Background In recent years, differentiation of human induced pluripotent stem cells (hiPSCs) into brain-specific microvascular endothelial cells (iBMECs) has frequently been used to model the blood–brain barrier (BBB). However, there are limitations in the use of iBMECs for in vitro studies, such as transendothelial electrical resistance (TEER) instability, weak junctional expression of VE-cadherin, and lack of proper fluid shear stress response. In vivo, the basement membrane (BM) composition of the BBB evolves throughout development, and laminins become the dominant component of the mature vascular BM. However, laminin isoforms of the endothelial BM have not been used for culture of differentiated iBMECs. The main goal of this study is to investigate the effect of different laminin isoforms of the endothelial BM on iBMEC functionality and to determine whether better recapitulation of the physiological BM in vitro can address the aforementioned limitations of iBMECs. Methods Using a previously reported method, hiPSCs were differentiated into iBMECs. The influence of main laminins of the endothelial BM, LN 411 and LN 511, on iBMEC functionality was studied and compared to a collagen IV and fibronectin mixture (CN IV-FN). Quantitative RT-PCR, immunocytochemistry, and TEER measurement were utilized to assess gene and protein expression and barrier properties of iBMECs on different extracellular matrices. Single-channel microfluidic devices were used to study the effect of shear stress on iBMECs. Results LN 511, but not LN 411, improved iBMEC barrier properties and resulted in more sustained TEER stability. Immunocytochemistry showed improved junctional protein expression compared to iBMECs cultured on CN IV-FN. iBMECs cultured on LN 511 showed a reduction of stress fibers, indicating resting endothelial phenotype, whereas gene expression analysis revealed upregulation of multiple genes involved in endothelial activation in iBMECs on CN IV-FN. Finally, culturing iBMECs on LN 511 enhanced physiological responses to shear stress, including morphological changes and enhanced junctional protein association. Conclusion LN 511 improves the functionality and long-term barrier stability of iBMECs. Our findings suggest that incorporation of physiologically relevant LN 511 in iBMEC culture would be beneficial for disease modeling applications and BBB-on-a-chip platforms that accommodate fluid flow.

中文翻译：

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化学定义的人血管层粘连蛋白用于 hiPSC 来源的脑微血管内皮细胞的生物学相关培养

背景近年来，人类诱导多能干细胞（hiPSC）分化为脑特异性微血管内皮细胞（iBMEC）经常被用来模拟血脑屏障（BBB）。然而，使用 iBMEC 进行体外研究存在局限性，例如跨内皮电阻 (TEER) 不稳定、VE-钙粘蛋白的连接表达较弱以及缺乏适当的流体剪切应力响应。在体内，BBB 的基底膜 (BM) 组成在整个发育过程中不断演变，层粘连蛋白成为成熟血管 BM 的主要成分。然而，内皮 BM 的层粘连蛋白亚型尚未用于分化 iBMEC 的培养。本研究的主要目的是研究内皮 BM 的不同层粘连蛋白亚型对 iBMEC 功能的影响，并确定体外更好地重现生理 BM 是否可以解决 iBMEC 的上述局限性。方法 使用先前报道的方法，hiPSC 分化为 iBMEC。研究了内皮 BM 的主要层粘连蛋白 LN 411 和 LN 511 对 iBMEC 功能的影响，并与 IV 型胶原和纤连蛋白混合物 (CN IV-FN) 进行比较。利用定量 RT-PCR、免疫细胞化学和 TEER 测量来评估 iBMEC 在不同细胞外基质上的基因和蛋白质表达以及屏障特性。使用单通道微流体装置研究剪切应力对 iBMEC 的影响。结果 LN 511（而非 LN 411）改善了 iBMEC 阻隔性能，并导致更持久的 TEER 稳定性。免疫细胞化学显示，与 CN IV-FN 上培养的 iBMEC 相比，连接蛋白表达有所改善。在 LN 511 上培养的 iBMEC 显示应力纤维减少，表明静息内皮表型，而基因表达分析显示 CN IV-FN 上的 iBMEC 中参与内皮激活的多个基因上调。最后，在 LN 511 上培养 iBMEC 增强了对剪切应力的生理反应，包括形态变化和增强的连接蛋白关联。结论 LN 511 改善了 iBMEC 的功能和长期屏障稳定性。我们的研究结果表明，在 iBMEC 培养物中加入生理相关的 LN 511 将有利于疾病建模应用和适应流体流动的 BBB 芯片平台。