3D Cell Culture Models Demonstrate a Role for FGF and WNT Signaling in Regulation of Lung Epithelial Cell Fate and Morphogenesis

FGF signaling plays an essential role in lung development, homeostasis, and regeneration. Several FGF ligands were detected in the developing lungs, however, their roles have not been fully elucidated. We employed mouse 3D cell culture models and imaging toex vivostudy ofa)the role of FGF ligands in lung epithelial morphogenesis andb)the interplay of FGF signaling with epithelial growth factor (EGF) and WNT signaling pathways. In non-adherent conditions, FGF signaling promoted formation of lungospheres from lung epithelial stem/progenitor cells (LSPCs). Based on their architecture, we defined three distinct phenotypes of lungospheres. Ultrastructural and immunohistochemical analyses showed that LSPCs produced more differentiated lung cell progeny. In 3D extracellular matrix, FGF2, FGF7, FGF9, and FGF10 promoted lung organoid formation with similar efficiency. However, FGF9 showed reduced capacity to promote lung organoid formation, suggesting that FGF9 has a reduced ability to sustain LSPCs survival and/or initial divisions. Analysis of lung organoid phenotypes revealed that FGF7 and FGF10 produce bigger organoids and induce organoid branching with higher frequency than FGF2 and FGF9. Higher FGF concentration and/or the use of FGF2 with increased stability and affinity to FGF receptors both increased lung organoid and lungosphere formation efficiency, respectively, suggesting that the level of FGF signaling is a crucial driver of LSPC survival and differentiation, and also lung epithelial morphogenesis. EGF signaling played a supportive but nonessential role in FGF-induced lung organoid formation. Moreover, analysis of tissue architecture and cell type composition confirmed that the lung organoids contained alveolar-like regions with cells expressing alveolar type I and type II cell markers, as well as airway-like structures with club cells and ciliated cells. WNT signaling enhanced the efficiency of lung organoid formation, but in the absence of FGF10 signaling, the organoids displayed limited branching and less differentiated phenotype. In summary, we present lung 3D cell culture models as useful tools to study the role and interplay of signaling pathways in lung development and we reveal roles for FGF ligands in regulation of mouse lung morphogenesisex vivo.