Differentiated and functional human airway epithelium regeneration in tracheal xenografts

F. Dupuit, D. Gaillard, J. Hinnrasky, E. Mongodin, S. de Bentzmann, E. Copreni, and E. Puchelle

Institut National de la Santé et de la Recherche Médicale Unité 514, Université de Reims, Institut Federatif de Recherche 53, Centre Hospitalier Universitaire Maison Blanche, 51092 Reims Cedex, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To investigate the regeneration process of a well-differentiated and functional human airway epithelium, we adapted an in vivo xenograft model in which adult human nasal epithelial cells adhere and progressively repopulate denuded rat tracheae grafted in nude mice. The proliferating activity, the degree of differentiation, and the barrier integrity of the repopulated epithelium were studied during the regeneration process at optical and ultrastructural levels with immunocytochemistry and a permeability tracer. Three days after implantation in nude mice, tracheal xenografts were partially repopulated with a flattened nonciliated and poorly differentiated leaky epithelium. By the end of the first week after the graft, cell proliferation produced on the entire surface of the rat trachea an epithelium that was stratified into multiple layers and tightly sealed. During successive weeks, cell proliferation dramatically decreased. Moreover, the epithelium became progressively columnar, secretory, ciliated, and transiently leaky. At 4-5 wk, a fully differentiated pseudostratified functional epithelial barrier impermeable to a low-molecular-weight tracer was reconstituted. The regeneration of a well-differentiated and functional human airway epithelium in rat tracheae grafted in nude mice includes several steps that mimic the regeneration dynamics of airway epithelium after injury.

cell proliferation; cell differentiation; epithelial barrier integrity; nude mice


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE EPITHELIAL CELLS LINING the airways contribute to lung defense through the process of mucociliary clearance and the presence of a variety of intercellular junctional complexes creating a barrier against the diffusion of exogenous injurious agents from the lumen to the underlying lamina propria (33). Nevertheless, acute airway injury may lead to transient or permanent alterations in the structure and function of the epithelium and thus plays an important role in the pathogenesis of respiratory diseases. Many extensive studies (22; for a review, see Ref. 31) on animal models have been carried out to examine the regenerative process or the pathological changes of airway epithelium resulting from injury. These studies and in vitro investigations on human airway cells (16) have shown that the regenerative process to repair airway epithelium involves dedifferentiation, migration, proliferation, and subsequent redifferentiation of epithelial cells. However, in humans, the mechanism of airway regeneration associated with specific sequences of cell proliferation, differentiation, and restoration of the normal function of lung defense has not been fully investigated and is still not well understood.

In previous reports (17, 20, 24, 32), a denuded tracheal xenograft model has been developed to analyze the regeneration potential of airway cell subpopulations from rabbit or rat airways. Isolation of specific cells followed by xenograft reconstitution has been used to define airway progenitor cells in which the phenotypes in human airways still remain unknown. Rat tracheae denuded of their surface epithelium by repeated cycles of freezing and thawing were seeded with adult airway epithelial cells of interest and were subcutaneously implanted into immunodeficient nude mice. It has been shown that airway cells could adhere to the tracheal extracellular matrix, grow, differentiate, and progressively develop an epithelial structure similar in morphology and function to proximal fully differentiated conducting airways. Using rat airway epithelial cells, Shimizu and colleagues (36, 37) demonstrated that the regenerative process described in this tracheal graft model is similar to that observed after a focal denuding mechanical injury. This suggests that the dedifferentiation, migration, proliferation, and redifferentiation pathways may be the regular pathways of epithelial regeneration at sites of injury. Such a xenograft model has been adapted by Engelhardt et al. (8) with human bronchial epithelial cells, offering a unique opportunity to perform studies either on regenerating or on fully differentiated human airway epithelium within an in vivo experimental environment. With this humanized xenograft model, differences between cystic fibrosis (CF) and non-CF airway secretions have been highlighted (12, 39). It has been also shown that regenerating human airway epithelium is more prone than fully differentiated airway epithelium to be transduced by viral gene vectors (13, 14). Despite these data, the sequence of events leading to fully differentiated human airway epithelium in tracheal xenografts has not been studied in detail to the present time.

In this study, we adapted the tracheal xenograft model in nude mice developed by Engelhardt et al. (8) by using human nasal epithelial cells to mimic regeneration of human proximal airway epithelium up to the fully differentiated state. Three main questions were addressed regarding the regenerative process: 1) what is the timing of cell proliferation, 2) what are the cellular pathways of normal differentiation, and 3) what are the sequential events involved in the restoration of the barrier integrity? To answer these questions, 3-day- to 5-wk-old xenografts were recovered for morphological observations at the optical and ultrastructural levels. Immunohistochemical staining for specific markers [Ki-67, cytokeratins (CKs), desmoplakin (DP) 1, DP2, zonula occludens (ZO)-1, and CF transmembrane conductance regulator (CFTR)] was also performed to quantify airway cell proliferation and analyze the different steps involved in the process of differentiation. In parallel with these experiments, the integrity and permeability of the epithelium were studied at the ultrastructural level with a lanthanum nitrate tracer.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Human Airway Tissue

Fresh human airway tissue collected from nasal polyps or nasal mucosa (turbinates) was immediately transferred to the laboratory in RPMI 1640 medium (Seromed, Biochrom, Berlin, Germany) supplemented with 20 mM HEPES and antibiotics (200 U/ml of penicillin and 200 µg/ml of streptomycin).

Human Airway Cell Dissociation

Dissociated human airway epithelial cells were obtained by an enzymatic procedure (4). As previously described, the cellular pellets contained isolated goblet, ciliated, or basal cells, small clumps of reaggregated epithelial cells, small epithelial sheets, and inflammatory cells (mainly eosinophils and lymphocytes). These inflammatory cells represented <1% of the total number of isolated cells (mean 0.97%, range 0.35-1.55%; n = 5 nasal polyps). Cell viability as assessed with the trypan blue exclusion procedure was >90%. The cells were resuspended at a density of 10 × 106 cells/ml in a hormonally defined medium (RPMI 1640 culture medium supplemented with 1 µg/ml of insulin, 1 µg/ml of transferrin, 10 ng/ml of vitamin A, 200 U/ml of penicillin, 200 µg/ml of streptomycin, 50 µg/ml of gentamicin, and 2.5 µg/ml of amphotericin). Fifteen separate cell preparations from 15 different airway tissue specimens (13 nasal polyps and 2 nasal mucosa) were used to inoculate 47 xenografts. Forty-one of the xenografts were obtained from nasal polyps.

