum-free differentiation medium (formulated in the laboratory) containing CSF-1, IL-34, and TGFβ1. By day 14, the cells expressed the myeloid-associated transcription factor PU.1 and the microglia-enriched protein TREM2 (Figure 1A iii), demonstrating early commitment to a microglial fate. Because this protocol yielded a large number of iMGLs, their development was followed by in vitro characterization of iMGLs every 4 days by flow cytometry. Early iMGLs at day 14 were c-kit- / CD45 + (Fig. 1C), suggesting myeloid lineage restriction. + / CX3CR1 - (A1) and CD45 + / CX3CR1 + (A2) and (B3). CD45 expression was consistently monitored in developing iMGLs and compared with monocyte-derived macrophages (MD-Mφ). CD45 expression increased with maturation, but levels never reached those of macrophages (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 exhibited high purity, as assessed by colocalization and quantification (>96%) of the purinergic receptors P2RY12 and TREM2 (Figure 1H). One million iPSCs produced 30–40 million iMGLs by 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 mouse microglial development in vivo, iMGLs developed in vitro expressed PU.1, TREM2, and CD11b. int / CD45 low As iMGLs matured in vitro, they became more ramified, similar to in vivo microglia (Figure 1A iv). [Example]

[0083] Transcriptome analysis of iMGL The transcriptome of iMGL was profiled in comparison with human primary cultured fetal microglia (fetal MG) and adult microglia (adult MG). + / CD16 - Monocytes (CD14 M), CD14 + / CD16 + Inflammatory monocytes (CD16 M), myeloid dendritic cells (blood DCs), iHPCs, and iPSCs were also examined for comparison with stem cell and other myeloid molecular signatures. Correlation analysis and principal component analysis (PCA) revealed striking similarities of iMGL with fetal and adult MG (fetal 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, Figure 2A arrow), defined the timeline of differentiation from iPSCs to iHPCs to iMGL cells, while PC2 and PC3 defined the trajectories of dendrites and monocytes, respectively.

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

[0085] [Table 1]

[0086] [Table 2]

[0087] [Table 3]

[0088] [Table 4] [Example]

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

[0090] Because iMGL express the purinergic receptor P2ry12, which is enriched in microglia and has been shown to be important for microglial homeostatic function (Figures 1H and 2C), ADP-P2ry12-mediated chemotaxis and calcium translocation were assessed. It was determined that iMGL robustly migrated in response to ADP and that ADP induced calcium translocation (Figures 3D and 3E), which could be abolished by the P2ry12-specific inhibitor PSB0739. These physiological findings further clarify that iMGL express functional surface receptors, enabling quantitative analysis of microglial physiology.

[0091] Microglia, along with astrocytes, also play a critical role in synaptic pruning. Because the in vitro synaptosome phagocytosis assay is an established surrogate for testing pruning, we quantitatively assessed the ability of iMGL to phagocytose human synaptosomes (hS). Compared to MD-Mφ, phagocytosis of pHrodo-labeled hS by iMGL was less robust (Figures 3F and 3G). However, iMGL preferentially internalized hS compared to E. coli particles, which, when normalized to MD-Mφ (Figures 10D and 10E), supports the notion that iMGL and microglia are more biased toward homeostatic functions than MD-Mφ.

[0092] Because microglia (and iMGL) express both C1q and CR3 (CD11b / CD18 dimer), we used iMGL to assess whether synaptic pruning in human microglia primarily involves this pathway. Using unscented CD11b antibodies 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 studies, the data indicate that MERTK plays a minor role in human microglia-mediated synaptic pruning, demonstrating that C1q / CR3 is essential for human microglia-mediated synaptic pruning.

[0093] [Table 5] [Example]

[0094] Validating the utility 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 AD. Therefore, we examined iMGL to determine whether it could phagocytose Aβ or tau, two hallmark AD pathologies. Similar to primary cultured microglia, iMGL internalized fluorescently labeled fibrillar Aβ (Figure 4B, bottom). iMGL also recognized and internalized pHrodo-labeled brain-derived tau oligomers (BDTO) (Figure 4B, top). The emitted fluorescence indicated transport of pHrodo-conjugated BDTO to acidic lysosomal compartments, indicating that iMGL can actively ingest extracellular tau that may be released during neuronal death. These data support recent findings that microglia may play a role in tau propagation in AD and other tauopathies. Collectively, these findings suggest that iMGL can be utilized in high-throughput drug screening assays to identify compounds that enhance Aβ degradation or block exosome-mediated tau release.

[0095] Microglial genes are implicated in late-onset AD, but little is known about how they modify disease risk. Therefore, we investigated iMGL to determine how these genes may affect microglial function and AD risk. Hierarchical clustering using only these 25 AD-GWAS genes demonstrated that iMGL resembled microglia but not peripheral myeloid cells (Figure 4A). In the basal conditions examined, iMGL and microglia expressed many AD-GWAS-associated genes, including those without mouse orthologs, namely CD33, MS4A4A, and CR1. Therefore, iMGL can be used to test how changes in the expression of these genes affect microglial phenotype in a manner that cannot be recapitulated with transgenic mice. Next, we investigated fAβ or BDTO treatment to determine how they affect AD-GWAS gene expression in microglia. After 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), genes involved in Aβ clearance / degradation. BDTO increased the expression of four genes, including CD2AP (4.62 ± 0.45), previously implicated in tau-mediated toxicity. Additionally, six genes were differentially elevated in fAβ compared with BDTO (Table 6). Furthermore, genes that were more enriched in other myeloid cells at baseline, CD33, TYROBP, and PICALM, were upregulated by fAβ and BDTO, suggesting that proteinopathy may alter the phenotype of microglia to resemble infiltrating peripheral myeloid cells (Stalder et al., 2005, Prinz et al., 2011, Chan et al., 2007). In addition to AD-GWAS genes, iMGLs express C9orf72, GRN, LRRK2, and TARDBP, and can be used to test other neurological diseases in which microglia play prominent roles in pathogenesis, such as ALS, FTD, and DLB (Figure ​(Figure11). 11).

