Human G795A iPSC-derived microglia exhibit robust viability and minimal alterations to gene expression in response to CSF1R inhibitors. (A) Crystal structure of hCSF1R-PLX3397 and hCSF1R-PLX5622 highlighting G795 residue in the kinase domain. (B) Molecular modeling of G795 variants G795A, G795C, and G795V demonstrating increasing R-group sizes. (C and D) Caspase 3/7 levels imaged over 24 h in iMG cultured in complete medium treated with 0.1% DMSO, 250 nM, 500 nM, or 1 µM PLX3397 (C) and equivalent concentrations of PLX5622 (D). Data captured from four images/well from n = 6 independent wells. Data represented as mean values ± SEM. (E and F) Quantification of cell death by fluorescent Caspase 3/7 Dye for Apoptosis after 24 h in culture with PLX3397 (E) and PLX5622 (F). Data captured from four images/well from n = 6 independent wells. Data represented as mean values ± SEM. P value, one-way ANOVA (P < 0.0001). Tukey’s HSD; ****P < 0.0001. (G–J) Bulk RNA-seq analysis of WT-, G795A-, and G795C-iMG. (G) Principal component analysis using the top 2,000 most variably expressed genes between WT-, G795A-, and G795C-iMG cultured for 24 h with 0.1% DMSO or 250 nM PLX5622 (n = 4 replicates per line, per condition) revealing that the primary source of variation is the WT response to PLX5622. (H) Linear regression analysis and the coefficient of determination between DMSO-treated G795A or G795C and WT microglia, confirming a high degree of concordance when comparing either the full transcriptome (G795A, R² = 0.96–0.99; G795C, R² = 0.97–0.98) or a 249-gene microglia signature (G795A, R² = 0.97–0.99; G795C, R² = 0.97–0.98). (I–K) Volcano plots comparing 24-h treatment with 250 nM PLX5622, revealing significant transcriptomic alterations (Log₂(FC) ≥ 1; FDR ≤ 0.05) in WT microglia (I), but no significant changes in G795A microglia compared with DMSO-treated cells (J) nor G795C microglia compared with DMSO-treated cells (K).