Temporal dynamics of ovine airway epithelial cell differentiation at an air-liquid interface

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Abstract

The respiratory tract and lungs are subject to diverse pathologies with wide-ranging implications for both human and animal welfare. The development and detailed characterization of cell culture models for studying such forms of disease is of critical importance. In recent years the use of air-liquid interface (ALI)-cultured airway epithelial cells has increased markedly, as this method of culture results in the formation of a highly representative, organotypic in vitro model system. In this study we have expanded on previous knowledge of differentiated ovine tracheal epithelial cells by analysing the progression of differentiation over an extensive time course at an ALI. We observed a pseudo-stratified epithelium with ciliation and a concurrent increase in cell layer thickness from 9 days post-ALI with ciliation approaching a maximum level at day 24. A similar pattern was observed with respect to mucus production with intensely stained PAS-positive cells appearing at day 12. Ultrastructural analysis by SEM confirmed the presence of both ciliated cells and mucus globules on the epithelial surface within this time-frame. Trans-epithelial electrical resistance (TEER) peaked at 1049 Ω x cm2 as the cell layer became confluent, followed by a subsequent reduction as differentiation proceeded and stabilization at ~200 Ω x cm2. Importantly, little deterioration or de-differentiation was observed over the 45 day time-course indicating that the model is suitable for long-term experiments.

Figures

  • Fig 1. Histological assessment of ovine tracheal epithelial cell culture differentiation over time. Ovine tracheal epithelial cell cultures were grown at an ALI for the indicated number of days (relative to establishment of the ALI), fixed and paraffin embedded using standard histological techniques. Samples of ex vivo tissue were dissected from the center of each trachea prior to cell extraction, fixed, embedded and processed. Sections were taken, deparaffinized and stained as follows. (A) H&E staining of tissue layers at the indicated time points; selected ciliated cells are indicated by arrowheads. (B) PAS staining to detect mucus-containing/secreting cells (indicated by arrows and arrowheads). (C) Labelling of the transcription factor p63 to detect basal stem cells (positively labelled cells possess brown labelled nuclei). (D) Cell layer thickness was measured using ImageJ. Five images (400×magnification) were taken per insert with three points being measured per image. (E) The numbers of ciliated cells per field were counted from five images per insert. Three inserts were analysed per time point and the data represented is the mean +/- standard deviation from tissues derived from three independent animals (D and E). One-way ANOVA with post-test for linear trend was performed on data with significant (P<0.001) increasing trends being observed for both thickness (D) and ciliation (E).
  • Fig 2. Ovine tracheal epithelial cell cultures display a time-dependent increase in apical surface ciliation. (A) Ovine tracheal epithelial cell cultures were grown at an ALI for the indicated number of days (relative to establishment of the ALI), fixed and immunostained using an anti-β tubulin antibody to detect cilia and rhodamine-phalloidin to stain the actin cytoskeleton. (B) Z-stack orthogonal representation of 21-day post-ALI tissue layer. (C) 3-dimensional representation of the Z-stack in panel B. (D) Ciliation was quantified by measuring the area above a manual fluorescence intensity threshold in ImageJ. For each time point, five regions on three independent cell cultures were measured. Results displayed are the mean +/- standard deviation from tissue layers derived from three animals.
  • Fig 3. Ultrastructural analysis of the apical surface of ovine tracheal epithelial cell cultures by SEM. Ovine tracheal epithelial cell cultures were grown at an ALI for the indicated number of days (relative to establishment of the ALI), fixed and processed for SEM. (A) Images were taken at 1500×magnification. (B) Images were taken at 5000×magnification. Ciliated epithelial cells were observed from day 12 onwards.
  • Fig 4. Mucus production by differentiated ovine tracheal epithelial cell cultures. (A) Ovine tracheal epithelial cell cultures were grown at ALI for the indicated number of days (relative to establishment of the ALI), fixed and processed for SEM. (B) Ovine tracheal epithelial cell cultures were grown for the indicated number of days, fixed and stained with jacalin-FITC (green), rhodamine-phalloidin (red) and DAPI (blue). Mucus globules are indicated by white arrows, carpets of amorphous mucus are indicated by white arrowheads and jacalin-labelled mucin-positive cells are indicated by yellow arrows.
  • Fig 5. Ovine tracheal epithelial cell cultures display stable barrier function and junctional integrity. (A) Ovine tracheal epithelial cell cultures were grown at ALI for the indicated number of days (relative to establishment of the ALI) and tissue layers were fixed and immunostained using an anti-ZO1 antibody at the indicated time points (relative to establishment of the ALI). (B) Orthogonal representation of ALI culture at 24 days postALI. (C) 3-dimensional model of the Z-stack shown in panel B. (D) TEER measurements from four independent cell culture inserts at each timepoint. Results for ALI cultures derived from three independent animals are shown (mean +/- standard deviation).

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Basal cells as stem cells of the mouse trachea and human airway epithelium

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O’Boyle, N., Sutherland, E., Berry, C. C., & Davies, R. L. (2017). Temporal dynamics of ovine airway epithelial cell differentiation at an air-liquid interface. PLoS ONE, 12(7). https://doi.org/10.1371/journal.pone.0181583

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