Fig. 5: DNA damage accumulation in C9 hiPSCs and ALI-COs.

a, Control and C9 hiPSC survival curves in response to topotecan, a TOP1i, in cultures of healthy control H-L2/C9-L1 hiPSC pairs (left) and genetically corrected isogenic control ISO-L2/C9-L2 hiPSC pairs (right). Data represent the percent of surviving cells as the mean ± s.e.m.; n = 3 independent cultures for each line; extra sum of square F-test. b, Quantification of comet assays for corresponding control and C9 hiPSCs (as seen for a) in response to TOP1i treatment. Graph represents the percentage of comet tails for untreated hiPSCs (U) and 1 h after TOP1i-induced DNA damage (D) or 1 h after TOP1i removal (R). Data show the mean ± s.e.m.; n = 3 independent cultures per group (for untreated H-L2, C9-L1 cells, n = 6 independent cultures per group; >30 cells for each). Two-tailed unpaired t-test. c,d, Confocal microscopy immunofluorescence images (upper) displaying γ-H2AX⁺ foci (green) in the nuclei of CTIP2⁺ DLNs (c) and SOX9⁺ astroglial cells (d) in control and C9 ALI-COs at 150 DIV and quantification (lower). Dashed circles illustrate nuclei defined by DAPI staining (blue in the merged images); γ-H2AX⁺nuclei are labelled by arrowheads. Corresponding bar plot graphs represent γ-H2AX⁺ foci for DLNs (c) and astroglia (d). Data expressed as the mean ± s.e.m. foci per cell per ALI-CO slice; n = 6 (left graphs) and n = 3 independent ALI-COs (right graphs) in 3 experiments; two-tailed unpaired t-test. Scale bars, 10 μm (c,d). See Supplementary Table 3 for detailed statistics.