Fig. 1: Morphological and functional maturation of synchronized cortical neurons derived from hPSCs.

a, Experimental paradigm. hPSC-derived cortical NPCs were induced for synchronized neurogenesis at d20, and neurons were analysed at d25, d50, d75 and d100. b, Representative images of neuronal cultures stained for TBR1. c, Expression of MKI67 and MAP2 throughout the differentiation by quantitative PCR with reverse transcription (RT–qPCR). n = 2 independent experiments. d–f, Representative reconstructions of neuronal morphologies (d) and quantification of neurite length (e) and complexity (Sholl analysis) (f). d25: n = 16; d50: n = 20; d75: n = 23; d100: n = 18 (neurons from 2 independent experiments). g, Representative traces of electrophysiological recordings of evoked action potentials. h, Quantification of electrophysiological measurement of action potential amplitude and rise slope in neurons over time. d25: n = 25; d50: n = 33; d75: n = 43; d100: n = 29 (neurons from 10 independent experiments). i, Representative trace of mEPSCs at d75. j, Representative maximal intensity projection of Ca²⁺ imaging at d70. k, Representative traces of normalized GCaMP6m intensity in d40 and d70 neurons l, Quantification of amplitude and frequency of spontaneous Ca²⁺ spikes. d40: n = 395; d60: n = 418; d70: n = 299; d80: n = 239 (neurons from 2 independent experiment). m, Synchronous firing rate per min of imaging. n = 10 fields of view (FOV) per timepoint from 2 independent experiments. n, Representative images of SYNI and MAP2 staining in maturing neurons. Regions highlighted in the main image are enlarged on the right. o, Heat map for the normalized expression of selected transcripts important for neuronal functionality by RNA-seq. n = 3 independent experiments. CaMK, Ca²⁺/calmodulin-dependent protein kinase. Data are mean ± s.e.m. Scale bars: 50 μm (b); 100 μm (d,j); 50 μm (n) and 20 μm (n, enlarged view). Two-tailed unpaired t-test (e,h,m). Welch’s one-way ANOVA with Games–Howell’s correction (l).

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