Extended Data Fig. 10: NOTCH3 expression and signalling in vascular organoids.

a, Vascular organoids were cultured under nondiabetic or diabetic conditions and costained for NOTCH3 expression and markers of endothelial cells (CD31) and pericytes (PDGFRβ). Predominant localization of NOTCH3 in pericytes is indicated by the white arrowheads. Experiments were repeated independently n = 3 times with similar results.b, RT–qPCR of endothelial cells (CD31⁺), pericytes (PDGFRβ⁺) and the remaining CD31⁻PDGFRβ⁻ cells shows the highest expression of NOTCH3 in pericytes. Data are mean ± s.e.m. of 3 independent experiments. c–h, Human vascular organoids transplanted into nondiabetic control or diabetic STZ-treated mice were analysed for NOTCH3 expression and the Notch downstream targets HES5 and HEY1. c, e, g, Histological sections of transplanted human blood vessels were stained for NOTCH3 (c), HES5 (e) or HEY1 (g) and costained for SMA (pericytes) and hCD31 (human endothelium). Note the localization of NOTCH3, HES5 as well as HEY1 in SMA⁺ pericytes. Experiments were repeated independently on n = 3 biological samples with similar results. d, f, h, Quantification of NOTCH3 (d), HES5 (f) and HEY1 (h) expression. Pericytes were segmented using SMA and the intensity of immunofluorescence signal for each marker was measured. Data are mean ± s.e.m. of n = 3 mice. The P value was calculated using a two-tailed Student’s t-test. i, Diabetic mice transplanted with human vascular organoids (H9 ES cells) were treated with a NOTCH3-blocking antibody. Basement membrane thickness of individual human blood vessels (hCD31⁺) of nondiabetic (n = 223), diabetic and vehicle-treated (n = 212), and diabetic and anti-NOTCH3 antibody-treated (n = 143) mice was determined based on collagen type IV staining. n = 3 nondiabetic and diabetic and vehicle-treated mice; n = 2 diabetic, anti-NOTCH3 antibody-treated mice. Data are mean ± s.d. Scale bars, 50 μm (a, c, e, g, i).