Combinatorial Modulation of Wnt, STAT3, TGF-β, and Tie2 Pathways Drives Brain Endothelial Cell–Like Differentiation from hiPSCs

During development, endothelial cells (ECs) migrate into the brain and acquire blood–brain barrier (BBB) properties such as tight junctions, limited transcellular transport, and high electrical resistance. Although key signaling pathways that are active in vivo have been identified, factors critical in inducing brain EC differentiation in vitro remain unclear. Here, we describe conditions that promote brain EC–like gene expression in human pluripotent stem cell (hiPSC)–derived ECs. Activation of Wnt/β-catenin signaling upregulates the brain EC marker GLUT1 (SLC2A1) while suppressing the peripheral EC marker PLVAP. Simultaneously, stimulation of STAT3 by CNTF together with TGF-β inhibition increases CLDN5 expression. We further found that hiPSC-derived ECs secrete high levels of angiopoietin-2 (ANGPT2) and that razuprotafib (AKB-9778), a PTPRB (VE-PTP) inhibitor, inhibits ANGPT2 and improves monolayer integrity. These results suggest that combinatorial modulation of specific signaling pathways stimulates the differentiation of human brain ECs in vitro .