Extracellular vesicles and soluble factors secreted by lung fibroblasts support alveolar organoid formation

Abstract

<b>Rationale:</b> COPD is characterized by progressive and irreversible airflow limitation as a result of enhanced tissue destruction and defective tissue repair. As current therapeutics do not alter disease progression, new therapies that reactivate lung repair are needed. The secretome of fibroblasts, composed of Extracellular Vesicles (EVs) and other soluble factors (SF), has been linked to alveolar regeneration. We aimed to elucidate the supportive function of lung fibroblast-derived EVs and SF on the regenerative potential of alveolar epithelial progenitor cells in an organoid model. <b>Methods:</b> EVs and SF were purified using ultrafiltration and size exclusion chromatography. Mouse organoids were obtained by co-culturing 10,000 alveolar EpCAM+ cells with 2,500 lung fibroblasts, and then treated with EVs (10⁹&nbsp;EVs/ml) or SF (30 µg/ml). On day 14, number and size of the organoids was determined, as was the number of differentiated alveolar organoids. <b>Results:</b> Single treatment with EVs or SF increased organoid count, i.e. a 29.50% ± 8.11% increase for EVs and 33.00% ± 20.34% for SF. Neither treatment with EVs nor SF affected organoid size. Immunostaining for prosurfactant protein C revealed that the alveolar organoid count was significantly enhanced upon single treatment with EVs or SF. In addition, consecutive treatment for 14 days with EVs or SF resulted in enhanced organoid count (i.e. a 58.17% ± 39.60% and 91.67% ± 33.28% increase respectively) and size (i.e. a 36.50% ± 10.46% and 37.50% ± 27.02% increase respectively). <b>Conclusions:</b> Lung fibroblast-derived EVs and SF support alveolar epithelial organoid formation, making them an interesting potential treatment to pursue for COPD.