[0096] [Table 6] [Example]

[0097] Interactions and functions of iMGLs in the neural environment In the brain, neurons and glia interact with microglia, influencing their function and gene expression. Therefore, we cultured iMGLs with rat hippocampal neurons (21 div) to assess 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 neuron coculture upregulated 156 iMGL genes and downregulated 244 iMGL genes (Figure 5C and 5D). FFAR2 and COL26A1 were two genes differentially expressed in iMGLs cultured with defined factors alone, indicating a developmentally primed microglial profile. In contrast, coculture of 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 the microglial genes CABLES1, TRIM14, MITF, MMP2, and SLCA25 implicates both neuronal surface cues and soluble factors in microglial maturation (Figures 5E and 12).

[0098] A fundamental property of microglia is their surveillance of the CNS environment through highly branched processes. To investigate how iMGL may interact in the CNS environment, we cultured iMGL with human iPSC (hiPSC) 3D cerebral organoids (BORGs). BORGs contain neurons and astrocytes that self-organize into cortical-like networks but lack microglia (Figure 6, Panel B). To test whether iMGL invades BORGs, similar to how microglia enter the developing neural tube, iMGL was added to BORG cultures. By day 3, iMGLs were embedded in BORGs and were no longer detectable in the medium, suggesting rapid iMGL chemotaxis toward neuronal cues (Figure 6, Panel A). Furthermore, iMGLs tiling and extending branched processes of varying degrees in the 3D organoid environment (Figure 6, Panel B). iMGL protrusions were observed in the majority of cells and displayed a morphology similar to in vivo microglia (Figure 6, Panel B). IMARIS 3D image reconstructions of some iMGLs highlight the development of ramified iMGLs in the BORG. To determine whether iMGLs respond to neuronal injury, a 25-gauge needle was pierced into the BORG (long white arrow, Figure 6, Panel C). After injury, iMGLs clustered near the injury site and at the edge of the BORG (Figure 6, Panel C) and adopted a more amoeboid morphology, resembling "activated" microglia found in injured or diseased brains (Figure 6, Panel C). Together, these data demonstrate that iMGLs can integrate within the 3D brain environment, mature, ramify, and respond to injury similar to brain microglia. [Example]

[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 recapitulate 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). The glial-derived cytokine TGFβ is required for the in vivo development of mouse microglia and the maintenance of a microglia-specific transcriptome signature. Differential gene expression analysis confirmed the role of TGFβ in maintaining the transcriptome signature of human microglia. TGFβ differentially expressed 1,262 genes in iMGL cells, whereas 1,517 genes were differentially expressed after TGFβ removal (24 h). Many of the differentially expressed genes were identified as core microglial signature targets, including P2RY12, TGFβR1, and CD33, transcription factors EGR1 and ETV5, and APOE (Figure 16A-C). Gene ontology examination highlighted TGFβ-dependent neurodegenerative disease pathways, including AD, Parkinson's disease, and Huntington's disease (Figure 16D). Furthermore, TGFβ removal resulted in significant changes in many human microglial homeostasis targets also identified as AD GWAS locus genes (Karch et al., 2016), including TREM2, APOE, ABCA7, SPI1 (CELF1 locus), PILRA (ZCWPW1 locus), and the HLA-DR and MS4A gene clusters, suggesting that many of the identified AD GWAS genes function in maintaining microglial homeostasis (Figure 16E), highlighting the utility of iMGL for investigating AD GWAS gene function. [Example]

[0100] Effect of CX3CL1 and CD200 on iMGL phenotype CX3CL1 and CD200 are neuron- and endothelium-derived cues that can further educate iMGLs toward an endogenous microglial phenotype. We tested CX3CL1 and CD200 to determine how the inclusion or exclusion of these factors modulates iMGL phenotype. Addition of CD200 and CX3CL1 to iMGLs increased the expression of select genes, such as COMT (Figure 16B), CD52, a cell surface receptor that binds Siglec-10 and interacts with DAP12 as part of the microglial sensome, and HLADRB5, a member of the MHC II complex implicated in AD, while maintaining similar expression levels of core microglial genes (e.g., P2RY12, TYROBP, OLFML3) and AD risk genes (Figure 17A). Results indicated that CD200 and CX3CL1 modulate the response of iMGLs 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 proteins, surface receptors, or anti-apoptotic events, such as CLU(APOJ). Cells exposed to these two factors differentially responded to fAβ, increasing the expression of genes involved in cell surface recognition of neuronal motifs or phagocytosis of CNS substrates, including MS4A genes, TREM2, TYROBP, CD33, and ABCA7 (Figure 178B). These studies further support the notion that the CD200-CD200R1 and / or CX3CL1-CX3CR1 axes can regulate microglia to respond to neurodegenerative conditions. Therefore, exposure to soluble CNS factors such as CD200 and CX3CL1 may enable access to microglia-specific transcriptional regulatory elements. [Example]

[0101] To examine the effect of direct contact of iMGLs with the CNS environment on iMGL maturation Next, we examined whether iMGL maturation could be achieved through direct contact with the CNS environment. iMGLs were co-cultured with rat hippocampal neurons (21 DIV) to assess how iMGLs respond to neuronal surface 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 neuron co-culture upregulated 156 iMGL genes and downregulated 244 iMGL genes (Figure 5C and D). FFAR2 and COL26A1 were two genes differentially expressed in iMGLs cultured with defined factors alone, indicating a developmentally primed microglial profile. In contrast, co-culture of microglia with neurons increased the expression of Siglec11 and 12, human-specific sialic acid-binding proteins that interact with the neuronal glycocalyx and function in neuroprotection, suppressing pro-inflammatory signaling, thereby maintaining the homeostatic state of microglia. Furthermore, increased expression of the microglial genes CABLES1, TRIM14, MITF, MMP2, and SLC2A5 was observed. Overall, these results implicate both soluble and surface CNS cues as factors in microglial maturation (Figure 5A-F). [Example]

