bREATH-EASy: UNDERSTANDING THE ROLE OF INFECTION & EXTRACELLULAR MATRIX IN IDIOPATHIC PULMONARY FIBROSIS USING STEM CELL DERIVED ALVEOLAR CELLS

在英国，四分之一的人死于呼吸道疾病。很难获得肺样本，无法培养肺细胞，以及缺乏复制人类肺部疾病的动物模型，这让我们对如何诊断和治疗肺部疾病缺乏充分的了解。特发性肺纤维化(IPF)就是这些问题的例证，这种疾病的快速疤痕形成会降低肺功能，导致平均3年内死亡。IPF的病因不明，预后难以预测。需要对包括IPF在内的肺部疾病有更多的了解，这样才能改善诊断、预后和治疗。人类多能干细胞正在提供新的方法来建模和理解疾病。然而，直到最近，还没有肺部疾病的模型可用。我们最近发表的工作解决了这一需求，我们现在可以产生大量的肺泡细胞，这是受IPF影响的一种细胞类型。我们现在可以制作3D肺器官，从而能够对复杂的生物功能和相互作用进行建模，并可以利用这些技术来了解IPF。为此，我们组建了一个国际团队来解决3个相互关联的目标：1)建立IPF的HiPSC模型，以了解SFTPC突变如何影响AE2细胞的细胞表型和基因表达。目前还没有已知的导致IPF的突变，但表面活性蛋白C(SFTPC)突变可导致遗传性IPF，也称为家族性IPF，但其机制仍不清楚。我们将通过对SFTPC突变患者的皮肤活检，然后使用我们建立的方案将他们的皮肤细胞转化为肺细胞，来生成家族性IPF的干细胞模型。然后，我们将使用这些细胞来研究这种突变如何导致肺细胞损伤和纤维化，并确定新的治疗靶点来治疗IPF体外模型呼吸道感染，以了解感染对携带SFTPC突变的AE2细胞的影响。IPF患者有更频繁的呼吸道感染，每次感染的死亡率为90%，细菌感染如何加剧IPF尚不清楚。使用在OBJ中创建的SFTPC突变细胞。1我们将使用我们的3D培养平台来培养肺器官，然后用细菌或病毒感染它们。我们将创建一个响应感染的基因表达变化的概况，并将其与未感染的细胞进行比较。这将有助于深入了解呼吸道感染如何影响正常的肺细胞功能，并重要地有助于深入了解感染加剧IPF.3)细胞外基质成分的模型变化，以了解细胞外基质如何影响细胞表型。随着IPF的进展，将肺连接在一起的支架--细胞外基质(ECM)--发生了巨大的变化，导致肺变得僵硬和缺乏弹性，同时减少了氧气的吸收。细胞外基质的变化如何影响肺细胞的功能，以及肺细胞是否积极促进进一步的细胞外基质变化还没有很好的特征。我们将通过培养在OBJ中产生的细胞来加深对这一过程的理解。1在简单的3D凝胶中，这将使我们能够模拟细胞外基质在IPF早期和后期的变化。然后我们将评估这些变化如何影响细胞功能和基因表达。这将提供对ECM如何影响肺细胞功能的洞察，并重要地提供旨在防止或逆转IPF中ECM变化的潜在治疗靶点。这些目标将解决难以获得人类IPF疾病模型的问题，并为IPF中肺纤维化和感染的发病机制提供洞察力，为开发令人兴奋的新药靶点以及疾病的诊断和预后标记物提供机会。重要的是，这个平台可以被改装成其他纤维化肺部疾病的模型，以及其他含有纤维化成分的疾病的模型。

In the UK 1 in 4 people die from respiratory disease. Poor access to lung samples, an inability to culture lung cells and a lack of animal models that reproduce human lung disease has left us with an inadequate understanding of how to diagnose and treat lung disease. These issues are exemplified by idiopathic pulmonary fibrosis (IPF), a disease where rapid scarring reduces lung function leading to death within an average of 3 years. The cause of IPF is unknown and prognosis is difficult to predict. A greater knowledge of lung disease including IPF is needed so diagnosis, prognosis and treatment can be improved.Human pluripotent stem cells are providing new ways to model and understand disease. Until recently however, models for lung diseases were not available. Our recently-published work has addressed this need and we can now produce large numbers of lung alveoli cells, one of the cell types affected by IPF. We can now make 3D lung organoids allowing modelling of complex biological functions and interactions, and can utilise these technologies to understand IPF.To this end we have assembled an international team to address 3 interlinked objectives:1) Generate a hIPSC model of IPF to understand how SFTPC mutations affect cellular phenotype and gene expression in AE2 cells. There are no known mutations that cause of IPF, however mutations in surfactant protein C (SFTPC) can cause inherited IPF also known as familial IPF, the mechanism however remains elusive. We will generate a stem cell model of familial IPF by taking a skin biopsy from patients with a SFTPC mutation and then turning their skin cells into lung cells using our established protocol. We will then use the cells to study how the mutation causes lung cell damage, fibrosis and to identify new therapeutic targets to treat IPF.2) Model respiratory infection in-vitro to understand the effect of infection on AE2 cells carrying SFTPC mutation. IPF patients have more frequent respiratory infections and >90% mortality rate per infection, how bacterial infection exacerbates IPF is not well understood. Using the SFTPC mutant cells created in Obj. 1 we will use our 3D culture platform to grow lung organoids and then infect them with bacteria or viruses. We will create a profile of changes in gene expression in response to infection and compare this to uninfected cells. This will provide insight into how respiratory infection affects normal lung cell function and importantly deliver insight into the mechanism by which infection exacerbates IPF.3) Model change in ECM composition to understand how ECM impacts cell phenotype. As IPF progresses, the scaffold that holds the lungs together, the extracellular matrix (ECM), dramatically changes, causing the lungs to become stiff and less elastic as well as reducing oxygen absorption. How the changes in ECM affect lung cell function, and whether lung cells actively contribute to further ECM changes has not been well characterised. We will develop our understanding of this process by growing cells created in Obj. 1 in simple 3D gels that will allow us to model how the ECM changes during early and late stages of IPF. We will then assess how these changes affect cell functionality and gene expression. This will provide insight into how ECM influences lung cell function and importantly deliver potential therapeutic targets that aim to prevent or reverse changes to ECM in IPF. These objectives will address poor access to human IPF disease models, and provide insight to the pathogenesis of lung fibrosis and infection in IPF, offering the opportunity to develop exciting new drug targets as well as diagnostic and prognostic markers for the disease. Importantly, this platform could be modified to model other fibrotic lung diseases, and other diseases that have a fibrotic component.