Serna Valverde, Ana Lilia
(2023)
In vitro modelling of respiratory infections in idiopathic pulmonary fibrosis (IPF) using human induced pluripotent stem cell (hiPSCs)-derived alveolar epithelial type II cells.
PhD thesis, University of Nottingham.
Abstract
Idiopathic pulmonary fibrosis (IPF) is a chronic, interstitial lung disease estimated to affect 4.5 individuals per 10,000[1]. It is characterised by the accumulation of extracellular matrix in the lung interstitium, which disrupts the normal architecture and impairs lung function resulting in severe respiratory failure and death. The rapid progression of IPF and the lack of curative treatments is often accompanied by a median survival of 2-3 years [2]. Its aetiology is unclear; however, environmental and genetic risk factors indicate that alveolar epithelial type 2 cells (AT2s), the facultative progenitor of the alveoli, play a pivotal role [3, 4]. Mutations in surfactant protein (SFTPC) genes compromise AT2s function and viability, reducing its capacity to regenerate damaged alveolar epithelial tissue. This function is further compromised by infection-induced lung injury. Integrating such environmental stimuli and genetic predisposition in IPF patients results in an aberrant fibrotic response driven by AT2s [5, 6].
The study of this host-pathogen interaction has been hampered by the lack of adequate human IPF models.[6]. Therefore, the differentiation of patient-derived human induced pluripotent stem cells (hiPSC) into alveolar organoids represents a valuable tool for studying genotype-phenotype relationships of IPF in vitro. For this, an organoid platform using IPF patient-derived hiPSCs carrying a BRICHOS-domain SFTPC mutation was generated. Furthermore, using base editing techniques, the platform was also adapted to be used with corrected isogenic hiPSCs [7]. A thoroughly optimised directed differentiation protocol was generated to differentiate lung progenitors in monolayer cultures. These cells were further enriched and transferred to 3D Matrigel droplets in maturation media to obtain a population of AT2 cells expressing SFTPC that was further enriched using a surrogate surface marker [8]. Transcriptome profiling of differentiating cells at several stages of the protocol confirmed the expression of key markers at the definitive endoderm, lung progenitor, and alveolar stages of the differentiation.
Finally, the SFTPC-mutant and control AT2 cells were enriched and exposed to H1N1 influenza A virus to evaluate their capacity to mount a response to immune stimuli and serve as an in vitro model for human respiratory infections. The infected and uninfected samples were analysed using RNA sequencing. Upon infection, it was found that AT2 cells induced an inflammatory and antiviral response led by the activation of interferon signalling pathways. Furthermore, overrepresented processes in mutant IPF AT2 cells included “extracellular matrix organisation", "extracellular matrix disassembly", and "collagen catabolic process” when compared with the wild-type cells. The analysis also revealed differentially expressed genes linked to IPF, such as MMP7, IL1B, MUC5B and TERC [9–11]. These data validate IPF-patient-derived AT2 cells as a robust in vitro model to better understand the connection between genotype and environmental interactions in IPF disease progression.
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