[0102] iMGL interactions in the human brain environment A fundamental property of microglia is their surveillance of the CNS environment through highly branched processes. To investigate how iMGL may interact in the human brain environment, we cultured iMGL with hiPSC-derived 3D cerebral organoids (BORGs). BORGs contain neurons, astrocytes, and oligodendrocytes that self-organize into cortical-like networks but lack microglia (Figure 6). To test whether iMGL invade BORGs, similar to how microglia enter the developing neural tube, iMGL was added to BORG cultures. By day 3, iMGLs were embedded in BORGs and were no longer detectable in the medium, suggesting rapid iMGL chemotaxis toward CNS cues (Figure 6, panels A–C). Moreover, by day 7, iMGLs tiled and extended various degrees of branched processes in the 3D organoid environment (Figure 6, panels D–F). To determine whether iMGLs respond to neuronal injury, BORGs were pierced with a 25-gauge needle. After injury, iMGLs cluster near the injury site and adopt a more amoeboid morphology, resembling "activated" microglia found in injured or diseased brains (Figure 6, panels G-I). Collectively, these data demonstrate that iMGLs are incorporated within the in vitro 3D brain and CNS environment, where they can mature, ramify, and respond to injury similar to brain microglia. [Example]

[0103] Investigating iMGL in the context of the in vivo CNS environment iMGLs were tested in the context of an in vivo CNS environment. iMGLs (day 38) were Rag2- and IL2rγ-deficient mice, and were treated with four knock-in cytokines (M-CSF, M-CSF, and M-CSF). h IL-3 / GM-CSF h ;TPO hWe also expressed human forms of iMGLs and transplanted them into the cortex of MITRG mice, enabling xenografting and survival of bone marrow and other leukocytes (Figure 13). Two months after transplantation, the extent of engraftment in the MITRG cortex was assessed by immunohistochemistry. Human-specific nuclear and cytoplasmic markers, ku80 (hNuclei) and SC121 (hCyto), respectively, were used to distinguish human iMGLs from endogenous microglia. P2ry12 and human-specific Tmem119 antibodies were used to assess the homeostatic status and identity of transplanted microglia. Transplanted human iMGLs coexpressing both ku80 and P2ry12 were abundant in the MITRG brain, suggesting long-term engraftment potential (Figure 13, panels A-D). Higher magnification images showed P2ry12 expression in highly ramified iMGLs, similar to quiescent cortical microglia with a membrane distribution highlighting finer outgrowth processes (Figure 13, panels B-D). Tmem119 also binds to hCyto + hCyto was expressed in both the cell body and the highly arborized processes of the iMGL (Figure 13, panels E-H). + High-magnification images of the cells show that Tmem119 is primarily membrane-bound, consistent with published studies. Collectively, these findings suggest that long-term survival and engraftment of iMGLs give rise to highly ramified 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 neural environment, which translates to potential use in testing human microglia in mouse CNS disease models. [Example]

[0104] Transplant iMGL into the hippocampus to determine how iMGL interacts 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, similar to developing microglia, engrafted and migrated along white matter tracts (Figure 13, panel M). In many cases, iMGLs migrated toward Aβ plaques, extending processes and beginning to wall them off (Figure 13, panels N–P). Numerous iMGLs also began phagocytosing fibrillar 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, extending 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 approval from the UC Irvine Human Stem Cell Research Oversight (hSCRO) and IBC Committees. The use of human tissue was in compliance with and approved by the Institutional Review Board (IRB). Human iPSC cell lines ADRC F5 and ADRC F14 (control subjects) were generated by the UCI ADRC Induced Pluripotent Stem Cell Core using non-integrating Sendai virus (Cytotune). iPSCs were confirmed to be karyotypically normal by G-banding, sterile, and pluripotent by Pluritest (UCLA) analysis. iPSCs were maintained feeder-free on Matrigel (MTG) in complete TeSR-E8 medium (Stemcell Technologies) in a humidified incubator (5% CO2, 37°C).

[0107] Differentiation of iPSCs into hematopoietic progenitor cells (iHPCs) Human iPSC-derived hematopoietic progenitor cells were generated using defined conditions based on previously published protocols with some modifications (Kennedy et al., 2007; Sturgeon et al., 2014). Briefly, iPSCs were triturated to generate a single-cell suspension and plated at 1–6 × 10 cells per well in E8 medium plus Y-27632 ROCK inhibitor (10 μM; R&D Systems). 5 Cells were seeded in 6-well plates. In some embodiments, Y-27632 is replaced with thiazovivin (R&D systems). After culturing the cells under normoxic (20% O2) conditions for 24 hours, the E8 medium was replaced with differentiation medium consisting of IMDM / F12 (50:50), insulin (0.02 mg / ml), holotransferrin (0.011 mg / ml), sodium selenite (0.0134 mg / ml), L-ascorbic acid 2-magnesium phosphate (64 μg / ml; Sigma), monothioglycerol (400 μM), PVA (5 mg / ml; Sigma), L-alanyl-L-glutamine (2 mM), chemically defined lipid concentrate (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) in hypoxic (5% O2) conditions. After 2 days, the medium was changed to basal medium supplemented with FGF2 (50 ng / ml) and VEGF (50 ng / ml). On day 4, the medium was changed to 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 medium was replenished with the above medium. After culturing the cells for an additional 4 days (10 days total), CD43 expression was detected by FACS for iMGL differentiation. + Furthermore, iPSC-derived HPCs (Cellular Dynamics) were isolated from the CD43 + It has been identified as a commercial source of progenitor cells.

