RESUMEN
The human airway mucociliary epithelium can be recapitulated in vitro using primary cells cultured in an Air-Liquid Interface (ALI), a reliable surrogate to perform pathophysiological studies. As tremendous variations exist between media used for ALI-cultured human airway epithelial cells, our study aimed to evaluate the impact of several media (BEGM, PneumaCult, "Half&Half" and "Clancy") on cell type distribution using single-cell RNA sequencing and imaging. Our work revealed the impact of these media on cell composition, gene expression profile, cell signaling or epithelial morphology. We found higher proportions of multiciliated cells in PneumaCult-ALI and Half&Half, stronger EGF signaling from basal cells in BEGM-ALI, differential expression of the SARS-CoV-2 entry factor ACE2, and distinct secretome transcripts depending on media used. We also established that proliferation in PneumaCult-Ex Plus favored secretory cell fate, showing the key influence of proliferation media on late differentiation epithelial characteristics. Altogether, our data offer a comprehensive repertoire for evaluating the effects of culture conditions on airway epithelial differentiation and will help to choose the most relevant medium according to the processes to be investigated such as cilia, mucus biology or viral infection. We detail useful parameters that should be explored to document airway epithelial cell fate and morphology.
Asunto(s)
VirosisRESUMEN
Background: Wastewater surveillance has been proposed as an epidemiological tool to define the prevalence and evolution of the SARS-CoV-2 epidemics. However, most implemented SARS-CoV-2 wastewater surveillance projects were relying on qPCR measurement of virus titers and did not address the mutational spectrum of SARS-CoV-2 circulating in the population. Methods: We have implemented a nanopore RNA sequencing monitoring system in the city of Nice (France, 550,000 inhabitants). Between October 2020 and March 2021, we monthly analyzed the SARS-CoV-2 variants in 113 wastewater samples collected in the main wastewater treatment plant and 20 neighborhoods. Findings: We initially detected the lineages predominant in Europe at the end of 2020 (B.1.160, B.1.177, B.1.367, B.1.474, and B.1.221). In January, a localized emergence of a variant (Spike:A522S) of the B.1.1.7 lineage occurred in one neighborhood. It rapidly spread and became dominant all over the city. Other variants of concern (B.1.351, P.1) were also detected in some neighborhoods, but at low frequency. Comparison with individual clinical samples collected during the same week showed that wastewater sequencing correctly identified the same lineages as those found in COVID-19 patients. Interpretation: Wastewater sequencing allowed to document the diversity of SARS-CoV-2 sequences within the different neighborhoods of the city of Nice. Our results illustrate how sequencing of sewage samples can be used to track pathogen sequence diversity in the current pandemics and in future infectious disease outbreaks.