Human Brain Microvascular Endothelial Cells Derived from the BC1 iPS Cell Line Exhibit a Blood-Brain Barrier Phenotype

The endothelial cells that form capillaries in the brain are highly specialized, with tight junctions that minimize paracellular transport and an array of broad-spectrum efflux pumps that make drug delivery to the brain extremely challenging. One of the major limitations in blood-brain barrier research and the development of drugs to treat central nervous system diseases is the lack of appropriate cell lines. Recent reports indicate that the derivation of human brain microvascular endothelial cells (hBMECs) from human induced pluripotent stem cells (iPSCs) may provide a solution to this problem. Here we demonstrate the derivation of hBMECs extended to two new human iPSC lines: BC1 and GFP-labeled BC1. These hBMECs highly express adherens and tight junction proteins VE-cadherin, ZO-1, occludin, and claudin-5. The addition of retinoic acid upregulates VE-cadherin expression, and results in a significant increase in transendothelial electrical resistance to physiological values. The permeabilities of tacrine, rhodamine 123, and Lucifer yellow are similar to values obtained for MDCK cells. The efflux ratio for rhodamine 123 across hBMECs is in the range 2–4 indicating polarization of efflux transporters. Using the rod assay to assess cell organization in small vessels and capillaries, we show that hBMECs resist elongation with decreasing diameter but show progressive axial alignment. The derivation of hBMECs with a blood-brain barrier phenotype from the BC1 cell line highlights that the protocol is robust. The expression of GFP in hBMECs derived from the BC1-GFP cell line provides an important new resource for BBB research.构成大脑毛细血管的内皮细胞具有高度特化的特性，其紧密连接可最大限度减少细胞旁转运，且表达一系列广谱外排泵，这使得向大脑递送药物极具挑战性。血脑屏障（blood-brain barrier, BBB）研究以及中枢神经系统疾病治疗药物开发面临的主要局限之一，便是缺乏合适的细胞系。近期研究表明，从人类诱导多能干细胞（human induced pluripotent stem cells, iPSCs）诱导分化获得人类脑微血管内皮细胞（human brain microvascular endothelial cells, hBMECs）或可解决这一难题。本研究成功将hBMECs的分化体系拓展至两种全新的人类iPSC细胞系：BC1与GFP标记的BC1。此类hBMECs可高表达黏着连接与紧密连接蛋白VE-钙粘蛋白（VE-cadherin）、ZO-1、闭合蛋白（occludin）以及克劳丁-5（claudin-5）。视黄酸（retinoic acid）的添加可上调VE-钙粘蛋白的表达，并使跨内皮细胞电阻显著升高至生理水平。他克林（tacrine）、罗丹明123（rhodamine 123）与荧光黄（Lucifer yellow）的通透系数与马-达二氏犬肾（MDCK）细胞测得的数值相近。罗丹明123在hBMECs两侧的外排比介于2~4之间，表明外排转运蛋白具有极性分布特性。本研究采用杆状检测实验（rod assay）评估小血管与毛细血管中的细胞组织形态，结果显示，随着管径减小，hBMECs可抵抗伸长，但呈现出渐进性的轴向排列。从BC1细胞系诱导分化获得具有血脑屏障表型的hBMECs，证明该分化方案具有良好的稳健性。从BC1-GFP细胞系诱导分化得到的hBMECs表达GFP，这为血脑屏障研究提供了一项极具价值的全新研究资源。