Lee, D. D. H. | bioRxiv Science

bioRxiv2020

Cell-intrinsic differences between human airway epithelial cells from children and adults

The airway epithelium is a key protective barrier, the integrity of which is preserved by the self-renewal and differentiation of basal stem cells. Epithelial cells are central to the pathogenesis of multiple lung diseases. In chronic lung diseases, increasing age is a principle risk factor. Few studies have explored the differences between airway epithelial cells in children and adults and how the function of basal stem cells changes during ageing is poorly understood. Here, we analyze airway epithelial cells from children and adults in homeostatic conditions (laser capture-microdissected whole epithelium and fluorescence-activated cell-sorted basal cells) and in proliferation-inducing cell culture conditions. We find that, while the cellular composition of the pediatric and adult tracheobronchial epithelium is broadly similar, in cell culture, pediatric airway epithelial cells displayed higher colony-forming ability, sustained in vitro growth and outcompeted adult cells in competitive proliferation assays. In RNA sequencing experiments, we observed potentially important differences between epithelium from children and adults, including higher baseline interferon-associated gene expression in pediatric epithelium. Our results demonstrate cell-intrinsic differences in transcriptional profile and regenerative capacity between proximal airway epithelial cells of children and adults. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/027144v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@b7a680org.highwire.dtl.DTLVardef@1145543org.highwire.dtl.DTLVardef@1e9a86aorg.highwire.dtl.DTLVardef@12fe0bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2020

High-content screening for rare respiratory diseases: readthrough therapy in primary ciliary dyskinesia

Development of therapeutic approaches for rare respiratory diseases is hampered by the lack of systems that allow medium-to-high-throughput screening of fully differentiated respiratory epithelium from affected patients. This is a particular problem for primary ciliary dyskinesia (PCD), a rare genetic disease caused by mutations in genes that adversely affect ciliary movement and consequently mucociliary transport. Primary cell culture of basal epithelial cells from nasal brush biopsies, followed by ciliated differentiation at air-liquid interface (ALI) has proven to be a useful tool in PCD diagnostics but the techniques broader utility, including in pre-clinical PCD research, has been limited by the number of basal cells that it is possible to expand from such biopsies. Here, we describe a high-content, imaging-based screening method, enabled by extensive expansion of PCD patient basal cells and their culture into differentiated human respiratory epithelium in miniaturised 96-well transwell format ALI cultures. Analyses of ciliary beat pattern, beat frequency and ultrastructure indicate that a range of different PCD defects are retained in these cultures. We perform a proof-of-principle personalized investigation in reduced generation of motile cilia (RGMC), a rare and very severe form of PCD, in this case caused by a homozygous nonsense mutation (c.441C>A; p.Cys147*) in the MCIDAS gene. The screening system allowed multiple drugs inducing translational readthrough to be evaluated alone or in combination with inhibitors of nonsense-mediated decay. Restoration of basal body formation in the patients nasal epithelial cells was seen in vitro, suggesting a novel avenue for drug evaluation and development in PCD. SummaryWe describe primary cell culture of nasal epithelial cells from patients with primary ciliary dyskinesia including differentiatiation of these to a ciliary phenotype and high-content screening in miniaturised air-liquid interface cultures.

cell biology↗