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

开发气道上皮的 iPSC 模型以了解宿主与病毒的相互作用

• 批准号: 2434526
• 金额: --
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2434526
• 关键词: Developing; iPSC; models; airway; epithelium

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)

气道上皮细胞作为环境和器官生理之间的关键界面。它作为潜在病原体和外来颗粒的屏障，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、分泌粘液的杯状细胞和分泌表面活性物质的Clara细胞组成。包括呼吸道合胞病毒（RSV）、流感病毒和鼻病毒（RV）在内的呼吸道病毒是常见的，在儿童中引起严重疾病，并且还加剧现有的肺部疾病。更深入地了解不同遗传背景或不同环境背景的供体中病毒-气道上皮相互作用的分子基础将提供机制性理解。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CDHR 3变异和RV-C（PMID：24241537，PMID：30930175）。 在人类环境中研究支气管上皮细胞-病毒相互作用的最常见方法是使用支气管镜刷技术分离气道上皮细胞，然后使用气液界面（ALI）分化培养细胞（PMID：22287976）。ALI细胞形成与体内结构非常相似的上皮屏障，由基底细胞、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，对个体具有威胁生命的风险，几乎不能收集足够数量的细胞，并且细胞的寿命有限。这些因素使得非常需要开发一种非侵入性的、可持续的人气道上皮模型，该模型结合了用于机理研究的人供体的遗传复杂性。诱导多能干细胞（iPSC）分化为成熟细胞类型显示出提供个性化疾病建模（包括机制研究）的巨大前景。最近已经开发了将成熟的多纤毛细胞包封在功能性气道上皮中的iPSC模型，其中clara、杯状细胞和基底细胞指示极化的上皮细胞层（PMID：24706852）。然而，需要更多的工作来推进这一领域，提供一个鲁棒的代表性模型，该模型捕获气道上皮的复杂性，并可用于研究代表人肺中发生的宿主上皮-病毒相互作用。 该提议的假设是i）iPSC模型可以捕获气道上皮的复杂性，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洞察，以及iii）通过研究来源于携带特异性遗传变体和/或使用CRISPR/PCR引入变化的供体的细胞中的病毒诱导的变化，Cas9，我们将确定潜在的新的/新的理解的途径，可能是新的抗病毒药物开发的目标。 项目的关键阶段：1。开发和优化培养条件，利用iPSC和ALI分化在体外生成气道上皮，包括克拉拉细胞、杯状细胞和基底细胞。2.在功能（屏障）、形态、蛋白质和转录组（RNA-seq）水平上比较和对比这些iPSC衍生的气道上皮层与目前的金标准，所述金标准衍生自通过支气管镜检查分离并在ALI下生长的供体支气管上皮细胞。3.研究iPSC和HBEC模型中的病毒-上皮细胞相互作用，使用来自对照和患有呼吸系统疾病（例如哮喘）的患者的细胞，以了解遗传变异（在基因型供体中和/或由CRISPR/Cas9引入）对这些反应的影响。 该项目代表了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，包括使用生理相关的气道模型，遗传学（塞耶斯）