Lineage tracing of alveolar fibroblasts during delayed resolution of pulmonary fibrosis in aged mice

Abstract

Organoid models have become an integral part of the research methodology in the lung field. Distinct models of lung organoids mimicking various anatomical regions of mature lungs. In the distal lung, AEC2s represent a stem cell population that is engaged in regenerative mechanisms in response to various insults. These cells self-renew and give rise to AEC1s that carry out gas exchange. Multiple experimental protocols allowing the generation of alveolar organoids from murine lungs have been described. Among the drawbacks have been the requirement of transgenic mice allowing the isolation of AEC2s with high viability and purity, and the occasional emergence of bronchiolar and bronchioalveolar organoids. Here, we provide a refined gating strategy and an optimized protocol for the generation of alveolar organoids from wild-type mice. Our approach not only overcomes the need for transgenic mice to generate such organoids but also yields a pure culture of alveolar organoids that is devoid of bronchiolar and bronchioalveolar organoids. Our protocol contributes to the standardization of this important research tool. IPF is an aging-associated, progressive and lethal lung disease that affects older adults. While lung fibrosis resolves in younger mice, aged mice developed non-resolving fibrosis in response to bleomycin-induced injury. In this study, we employed 52–to-56-week-old female Fgf10Cre-ERT2/+; tdTomatoflox mice to establish a pulmonary fibrosis model with delayed resolution and recapitulate pathological features of IPF. Histological analyses and single-cell transcriptomics on sorted lineage-labeled cells were performed at the peak of fibrosis (Bleo d14) as well as early (Bleo d30) and late (Bleo d60) resolution. The lineagetracing approach identified Fgf10+ AFs with LIF signature as a cellular source of not only MyoFBs that arise during fibrogenesis but also Adamts4+ cells that persist during delayed fibrosis resolution. Datamining and ex vivo therapeutic interventions indicated strong clinical relevance for our findings and highlighted ADAMTS4 as a promising therapeutic target in pulmonary fibrosis. Functional intervention using ex vivo assays revealed strong clinical relevance. Our study provides valuable insights into the cellular origins and trajectories of AFs and MyoFBs and sets the stage for identifying further targets for future interventions.