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 co-cultured iN cells with mouse glial cells (Pang et al., 2011). The iN cells reliably produced robust action potentials, and exhibited voltage-gated Na+- and K+-currents that were indistinguishable between iN cells derived from H1 ES and different iPS cell lines (Figs. 4A-4C and S4A). iN cells exhibited massive spontaneous synaptic activity that was blocked by the AMPA-receptor antagonist CNQX (Fig. 4D). Extracellular stimulation evoked EPSCs of large amplitudes, documenting abundant synapse formation (Figs. 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. Thus, consistent with the gene expression profile described in Fig. 3A, EPSCs are entirely due to activation of AMPA-type and not of NMDA-type glutamate receptors, although we did observe small NMDA-receptor mediated synaptic currents in iN cells after more than 4 weeks of culture (Fig. S4B). When we quantified the frequency and amplitude of spontaneous
