Supplementary Figure 5: Morphometric assessment of subject-derived neurons throughout maturation.

a, Representative confocal images of the morphological development of ASD and control neurons at different days post retroviral infection (dpi) expressing GFP, early neuronal genes (DCX, PSA-NCAM) and axonal filaments (Smi312). Scale bars 50 μm. Immunohistochemistry was performed in all 13 subject lines and 2 cell culture replicates per time point with similar results. b, ASD neurons were significantly more complex at 4, 7 and 14 dpi (two-way ANOVA with Sidak correction, 4dpi: ***P = 0.0006, 7dpi: *P = 0.0147, 14dpi: ****P < 0.0001). Values show means ± s.d.; ASD (n = 8; 320 technical tracing replicates total), control (n = 5; 244 technical tracing replicates total); n refers to biologically independent subject lines. c, Sholl analysis of neurite length from ASD and control neurons at 4 dpi. Total sholl neurite length complexity was larger in the ASD group as compared with controls (two-way ANOVA with Sidak correction, *P < 0.05, **P < 0.001, ***P < 0.001, ****P < 0.0001). Values show means ± s.e.m.; ASD (n = 8), control (n = 5); n refers to biologically independent subject lines. d, Flow cytometry-based gating strategy for the assessment of hDCX:GFP reporter activity during defined neurodevelopmental stages. Experiments were repeated in all 13 subject lines at 5 different time points with similar results; numeric values represent percentages. e, ASD neurons showed altered dynamics in sustaining premature activation of the inserted hDCX promoter with significantly lower fold-changes required to reach maximum activity at 2 and 5 dpi (two-way ANOVA with Sidak correction, 3 dpi: ***P = 0.0002, 5 dpi: **P = 0.0043). The data were normalized to the NSC stage activity for each line as shown in Fig. 2k. Values represent means ± s.e.m.; ASD (n = 8), control (n = 5); n refers to biologically independent subject lines.