Generation of Tracheal Xenografts in Nude Mice

Humanized tracheal xenografts, one or two per mouse, were prepared as previously described (8). Briefly, tracheae of male Wistar rats (weighing 220-250 g; Charles River France, Saint-Aubin-Lès-Elbeuf, France) were frozen at -80°C and thawed two times to remove the surface epithelium. Their inner diameter was ~2 mm, and their length varied between 1 and 1.4 cm, including 15-20 cartilaginous rings. The rat tracheae were then aseptically tied at their distal end to sterile polyethylene tubing. The tracheae were then stored at -80°C until inoculation with dissociated human airway cells (0.75 × 106 cells in 75 µl of the defined culture medium) and subcutaneous implantation in the flanks of female recipient nude mice anesthetized with an intraperitoneal injection of pentobarbital sodium (40 mg/kg). The mice were housed under pathogen-free conditions and used for experimentation after 8 wk of age. The xenografts were flushed weekly to clear the lumen of cell debris. To remove tracheal xenografts at various times after implantation, the mice were killed with an injection of an overdose of pentobarbital sodium.

Immunocytochemistry and Light Microscopy

To characterize the proliferation, phenotype, and differentiation state of cells repopulating the xenografts, small portions of freshly removed tracheal xenografts were embedded in optimum cutting temperature compound (Tissue-Tek, Miles) and stored at -80°C. Transverse cryosections (5 µm thick) were prepared on a microtome 2800 Frigocut-N (Reichert-Jung, Germany), transferred onto gelatin-coated glass slides, air-dried, and stored at -20°C. Immediately before immunocytological studies, the frozen tissue sections were rehydrated in 0.1 M phosphate-buffered saline (PBS) at pH 7.2 for 10 min. After incubation in PBS supplemented with 1% bovine serum albumin (BSA) for 30 min, the sections were incubated with a primary antibody at the appropriate dilution in PBS-BSA for 60 min at room temperature. The following primary monoclonal antibody solutions were used: mouse antibody to Ki-67 (nuclear marker of proliferating cells expressed during the G1, S, G2, and M phases of the cell cycle; 1:25 working dilution; MIB-one, Immunotech, Marseille, France), mouse antibody to cytokeratin (CK) 13 (a marker of basal and squamous metaplasia cells; clone KS-1A3; 1:800 dilution; Sigma, St. Louis, MO), mouse antibody to CK14 (marker of basal cells; 1:10 dilution; provided by Dr. E. B. Lane, CRC Cell Structure Research Group, Dundee, UK), mouse antibody to CK18 (marker of ciliated and secretory cells; clone CY-90; 1:2,000 dilution; Sigma), mouse antibody to desmoplakin (DP) 1 and DP2 (markers of desmosomes; clones DP1 and DP2-2.15, respectively; 1:10 dilution, e.g., antibody concentration 2 µg/ml; Boehringer Mannheim France, Meylan, France), rat antibody to zonula occludens-1 (ZO-1; a marker of tight junction; clone 6A1; 1:20 dilution; Biogenesis, Poole, UK), and mouse anti-human CFTR monoclonal antibody 24-1 raised against a synthetic peptide in the carboxy-terminal domain of the CFTR protein (1:50 dilution; Genzyme, Cambridge, MA). After two washes in PBS for 5 min and one in PBS-BSA for 5 min, the sections were incubated with goat biotinylated anti-mouse IgG solution (Boehringer Mannheim France) or goat anti-rat IgG solution (Amersham International) at a 1:50 dilution in PBS-BSA solution for 60 min and were then incubated with streptavidin-FITC (Amersham International) at a 1:50 dilution in PBS for 30 min. The sections were counterstained with Harris hematoxylin solution for 30 s, mounted in citifluor antifading solution (Agar Scientific, Stansted, UK), and observed with a Zeiss Axiophot microscope (Zeiss, Le Pecq, France) with epifluorescence and Nomarski differential interference illumination.

Quantification of Airway Epithelial Cell Proliferation

To evaluate the proliferating activity of the cells repopulating xenografts as a function of time after implantation in nude mice, at least three xenografts inoculated with nasal cells from three different patients were analyzed for each specific time point of our study. For each xenograft specimen, nine 5-µm frozen sections were used for immunodetection of Ki-67, hematoxylin staining, and observation by one investigator using the Zeiss Axiophot microscope with a ×40 objective lens. Microscopic fields were randomly chosen in the nine different sections because of the regional variation in Ki-67 immunoreactivity. The number of Ki-67-positive cell nuclei was counted as well as the total number of hematoxylin-stained nuclei. To quantify cell proliferation in the surface epithelium, at least 1,000 epithelial cells/xenograft were counted, with the number of Ki-67-positive cell nuclei then divided by the total number of nuclei counted.

Alcian Blue-Periodic Acid Schiff Cytochemistry

Five-micrometer-thick transverse cryosections of tracheal xenografts were stained with Alcian blue (AB; pH 2.5)-periodic acid Schiff (PAS) for identification of acidic (blue) and neutral (red) mucosubstances, respectively.

Scanning and Transmission Electron Microscopy

Xenografts were fixed with 2.5% glutaraldehyde in 0.1 M PBS at pH 7.2 for 2 h, postfixed with 2% osmium tetroxide, and dehydrated in a graded ethanol series. For transmission electron microscopy (TEM), tracheae were embedded in Epon resin. Ultrathin sections were stained with uranyl acetate and lead citrate. Grids were observed with a Hitachi H300 electron microscope at 75 kV (Elexience, Verrières Le Buisson, France). For scanning electron microscopy, ethanol dehydration was followed by critical point drying with carbon dioxide and coating with 15-nm-diameter gold-palladium beads. The specimens were then observed with a Philips XL 30 scanning electron microscope at 10 kV.

