CSF1R inhibition enables the replacement of murine microglia with human G795A microglia in adult mice. (A) Schematic depicting timing of stereotactic iMG injection, PLX3397 treatment (red, 600 mg/kg chow) and endpoints at 90 (10 d of treatment or no treatment [black]), 120 d (30 d of treatment), 120 d (30 d on/30 d off treatment), and 150 d (60 d of treatment). (B) IBA1+ (green) and Ku80+ (red) G795A (top) and WT (bottom) human microglia exhibit limited engraftment within the adult murine brain without PLX3397 treatment (left). G795A microglia remain viable and progressively expand from the injection site across 10–60 (left-right) days of PLX3397 treatment while endogenous murine (Ku80−/IBA1+) and transplanted WT human microglia (bottom) are depleted by CSF1R inhibition. Brain stitch scale bar, 500 µm. Representative 40× scale bar, 50 µm. (C) Quantification of percent human (Ku80+/IBA1+) versus mouse (Ku80−/IBA1+) microglia reveal a progressive replacement of endogenous microglia, with 99% of microglia expressing the human nuclear marker Ku80 within 60 d. Counts calculated from three matched coronal sections per animal (n = 3 biological replicates per condition). (D) G795A microglia proliferate from the initial injection sites throughout the murine brain with a distinctive Ki67+ wavefront (blue, arrows). Scale bar, 100 µm. (E) G795A microglia persist in engrafted regions after cessation of 30 d PLX3397 treatment and immunostain for homeostatic microglia marker P2RY12 (purple). Brain stitch scale bar, 500 µm. Representative 20× scale bar, 100 µm.