Extended Data Fig. 2: Differentiation of iCoMoNSCs.

a, Schematics of iCoMoNSCs differentiation protocol. b, Immunofluorescence of neuronal markers MAP2/NFM, NEUN and DCX at 4 weeks of differentiation. c, Quantification of NEUN-positive cells over time in differentiation reaching approx. 40% at 12 weeks. n = 8 independent fields of view normalized to DAPI⁺ nuclei. Unpaired t tests. Data are from one differentiation and are shown as mean with SEM and p values are two-tailed. 4 weeks vs 12 weeks p = 0.01 d, KI67 immunofluorescence of iCoMoNSCs at passage 6. e, Quantification of KI67-positive cells over time in differentiation showing a drop to 5% at 12 weeks. n = 13 independent fields of view normalized to DAPI⁺ nuclei. Unpaired t tests. Data are from one differentiation and are shown as mean with SEM and p values are two-tailed. 4 weeks vs 8 and vs 12 weeks p < 0.0001. f, 2-photon calcium imaging of iNets bolus loaded with the AM ester form of Oregon Green BAPTA-1 showed typical firing neurons (neurons 1. and 3.). Calcium imaging was repeated twice; once with 2-photon confocal, once with epifluorescent microscope. g, Infrared DIC image of whole-cell patch clamped human neuron at 4 months (representative image from one patch session). h, Example voltage responses of a neuron elicited by tonic current injection. i, Average action potential and input resistance. Box plots indicate distribution median value and the first and third quartiles, and whiskers extend to minimum/maximum. n = 10 individually patched neurons. Data shown are mean with SEM. j, Example current evoked by voltage steps from –60 mV to +40 mV (duration, 100 ms) from a holding voltage of –80 mV. Data recorded from the neuron in (g, h). k, Peak Na+ and K+ current densities plotted vs. voltage. n = 10 individually patched neurons. Data shown are mean with SEM. Scale bars, (b,d) 50 µm, (f) 100 µm, (g) 20 µm.

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