Characterization of blood-brain barrier-like structures in vhCOs. (a) The EGFP transgene template used to generate cells overexpressing EGFP. (b) EGFP-expressing blood vessel organoids co-cultured with wild-type cerebral organoids. (c) A few EGFP+ cells expressed the CD31+ endothelial marker in the vhCOs. (d) In magnificent images, CD31-expressing cells co-localized with EGFP+ cells. (e) ZO-I+ tight junction proteins detected between endothelial cells. (f) Expression of blood–brain barrier markers (occludin, claudin-5, glucose transporter1, ABCB1 and tight junction protein1) detected by RT-qPCR. Day 15 BVOs, day 18 hCOs, and day 18 vhCOs were analyzed (n = 8). * p < 0.05, ** p < 0.01, *** p < 0.001; unpaired, two-tailed student’s t-test. (g) GFAP+ astrocyte-like cells were non-covered CD31+ endothelial tubes.