A few striking differences between our NPC-based and previous hiPSC-based neuronal induction methods should be noted. First, we were able to identify SYN1-positive synaptic puncta in mNgn2-induced neurons without astrocyte co-culture, perhaps indicating that prior neural commitment of NPCs, unlike hiPSCs, permits enhanced neuronal maturation via mNgn2-induction. Nonetheless, we expect that co-culture of Ngn2-induced neurons with human astrocytes would enhance synaptogenesis of Ngn2-induced neurons from NPCs, perhaps permitting detection of puncti positive for additional synaptic markers. Second, we found it surprising that our Ngn2-induced neurons showed elevated expression of presynaptic proteins such as SYN1 and vGLUT1, but not the postsynaptic marker PSD95, contrary to previous hiPSCbased induction studies [19–21] and may suggest that Ngn2-induction primarily accelerates the gene networks relevant to presynaptic maturation or that astrocyte co-culture is required for postsynaptic maturation. Third, neuronal induction methods to date maintain transgene expression throughout neuronal maturation [17,19]. Ngn2 decreases during neurogenesis in vivo [38]; here we demonstrate that transient Ngn2-induction, for a period as short as two days, is sufficient to induce functional neurons.
