At 4 weeks post-exogenous gene induction (WPI), 93.2 ± 3.4% of all human cells were MAP2 positive, indicating successful neuronal conversion of almost the entire ES cell population (Fig. 1b). More importantly, the vast majority of iN cells expressed markers for GABAergic neurons, including the pan-DLX proteins (91.9% ± 6.6%), GABA (81.4% ± 4.7%), GAD1/2 (GAD67/65) (83.5% ± 6.7%) and vesicular GABA transporter vGAT (SLC32A1) (Fig. 1c–g). Whole-cell patch–clamp recordings showed that the cells were able to fire repetitive action potentials with prominent spike adaptation upon depolarization, and they exhibited both active and passive membrane properties indicating functional maturation (Fig. 1h,i). The AMD-induced neurons displayed spontaneous IPSCs and generated large GABAA-receptor-mediated evoked IPSCs upon extracellular field stimulations, confirming the predominant formation of inhibitory synapses (Fig. 1j,k). Moreover, when cocultured with Ngn2-iN cells, the AMD-induced neurons displayed both EPSCs and IPSCs, which indicated that despite having a presynaptic specification for GABAergic outputs, the AMD-iN cells are fully competent to receive excitatory synaptic inputs from glutamatergic Ngn2-iN cells (Fig. 1l,m).
