Airway Progenitor Clone Formation Is Enhanced by Y-27632-Dependent Changes in the Transcriptome

Am J Respir Cell Mol Biol. 2016 Sep;55(3):323-36. doi: 10.1165/rcmb.2015-0274MA.


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.

Keywords: Y-27632; airway stem progenitor; clone-forming cell frequency; conditionally reprogrammed cells.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • Amides / pharmacology*
  • Animals
  • Bronchi / cytology
  • Cell Communication / drug effects
  • Cell Culture Techniques
  • Cell Differentiation / drug effects
  • Cell Differentiation / genetics
  • Cell-Matrix Junctions / drug effects
  • Cell-Matrix Junctions / metabolism
  • Cellular Reprogramming / drug effects
  • Cellular Reprogramming / genetics
  • Clone Cells
  • Culture Media / pharmacology
  • Epithelial Cells / cytology
  • Epithelial Cells / drug effects
  • Epithelial Cells / metabolism
  • Extracellular Matrix / drug effects
  • Extracellular Matrix / metabolism
  • Fibroblasts / cytology
  • Fibroblasts / drug effects
  • Gene Expression Regulation / drug effects
  • Humans
  • Lung / cytology*
  • Mice
  • NIH 3T3 Cells
  • Nose / cytology
  • Pyridines / pharmacology*
  • Stem Cells / cytology*
  • Transcriptome / drug effects
  • Transcriptome / genetics*


  • Amides
  • Culture Media
  • Pyridines
  • Y 27632