No 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.

The lung's unique extracellular matrix (ECM), while providing structural support for cells, is critical in the regulation of developmental organogenesis, homeostasis and injury-repair responses. The ECM, via biochemical or biomechanical cues, regulates diverse cell functions, fate and phenotype. The composition and function of lung ECM become markedly deranged in pathological tissue remodeling. ECM-based therapeutics and bioengineering approaches represent promising novel strategies for regeneration/repair of the lung and treatment of chronic lung diseases. In this review, we assess the current state of lung ECM biology, including fundamental advances in ECM composition, dynamics, topography, and biomechanics; the role of the ECM in normal and aberrant lung development, adult lung diseases and autoimmunity; and ECM in the regulation of the stem cell niche. We identify opportunities to advance the field of lung ECM biology and provide a set recommendations for research priorities to advance knowledge that would inform novel approaches to the pathogenesis, diagnosis, and treatment of chronic lung diseases.

The regenerative capacity of lung and liver is sometimes impaired by chronic or overwhelming injury. Orthotopic transplantation of parenchymal stem cells to damaged organs might reinstate their self-repair ability. However, parenchymal cell engraftment is frequently hampered by the microenvironment in diseased recipient organs. Here, we show that targeting both the vascular niche and perivascular fibroblasts establishes “hospitable soil” to foster incorporation of “seed”, in this case the engraftment of parenchymal cells in injured organs. Specifically, ectopic induction of endothelial cell (EC)-expressed paracrine/angiocrine hepatocyte growth factor (HGF) and inhibition of perivascular NADPH Oxidase 4 (NOX4) synergistically enabled reconstitution of mouse and human parenchymal cells in damaged organs. Reciprocally, genetic knockout of Hgf in mouse ECs (HgfiΔEC/iΔEC) aberrantly upregulated perivascular NOX4 during liver and lung regeneration. Dysregulated HGF and NOX4 pathways subverted the function of vascular and perivascular cells from an epithelially-inductive niche to a microenvironment that inhibited parenchymal reconstitution. Perivascular NOX4 induction in HgfiΔEC/iΔEC mice recapitulated the phenotype of human and mouse fibrotic livers and lungs. Consequently, EC-directed HGF and NOX4 inhibitor GKT137831 stimulated regenerative integration of mouse and human parenchymal cells in chronically injured lung and liver. Our data suggest that targeting dysfunctional perivascular and vascular cells in diseased organs can bypass fibrosis and enable reparative cell engraftment to reinstate lung and liver regeneration.