Fig. 2. Establishing a human model of the BBB using iPSC and microfluidic technologies.

A Model showing the BBB chip and experimental design. Inlet arrows indicate where the cells and ECM gel are loaded. B Gene expression validation by RT-qPCR of markers of endothelial cell identity in primary human brain microvascular endothelial cells (hBMEC) or BMEC-like cell monolayers. The heatmap represents the log₂(fold change) values of hBMEC or control BMEC-like cells vs. control iPSCs. C Western blot-based quantification of tight junction proteins ZO-1, CD31, VE-cadherin and claudin 5 in hBMEC and BMEC-like cell monolayers normalized to GAPDH loading control. Data are represented as the combined protein levels for three different control iPSC lines independently differentiated into BMEC-like cells. D Confocal images of immunostained control BMEC-like cells illustrate the expression and localization of tight junction proteins VE-cadherin (green), occludin (green), claudin-5 (green), ZO-1 (red) and merged claudin-5 (red) and ZO-1 (green) with the nuclear marker DAPI represented in blue. E Confocal images of an immunostained BBB chip indicate expression of ZO-1 and Glut1 (white, bottom center and right panels), and GFAP and αSMA (white, left panels). Blue structures in all images represent the nuclear marker DAPI. F Retention of fluorescein, 4.4 kDa dextran-TMRE and p-glycoprotein substrate rhodamine over a 40-min incubation in BBB^CTL vessels cultured for 6 days in vitro (6 DIV). G Graphs showing BBB^CTL vessel permeability to IgG. H–K Graphs showing vessel permeability to rhodamine (H, J) and 4.4 kDa dextran-TMRE (I, K) at 6 DIV when a BBB^CTL is prepared with pericytes only (P only) in the absence of iPSC-derived astrocytes (H, I), or with astrocytes only (A only) (J, K). Data are from three (B, F–K) biological replicates; in (C), data were produced using a total of six biological BMEC-like cell replicates originating from three independent iPSC lines. Error bars represent mean + SEM. Statistical analysis was performed using two-tailed unpaired Student’s t test with equal standard deviation (s.d.), Scale bars: 20 µm (D), 50 and 200 µm (E). The BBB^CTL and BBB^G2019S nomenclature refers to the presence of either control or LRRK2 G2019S astrocytes in the brain compartment of the BBB chip. P_app apparent permeability, s seconds. Source data are provided as Source Data file.