Tziouvelis, M. | bioRxiv Science

bioRxiv2026

Personalized multi-assay profiling of respiratory motile ciliopathies and mRNA therapy

IntroductionImpaired motile cilia function contributes to many respiratory disorders, but therapies targeting this cellular defect are currently lacking. Personalized airway epithelial models combined with quantitative, complementary ciliary assays can pave the way for the development of such therapies. However, existing airway epithelial cultures often show variable ciliogenesis, and ciliary function is frequently assessed using a single assay that does not capture the phenotypic heterogeneity of ciliary dysfunction. Here, we established a personalized, multi-assay in vitro platform using human nasal epithelial cells (HNECs) to assess ciliary function and therapeutic response, using primary ciliary dyskinesia (PCD) as a model disease. MethodsHNECs from 8 healthy individuals and 13 individuals with PCD carrying distinct disease-associated variants were obtained by nasal brushing. Cells were differentiated under optimized conditions, including {gamma}-secretase/Notch and BMP pathway inhibitors and a low liquid-liquid interface, to generate highly ciliated 2D epithelial cultures. Ciliary function was assessed using ciliary beat frequency, bead transport, and apical-out nasal organoid rotation assays. Therapeutic rescue was assessed in HNECs harboring DNAI1 alterations using DNAI1 mRNA-loaded lipid nanoparticles. ResultsOptimized differentiation yielded reproducibly multiciliated HNEC cultures. The multi-assay platform distinguished healthy from PCD-derived HNECs and revealed individual- and genotype-specific patterns of ciliary dysfunction not captured by a single assay. Basolateral administration of DNAI1 mRNA-loaded lipid nanoparticles resulted in partial, dose-dependent recovery of ciliary function in DNAI1-deficient HNECs. ConclusionThis study establishes a standardized, individual-specific multi-assay nasal epithelial platform for functional phenotyping of motile cilia and preclinical evaluation of emerging therapies, with demonstrated utility in PCD.

cell biology↗

bioRxiv2025

Immortalization of human nasal and bronchial airway epithelial cells for genome editing applications

In vitro air-liquid interface culture of airway epithelial cells is used as a model system to study respiratory diseases. This culture system not only overcomes the need for animal models or continuous biopsies from individuals but also enables studies of pathophysiology associated with the disease in a patient background. Human airway basal cells serve as progenitor cells for a functional pseudostratified airway epithelium composed mainly of multiciliated and secretory cells. However, due to the limited ability of basal cells to proliferate and differentiate, the long-term use of primary material in culture is restricted. This challenges research that requires genome editing. Here, we describe airway stem cells from nasal and bronchial origin immortalized by hTERT overexpression followed by polyclonal expansion. We demonstrate that this diverse panel of cell lines shows differentiation patterns similar to primary stem cells and can be used for lentiviral and CRISPR/Cas9 genome editing. These cell lines and optimized protocols facilitate airway biology research and disease phenotyping.

cell biology↗