Fig. 2

Differentiation of mESC-D3 into mouse brain endothelial like-cells (mBECs). Flow cytometry analysis assessing transitional differentiation stages of endothelial differentiation. A Brachyury expression was assessed after 24 h of mesodermal induction with 5 ng/ml BMP4 and 30 ng/ml VEGF and B endothelial progenitor Flk1 expression was assessed after 5 days in endothelial differentiation media. C–F GLUT1, CD31, VE-Cadherin and CLAUDIN 5 expression was assessed at the end of the differentiation period (Day 8). Red = unstained controls. G Temporal GLUT1 expression was assessed during the entire differentiation period. Increasing GLUT1 expression is shown with MFI (inset) at Day 1, 3, 5 and 7. The terminally differentiated mBECs exhibit cobblestone monolayer morphology and stained positive for key H endothelial (CD31, green) and (I–K) BBB-specific tight junction proteins (Claudin 5, Occludin and ZO-1; all green). Hoechst counterstain (blue); Scale bar = 20 µm. L In the presence of VEGF, the mBEC formed vascular–like structures in Matrigel assay within 24 h. Green = CFDA staining; Scale bar = 100 µm. M Following stimulation with inflammatory cytokines, the mBECs expressed immune adhesion molecule VCAM-1. Blue = unstained, Purple = non-stimulated; Orange = TNF-α  and INF-γ stimulated. Representative images shown of 5 independent differentiations