Laboratory of Biomedical Innovation, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Laboratory of Cell Model for Drug Discovery, National Institutes of Biomedical Innovation, Health, and Nutrition, Saito-Asagi 7-6-8, Ibaraki, Osaka, 567-0085, Japan.
Fluids Barriers CNS. 2023 Feb 2;20(1):10. doi: 10.1186/s12987-023-00408-5.
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.
脑微血管内皮细胞（BMEC）在血脑屏障（BBB）中发挥主要作用，对于建立体外 BBB 模型至关重要。目前，已将诱导多能干细胞（iPSC）衍生的 BMEC（iBMEC）用于构建具有生理屏障功能的体外 BBB 模型，例如高跨内皮电阻（TEER）和转运蛋白的表达。然而，iBMEC 中相对较低的 P-糖蛋白（P-gp）水平以及其底物的外排率降低表明它们的不成熟性。因此，通过优化分化诱导方案使 iBMEC 更加成熟，有利于建立更可靠的用于研究中枢神经系统（CNS）药物转运的体外 BBB 模型。
为了鉴定人类脑内皮细胞命运诱导因子，我们使用 Zic3A、Zic3B 和 Sox18 表达的慢病毒载体转染 HUVEC。由于发现 SOX18 诱导 BMEC 特性，我们在 iBMEC 分化过程中使用 Dox 诱导的 Tet-on 系统表达 SOX18，并探讨了 SOX18 表达对 iBMEC 成熟的影响。
Sox18 介导的 iBMEC 达到了比正常 iBMEC 更高的 TEER 值（>3000 Ω cm）。在第 6 天至第 10 天（d6-10 组），表达 SOX18 的 iBMEC 表达了一系列紧密连接标记物，并上调了 BBB 的特异性标记物 Mfsd2a。d6-10 组还表达了与正常 iBMEC 一样高的 SLC2A1/Glut1，上调了 ABCB1/P-gp 和 ABCC1/MRP1 的表达。此外，与正常 iBMEC 相比，Sox18 介导的 iBMEC 在经过嘌呤霉素处理后显示出更高的活力，表明 SOX18 表达可以上调 iBMEC 中的 P-gp 活性。
在 iBMEC 中诱导性表达 SOX18 获得了 BBB 表型，包括高 TEER 值和上调紧密连接相关基因、内皮细胞（EC）标记物、BBB 转运蛋白以及经嘌呤霉素处理后的更高细胞活力。总之，我们提供了一种使用 SOX18 表达诱导人 iPS 细胞衍生的 BMEC 成熟的分化方法，描述了它在形成用于 CNS 药物转运研究的体外 BBB 模型中的贡献。
