In vitro differentiation of ciliated cells in ALI-cultured human airway epithelium - The framework for functional studies on airway differentiation in ciliopathies

Abstract

Primary cultures of the human airway epithelium (AE) cells are an indispensable tool in studies of pathophysiology of genetic and environmental pulmonary diseases, including cystic fibrosis (CF), primary ciliary dyskinesia (PCD) and chronic obstructive pulmonary disease (COPD). Air-liquid interface (ALI) culture is the best method to follow the differentiation of ciliated cells, whose dysfunction forms the basis of PCD. Here, we used custom-designed Taqman Low Density Array (TLDA), qRT-PCR-based assay, to analyze expression of 14 AE genes in cells from healthy donors, cultured in ALI settings using Pneumacult medium, with the focus on genes involved in cilia differentiation and in PCD pathogenesis. The results of TLDA assay were compared with the bulk RNAseq analysis, and placed in the cellular context using immunofluorescent staining (IF) of ALI cultured cells. Expression analysis revealed culture time-related upregulation of the majority of cilia-related genes, followed by the appearance of respective protein signals visualized by IF. Strong correlation of TLDA with RNAseq results indicated that TLDA assay is a reliable and scalable approach to analyze expression of selected genes specific for different AE cell types. Characterization of temporal and inter-donor changes in the expression of these genes, performed in healthy donors and in well-defined ALI/Pnemacult culture conditions, provides a useful reference relevant for a broad spectrum of functional studies where the in vitro AE differentiation is in focus.

Keywords: Airway tissue differentiation, genetic disorders, ciliogenesis; Bulk RNAseq; Gene expression; Immunofluorescence; Primary ciliary dyskinesia genes; Protein expression; TLDA; qRT-PCR.