Temporal Whole-Transcriptomic Analysis of Characterized In Vitro and Ex Vivo Primary Nasal Epithelia

Air-liquid interface (ALI) cell culture of primary airway progenitors enables the differentiation and recapitulation of a pseudostratified epithelium , providing a highly useful tool for researching respiratory health and disease. Previous studies into gene expression in ALI-cultures compared to nasal brushings have been limited in the number of time-points and/or the number of genes studied. In this study physiological and global transcriptomic changes were assessed in an extended 63-day human healthy nasal epithelium ALI-culture period and compared to nasal brushing samples. nasal brushing samples formed distinct transcriptome clusters to ALI-cultured nasal epithelia, with from day 14 onwards ALI samples best matching the samples. Immune response regulation genes were not expressed in the ALI-culture compared to the nasal brushing samples, likely because the cultures lack an airway microbiome, lack airborne particles stimulation, or did not host an immune cell component. This highlights the need for more advanced co-cultures with immune cell representation to better reflect the physiological state. During the first week of ALI-culture genes related to metabolism and proliferation were increased. By the end of week 1 epithelial cell barrier function plateaued and multiciliated cell differentiation started, although widespread ciliation was not complete until day 28. These results highlight that time-points at which ALI-cultures are harvested for research studies needs to be carefully considered to suit the purpose of investigation (transcriptomic and/or functional analysis).