TGF-β activation impairs fibroblast ability to support adult lung epithelial progenitor cell organoid formation

Transforming growth factor-beta (TGF-beta)-induced fibroblast-to-myofibroblast differentiation contributes to remodeling in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis, but whether this impacts the ability of fibroblasts to support lung epithelial repair remains little explored. We pretreated human lung fibroblasts [primary (phFB) or MRC5 cells] with recombinant human TGF-beta to induce myofibroblast differentiation, then cocultured them with adult mouse lung epithelial cell adhesion molecule-positive cells (EpCAM(+)) to investigate their capacity to support epithelial organoid formation in vitro. While control phFB and MRC5 lung fibroblasts supported organoid formation of mouse EpCAM(+) cells, TGF-beta pretreatment of both phFB and MRC5 impaired organoid-supporting ability. We performed RNA sequencing of TGF beta-treated phFB. which revealed altered expression of key Wnt signaling pathway components and Wnt/beta-catenin target genes, and modulated expression of secreted factors involved in mesenchymal-epithelial signaling. TGF-beta profoundly skewed the transcriptional program induced by the Wnt/beta-catenin activator CHIR99021. Supplementing organoid culture media recombinant hepatocyte growth factor or fibroblast growth factor 7 promoted organoid formation when using TGF-beta pretreated fibroblasts. In conclusion, TGF-beta-induced myofibroblast differentiation results in Wnt/beta-catenin pathway skewing and impairs fibroblast ability to support epithelial repair likely through multiple mechanisms, including modulation of secreted growth factors.