Airway epithelial cells in healthy lungs produce mucus to protect the body from outside particles and infectious microorganisms. However, during infection and in chronic lung diseases such as cystic fibrosis, this mucus production and subsequent airway clearance is disrupted. In cystic fibrosis, this is due to variants of the CFTR gene, which cause the build-up of abnormally sticky mucus. Other genes have been proposed to influence the severity of cystic fibrosis, including the transcription factor EHF. Studies have suggested that EHF has a function in the lung airway epithelium; however, its exact role in disease is currently unclear.
Here, Pinte and colleagues developed a platform to investigate the role of EHF in lung homeostasis and disease. They differentiated human induced pluripotent stem cells (hiPSCs) into lung airway epithelial cells and seeded them in an air–liquid interface culture system that mimics aspects of the respiratory tract environment. Single-cell RNA sequencing (scRNAseq) validated the presence of multiple airway epithelial cell subtypes and confirmed expression of EHF across all of these. The authors then generated EHF knockout hiPSCs and used them to generate airway epithelial cells. Further scRNAseq showed that loss of EHF did not impact cellular differentiation but led to some transcriptional differences. Among the differentially expressed genes identified in the air–liquid interface cultures following EHF loss were ADGRL3, GOPC and PDE3, known to affect CFTR. The authors subsequently investigated CFTR function and found it to be increased in EHF knockout airway epithelial cells, but without any change to CFTR gene expression. This supports the idea that EHF can modulate cystic fibrosis severity by influencing CFTR function.
Airway obstruction, inflammation and mucus accumulation in lung disease can often lead to decreased oxygen availability, so the authors additionally investigated the role of EHF in hypoxia in the air–liquid interface system. Loss of EHF did not impact cell survival or the range of cell types present in the system. However, it did significantly decrease hypoxia-driven acidification of the culture, suggesting that EHF could have a role in hypoxia response in the airway epithelium.
Overall, this differentiation system paves the way for further studies of airway epithelial homeostasis and provides greater understanding of the role that EHF might play in airway clearance and hypoxia in cystic fibrosis and other lung disease contexts.
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