Fig. 5: Ca²⁺ activity of two-layer cortical tissue and integration with the host explant.

a Tiled fluorescence confocal image of implanted explant with RFP-labelled UNPs and unlabelled DNPs at 3 DPI. Dashed lines delineate cerebral cortex of the mouse and the implanted tissue. The human-specific neural marker HNCAM labels both layers of the implanted tissue while RFP only labels the upper layer. b Quantitative analysis of process outgrowth and neuron migration from the two-layered tissues into the brain explant on 1, 3 and 5 DPI (n = 3, 3, 4, 4, 6, 6 biological replicates from left to right; mean ± SEM; ns not significant; **P < 0.01, ***P < 0.001; ****P < 0.0001; two-sided unpaired Student’s t tests). c A tiled fluorescence confocal image of a two-layer implant with RFP-labelled UNPs and unlabelled DNPs at 5 DPI. d Fluorescence-intensity profiles showing HNCAM expression in both layers of the printed tissue, while RFP expression falls off in the deep layer (n = 5 biological replicates; mean ± SD). e Heatmap showing marker expression by grey value in the upper and deep layers of a tissue implant and in the host at 5 DPI (n = 6 biological replicates). f Video frames of Fluo-4 calcium imaging of the implanted explants reveals spontaneous calcium oscillations (coloured as fire) in individual adjacent cells at 5 DPI. g Single-cell Fluo-4 calcium traces. Regions of interest (ROIs) indicated by arrows in f. h Fluo-4 calcium imaging of an explant implanted with unlabelled DNPs only at 5 DPI. Dashed line shows the interface of the implant and the host (see Supplementary Fig. 8a). i Correlated single-cell calcium traces between implant and the host explant, colour-coded according to their ROIs as indicated in h Scale bar: ΔF/F₀ = 0.8. j Similarity matrix showing communities of neurons with correlated firing patterns. Dashed boxes show neurons that tend to activate together. Colour indicates normalised correlation. k Fluo-4 images of an explant implanted with RFP-labelled UNPs only at 5 DPI. The dashed line shows the interface between the implant and the host (see Supplementary Fig. 8c). l Correlated neuron pairs within k, between the implant and the host at 5 DPI by network analysis. Points representing neurons and the lines between them indicate correlated calcium signals. m Single-cell calcium traces, colour-coded ROIs as indicated in k. Scale bar: ΔF/F₀ = 0.05. n Quantitative analysis of the correlated cell pairs within the host, within the implant and between the host and the implant. Signal pairs with Pearson’s correlations over 0.1 are counted (n = 3 biological replicates; mean ± SEM). Slice thickness: 300 µm. For a, c, h and k: scale bars, 200 µm. For i and m. Pearson’s correlation: *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.