In this study, we describe a Ngn2-mediated neuronal induction protocol that begins with hiPSC-derived NPCs, rather than hiPSCs (Fig. 1A). This approach has a number of advantages, as NPCs proliferate robustly and are relatively straightforward to maintain in vitro as they require less frequent feeding and passaging. In addition, NPCs are more amenable to parallel culture of dozens of cell lines and are highly adaptable to automated methods. This makes NPCs an ideal cell source for both the large patient cohort studies required for studying a complex genetic disease, as well as adaptation to high throughput drug and phenotypic screens. Moreover, NPCs are a relatively uniform population committed to forebrain neural fate, potentially reducing methodological variability. Here we report that lentiviral transduction of doxycycline-inducible mouse Ngn2 (mNgn2) or human NGN2 (hNGN2) rapidly yields MAP2AB-positive neurons (Figs. 1 and 2). Ngn2-induced neurons exhibit electrical activity within two weeks and show accelerated formation of SYN1-positive puncta and increased expression of glutamatergic genes, even in the absence of co-culture with mouse cortical neurons or glial cells (Figs. 3 and 4). Moreover, transient mNgn2-expression