Permeability to Lanthanum Nitrate

Epithelial barrier integrity was examined by visualizing diffusion into the intercellular spaces of a low-molecular-weight electron-dense tracer (lanthanum nitrate) instilled into the lumens of the tracheal xenografts. Lanthanum nitrate incubation was performed as previously described (16, 34). Briefly, tracheae were fixed with 2.5% glutaraldehyde in 0.1 M PBS at pH 7.2 for 2 h and intensively washed in PBS. The specimens were then rinsed in a 0.2 M S-collidine-HCl buffer at pH 7.4 (Sigma) and postfixed with a 1:1 (vol/vol) mixture of 0.2 M lanthanum nitrate (Sigma) in S-collidin-HCl buffer plus 2% osmium tetroxide at pH 7.8 for at least 2 h at room temperature. The tracheae were immediately dehydrated in graded solutions of ethanol and embedded in Epon resin for subsequent TEM observations.

Evaluation of Airway Epithelial Cell Types

To evaluate the proportions of the various airway epithelial cell types constituting the surface epithelium of 4- to 5-wk-old xenografts, toluidine blue-stained Epon semithin sections of three specimens (repopulated with cells from three different human nasal tissues) were observed with the Zeiss Axiophot microscope with a ×63 objective lens. At least 350 epithelial cells/xenograft were counted. As described by Engelhardt et al. (7), the cells were categorized into basal cells, ciliated cells, goblet cells, and intermediate cells. The relative proportions of the various cell types (mean of the three values obtained from the three xenografts) were then compared with those of normal human proximal airway epithelium with the chi 2 test. Normal bronchial tissues were obtained from one patient who died from nonpulmonary diseases and from one patient undergoing surgery for bronchial carcinoma (microscopically normal area distant from the tumor). Data are means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Sequential Events of Human Airway Regeneration in Tracheal Xenografts

Morphological observations of tracheal xenograft lumens at various times after implantation in nude mice indicated that 40 of the 47 denuded rat tracheae (85%) were successfully repopulated with human airway epithelial cells. Even though the composition of airway cell inocula was not standardized, no difference in cell repopulation was noticed in tracheal xenografts from experiment to experiment. In comparing xenograft morphologies maintained from 3 days to 5 wk in mice, we selected four specific time points that corresponded to up to 1, 1-2, 2-4, and 4-5 wk. These four successive characteristic morphologies of human airway surface epithelium regenerated in xenografts are shown at the optical and scanning electron microscopy levels in Fig. 1. The isolated populations of human airway epithelial cells were also capable of invaginating into the underlying lamina propria. Such structures were clearly observed in 11 of the 47 xenografts. One to two weeks after implantation, the epithelium of some xenografts exhibited slight invaginations penetrating into the underlying lamina propria (Fig. 2, A and B). After 4 wk of implantation, evidence of AB-positive submucosal glandular structures could be observed (Fig. 2, C and D).


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Fig. 1.   Morphology of human airway surface epithelium regenerated in tracheal xenografts. At 3-5 days after implantation, xenograft is partially repopulated with flattened nonciliated cells morphologically similar to those identified in repairing regenerating airway epithelium (step I of regeneration process; A). At 1-2 wk, entire surface of rat tracheal lumen is lined by multiple epithelial layers with cuboid cells and more superficial flattened cells (step II; B). At 2-4 wk, cells become progressively columnar, and on rare occasions, ciliated cells are observed (step III; C). At 4-5 wk, cell lining is similar to that of a normal human pseudostratified mucociliary airway surface epithelium (step IV; D). Left: light micrographs of cross sections stained by hematoxylin. Bar, 50 µm. Right: scanning electron microscopy micrographs. Bar, 10 µm.



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Fig. 2.   Human airway surface epithelium penetrating into underlying lamina propria of tracheal xenografts. Light micrographs of a 1 (A)- or 2 (B)-wk-old xenograft (step II of regeneration process) show slight epithelial invaginations. Bars, 50 µm. Alcian blue-periodic acid Schiff (AB-PAS) staining shows blue mucus in lumen of a glandular submucosal structure in a 4-wk-old xenograft (step IV; C). Bar, 100 µm. Under scanning electron microscopy, such a glandular structure exhibits a collecting duct (arrow) from which mucus (m) is flowing (D). Bar, 20 µm.

Cell Proliferation, Cell Differentiation, and Epithelial Barrier Integrity During Human Airway Regeneration in Tracheal Xenografts

Figure 3, Table 1, and Fig. 4 summarize our data concerning 1) the variation of cell proliferation, 2) the sequential expression of markers of epithelial differentiation, and 3) the epithelial barrier integrity found in xenografts, respectively, according to time after implantation in nude mice. Although one portion of one xenograft could be, in some cases, different in its morphology, differentiation or proliferation state, or barrier integrity function from one another, the xenografts underwent a similar sequence of changes with time in all experiments.


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Fig. 3.   Percentage of proliferating airway epithelial cells through steps of regeneration process. Quantification was performed with tracheal xenografts maintained 5 days (step I of regeneration process), 8 days (step IIa), 13 days (step IIb), 2-4 wk (step III), and 4-5 wk (step IV) after implantation in nude mice. Data are means ± SE.


                              
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Table 1.   Changes in expression of epithelial differentiation markers during regeneration of airway surface epithelium in tracheal xenografts



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Fig. 4.   Transmission electron microscopy (TEM) micrographs visualizing permeability to lanthanum nitrate (arrows) of airway surface epithelium regenerated in tracheal xenografts. A: lanthanum nitrate is rarely observed (step I). On occasions, it is sparsely deposited on the apical membrane of poorly differentiated cells. Original magnification, ×7,000. B: when epithelium is stratified, lanthanum nitrate deposits are strongly accumulated along apical plasma membrane of cells in upper layer (step II). Original magnification, ×15,000. C: during cell differentiation, lanthanum nitrate is concentrated in lateral cell borders between adjacent columnar cells (step III). Original magnification, ×20,000. D: when epithelium is fully differentiated, lanthanum nitrate diffusion in intercellular spaces is halted by tight junctions present at apical ends of lateral borders between adjacent columnar cells (step IV). Original magnification, ×20,000.