[0108] Generation of microglia-like cells from iHPCs CD43 +iHPCs were cultured at 1–2 × 10 per well. 5 Cells were seeded onto Matrigel-coated 6-well plates (BD Biosciences) containing serum-free complete differentiation medium at a density of 1000 x g. Differentiation medium consisted of 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) just before addition to the cells), supplemented with M-CSF (25 ng / ml), IL-34 (100 ng / ml; Peprotech), and TGFβ-1 (50 ng / ml; Militenyi). Cells were replenished with complete differentiation medium every 2 days. On day 12, primary iMGLs were harvested (300 × g for 5 min at 25°C) and a 50% medium change was performed. After 25 days of microglial differentiation (day 35 from iPSCs), iMGLs were cultured for an additional 3 days in complete differentiation medium supplemented with CD200 (100 ng / ml, Novoprotein) and CX3CL1 (100 ng / ml; Peprotech) and cocultured with hippocampal neurons or human cerebral 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 layered on top of Ficoll-Paque and centrifuged in a swinging bucket rotator without the brake (400 × g, 40 min, 18°C). After centrifugation, the plasma and upper layer were removed, and PBMCs were isolated from the interlayer. Cells were then washed once with ice-cold PBS and used immediately.

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

[0111] Monocyte-derived macrophages Isolated monocytes were cultured at 2 × 10 in RPMI-1640 medium in a 37°C, 5% CO2 incubator. 6 Cells were seeded onto tissue-culture-treated 6-well plates at 1000 cells / ml. After 2 hours, the medium was aspirated and discarded, and the adherent 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 cells, M-CSF (25 ng / ml) was added to the wells, and the cells were allowed to differentiate for 5 days.

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

[0113] RNA-seq analysis RNA-seq reads were mapped to the hg38 reference genome using the STAR aligner and to Gencode version 24 gene annotations using RSEM. Genes with expression across all samples (<1 FPKM) were filtered from all subsequent analyses. Differential gene expression analysis was performed on TMM normalized counts using EdgeR (Robinson et al., 2010). Multiple biological replicates were used for all comparative analyses. A p-value ≤ 0.001 and a 2-fold change in expression were used to determine significantly differentially expressed genes for each comparison. 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 Transwell migration assays to ADP were performed as previously described ( De Simone et al., 2010 ; Moore et al., 2015 ). iMGLs (5.5 × 10 4iMGLs (cells / well) were cultured in serum-free basal medium without cytokines for 1 hour. Next, iMGLs were pre-exposed to DMSO or PSB0739 (50 μM, Tocris) for 1 hour at 37°C in a 5% CO2 cell culture incubator. Cells were then washed three times with basal medium and seeded into transwell migration chambers (24-well, 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, cells were washed three times and fixed in PFA (4%) for 15 minutes at room temperature. Cells were stained with Hoechst stain for 10 minutes to visualize cell nuclei. A blinded observer counted the total number of cells per slide, then scraped the cells off the surface, washed them with PBS, and recounted to record the migrated cells. Migration was reported as the number of migrated cells relative to the total number of cells per well. Fluorescent images of the cells were recorded using an Olympus IX71 inverted microscope.

[0115] For calcium imaging, iMGLs were seeded onto poly-L-lysine-coated coverslips and incubated for 1 h with Fura-2-AM (Molecular Probes) calcium dye diluted in Ringer's solution, pH 7.4, containing (in mM): NaCl 140, KCl 4.5, CaCl2 2, MgCl2 1, HEPES 10, glucose 10, and sucrose 5. After 1 h of incubation, the dye was washed three times using Ringer's solution and treated with the P2RY12 inhibitor PSB0739 (50 μM, Tocris) or vehicle (DMSO) for 1 h before use. Baseline Ca was measured. 2+ Signal (I 340 / I 380 ) was measured over 100 seconds, and after a baseline measurement, ADP (10 μM) was introduced under steady flow. 2+Recordings were performed with a Zeiss (Axiovert 35)-based imaging setup, and data acquisition was performed with 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 and fixed with cold PFA (4%) for 20 minutes at 25°C, followed by three washes with PBS. The cells were blocked with PBS containing either 0.05% goat serum or donkey serum and Triton X100 (0.01%) for 1 hour at 25°C. Primary antibodies (1:500) were added in blocking solution overnight at 4°C. The cells were then washed three times with PBS and stained with 1:400 AlexaFluor®-conjugated secondary antibodies for 1 hour at 25°C. After secondary staining, the cells were washed three times and then coverslipped with DAPI-counterstain mounting medium (Fluoromount, Southern Biotech). Primary antibodies used for immunocytochemical analysis included: 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) and fixed with cold PFA (4% w / v) at room temperature for 20 minutes, followed by washing three times with PBS (1x). Cells were blocked with blocking solution (1x PBS, 5% goat or donkey serum, 0.2% Triton X-100) at room temperature for 1 hour. ICC primary antibodies were added at the appropriate dilutions (see below) in blocking solution and incubated overnight at 4°C. The next day, cells were washed three times for 5 minutes with PBS and stained with 1:400 AlexaFluor®-conjugated secondary antibodies at room temperature for 1 hour in the dark.

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

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

[0120] After secondary staining, slides were washed five times with PBS for 5 minutes each. Slides were coverslipped using Fluoromount (Southern Biotech). For mouse brain IHC, brains were collected, fixed, and processed as described above. Free-floating sections were blocked in blocking solution (1x PBS, 0.2% Triton X-100, and 10% goat serum) at room temperature for 1 hour with gentle agitation. For human TMEM119 staining, heat-mediated antigen retrieval was performed before blocking, as previously described (Bennett et al., 2016). Free-floating tissue antigen retrieval was performed by placing free-floating sections in a 1.5 ml microcentrifuge tube containing 1 ml of citrate buffer and placing them in a preheated temperature block set at 100°C. The tissue was heated at 100°C for 10 minutes, then removed and allowed to return to room temperature for 20 minutes. Then, it was washed three times with PBS for 5 minutes before proceeding with the blocking step. For AD mouse brain staining for amyloid plaques, free-floating sections were placed in 1× Amylo-Glo® RTD™ (Biosensis) staining solution for 10 minutes at room temperature without shaking.

