Endres Leo M, Hathcock Sarah F, Schubert-Unkmeir Alexandra, Kim Brandon J
Institute for Hygiene and Microbiology, University of Würzburg.
Institute for Hygiene and Microbiology, University of Würzburg; Department of Biological Sciences, University of Alabama;
J Vis Exp. 2020 Jul 14(161). doi: 10.3791/61400.
Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central nervous system (CNS) by penetrating highly specialized brain endothelial cells (BECs). As Nm is a human-specific pathogen, the lack of robust in vivo model systems makes study of the host-pathogen interactions between Nm and BECs challenging and establishes a need for a human based model that mimics native BECs. BECs possess tighter barrier properties when compared to peripheral endothelial cells characterized by complex tight junctions and elevated trans-endothelial electrical resistance (TEER). However, many in vitro models, such as primary BECs and immortalized BECs, either lack or rapidly lose their barrier properties after removal from the native neural microenvironment. Recent advances in human stem-cell technologies have developed methods for deriving brain-like endothelial cells from induced pluripotent stem-cells (iPSCs) that better phenocopy BECs when compared to other in vitro human models. The use of iPSC-derived BECs (iPSC-BECs) to model Nm-BEC interaction has the benefit of using human cells that possess BEC barrier properties, and can be used to examine barrier destruction, innate immune activation, and bacterial interaction. Here we demonstrate how to derive iPSC-BECs from iPSCs in addition to bacterial preparation, infection, and sample collection for analysis.
脑膜炎球菌性脑膜炎是一种危及生命的感染性疾病，当脑膜炎奈瑟菌（脑膜炎球菌，Nm）通过穿透高度特化的脑内皮细胞（BECs）进入中枢神经系统（CNS）时就会发生这种感染。由于Nm是一种人类特异性病原体，缺乏强大的体内模型系统使得研究Nm与BECs之间的宿主-病原体相互作用具有挑战性，因此需要一种模仿天然BECs的基于人类的模型。与具有复杂紧密连接和升高的跨内皮电阻（TEER）的外周内皮细胞相比，BECs具有更紧密的屏障特性。然而，许多体外模型，如原代BECs和永生化BECs，在从天然神经微环境中分离后要么缺乏屏障特性，要么会迅速丧失屏障特性。人类干细胞技术的最新进展已经开发出从诱导多能干细胞（iPSCs）中衍生出类脑内皮细胞的方法，与其他体外人类模型相比，这些细胞能更好地模拟BECs。使用iPSC衍生的BECs（iPSC-BECs）来模拟Nm与BECs的相互作用，其好处在于使用具有BEC屏障特性的人类细胞，并且可用于检查屏障破坏、先天免疫激活和细菌相互作用。在这里，我们展示了如何从iPSCs中衍生出iPSC-BECs，以及细菌制备、感染和样本收集用于分析的方法。
