Fig. 2: The patient-derived cerebral organoids display premature cell cycle exit and precocious neuronal differentiation of NPCs.

A The schematic of generation of cerebral organoids from iPSCs. B Bright-field images of the patient-derived cerebral organoids (Patient 1) and the isogenic control organoids (Correct-P1) at different time points. D, day. Scale bar, 400 μm. Quantifications of the area (C) and the perimeter (D) of organoids at different time points. More than 15 organoids from three independent experiments were analyzed for each line. Representative images of SOX2 and TUJ1 staining of day-30 (E) and day-55 (F) organoids. Scale bar, 200 μm in the left column and 50 μm in the right column. G Quantification of the thickness of SOX2⁺ layers and ventricle area in organoids. 18 to 24 images from four individual organoids from each group were counted. H Representative images of SOX2 and Ki67 staining on day-55 organoids. Scale bar, 50 μm. The bar graph shows the percentage of Ki67⁺ cells in SOX2⁺ cells. 14 to 18 images from four individual organoids from each group were counted. I Representative images of the isogenic control (Correct) and the patient-derived (Patient) cortical organoids immunostained with CTIP2, TBR2 and PAX6 antibodies. Scale bar, 50 μm. The inferred VZ-, SVZ-, and CP-like regions are delineated based on the combination of DAPI, PAX6, TBR2, and CTIP2 staining patterns. The bar graph shows the percentages of the indicated cells in Correct (12 total organoids from 2 isogenic control lines) and Patient (12 organoids from 2 patient-derived lines) organoids. J DCX staining (green) of day-28 organoids at 48 h after EdU labeling (red). Scale bar, 50 μm. The bar graph shows the percentage of EdU⁺ DCX⁺ cells in all EdU⁺ cells. N = 10 sections from five organoids each line. K Ki67 staining (green) of day-28 organoids at 48 h after EdU labeling (red). Scale bar, 50 μm. The bar graph shows the percentage of EdU⁺ Ki67⁻ cells in all EdU⁺ cells. N = 12 sections from six organoids each line. Data are presented as the mean ± SEM. The statistical significance was determined using two-tailed unpaired t-test. *: P < 0.05; **: P < 0.01; ***: P < 0.001. ns: no significance.