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Stem Cells Vol. 23 No. 7 August 2005, pp. 992 -1001
doi:10.1634/stemcells.2004-0197; www.StemCells.com
© 2005 AlphaMed Press

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Aquaporin-3 Expression in Human Fetal Airway Epithelial Progenitor Cells

Aurélie Avril-Delplanquea, Ibrahim Casala, Nicolas Castillonb, Jocelyne Hinnraskyb, Edith Puchelleb, Bruno Péaulta,c

a INSERM U506, Hôpital Paul Brousse, Villejuif, France;
b INSERM UMR514, IFR 53, Université de Reims, Reims, France;
c Rangos Research Center, Children’s Hospital, Pittsburgh, Pennsylvania, USA

Key Words. Aquaporin • Airway • SCID mouse • Epithelium

Correspondence: Bruno Péault, Ph.D., Children’s Hospital of Pittsburgh, Rangos Research Center, 3460 Fifth Avenue, Pittsburgh, PA 15213-2583, USA. Telephone: 412-692-6509; Fax: 412-692-5837; e-mail: bruno.peault{at}chp.edu


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Airway epithelium stem cells have not yet been prospectively identified, but it is generally assumed that both secretory and basal cells have the capacity to divide and differentiate. Previously, we developed a test for progenitor cells of the human airway epithelium, relying on the transplantation of fetal respiratory tissues into immunodeficient mice. In this study, we hypothesized that airway-repopulating epithelial progenitors can be marked with surface antigens, and we screened an array of such candidate markers, including lectin ligands, the CD44 and CD166 adhesion molecules, and the aquaporin-3 (AQP3) water channel. We observed that AQP3 is selectively expressed on the surface of basal cells, allowing the separation by flow cytometry of AQP3+ basal cells and AQP3– ciliated and secretory cells. Functional evaluation of sorted cells in vivo showed that AQP3+ cells can restore a normal pseudostratified, mucociliary epithelium as well as submucosal glands. AQP3– cells are also endowed with a similar potential, although faster engraftment suggests their inclusion of more committed progenitors. These results show that stem cell candidates in the human tracheo-bronchial mucosa can be positively selected with a novel marker but also, for the first time, that epithelial progenitors exist among both basal and suprabasal cell subsets within the human airway.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
The pseudostratified surface epithelium that lines human airways is slowly renewed under normal circumstances, but airway epithelial cells can also proliferate extensively to repair an injury [1]. Similar to other permanently renewing epithelial tissues such as the intestine and epidermis [2, 3], renewal in the steady state and repair ability suggest the existence of stem cells in the airways [4, 5]. It is generally accepted that of the three major cell types present in the upper and lower airway surface epithelia, both secretory and basal cells retain the capacity to divide and differentiate. Ciliated cells, by contrast, are considered to be irreversibly differentiated and unable to divide [6, 7].

Several approaches have been developed to identify progenitor cells in the normal or injured airway epithelium such as 3H-thymidine radiolabeling [8, 9], in vitro or in vivo development of sorted subsets of cells [10–19], and cell-lineage tracking with recombinant retroviruses [20–22]. Studies of cell turnover in the normal respiratory epithelium designate basal cells as potential stem cells [23]. However, the strong proliferation of secretory cells after chemical or physical injury [5, 7] suggests that these may also represent candidate progenitor cells. Cells constituting the airway mucosa have been separated on differences in density [10–12, 15], or by flow cytometry on the basis of cell size [13, 14, 16] or surface-antigen expression [17–19]. Divergent conclusions have been reached regarding the role of sorted basal and small-granule secretory cells in airway epithelium regeneration. Several studies suggested that both cell subpopulations could regenerate a complete mucociliary epithelium in rat xenografts in nude mice [20, 24, 25]. The analysis of gland development in that model suggested that only a subset of basal cells could form both surface epithelium and glands [20]. The existence of multipotent epithelial stem cells in a niche located within submucosal gland ducts has been retrospectively suggested [26]. On the other hand, recent experiments in which cytokeratin (CK) 14–expressing cells were tracked in double-transgenic mice (cre/lox system) during airway epithelium repair show that basal cells include both stem cells and more committed progenitors and are also involved in postinjury regeneration [27, 28]. Accordingly, an equally recent report has assigned stem cell activity to a subset of basal cells in the mouse tracheal epithelium that express high levels of the CK 5 promoter and yield large, multilineage clonal colonies in culture [29]. Despite these advances, a definitive model for the progenitor-progeny relationship in the normal or injured airway has not yet been established, and epithelial stem cells have not been identified at all in human airways.

