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. 2022 Sep 8;2(9):100289. doi: 10.1016/j.crmeth.2022.100289

Figure 2.

Figure 2

Amyloid-β and tau pathology in day 120 fAD FBOs

(A) Immunohistochemical staining for amyloid-β (4G8: red), MAP2 (green), and GFAP (magenta) in day 120 414C2 (control) FBOs and PS1-2 (fAD) FBOs. Images are representative of 414C2 (control) FBOs and PS1-2 (fAD) FBOs that were imaged with these markers (n = 3). The nuclei were stained with Hoechst 33258 (blue). Scale bar, 50 μm. See also Figures S3A–S3C.

(B) The Aβ42/Aβ40 ratio was significantly different between control FBOs (iPSCs: 414C2, ESCs: KhES1) and fAD-FBOs (PS1-2, PS2-2). The concentrations of Aβ42 and Aβ40 in the culture medium of day 120 control FBOs and fAD FBOs with 2–3 mm in diameter were measured by ELISA (n = 3). Error bars represent ± SEM of three organoids. Statistical significance was determined using unpaired Student’s t test (∗p < 0.05).

(C) Immunostaining for phospho-tau (AT8: magenta) and total tau (cyan) in day 120 414C2 (control) FBOs and PS1-2 (fAD) FBOs. Images are representative of 414C2 (control) FBOs and PS1-2 (fAD) FBOs that were imaged with these markers (n = 3). The nuclei were stained with Hoechst 33258. Scale bar, 50 μm.

(D) Western blot analysis of RIPA buffer-soluble fractions using tau antibody and AT8 for phospho-tau. Quantification of ratio of phospho-tau/total tau protein revealed a significant difference between 414C2 (control) and PS1-2 (fAD) FBOs. Error bars represent ± SEM of three organoids. Statistical significance was determined using an unpaired Student’s t test (∗p < 0.05) (n = 3).

(E) Immunostaining for aggregated tau (MC1: magenta) and total tau (cyan) in day 120 414C2 (control) FBOs and PS1-2 (fAD) FBOs. Images are representative of 414C2 (control) FBOs and PS1-2 (fAD) FBOs that were imaged with these markers (n = 3). The nuclei were stained with Hoechst 33258. Scale bar, 50 μm.

(F) Schematic representation of phenotypes in fAD FBOs. Amyloid-β pathology and phospho-tau were observed, but tau aggregation was not developed in fAD FBOs.

(G) Representative images of PS1-2 (fAD) FBOs overexpressing GFP (left panels) or Tau-P301L (right panels) by AAV injection immunostained for aggregated tau (MC1: magenta) and total tau (RTM38: cyan). The nuclei were stained with Hoechst 33258 (blue). The FBOs were analyzed 5 weeks after AAV injection. Images are representative of PS1-2 (fAD) FBOs overexpressing GFP or Tau-P301L that were imaged with these markers (n = 2, GFP: n = 3, Tau-P301L). Scale bar, 200 μm.