In summary, the signatures that emerged from our transcriptome analyses as significantly perturbed in probands were transcriptional regulation of cell proliferation/cell fate, neuronal differentiation/process outgrowth, and synaptic transmission. To functionally validate these signatures, we performed morphometric cellular analyses and immunostaining for cell fate markers. We first studied the dynamics of the cell cycle in undifferentiated iPSCs and neuronal progenitors (TD11) by BrdU incorporation (Figure 3A, B). These experiments revealed a significant decrease in cell cycle length in ASD-derived iPSCs (Figure 3A). We saw a similarly strong trend in early neuronal progenitors cultured as monolayers (Figure 3B). However, when we estimated the proportion of proliferating cells in more mature organoids (TD31), there was no significant difference in proliferation between ASD- and control-derived organoids (Figure 3C, D). Taken together, these results suggest that a decrease in cell cycle length could be an early event that is present at the iPSC undifferentiated stage and during the early stages of neuronal differentiation, which is consistent with the transient increase in organoid size at TD11 (Figure S3B).
