Fig. 1

Induction of barrier properties brain microvascular endothelial cells (BMECs) following co-culture with pericytes, neurons, and/or astrocytes. a A completely isogenic blood brain barrier model was obtained by differentiating BMECs, neurons, astrocytes and pericytes from IMR90C4 induced pluripotent stem cells (iPSCs). BMECs cultured in monoculture conditions (MC) were directly compared to BMECs in co-culture up to 96 h past initial seeding onto Transwell filters. BMECs were co-cultured with mouse 3T3 fibroblasts (F), a mixture of iPSC-derived neurons and astrocytes (1:3; NA) or Pericytes (P). BMECs were initially co-cultured with pericytes for 24 h and then transferred to co-culture with a mixture of neurons and astrocytes (1:3) for the remainder of the experiment (PNA). b Trans-endothelial electrical resistance (TEER) profile of the experimental groups defined in panel (a) over a 96 h period. Statistical significance calculated using ANOVA. *p < 0.05 versus MC, F, ^#p < 0.05 versus P, ^†p < 0.05 versus NA. Values are mean ± SD of three replicates from a single differentiation and experiments were repeated for two additional differentiations for verification of reported statistical trends. c TEER profile of retinoic acid- treated BMECs over a 96 h period. *p < 0.05 versus MC, F, ^#p < 0.05 versus P, ^†p < 0.05 versus NA. Values are mean ± SD of three replicates from a single differentiation and experiments were repeated for two additional differentiations for verification of reported statistical trends. d Immunostaining of iPSC-derived BMECs following co-culture for 48 h in the PNA experimental condition probing for tight junction proteins, claudin-5 and ZO-1, endothelial cell markers, PECAM and VE-Cadherin, and glucose transporter, GLUT1. Scale bar = 100 μm. e Immunostaining of iPSC-derived neurons, astrocytes, and pericytes following co-culture with RA-treated BMECs for 48 h probing for β-tubulin III, GFAP, PDGFR-β, and NG2. Scale Bar = 100 μm