title: Airway Progenitor Clone Formation Is Enhanced by Y-27632-Dependent Changes in the Transcriptome.
abstract: The application of conditional reprogramming culture (CRC) methods to nasal airway epithelial cells would allow more wide-spread incorporation of primary airway epithelial culture models into complex lung disease research. In this study, we adapted the CRC method to nasal airway epithelial cells, investigated the growth advantages afforded by this technique over standard culture methods, and determined the cellular and molecular basis of CRC cell culture effects. We found that the CRC method allowed the production of 7.1 × 10(10) cells after 4 passages, approximately 379 times more cells than were generated by the standard bronchial epithelial growth media (BEGM) method. These nasal airway epithelial cells expressed normal basal cell markers and could be induced to form a mucociliary epithelium. Progenitor cell frequency was significantly higher using the CRC method in comparison to the standard culture method, and progenitor cell maintenance was dependent on addition of the Rho-kinase inhibitor Y-27632. Whole-transcriptome sequencing analysis demonstrated widespread gene expression changes in Y-27632-treated basal cells. We found that Y-27632 treatment altered expression of genes fundamental to the formation of the basal cell cytoskeleton, cell-cell junctions, and cell-extracellular matrix (ECM) interactions. Importantly, we found that Y-27632 treatment up-regulated expression of unique basal cell intermediate filament and desmosomal genes. Conversely, Y-27632 down-regulated multiple families of protease/antiprotease genes involved in ECM remodeling. We conclude that Y-27632 fundamentally alters cell-cell and cell-ECM interactions, which preserves basal progenitor cells and allows greater cell amplification.
authors.affiliation: 1 Center for Perinatal Research; Nationwide Children's Hospital, Columbus, Ohio.
authors.forename: Susan D
authors.initials: SD
authors.lastname: Reynolds
authors.affiliation: 2 Center for Genes, Environment, and Health, and.
authors.forename: Cydney
authors.initials: C
authors.lastname: Rios
authors.affiliation: 2 Center for Genes, Environment, and Health, and.
authors.forename: Agata
authors.initials: A
authors.lastname: Wesolowska-Andersen
authors.affiliation: 2 Center for Genes, Environment, and Health, and.
authors.forename: Yongbin
authors.initials: Y
authors.lastname: Zhuang
authors.affiliation: Departments of 3 Pediatrics and.
authors.forename: Mary
authors.initials: M
authors.lastname: Pinter
authors.affiliation: Departments of 3 Pediatrics and.
authors.forename: Carrie
authors.initials: C
authors.lastname: Happoldt
authors.affiliation: 1 Center for Perinatal Research; Nationwide Children's Hospital, Columbus, Ohio.
authors.forename: Cynthia L
authors.initials: CL
authors.lastname: Hill
authors.affiliation: 1 Center for Perinatal Research; Nationwide Children's Hospital, Columbus, Ohio.
authors.forename: Scott W
authors.initials: SW
authors.lastname: Lallier
authors.affiliation: 4 Medicine, National Jewish Health, Denver, Colorado.|5 Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado School of Medicine, Denver, Colorado.
authors.forename: Gregory P
authors.initials: GP
authors.lastname: Cosgrove
authors.affiliation: 6 Department of Medicine, University of Alabama-Birmingham, Birmingham, Alabama; and.
authors.forename: George M
authors.initials: GM
authors.lastname: Solomon
authors.affiliation: Departments of 3 Pediatrics and.|4 Medicine, National Jewish Health, Denver, Colorado.|7 University of Colorado School of Medicine, Denver, Colorado.
authors.forename: David P
authors.initials: DP
authors.lastname: Nichols
authors.affiliation: 2 Center for Genes, Environment, and Health, and.|Departments of 3 Pediatrics and.|5 Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado School of Medicine, Denver, Colorado.
authors.forename: Max A
authors.initials: MA
authors.lastname: Seibold
mesh_terms: D000577:Amides / Q000494:pharmacology*; D000818:Animals; D001980:Bronchi / Q000166:cytology; D002450:Cell Communication / Q000187:drug effects; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D021982:Cell-Matrix Junctions / Q000187:drug effects / Q000378:metabolism; D065150:Cellular Reprogramming / Q000187:drug effects / Q000235:genetics; D002999:Clone Cells; D003470:Culture Media / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008168:Lung / Q000166:cytology*; D051379:Mice; D041681:NIH 3T3 Cells; D009666:Nose / Q000166:cytology; D011725:Pyridines / Q000494:pharmacology*; D013234:Stem Cells / Q000166:cytology*; D059467:Transcriptome / Q000187:drug effects / Q000235:genetics*
chemical_list: D000577:Amides; D003470:Culture Media; D011725:Pyridines; C108830:Y 27632
keywords: Y-27632; airway stem progenitor; clone-forming cell frequency; conditionally reprogrammed cells