FIGURE 3.

Characterization of ChR2‐engineered forebrain organoids at the early stage. (a) Experimental scheme to generate ChR2‐engineered forebrain organoids. (b) Representative images of forebrain organoids at day 35 immunostained for the NPC marker SOX2 and the neuronal marker TUJ1. Scale bar, 100 μm. (c) Quantification of the diameter of control and ChR2‐engineered forebrain organoids. Forebrain organoids were imaged with brightfield and evaluated (n = 15). Significance was calculated using an unpaired t‐test. (d) Quantification of the number of rosettes in control and ChR2‐engineered forebrain organoids. Two sections per sample, four biological replicates in each group were evaluated (n = 8). Significance was calculated using an unpaired t‐test. (e) Quantification of the relative VZ thickness of control and ChR2‐engineered forebrain organoids. Five rosettes per sample, three biological replicates were analyzed (n = 15). Significance was calculated using an unpaired t‐test. (f) Representative images of forebrain organoids at day 35 immunostained for the proliferation marker Ki67. Scale bar, 100 μm. (g) Quantification of the proliferation rate using Ki67⁺ cells of DAPI⁺ cells. Five rosettes per sample, three biological replicates in each group were evaluated (n = 15). Significance was calculated using an unpaired t‐test. (h) Representative images of forebrain organoids at day 35 immunostained for ChR2. Scale bar, 50 μm. (i) Quantification of ChR2 intensity in control, ChR2‐2, and ChR2‐3 forebrain organoids. Five sections per sample, three biological replicates in each group were evaluated (n = 15). Significance was calculated using an unpaired t‐test.