Step I: Cell migration and cell proliferation. From 3 to 5 days after implantation, tracheal lumen surfaces presented areas of adherent cells neighboring areas of denuded extracellular matrix. Generally, rat tracheae were partly covered with a single flattened nonciliated cell lining generated by airway epithelial cells adhering rapidly to the denuded basal lamina after inoculation. The cells at the periphery of this lining were very similar to migrating flattened epithelial cells involved in airway wound repair, exhibiting no or very few short microvilli on their apical surface but having cytoplasmic expansions on the free edge of the cell-cell contacts (Fig. 1A). Cross sections indicated that the cell lining was one to three layers thick (Figs. 1A and 5A). Concerning the results obtained from three 5-day-old xenografts inoculated with three different human nasal tissue specimens, proliferating epithelial cells were found in all cell layers and represented 24.11 ± 12.28% of the total number of cells examined (Fig. 3). All of these cells were nonciliated poorly differentiated flattened cells and expressed CK13, thus confirming their epithelial origin (Table 1). When the cell lining was a monolayer, epithelial cells also expressed CK14 and CK18. When the cells were arranged in a multilayer, only the more superficial cells covered by a few short rudimentary microvilli expressed CK18, whereas cells in contact with the rat trachea (basal cells) and intermediate cells expressed CK14. CFTR labeling was undetectable. Many basal cells displayed osmiophilic cytoplasmic granules corresponding to lipid droplets. Negative AB-PAS staining indicated that the cells synthesized neither acidic nor neutral mucosubstances. Under TEM, characteristic features of basal, secretory, or ciliated cell differentiation were never observed (Fig. 5A). The immunocytochemical labeling of DP1, DP2, and ZO-1 was absent. However, under TEM, some cells displayed patchy intercellular contacts with occasional desmosomes. There was no ultrastructural evidence of tight junctions. Lanthanum nitrate precipitates were rarely visualized in the TEM experiments, and the electron-dense tracer did not accumulate on any specific structures. On occasion, it was sparsely deposited on the apical membranes of poorly differentiated cells (Fig. 4A). These data indicate that tracheal xenografts did not exhibit cell contacts that were efficient enough to oppose the passage of ions or small molecules through intercellular spaces.


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Fig. 5.   Transmission electron microscopy (TEM) micrographs of human airway surface epithelium regenerated in tracheal xenografts. A: flattened cells exhibit no characteristic features of basal, secretory, or ciliated differentiation (step I). Original magnification, ×3,000. B: squamous epithelial cells exhibit wide intercellular spaces, abundant interdigitating cytoplasmic protusions, and numerous desmosomes (step II). Original magnification, ×3,000. C: columnar epithelium contains abundant secretory cells(s) (step III). Original magnification, ×3,000. Inset: Preciliated cell presenting both secretory granules (sg) and cilia (c) is also observed. Original magnification, ×5,000. D: in fully differentiated epithelium, ciliated cells are rich in mitochondria, secretory cell exhibits granules with heterogeneous patterns of electron density, intercellular spaces are narrow, and apical intercellular junctional complexes are present between adjacent cells (step IV). Original magnification, ×5,000.

Step II: Cell proliferation and stratification. One to two weeks after xenograft implantation, the entire surface of the rat trachea was covered with a squamous epithelium composed of two to seven layers (Fig. 1B). As shown in Fig. 3, a large variation in epithelial cell proliferation was observed between 1- and 2-wk-old xenografts. For 8-day-old xenografts (n = 2), proliferating cells were observed in all cell layers and represented 28.63 ± 5.31% of the total number of epithelial cells. In 13-day-old xenografts (n = 3), squamous morphology persisted, and cell proliferation was markedly decreased, with 3.80 ± 2.73% of the cells analyzed being proliferating cells. The more superficial cells were large and flattened. These cells exhibited more microvilli than during the first week (Fig. 1, A and B). As described in Table 1, all cells expressed CK13 with a similar intensity of staining. The airway cells of the upper layers were characterized by CK18 expression, whereas nearly all the basal cells as well as cells from the intermediate layers expressed CK14. CFTR labeling was undetectable. Some of the superficial cells were stained with PAS. Under TEM, there was no evidence of secretory or ciliated differentiation, but a discontinuous newly synthesized basal lamina was observed beneath some basal cells. Although ZO-1 could not be immunodetected, DP1 and DP2 labeling was clearly evident as a punctuated pattern along the lateral borders of all repopulated airway cells. Although wide intercellular spaces were often observed between adjacent cells from the different cell layers, numerous cell-cell contacts existed and took place via abundant interdigitating cytoplasmic protusions and desmosomes (Fig. 5B). All cells from the upper layers were connected to each other by junctional complexes identified as desmosomes. Lanthanum nitrate incorporated into the xenograft lumens was excluded from the spaces between the multilayered cells and was strictly located at the apical surface of the upper layer, indicating the efficiency of the epithelial barrier function (Fig. 4B). In very few cases, the tracer penetrated into some altered cells, which consequently appeared more electron dense. In a small number of epithelial areas, the tracer filled intercellular spaces, indicating that the integrity of the epithelium was incomplete.

Step III: Cell differentiation. Two to four weeks after xenograft implantation, the rat tracheae had repopulated with an epithelium exhibiting a heterogeneous structure, with fields of cells organized in squamous multilayer neighboring areas of columnar epithelium. As shown in Fig. 1C, areas of columnar epithelium were characterized by the presence of two cell layers with vertically oriented nuclei. Most of the superficial cells were covered with long microvilli, and ciliated cells were occasionally observed (Fig. 1C). The proportion of proliferating cells (1.74 ± 1%) was low (Fig. 3). Secretory differentiation could be identified in abundant superficial cells by TEM (Fig. 5C). The apical surface of these cells bulged into the lumen and exhibited elongated microvilli and apical cytoplasmic protrusions. Throughout this step in the regeneration process, ciliogenesis could also be observed. Some cells presented the features of preciliated cells (i.e., simultaneous presence of sparse irregularly oriented cilia and secretory granules in apical domains; Fig. 5C, inset). Some epithelial cells were seen to exhibit formation of centrioles in their cytoplasm. Ciliated cells were observed to contain either mature, short, or compound cilia (megacilia) composed of several axonema enclosed within a single plasma membrane projection. Basal cells were typical of basal cells from differentiated airways, with prominent nuclear heterochromatin and the presence of hemidesmosomes between their basal plasma membrane and the substrate. Evidence of a newly synthesized basal lamina, complete or incomplete, could be observed in some areas. Although the CK pattern of expression in squamous areas was similar to the one detected during the second step of the regeneration process (i.e., cell stratification), some differences appeared concerning the pattern of CK13 expression in areas of columnar epithelium. CK13 was detected in all epithelial cells of the repopulated surface epithelium. However, the immunostaining was higher in basal cells than in superficial columnar cells. Thus immunoreactivity with CK13 antibody was predominant in basal cells, whereas CK14 expression was restricted to the basal cells and CK18 was localized to superficial cells. CFTR was detected as a diffuse, very faint labeling in the cytoplasm of superficial cells. A similar pattern of distribution was observed for ZO-1 protein. DP1 and DP2 labeling was detected along the lateral border of all cells. In agreement with these data, desmosomes were visualized by TEM. As shown in Fig. 4C, lanthanum nitrate penetrated the intercellular spaces. Electron-dense deposits were associated with the basolateral membrane of the more superficial cells. The junctional permeability tracer occasionally reached the basal cells. Lanthanum nitrate was never observed in contact with the basal lamina.

