Fig. 4: enTsOrg grafts show the composition and functional maturation of principal neurons associated with motor function.

a, UMAP analysis of projections of neurotransmitter-classified neurons with pie diagrams showing the subtype proportions by ST-seq at 7 weeks; n = 2 biological replicates. b, Heat map of neurotransmitter-related gene expression in the enTsOrg and sOrg groups at 7 weeks by ST-seq. c, Representative immunofluorescence images of interneurons (Calb2), cholinergic neurons (ChAT), GABAergic neurons (GAD1) and glutaminergic neurons (vGlut2) in the enTsOrg and sOrg groups at 7 weeks. The magnified areas are shown in the white boxes; white arrowheads indicate signal-positive cells. d, Heat map showing the qualification analysis of Calb2⁺, ChAT⁺, GAD1⁺ and vGlut2⁺ cells in organoid grafts of sOrg and enTsOrg by immunostaining. e, Representative immunofluorescence timeline showing the changes of cholinergic neurons (ChAT⁺) number in organoid grafts after in vivo transplantation; n ≥ 3. f, Statistic analysis of ChAT-positive cells in sOrg and enTsOrg at different time points. The folded line showing the trend of the population of cells. Two-way ANOVA followed by uncorrected Fisher’s LSD (3 wpi, n = 3 biological replicates; 5 wpi, n = 4 biological replicates; 7 wpi, n = 6 biological replicates; 14 wpi, n = 5 biological replicates). g, Schematic diagram of the in vivo optogenetics experimental set-up with representative immunofluorescence image showing the co-localization of virus fluorescent signals HNA, and ChAT-positive cholinergic neurons in the transplanted area of enTsOrg group. h, Effect of optogenetic inhibition of labelled ChAT⁺ neurons on the relative firing frequency (spike count/total count) during a 200 s light on (590 nm, 10 mA, 100% duty cycle, yellow shaded) or light off period in the enTsOrg and sOrg groups at 14 weeks (n = 3 biological replicates). i, Peri-event raster plots and histogram of an example unit in a 5 s light on (590 nm, 10 mA). j, Peri-event spectrogram analysis of an example unit in a 200 s light on (590 nm, 10 mA, 50% duty cycle). k,l, Representative MEP waves (k) and amplitude statistics (l) of each group before, during and after optogenetic inhibition (14 weeks); one-way ANOVA followed by Welch’s correction, n = 4 biological replicates. Scale bars, 100 μm in c and e, and 50 μm in g. Data represent the mean ± s.e.m. in f. Data in l are presented as box-and-whisker plots showing the median (centre line), the 25th–75th percentiles (box limits) and the minimum and maximum values (whiskers); all individual measurements are shown as points overlaid on the plot. Calb2, calbindin 2; GAD1, glutamate decarboxylase 1; vGlut2, vesicular glutamate transporter 2; HNA, human nuclear antigen.

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