[0121] After staining, sections were washed three times with PBS for 5 minutes each, briefly rinsed with MiliQ DI water, and then returned to PBS before blocking. Primary antibodies were added to staining solution (1x PBS, 0.2% Triton X-100, 1% goat serum) at the appropriate dilution (see below) and incubated overnight at 4°C with slight shaking. The next day, sections were washed three times with PBS and stained with 1:400 AlexaFluor®-conjugated secondary antibodies in the dark at room temperature for 1 hour with slight shaking. After secondary staining, sections were washed three times with PBS for 5 minutes each and mounted on glass slides. After mounting, slides were cover-slipped with DAPI counterstain mounting media (Fluoromount, Southern Biotech). Primary antibodies: Rabbit anti-amyloid fibril (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 nuclei (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) *Recommended for use with Alexa Fluor 488 or 555 secondary antibodies only. 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 anti-Pros (1:500; Abcam, ab97387) rabbit anti-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 brightfield imaging Immunofluorescent sections were visualized and images were recorded using an Olympus FX1200 confocal microscope. To avoid nonspecific bleed-through, each laser line was excited and detected independently. All images shown represent single confocal z-slices or z-stacks. Bright-field images of cell cultures were recorded with an Evos XL Cell Imaging microscope.

[0123] Flow cytometer analysis Cells were suspended in FACs buffer (DPBS, 2% BSA, and 0.05 mM EDTA) and incubated with human Fc block (BD Bioscience) for 15 minutes at 4°C. For 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 gated using ZombieViolet™ Live / Dead stain, all from Biolegend (San Diego, CA). Cells were run on a FACs Aria II (BD Biosciences) and analyzed with FlowJo software (FlowJo).

[0124] Cytospins and May-Grünwald-Giemsa staining 1×10 5 Cells were suspended in 100 μl of FACs buffer, added to a Shandon slide (Biomedical Polymers), and assembled using a cytology funnel. The assembled slide containing the cells was loaded onto a cytospin instrument and centrifuged (500 rpm, 5 min). The slide was air-dried for 2 min and immediately stained with 100% May-Grünwald stain (Sigma) for 5 min. The slide was then washed with PBS for 1.5 min and immediately placed in 4% Giemsa stain (Sigma) for 20 min at room temperature. The slide was washed six times with double-distilled H2O and air-dried for 10 min. The slide was preserved using glass coverslips and Permount (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. Analysis of AD-GWAS genes utilized custom Taqman low-density array cards using the primers listed below.

[0126] Isolation of rat cortical and hippocampal neurons All procedures were performed under IUCAC-approved protocols. Primary cortical and hippocampal neuronal cultures were derived from embryonic rats (E18). Briefly, dissected tissues were dissociated with trypsin, triturated, and seeded onto poly-L-lysine-coated 6-well plates in serum-free Neurobasal medium (NB medium) supplemented with B27 (1% v / v). Cells were cultured at 5 × 10 6 Cells were seeded at a density of 1000 cells / ml and maintained in culture until use.

[0127] Co-culture of iMGLs with rat neurons Rat hippocampal or cortical neurons were cultured for 21 days with a 50% medium change every 3–4 days. iMGL was cultured with neurons (1 × 10) at a ratio of 1:5 in 50% iMGL and 50% NB medium. 6 iMGL vs 5x10 6 After 3 days, iMGLs were harvested for RNA isolation.

[0128] Mesoscale multiplex cytokine and chemokine assays iMGL culture medium was replaced with basal medium for 2 hours before stimulation with IFNγ (20 ng / ml), IL1β (20 ng / ml), and LPS (100 ng / ml) for 24 hours, after which cells were harvested for RNA and culture supernatants were assessed for cytokine secretion. To simultaneously assess multiple cytokine and chemokine analytes from iMGL culture supernatants, culture supernatants from each treatment group were 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 as previously described ( Lancaster et al., 2013 ) with some modifications, which are detailed in the Supplementary Information.

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

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

[0132] Human synaptosomes Human tissue samples were obtained at autopsy, minced, and slowly frozen in 0.32 M sucrose containing 10% DMSO and stored at -80°C. To obtain crude synaptosomal fractions, tissues were thawed in a 37°C water bath and homogenized in 10 mm Tris buffer (pH 7.4) containing proteinase inhibitors (Roche) and phosphatase inhibitors (Sigma-Aldrich) using a glass / Teflon homogenizer (0.1-0.15 mm clearance). The homogenate was centrifuged at 1,000 g for 10 min at 4°C, the supernatant removed, and centrifuged again at 10,000 g for 20 min 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 iMGLs and MD-Mφs were incubated with mouse anti-CD16 / 32 Fc receptor block (2 mg / ml; BD Biosciences) for 15 minutes at 4°C. Cells were then stained with anti-CD45-APC clone (mouse cells; Tonbo Biosciences; San Diego, CA) at 1:200 in flow cytometer buffer. Imaging was then performed using an Amnis Imagestreamer. 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. A pure anti-CD11b antibody (Biolegend) was used for CD11b blockade.

