Functional maturation and synaptic integration of AD-iN cells in vitro and long-term stability of GABAergic fate in vivo. (a) Current injection-induced action potentials recorded from AD-iN cells as replated on mouse glia for 4 to 6 weeks (red and blue, respectively) or when cocultured with Ngn2-induced excitatory neurons for 6 weeks (brown). (b) AP-generation properties (i) measured as number of APs generated with current-pulse amplitude (left) and as resting membrane potential (Vrest), AP threshold (APt), AP height (APh) and after-hyperpolarization potentials (AHP) (right); and intrinsic properties (ii) measured as membrane capacitance (Cm, left) and input resistance (Rm, right). All values are mean ± s.e.m. (n = 14 cells, 5 WPI; n = 12 cells, 7 WPI;n = 32 cells, 7 WPI Ngn2-iN coculture; Student’s t-test; *, P > 0.05). (c) Sample traces (left) of spontaneous IPSCs and event amplitude and frequency (mean ± s.e.m., right), as recorded from AD-iN cells cocultured with mouse glia for 4 weeks (red) or 6 weeks (blue). (d) Sample traces of train-stimulation-induced evoked IPSCs (left) and first IPSC amplitude and total charge transfer from IPSC trains (mean ± s.e.m., right) recorded from AD-iN cells when coculturedwith mouse glia for 4 weeks (red) or 6 weeks (blue). (c,d) Filled circles represent individual cells (n = 15 cells per condition; Student’s t-test; *, P > 0.05). (e) Representative traces (left) of spontaneous EPSCs and IPSCs recorded from GABAergic AD-iN cell when cocultured with Ngn2-induced glutamatergic neurons for 7 weeks. Arrowheads, network activity. Inset, expanded view of boxed area. Asterisks, EPSC-like events with fast kinetics (magenta) and IPSC-like events with slow kinetics (blue). Pie charts indicate cell fraction with EPSCs (magenta), IPSCs (blue) or both types of responses (mixed, green; n = 54 cells). (f) Representative image of grafted AD-iN cells identified by human nuclei antibodies (magenta) and expressed GABAergic neuron markers including GABA, CR, CB, SST, NPY and PV (green). Histogram shows percentage of human cells that express different markers.
