Extended Data Fig. 2: Characterization of human iBBB.

a, Three-dimensional vascular network of endothelial cells stained with CD144 scale bar = 200 μm. b, one week after formation iMural cells labeled with SM22 are homogeneously dispersed and rudimentary vessels started forming. After two weeks endothelial vessels formed and iMural cells have homed to perivascular space. c, Astrocytes are dispersed throughout iBBB cultures. Experiments in a-c were repeated at least 3 times. d, mRNA expression of AQP4 in each cell type alone, pair-wise and combined. One-way ANOVA with Bonferroni’s multiple comparison. **, p = 0.0013, 0.002, 0.0035, and 0.0076 for BECs, iMCs, Astrocytes, and BECs + iMCs respectively. ****, p < 0.0001. Center values and error bars are means and SDs from 6 independent RNA isolations. e, iBBB without astrocytes do not stain for AQP4 (green). In iBBBs with astrocytes AQP4 densely stains along endothelial vessels. f, Immunostaining for LAMA4 showing that Matrigel does not contain LAMA4 however iBBB cultures remodel basement membrane surrounding endothelial vessels to contain LAMA4. Experiments in e-f were repeated at least 3 times. g, PLVAP mRNA expression is upregulated in BECs from iBBB cultures compared to BECs cultured alone. Center values and error bars are mean and SD from RNA harvested from six independent wells. h, PLVAP mRNA expression is downregulated in BECs from iBBB upon removal of VEGFA from culture media. Center values and error bars are mean and SD from RNA harvested from six independent wells. Two-sided unpaired student t-test p < 0.0001. i, iBBB cultured in trans-well format express high levels of BBB marker CLDN5 (green) and ZO1 (red). Experiments in e-f were repeated at least 2 times. j, Polarization of ABCG2 was measured by Hoechst transport for both a BECs monolayer and the iBBB from the apical to the basolateral surface and vice versa. Samples treated with the ABCG2 specific inhibitor KO143 were normalized to each respective non-inhibitor treated sample. Stars represent significance determined by two-sided multiple student’s t-test (FDR = 0.01; p = 0.0011) Center values and error bars are mean and SD from 6 independent transwells.