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

[0135] ADP migration and calcium imaging assay iMGL(5.5×10 4 iMGLs (cells / well) were cultured in serum-free basal medium without cytokines for 1 hour. Next, iMGLs were pre-exposed to DMSO or PSB0739 (50 μM, Tocris) for 1 hour at 37°C in a 5% CO2 cell culture incubator. Cells were then washed three times with basal medium and seeded into transwell migration chambers (24-well, 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, cells were washed three times and fixed in PFA (4%) for 15 minutes at room temperature. Cells were stained with Hoechst stain for 10 minutes to visualize cell nuclei. A blinded observer counted the total number of cells per slide, then scraped the cells off the surface, washed them with PBS, and recounted to record the migrated cells. Migration was reported as the number of migrated cells relative to the total number of cells per well. Fluorescent images of the cells 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) in 6-well tissue culture-treated plates (BD Falcon) in TeSR-E8 medium (Stem Cell Technologies) at 37°C with 5% CO2 daily. At approximately 80% confluency, iPSCs were detached from the Vitronectin XF substrate using the standard ReLeSR protocol (Stem Cell Technologies), centrifuged, pelleted, and suspended in embryoid body (EB) medium consisting of KO DMEM / F12 (Invitrogen), KOSR (20% v / v), L-alanyl-L-glutamine (2 mM), NEAA (1x), 2-mercaptoethanol (0.1 mM), rhubarb FGF (4 μg / ml), HSA (0.1% v / v), and ROCK inhibitor (50 μM) to form EBs. Approximately 1 × 10 EBs per well of a standard V-bottom 96-well plate coated with Lipidure (1% v / v; AMS Bio) to prevent EBs from adhering to the 96-well plate. 4Cerebral organoids were seeded in EB medium containing bFGF (4 ng / ml) and ROCK inhibitor (50 μM). 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 cerebral organoids were left in basic EB medium for an additional 3 days (7 days total). 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 Millipore). Cerebral organoids were transferred to ultra-low attachment 24-well plates (Corning) at 1–2 EBs per well in 1 ml of NE medium using a cut P200 pipette tip. EBs were neuronated in NE medium for 5 days and then transferred to Matrigel (Corning) using molds made from siloed parafilm and a sterile, empty P200 box. Brain organoids were maintained in 6 cm suspension Petri dishes in differentiation medium consisting of KO DMEM / F12 (50%), neurobasal 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 5 days of exposure to differentiation medium containing vitamin A-free B27, the differentiation medium was changed to an identical formulation except that vitamin A-free B27 was replaced with vitamin A-containing B27, and at this point, cerebral organoids were also transferred to Sigmacote (Sigma) siliconized 125 ml spinner flask bioreactors (Corning) where they were fed weekly for 8 weeks with differentiation medium containing vitamin A. After 12 weeks, Borg were utilized for iMGL coculture studies.

[0137] Isolation of human adult and fetal microglia Briefly, normal-appearing cortical tissue was excised from a pharmacologically refractory, nonmalignant case of temporal lobe epilepsy. The tissue was extensively washed and mechanically dissociated. After gentle enzymatic digestion using trypsin and DNAse, a single-cell suspension was generated before passing through a nylon mesh filter. The single-cell suspension underwent a fickle ultracentrifugation step to remove myelin. Dissociated cells were centrifuged, counted, and plated at 2 × 10 in MEM supplemented with 5% FBS, 0.1% P / S, and 0.1% glutamine. 6 Microglia were grown for 3 days, harvested, and seeded at 1 × 10 cells / mL. 5 Cells were seeded at 0.1 cells / mL and maintained in culture for 6 days, during which time they received two treatments with TGFβ (20 ng / mL) on days 3 and 5. Human fetal brain tissue was obtained from the Fetal Tissue Repository (Albert Einstein College of Medicine, Bronx, NY). Total RNA was isolated using standard Trizol (Invitrogen) protocols and stored at -80°C. In some embodiments, a suspension of small clumps of cells is produced and used in a similar manner to a single-cell suspension.

[0138] Transplantation of iMGL in MITRG and Rag5 × fAD brains All animal procedures were performed in accordance with 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 have been previously characterized (Rongvaux et al., 2014). MITRG mice are designed to allow xenotransplantation and support human bone marrow engraftment. iMGLs were harvested on day 38 and suspended in 1x HBSS containing injection buffer: M-CSF (10 ng / ml), IL-34 (50 ng / ml), and TGFβ-1 (25 ng / ml). iMGLs were delivered using the following coordinates: AP: -0.6, ML: ±2.0, DV: -1.65, using stereotactic surgery as previously described (Blurton-Jones, et al., 2009). Brains were collected from mice 60 days after transplantation according to established protocols (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 and allow for human cell xenografting. iMGLs were transplanted into the hippocampus using the following coordinates: AP: -2.06, ML: ±1.75, DV: -1.95. After transplantation, mice were sacrificed and brains were collected using previously established protocols. Briefly, mice were anesthetized using sodium barbiturate and perfused into the left ventricle with cold 1x HBSS for 4 minutes. Perfused mice were decapitated, and brains were removed and drop-fixed in PFA (4% w / v) at 4°C for 48 hours. Brains were then washed three times with PBS and submerged in sucrose (30% w / v) solution for 48 h before coronal sectioning (40 μm) using a microtome (Leica). Free-floating sections were stored in PBS sodium azide (0.05%) at 4°C until IHC was performed.

[0139] Dot Blot Serial dilutions of protein (2 μl) were blotted onto pre-wetted nitrocellulose paper and allowed to dry. After drying, the blots were blocked with 5% BSA in 1x Tris-buffered saline containing Tween 20 (TBST) for 1 hour at room temperature with gentle agitation. The blots were then incubated with primary antibodies (see below) for 1 hour at room temperature. The blots were then washed three times with TBST for 5 minutes each. The blots were then incubated with HRP-conjugated secondary antibodies (Santa Cruz) at 1:10,000 for 1 hour at room temperature with gentle agitation. After 1 hour, the blots were washed three times with TBST for 5 minutes each. After washing, the blots were dried on filter paper and incubated with Pierce ECL Western Blotting Development Substrate (Thermo Fisher) for 10 minutes in the dark. The blots were 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] It is contemplated that various combinations or subcombinations of the specific features and aspects of the embodiments disclosed above may be made and still fall within the scope of one or more of the present inventions. Furthermore, any specific feature, aspect, method, property, characteristic, quality, attribute, element, etc. disclosed herein in connection with an embodiment may be used in all other embodiments described herein. It is therefore to be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for one another to form varying modes of the disclosed invention. Therefore, it is not intended that the scope of the invention disclosed herein be limited by the specific disclosed embodiments described above. Furthermore, the invention is susceptible to various modifications and alternative forms, specific examples of which have been shown in the drawings and are described in detail herein. However, it is to be understood that the invention is not limited to the specific forms or methods disclosed; on the contrary, the invention encompasses all modifications, equivalents, and alternatives falling within the spirit and scope of the various described embodiments and the appended claims. Any method disclosed herein does not have to be performed in the order described. The methods disclosed herein include specific actions taken by a practitioner, but can also include, explicitly or implicitly, any third-party direction of those actions. For example, an action such as "administering a population of expanded NK cells" includes "directing administration of the population of expanded NK cells." Furthermore, when features or aspects of the present disclosure are described in terms of a Markush group, one of skill in the art will understand that the present disclosure is also thereby described in terms of any individual member of the Markush group, or subgroup of members of the Markush group.

