Developing iPSC models of the airway epithelium to understand host - virus interactions

气道上皮在环境和器官生理之间起着重要的接口作用。它作为潜在病原体和外来颗粒的屏障，帮助调节宿主防御机制，包括炎症过程。正常情况下，支气管上皮由纤毛柱状、分泌黏液的杯状细胞和分泌表面活性剂的克拉拉细胞组成。呼吸道病毒包括呼吸道合胞病毒（RSV）、流感病毒和鼻病毒（RV）是常见的，可引起儿童重大疾病，也会加重现有的肺部疾病。更好地了解不同遗传背景或不同环境下供体中病毒-气道上皮相互作用的分子基础将提供机制理解。越来越多的证据表明，遗传变异介导了病毒相互作用的程度和性质，例如CDHR3变异和RV-C （PMID: 24241537, PMID: 30930175）。研究人类支气管上皮细胞-病毒相互作用最常见的方法是使用支气管镜刷法分离气道上皮细胞，然后使用气液界面（ALI）分化培养细胞（PMID: 22287976）。ALI细胞形成的上皮屏障与体内结构非常相似，由基底细胞、杯状细胞和纤毛细胞组成。然而，支气管镜手术是侵入性的，对个体有生命危险，很难收集到足够数量的细胞，细胞的寿命有限。这些因素使得开发一种非侵入性的、可持续的人类气道上皮模型，该模型结合了人类供体的遗传复杂性，非常值得进行机制研究。诱导多能干细胞（iPSCs）向成熟细胞类型的分化显示了提供个性化疾病模型包括机制研究的巨大希望。最近，研究人员开发了iPSC模型，将成熟的多纤毛细胞包埋在功能性气道上皮中，其中含有指示极化上皮细胞层的clara、杯状细胞和基底细胞（PMID: 24706852）。然而，需要更多的工作来推进这一领域，提供一个强大的代表性模型来捕捉气道上皮的复杂性，并可用于研究宿主上皮-病毒相互作用，代表发生在人肺中的相互作用。这一提议背后的假设是：i) iPSC模型可以捕捉气道上皮的复杂性，ii)这些模型将允许研究RSV，流感和RV诱导的应答提供了对特异性和重叠病毒驱动效应的分子基础的前所未有的见解。iii)通过研究携带特定遗传变异和/或使用CRISPR/Cas9引入变化的供体细胞中病毒诱导的变化，我们将确定对可能成为新抗病毒药物开发目标的途径的潜在新/新理解。项目关键阶段：1；利用iPSC和ALI分化，开发并优化体外培养具有clara、杯状细胞和基底细胞的气道上皮的培养条件。2. 在功能（屏障）、形态学、蛋白质和转录组学（RNA-seq）水平上，将这些iPSC衍生的气道上皮与目前的金标准进行比较和对比，这些标准来自于经支气管镜分离并在ALI培养的供体支气管上皮细胞。3. 研究iPSC和HBEC模型中病毒-上皮细胞的相互作用，使用来自对照组和呼吸系统疾病（如哮喘）患者的细胞，以了解遗传变异（在基因型供体和/或由CRISPR/Cas9引入）对这些反应的影响。这个项目代表了一个令人兴奋的博士机会，汇集了不同学校和学科的重要专业知识，包括生理学相关气道模型的使用，遗传学（Sayers）

The airway epithelium acts as the critical interface between the environment and organ physiology. It acts as a barrier to potential pathogens and extraneous particles and helps regulate host defence mechanisms, including the inflammation process. Under normal conditions, the bronchial epithelium is composed of ciliated columnar, mucus-secreting goblet and Clara cells that secrete surfactant. Respiratory viruses including respiratory syncytial virus (RSV), influenza virus and rhinovirus (RV) are common and cause significant illness in children and also exacerbate existing lung diseases. A greater understanding of the molecular basis of the virus-airway epithelial interactions in donors of different genetic backgrounds or in different environmental context will provide mechanistic understanding. Accumulating evidence suggest that genetic variants mediate the extent and nature of the virus interaction, e.g. CDHR3 variants and RV-C (PMID: 24241537, PMID: 30930175). The most common approach to study bronchial epithelial cell - virus interactions in human context is to isolate airway epithelial cells using bronchoscopic brush technique and then culture the cells using air-liquid interface (ALI) differentiation (PMID: 22287976). ALI cells form an epithelial barrier that closely resembles the in vivo architecture and is composed of basal, goblet, and ciliated cells. However, the bronchoscopic procedure is invasive with life threatening risk to the individual, adequate numbers of cells are hardly collected and cells have a limited lifespan. These factors make the development of a non-invasive, sustainable model of the human airway epithelium that incorporates the genetic complexity of human donors for mechanistic studies highly desirable. The differentiation of induced pluripotent stem cells (iPSCs) to mature cell types shows great promise to provide personalized disease modelling including mechanistic studies. Development of iPSC models that encapsulate mature multiciliated cells in a functional airway epithelium with clara, goblet, and basal cells indicative of a polarized epithelial-cell layer have recently been developed (PMID: 24706852). However, more work is needed to advance this area providing a robust representative model the captures the complexity of the airway epithelium and can be used to investigate the host epithelial - virus interactions representative of that occurs in the human lung. The hypotheses underlying this proposal are i) iPSC models can capture the complexity of airway epithelium, ii) these models will allow the study of RSV, influenza and RV induced responses providing unprecedented insight into the molecular basis of specific and overlapping virus driven effects and iii) by studying viral induced changes in cells derived from donors that carry specific genetic variants and/or introduce changes using CRISPR/Cas9 we will identify potentially new/novel understanding of pathways that could be the target of new anti-viral drug development. Key stages to the project: 1. Develop and optimise culture conditions for the generation of airway epithelium in vitro complete with clara, goblet, and basal cells using iPSC and ALI differentiation. 2. Compare and contrast at the functional (barrier), morphological, protein and transcriptomic (RNA-seq) level these iPSC derived airway epithelial layers with the current gold standard derived from donor bronchial epithelial cells isolated by bronchoscopy and grown at ALI. 3. Investigate virus - epithelial cell interactions in both the iPSC and HBEC models using cells from controls and patients with respiratory conditions e.g. asthma to understand the effects of genetic variation (in genotype donors and/or introduced by CRISPR/Cas9) on these responses. This project represents an exciting PhD opportunity and brings together significant expertise across schools and disciplines including the use of physiological relevant airway models, genetics (Sayers)