Here we report a novel adaptation of Ngn2-induction strategies, whereby hiPSC derived NPCs can be rapidly induced to functional neurons within two weeks. Moreover, transient exogenous transgene expression is sufficient to accelerate neuronal differentiation, synaptogenesis and neuronal activity. This protocol provides a rapid and scalable platform for inducing functional neurons from NPCs, one that is highly amenable to high throughput drug screenings. It remains unresolved why neuronal induction methods, in which pro-neuronal transcription factors are overexpressed, so greatly exceed neuronal differentiation protocols in terms of purity and speed, but we speculate that this may reflect: (i) increased expression levels of key neuronal genes via overexpression relative to levels achieved by growth factor signaling; (ii) positive selection for cells with transgene expression; (iii) reduced heterogeneity owing to spontaneous growth factor secretion by poorly patterned cells; (iv) inconsistent dosing or batch effects by biological protein and small molecules with; (v) variable growth factor receptor expression across independent hiPSC lines derived from different individuals. This is not to say that we believe that there is an inherent biological limitation in directed differentiation protocols, only that in this case, Ngn2-overexpression improves the purity, yield and rate of maturation of excitatory neurons from NPCs.
