Specifically, Ngn2-iN cells expressed at high levels the telencephalic markers Brn-2, Cux1, and FoxG1, which are characteristic of layer 2/3 excitatory cortical neurons, but lacked other prominent forebrain transcription factors (e.g., Tbr1 and Fog2). iN cells consistently expressed AMPA-type glutamate receptors GluA1, A2, and A4, but lacked NMDA-type glutamate receptors 3 weeks after induction (Figure 3A). Moreover, nearly all iN cells expressed vGlut2, and approximately 20% of iN cells expressed vGlut1. iN cells highly expressed GABAA receptors but lacked the vesicular GABA transporter vGAT or the GABA-synthetic enzyme glutamate decarboxylase

(GAD). Ngn2 iN cells expressed all panneuronal find more markers tested, but lacked expression of markers for various glia cell types or for stem cells (Figures 3A and S3B). These measurements show that Ngn2 iN cells are relatively homogeneous and that they constitute excitatory neurons that express telencephalic markers suggestive of

cortical layers 2/3. Arguably the most important question in the production of iN cells—in fact, in the in vitro production of all human neurons—is reproducibility between lines. We therefore assessed this question for the Ngn2-based protocol in great detail. Comparison of the gene expression profiles between iN cells produced by forced differentiation of H1 ESCs and of two independent

lines of iPSCs revealed a striking concordance Bleomycin in vitro in expression patterns (Figures 3B and S3D). There was no major difference between stem cells in the expression of the genes tested. The highly similar transcriptional effects of Ngn2 indicate that forced expression of Ngn2 can override presumptive epigenetic differences between various pluripotent stem cell lines to induce differentiation of a single homogenous population of excitatory forebrain neurons. We next probed the ability of Ngn2-induced iN cells to differentiate into electrophysiologically active neurons and to form synapses. To promote synapse formation, we cocultured iN cells with mouse glial cells (Pang et al., 2011). The iN cells reliably produced robust action potentials, Farnesyltransferase and exhibited voltage-gated Na+ and K+ currents that were indistinguishable between iN cells derived from H1 ESC and different iPSC lines (Figures 4A–4C and S4A). iN cells exhibited massive spontaneous synaptic activity that was blocked by the AMPA-receptor antagonist CNQX (Figure 4D). Extracellular stimulation evoked EPSCs of large amplitudes, documenting abundant synapse formation (Figures 4E and 4F). The kinetics of evoked EPSCs were identical at −70 mV and +40 mV holding potentials, and EPSCs were blocked by CNQX at both holding potentials.