Because the effects of NeuroD1 and Ngn2 were similar, we decided to focus only on one factor and chose Ngn2. To selectively culture only cells expressing the transcription factor, we co-expressed a puromycin resistance gene with Ngn2 (allowing us to select for cells expressing Ngn2), and we additionally co-expressed EGFP (allowing us to identify lentivirally transduced cells). In the standard protocol (Fig. 1A), ES or iPS cells were plated on day −2, infected with lentiviruses on day −1, and Ngn2 expression was induced with doxycyclin on day 0. A 24 hr puromycin selection period was started on day 1, and mouse glia (primarily astrocytes) were added on day 2 to enhance synapse formation (Fig. 1B; Vierbuchen et al., 2010). Strikingly, forced Ngn2 expression converted ES and iPS cells into neuron-like cells in less than one week, and produced an apparently mature neuronal morphology in less than two weeks (Figs. 1C and 1D). This is faster than any currently available method for generating neurons from human ES or iPS cells (Table 1).
