背景:基于原发性小鼠的血脑屏障(BBB)模型,牛,和猪脑毛细血管内皮细胞培养一直被认为是具有适当特性的可靠模型,以检查小分子的功能运输。然而,物种差异有时会使将这些模型的结果转换为人类环境变得复杂。在过去的十年里,已经从干细胞来源产生了脑毛细血管内皮样细胞(BCECs),以体外模拟人BBB。本研究的目的是使用来自hIPSC系SBAD0201的人诱导多能干细胞(hiPSC)衍生的BCECs来建立和表征人BBB模型。
方法:使用转录组学评估模型,蛋白质组学,免疫细胞化学,跨内皮电阻(TEER)测量,and,最后,用于评估选定转运蛋白和受体(GLUT-1,LAT-1,P-gp和LRP-1)的功能的转运测定法。
结果:所得的BBB模型显示出平均TEER为5474±167Ω·cm2,并且在紧密连接区形成了claudin-5,ZO-1和occludin表达的细胞单层。细胞单层表达典型的BBB标记VE-cadherin,VWF,PECAM-1转录组学和定量靶向绝对蛋白质组学分析显示,溶质载体(SLC)转运蛋白的丰度很高,而外排转运蛋白的表达相对较低。使用GLUT-1,LAT-1和LRP-1底物和抑制剂的转运测定证实了这些转运蛋白和受体在模型中的功能活性。转运试验表明,P-gp在模型中没有功能性表达,尽管抗体染色显示P-gp位于腔膜。
结论:结论:新的SBAD0201衍生的BBB模型形成紧密的单层,被证明可用于研究GLUT-1,LAT-1和LRP-1介导的跨BBB转运.然而,该模型不表达功能性P-gp,因此不适合进行药物外排P-gp相关研究。
BACKGROUND: Blood-brain barrier (BBB) models based on primary murine, bovine, and porcine brain capillary endothelial cell cultures have long been regarded as robust models with appropriate properties to examine the functional transport of small molecules. However, species differences sometimes complicate translating results from these models to human settings. During the last decade, brain capillary endothelial-like cells (BCECs) have been generated from stem cell sources to model the human BBB in vitro. The aim of the present study was to establish and characterize a human BBB model using human induced pluripotent stem cell (hiPSC)-derived BCECs from the hIPSC line SBAD0201.
METHODS: The model was evaluated using transcriptomics, proteomics, immunocytochemistry, transendothelial electrical resistance (TEER) measurements, and, finally, transport assays to assess the functionality of selected transporters and receptor (GLUT-1, LAT-1, P-gp and LRP-1).
RESULTS: The resulting BBB model displayed an average TEER of 5474 ± 167 Ω·cm2 and cell monolayer formation with claudin-5, ZO-1, and occludin expression in the tight junction zones. The cell monolayers expressed the typical BBB markers VE-cadherin, VWF, and PECAM-1. Transcriptomics and quantitative targeted absolute proteomics analyses revealed that solute carrier (SLC) transporters were found in high abundance, while the expression of efflux transporters was relatively low. Transport assays using GLUT-1, LAT-1, and LRP-1 substrates and inhibitors confirmed the functional activities of these transporters and receptors in the model. A transport assay suggested that P-gp was not functionally expressed in the model, albeit antibody staining revealed that P-gp was localized at the luminal membrane.
CONCLUSIONS: In conclusion, the novel SBAD0201-derived BBB model formed tight monolayers and was proven useful for studies investigating GLUT-1, LAT-1, and LRP-1 mediated transport across the BBB. However, the model did not express functional P-gp and thus is not suitable for the performance of drug efflux P-gp reletated studies.