over time after the in vitro stage equivalent to the early mid-fetal period, as shown in the human neocortex13. In contrast, superficial-layer cortical markers increased up to sevenfold from day 52 to day 137 in vitro. The transcription factor BRN2 (or POU3F2), which is expressed in late cortical progenitors and migrating neurons23, reached a peak of expression earlier than SATB2, which defines corticocortical projecting neurons21,22. The relative proportion of superficial-layer neurons was also confirmed by the expression of the homeodomain family proteins CUX1 and CUX2, whose expression is mostly localized to layers II–IV20,24. We found that the generation of these neurons is highly reproducible between hiPSC lines and within and across differentiations of the same hiPSC line (Supplementary Fig. 4). To provide independent validation of the quantification of neural populations from cryosections, we dissociated hCSs into single-cell suspensions and used flow cytometry to quantify the proportion of distinct cell populations (Supplementary Fig. 5).