Step IV: Full differentiation. After four to five weeks, the entire surface of the rat trachea was covered by a pseudostratified columnar epithelium where evidence of ciliated and secretory differentiation could be observed. Some xenografts also displayed occasional patches of less-differentiated epithelium corresponding to step III of the regeneration process, a basal cell hyperplasia phenotype, or squamous metaplasia. Proliferating cells, representing 1.46 ± 0.46% of the total number of cells, consisted of basal or epithelial cells located just above the basal cells. The morphology of this pseudostratified epithelium was indistinguishable from that of a normal human fully differentiated airway epithelium. Moreover, the relative proportion of each airway cell type (40 ± 1% basal cells, 39 ± 1.5% ciliated cells, 9 ± 2.5% goblet cells, and 12 ± 2% intermediate cells) was not significantly different from that reported in native human bronchi (P = 0.055) by Engelhardt et al. (7) or from what we found in human bronchial surface epithelium (34 ± 2% basal cells, 39 ± 6.5% ciliated cells, 18 ± 5% goblet cells; 9 ± 1% intermediate cells; p = 0.09). Ciliated cells were well developed and rich in mitochondria (Fig. 5D). As shown in Fig. 6H, AB-PAS-positive secretory cells were present. These cells exhibited granules with heterogeneous patterns of electron density (Fig. 5D). The pattern of distribution for CK13, CK14, CK18, DP1, DP2, ZO-1, and CFTR was similar in 4- to 5-wk-old xenografts and in fully differentiated human airway surface epithelium (2, 27; unpublished data). CK13 expression was characteristic of basal cells and CK14 was found on rare occasions in basal cells, whereas CK18 expression was restricted to ciliated and secretory cells (Fig. 6). The CFTR protein was markedly detected at the apical membrane of the ciliated cells (Fig. 6G). DP1, DP2, and ZO-1 were clearly detected by immunohistochemistry (Fig. 6, E and F). DP1 and DP2 labeling presented a characteristic punctuate pattern observed along the lateral borders of all the epithelial cells, with stronger labeling at the interface between superficial and basal cells and between basal cells. ZO-1 protein was detected at the apex of all the columnar superficial cells. In agreement with these results concerning DP1, DP2, and ZO-1 immunodetection, desmosomes (at lateral borders) and tight junctions (at the apical ends of lateral borders between adjacent superficial cells) were identified by TEM. The intercellular spaces were generally narrow. As shown in Fig. 4D, no specific deposits of lanthanum nitrate could be visualized on the apical plasma membrane of differentiated ciliated or secretory cells. The detection of lanthanum nitrate precipitates was restricted to the apical ends of lateral borders between adjacent columnar cells so that its penetration was rapidly halted along the basolateral membranes. At this stage, the integrity of the epithelial barrier was completely restored.


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Fig. 6.   Immunocytochemical detection of epithelial differentiation markers and AB-PAS staining in a fully differentiated airway surface epithelium from tracheal xenografts maintained for 4-5 wk in nude mice. A: Nomarski picture. Cytokeratin (CK) 13 labeling was mainly detected in basal cells. B: very few columnar cells were CK13 positive. C: CK14 labeling was restricted to rare basal cells (arrows). D: CK18 was expressed in columnar ciliated and secretory cells. E: desmoplakin (DP) 1 and DP2 were distributed at lateral borders of all cells. F: zonula occludens-1 was detected at apex of columnar cells (arrows). G: cystic fibrosis transmembrane conductance regulator was localized at apical plasma membrane of ciliated cells (arrows). H: some AB-PAS-positive secretory cells were observed.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The aim of the present study was to characterize, in terms of cell proliferation, differentiation, and integrity of the epithelial barrier, the sequence of events involved in the regeneration of human airway surface epithelium in a model of tracheal xenograft in nude mice. Many authors have previously shown that such a humanized model has important applications, particularly in CF gene therapy (8, 13, 14, 40), the analysis of airway secretions (12, 39), and the characterization of airway progenitor cells (6, 38). However, for the first time, this model of a denuded tracheal repopulation with adult human airway epithelial cells is described in detail. In full agreement with previous data (36, 37) obtained with a similar tracheal xenograft model inoculated with rodent airway cells, we discriminated characteristic successive events similar to the sequences of events described in airway epithelium regeneration after injury. These events include cell migration and proliferation, cell differentiation, and restoration of a fully differentiated epithelium. Interestingly, we also observed that the barrier function of the airway epithelium 1) was rapidly restored after a wave of proliferation, 2) was transiently altered during cell differentiation, and 3) recovered once differentiation is achieved.

Proliferation of the airway epithelium was investigated by employing Ki-67 antigen immunoreactivity. In agreement with data reported by Lane and Gordon (23) and Keenan et al. (21) on rodent tracheal epithelium after mechanical injury, we found that the regenerative process of human airway cells in tracheal xenografts first involved a wave of cell proliferation leading to a multilayered epithelium after 1 wk. We also observed that this wave of proliferation rapidly declined before the appearance of secretory and ciliated cell differentiation. This result supports the concept that differentiation is associated with a decreasing capacity for cell division (23). When the airway epithelium of the xenografts was fully differentiated, the proportion of proliferating cells (1.46 ± 0.46%) was identical to that evaluated for the normal human airway epithelium (1).

