Abstract Rationale Impaired CFTR function in airway submucosal glands (SMGs) and surface epithelium leads to airway obstruction in cystic fibrosis (CF). Defining stem cell populations within these compartments is critical for developing regenerative and gene editing-based therapies. Unlike rodents, ferrets possess SMGs throughout their cartilaginous airways, similar to humans, and develop human-like CF lung disease. SMG myoepithelial cells (MECs) are a reserved stem cell population in the proximal trachea of mice, capable of regenerating both SMG and surface airway epithelial cell types. However, the behavior and regenerative potential of MECs within the intralobar cartilaginous airways, and in the context of CF disease, have yet to be investigated. Methods We generated ACTA2-CreERT2: ROSA-mTmG transgenic ferrets to enable fate-mapping of MECs and their progeny via EGFP expression. Tamoxifen was administered to trace MECs during postnatal development, CF disease progression, or following polidocanol-induced airway epithelial injury in adult ferrets. Labeled MECs were isolated and tested in organoid and air-liquid interface (ALI) cultures to evaluate multipotency. CFTR-mediated ion transport was evaluated using Forskolin-induced swelling (FIS) assays on organoids and Ussing chamber recordings in ALI epithelia. Results Lineage tracing confirmed MECs contribute to the development of SMGs and can migrate to regenerate the surface airway epithelium after severe injury, where they differentiate into all major epithelial cell types, including CFTR-enriched ionocytes. EGFP-labeled MEC lineage contributed to MUC5B+ mucous cells and Lysozyme+ serous cells within SMGs when ACTA2-CreERT2: ROSA-mTmG MECs were traced at 2 weeks of age during active gland development or in CF ferrets (CFTR-G551D:ACTA2-CreERT2: ROSA-mTmG) with lung disease. This latter observation suggests that MECs may contribute to pathologic SMG hypertrophy and remodeling. FIS assay showed that MEC-derived organoids exhibited significant forskolin-induced swelling, demonstrating robust fluid secretion dependent on a functional CFTR channel, which was absent in CF organoids. Ussing chamber analysis of MEC-derived ALI cultures also revealed substantial forskolin-stimulated CFTR-mediated currents. These CFTR-specific functions were lost in epithelia derived from MECs isolated from CFTR-G551D CF ferrets. Conclusion These results demonstrate that SMG MECs act as progenitors capable of regenerating a functional airway epithelium with proper CFTR-mediated ion transport. MECs also contribute to pathological glandular remodeling in CF. Together, these findings identify MECs as a potential therapeutic target for CF and other airway obstructive diseases. This abstract is funded by: No
Ma et al. (Fri,) studied this question.