Extended Data Fig. 1: Phenotypical characterization of vascular organoids.

a, Calponin 1⁺ pericytes are tightly associated with endothelial networks (CD31). Collagen type IV staining is used to visualize the basement membrane. b, Formation of a basement membrane is shown by abluminal collagen type IV deposition. a, b, Experiments were repeated independently n = 10 times with similar results.c, Coculture of differentiated (NC8) endothelial cells and pericytes in a collagen 1:Matrigel matrix. The formed endothelial networks (CD31⁺), formed only weak interactions with pericytes (PDGFRβ⁺) and were not enveloped by a collagen type IV⁺ basement membrane. Experiments were repeated independently n = 3 times with similar results. d, Successful generation of vascular networks from embryonic stem cells (H9) and two independent iPS cell lines. Note how PDGFRβ⁺ pericytes are in close proximity to the endothelial tubes (CD31⁺) and the formation of a collagen type IV⁺ basement membrane. Experiments were repeated independently n = 3 times with similar results. e, Vascular organoid generation from H9 cells. Endothelial networks are shown by CD31 staining and pericytes are shown by PDGFRβ. Experiments were repeated independently n = 10 times with similar results. f, Representative electron microcopy images of vascular organoids (NC8). Note the generation of lumenized, continuous capillary-like structures with the appearance of tight junctions (white arrowheads) and a basement membrane (black arrowheads). Experiments were repeated independently n = 3 times with similar results. g, Tip cells (arrowheads) identified by CD31⁺ filopodia mark newly forming vessels. The collagen type IV⁺ basement membrane is absent at the site of active angiogenesis. Experiments were repeated independently n = 10 times with similar results. Scale bars, 2 μm (f), 10 μm (e (right)), 20 μm (g), 50 μm (a, b, d, e (left)), 100 μm (c (right)) and 500 μm (c (left)).