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

呼吸道上皮在环境和器官生理学之间起着至关重要的作用。它对潜在的病原体和外来颗粒起到屏障作用，并有助于调节宿主防御机制，包括炎症过程。正常情况下，支气管上皮由纤毛柱状结构、分泌粘液的杯状结构和分泌表面活性物质的Clara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，会导致儿童重大疾病，也会加剧现有的肺部疾病。更好地理解不同遗传背景或不同环境背景的供体中病毒-呼吸道上皮相互作用的分子基础将提供机制上的理解。越来越多的证据表明，遗传变异调节病毒相互作用的程度和性质，例如CDHR3变异和RV-C(PMID：24241537，PMID：30930175)。在人类环境中研究呼吸道上皮细胞与病毒相互作用最常用的方法是用支气管镜毛刷技术分离呼吸道上皮细胞，然后用气-液界面(ALI)分化培养细胞(PMID：22287976)。ALI细胞形成与体内结构非常相似的上皮屏障，由基细胞、杯状细胞和纤毛细胞组成。然而，支气管镜检查程序是侵入性的，对个人有生命威胁，很难收集足够数量的细胞，而且细胞的寿命有限。这些因素使得开发一种非侵入性的、可持续的人类呼吸道上皮模型非常必要，该模型包含了用于机械研究的人类供体的遗传复杂性。诱导多能干细胞(IPSCs)向成熟细胞类型的分化显示了提供包括机制研究在内的个性化疾病模型的巨大希望。最近开发的IPSC模型将成熟的多纤毛细胞包裹在具有克拉拉、杯状和指示极化上皮细胞层的基底细胞的功能性呼吸道上皮中(PMID：24706852)。然而，需要更多的工作来推动这一领域的发展，提供一个强大的代表性模型，该模型捕捉到呼吸道上皮的复杂性，并可用于研究发生在人类肺部的宿主上皮与病毒的相互作用。这一建议所依据的假设是：i)IPSC模型可以捕捉到呼吸道上皮细胞的复杂性；ii)这些模型将使我们能够研究RSV、流感和RV诱导的反应，从而为特定和重叠的病毒驱动效应的分子基础提供前所未有的见解；iii)通过研究病毒诱导的携带特定基因变体的捐赠者细胞的变化和/或使用CRISPR/Cas9引入变化，我们将确定可能成为新抗病毒药物开发靶点的通路的潜在新的/新的理解。该项目的关键阶段：1.建立和优化培养条件，在体外生成具有Clara细胞、杯状细胞和基底细胞的呼吸道上皮细胞，并使用IPSC和ALI分化。2.在功能(屏障)、形态、蛋白质和转录(RNA-seq)水平上，将IPSC来源的呼吸道上皮层与目前以支气管镜下分离的供体支气管上皮细胞为来源的金标准进行对比。3.研究IPSC和HBEC模型中病毒与上皮细胞的相互作用，使用来自对照组和患有呼吸道疾病(如哮喘)的患者的细胞，以了解基因变异(基因供体和/或CRISPR/Cas9引入)对这些反应的影响。这个项目代表着一个令人兴奋的博士机会，并汇集了跨学校和学科的重要专业知识，包括使用与生理相关的呼吸道模型、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)