Cell differentiation was assessed by immunodetection of epithelial cell differentiation markers and morphological analyses by TEM. During the process of cell migration and proliferation, we could not precisely identify the potential origin of the cells able to adhere to and regenerate on the denuded rat tracheae. Thus, CK14 (regarded as a marker of the basal cell phenotype) and CK18 (a marker of columnar ciliated and secretory cells in mature human airways) were both expressed in 3- to 5-day-old xenografts. No evidence of secretory, ciliated, or basal cell differentiation was observed at the ultrastructural level of the flattened cells. It can be hypothesized that dedifferentiation is associated with the xenograft regeneration process as previously described for airway repair after injury (24, 28, 36). It can be also hypothesized that cells contributing to regeneration come from small foci of actively dividing pluripotent cells that never exhibit features of differentiated airway cells. Up to now, this preexisting stem cell population in human airway epithelium still remains unknown. Because the pattern of distribution for the epithelial markers was similar during steps II and III of the regeneration process, only ultrastructural investigations permitted us to demonstrate differences in cell phenotypes. These included 1) the squamous architecture of an undifferentiated epithelium exhibiting abundant desmosomes and 2) the abundance of secretory cells before active ciliogenesis and replacement by ciliated cells as previously described during fetal airway development (10). Interestingly, we also provided evidence that the distribution of epithelial markers, the ultrastructural morphology, and the relative proportions of epithelial cells were strictly similar in 4- to 5-wk-old xenografts and normal mature adult airways (2).

The most original finding of this study concerns the sequential changes in epithelial barrier function during airway epithelial regeneration. As far as we know, this is the first time that the barrier function of human airway surface epithelium has been analyzed in a model of regeneration from a state of poorly differentiated cell migration through to full differentiation. This barrier function is known to be maintained by a variety of cellular junctional complexes. The desmosomes and the intermediate junctions (zonula adherens) are the most effective adhesive structures for maintaining cell-to-cell adhesion and tissue integrity, whereas tight junctions (ZOs) are important in the regulation of opening and occluding intercellular spaces and facilitating intercellular communication (27). The absence of epithelial barrier function early after xenograft implantation in nude mice correlates with the absence or the scarcity of junctional complexes between the poorly differentiated flattened cells. This is consistent with previous data (15, 16, 25) obtained in vivo on rat tracheal epithelium or in vitro on a wound model of human airway epithelium during the initial hours after injury. Interestingly, 1-2 wk after xenograft implantation, the airway surface epithelium was squamous, exhibited numerous desmosomes along the lateral borders of all cells, and excluded lanthanum nitrate from diffusion in the intercellular spaces. This suggests that restoration of the airway epithelial barrier is an adaptive step in the regenerative process. This result confirms the idea that the squamous epithelium phenotype represents a "highly protective" phenotype against injurious agents. This metamorphism is also observed in normal development when the columnar ciliated epithelium of the esophagus is transformed into squamous epithelium at 3-4 mo of human fetal life (26). With regard to the junctional complexes involved, it has been shown in models of airway injury that a rapid development of effective tight junctions is responsible for such a rapid restoration of epithelial barrier integrity (9, 16, 25). There is an apparent discrepancy between these data and our present study because we did not detect either by ZO-1 immunodetection or by TEM the presence of tight junctions in squamous airway epithelium. Although they were not detected, some of these junctional complexes may be actually present and effective. Analysis by freeze-fracture would be undoubtedly more sensitive in demonstrating the presence or absence of tight junctions (3, 5). It can be also hypothesized that the presence of abundant desmosomes along the cell borders is responsible for such barrier integrity. Interestingly, we observed that this epithelial tightness was transient and rapidly disappeared when the squamous epithelium was replaced by a columnar and more differentiated epithelium. When xenografts were rinsed at this time, numerous exfoliated squamous cells were observed in the flush medium. Thus, in concordance with previous data (22), we assume that the upper cells of the squamous epithelium slough and that the remaining cells provide a new columnar epithelium. Our data suggest that the regenerating airway epithelium is fragile during the cell differentiation step before acting as an efficient barrier by the presence of effective apical tight junctions when cell differentiation is restored. Although no tight junction was detected before cell differentiation achievement, such junctional complexes may actually be present. In this case, our results could be consistent with the results reported by Carson et al. (3) concerning the postnatal development of ferret airways. These authors have described the decrease in tight junction permeability in relation to cell differentiation and have observed on freeze-fracture replicas that such alterations are associated with configurational changes in tight junction architecture.

As previously described by Engelhardt et al. (6), formations of submucosal glands can be observed in humanized tracheal xenografts. For xenografts harvested 4-5 wk after implantation in nude mice, the mucus secretory function of these glandular structures was verified by AB-PAS staining. However, neither lysozyme (a glandular serous cell marker) nor CFTR [highly expressed in human airway glands (18)] was immunodetected here (data not shown). In fetal human airways, achievement of full submucosal gland development occurs after surface epithelium differentiation, and mucous acini differentiate several weeks before serous cells (19, 29). Thus it can be suggested, as previously reported by Engelhardt et al. (6), that such submucosal glandular structures are not fully differentiated in tracheal xenografts exhibiting a fully differentiated surface epithelium.

With regard to CF research, we examined CFTR protein expression in our model of human airway regeneration. In normal adult airway surface epithelium, this cAMP chloride channel is localized to the apex of ciliated cells (30). In CF, CFTR has a decreased expression or presents an abnormal cytoplasmic distribution likely to lead to the significant disorders associated with the disease (30). Our present results indicate that the level of expression or the localization of CFTR in non-CF human airway surface epithelium is related to differentiation of this epithelium. CFTR was only immunodetected in apical domains when mature ciliated cells were present. This is in agreement with previous immunohistochemical studies performed on remodeled human nasal epithelium (2) or human airways during fetal development (11). CFTR in situ hybridization in ferret airways during the postnatal development has also demonstrated an increase in CFTR mRNA within the surface epithelium, mirroring an increase in ciliogenesis (35).

