Figure 7. Re-introduction into the CSF1R^−/− CNS rescues microglial character.

(A) Schematic showing experimental paradigm, left to right: isolation of microglia by magnetic bead separation, culture for 4 hours in TIC + 5% FCS, intracranial injection into CSF1R^−/−pups (P0 to P2), 2 weeks incubation in vivo, analysis of engrafted cells. (B) Cultured mouse microglia demonstrate loss of signature gene expression by 4 hours that is sustained at 6 days. (C) Cultured microglia (blue) show reduced immunoreactivity to Tmem119 after 4 hours (left) or 16 hours (right) in culture compared to acutely purified cells (black) by flow cytometry. (D) FACS histograms of CD11b⁺CD45^Lo gated cells from microglia-injected CSF1R^−/− brains or WT controls showing that engrafted microglia show near-WT levels of Tmem119 immunoreactivity two weeks after injection. (E) The geometric mean florescence intensity for Tmem119 immunostaining rapidly decreases in culture, but returns to WT levels in cells cultured for 16 hr then re-engrafted. (F) Engrafted brains have Tmem119 immunopositive ramified microglia, whereas non-engrafted CSF1R^−/− brains lack these cells. Averages are mean ± sem. Scale bar = 50 μm. * P < 0.001 one-way ANOVA with Dunnett’s comparison to 0 hr.