Tracheal Basal Cells Research Articles

Differentiation of Tracheal Basal Cells to Ciliated Cells and Tissue Reconstruction on the Synthesized Basement Membrane Substratum In Vitro

Although lung epithelial cells directly attach to the basement membrane underneath in vivo, harvested epithelial cells are typically cultured on type I collagen gel (Col I-gel) in vitro. Recently we developed new culture substratum, designated as “synthesized Basement Membrane” (sBM), that has bared lamina densa on fibrillar collagen. To validate the usefulness of sBM substratum in airway tissue reconstitution in vitro, we cultured rat tracheal epithelial cells on sBM substratum and Col I-gel. When starting the air-liquid interface culture, most of the epithelial cells were squamous and positive for the basal cell marker cytokeratin 14 (CK14). After 14 days on sBM substratum, CK14-positive cells differentiated not only to Clara and mucous cells, but also to ciliated cells. Those differentiated cells formed pseudostratified-like epithelium and the remaining CK14-positive cells were polarized to the basal side. However, on Col I-gel, the CK14-positive cells were still squamous and not polarized, and ciliated cells did not appear. In conclusion, we established a new culture model on sBM substratum in which basal cells could differentiate to ciliated cells. The application of sBM substratum is useful in the study of the airway epithelial cell differentiation in vitro.

A subset of mouse tracheal epithelial basal cells generates large colonies in vitro.

Airway epithelial stem cells are not well characterized. To examine clonal growth potential, we diluted single, viable B6.129S7-Gtrosa26 (Rosa26) mouse tracheal epithelial cells that constitutively express -galactosidase into non-Rosa26 cells in an air-liquid interface cell culture model; 1.7% of the cells formed colonies of varying size, and, on average, 0.1% of the cells formed large colonies. Thus only a small subset of cells displayed progenitorial capacity suggestive of stem or early transient amplifying cells. Prior studies identified cells with high keratin 5 (K5) promoter activity in specific niches in the mouse trachea and these cells corresponded to the location of bromodeoxyuridine label-retaining cells, thought to be stem cells (Borthwick DW, Shahbazian M, Todd KQ, Dorin JR, and Randell SH, Am J Respir Cell Mol Biol: 24: 662-670, 2001). To explore the hypothesis that stem cells were present in the K5-expressing compartment, we created transgenic mice in which enhanced green fluorescent protein (EGFP) was driven by the K5 promoter. These mice expressed EGFP in most basal cells of the body including a subset of tracheal basal cells apparently located in positions similar to previously identified stem cell niches. Flow cytometrically purified EGFP-positive cells had an overall colony-forming efficiency 4.5-fold greater than EGFP-negative cells, but the ability to generate large colonies was 12-fold greater. Thus adult mouse tracheal epithelial cells with progenitorial capacity sufficient to generate large colonies reside in the basal cell compartment. These studies are a first step toward purification and characterization of airway epithelial stem cells.

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

The composition of the conducting airway epithelium varies significantly along the proximal to distal axis, with that of the tracheal epithelium exhibiting the greatest complexity. A number of progenitor cells have been proposed to contribute to the maintenance of this cellular diversity both in the steady state and in response to injury. However, individual roles for each progenitor cell type are poorly defined in vivo. The present study was undertaken to investigate the hypothesis that basal cells represent a multipotent progenitor cell type for renewal of the injured tracheal epithelium. To understand their contribution to epithelial repair, mice were exposed to naphthalene to induce airway injury and depletion of the secretory cell progenitor pool. Injury resulted in a rapid induction of cytokeratin 14 (K14) expression among the majority of GSI-B4-reactive cells and associated hyperplasia of basal cells. Restoration of depleted secretory cells occurred after 6 days of recovery and was associated with regression of the basal cell hyperplasia, suggesting a progenitor-progeny relationship. Multipotent differentiation of basal cells was confirmed using a bitransgenic ligand-regulated Cre-loxP reporter approach in which expression of a ubiquitously expressed LacZ reporter was activated within K14-expressing progenitor cells during airway repair. With the use of this approach, it was determined that K14-expressing cells include subsets capable of either multipotent or unipotent differentiation in vivo. We conclude that basal cells have the capacity for restoration of a fully differentiated epithelium.