Previously, we developed an in vivo assay for progenitor cells of the human airway epithelium, relying on the transplantation of human fetal respiratory tissues into severe combined immunodeficiency (SCID) mice [27]. Donor human epithelial cells dissociated from either mature airway xenografts or embryonic lung primordia were seeded in epithelium-denuded host airway grafts and implanted back into SCID mice. All grafts seeded with donor epithelial cells restored a normal mucociliary surface epithelium maintained on the long term and expressing expected markers.

In this study, we aimed at using this assay to further investigate the progenitor potentialities of human fetal basal and suprabasal airway epithelial cells. We hypothesized that cell-surface antigens could be used to select airway epithelium cell subset by flow cytometry. To this end, we tested an array of cell-surface antigens, including lectin ligands, the CD44 and CD166 adhesion molecules, and the aquaporin-3 (AQP3) water channel. We observed that only basal airway epithelial cells express AQP3 at their surface. We show that in vivo both AQP3+ basal cells and AQP3– suprabasal cell subpopulations can restore a well-differentiated, pseudostratified mucociliary surface epithelium and functional submucosal glands. The AQP3– suprabasal cells, however, restore the mucosa much faster than their AQP3+ basal counterparts, suggesting their inclusion of late committed progenitors. One of the primary interests of this work is that it demonstrates the feasibility of prospectively identifying and physically isolating, by flow cytometry, a subset of human airway epithelial cells endowed with significant progenitor potential.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
SCID-hu Mice
C.B.17 scid/scid (SCID) mice were bred in our facility in isolators supplied with sterile-filtered, temperature-controlled air. Cages, bedding, and drinking water were autoclaved. Food was sterilized by x-ray irradiation. All experiments on animals were performed under laminar flow hoods.

Human fetal tissues were collected following therapeutic abortions performed in compliance with the current French legislation and with approval of both national (Comité Consultatif National d’Ethique) and institutional (Comité Opérationnel pour l’Ethique) ethics committees. Developmental stages were estimated from the duration of amenorrhea. Engrafted tissues ranged from 8–32 weeks postconception.

Large proximal airways were dissected under a microscope in phosphate-buffered saline (PBS) containing penicillin and streptomycin. As previously described, segments of human fetal trachea or stem bronchus were implanted subcutaneously in the flanks of 6- to 8-week-old SCID mice anesthetized by intraperitoneal injection of 0.4 ml Hypnomidate (Janssen-Cilag, Issyles-Moulineaux, France, http://www.janssen-cilag.com). To harvest xenografts, host mice were euthanized by cervical dislocation.

Immunohistochemistry
We previously described that human fetal airways enlarge remarkably and reach complete anatomic and functional maturation after 6–8 weeks of engraftment in the SCID mouse [28].

In this study, well-differentiated xenografts from 24 fetuses (gestational age 19.5 weeks ± 7.9, range 8–32; duration of engraftment 28.7 weeks ± 11.5, range 10–55) were harvested, and surface epithelium was studied by immunohistochemistry. Tissues were rapidly dissected, fixed in 3.7% formaldehyde (Merck, Haar, Germany, http://www.merck.com) in PBS for 2 hours, immersed overnight in PBS 30% sucrose, embedded in Cryo-M-Bed medium (Bright Instruments Co., Huntingdon, U.K., http://www.brightinstruments.com), then frozen in liquid nitrogen and stored at –20°C. Five-µm serial frozen sections were cut, and slides were dried for 1 hour at room temperature before storage at –20°C. Mouse monoclonal antibodies to (a) CKs 7 and 13 were purchased respectively from Dako Cytomation (Trappes, France, http://www.dakocytomation.us) (OV-TL 12/30; IgG1) and Sigma (Lyon, France, http://www.sigmaaldrich.com) (KS-1A3; IgG1); (b) integrin subunits were a gift from Dr. F. Watt (London), (anti{alpha}6: MP4F10, IgG1) or purchased from Gibco (Issyles-Moulineaux, France) (antiß4: 3E1; IgG); (c) surface molecules were a gift from Dr. W. Stallcup (La Jolla, CA) (antiHCA: F84.1; IgG) or purchased from Sigma (anti CD44: A3D8; IgG1), from R&D Systems (Abingdon, U.K., http://www.rndsystems.com) (anti CFTR:24–1, IgG1) or from Zymed Laboratories (San Francisco, http://www.zymed.com) (ZO-1, IgG1); and (d) glandular components were gifts from Dr. J.P. Aubert (Lille, France) (anti MUC4, IgG1) and from Dr. V. Godding and J.P. Vaerman (Louvain, Belgium), (antisecretory component of IgA, IgG1). Rabbit polyclonal antibody to aquaporin channel 3 (antiAQP3) was purchased from Alomone (Euromedex, Mundolsheim, France, http://www.euromedex.com). Rabbit polyclonal antibody to glandular components was a gift from Dr. J.P. Aubert (Lille, France) (anti MUC5AC) and purchased from DakoCytomation (antilysozyme). The lectins Ulex europaeus agglutinin (UEA-1), wheat germ agglutinin (WGA), peanut agglutinin (PNA), and Griffonia simplicifolia agglutinin isolectin B4 (GSI-B4) were purchased from Sigma, each one being coupled to fluorescein isothiocyanate (FITC).

