Dreyer, H. H. M. | Search bioRxiv

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↗

bioRxiv2024

Small molecule-directed differentiation of submerged-cultured human nasal airway epithelia for respiratory disease modelling

Submerged cultures of undifferentiated or transformed epithelial cells are widely used in respiratory research due to their ease of use and scalability. However, these systems fail to capture the cellular diversity of the human airway epithelium. In this study, we developed an in vitro model where cryopreserved human nasal epithelial cells, collected by brushings, are differentiated under submerged conditions on standard plastic cultureware. By applying small-molecule inhibitors targeting Notch and BMP signaling, we achieved efficient differentiation of cultures containing basal, secretory, and ciliated cells. This approach supports scalable culturing of both 2D epithelial monolayers and 3D organoids, validated as (personalized) disease models for primary ciliary dyskinesia, cystic fibrosis, and respiratory syncytial virus infection. This model offers a cost-effective, scalable platform that combines the simplicity of traditional cultures with the cellular complexity of the human airway epithelium, providing a valuable tool for respiratory disease research.

cell biology↗