Abstract
BackgroundThe pathology of chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF) and most lung cancers involves the small airway epithelium (SAE), the single continuous layer of cells lining the airways ≥ 6th generations. The basal cells (BC) are the stem/progenitor cells of the SAE, responsible for the differentiation into intermediate cells and ciliated, club and mucous cells. To facilitate the study of the biology of the human SAE in health and disease, we immortalized and characterized a normal human SAE basal cell line.MethodsSmall airway basal cells were purified from brushed SAE of a healthy nonsmoker donor with a characteristic normal SAE transcriptome. The BC were immortalized by retrovirus-mediated telomerase reverse transcriptase (TERT) transduction and single cell drug selection. The resulting cell line (hSABCi-NS1.1) was characterized by RNAseq, TaqMan PCR, protein immunofluorescence, differentiation capacity on an air-liquid interface (ALI) culture, transepithelial electrical resistance (TEER), airway region-associated features and response to genetic modification with SPDEF.ResultsThe hSABCi-NS1.1 single-clone-derived cell line continued to proliferate for > 200 doubling levels and > 70 passages, continuing to maintain basal cell features (TP63+, KRT5+). When cultured on ALI, hSABCi-NS1.1 cells consistently formed tight junctions and differentiated into ciliated, club (SCGB1A1+), mucous (MUC5AC+, MUC5B+), neuroendocrine (CHGA+), ionocyte (FOXI1+) and surfactant protein positive cells (SFTPA+, SFTPB+, SFTPD+), observations confirmed by RNAseq and TaqMan PCR. Annotation enrichment analysis showed that “cilium” and “immunity” were enriched in functions of the top-1500 up-regulated genes. RNAseq reads alignment corroborated expression of CD4, CD74 and MHC-II. Compared to the large airway cell line BCi-NS1.1, differentiated of hSABCi-NS1.1 cells on ALI were enriched with small airway epithelial genes, including surfactant protein genes, LTF and small airway development relevant transcription factors NKX2–1, GATA6, SOX9, HOPX, ID2 and ETV5. Lentivirus-mediated expression of SPDEF in hSABCi-NS1.1 cells induced secretory cell metaplasia, accompanied with characteristic COPD-associated SAE secretory cell changes, including up-regulation of MSMB, CEACAM5 and down-regulation of LTF.ConclusionsThe immortalized hSABCi-NS1.1 cell line has diverse differentiation capacities and retains SAE features, which will be useful for understanding the biology of SAE, the pathogenesis of SAE-related diseases, and testing new pharmacologic agents.
Highlights
The small airway epithelium (SAE), comprised of basal, intermediate, club, mucous and ciliated cells, plays a central role in the pathogenesis of chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), cystic fibrosis (CF) and most lung cancers [1,2,3,4,5,6]
Based on methods to sample SAE and to isolate basal cells (BC) from the SAE brushes obtained by fiberoptic bronchoscopy [9, 12,13,14], we have characterized SAE and SAE BC in normal non-smokers, asymptomatic smokers, and individuals with COPD using mRNA microarrays, RNAseq, single cell transcriptome, methylation, microRNA, metabolome and protein levels, characterizing the BC in health and dysregulation of the BC population associated with smoking and COPD [13,14,15,16,17,18,19,20]
We have generated hSABCi-NS1.1, an immortalized human small airway basal cell line from the brushed epithelium of a healthy non-smoker. hSABCi-NS1.1 cells can be passaged for at least 200 population doublings, have the capacity to differentiate into the major differentiated SAE club, mucous and ciliated cells, as well as rare SAE cell types, including surfactant protein positive cells and novel ionocytes, and have the capacity to recapitulate SAE disease-relevant biology when stressed with relevant signals
Summary
The small airway epithelium (SAE), comprised of basal, intermediate, club, mucous and ciliated cells, plays a central role in the pathogenesis of chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), cystic fibrosis (CF) and most lung cancers [1,2,3,4,5,6]. While isolation of human SAE and SAE BC by bronchoscopy and brushing permits assessment of primary BC, the procedure is invasive, time consuming and expensive, and the primary BC can be cultured only for 3 to 4 passages before becoming senescent In this context, if it is possible to immortalize normal human SAE BC that retain the capacity to differentiate to ciliated, secretory, and other differentiated cell types in vitro on air-liquid interface (ALI) culture, it would be very useful to the investigation of SAE biology in health and disease, and the assessment of pharmacologic agents targeted to modify dysregulated BC biology relevant to the pathogenesis of human lung disease. To facilitate the study of the biology of the human SAE in health and disease, we immortalized and characterized a normal human SAE basal cell line
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