After rehydration and blocking of endogenous peroxidases with hydrogen peroxide and nonspecific binding sites with 2% fetal calf serum (FCS), sections were exposed to the primary antibody overnight at 4°C. Biotinylated rabbit anti-mouse IgG Antibody (DakoCytomation) or horseradish peroxidase-goat anti-rabbit IgG antibody (Citifluor, London, http://www.citifluor.co.uk) was added during 1 hour at room temperature. For the former antibody, sections were then incubated 1 hour at room temperature with a streptavidin-biotin complex (Dako-Cytomation), whereas for the latter, sections were preserved in PBS. The reaction was visualized with aminoethylcarbazole (AEC) or diaminobenzidine (DAB) (Sigma). Sections were counterstained with Gill’s hematoxylin and mounted in aqueous medium (Aqueous Mounting Media, Bio Genex, San Ramon, CA, http://www.biogenex.com). Alternatively, FITC-coupled goat anti-mouse or -rabbit IgG antibodies were used, in which case slides were mounted in PBS-glycerol-agar (Citifluor). Observations and photographs were made on an epifluorescence photomicroscope (Nikon, Champigny Sur Marne, France, http://www.nikon.fr).

Human Airway Epithelium Dissociation and Processing by Flow Cytometry
Grafts developed in SCID mice were used as cell donors in all experiments. For isolation of respiratory epithelial cells, 37 well-differentiated human fetal airway xenografts (gestational age 19.2 weeks ± 6.5, range 8–32; duration of engraftment 24.6 weeks ± 13.2, range 5–60) were harvested, filled intraluminally with 1% pronase (Sigma) in RPMI medium-hepes buffer 20 mM and left at 4°C overnight. Epithelial cells were then flushed out with PBS without Ca2+, Mg2+ (PBS-CaMg) and supplemented with 10% FCS to produce a single-cell suspension. One to 2 x 106 cells were recovered per xenograft. For sorting experiments, 2–4 xenografts of the same donor origin were dissociated, and cells were pooled. Cells were counted using a haemocytometer and adjusted to 106 cells per ml. The cell viability was assessed by trypan blue exclusion. Cells were used for sorting experiments when viability was greater than 90%.

A Portion of Each Dissociated Graft Was Frozen and Analyzed by Immunohistochemistry
Cells in suspension were incubated with the primary antibody for 45 minutes at 4°C. After three rinses in PBS-CaMg, FITC-conjugated secondary antibody was added for 20 minutes at 4°C. Cells incubated with no primary antibody or with isotype-matched unspecific antibodies served as negative controls.

Cells were analyzed on a FACScan flow cytometer (Becton, Dickinson, Le Pont-De-Claix, France, http://www.bd.com) and sorted with an Epics Elite cell sorter (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com). Cells were examined using a single laser (488 nm wavelength, 0.4 mW) to generate 2° and 90° light scatter signals and green fluorescence. Data were processed as 2-parameter dot plots and individual frequency distribution histograms. Five thousand ungated events per sample were analyzed using the FACS can Research Software (Becton, Dickinson). Eight sorting experiments were conducted with 20 mature xenografts (gestational age 21.4 weeks ± 4.1, range 14–27; duration of engraftment 27.4 weeks ± 14.2, range 10–44). Basal and apical cells were segregated on antiAQP3 staining. Two sort gates were set to select the negative fraction, and high-expressing cells were sorted at a rate of 400 cells per second. Sorted populations were reanalyzed by flow cytometry, and 5 x 104 cells were cytospun onto glass slides and processed for CK staining by the immunofluorescent method previously described.