[0142] Ranges disclosed herein also encompass any and all overlaps, subranges, and combinations thereof. Words such as "at most," "at least," "greater than," "less than," and "between" are inclusive of the referenced number. Numbers preceded by terms such as "about" or "approximately" are inclusive of the referenced number. For example, "about 10 nanometers" includes "10 nanometers." [Example]

[0143] iHPC transplantation allows for the testing of human microglial 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 the mouse brain (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. 1. A method for producing human microglia-like cells (iMGLs) from pluripotent stem cells (PSCs), comprising: (i) differentiating PSCs using a medium supplemented with hematopoietic differentiation factors to produce induced hematopoietic progenitor cells (iHPCs); (ii) Microglial differentiation medium was used to differentiate CD43 ＋ differentiating the iHPCs into human microglia-like cells (iMGLs); A method comprising:

2. 2. The method of claim 1, wherein step (i) comprises an incubation period of between 3 and 21 days.

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

4. 4. The method of claim 3, wherein the PSCs are incubated in a hypoxic or normoxic environment on days 1 to 10 of the incubation period.

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

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

7. the concentration of each of FGF2, BMP4, VEGF, TPO, SCF, IL-3, and IL-6 in the medium is about 50 ng / ml; the concentration of activin A in the medium is about 12.5 ng / ml; The concentration of LiCl is about 2 mM; The method of claim 6.

8. CD43 using fluorescence-activated cell sorting (FACS) ＋ 10. The method of claim 1, further comprising isolating the iHPC.

9. CD43 ＋ Isolating iHPCs may be performed using CD43 ＋ 9. The method of claim 8, which is carried out by selecting a marker.

10. 10. The method of claim 9, wherein the isolated iHPC is greater than 80% pure.

11. 11. The method of claim 10, wherein the isolated iHPC is greater than 90% pure.

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

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

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

15. 15. The method of claim 14, wherein the microglial differentiation medium is serum-free.

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

17. The concentration of CSF-1 is about 25 ng / ml; the concentration of IL-34 is about 100 ng / ml; The concentration of TGFβ1 is about 50 ng / ml; 17. The method of claim 16.

18. 2. The method of claim 1, wherein step (ii) comprises maturing the iMGL for an incubation period of between 1 and 5 days.

19. 19. The method of claim 18, wherein step (ii) comprises maturing the iMGL for an incubation period of about 3 days.

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

21. The method of claim 20, wherein the CD200 is human recombinant CD200 and the CX3CL1 is human recombinant CX3CL1.

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

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

24. The produced iMGL is c-kit － / CD45 ＋ The method of claim 1, wherein

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

26. 26. The method of claim 25, further comprising testing for expression of factors that are known markers for commitment to a microglial fate.

27. 27. The method of claim 26, wherein the expression of factors tested includes PU.1 and TREM2.

28. The iMGLs are classified into two distinct populations of iMGLs: (1) CD45 ＋ / CX3CR1 － and (2) CD45 ＋ / CX3CR1 ＋ The method of claim 1 , comprising:

29. 2. The method of claim 1, wherein the iMGL produced is at least 70% pure.

30. 30. The method of claim 29, wherein the iMGL produced is at least 96% pure.

31. 31. The method of claim 29 or 30, wherein the purity level is assessed using expression of a purinergic receptor, P2ry12 expression, Trem2 expression, Iba1 expression, or Pu1 expression.

32. CD43 ＋ iHPC is CD235a ＋ / CD41a ＋ The method of claim 1, wherein

33. 1. A method for producing human microglia-like cells (iMGLs) from pluripotent stem cells (PSCs), comprising: (i) differentiating PSCs into induced hematopoietic progenitor cells (iHPCs); (ii) differentiating the iHPCs to produce iMGLs; A method comprising:

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

35. The iHPC is CD43 ＋ / CD235a ＋ / CD41 ＋ 34. The method of claim 33, wherein:

36. 34. The method of claim 33, wherein the iHPCs are differentiated into the iMGLs in step (ii) by placing the iHPCs in a serum-free differentiation medium.

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

38. The produced iMGL is c-kit － / CD45 ＋ 34. The method of claim 33, wherein:

39. The iMGLs are classified into two distinct populations of iMGLs: (1) CD45 ＋ / CX3R1 － and (2) CD45 ＋ / CX3R1 ＋ 34. The method of claim 33, comprising:

40. 34. The method of claim 33, wherein step (i) comprises an incubation period of between 5 and 15 days and step (ii) comprises an incubation period of between 20 and 30 days.

41. 41. The method of claim 40, wherein step (i) comprises an incubation period of 10 days and step (ii) comprises an incubation period of 25 days.

42. 35. The method of claim 34, wherein step (iii) comprises an incubation period of between 1 and 5 days.

43. 43. The method of claim 42, wherein step (iii) comprises an incubation period of 3 days.

44. A composition of iMGL comprising 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. 45. The composition of claim 44, wherein the TREM2 and P2RY12 are co-localized.

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

47. A composition of iMGLs, comprising expression of CD33, MS4A4A, and CR1 when the iMGLs are in a basal state.

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

49. The composition of any one of claims 44 to 47, wherein the iMGLs migrate in response to ADP.

50. The composition of any of claims 44 to 47, wherein ADP stimulation results in the generation of a calcium transient in said iMGL.

51. The composition according to any one of claims 44 to 47, wherein the iMGL is capable of phagocytosing human synaptosomes.

52. The composition of any one of claims 44 to 47, wherein the iMGL is capable of synaptic pruning.

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

54. The composition of any of claims 44 to 47, wherein the iMGL is capable of phagocytosing Aβ and tau.

55. The composition of any one of claims 44 to 47, wherein the iMGL is capable of phagocytosing fluorescently labeled fibrillar Aβ and pHrodo-labeled brain-derived tau oligomers.

