Fig. 1

Three-dimensional BBB model incorporating iBMECs displays stable and low paracellular permeability. A Schematic illustration of the differentiation protocol. WTC hiPSCs were sequentially treated with mTESR1 media (2 days), unconditioned media without bFGF (UM/F−; 6 days) and retinoic acid-supplemented endothelial media (EC + RA; 2 days). iBMECs were purified by a one-hour sub-culture on collagen IV and fibronectin-coated plates, and then used within 2D or 3D models. B Fabrication and imaging of iBMEC microvessels. 150 μm diameter channels patterned in collagen I were seeded with iBMECs to form a confluent endothelium. Insets below show phase contrast and fluorescence images along the experimental timeline. C Representative images show organization of iBMEC microvessels with localized tight junctions (TJ), plasma membrane (PM), and actin cytoskeleton (ACTB) expression under confocal imaging (max. intensity projection shown). D Representative images following perfusion with Lucifer yellow. E Permeability of Lucifer yellow on day 2 across microvessel conditions: iBMEC microvessels (n = 14), iBMEC Transwells (n = 11), isogenic iEC microvessels (n = 4 microvessels), and iEC Transwells (n = 4). F Time course of Lucifer yellow permeability over one week for iBMEC microvessels (n = 14 and 4, respectively) and iBMEC Transwells (n = 11). G Confocal images of iBMEC-PM microvessels highlighting the glycocalyx and showing solute perfusion in the lumen. (Left) Wheat germ allglutinin staining of iBMEC glycocalyx. (Right) 10 kDa dextran did not accumulate within glycocalyx or iBMECs (60 min after exposure). Data are presented as mean ± SD. Significance calculated using a Mann–Whitney test. * < p 0.05, ***p < 0.001. See also Additional file 1: Figs. S1–S3); permeability experiments were conducted across TJ, PM, and ACTB-labeled source cells