Fig. 3: Clonal FAD hNPCs with high Aβ42/40 ratio showed robust accumulation of detergent-resistant Aβ42 aggregates.

a Comparison of accumulated Aβ levels between differentiated mixed and clonal AD hNPC lines in 3D gels (top panel) and media (bottom panel). Mixed and clonal hNPCs (1.2 × 10⁶) were 3D-differentiated in 24-well plates for 3 and 7 weeks. SDS-soluble protein lysates were prepared from differentiated 3D cultures. The levels of Aβ peptides in both 3D gels and media were measured by Western blot analysis using anti-Aβ antibody (6E10). The overexposed images, in dashed line rectangles (blue: 3 weeks, red: 7 weeks), are presented on the right. Asterisk represents unidentified bands which might be partially cleaved APP fragments, large Aβ, or nonspecific bands detected by 6E10 antibody. b–e The clonal control and FAD hNPCs (3 × 10⁵) were 3D-differentiated in 96-well plates for 6 weeks. Concentrations of Aβ species (Aβ38, Aβ40 and Aβ42) in media (b) and in 3D gels (c) were analyzed by MSD Aβ assay. Aβ42/40 ratios based on the Aβ concentrations in media (d) and in 3D gels (e) were presented. Protein extracts from 3D-differentiated clonal hNPCs were prepared using 5 M guanidine hydrochloride (GuHCl). All data are expressed as mean ± SEM of four independent repeats (black dots). Statistical significances were determined by one-way ANOVA with Tukey’s multiple comparisons test. Aβ values below the detection limit of the MSD Aβ assay were excluded from calculation of Aβ42/40 ratio. Graphs represent only data validated by MSD Aβ assay. f Clonal hNPCs (8 × 10⁶) were 3D-differentiated in 6-well plates for 6 weeks. Samples were harvested and dissolved in 1% sarkosyl lysis buffer. Sarkosyl-insoluble fractions reveal aggregated Aβ species. g ReN-mGAP10#D4 cells were differentiated in 3D cultures for 6 weeks. After fixing with 4% paraformaldehyde, cells were immunostained with antibodies against Aβ (3D6) and MAP2. The extracellular Aβ deposition and MAP2-positive cells were imaged by confocal microscopy. h Representative electron micrographs show Aβ fibrils. Detergent-insoluble fractions were purified from 3D-differentiated ReN-mGAP10#D4 for 9 weeks. Aβ fibrils were labeled with anti-Aβ antibody (3D6)-conjugated gold nanoparticles.