3D cell culture models demonstrate a role for FGF and WNT signaling in regulation of lung epithelial cell fate and morphogenesis

Abstract

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...

R-SPONDIN2 mesenchymal cells form the bud tip progenitor niche during human lung development

“…We observed that two mesenchymal cell clusters identified by scRNA-seq are enriched for expression of the WNT agonist RSPO2 throughout the developmental time frame analyzed, and spatial localization showed that RSPO2 is expressed adjacent to the bud tip domain. It is known that high WNT signaling conditions are necessary for the maintenance of the bud tip progenitor cell state in vitro Miller et al, 2018;Rabata et al, 2020), which made this cell population a strong bud tip-associated mesenchymal cell candidate.…”

Stable iPSC-derived NKX2-1+ Lung Bud Tip Progenitor Organoids Give Rise to Airway and Alveolar Cell Types

“…Based on this observation, there is an opportunity to further refine the signaling pathways that are manipulated to expand and maintain NKX2-1 + lung lineages; however, this may prove challenging given the redundant use of some pathways (i.e. WNT) to maintain stem/progenitor cells from both lineages (Pinto and Clevers, 2005; Kapoor, Li and Leiter, 2007; Volckaert and De Langhe, 2015; Chin et al ., 2016; Nikolić and Rawlins, 2017; Yan et al ., 2017; Miller et al ., 2018; Rabata et al ., 2020; Aros, Pantoja and Gomperts, 2021). We observed that hindgut lineages expand after passaging organoids using the standard approach of mechanically shearing them into small fragments.…”