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（BMEC），排列在中枢神经系统的血管网络中 (CNS)与血管周围细胞一起形成称为血脑屏障（BBB）的专门屏障， 调节选定分子进出中枢神经系统的动态交通。BBB功能障碍的研究已经 由于无法在患者中进行直接测试和缺乏体外模型而受到阻碍。分析现有 来自多能干细胞来源的推定BMEC揭示它们不具有真正的脑EC分子， 签名.使用转录因子为基础的重编程策略，我们证明，持久的， 可以得到具有BBB特征的功能性真内皮细胞。我们将使用这些经过验证的BMEC， 同基因血脑屏障/片上器件的开发。源自患者特异性诱导的皮质脑类器官 多能细胞（IPSC）将在微流体装置中通过匹配的IPSC衍生的真实BMEC血管化。 为了探索这项新技术的潜力，我们将研究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。感染疟原虫的红细胞 恶性疟原虫粘附于脑微血管系统，引起功能障碍。我们假设寄生虫 通过小的细胞外囊泡的“教育”， 外来体这是癌症生物学中的一个既定范例，其中恶性细胞培养转移性细胞。 “微环境”，以启动和提高肿瘤存活率。外泌体介导局部和全身细胞间 通过蛋白质组中信息的水平传递进行交流。我们最近发现 通过外泌体细胞摄取的研究，乳腺癌脑转移的关键预后生物标志物 和蛋白质组学分析。我们将分析脑型疟疾患儿的外泌体蛋白质组， 将它们注入我们创新的基于微流体的血脑屏障模型。我们将在真实的时间监测外泌体如何触发 BBB功能障碍并随后影响来自受CM影响的患者的IPS衍生的脑类器官。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.