Fig. 4

Timing-dependent neuroprotection by hiMG in hiNS(+) during two different chronic Aβ treatment protocols indicating hiMG are effective in phagocytosing Aβ A Schematic illustrating 3-weeks (3w Aβ hiNS(+)) and 5-weeks (5w Aβ hiNS(+)) long chronic Aβ protocols. In each, hiMG were transferred 10 days before the end of both protocols. Tissue was harvested at DIV 60 for subsequent imaging. B Quantification of hiNS areas covered by Aβ (stained for 4G8) reveals less Aβ burden after 3w than 5w with a significant reduction in Aβ levels in 3w Aβ hiNS(+) compared to 3w Aβ hiNS(−) (N = 6 each). Statistical testing performed using two-way ANOVA followed by Holm-Sidak post-hoc tests on logarithmically transformed data. C Co-localization of Aβ in IBA1 or lectin-stained hiMG indicates phagocytosis of highly aggregated (mOC87 positive) Aβ D CD68 immunofluroescence confirms phagocytic microglia-like cells (white arrows) in both, 3w/5w Aβ treated hiNS(+). E In both, 3w/5w Aβ treated hiNS(+), Aβ co-localizes with hiMG cell bodies (see white arrows). Only at 5w, hiMG appear to clear volumes of tissue from Aβ leaving dark shadows in the Aβ staining pattern (see red arrows). F Conjugation of Aβ oligomers with pHrodo green (pHrodo-Aβ) confirms functional phagocytosis of Aβ in hiMG. Successful microglial uptake of pHrodo-Aβ was first tested in 2D hiMG. G: 3w Aβ hiNS(+) were treated for 1w with pHrodo-Aβ to confirm microglial Aβ phagocytosis in 3D. Additional co-staining for IBA1 and GFAP reveals that microglia are the main cell type phagocytosing Aβ in hiNS (N = 6 hiNS, IBA1 co-stainings; N = 3 hiNS, GFAP co-stainings). Statistical testing was performed using an unpaired student’s t-test