We next explored whether the order of in vitro generation of cortical-layer neurons reflected the in vivo developmental program by labeling cells with EdU for 48 h at day 55 (Fig. 2o,p). Three weeks later, we observed that although the vast majority of SATB2+ cells colocalized with EdU, almost no TBR1+ cells showed EdU labeling. This indicates that most of the superficial-layer neurons are formed after 8 weeks of differentiation in vitro. This process continues for at least another 7 weeks, until the proportion of superficial- and deep-layer neurons is approximately equal (Fig. 2j).
