During embryonic and adult neurogenesis in vivo, Ngn2 is highly expressed at the neuroblast stage, rather than in mature excitatory neurons [38]. Moreover, the Allen Brain Span Atlas shows high NGN2 expression in early fetal human cortical tissue with gradual decreases during embryonic development [39]. Thus, we hypothesized that transient induction of mNgn2 would be sufficient to induce functional excitatory neurons from hiPSC forebrain NPCs. To test this, we removed doxycycline at different time points, and then assayed three-week-old mNgn2-induced neurons (Fig. 5A) by immunofluorescence and qPCR.
