Fig. 4

Cellular changes of iPSC-derived neurons in the co-culture system. a Ca²⁺ oscillations (frequency (Spikes/Min) and amplitude (ΔF/F₀)) of the neurons measured by Ca.²⁺ imaging using the Fluo-8 indicator in the mono-culture model (n = 50) and co-culture model (n = 50) (three independent experiments). ****p < 0.0001 (Unpaired t-test). b PSD-95 staining and the quantification of PSD-95 density (PSD-95 intensity/PSD-95 dot after overlapping with VGLUT2) of the iPSC-derived neurons in the mono-culture model (n = 1,468) and co-culture model (n = 1,242) (three independent experiments). Scale bar; 5 μm. Bars, mean ± SEM. ****p < 0.0001 (Mann-Whitney test). c Quantification of the number of synapses between mono-culture (n = 9) and co-culture models (n = 9) (three independent experiments). Bars, mean ± SEM. ****p < 0.0001 (Unpaired t-test). d Representative transmission electron microscopy (TEM) images of synaptic structures in the mono-culture model and in the co-culture model. Scale bar; 500 nm. e Quantification of the number of synaptic vesicles in the presynaptic terminal of neurons in co-culture models (n = 90) compared to the mono-culture models (n = 90) (three independent experiments). Bars, mean ± SEM. ****p < 0.0001 (Mann-Whitney test). f Representative images of neurite branching of the iPSC-derived neurons in the mono- and co-culture model labeled with pCl-DsRed protein and supplementary stained with anti-RFP antibody. Scale bar; 100 μm. g Sholl analysis showing the complexity of neurites of the iPSC-derived neurons in the mono-culture model (n = 50) and co-culture model (n = 50) (three independent experiments). Bars, mean ± SEM. ****p < 0.0001 (Two-way ANOVA)