Although we validated this method predominantly using isogenic comparisons of the effect of Ngn2-overexpression in one NPC line (NSB553-3-C); transduction efficiencies are comparable across NPCs derived from independent hiPSC lines from the same individual, as well as from six independent individuals (Fig. 1E). Over the time course of neuronal induction (described in Fig. 1C), exogenous expression of mNgn2 and hNGN2 in NPCs resulted in accelerated acquisition of neuronal morphology relative to NPCs transduced with GFP alone (Fig. 1F); this effect was robust in NPCs derived from six independent individuals (Fig. 7). After two weeks of induction, MAP2AB levels in mNgn2- and GFP-transduced (as well as hNGN2- and GFP-transduced) NPCs were compared. Immunofluorescence analysis showed increased MAP2AB staining with an enhanced neuronal morphology (1.3-fold, p < 0.001) in mNgn2-induced NPCs (Fig. 2A and B). Consistent with this, we observed increased MAP2AB mRNA expression in mNgn2-induced NPCs, relative to GFP-transduced NPCs (2.3-fold, p < 0.001) (Fig. 2C). Moreover, three-week-old mNgn2-induced neurons showed elevated MAP2AB protein relative to two-week-old mNgn2-induced neurons (1.4-fold, p < 0.001), indicating that neuronal differentiation increased with continued culture (Fig. 2D and E).
