Gene targeting and experimental strategy for hMGL differentiation and characterization. (A) Schematic representation of the genomic location and intron/exon schematic of AD risk SNPs CD33, INPP5D, TREM2, and SORL1 in this study. (B) Schematic diagram of the analytical workflow for this study. RNA-seq datasets from the hMGL lines (1) are analyzed for cross-regulatory interactions to generate an epistatic model (2) and identify potential pathogenic effectors or signatures. hMGL lines are characterized for physiological microglial function (3) and interactions with Aβ in immunodeficient human MCSF knockin mouse brain xenotransplants (4). (C) Representative sequences of various isogenic clones in AD-associated mutant ESC lines and H9-WT sequences. Repair single-strand donor (ssODN) templates, sgRNA (gray), corresponding amino acids, DNA directionality (arrow, 5′ to 3′) and nucleotide substitutions are shown. For TREM2^R47H, two synonymous mutations were introduced in the repair ssODN, generating a new HindIII restriction site (lowercase) for consequent clone screening. (D) Sanger sequencing and validation of CD33 SNP, INPP5D SNP, TREM2 KO, TREM2^R47H, SORL1 KO, and SORL1^A528T lines and isogenic controls (nontargeting sgRNA). The WT H9 ESC line is heterozygous for G/A INPP5D SNPs; CRISPR-Cas9 editing was performed to convert H9 homozygously to the INPP5D “A” allele. All other modifications were converted homozygously in the H9 ESC lines. (E) After maturation induced by exposure to CD200 and CX3CL1, hMGLs were stained for CX3CR1 (red), CD43 (green), Iba1 (purple), and DAPI (blue) as indicated. Scale bar, 20 µm. (F) Representative inward currents from WT hMGLs; hyperpolarizing voltage steps from −160 mV to −60 mV were applied in the absence (top) or presence of Cs⁺ (bottom). At right panel, quantification of inward currents as measured in the absence (black) or presence of Cs⁺ (gray). (G) Induction of cytokines and chemokines in WT hMGLs stimulated with IL-1β (20 ng/ml) and IFN-γ (20 ng/ml) as determined by ELISA multiplex assay. Heatmaps indicate log₂ fold change of cytokines/chemokines indicated (MCP-1, GPOa, HGF, TNFα) above vehicle treatment. Results are from three replicate cultures in three independent experiments. (H) Representative time-lapse images showing WT hMGL migration toward to ATP source (a pipette tip). (I) Representative images of calcium imaging over the time periods as indicated with 100 µM ATP stimulation. Scale bar, 25 µm. Graphs (right) depict Ca²⁺ traces depicting changes in Fluo-4 fluorescence over the baseline (ΔF/F0) in response to 100 µM ATP in the WT hMGLs. Results are derived from averaged values in three replicate cultures and three experiments. (J) Representative time-lapse images of fluorescent Aβ_1-42 oligomers (red) bound to WT hMGLs, imaged by automated live-cell microscopy. In the adjacent graph, phagocytosis of Aβ_1-42 oligomers in WT hMGLs over time was quantified, as depicted on the left. PI was determined by measuring average fluorescence intensity at each time point in comparison to the 15-min time point (set to 1.0). Images in E–J are representative of three independent experiments. Values represent mean ± SEM from n = 3 independent experiments.