Human Airway Epithelium Reconstitution in SCID-hu Mice
The experimental protocol has been previously described in detail [27]. Briefly, host human fetal xenografts (gestational age 19.94 weeks ± 7.1, range 8–33; duration of engraftment 21.87 weeks ± 9.5, range 6–41) were harvested from mice and deprived of their own native epithelium by three rounds of freezing and thawing in FCS, 10% dimethylsulfoxyde (DMSO). For each repopulation experiment (n = 6), 1 x 105 unsorted and sorted cells were then inoculated intraluminally into distinct conditioned host grafts. Control grafts were denuded but received PBS-CaMg containing no epithelial cells. After 24 hours at 37°C, 5% CO2, all grafts were implanted back into SCID mice. Reconstituted and control grafts were harvested after several weeks for short- (4 weeks) and long-term reconstitution (20 weeks) and processed for immunohistochemistry.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
Antigenic Discrimination of Epithelial Cell Subsets in Human Airway Xenografts
As described previously, human airway grafts in SCID mice were eventually terminally differentiated and lined entirely with a ciliated and secretory pseudostratified epithelium [27]. Such human tissues developed in a SCID mouse display epithelial cell markers similar to those described in airways developed in utero. We herein further focused the analysis of these grafts on the identification of markers expressed differentially by epithelial cell subpopulations.

We first examined the binding to graft sections of lectins described as markers in the rat airway epithelium [29]. The U. europaeus agglutinin-1 (UEA 1) lectin recognized all epithelial cells in the xenografts, but G. simplicifolia isolectin (GSI-B4), wheat germ agglutinin (WGA), and peanut agglutinin (PNA) showed no affinity for human airway epithelial cells (data not shown). CK 7 was identified in apical ciliated and secretory cells (Fig. 1AGo), whereas CK 13 was restricted to the basal cell layer (Fig. 1BGo). {alpha}6 and ß4 integrin subunits showed a very restricted distribution on the basal membrane of basal cells (Figs. 1C, 1DGo). Among adhesion molecules, the hyaluronate receptor CD44 was located on all basal cell membranes (Fig. 1EGo), and CD166, expressed otherwise by embryonic neuroblasts and by hematopoietic stem cells and stromal cells [30–32], was found on the apical and basolateral membranes of ciliated and secretory cells (Fig. 1FGo). Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein was, as expected, restricted to the apical membrane of ciliated cells (Fig. 1GGo). Interestingly, while systematically screening the expression in the human fetal airway of antigens present in other epithelial tissues, we observed that the AQP3 cell-surface water channel was expressed by basal cells and rare intermediate cells in the human surface epithelium, but absent from apical, well-differentiated suprabasal airway cells (Fig. 1HGo). Of note, AQP3 was not detected at all in epithelial cells lining gland ducts and within submucosal glandular cells.



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  		Figure 1. Epithelial cell marker expression in a 20-week fetal human trachea implanted 15 weeks in the SCID mouse. A mature pseudostratified mucociliary epithelium lines the whole inner surface of the graft and rests on a thick mesenchyme. CK 7 is present in apical epithelial cells (A), whereas CK 13 is found only in basal cells (B). Integrin subunits {alpha}6 (C) and ß4 (D) are clearly restricted to basal membranes of basal cells. The CD44 adhesion molecule is also expressed by all basal cells (E), whereas the hematopoietic cell antigen CD166 is located on apical ciliated and secretory cells (F). This epithelial structure expresses the CFTR protein at the apical side of ciliated cells (arrows) (G) and the aquaporin-3 water channel solely on basal cells (H). Scale bars = 50 µm. Abbreviations: CFTR, cystic fibrosis transmembrane conductance regulator; CK, cytokeratin; SCID, severe combined immunodeficiency.

 
The restricted expression of CD166 and AQP3 on apical and basal epithelial cells, respectively, prompted us to assay these markers on cells dissociated from human airway grafts.

Dissociation and Characterization by Flow Cytometry of Human Airway Epithelial Cells
Similar to native human airway tissues, the surface epithelium of human airway grafts in SCID mice lies on a basal lamina and a thick mesenchyme that surrounds secretory glands, cartilaginous rings, and blood vessels [28]. Referring to previous studies, we assayed different enzyme solutions (i.e., pronase, collagenase-dispase, amylase, and trypsin) with the aim of combining optimal cell viability, surface antigen integrity, and dissociation efficiency. Intraluminal administration of 1% pronase at 4°C overnight resulted in complete separation of the surface epithelium from underlying connective tissue. Further dispersion of the epithelium by gentle repeated passages through a syringe yielded an epithelial single-cell suspension that reached a viability of nearly 99% (98.4% ± 0.56%, n = 37 grafts). This cell preparation was expectedly heterogeneous and composed of small, presumably basal cells and larger cells with secretory granules or actively beating cilia (not shown). Cell viability at 4°C, 48 hours after the enzyme treatment, was still 80%. The tendency of epithelial cells to reaggregate in the suspension was limited by using PBS-CaMg and frequent gentle agitation. An average of 1.45 ± 0.85 x 106 epithelial cells were collected from each dissociated graft, two to four of which were pooled for each experiment. By contrast, when other enzyme solutions were used, clumps of cells always remained and could not be dispersed. Hence, pronase digestion was chosen to perform all subsequent epithelium dissociations.