56. 1. A method for profiling the secretion of inflammatory molecules from iMGLs, comprising: (i) treating the iMGL with lipopolysaccharide, IFNγ, TNFα, or IL-1β; (ii) measuring inflammatory markers secreted by said iMGL; A method comprising:

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

58. 1. A method for assessing iMGL migration, comprising: (i) treating the iMGL with ADP; (ii) assessing the motility and migration of iMGLs in response to chemical stimuli; A method comprising:

59. 1. A method of producing calcium transients in iMGLs, comprising: (i) treating the iMGL with ADP; (ii) examining calcium flux signals in the iMGL; wherein the calcium flux signal occurs in response to an electrical, biological, or chemical stimulus.

60. 1. A method for testing microglial phagocytosis of a compound, comprising: (i) exposing iMGLs to a compound selected from the group consisting of Aβ, tau, fluorescently labeled Aβ, and pHrodo-labeled brain-derived tau oligomers, and allowing said compound to be phagocytosed, endocytosed, or ingested by said iMGLs; (ii) measuring said phagocytosis, endocytosis, or uptake of said compound; A method comprising:

61. 1. A method for establishing an iMGL gene expression profile that resembles the in vivo state of iMGL, comprising: co-culturing iMGLs with neurons, astrocytes, or other cells of the central nervous system, thereby recapitulating in vivo conditions for the iMGLs more than exist for the iMGLs when the iMGLs are not co-cultured with the neurons, astrocytes, or other cells of the central nervous system; A method comprising:

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

63. 63. The method of claim 61 or 62, wherein the differentially regulated genes are CABLES, TRIM4, MITF, MMP2, and SLCA25, and said differentially regulated genes are upregulated in iMGLs.

64. A method for incorporating iMGL into a 3D CNS environment, comprising: A method comprising co-culturing iMGLs with hiPSC 3D cerebral organoids (BORGs), wherein the iMGLs migrate to, assemble into, or are integrated into the BORGs.

65. 1. A method for studying 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; (ii) profiling an iMGL omic signature selected from RNA-seq, proteomics, metabolomics, and lipidomics; A method comprising:

66. 66. The method of claim 65, wherein the differentially regulated genes are CD33, TYROPB, and PICALM, and said differentially regulated genes are upregulated in iMGLs.

67. A method for investigating the role of microglia in synaptic pruning and synaptic plasticity, comprising: (i) exposing iMGLs to human synaptosomes; and (ii) assessing phagocytosis of human synaptosomes by said iMGLs.

68. 1. A method for assessing gene regulation in iMGLs in response to neuronal cues, comprising: (i) exposing iMGLs to a factor present in the central nervous system selected from the group consisting of CX3CL1, CD200, and TGFβ; (ii) assessing 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, C9ORF72, LRRK2, and SOD1; A method comprising:

69. 1. A method for assessing the engraftment of iMGLs into the cortex, comprising: (i) transplanting iMGLs into the cortex; (ii) assessing the engraftment of the iMGLs into the cortex; A method comprising:

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

71. 71. The method of claim 69 or 70, further comprising transplanting the iMGL into the cortex of the mouse.

72. 72. The method of claim 71, wherein the mouse is a MITRG mouse.

73. 1. A method for assessing the interaction of iMGL with AD neuropathy, comprising: (i) transplanting iMGL into the brain of a mouse; (ii) assessing iMGL interaction with the mouse brain; A method comprising:

74. 74. The method of claim 73, wherein step (ii) comprises assessing migration of iMGLs into the plaque.

75. 74. The method of claim 73, wherein step (ii) comprises assessing phagocytosis of fibrillar Aβ by iMGL.

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

77. 77. The method of claim 76, wherein the mammalian brain is from a mouse.

78. 78. The method of claim 77, wherein the iMGL is transplanted into the hippocampus of the mouse.

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

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

81. 4. The method of claim 3, wherein the PSCs are incubated in a 5% oxygen environment on days 1-4 of the incubation period, and the PSCs are incubated in a 20% oxygen environment on days 5-10 of the incubation period.

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

83. the concentration of CSF-1 in the medium is between 15 ng / ml and 35 ng / ml; the concentration of IL-34 in the medium is between 80 ng / ml and 120 ng / ml; the concentration of TGFβ1 in the medium is between 30 ng / ml and 70 ng / ml; 16. The method of claim 14 or 15.

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

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

86. 2. The method of claim 1, wherein the microglia differentiation medium of step (iii) comprises the factors CSF-1, IL-34, and a TGFβ mimetic.

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

88. 10. The method of claim 1, wherein the PSCs comprise single cell PSCs or small clumps of PSCs.

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

90. 34. The method of claim 1 or 33, wherein the PSCs are not derived from embryoid bodies.

91. The c-kit － / CD45 ＋ 25. The method of claim 24, wherein iMGL is detected using flow cytometry, immunofluorescence microscopy, qPCR, RNA-seq, or proteomics.

92. 34. The method of claim 1 or 33, wherein the PSCs comprise induced PSCs (iPSCs).

93. 34. The method of claim 1 or 33, wherein the PSCs comprise embryonic stem cells (ESCs).

94. 94. The method of any one of claims 1, 33, 92, or 93, wherein the PSCs are mammalian PSCs.

95. 94. The method of any one of claims 1, 33, 92, or 93, wherein the PSCs are human PSCs.

96. 94. The method of any one of claims 1, 33, 92, or 93, wherein the PSCs are mouse PSCs.

97. The iHPC is CD34 ＋ , CD31 ＋ or CD45 ＋ 36. The method of claim 35, wherein:

98. The PSCs, prior to differentiation, are ＋ , CD235a ＋ , CD41 ＋ , CD34 ＋ , CD31 ＋ , and CD45 ＋ 34. The method of claim 1 or 33, wherein the method is neither of the above.

99. 1. A method of producing human microglia-like cells (iMGLs) from a first type of cell, comprising: (i) differentiating the first type of cells into induced hematopoietic progenitor cells (iHPCs); (ii) differentiating the iHPCs to produce iMGLs; A method comprising:

100. A 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), the culture medium comprising FGF2 and VEGF.

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

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

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

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

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

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

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

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

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