C19-01 KRT7 Cells of Ferret Airway Submucosal Glands Exhibit Progenitor Potential for Epithelial Regeneration

Abstract Rationale Airway submucosal glands (SMGs) play a critical in the pathogenesis of mucus obstructive airway diseases, including cystic fibrosis (CF). A subset of glandular ductal cells serves as stem cells for airway epithelial regeneration following injury. Keratin 7 (KRT7) expression is activated in proliferative epithelial cells and expressed in SMG ductal cells, indicating it may identify such a progenitor population. Because the ferret lung closely resembles the human lung in both anatomy and physiology, particularly in the abundance and distribution of SMGs, ferrets represent a highly relevant model for studying pulmonary disease. However, whether KRT7-expressing cells within ferret SMGs serve as airway progenitor cells capable of regenerating epithelium with CFTR functions has not been investigated. Methods Immunolocalization assays were used to examine the expression of KRT7 and other marker genes. KRT7-CreERT2:ROSA26-mTmG transgenic ferrets were generated for in vivo lineage tracing, in which the KRT7-expressing cells and their progeny were genetically labeled with EGFP after tamoxifen induction. Epithelial injury was induced via airway polidocanol administration. Primary ferret EGFP+ KRT7-lineage cells were isolated from SMGs and cultured as organoids or on an air-liquid interface (ALI) for in vitro validation. CFTR function was assessed using a Forskolin-induced swelling (FIS) assay on organoids and by Ussing chamber analysis of ALI cultures. Results A subset of airway glandular epithelial cells highly expresses KRT7 across human, ferret, and mouse, which was restricted to gland ducts and acini. These KRT7high glandular cells also expressed the ductal stem cell known markers KRT19 and CD166. After tamoxifen induction, a subpopulation of airway SMG epithelial cells was EGFP-labeled and colocalized with CD116 and KRT19 in KRT7-CreERT2:ROSA26-mTmG ferrets. Following polidocanol-induced injury, these EGFP+ KRT7-lineage cells migrated from the glands into the regenerating surface epithelium, where they differentiated into major airway epithelial cell types, including ciliated, goblet, club, and basal cells. In vitro, primary EGFP+ KRT7-lineage cells isolated from airway SMGs exhibited robust proliferation and differentiation in both organoid and air-liquid interface cultures. Importantly, functional assays confirmed that the regenerated KRT7-lineage epithelia exhibited CFTR-mediated ion transport, validated by positive forskolin-induced swelling and short-circuit current responses in Ussing chamber measurements. Conclusion This study identifies KRT7 as a potential marker for progenitors within SMG ducts capable of regenerating a fully differentiated and functional airway epithelium. These cells represent an endogenous source for epithelial repair and a promising target for cell-based or gene-based therapies to correct defects in mucus-obstructive airway diseases like CF. This abstract is funded by: None