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.

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

Figure 1.

The conditional reprogramming culture (CRC) method enhances preservation of nasal airway basal progenitor cells. (A) Phase-contrast microscopy of nasal epithelial cells grown using the bronchial epithelial growth media (BEGM) and CRC techniques reveals “scattered” and “colony” growth patterns, respectively. Black arrows indicate individual cells (A) or the colony edge (B). Red arrows in A indicate feeder cells. Scale bars: 200 μm. Values represent the mean (±SEM). (B) Frequency of cytokeratin-5– and -14–expressing passage (P) 2 cells recovered from BEGM and CRCs. Representative of studies done in three donors. (C) The surface phenotype of P2 cells recovered from BEGM cultures or CRCs was determined by flow cytometry. Bivariate FACS plots of cells stained for the tetraspanin (CD151) and tissue factor or α6 integrin (CD49f). Representative of three to four donors. (D) Quantification of cell subtype frequency as determined by flow cytometry (n = 3–4 donors). Values represent the mean (±SEM). (E) The cell cycle distribution of P2 nasal cells recovered from CRC or BEGM cultures was determined by saponin/propidium iodide staining and flow cytometry. G1, gap 1; G2M, gap 2 mitosis; S, DNA synthesis (n = 3–4 donors). Values represent the mean (±SEM). (F) The effect of culture technique on clone-forming cell frequency (CFCF). Nasal brushings were cultured at P2 using the CRC or BEGM method. At P3, cells were cultured in the same medium or switched to the alternative medium. CFCF was determined by limiting dilution analysis. Values represent the mean (±SEM). Representative of studies done in three donors.

Figure 2.

CRC permits greater nasal airway basal cell amplification compared with BEGM culture. Comparison of cell amplification metrics for paired cell cultures grown using the CRC and BEGM culture methods. (A) Number of airway basal cells recovered at each of four consecutive passages. Median, interquartile range. (B) Airway basal cell burst size at each passage, calculated as the number of cells recovered after culture divided by the number of cells plated. Values represent the mean (±SEM). (C) The total number of airway basal cells that could be generated at each passage based on the empirically determined cell burst size and starting with 8 × 10⁵ P0 cells. Median, interquartile range. Data shown for four donors. Asterisks represent Mann–Whitney test P value < 0.05.

Figure 3.

P4 CRC amplified airway basal cells retain the ability to generate a mucociliary epithelium in vitro. (A) Confocal imaging analysis of epithelial structure. Arrows indicate the orientation of the X-, Y-, and Z-planes. Scale bar: 25 μm. (B) Immunohistochemical analysis of air–liquid interface (ALI) membranes for markers of mucociliary differentiation (images from a single donor, representative of four donors). Scale bar: 100 μm. (C–E) Morphometric analysis of mucin (MUC) 5B (C), MUC5AC (D), and acetylated α-tubulin (E) immunopositive cell frequency on ALI membranes. Values represent the mean (±SEM). Data from three membranes across four donors. (F) Relative gene expression for markers of mucociliary differentiation during CRC expansion (EXP) and after differentiation (ALI). Lines connect data from a single donor. Marker gene expression is presented relative to β-glucuronidase gene expression. Data are from two donors. Average fold changes of gene expression in the ALI phase relative to the EXP phase are shown. * <0.05, ** <0.01, *** <0.001, **** <0.0001 for Mann–Whitney or t test P values. DAPI, 4′,6-diamidino-2-phenylindole.

Figure 4.

Impact of fibroblasts on nasal airway basal progenitor frequency. (A) CFCF was determined for nasal airway basal cells plated on embryonic mouse (NIH3T3), adult human lung fibroblasts from normal (NHLF), or idiopathic pulmonary fibrosis (IPF) 11 or normal human lung smooth muscle (SM). Mean (±SEM) (n = 3). Representative of four subjects. (B) CFCF was determined for nasal airway basal cells cultured with or without Y-27632 in the presence or absence of NIH3T3 feeders. Mean (±SEM) (n = 3). Representative of four subjects. (C–F) Phase-contrast images of Geimsa-stained nasal airway basal cells grown with Y-27632 and NIH3T3 feeders (C), Y-27632 without feeders (D), without Y-27632 with feeders (E), and without Y-27632 or feeders (F). Solid lines in C, D, and E demarcate clones. Arrows in F indicate single cells. Scale bars: 200 μm. (G) CFCF was determined for nasal airway basal cells that were cultured with NIH3T3 fibroblast feeders for 2, 3, 4, 5, 6, or 8 days and assayed on Culture Day 8. Values represent the mean (±SEM). (H) Cells per colony were determined for nasal airway basal cells that were cultured with NIH3T3 fibroblast feeders for 2, 3, 4, 5, 6, or 8 days and assayed on Culture Day 8. Values represent the mean (±SEM). (I–N) Phase-contrast images of Geimsa-stained nasal airway basal cells that were cultured with NIH3T3 fibroblast feeders for 2 (I), 3 (J), 4 (K), 5 (L), 6 (M), or 8 (N) days and assayed on Culture Day 8. Scale bars: 200 μm.

Figure 5.

CRC amplification and increase in nasal airway basal progenitor cell clone-forming frequency is dependent on Y-27632 stimulation. (A) Comparison of burst size for airway basal cells grown with the CRC methods with and without Y-27632. (B–D) Effect of Y-27632 supplementation on cell behavior in CRCs: none, Y-27632 was not added; 0–6, 0–4, 0–2 indicate the intervals (in days) during which Y-27632 was present. (B) Effect of Y-27632 supplementation on the number of clones per cell plated. (C) Effect of Y-27632 supplementation on the CFCF. (D) Effect of Y-27632 supplementation on the number of cells per clone. * <0.05, ** <0.01, *** <0.001 for Mann–Whitney or t test P values.

Figure 6.

The effects of Y-27632 supplementation of CRC on the airway basal cell transcriptome. (A) Multidimensional scaling plot of whole-transcriptome data from three subjects’ cultures grown with and without Y-27632. Dotted lines connect the subject pairs and show similar directional change in transcriptome expression between Y-27632–treated and untreated cultures for all donors. Red, +Y-27632; blue, −Y-27632. (B and C) Differentially expressed (B) cellular component and (C) molecular function gene ontology categories with Y-27632 stimulation are shown. Box plot of fold changes for genes within each set are shown. Individual gene fold changes shown by points. Red points indicate genes, the expression of which is differentially regulated by Y-27632. (D) Fold changes with Y-27632 treatment for genes in select functional groups (median, interquartile range). Genes represented by points are shown in Table E5. (E) In situ analysis of matrix metalloproteinase activity. Solid lines in E demarcate clones; arrows point toward areas of matrix metalloproteinase activity. Scale bars: 200 μm. (F) Quantification of cells with matrix metalloproteinase activity. Note: Gene Ontology (GO) term 0005882 intermediate filament was also significant in the cellular component analysis, but greater than 98% of genes within this term were also part of the GO:0045111 term, intermediate filament cytoskeleton, displayed. ADAM, a disintegrin and metalloproteinase ADAM; MMP, matrix metalloproteinase; SERPIN, serine protease inhibitor.