Fibroblast growth factor 2 (FGF2) plays important roles in tissue development and repair. Using heparan sulfates (HS)/heparin as a cofactor, FGF2 binds to FGF receptor (FGFR) and induces downstream signaling pathways, such as ERK pathway, that regulate cellular behavior. In most cell lines, FGF2 signaling displays biphasic dose-response profile, reaching maximal response to intermediate concentrations, but weak response to high levels of FGF2. Recent reports demonstrated that the biphasic cellular response results from competition between binding of FGF2 to HS and FGFR that impinge upon ERK signaling dynamics. However, the role of HS/heparin in FGF signaling has been controversial. Several studies suggested that heparin is not required for FGF-FGFR complex formation and that the main role of heparin is to protect FGF from degradation. In this study, we investigated the relationship between FGF2 stability, heparin dependence and ERK signaling dynamics using FGF2 variants with increased thermal stability (FGF2-STABs). FGF2-STABs showed higher efficiency in induction of FGFR-mediated proliferation, lower affinity to heparin and were less dependent on heparin than wild-type FGF2 (FGF2-wt) for induction of FGFR-mediated mitogenic response. Interestingly, in primary mammary fibroblasts, FGF2-wt displayed a sigmoidal dose-response profile, while FGF2-STABs showed a biphasic response. Moreover, at low concentrations, FGF2-STABs induced ERK signaling more potently and displayed a faster dynamics of full ERK activation and higher amplitudes of ERK signaling than FGF2-wt. Our results suggest that FGF2 stability and heparin dependence are important factors in FGF-FGFR signaling complex assembly and ERK signaling dynamics.

FGF signaling plays an essential role in lung development, homeostasis, and regeneration. We employed mouse 3D cell culture models and imaging to study ex vivo the role of FGF ligands and the interplay of FGF signaling with epithelial growth factor (EGF) and WNT signaling pathways in lung epithelial morphogenesis and differentiation. In non-adherent conditions, FGF signaling promoted formation of lungospheres from lung epithelial stem/progenitor cells (LSPCs). Ultrastructural and immunohistochemical analyses showed that LSPCs produced more differentiated lung cell progeny. In a 3D extracellular matrix, FGF2, FGF7, FGF9, and FGF10 promoted lung organoid formation. FGF9 showed reduced capacity to promote lung organoid formation, suggesting that FGF9 has a reduced ability to sustain LSPC survival and/or initial divisions. FGF7 and FGF10 produced bigger organoids and induced organoid branching with higher frequency than FGF2 or 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 non-essential role in FGF-induced lung organoid formation. 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. FGF ligands showed differences in promoting distinct lung epithelial cell types. FGF9 was a potent inducer of more proximal cell types, including ciliated and basal cells. FGF7 and FGF10 directed the differentiation toward distal lung lineages. 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 postnatal lung development and homeostasis, and we reveal distinct roles for FGF ligands in regulation of mouse lung morphogenesis and differentiation ex vivo .

Lung epithelium contains distinctive subpopulations of lung stem/progenitor cells (LSPCs) that are essential for lung epithelial maintenance and repair in vivo. Hence, LSPCs are in the center of interest of lung biology due to their promising therapeutic applications. To reach this goal, proper characterization of LSPCs, understanding of their proliferation and differentiation potentials and elucidation of mechanisms that control them are necessary. Therefore, development of reliable in vitro clonogenic assays has been needed. We established lungosphere assay, an in vitro sphere-forming 3D culture assay that enables to evaluate stem/progenitor cell activity, self-renewal and differentiation capacity of LSPCs and to conveniently test the effect of various treatments on LSPCs. Here we provide a detailed description of procedures for isolation of adult mouse lung epithelial cells, their culture in non-adherent conditions to form LSPC-derived spheroids (lungospheres) and for embedding of lungospheres into 3D extracellular matrix to model processes of lung tissue maintenance in a physiologically relevant microenvironment.

