背景:脑微血管内皮细胞(BMEC)在血脑屏障(BBB)中起主要作用,并且对于建立体外BBB模型至关重要。目前,iPSC来源的BMECs(iBMECs)已用于构建具有生理屏障功能的体外BBB模型,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)水平和其底物外排率的降低表明其不成熟的性质。因此,通过优化分化诱导方案,更成熟的iBMEC有利于建立更可靠的体外BBB模型,用于研究中枢神经系统(CNS)药物运输。
方法:为了鉴定人脑内皮细胞命运诱导因子,用Zic3A-转染HUVEC,Zic3B-,和表达Sox18的慢病毒载体。由于SOX18被发现能诱导BMEC特性,我们使用Dox诱导的Tet-on系统在iBMEC分化过程中表达SOX18,并探索SOX18表达对iBMEC成熟的影响。
结果:Sox18介导的iBMEC获得了比正常iBMEC更高的TEER值(>3000Ωcm2)。从第6天到第10天(d6-10组),具有SOX18表达的iBMEC表达了一系列紧密连接标记,并显示Mfsd2a的上调,BBB的特异性标记。d6-10组也表达SLC2A1/Glut1的水平与正常iBMEC一样高,并上调ABCB1/P-gp和ABCC1/MRP1表达。此外,Sox18介导的iBMEC在嘌呤霉素治疗后显示出比正常iBMEC更高的活力,表明SOX18表达可以上调iBMEC中P-gp的活性。
结论:iBMECs中可诱导的SOX18表达获得了BBB表型,包括高TEER值和紧密连接相关基因的上调,内皮细胞(EC)标志物,BBB运输机,和更高的细胞活力后,用嘌呤霉素处理。总的来说,我们为具有SOX18表达的人iPS细胞来源的BMECs的成熟提供了分化方法,描述其对形成用于CNS药物转运研究的体外BBB模型的贡献。
BACKGROUND: 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.
METHODS: 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.
RESULTS: Sox18-mediated iBMECs achieved a higher TEER value than normal iBMECs (> 3000 Ω cm2). 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.
CONCLUSIONS: 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.