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.
Highlights
The human airway epithelium, a complex pseudostratified multicellular layer that lines the bronchial tree, is comprised of ciliated, secretory, intermediate/undifferentiated and basal cells (BC) [1,2,3]
Understanding the mechanisms by which the differentiation capacity of the BC is regulated and how the cells respond to specific environmental stimuli is central to the understanding of diseases characterized by airway epithelial remodeling such as chronic obstructive pulmonary disease (COPD) and asthma, and the biology of the malignant transformation of the epithelium into bronchogenic carcinoma [3,8,26,27,28,29,30]
Immortalization of primary human airway basal cells To establish an immortalized human airway basal cell (BC) cell line, primary airway BC were isolated with selective culture methods from the large airway epithelium of a male healthy nonsmoker
Summary
The human airway epithelium, a complex pseudostratified multicellular layer that lines the bronchial tree, is comprised of ciliated, secretory, intermediate/undifferentiated and basal cells (BC) [1,2,3]. Alternative strategies to viral oncoproteins have used retroviral gene transfer-mediated expression of hTERT alone or together with cyclin dependent kinase 4 (CDK4) or B-cell Moloney murine leukemia retrovirus-specific integration site 1 (Bmi-1). Cells produced by these strategies have an extended life span far beyond normal senescence and retain characteristics of the primary cells [42,43,44,45,46]. 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
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