Extracellular matrix (ECM) is an essential component of the tissue microenvironment, actively shaping cellular behavior. In vitro culture systems are often poor in ECM constituents, thus not allowing for naturally occurring cell-ECM interactions. This study reports on a straightforward and efficient method for the generation of ECM scaffolds from lung tissue and its subsequent in vitro application using primary lung cells. Mouse lung tissue was subjected to decellularization with 0.2% sodium dodecyl sulfate, hypotonic solutions, and DNase. Resultant ECM scaffolds were devoid of cells and DNA, whereas lung ECM architecture of alveolar region and blood and airway networks were preserved. Scaffolds were predominantly composed of core ECM and ECM-associated proteins such as collagens I-IV, nephronectin, heparan sulfate proteoglycan core protein, and lysyl oxidase homolog 1, among others. When homogenized and applied as coating substrate, ECM supported the attachment of lung fibroblasts (LFs) in a dose-dependent manner. After ECM characterization and biocompatibility tests, a novel in vitro platform for three-dimensional (3D) matrix repopulation that permits live imaging of cell-ECM interactions was established. Using this system, LFs colonized the ECM scaffolds, displaying a close-to-native morphology in intimate interaction with the ECM fibers, and showed nuclear translocation of the mechanosensor yes-associated protein (YAP), when compared with cells cultured in two dimensions. In conclusion, we developed a 3D-like culture system, by combining an efficient decellularization method with a live-imaging culture platform, to replicate in vitro native lung cell-ECM crosstalk. This is a valuable system that can be easily applied to other organs for ECM-related drug screening, disease modeling, and basic mechanistic studies.

15FGF signaling plays an essential role in lung development, homeostasis, and regeneration. Several 16 FGF ligands were detected in the developing lungs, however, their roles have not been fully elucidated. 17 We employed mouse 3D cell culture models and imaging to ex vivo study of a) the role of FGF ligands 18 in lung epithelial morphogenesis and b) the interplay of FGF signaling with epithelial growth factor 19 (EGF) and WNT signaling pathways. In non-adherent conditions, FGF signaling promoted formation 20 of lungospheres from lung epithelial stem/progenitor cells (LSPCs). Based on their architecture, we 21 defined three distinct phenotypes of lungospheres. Ultrastructural and immunohistochemical analyses 22 showed that LSPCs produced more differentiated lung cell progeny. In 3D extracellular matrix, FGF2, 23 FGF7, FGF9, and FGF10 promoted lung organoid formation with similar efficiency. However, FGF9 24 showed reduced capacity to promote lung organoid formation, suggesting that FGF9 has a reduced 25 ability to sustain LSPCs survival and/or initial divisions. Analysis of lung organoid phenotypes 26 revealed that FGF7 and FGF10 produce bigger organoids and induce organoid branching with higher 27 frequency than FGF2 and FGF9. Higher FGF concentration and/or the use of FGF2 with increased 28 stability and affinity to FGF receptors both increased lung organoid and lungosphere formation 29 efficiency, respectively, suggesting that the level of FGF signaling is a crucial driver of LSPC survival 30 and differentiation, and also lung epithelial morphogenesis. EGF signaling played a supportive but 31 nonessential role in FGF-induced lung organoid formation. Moreover, analysis of tissue architecture 32 and cell type composition confirmed that the lung organoids contained alveolar-like regions with cells 33 expressing alveolar type I and type II cell markers, as well as airway-like structures with club cells and 34 ciliated cells. WNT signaling enhanced the efficiency of lung organoid formation, but in the absence 35 of FGF10 signaling, the organoids displayed limited branching and less differentiated phenotype. In 36 summary, we present lung 3D cell culture models as useful tools to study the role and interplay of 37 signaling pathways in lung development and we reveal roles for FGF ligands in regulation of mouse 38 lung morphogenesis ex vivo. 39 65 amplification of Fgf10 expressing airway smooth muscle cell progenitors in the distal mesenchyme 66 (Volckaert and De Langhe, 2015). In adult lung, FGF10 and WNT signaling regulate the activity of 67 basal cells, the lung epithelial stem/progenitor cells (LSPCs) that ensure lung epithelial homeostasis 68 and repair after injury (Volckaert et al., 2013). However, the exact functions of FGF and WNT 69 signaling in LSPCs have not been fully elucidated. 70In this study, we investigated the role of FGF and WNT signaling in regulation of epithelial 71 morphogenesis from LSPCs. To this end, we developed and used several 3D cell culture techniques, 72 including lungosphere an...