Extended Data Fig. 11: Macrophages support prenatal skin and skin organoid angiogenesis.

(a) Dot plot showing variance-scaled, mean expression (dot colour) and percent of expressing cells (dot size) of pro- and anti-angiogenic factors and of corresponding receptors in prenatal skin and SkO endothelial cells. Genes encoding the main pro-angiogenic factors secreted by macrophages in prenatal skin are highlighted. (b) Dot plot showing variance-scaled, mean expression (dot colour) and percent of expressing cells (dot size) of vascular endothelial growth factors in prenatal skin and SkOs. (c) Dot plot showing variance-scaled, mean expression (dot colour) and percent of expressing cells (dot size) of genes (vascular endothelial growth factor receptors and endothelial differentiation) in prenatal skin and SkO capillary arteriole cells. (d) Comparison of regulon activity between prenatal skin (x-axis) and SkO (y-axis) capillary arterioles. (e) Gene regulation network for regulons with high specificity score in prenatal skin and/or SkO capillary arterioles. Arrows indicate the direction of regulation from transcription factor to target gene. Edges show the proportion of genes shared by two regulons (colour for proportion in the larger regulon and thickness for proportion in the smaller regulon). (f) Gene network for four regulons with high specificity score in prenatal skin and/or SkO capillary arterioles (GATA2, GATA1, NFATC1, SOX7), and selected GATA2 target genes. The proportion of red in the ring around nodes indicates the proportion of gene ontology terms associated with angiogenesis in the gene set enrichment analysis performed with genes in the network. (g) Tree diagram showing network of interactions (NicheNet) linking the ligand VEGFA (red) to GATA2 as target gene (blue) through identified signalling mediators and transcriptional regulators (grey). Edges representing signalling interactions are coloured red and gene regulatory interactions in blue; edge thickness is proportional to the weight of the represented interaction. (h) Gating strategy used on iPSC-derived macrophages before co-culture (n = 3 batches) to isolate single live cells, analyse expression of macrophage markers (CD45, CD14, CD16, CD206) and exclude dendritic cells (CD1c). (i) Representative images of angiogenesis assay of endothelial cells without (top) and with macrophages (bottom), red arrows indicate disorganised network. Quantification of endothelial density in 2D cultures of iPSC-derived endothelial cells without (n = 6, grey) and with macrophages (n = 6; magenta) at 24, 48 and 72 h of culture from 2 independent differentiation batches. Data are mean ± SD and statistics generated with an unpaired t-test. For details on statistics and reproducibility, see Methods

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