Goldeman C, Andersen M, Al-Robai A, Buchholtz T, Svane N, Ozgür B, Holst B, Shusta E, Hall V J, Saaby L, Hyttel P, Brodin B
Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
Bioneer A/S, Hørsholm, Denmark.
Eur J Pharm Sci. 2021 Jan 1;156:105577. doi: 10.1016/j.ejps.2020.105577. Epub 2020 Oct 1.
The barrier properties of the brain capillary endothelium, the blood-brain barrier (BBB) restricts uptake of most small and all large molecule drug compounds to the CNS. There is a need for predictive human in vitro models of the BBB to enable studies of brain drug delivery. Here, we investigated whether human induced pluripotent stem cell (hiPSC) line (BIONi010-C) could be differentiated to brain capillary endothelial- like cells (BCEC) and evaluated their potential use in drug delivery studies. BIONi010-C hIPSCs were differentiated according to established protocols. BCEC monolayers displayed transendothelial electrical resistance (TEER) values of 5,829±354 Ω∙cm, a P, of 1.09±0.15 ∙ 10 cm∙s and a P of 85.7 ± 5.9 ∙ 10 cm ∙s. The P/P ratio of ~80, indicated a large dynamic passive permeability range. Monolayers maintained their integrity after medium exchange. Claudin-5, Occludin, Zonulae Occludens 1 and VE-Cadherin were expressed at the cell-cell contact zones. Efflux transporters were present at the mRNA level, but functional efflux of substrates was not detected. Transferrin-receptor (TFR), Low density lipoprotein receptor-related protein 1 (LRP1) and Basigin receptors were expressed at the mRNA-level. The presence and localization of TFR and LRP1 were verified at the protein level. A wide range of BBB-expressed solute carriers (SLC's) were detected at the mRNA level. The presence and localization of SLC transporters GLUT1 and LAT1 was verified at the protein level. Functional studies revealed transport of the LAT1 substrate [H]-L-Leucine and the LRP1 substrate angiopep-2. In conclusion, we have demonstrated that BIONi010-C-derived BCEC monolayers exhibited, BBB properties including barrier tightness and integrity, a high dynamic range, expression of some of the BBB receptor and transporter expression, as well as functional transport of LAT1 and LRP1 substrates. This suggests that BIONi010-C-derived BCEC monolayers may be useful for studying the roles of LAT-1 and LRP1 in brain drug delivery.
脑毛细血管内皮的屏障特性，即血脑屏障（BBB），限制了大多数小分子和所有大分子药物化合物进入中枢神经系统。因此需要有预测性的血脑屏障体外模型来开展脑药物递送研究。在此，我们研究了人诱导多能干细胞（hiPSC）系（BIONi010-C）是否可分化为脑毛细血管内皮样细胞（BCEC），并评估了其在药物递送研究中的潜在用途。BIONi010-C hiPSC按照既定方案进行分化。BCEC单层细胞的跨内皮电阻（TEER）值为5,829±354 Ω∙cm，通透系数（P）为1.09±0.15 ∙ 10 cm∙s，通透系数（P）为85.7 ± 5.9 ∙ 10 cm∙s。P/P比值约为80，表明具有较大动态被动通透性范围。换液后单层细胞保持其完整性。Claudin-5、Occludin、紧密连接蛋白1（ZO-1）和血管内皮钙黏蛋白在细胞间接触区域表达。外排转运蛋白在mRNA水平存在，但未检测到底物的功能性外排。转铁蛋白受体（TFR）、低密度脂蛋白受体相关蛋白1（LRP1）和碱性磷酸酶受体在mRNA水平表达。TFR和LRP1的存在及定位在蛋白水平得到验证。在mRNA水平检测到多种血脑屏障表达的溶质载体（SLC）。SLC转运蛋白葡萄糖转运蛋白1（GLUT1）和L型氨基酸转运体1（LAT1）的存在及定位在蛋白水平得到验证。功能研究揭示了LAT1底物[H]-L-亮氨酸和LRP1底物血管活性肠肽-2的转运。总之，我们已证明源自BIONi010-C的BCEC单层细胞表现出血脑屏障特性，包括屏障紧密性和完整性、高动态范围、一些血脑屏障受体和转运体表达，以及LAT1和LRP1底物的功能性转运。这表明源自BIONi010-C的BCEC单层细胞可能有助于研究LAT-1和LRP1在脑药物递送中的作用。
