Generation of a human airway epithelium derived basal cell line with multipotent differentiation capacity

Abstract

BackgroundAs the multipotent progenitor population of the airway epithelium, human airway basal cells (BC) replenish the specialized differentiated cell populations of the mucociliated airway epithelium during physiological turnover and repair. Cultured primary BC divide a limited number of times before entering a state of replicative senescence, preventing the establishment of long-term replicating cultures of airway BC that maintain their original phenotype.MethodsTo generate an immortalized human airway BC cell line, primary human airway BC obtained by brushing the airway epithelium of healthy nonsmokers were infected with a retrovirus expressing human telomerase (hTERT). The resulting immortalized cell line was then characterized under non-differentiating and differentiating air-liquid interface (ALI) culture conditions using ELISA, TaqMan quantitative PCR, Western analysis, and immunofluorescent and immunohistochemical staining analysis for cell type specific markers. In addition, the ability of the cell line to respond to environmental stimuli under differentiating ALI culture was assessed.ResultsWe successfully generated an immortalized human airway BC cell line termed BCi-NS1 via expression of hTERT. A single cell derived clone from the parental BCi-NS1 cells, BCi-NS1.1, retains characteristics of the original primary cells for over 40 passages and demonstrates a multipotent differentiation capacity into secretory (MUC5AC, MUC5B), goblet (TFF3), Clara (CC10) and ciliated (DNAI1, FOXJ1) cells on ALI culture. The cells can respond to external stimuli such as IL-13, resulting in alteration of the normal differentiation process.ConclusionDevelopment of immortalized human airway BC that retain multipotent differentiation capacity over long-term culture should be useful in understanding the biology of BC, the response of BC to environmental stress, and as a target for assessment of pharmacologic agents.

Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology

“…However, recent advances involving induced expression of human telomerase reverse transcriptase (hTERT) to prevent replicative senescence and expression of the cell cycle protein cdk4 (Ramirez et al, 2004) has enabled immortalization of epithelial cells in culture without loss of their ability to differentiate when taken to an ALI (Vaughan et al, 2006). More recently, a human airway epithelium-derived basal cell line with multipotent differentiation capacity has been established by immortalization using a retrovirus expressing hTERT (Walters et al, 2013). Such an approach might be useful to generate cell lines from asthma-derived cells, although any such lines would need careful phenotypic characterization.…”

Endothelial MMP14 is required for endothelial dependent growth support of human airway basal cells

“…The expression of MMP14 in endothelial cells was assessed using TaqMan quantitative PCR as described previously (Walters et al, 2013). Relative expression levels were determined using the ΔCt method with 18S ribosomal RNA (TaqMan ® Ribosomal RNA Control, VIC, number 4308329, Applied Biosystems) as the endogenous control.…”

“…Immunohistochemistry was performed as described previously (Walters et al, 2013). The primary antibody against FGF2 was from Cell Signaling Technology (2 µg/ml; catalog number 3196), and that against FGF5 from Abcam (0.2 µg/ml; catalog number ab88118).…”

“…The secretion of FGF2 and FGF5 by basal cells was assessed by ELISA (FGF2, catalog number ab99979, Abcam and FGF5, catalog number ELH-FGF5-1, RayBiotech, Inc., Norcross, GA) following incubation of basal cells overnight in BEBM as described previously (Walters et al, 2013).…”

Target cell types with stem/progenitor function to isolate for in vitro reconstruction of human bronchiolar epithelia

“…In addition, the choice of medium (components) also appears to have a great deal of influence on the reconstruction process of airway epithelia. A third possibility may be to resort to immortalizing stem/progenitor cells or those possessing a comparable status (Vaughan et al, 2006;Delgado et al, 2011;Walters et al, 2013), whereas this technology is at a stage of development where many improvements are still required.…”