当前位置: X-MOL 学术Fluids Barriers CNS › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
Laminin 221 fragment is suitable for the differentiation of human induced pluripotent stem cells into brain microvascular endothelial-like cells with robust barrier integrity
Fluids and Barriers of the CNS ( IF 7.3 ) Pub Date : 2020-03-30 , DOI: 10.1186/s12987-020-00186-4
Hiromasa Aoki , Misaki Yamashita , Tadahiro Hashita , Takahiro Iwao , Tamihide Matsunaga

Background In vitro blood–brain barrier (BBB) models using human induced pluripotent stem (iPS) cell-derived brain microvascular endothelial-like cells (iBMELCs) have been developed to predict the BBB permeability of drug candidates. For the differentiation of iBMELCs, Matrigel, which is a gelatinous protein mixture, is often used as a coating substrate. However, the components of Matrigel can vary among lots, as it is obtained from mouse sarcoma cells with the use of special technics and also contains various basement membranes. Therefore, fully defined substrates as substitutes for Matrigel are needed for a stable supply of iBMELCs with less variation among lots. Methods iBMELCs were differentiated from human iPS cells on several matrices. The barrier integrity of iBMELCs was evaluated based on transendothelial electrical resistance (TEER) values and permeability of fluorescein isothiocyanate-dextran 4 kDa (FD4) and Lucifer yellow (LY). Characterization of iBMELCs was conducted by RT-qPCR and immunofluorescence analysis. Functions of efflux transporters were defined by intracellular accumulation of the substrates in the wells of multiwell plates. Results iBMELCs differentiated on laminin 221 fragment (LN221F-iBMELCs) had higher TEER values and lower permeability of LY and FD4 as compared with iBMELCs differentiated on Matrigel (Matrigel-iBMELCs). Besides, the gene and protein expression levels of brain microvascular endothelial cells (BMEC)-related markers were similar between LN221F-iBMELCs and Matrigel-iBMELCs. Moreover, both Matrigel- and LN221F-iBMELCs had functions of P-glycoprotein and breast cancer resistance protein, which are essential efflux transporters for barrier functions of the BBB. Conclusion The fully defined substrate LN221F presents as an optimal coating matrix for differentiation of iBMELCs. The LN221F-iBMELCs had more robust barrier function for a longer period than Matrigel-iBMELCs with characteristics of BMECs. This finding will contribute the establishment of an iBMELC supply system for pharmacokinetic and pathological models of the BBB.

中文翻译:

Laminin 221 片段适用于将人诱导多能干细胞分化为具有强大屏障完整性的脑微血管内皮样细胞

背景 已经开发出使用人诱导多能干 (iPS) 细胞衍生的脑微血管内皮样细胞 (iBMELC) 的体外血脑屏障 (BBB) 模型来预测候选药物的 BBB 通透性。对于 iBMELCs 的分化,基质胶是一种凝胶状蛋白质混合物,通常用作涂层基质。然而,Matrigel 的成分可能因批次而异,因为它是从小鼠肉瘤细胞中使用特殊技术获得的,并且还包含各种基底膜。因此,需要完全定义的底物作为基质胶的替代品,以稳定供应 iBMELC,批次之间的差异较小。方法 iBMELCs 在几种基质上从人 iPS 细胞分化而来。基于跨内皮电阻 (TEER) 值和异硫氰酸荧光素-葡聚糖 4 kDa (FD4) 和荧光黄 (LY) 的渗透性评估 iBMELCs 的屏障完整性。iBMELCs 的表征是通过 RT-qPCR 和免疫荧光分析进行的。外排转运蛋白的功能由底物在多孔板孔中的细胞内积累来定义。结果 与在基质胶上分化的 iBMELCs (Matrigel-iBMELCs) 相比,在层粘连蛋白 221 片段上分化的 iBMELCs (LN221F-iBMELCs) 具有更高的 TEER 值和更低的 LY 和 FD4 渗透性。此外,脑微血管内皮细胞(BMEC)相关标志物的基因和蛋白质表达水平在 LN221F-iBMELCs 和 Matrigel-iBMELCs 之间相似。而且,Matrigel- 和 LN221F-iBMELCs 都具有 P-糖蛋白和乳腺癌抗性蛋白的功能,它们是 BBB 屏障功能必不可少的外排转运蛋白。结论 完全定义的基底 LN221F 是区分 iBMELC 的最佳涂层基质。LN221F-iBMELCs 比具有 BMECs 特征的 Matrigel-iBMELCs 具有更强大的屏障功能。这一发现将有助于建立用于 BBB 药代动力学和病理模型的 iBMELC 供应系统。LN221F-iBMELCs 比具有 BMECs 特征的 Matrigel-iBMELCs 具有更强大的屏障功能。这一发现将有助于建立用于 BBB 药代动力学和病理模型的 iBMELC 供应系统。LN221F-iBMELCs 比具有 BMECs 特征的 Matrigel-iBMELCs 具有更强大的屏障功能。这一发现将有助于建立用于 BBB 药代动力学和病理模型的 iBMELC 供应系统。
更新日期:2020-03-30
down
wechat
bug