Transcriptome analysis of ciliary differentiation in airway epithelium

Fresh nasal brushings and air-liquid interface (ALI) cultures of nasal epithelial cells are frequently used as a model for airways research but the process of cell differentiation and ciliation in ALI cultures is poorly understood. We aimed to characterise ALI culture, in terms of gene expression and cell phenotype, over 63 days.ALI cultures of three healthy volunteers were harvested at 1, 4, 8, 14, 21, 28 and 63 days. We assessed the transcriptome and morphology of ciliary differentiation using RNA-seq, high-speed video microscopy (HSVM) and scanning electron microscopy (SEM). Specific gene markers were used to assess cell type changes throughout the culturing process. Differential gene expression analysis and functional enrichment analysis were done to identify changes in biological processes during culturing. Furthermore, the transcriptomes of ALI-cultures were compared to ‘fresh’ nasal epithelium in RNAlater.We present the ciliary differentiation from proliferate (day 4), to deuterosomal (day 8), to multi-ciliated cells (day 14) in ALI culture. Motile cilia were first detected on day 7 with HSVM, and SEM showed widespread ciliation from day 24 onwards. In addition, we present changes in biological processes involved with e.g. the cell cycle (day 4) and cilium assembly (day 8). It was found that ‘fresh’ nasal epithelium in RNAlater showed more similarity with ALI cultures from day 14 onwards.This work gives insights into the transcriptomic changes and ciliary differentiation throughout ALI cell culturing providing the timepoint of fully differentiated multi-ciliated cells. In addition, we determined which ALI culture time point showed the most in vivo similarity compared to nasal brushings