Department of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, 467-8603, Japan.
Fluids Barriers CNS. 2020 May 26;17(1):36. doi: 10.1186/s12987-020-00197-1.
The blood-brain barrier (BBB) plays an important role as a biological barrier by regulating molecular transport between circulating blood and the brain parenchyma. In drug development, the accurate evaluation of BBB permeability is essential to predict not only the efficacy but also the safety of drugs. Recently, brain microvascular endothelial-like cells derived from human induced pluripotent stem cells (iPSCs) have attracted much attention. However, the differentiation protocol has not been optimized, and the enhancement of iPSC-derived brain microvascular endothelial-like cells (iBMELCs) function is required to develop highly functional BBB models for pharmaceutical research. Thus, we attempted to improve the functions of differentiated iBMELCs and develop a versatile BBB model by modulating TGF-β signaling pathway without implementing complex techniques such as co-culture systems.
iPSCs were differentiated into iBMELCs, and TGF-β inhibitor was used in the late stage of differentiation. To investigate the effect of TGF-β on freezing-thawing, iBMELCs were frozen for 60-90 min or 1 month. The barrier integrity of iBMELCs was evaluated by transendothelial electrical resistance (TEER) values and permeability of Lucifer yellow. Characterization of iBMELCs was conducted by RT-qPCR, immunofluorescence analysis, vascular tube formation assay, and acetylated LDL uptake assay. Functions of efflux transporters were defined by intracellular accumulation of the substrates.
When we added a TGF-β inhibitor during iBMELCs differentiation, expression of the vascular endothelial cell marker was increased and blood vessel-like structure formation was enhanced. Furthermore, TEER values were remarkably increased in three iPSC lines. Additionally, it was revealed that TGF-β pathway inhibition suppressed the damage caused by the freezing-thawing of iBMELCs.
We succeeded in significantly enhancing the function and endothelial characteristics of iBMELCs by adding a small molecular compound, a TGF-β inhibitor. Moreover, the iBMELCs could maintain high barrier function even after freezing-thawing. Taken together, these results suggest that TGF-β pathway inhibition may be useful for developing iPSC-derived in vitro BBB models for further pharmaceutical research.
血脑屏障（BBB）通过调节循环血液与脑实质之间的分子转运，起着重要的生物屏障作用。在药物开发中，准确评估 BBB 通透性对于预测药物的疗效和安全性至关重要。最近，源自人诱导多能干细胞（iPSC）的脑微血管内皮样细胞引起了广泛关注。然而，其分化方案尚未得到优化，并且需要增强 iPSC 衍生的脑微血管内皮样细胞（iBMELC）的功能，以开发用于药物研究的高度功能性 BBB 模型。因此，我们试图通过调节 TGF-β 信号通路来改善分化的 iBMELC 的功能，并开发一种多功能的 BBB 模型，而无需实施复杂的技术，如共培养系统。
iPSC 分化为 iBMELC，在分化后期使用 TGF-β 抑制剂。为了研究 TGF-β 对冻融的影响，将 iBMELC 冷冻 60-90 分钟或 1 个月。通过跨内皮电阻（TEER）值和 Lucifer yellow 的通透性评估 iBMELC 的屏障完整性。通过 RT-qPCR、免疫荧光分析、血管管腔形成测定和乙酰化 LDL 摄取测定来进行 iBMELC 的特征分析。通过细胞内底物积累来定义外排转运蛋白的功能。
当我们在 iBMELC 分化过程中添加 TGF-β 抑制剂时，血管内皮细胞标志物的表达增加，血管样结构形成增强。此外，三种 iPSC 系的 TEER 值显著增加。此外，研究表明，TGF-β 通路抑制可抑制 iBMELC 冻融损伤。
我们通过添加小分子化合物 TGF-β 抑制剂，成功地显著增强了 iBMELC 的功能和内皮特性。此外，iBMELC 即使在冻融后仍能保持高屏障功能。综上所述，这些结果表明，TGF-β 通路抑制可能有助于开发用于进一步药物研究的 iPSC 衍生体外 BBB 模型。
