Fig. 3: Hi-Q brain organoids mature over time.

A, B Tissue clearing and wholemount staining of day 20 (Ai-iv) and 60-day (Bi-iv) old organoids show that Hi-Q organoids mature over time. SOX2 and Nestin label NPCs at the ventricular zone (VZ), DCX, Acetylated tubulin, MAP2, TUJ-1, PCP4, and Tau mark neurons at the cortical plate (CP). Note cell body localization of acetylated α -tubulin, MAP2, and TUJ-1 (Arrows in Aii-iii) on day 20, organoids are remodeled into defined cortical plates (CP) on day 60 organoids (Bi-iii). Likewise, the cortical neuronal markers Tau and PCP4 were remodeled into distinct cortical plates in day 60 Hi-Q brain organoids (Aiv-Biv). Therefore, CPs are primitive or thin on day 20, and thick and distinct on day 60. Representative images are shown, and panels show scale bars. Diagrams quantify size differences between time points derived from at least 300 organoids (n = 300) from three independent batches (C), VZ diameter (D), and CP thickness (E). At least 100 organoids (n = 100) across several batches have been sampled for size comparison. At least 25 independent brain organoids (n = 25) have been sampled for VZ and CP thickness. Statistical analysis was carried out by One-way ANOVA, followed by Tukey’s multiple comparisons test, ***P < 0.001. Data presented as mean ± SEM.