In vitro modeling of blood brain barrier dysfunction on a chip to elucidate the pathogenesis of cerebral malaria

项目摘要 脑微血管内皮细胞(BMECs)排列在中枢神经系统血管网络中 (CNS)与血管周围细胞一起形成一种特殊的屏障，称为血脑屏障(BBB)， 调节特定分子进出中枢神经系统的动态交通。关于血脑屏障功能障碍的研究已经被 由于无法在患者身上进行直接测试和缺乏体外模型而受到阻碍。分析可用的 从多能干细胞来源推测的BMEC表明它们并不含有真正的脑EC分子 签名。使用基于转录因子的重新编程策略，我们证明了耐用性和 具有血脑屏障特性的功能性真内皮细胞可能被衍生出来。我们将使用这些经过验证的BMEC在 同基因BBB/片上器件的开发。从患者特异性诱导产生的皮质脑器官 多能细胞(IPSC)将由匹配的IPSC来源的真诚的BMEC在微流体设备中血管化。 为了探索这项新技术的潜力，我们将研究一种严重的脑型疟疾的病理生物学 导致20%幸存者神经残疾的疾病综合征。感染了疟原虫的红细胞 恶性寄生虫附着在脑微血管系统上，导致功能障碍。我们假设这种寄生虫 通过称为细胞外小泡的小细胞外小泡，为脑内皮细胞的黏附做好准备 外显体。这是癌症生物学中的一个既定范例，在这个范例中，恶性细胞培养转移 为肿瘤生存做好准备并提高存活率的“微核”。外切体介导局部和系统的细胞间。 通过蛋白质组中信息的水平传递进行交流。我们最近能够识别出 通过胞外细胞摄取研究乳腺癌脑转移的关键预后生物标志物 和蛋白质组分析。我们将分析脑型疟疾儿童外显子的蛋白质组和 将它们注入我们创新的基于微流控的BBB模型。我们将实时监测Exosome如何触发 血脑屏障功能障碍，并随后影响受CM影响患者的IPS来源的脑有机物质。这些 研究将为疾病发病机制提供新的见解，并可能确定新的预后生物标志物。

Project Summary Brain microvascular endothelial cells (BMECs) which line the vascular network of the central nervous system (CNS) in conjunction with perivascular cells form a specialized barrier termed the blood-brain barrier (BBB) that regulates the dynamic traffic of select molecules into and out of the CNS. Studies of BBB dysfunction have been hampered by an inability to perform direct testing in patients and a lack of in vitro models. Analysis of available putative BMECs from pluripotent stem cell sources reveals that they do not harbor bona fide brain EC molecular signatures. Using a transcription factor-based reprogramming strategy, we demonstrate that durable and functional true endothelial cells with BBB traits may be derived. We will use these verified BMECs in the development of isogenic BBB/on-a-chip devices. Cortical brain organoids derived from patient specific induced pluripotent cells (IPSC) will be vascularized by matched IPSC derived bona fide BMECs in a microfluidic device. To explore the potential of this new technology we will investigate the pathobiology of cerebral malaria a severe disease syndrome that causes neurodisability in 20% of survivors. Red blood cells infected with the Plasmodium falciparum parasite adhere to the brain microvasculature causing dysfunction. We hypothesize that the parasite primes the brain endothelial cells for adherence through their “education” by small extracellular vesicles termed exosomes. This is an established paradigm in cancer biology in which malignant cells educate metastatic “microniches” to prime for and enhance tumor survival. The exosomes mediate local and systemic intercellular communication through the horizontal transfer of information in their proteomes. We recently were able to identify a critical prognosis biomarker for brain metastasis in breast cancer through studies of exosomal cellular uptake and proteomic analysis. We will analyze the proteomes of exosomes from children with cerebral malaria and infuse them into our innovative microfluidic-based BBB model. We will monitor in real time how exosomes trigger BBB dysfunction and subsequently affect IPS-derived brain organoids from patients affected by CM. These studies will provide new insights into disease pathogenesis and potentially identify new prognosis biomarkers.