Rapid and efficient generation of human inhibitory iN cells with Ascl1, Dlx2 and Myt1l. (a) Identification of transcription factor (TF) combinations to generate GABAergic neurons. TF combinations give rise to different fractions of iN cells (pie charts) with spontaneous EPSCs (magenta), IPSCs (blue) or mixed postsynaptic currents (green) and varying frequencies (mean ± s.e.m.; n = 5–7 cells) of EPSCs and IPSCs (histograms). Filled circles represent values measured from individual cells. (b) Representative images of AMD-iN cells 5 weeks after infection (4 weeks after coculture with mouse glia). iN cells were detected with antibodies against MAP2 and Human Nuclear Antigen (HuNu). Bar graphs show percentage of HuNu-positive cellsthat express neuronal marker MAP2 (n = 3 independent experiments). (c–g) MAP2-expressing AMD-iN cells colabel with additional GABAergic markers including DLX proteins, GAD1/2 (GAD67/GAD65), GABA and vGAT. (h) Repetitive series of action potentials (APs) in response to step-current injections in AMD-iN cells. (i) Intrinsic and active membrane properties of AMD-iN cells observed as AP generation in response to amplitude of step-current stimulation (left); resting membrane potential (Vrest), AP threshold (APt), AP height (APh) and after hyperpolarization potentials (AHP) (second left); membrane capacitance (Cm, second right) and input resistance (Rm, right). Values are mean ± s.e.m. (n = 8–18 cells). (j) Spontaneous IPSCs recorded from AMD-iN cells. (k) Evoked IPSCs in AMD-iN cells as elicited by single (top left) or a train (top right) of stimulation (triangles). Inset, expanded view of asynchronous delayed IPSCs. Evoked IPSC amplitude (bar graph) and synaptic depression (filled circles) are presented as mean ± s.e.m. (l,m) AMD-iN cells received spontaneous excitatory (red asterisk and example trace) and inhibitory (blue asterisk and example trace) synaptic inputs when cocultured with Ngn2-iN cells. Pie chart indicates percentages of synaptic event types (IPSC, blue; EPSC, red) recorded from an AMD-iN cell cocultured with Ngn2-induced neurons. Scale bars, 50 um (b,c–f,l).
