Fig. 6. Human iPSC microglia internalizes soluble Aβ species to form extracellular plaques.

a Schematic showing sAβ42s labeled by HiLyte-555 and pHrodo Green continuously fluoresce red, but only fluoresce green under intracellular pH 5 conditions. b Quantitative analysis of red Aβ plaque area and green internalized Aβ. Internalized green Aβ outpace the red extracellular Aβ plaque formation, indicating active Aβuptake throughout the 7 days and proceed before the appearance of red Aβ plaques. c Example images of the plaque formation time-lapse movie. Four different plaque formations are retrospectively labeled. sAβ42s are first internalized by microglia (green) before plaque formation (red) in the center of the cultured microglia. Scale bar = 100 μm. d iPSC-derived microglia were treated with 5 μM sAβ42s, then fixed and stained after 30 min, 6 h, 1 day, and 4 days. Microglia (IBA1, red) internalize small Aβ puncta (green; white—the second row) indicated by white arrowheads (green) after 30 min, then externalize these puncta as large aggregates that are faintly X04 positive (blue; white—bottom row) indicated by white arrows, then form large, extracellular X04-positive plaque structures surrounded by microglia from 1 to 6 days. e Human iPSC-derived microglia were treated with 5 μM soluble Aβ species and various dynamin inhibitors (Dynasore, Dynole 4a, Dynole 34-2) at 0.6 μM for 24 h and plaque-like structures (Methoxy-X04-positive) were quantified as a percentage of control. Treating with dynamin inhibitors decreased plaque formation approximately fourfold in all conditions. f A summary of steps of microglia plaque formation observed. Data are presented as mean values +/− SEM and n = 4 wells. One-way ANOVA with Dunnett’s multiple comparisons test.