In summary, we have shown that the regeneration of a well-differentiated and functional human airway epithelium in rat tracheae grafted in nude mice includes several steps that mimic the regeneration dynamics of airway epithelium after injury. Our study has also documented the dynamic restoration of epithelial barrier integrity during human airway regeneration. We will extend this study to the analysis of the interactions between bacteria and epithelial cells during these different steps of regeneration in non-CF and CF human airway surface epithelium.


    ACKNOWLEDGEMENTS

We thank Dr. P. Moullier (Laboratoire de Thérapie Génique, Centre Hospitalier Universitaire Nantes, Nantes, France) and Dr. J.-M. Zahm (Institut National de la Santé et de la Recherche Médicale, Reims, France) for helpful assistance and comments. We are also grateful to Pr. J.-M. Klossek (Hôpital Jean Bernard, Poitiers, France) and Dr. X. Hannion (Polyclinique Courlancy, Reims, France) for providing human nasal tissues, Dr. S. H. Cheng (Genzyme Corporation, Cambridge, MA) and Prof. E. B. Lane (Skin Tumor Laboratory, London, UK) for the kind gift of anti-cystic fibrosis transmembrane conductance regulator monoclonal antibodies and anti-cytokeratin 14 monoclonal antibodies, respectively.


    FOOTNOTES

This work was supported by the Association Française de Lutte contre la Mucoviscidose, the Direction Générale des Armées, and European Community Biotechnology Grant BIO4CT95-0284.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: E. Puchelle, INSERM U514, CHU Maison Blanche, 45, rue Cognacq Jay, 51092 Reims Cedex, France (E-mail: epuche{at}worldnet.fr).

Received 25 January 1999; accepted in final form 25 August 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Boers, J. E., A. W. Ambergen, and F. B. J. M. Thunnissen. Number and proliferation of basal and parabasal cells in normal human airway epithelium. Am. J. Respir. Crit. Care Med. 157: 2000-2006, 1998[Abstract/Free Full Text].

2.   Brézillon, S., F. Dupuit, J. Hinnrasky, V. Marchand, N. Kälin, B. Tümmler, and E. Puchelle. Decreased expression of CFTR protein in remodeled human nasal epithelium from non-cystic fibrosis patients. Lab. Invest. 72: 191-200, 1995[ISI][Medline].

3.   Carson, J. L., A. M. Collier, T. M. Gambling, M. W. Leigh, R. C. Boucher, S.-C. S. Hu, and T. F. Boat. Development, organization, and function of tight junctional complexes in the tracheal epithelium of infant ferrets. Am. Rev. Respir. Dis. 138: 666-674, 1988[ISI][Medline].

4.   Chevillard, M., J. Hinnrasky, D. Pierrot, J.-M. Zahm, J.-M. Klossek, and E. Puchelle. Differentiation of human surface upper airway epithelial cells in primary culture on a floating collagen gel. Epithelial Cell Biol. 2: 17-25, 1993[ISI][Medline].

5.   Elia, C., C. Bucca, G. Rolla, E. Scappaticci, and D. Cantino. A freeze-fracture study of human bronchial epithelium in normal, bronchitic and asthmatic subjects. J. Submicrosc. Cytol. Pathol. 20: 509-517, 1988[ISI][Medline].

6.   Engelhardt, J. F., H. Schlossberg, J. R. Yankaskas, and L. Dudus. Progenitor cells of the adult human airway involved in submucosal gland development. Development 121: 2031-2046, 1995[Abstract/Free Full Text].

7.   Engelhardt, J. F., Y. Yang, L. D. Stratford-Perricaudet, E. D. Allen, K. Kozarsky, M. Perricaudet, J. R. Yankaskas, and J. M. Wilson. Direct gene transfer of human CFTR into human bronchial epithelia of xenografts with E1-deleted adenoviruses. Nat. Genet. 4: 27-34, 1993[ISI][Medline].

8.   Engelhardt, J. F., J. R. Yankaskas, and J. M. Wilson. In vivo retroviral gene transfer into human bronchial epithelia of xenografts. J. Clin. Invest. 90: 2598-2607, 1992[ISI][Medline].

9.   Erjefält, J. S., I. Erjefält, F. Sundler, and C. G. A. Persson. In vivo restitution of the airway epithelium. Cell Tissue Res. 281: 305-316, 1995[ISI][Medline].

10.   Gaillard, D. A., A. V. Lallement, A. F. Petit, and E. S. Puchelle. In vivo ciliogenesis in human fetal tracheal epithelium. Am. J. Anat. 185: 415-428, 1989[ISI][Medline].

11.   Gaillard, D., S. Ruocco, A. Lallemand, W. Dalemans, J. Hinnrasky, and E. Puchelle. Immunohistochemical localization of cystic fibrosis transmembrane conductance regulator in human fetal airway and digestive mucosa. Pediatr. Res. 36: 137-143, 1994[Abstract].

12.   Goldman, M. J., G. M. Anderson, E. D. Stolzenberg, U. P. Kari, M. Zasloff, and J. M. Wilson. Human beta -defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell 88: 553-560, 1997[ISI][Medline].

13.   Goldman, M. J., P.-S. Lee, J.-S. Yang, and J. M. Wilson. Lentiviral vectors for gene therapy of cystic fibrosis. Hum. Gene Ther. 8: 2261-2268, 1997[ISI][Medline].

14.   Goldman, M. J., and J. M. Wilson. Expression of alpha vbeta 5 integrin is necessary for efficient adenovirus-mediated gene transfer in the human airway. J. Virol. 69: 5951-5958, 1995[Abstract].

15.   Gordon, R. E., and B. P. Lane. Regeneration of rat tracheal epithelium after mechanical injury. II. Restoration of surface integrity during the early hours after injury. Am. Rev. Respir. Dis. 113: 799-807, 1976[ISI][Medline].

16.   Hérard, A.-L., J.-M. Zahm, D. Pierrot, J. Hinnrasky, C. Fuchey, and E. Puchelle. Epithelial barrier integrity during in vitro wound repair of the airway epithelium. Am. J. Respir. Cell Mol. Biol. 15: 624-632, 1996[Abstract].

17.   Inayama, Y., G. E. R. Hook, A. R. Brody, G. S. Cameron, A. M. Jetten, L. B. Gilmore, T. Gray, and P. Nettesheim. The differentiation potential of tracheal basal cells. Lab. Invest. 58: 706-717, 1988[ISI][Medline].

