SUMMARY Functional modeling of many adult epithelia is limited by the difficulty of maintaining relevant stem cell populations in culture. Here, we show that dual inhibition of SMAD signaling pathways enables robust expansion of primary epithelial basal cell populations. We found that TGFβ/BMP/SMAD pathway signaling is strongly activated in luminal and suprabasal cells of several epithelia, but suppressed in p63+ basal cells. In airway epithelium, SMAD signaling promotes differentiation, and its inhibition leads to stem cell hyperplasia. Using dual SMAD inhibition in a feeder-free culture system we were able to expand airway basal stem cells from multiple species. Expanded cells can produce functional airway epithelium that is physiologically responsive to clinically relevant drugs such as CFTR modulators. This approach is effective for clonal expansion of single human cells and for basal cell populations from epithelial tissues from all three germ layers, and may therefore be broadly applicable for modeling of epithelia.

CFTR modulator theratyping is a novel and rapidly evolving field that has the potential to identify rare CFTR variants that are responsive to approved drugs or drugs in development.

The gain-of-function MUC5B promoter variant rs35705950 is the dominant risk factor for developing idiopathic pulmonary fibrosis (IPF). Here we show in humans that MUC5B, a mucin thought to be restricted to conducting airways, is co-expressed with surfactant protein C (SFTPC) in type 2 alveolar epithelia and in epithelial cells lining honeycomb cysts, indicating that cell types involved in lung fibrosis in distal airspace express MUC5B. In mice, we demonstrate that Muc5b concentration in bronchoalveolar epithelia is related to impaired mucociliary clearance (MCC) and to the extent and persistence of bleomycin-induced lung fibrosis. We also establish the ability of the mucolytic agent P-2119 to restore MCC and to suppress bleomycin-induced lung fibrosis in the setting of Muc5b overexpression. Our findings suggest that mucociliary dysfunction might play a causative role in bleomycin-induced pulmonary fibrosis in mice overexpressing Muc5b, and that MUC5B in distal airspaces is a potential therapeutic target in humans with IPF.

Rationale:The cystic fibrosis (CF) modulator drug elexacaftor/tezacaftor/ivacaftor (ETI) proved highly effective in controlled clinical trials for individuals with ≥1 F508del allele, which occurs in at least 85% of people with CF (PwCF). Objective: PROMISE is a post-approval study to understand the broad effects of ETI through 30 months clinical use in a more diverse US patient population with planned analyses after 6 months. Methods: Prospective, observational study in 487 PwCF age ≥12 years with ≥1 F508del allele starting ETI for the first time. Assessments occurred before and 1, 3, and 6 months into ETI therapy. Outcomes included change in ppFEV 1 , sweat chloride concentration, body mass index, and selfreported respiratory symptoms. Results: average age was 25.1 years. 44.1% entered the study using tezacaftor/ivacaftor or lumacaftor/ivacaftor while 6.7% were using ivacaftor, consistent with F508del homozygosity and G551D allele, respectively. At 6 months into ETI therapy, ppFEV 1 improved 9.76 percentage points (95% CI 8.76, 10.76) from baseline, CFQ-R Respiratory Domain score improved 20.4 points (95% CI 18.3, 22.5), and sweat chloride decreased -41.7 mmol/L (95% ). BMI also significantly increased.Changes were larger in those naïve to modulators but substantial in all groups, including those treated with ivacaftor at baseline. Conclusions: ETI by clinical prescription provided large improvements in lung function, respiratory symptoms, and BMI in a diverse population naïve to modulator drug therapy, using existing two-drug combinations, or using ivacaftor alone. Each group also experienced significant reductions in sweat chloride concentration, which correlated with improved ppFEV 1 in the overall study population.

The application of conditional reprogramming culture (CRC) methods to nasal airway epithelial cells would allow more widespread incorporation of primary airway epithelial culture models into complex lung disease research. In this study, we adapted the CRC method to nasal airway epithelial cells, investigated the growth advantages afforded by this technique over standard culture methods, and determined the cellular and molecular basis of CRC cell culture effects. We found that the CRC method allowed the production of 7.1 3 10 10 cells after 4 passages, approximately 379 times more cells than were generated by the standard bronchial epithelial growth media (BEGM) method. These nasal airway epithelial cells expressed normal basal cell markers and could be induced to form a mucociliary epithelium. Progenitor cell frequency was significantly higher using the CRC method in comparison to the standard culture method, and progenitor cell maintenance was dependent on addition of the Rho-kinase inhibitor Y-27632. Wholetranscriptome sequencing analysis demonstrated widespread gene expression changes in Y-27632-treated basal cells. We found that Y-27632 treatment altered expression of genes fundamental to the formation of the basal cell cytoskeleton, cell-cell junctions, and cell-extracellular matrix (ECM) interactions. Importantly, we found that Y-27632 treatment up-regulated expression of unique basal cell intermediate filament and desmosomal genes. Conversely, Y-27632 down-regulated multiple families of protease/antiprotease genes involved in ECM remodeling. We conclude that Y-27632 fundamentally alters cell-cell and cell-ECM interactions, which preserves basal progenitor cells and allows greater cell amplification.