Cellular and subcellular localizations of annexins I, IV, and VI in lung epithelia.

The cellular and subcellular localizations of annexins I, IV, and VI in the rabbit tracheal and alveolar epithelia were studied by performing immunofluorescence labeling on thin frozen sections of these tissues, using specific monoclonal antibodies. Annexin I was highly expressed by ciliated cells, where it was concentrated in the cilia but was also present along the basolateral domain of the plasma membrane and in the nuclei. It was also abundant in the cytoplasm of type II pneumocytes and alveolar macrophages. Either one or both of these alveolar cells might be capable of secreting a very small amount of annexin I, which was found to be associated with the surfactant layer. Annexin IV was synthesized by all the lung epithelial cells. It was associated with the plasma membrane basolateral domain in ciliated cells and with either the apical or basal domain of plasma membrane in type I pneumocytes, whereas it was cytoplasmic in goblet cells and type II pneumocytes. Annexin VI was expressed only by alveolar endothelial cells, where it was probably cytoplasmic. None of these three annexins seem to be expressed in the nondifferentiated tracheal basal epithelial cells.

In vivo growth and differentiation potential of tracheal basal cells of rabbits in vitamin A deficiency.

The growth and differentiation potential of rabbit tracheal basal cells were investigated in vitamin A deficient mice. Denuded rat tracheal grafts were xenotransplanted into nude mice made vitamin A deficient by feeding them retinol-free pellets from mid-gestation. Rabbit tracheal epithelial cells harvested enzymatically or cells derived from a basal-cell-rich fraction obtained by elutriation (purity 93.3%) had previously been inoculated into the grafts (n = 8, each). The grafts were implanted into the vitamin A deficient or control mice aged about 10 weeks. Four weeks later, the grafts were retrieved for histological examination. The graft epithelium established by either basal cells or un-fractionated cells in vitamin A deficient hosts (groups 1 and 2, respectively) was atrophic, whereas grafts repopulated with both cell types in the controls had pseudostratified columnar epithelium. Group 1 and 2 grafts both showed squamous metaplasia; 10 metaplastic foci in 32 tracheal rings in group 1 (P < 0.02 or 0.002, compared with values for group 2 or controls, respectively), and 2 foci in 35 rings in group 2 (no statistical difference compared with controls). In conclusion, during vitamin A deficiency, rabbit tracheal epithelial cells, including the progeny of highly-purified basal cells, lost their potential for establishing a mucociliary epithelium and rather appeared to undergo squamous metaplasia.

Characterization of a marker for tracheal basal cells.

An IgM monoclonal antibody (1D9/B3) is characterized, which specifically recognizes basal cells of the upper airway epithelium. Although morphological features have been used to follow cell lineage and differentiation, an objective assessment of differentiation can be enhanced by characterizing the expression of specific antigens that form the phenotypic profile of specialized cells. Mice were immunized with rabbit tracheal basal cells that had been obtained by pronase digestion and purified into a subpopulation of basal cells by flow cytometry. Six immunization experiments produced five hybridomas specific to epithelial cells. A hybridoma whose supernatant immunocytochemically stained the basal cell subpopulation of rabbit tracheal cells was selected. The antibody reacted with tracheal basal cells in rabbit, rat, sheep, pig, and human tracheal sections, and in cultured monolayers of tracheal epithelial cells of the same species. The antibody did not react with the basal cells of other rabbit tissue, including the skin, or other rabbit epithelia. Confocal microscopy and exposure of tracheal epithelial cells to fluorescent-tagged monoclonal antibody 1D9/B3 prior to loading on to flow cytometry showed that the basal cell antibody recognized an intracellular epitope. The epitope for the 1D9/B3 antibody was characterized by Western blotting. The 1D9/B3 antibody appears to be a distinct and specific marker to the airway epithelial basal cell and will be useful in studies of airway epithelial differentiation, injury, and regeneration.

