Extended Data Fig. 9: Chimeroids comparison across protocols and donors.

a, UMAP showing overlapping neighbourhoods of cells, as calculated using Milo. Red and blue colours indicate neighbourhoods with significant enrichment for cells from single-donor or multi-donor Chimeroids, respectively. Point size indicates the number of cells in a neighbourhood, and edge thickness indicates the number of cells shared between pairs of neighbourhoods. b, Beeswarm plot showing shifts in the composition of neighbourhoods of cells, grouped by the cell-type identity of those neighbourhoods. Each point represents a neighbourhood of 50-200 cells with similar gene expression profiles. The vertical axis indicates the enrichment of single-donor cells within a neighbourhood, with positive log fold change values indicating more than expected single-donor cells, and negative values indicating fewer than expected single-donor cells. Neighbourhoods are coloured based on statistical significance of that enrichment: grey, not significantly different from random; red, significant over-enrichment; blue, significant under-enrichment. c-d, Volcano plots showing DEGs, overall and for each donor (c), and for each major cell type (d), between the MD- and SD-NSC Chimeroid protocols. Positive log2 fold change indicates higher expression in the SD protocol. e, Correlation plots comparing the DEGs between each pair of donors in MD-NSC Chimeroids to those between the same pair of donors in the SD-NSC Chimeroids. The x- and y-axies shows log2 fold change in MD and SD, respectively. Point size and colour indicate statistical significance in MD and SD, respectively. f, Heatmap showing the mean absolute value of the log2 fold change between each donor in the MD-NSC Chimeroid protocol (columns) and each donor in the SD-NSC Chimeroid protocol (rows). Lower values imply more similarity; samples from each donor were transcriptionally most similar to samples from the same donor in the other protocol. g, Aitchison distance measuring the dissimilarity in cell type composition between replicates within each protocol, split into comparisons within batches and between batches, and limited to cells derived from the PGP1 and Mito210 donors (as only these two donors are present in all organoid/Chimeroid protocols assayed here). (n = 23 PGP1, 23 Mito210, and 10 fetal samples; Boxes show upper and lower quartiles and median, whiskers show highest/lowest values within 1.5 interquartile range (IQR) of the nearest hinge). The dissimilarity in cell type composition between samples within each of three fetal cortical datasets is also shown, indicating natural variability between individuals. Dotted lines represent mean inter-sample distances within each fetal dataset^20,23,24. Some icons were created using BioRender.