Expression of phenotypic markers during regeneration of rat tracheal epithelium following mechanical injury.

Abstract

We examined epithelial regeneration in mechanically injured rat trachea using phenotypic markers that identify unique differentiated stages of epithelial cells. Following a focal denuding wound, the cells from the adjacent nonwounded epithelium flattened and migrated into the wounded site during the first 12 h. At 24 h, these cells dedifferentiated into poorly differentiated (PD) cells that did not precisely resemble any of the mature tracheal cells. Proliferation of PD cells produced a multilayered epithelium by 48 h. Mitotic activity, measured as mitotic rate (MR) following a 6-h colchicine metaphase blockade, was high at 24 h (MR 23.4%) and 48 h (MR 24.0%). These PD cells expressed keratin 14 and Griffonia simplicifolia I-isolectin B4 (GSI-B4) lectin binding sites, which are specific for basal cells in normal epithelium but did not react with secretory or ciliated cell markers. At 72 h, MR fell to 1.8% (control MR 0.38%). The wound was covered with a pseudostratified epithelium; secretory cell markers were present at the apex of differentiating columnar cells, and a few preciliated cells expressing ciliated cell markers appeared. Basal cells also became distinctly recognizable and expressed keratin 14 and GSI-B4 binding sites. Newly appearing secretory or ciliated cells also expressed these markers but lost them gradually as they acquired new sets of specific markers. During epithelial regeneration after mechanical injury, "dedifferentiation," "proliferation," and "redifferentiation" of epithelial cells occurred, and the PD cell was pivotal in this process.

Basal Cells Are a Multipotent Progenitor Capable of Renewing the Bronchial Epithelium

“…These findings are consistent with results of others in which basal-cell-like intermediates have been observed either following mechanical injury to the tracheal epithelium or during reconstitution of denuded tracheas by isolated tracheal epithelial cells. 7,33,45 However, the identity of precursor cells contributing to renewal of scrape wounds introduced into the rat trachea 7 or among isolated cells exhibiting multipotential differentiation potential when seeded into tracheal xenografts 23,43 could not be determined. Our findings characterizing cell types contributing to regeneration of the bronchial epithelium demonstrate that K14-expressing basal cells have multipotential differentiation potential and, in the context of Clara cell depletion, contribute to restoration of CE progenitor cell populations.…”

“…Tissue sections were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline containing 0.1 mmol/L CaCl 2 and 0.1 mmol/L MgCl 2 (LHB) for 30 minutes at room temperature and then incubated with 6.5 g/ml biotinylated Griffonia (Bandeiraea) simplicifolia isolectin B 4 (GSI-B 4 ; Sigma) 32,33 diluted in 3% BSA in LHB for 1 hour at room temperature. Sections were washed in LHB and then incubated with streptavidinconjugated horseradish peroxidase (S-HRP) (Zymed, South San Francisco, CA) diluted in 0.5% BSA in LHB for 30 minutes at room temperature.…”

Airway Epithelial Stem Cells and the Pathophysiology of Chronic Obstructive Pulmonary Disease

“…However, as discussed above, wound repair (55) and cell isolation studies (57) indicate that all cell types can be generated from columnar cells of the pseudostratified epithelium via an intermediate, poorly differentiated cell type (downward pointing, red arrows in Figure 4). Whether "dedifferentiated" transiently amplifying progenitors can achieve "stemness" equivalent to the normal putative basal stem cell subpopulation requires further study.…”

In vivo differentiation potential of tracheal basal cells: evidence for multipotent and unipotent subpopulations

“…GSI-B 4 is a plant-derived protein that shows specificity toward glycoconjugates present on the surface of basal cells of various mouse and rat epithelia, including tracheobronchial epithelium (10,21). Images of representative fields were acquired using an Olympus Provis AX70 microscope (Olympus, Lake Success, NY) equipped with a Spot RT color digital camera (Diagnostic Instruments, Sterling Heights, MI) linked to a PC running Image-Pro Plus (Media Cybernetics, Silver Spring, MD).…”