The maturation of iPS cell-derived brain microvascular endothelial cells by inducible-SOX18 expression

Brain microvascular endothelial cells (BMECs) play a major role in the blood-brain barrier (BBB), and are critical for establishing an in vitro BBB model. Currently, iPSC-derived BMECs (iBMECs) have been used to construct in vitro BBB models with physiological barrier functions, such as high trans-endothelial electrical resistance (TEER) and expression of transporter proteins. However, the relatively low p-glycoprotein (P-gp) level and a decrease in the efflux ratio of its substrates in iBMECs suggest their immature nature. Therefore, more mature iBMECs by optimizing the differentiation induction protocol is beneficial for establishing a more reliable in vitro BBB model for studying central nervous system (CNS) drug transport. To identify human brain endothelial cell fate-inducing factors, HUVEC was transfected with Zic3A-, Zic3B-, and Sox18-expressing lentivirus vector. Since SOX18 was found to induce BMEC properties, we used a Dox-inducible Tet-on system to express SOX18 during iBMEC differentiation and explored the impact of SOX18 expression on iBMEC maturation. Sox18-mediated iBMECs achieved a higher TEER value than normal iBMECs (> 3000 Ω cm ). From day 6 to day 10 (d6-10 group), the iBMECs with SOX18 expression expressed a series of tight junction markers and showed upregulation of Mfsd2a, a specific marker of the BBB. The d6-10 group also expressed SLC2A1/Glut1 at levels as high as normal iBMECs, and upregulated ABCB1/P-gp and ABCC1/MRP1 expression. Moreover, Sox18-mediated iBMECs showed higher viability than normal iBMECs after puromycin treatment, indicating that SOX18 expression could upregulate P-gp activity in iBMECs. Inducible SOX18 expression in iBMECs gained BBB phenotypes, including high TEER values and upregulation of tight junction-related genes, endothelial cell (EC) markers, BBB transporters, and higher cell viability after treatment with puromycin. Collectively, we provide a differentiation method for the maturation of human iPS cell-derived BMECs with SOX18 expression, describing its contribution to form an in vitro BBB model for CNS drug transport studies.