Biology subjects

Rodenburg, L. W.

Publications and source records attributed to Rodenburg, L. W..

4 recordsLinked to original sources

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↗

bioRxiv2022

Drug repurposing for Cystic Fibrosis: identification of drugs that induce CFTR-independent fluid secretion in nasal organoids

Individuals with Cystic Fibrosis (CF) suffer from severe respiratory disease due to a genetic defect in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene, which impairs airway epithelial ion and fluid secretion. New CFTR modulators that restore mutant CFTR function have been recently approved for a large group of people with CF (pwCF), but [~]19% of pwCF cannot benefit from CFTR modulators [1]. Restoration of epithelial fluid secretion through non-CFTR pathways might be an effective treatment for all pwCF. Here we developed a medium-throughput 384-wells screening assay using nasal CF airway epithelial organoids, with the aim to repurpose FDA-approved drugs as modulators of non-CFTR dependent epithelial fluid secretion. From a [~]1400 FDA-approved drug library, we identified and validated 12 FDA-approved drugs that induced CFTR-independent fluid secretion. Among the hits were several cAMP-mediating drugs, including {beta}2-adrenergic agonists. The hits displayed no effects on chloride conductance measured in Ussing chamber, and fluid secretion was not affected by TMEM16A as demonstrated by knockout (KO) experiments in primary nasal epithelial cells. Altogether, our results demonstrate the use of primary nasal airway cells for mediumscale drug screening, target validation with a highly efficient protocol for generating CRISPR-Cas9 KO cells and identification of compounds which induce fluid secretion in a CFTR- and TMEM16A-indepent manner.

cell biology↗

bioRxiv2021

Optical nanoscopy reveals SARS-CoV-2-induced remodeling of human airway cells

A better understanding of host cell remodeling by the coronavirus SARS-CoV-2 is urgently needed to understand viral pathogenesis and guide drug development. Expression profiling and electron microscopy have frequently been used to study virus-host interactions, but these techniques do not readily enable spatial, sub-cellular and molecular analysis of specific cellular compartments. Here, we use diffraction-unlimited fluorescence microscopy to analyze how SARS-CoV-2 infection exploits and repurposes the subcellular architecture of primary human airway cells. Using STED nanoscopy, we detect viral entry factors along the motile cilia of ciliated cells and visualize key aspects of the viral life cycle. Using Tenfold Robust Expansion (TREx) microscopy, we analyze the extensively remodeled three-dimensional ultrastructure of SARS-CoV-2-infected ciliated cells and uncover Golgi fragmentation, emergence of large and atypical multivesicular bodies enclosing viral proteins, ciliary clustering, and remodeling of the apical surface. These results demonstrate a broadly applicable strategy to study how viruses reorganize host cells with spatial and molecular specificity and provide new insights into SARS-CoV-2 infection in primary human cell models.

cell biology↗

bioRxiv2021

CFTR modulator response measurements in subjects with cystic fibrosis using 2D differentiated nasal epithelia converted into spheroids

Cystic Fibrosis (CF) is caused by genetic defects that impair the cystic fibrosis transmembrane conductance regulator (CFTR) channel in airway epithelial cells. These defects may be overcome by specific CFTR modulating drugs, for which the efficacy can be predicted in a personalized manner using 3D nasal-brushing-derived airway organoids in a forskolin-induced swelling assay. Despite of this, previously described application of 3D airway organoids in CFTR function assays have not been fully optimal. In this report we therefore describe an alternative method of culturing nasal brushing-derived airway organoids, which are created from an equally differentiated airway epithelial monolayer of a 2D air-liquid interface culture. In addition, we have defined organoid culture conditions, with the growth factor/cytokine combination neuregulin-1{beta} and interleukine-1{beta}, which enabled consistent detection of CFTR modulator responses in nasal airway organoids cultures from subjects with CF.

cell biology↗