In situ preparation of rat tracheal basal cells

The primary function of basal cells is to attach columnar epithelium to the basal lamina. They may also have limited stem cell potential, but very little is known of other biological functions. Basal cells lie on the basal lamina beneath the ciliated and secretory cells and do not reach the surface of the epithelium. The position of the cell beneath the ciliated and secretory cell epithelium makes their in situ study difficult. In order to further aid in the study of basal cells, we have developed an in situ preparation technique in which ciliated and secretory cells are removed. Treatment of rat tracheas with a 20 mM Na 2 EDTA solution, pH 7.4, results in partial removal of columnar epithelium from the basal lamina. The percent of denuded columnar epithelial cells per mm of basal lamina is 43.9 ± 7.8% at 60 min, 47.6 ± 8.4% at 90 min, and 52.6 ± 2.7% at 120 min. The viability of the exposed basal cells was the same at both 60 and 90 min of treatment (79.4 ± 7.8 and 78.0 ± 8.5, respectively). Morphologically, the exposed basal cells are attached to the basal lamina by hemidesmosomes and are similar to those in the intact animal.

Differentiation of anchoring junctions in tracheal basal cells in the growing rat.

A function of airway basal cells is to attach ciliated and nonciliated columnar cells to the basal lamina. The significance of the basal cell in attachment is related to the height of the columnar epithelium. In taller epithelia, basal cells are more numerous and differentiated with respect to anchoring junctional adhesion mechanisms (desmosomes, hemidesmosomes, and the cytoskeleton) than in shorter epithelia. In this study, we determined if basal cell anchoring junctional adhesion mechanisms differentiated during growth of the airway. Tracheas from five 3-day-old, five 30-day-old, and five 90-day-old rats were prepared for electron microscopy and morphometrically studied by standard techniques. The circumference of the trachea increased from 2.5 +/- 0.2 to 7.5 +/- 0.4 mm during growth. The height of the columnar cell increased from 13.4 +/- 1.5 to 24.6 +/- 3.9 microns, and the number of basal cells per millimeter increased from 3.2 +/- 0.7 to 9.6 +/- 1.8 during growth. The number of desmosomes per basal cell profile increased significantly from 1.5 +/- 0.1 to 2.1 +/- 0.1, as did keratin filament volume density from 0.046 +/- 0.05 to 0.098 +/- 0.032. The amount of hemidesmosome attachment per basal cell did not increase significantly during growth of the airway. These data demonstrate that as tracheas grow in circumference, the columnar cells increase in height, basal cells increase in number, and anchoring junctional adhesion mechanisms differentiate in the basal cells. These changes are closely related to the height of the epithelium and result in maintaining a constant amount of attachment between the columnar epithelium and the basal lamina as the epithelium increases in height.

Effect of cigarette smoke condensate and vitamin A depletion on keratin expression patterns in cultured hamster tracheal epithelium

Keratin expression in hamster tracheal epithelium was investigated during organ culture in serum-free, hormone-supplemented medium using monospecific monoclonal antibodies. Generally, tracheal basal cells expressed keratins detected by antibodies RCK102 and RCK103, while columnar epithelial cells were stained positively by RGE53, RCK103, RCK105 and HCK19. Metaplastic squamous cell foci reacted with antibodies RKSE60, RCK103 and HCK19. Early metaplastic alterations were more clearly RKSE60-positive than the mature lesions. In the vitamin A-depleted tracheas basal cells were clearly RCK102-positive. Superficial cells in the central part of areas of squamous metaplasia induced by cigarette smoke condensate expressed the basal cell keratins, and were negative for the columnar cell keratin 18 detected by the RGE53 antibody. This finding suggests that in cigarette smoke condensate-induced squamous metaplasia basal cells play an important role. The mucus-producing cells at the edges of metaplastic squamous cell foci expressed the keratins specific to columnar cells. Cigarette smoke condensate exposure accelerated epithelial keratinization compared to the vitamin A-depleted epithelium. It was concluded that not only small mucous granule cells, but also basal cells are involved in the development and maintenance of induced squamous metaplasia in tracheal epithelium. Furthermore, in vitro vitamin A-depleted epithelium did not coexpress vimentin in addition to the different keratins.