After 11 days of terminal differentiation (TD11), the organoids were composed of polarized, proliferating progenitors expressing the radial glial cell markers BLBP, NESTIN, PAX6, BRN2, SOX1/2. The radial glial cells underwent mitoses on the apical (luminal) side, whereas neuronal precursor cells expressing the immature neuronal proteins DCX and TUBB3, and more mature NeuN+ neurons after 31 days of terminal differentiation (TD31), accumulated on the basal side of the layer of radial glial cells (Figure 1A; Figure S3C, D). Both ASD-derived and unaffected family member-derived iPSCs had an equivalent potential to generate neuronal cells (Figure 1A, B; Figure S3C, D). We observed no significant change in either perimeter or area of the organoids between control and affected groups, except for a transient increase in ASD-derived organoid size at day 11 (Figure S3B). This is most likely due to intrinsic factors, since the starting number of iPS cells seeded per aggregate was the same in all experiments and the size of EBs at the beginning of the differentiation was not different between patients and controls (see Detailed Experimental Procedures).