18.   Jacquot, J., E. Puchelle, J. Hinnrasky, C. Fuchey, C. Bettinger, C. Spilmont, N. Bonnet, A. Dieterle, D. Dreyer, A. Pavirani, and W. Dalemans. Localization of the cystic fibrosis transmembrane conductance regulator in airway secretory glands. Eur. Respir. J. 6: 169-176, 1993[Abstract].

19.   Jeffery, P. K., D. Gaillard, and S. Moret. Human airway secretory cells during development and in mature airway epithelium. Eur. Respir. J. 5: 93-104, 1992[Abstract].

20.   Johnson, N. F., and A. F. Hubbs. Epithelial progenitor cells in the rat trachea. Am. J. Respir. Cell Mol. Biol. 3: 579-585, 1990[ISI][Medline].

21.   Keenan, K. P., J. W. Combs, and E. M. McDowell. Regeneration of hamster tracheal epithelium after mechanical injury. III. Large and small lesions: comparative stathmokinetic and single pulse and continuous thymidine labeling autoradiographic studies. Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 41: 231-252, 1982[ISI][Medline].

22.   Keenan, K. P., T. S. Wilson, and E. M. McDowell. Regeneration of hamster tracheal epithelium after mechanical injury. IV. Histochemical, immunocytochemical and ultrastructural studies. Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 43: 213-240, 1983[ISI][Medline].

23.   Lane, B. P., and R. Gordon. Regeneration of rat tracheal epithelium after mechanical injury. I. The relationship between mitotic activity and cellular differentiation. Proc. Soc. Exp. Biol. Med. 145: 1139-1144, 1974.

24.   Liu, J. Y., P. Nettesheim, and S. H. Randell. Growth and differentiation of tracheal epithelial progenitor cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 266: L296-L307, 1994[Abstract/Free Full Text].

25.   Marin, M. L., R. E. Gordon, and B. P. Lane. Development of tight junctions in rat tracheal epithelium during the early hours after mechanical injury. Am. Rev. Respir. Dis. 119: 101-106, 1979[ISI][Medline].

26.   Ménard, D. Morphological studies of the developing human esophageal epithelium. Microsc. Res. Tech. 31: 215-225, 1995[ISI][Medline].

27.   Montefort, S., C. A. Herbert, C. Robinson, and S. T. Holgate. The bronchial epithelium as a target for inflammatory attack in asthma. Clin. Exp. Allergy 22: 511-520, 1992[ISI][Medline].

28.   Pilewski, J. M., J. D. Latoche, S. M. Arcasoy, and S. M. Albelda. Expression of integrin cell adhesion receptors during human airway epithelial repair in vivo. Am. J. Physiol. Lung Cell. Mol. Physiol. 273: L256-L263, 1997[Abstract/Free Full Text].

29.   Plopper, C. G., J. L. Alley, and A. L. Weir. Differentiation of tracheal epithelium during fetal lung maturation in the rhesus monkey Macaca mulatta. Am. J. Anat. 175: 59-71, 1986[ISI][Medline].

30.   Puchelle, E., D. Gaillard, D. Ploton, J. Hinnrasky, C. Fuchey, M.-C. Boutterin, J. Jacquot, D. Dreyer, A. Pavirani, and W. Dalemans. Differential localization of the cystic fibrosis transmembrane conductance in normal and cystic fibrosis airway epithelium. Am. J. Respir. Cell Mol. Biol. 7: 485-491, 1992[ISI][Medline].

31.   Puchelle, E., and J.-M. Zahm. Repair process of the airway epithelium. In: Environmental Impact on the Airways, edited by J. Chretien, and D. Dusser. New York: Dekker, 1996, vol. 93, p. 157-182. (Lung Biol. Health Dis. Ser.)

32.   Randell, S. H., C. E. Comment, F. C. S. Ramaekers, and P. Nettesheim. Properties of rat tracheal epithelial cells separated based on expression of cell surface alpha -galactosyl end groups. Am. J. Respir. Cell Mol. Biol. 4: 544-554, 1991[ISI][Medline].

33.   Rennard, S. I., D. J. Romberger, J. H. Sisson, S. G. Von Essen, I. Rubinstein, R. A. Robbins, and J. R. Spurzem. Airway epithelial cells: functional roles in airway disease. Am. J. Respir. Crit. Care Med. 150: S27-S30, 1994[ISI][Medline].

34.   Revel, J. P., and M. J. Karnovsky. Hexagonal array of subunits in intercellular junctions of the mouse heart and liver. J. Cell Biol. 33: C7-C12, 1967[Free Full Text].

35.   Sehgal, A., A. Presente, and J. F. Engelhardt. Developmental expression patterns of CFTR in ferret tracheal surface airway and submucosal gland epithelia. Am. J. Respir. Cell Mol. Biol. 15: 122-131, 1996[Abstract].

36.   Shimizu, T., P. Nettesheim, F. C. S. Ramaekers, and S. H. Randell. Expression of "cell-type-specific" markers during rat tracheal epithelial regeneration. Am. J. Respir. Cell Mol. Biol. 7: 30-41, 1992[ISI][Medline].

37.   Shimizu, T., M. Nishihara, S. Kawaguchi, and Y. Sakakura. Expression of phenotypic markers during regeneration of rat tracheal epithelium following mechanical injury. Am. J. Respir. Cell Mol. Biol. 11: 85-94, 1994[Abstract].

38.   Zepeda, M. L., M. R. Chinoy, and J. M. Wilson. Characterization of stem cells in human airway capable of reconstituting a fully differentiated bronchial epithelium. Somatic Cell Mol. Genet. 21: 61-73, 1995[ISI][Medline].

39.   Zhang, Y., B. Doranz, J. R. Yankaskas, and J. F. Engelhardt. Genotypic analysis of respiratory mucous sulfation defects in cystic fibrosis. J. Clin. Invest. 96: 2997-3004, 1995[ISI][Medline].

40.   Zhang, Y., J. Yankaskas, J. Wilson, and J. F. Engelhardt. In vivo analysis of fluid transport in cystic fibrosis airway epithelia of bronchial xenografts. Am. J. Physiol. Cell Physiol. 270: C1326-C1335, 1996[Abstract/Free Full Text].


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