Strikingly, our approach requires only a single transcription factor and generates large FK228 nmr amounts of human iN cells with robust synapse formation capabilities. Moreover, we demonstrate that the resulting iN cells can be used for analysis of human neuronal short-term plasticity, large-scale Ca2+-imaging, or analysis of loss-of-function states mimicking a human genetic disorder. Thus, the approach we describe may
be generally useful not only to explore the cellular phenotype associated with neuropsychiatric disorders, but also for drug screening endeavors and for mechanistic studies. Following our initial observation that the combined expression of Brn2, Ascl1, and MytL1 induces functional neurons from human ESCs (Pang et al., 2011), we examined whether forced expression of a series of single transcription factors in ESCs and iPSCs might initiate iN cell differentiation. SAHA HDAC supplier As in previous studies (Vierbuchen et al., 2010; Pang et al., 2011), we used lentiviral delivery for constitutive expression of rtTA
(Urlinger et al., 2000) and tetracycline-inducible expression of exogenous proteins driven by a tetO promoter. Surprisingly, we found that overexpressing either neurogenin-2 (Ngn2) or NeuroD1 alone rapidly converted ESCs and iPSCs into neuronal cells (Figure 1 and Figure S1, available online). Since this conversion was based on forced expression Sodium butyrate of a lineage-specific transcription factor and appears to be a direct lineage conversion similar
to lineage conversion between somatic cells, we refer to the resulting neurons as iN cells as previously (Vierbuchen et al., 2010; Pang et al., 2011). 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 coexpressed a puromycin resistance gene with Ngn2 (allowing us to select for cells expressing Ngn2), and we additionally coexpressed EGFP (allowing us to identify lentivirally transduced cells). In the standard protocol (Figure 1A), ESCs or iPSCs were plated on day −2, the cells were 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 (Figure 1B; Vierbuchen et al., 2010). Strikingly, forced Ngn2 expression converted ESCs and iPSCs into neuron-like cells in less than 1 week and produced an apparently mature neuronal morphology in less than 2 weeks (Figures 1C and 1D). This is faster than any currently available method for generating neurons from human ESCs or iPSCs (Table 1).