Human iPSC-derived brain endothelial microvessels in a multi-well format enable permeability screens of anti-inflammatory drugs
Human iPSC-derived brain endothelial microvessels in a multi-well format enable permeability screens of anti-inflammatory drugs
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DOI：
10.1101/2021.05.03.442133
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发表时间：
2021-05
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通讯作者：
S. Fengler;B. Kurkowsky;S. Kaushalya;W. Roth;Eugenio Fava;Philip Denner
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作者：
S. Fengler;B. Kurkowsky;S. Kaushalya;W. Roth;Eugenio Fava;Philip Denner
优化用于人类血脑屏障（BBB）渗透的候选药物仍然是关键挑战之一，许多破坏性脑疾病（包括神经退行性疾病）仍然没有足够的治疗方法。到目前为止，很难建立最先进的人干细胞衍生的体外模型，其模拟生理屏障特性，包括3D微血管系统，其形式可扩展到足以在早期药物开发阶段筛选药物的BBB渗透。为了应对这一挑战，我们以标准化和可扩展的多孔板形式建立了人诱导多能干细胞（iPSC）衍生的脑内皮微血管。用原代细胞条件培养基补充iPSC衍生的脑微血管内皮细胞（BMEC），并在培养10天后生长成完整的微血管。所产生的微血管显示出典型的血脑屏障表型，包括内皮蛋白表达、紧密连接和外排转运蛋白的极化定位。微血管表现出生理相关的跨内皮电阻（TEER），10 kDa的葡聚糖-Alexa 647是密封的，并强烈限制荧光素钠（NaF）的渗透性。用参比化合物进行的渗透性测试证实了我们的模型作为鉴定潜在的BBB渗透性抗炎药物的平台的适用性。总之，本文提出的脑微血管平台概括了生理特性，并允许快速筛选BBB渗透性抗炎化合物，其已被认为是治疗迄今无法治疗的神经退行性疾病的有希望的物质。
Optimizing drug candidates for blood-brain barrier (BBB) penetration in humans remains one of the key challenges and many devastating brain diseases including neurodegenerative diseases still do not have adequate treatments. So far, it has been difficult to establish state-of-the-art human stem cell derived in vitro models that mimic physiological barrier properties including a 3D microvasculature in a format that is scalable enough to screen drugs for BBB penetration in early drug development phases. To address this challenge, we established human induced pluripotent stem cell (iPSC)-derived brain endothelial microvessels in a standardized and scalable multi-well plate format. iPSC-derived brain microvascular endothelial cells (BMECs) were supplemented with primary cell conditioned media and grew to intact microvessels in 10 days of culturing. Produced microvessels show a typical BBB phenotype including endothelial protein expression, tight-junctions and polarized localization of efflux transporter. Microvessels exhibited physiological relevant trans-endothelial electrical resistance (TEER), were leak-tight for 10 kDa dextran-Alexa 647 and strongly limited the permeability of sodium fluorescein (NaF). Permeability tests with reference compounds confirmed the suitability of our model as platform to identify potential BBB penetrating anti-inflammatory drugs. In summary, the here presented brain microvessel platform recapitulates physiological properties and allows rapid screening of BBB permeable anti-inflammatory compounds that has been suggested as promising substances to cure so far untreatable neurodegenerative diseases.