a, b, Analysis of vascular organoids transplanted into nondiabetic or diabetic STZ-treated mice treated with vehicle or DAPT. a, Human blood vessels were transplanted into diabetic STZ-treated mice treated with or without DAPT and were stained for expression of the human-specific endothelial markers VE-cadherin, CD34 (hCD34) and von-Willebrand factor (hvWF). Note the absence of signal in the adjacent mouse kidney. b, Human blood vessels were transplanted into diabetic STZ-treated mice treated with or without DAPT and were stained for the pericyte-specific markers PDGFRβ, NG2, SM22 and SMA. Costaining with a human-specific CD31 (hCD31) antibody was used to identify human blood vessels. a, b, Experiments were repeated independently on n = 3 biological samples with similar results. c, d, CRISPR-Cas9 genome editing was used to generate DLL4 and NOTCH3 knockout iPS cells (NC8). sgRNAs are indicated in the NOTCH3 and DLL4 sequences as well as the generated indels. c, Western blot shows ablation of NOTCH3 expression in target iPS cells. Clone 4 (red) was used for functional assays. FL, full-length NOTCH3; NTM, transmembrane NOTCH3 subunit. d, Immunostaining in control vascular organoids shows expression of DLL4 in endothelial cells (CD31+) but not in CRISPR-Cas9 genome-edited iPS cells. c, d, Experiments were repeated independently n = 2 times with similar results. e, Vascular organoids differentiated from DLL4 knockout (KO) and NOTCH3 knockout iPS cells (NC8) were stained for markers of endothelial cells (CD31) and pericytes (PDGFRβ). Experiments were repeated independently n = 3 times with similar results. f, Quantification of endothelial networks (CD31+ area) in DLL4 knockout and NOTCH3 knockout vascular organoids. Data are mean ± s.d. from n = 3 independent experiments. P values were calculated using a one-way ANOVA. g, Quantification of pericyte number in DLL4 knockout and NOTCH3 knockout vascular organoids. Data are mean ± s.d. from n = 3 independent experiments. P values were calculated using a one-way ANOVA. h, Representative images of basement membranes stained for collagen type IV from control, DLL4 knockout and NOTCH3 knockout vascular organoids (NC8 iPS cells) exposed to hyperglycaemia, IL-6 and TNF (diabetic) or maintained under standard culture conditions (nondiabetic). Experiments were repeated independently n = 3 times with similar results. i, Thickness of continuously surrounded lumina by collagen type IV was measured in optical cross-sections. Each individual measurement from a lumenized vessel is shown as a dot. A total of nondiabetic (control (n = 265), DLL4 knockout (n = 203) and NOTCH3 knockout (n = 215)) and diabetic (control (n = 214), DLL4 knockout (n = 187), NOTCH3 knockout (n = 206)) lumina were analysed for each experimental condition from 3 independent biological replicates with equal sample size. Data are mean ± s.d. ***P < 0.001; one-way ANOVA. Scale bars, 20 μm (a, b, e) and 50 μm (d, h).