Vasculogenesis in kidney organoids upon transplantation.

"both raw and processed sequencing data are available in arrayexpress under accession number e-mtab-11429. after species demultiplexing (for transplanted organoids) quality controls and filtering a total of 16290 high quality human organoid cells and 6866 high quality chicken host cells were retained for downstream analyses (fig 1d supplementary figs2a 3a and supplementary data 1 ).; unsupervised clustering of the organoid-derived human cells revealed three main cell populations which were identified as nephron mesenchymal and endothelial cells based on expression of established marker genes (fig 1d e and supplementary data 2 ).; upon transplantation however we observed an increase to 4% of ecs at d7+20 (fig 1f and supplementary data 3 ).; within the chicken cells collected with transplanted organoids for scrnaseq an angiogenic ec population was identified (supplementary fig 3b-d and supplementary data 4 ).; unsupervised subclustering of a total of 10198 high quality nephron cells obtained from untransplanted (3116 cells from d7+13; 3963 cells from d7+20) and transplanted (1816 cells from d7+13; 1303 cells from d7+20) organoids resulted in 16 subclusters which we identified based on the top marker genes for each cluster (fig 2a b supplementary fig 2b-d supplementary data 5 6 ).; more specifically 4 subclusters of tubular cells were distinguished: 2 types of proximal tubule cells (#1 and 2) loop of henle-like cells and distal tubule/collecting duct-like cells (fig 2b c supplementary fig 2c and supplementary data 5 7 ).; the immature podocyte early podocyte #1 and 2 and late podocyte clusters expressed increasing levels of markers for podocyte identity ( mafb wt1 ) and differentiation ( nphs1 and 2 podxl clic5 ) (fig 2b supplementary fig 2b and supplementary data 5 ).; this revealed 12 clusters which we identified as proliferating cells fibroblasts mural cells mesenchymal progenitor cells and off-target cell populations based on their top marker gene expression (fig 2e f and supplementary data 8 ).; in transplanted organoids at d7+20 the increase in off-target cell types and mesenchymal progenitors was much less pronounced but we did observe an increase in pericytes/mesangial cells and fibroblasts (fig 2h and supplementary data 3 ).; interestingly a gene set associated with cell-cell junction organization an important event in vessel wall stabilization as well as a gene set involved in the regulation of vasoconstriction reflecting maturation of the vasculature were significantly enriched (fig 3a and supplementary data 9 ).; in transplanted late podocytes we observed enrichment of gene sets associated with vegf production vasculogenesis organization of cell-cell junctions and establishment and maintenance of cell polarity (supplementary fig 6a and supplementary data 10 ).; the gsea for d7+20 transplanted tubular epithelial cells versus untransplanted controls demonstrated enrichment of gene sets associated with establishment and maintenance of cell polarity and of potassium ion transport (supplementary fig 6b and supplementary data 11 ).; key sequencing metrics are summarized in supplementary data 1 .; the following quality control steps were performed for each dataset: (i) genes expressed by less than 10 cells were removed; (ii) cells that expressed fewer than 600 genes (for human datasets) and 200 genes (for chicken datasets) were discarded as low-quality cells (supplementary data 1 ); (iii) cells with a detected number of genes exceeding a "doublet" threshold as listed in supplementary data 1 were excluded (determined by inspecting the cell frequency per total number of genes expressed for each sample); (iv) cells with a fraction of mitochondrial genes >15% (for human datasets) and >5% (for chicken datasets) were also removed (cells with compromised cell membrane/dying or dead cells).; supplementary information supplementary movie 1 supplementary movie 2 supplementary movie 3 supplementary movie 4 supplementary movie 5 supplementary movie 6 supplementary data 1 supplementary data 2 supplementary data 3 supplementary data 4 supplementary data 5 supplementary data 6 supplementary data 7 supplementary data 8 supplementary data 9 supplementary data 10 supplementary data 11 related manuscript file reporting summary publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."

"Competing interests The authors declare no competing interests."

"We are grateful to Christian Freund (hiPSC core facility, LUMC, Leiden, the Netherlands) for providing hiPSC lines (LUMC0072 and LUMC0020), and Melissa Little (Murdoch Children’s Research Institute, Melbourne, Australia) for iPSC-MAFB. We acknowledge the support of Saskia van der Wal-Maas (Department of Anatomy & Embryology, LUMC, Leiden, the Netherlands), Conny van Munsteren (Department of Anatomy & Embryology, LUMC, Leiden, the Netherlands), Manon Zuurmond (LUMC, Leiden, the Netherlands), George Galaris (LUMC, Leiden, the Netherlands), and Annemarie de Graaf (LUMC, Leiden, the Netherlands). This work is supported by the partners of Regenerative Medicine Crossing Borders (RegMedXB) and Health Holland, Top Sector Life Sciences & Health. M.Koning is supported by ‘Nephrosearch Stichting tot steun van het wetenschappelijk onderzoek van de afdeling Nierziekten van het LUMC’. S.J. Dumas is supported by a Marie Skłodowska-Curie fellowship (grant agreement No 846615) from the European Union’s Horizon 2020 research and innovation program. M. Borri is supported by the ‘Fonds voor Wetenschappelijk Onderzoek’ (FWO). L. Lin is supported by the DFF Sapere Aude Starting grant (8048-00072A). Y. Luo is supported by the Danish Research Council (9041-00317B) and European Union’s Horizon 2020 research and innovation program under grant agreement No 899417. H.S. Spijker is supported by a Kolff grant from the Dutch Kidney Foundation. P. Carmeliet is supported by Grants from Methusalem funding (Flemish government), the Fund for Scientific Research-Flanders (FWO-Vlaanderen), the European Research Council ERC Advanced Research Grant EU- ERC74307, and NNF Laureate Research Grant from Novo Nordisk Foundation (Denmark). C.W. van den Berg is supported by the Wiyadharma fellowship (Bontius stichting-LUMC). C.W. van den Berg and T.J. Rabelink are supported by The Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), The Novo Nordisk Foundation Center for Stem Cell Medicine is supported by Novo Nordisk Foundation grants (NNF21CC0073729)."