title: BMI-1 extends proliferative potential of human bronchial epithelial cells while retaining their mucociliary differentiation capacity.
abstract: Air-liquid interface (ALI) culture of primary airway epithelial cells enables mucociliary differentiation providing an in vitro model of the human airway, but their proliferative potential is limited. To extend proliferation, these cells were previously transduced with viral oncogenes or mouseBmi-1+hTERT, but the resultant cell lines did not undergo mucociliary differentiation. We hypothesized that use of humanBMI-1alone would increase the proliferative potential of bronchial epithelial cells while retaining their mucociliary differentiation potential. Cystic fibrosis (CF) and non-CF bronchial epithelial cells were transduced by lentivirus withBMI-1and then their morphology, replication kinetics, and karyotype were assessed. When differentiated at ALI, mucin production, ciliary function, and transepithelial electrophysiology were measured. Finally, shRNA knockdown ofDNAH5in BMI-1 cells was used to model primary ciliary dyskinesia (PCD).BMI-1-transduced basal cells showed normal cell morphology, karyotype, and doubling times despite extensive passaging. The cell lines underwent mucociliary differentiation when cultured at ALI with abundant ciliation and production of the gel-forming mucins MUC5AC and MUC5B evident. Cilia displayed a normal beat frequency and 9+2 ultrastructure. Electrophysiological characteristics ofBMI-1-transduced cells were similar to those of untransduced cells. shRNA knockdown ofDNAH5in BMI-1 cells produced immotile cilia and absence of DNAH5 in the ciliary axoneme as seen in cells from patients with PCD. BMI-1 delayed senescence in bronchial epithelial cells, increasing their proliferative potential but maintaining mucociliary differentiation at ALI. We have shown these cells are amenable to genetic manipulation and can be used to produce novel disease models for research and dissemination.
authors.affiliation: University College London Great Ormond Street Institute of Child Health, London, United Kingdom.
authors.forename: Mustafa M
authors.initials: MM
authors.lastname: Munye
authors.affiliation: Imperial College London, UK Electron Microscopy Department, Royal Brompton and Harefield NHS Foundation Trust, London, United Kingdom.
authors.forename: Amelia
authors.initials: A
authors.lastname: Shoemark
authors.affiliation: Primary Ciliary Dyskinesia Centre Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Hirst
authors.affiliation: University College London Great Ormond Street Institute of Child Health, London, United Kingdom.
authors.forename: Juliette M
authors.initials: JM
authors.lastname: Delhove
authors.affiliation: Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, United Kingdom.
authors.forename: Tyson V
authors.initials: TV
authors.lastname: Sharp
authors.affiliation: School of Healthcare Science, Manchester Metropolitan University, Manchester, United Kingdom; and.
authors.forename: Tristan R
authors.initials: TR
authors.lastname: McKay
authors.affiliation: University College London Great Ormond Street Institute of Child Health, London, United Kingdom.
authors.forename: Christopher
authors.initials: C
authors.lastname: O'Callaghan
authors.affiliation: Institute for Infection and Immunity, St George's, University of London, London, United Kingdom.
authors.forename: Deborah L
authors.initials: DL
authors.lastname: Baines
authors.affiliation: University College London Great Ormond Street Institute of Child Health, London, United Kingdom.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Howe
authors.affiliation: University College London Great Ormond Street Institute of Child Health, London, United Kingdom; s.hart@ucl.ac.uk.
authors.forename: Stephen L
authors.initials: SL
authors.lastname: Hart
mesh_terms: D000818:Animals; D056444:Axonemal Dyneins / Q000378:metabolism; D001980:Bronchi / Q000166:cytology*; D002454:Cell Differentiation; D049109:Cell Proliferation; D048430:Cell Shape; D002923:Cilia / Q000378:metabolism*; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism; D004398:Dyneins / Q000378:metabolism; D017097:Electric Impedance; D055724:Electrophysiological Phenomena; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D007619:Kartagener Syndrome / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D007621:Karyotyping; D051379:Mice; D008870:Microtubules / Q000378:metabolism; D008954:Models, Biological; D009093:Mucus / Q000378:metabolism*; D010641:Phenotype; D063150:Polycomb Repressive Complex 1 / Q000378:metabolism*; D014161:Transduction, Genetic
chemical_list: C069077:BMI1 protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D063150:Polycomb Repressive Complex 1; D056444:Axonemal Dyneins; C497507:DNAH5 protein, human; D004398:Dyneins
keywords: air-liquid interface; airway model; lung; mucociliary differentiation; primary ciliary dyskinesia