Pong Sovannarath, Lizano Paulo, Karmacharya Rakesh
Center for Genomic Medicine, Massachusetts General Hospital; Department of Psychiatry, Beth Israel Deaconess Medical Center; Chemical Biology and Therapeutic Science Program, Broad Institute of MIT and Harvard.
Center for Genomic Medicine, Massachusetts General Hospital; Department of Psychiatry, Beth Israel Deaconess Medical Center; Chemical Biology and Therapeutic Science Program, Broad Institute of MIT and Harvard; Department of Psychiatry, Harvard Medical School.
J Vis Exp. 2020 Nov 19(165). doi: 10.3791/61629.
Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 Ω x cm. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.
脑微血管内皮细胞（BMECs）可从人诱导多能干细胞（iPSCs）分化而来，以建立用于研究血脑屏障（BBB）功能的体外细胞模型。本改良方案提供了详细步骤，可使用与先前方案中报道的不同供体和试剂，从人iPSCs中获得、扩增和冻存BMECs。iPSCs用基础6培养基处理4天，然后用补充有碱性成纤维细胞生长因子、视黄酸和B27添加剂的人内皮无血清培养基处理2天。在第6天，将细胞传代培养到胶原蛋白/纤连蛋白基质上2天。在第8天进行免疫细胞化学分析，使用CLDN5、OCLN、TJP1、PECAM1和SLC2A1对BMEC标志物进行分析。进行蛋白质免疫印迹以确认BMEC标志物的表达，并检测内胚层标志物SOX17是否缺失。通过发芽试验证明血管生成潜力。从第7天开始，使用筷子电极和电压电阻计测量跨内皮电阻（TEER）。在第8天使用多板读数器测量ATP结合盒亚家族B成员1和ATP结合盒亚家族C成员1的外排转运蛋白活性。通过相关细胞标志物的存在、低水平的SOX17、血管生成潜力、转运蛋白活性和TEER值~2000Ω×cm证实成功获得了BMECs。在传代到新包被的胶原蛋白/纤连蛋白平板上或冻存之前，将BMECs扩增至第10天。本方案表明，iPSC来源的BMECs可以扩增并传代至少一次。然而，冻存后观察到较低的TEER值和BMEC标志物定位较差。BMECs可用于与其他细胞类型（神经元、神经胶质细胞、周细胞）的共培养实验、三维脑模型（器官芯片和水凝胶）、脑类器官的血管化以及研究神经精神疾病中的血脑屏障功能障碍。
