Promoter Hypomethylation Defines Airway Basal Cells with Impaired Capacity to Generate Ciliated Organoids

Background: Airway basal cells are the progenitors of the bronchial epithelium, and their ability to differentiate is crucial for the repair and function of the epithelium. Methods: Basal cells obtained via brush biopsies from twelve donors were expanded as 2D cultures and differentiated into 3D bronchial organoids. Genomic DNA was profiled using the MethylationEPIC v2.0 BeadChip and analysed using RnBeads. Gene expression was measured by bulk RNA sequencing. Results: Of twelve donor-derived basal cell populations, six generated large ciliated organoids, whereas six produced significantly smaller non-ciliated organoids. Epigenetic profiling revealed distinct promoter methylation patterns between the two groups. Principal component analysis and hierarchical clustering robustly separated ciliated cultures from non-ciliated cultures. Loss of ciliation was associated with an increased proportion of hypomethylated promoters. Promoters that were differentially hypomethylated in basal cells before differentiation included MMP3 , NREP , SLCO1B1 , and TM4SF19 . Significant hypomethylated promoters in non-ciliated organoids mapped to differentiation-related genes, including WWTR1 . However, WWTR1 was only weakly expressed in non-ciliated organoids, suggesting the presence of additional transcriptional regulation. Conclusion: Airway basal cells that fail to generate ciliated organoids display distinct promoter hypomethylation signatures. These findings highlight the critical role of epigenetic regulation in determining the differentiation potential of basal cells. Publication History Article published online: 04 March 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany