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

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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)