To characterize the neuronal subtypes present in hCSs and to study the timing of their generation, we validated a panel of antibodies in the human fetal cortex (36 PCW) as probes for deep- and superficial-layer cortical identify (Supplementary Fig. 3). On the surface of the hCSs, we observed a layer of horizontal cells expressing reelin (RELN), suggestive of a MZ19 (Fig. 2i). We then used the validated markers to quantify the relative proportion of cells on cryosections at three separate time points (Fig. 2j). The T-box homeobox protein TBR1 localized in SP and CP and later in layers V–VI20, and reached a peak of expression (40.7% ± 0.3 of all cells, mean ± s.e.m.) at day 76 (equivalent of SP and CPi). CTIP2, a transcription factor involved in specifying subcortical projection neurons21,22, was highly expressed early in hCSs, and decreased 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