Thirty minutes after complete dissociation, all epithelial cells had lost their native shape. On flow cytometry analysis, forward scatter versus side scatter dot plot representation did suggest that discrete cell subsets could be circumscribed and sorted on physical parameters (Fig. 2AGo). However, when subpopulations of epithelial cells were sorted on scatter properties and reanalyzed for CK expression, no enrichment of any fraction in a given cell type was observed (not shown).



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  		Figure 2. Flow cytometry analysis of human airway epithelial cell suspensions. (A): Side scatter versus forward scatter analysis of PI-negative events. (B): Staining with antiCD166 antibody reveals the presence of 60% brightly fluorescent cells, as compared with the threshold determined with a control unrelated antibody (dotted line). (C): Upon staining with an antiAQP3 antibody, 20% of the cells are brightly positive as compared with a negative control antibody (dotted line). Abbreviations: AQP3, aquaporin-3: PI, propidium iodide.

 
CKs 7 and 13 could be detected in epithelial cells by flow cytometry after fixation and permeabilization (data not shown). The whole suspension of human airway cells contained 70% of cells positive for CKs, either CK 7 or CK 13. As for membrane markers, integrin subunits and CD44 antigens appeared to be particularly sensitive to enzyme treatment, and no antibody staining was detected after cell dissociation and flow cytometry analysis.

The CD166 and AQP3 surface molecules, by contrast, were readily detected on dissociated epithelial cells. Eight sorts were performed on 20 grafts. An average of 58.6% ± 4.87% of the cells were brightly fluorescent upon CD166 staining (Fig.2CGo), whereas only 19.5% ± 5.16% were strongly positive for AQP3 antigen expression. AQP3+ cells exhibited a bimodal fluorescence distribution, indicating the presence of bright and dim positive cells. We selected bright cells only for fluorescent-activated cell sorting (FACS) (see below) (Fig. 2DGo).

Ability of Sorted Cells to Restore the Human Airway Epithelium in SCID Mice
Next, we used flow cytometry to sort suprabasal and basal cells from dissociated epithelium. We performed four sorting experiments based on AQP3 expression (Fig. 3Go, Table 1Go). Sorting gates were set on forward scatter versus side scatter dot plots and on AQP3 bright and AQP3– cells, as indicated in Figures 3A and 3BGo. Typical postsort cell recovery yields were 66% ± 4.2%. Among collected cells, positive and negative fractions accounted for 20.3% ± 3.8% and 59.6% ± 8.7% of the total cells, respectively. The percentage of positive cells appears to be in agreement with our observations in situ. Indeed the examination on tissue sections of epithelia lining mature grafts revealed that 34.0% ± 0.07% of the cells express the aquaporin channel. Reanalysis consistently revealed a purity higher than 95% for AQP3+ sorted cells (95.3% ± 2.5%, n = 3) (Figs. 3C, 3DGo). We aimed at further characterizing the sorted positive and negative cells by analyzing the expression of CK 13, which is specific to basal cells. Most AQP3+ sorted cells (85.51% ± 4.75%) contained CK 13, whereas very few CK 13 positive cells (5.5% ± 2.47%) were detected in the AQP3– fraction. We considered that the 95% purity observed for sorted cells allowed us to proceed with experiments in vivo.



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  		Figure 3. Flow cytometry sorting of human airway epithelial cell subsets. (A): PI-negative events to be sorted were first circumscribed within a double-scatter gate. (B): After indirect staining with anti-AQP3 antibody and fluorescein isothiocyanate, two populations of AQP3– and AQP3 brightly positive cells were selected. (C, D): Reanalyses of sorted AQP3– and AQP3+ cells, respectively . Abbreviations: AQP3, aquaporin-3; PI, propidium iodide.

 

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  		Table 1. SCID-hu airway graft repopulation with sorted AQP3+ and AQP3– human fetal airway epithelial cells
 
To determine the capacity of basal and suprabasal airway epithelial cells to restore the respiratory epithelium within the airway, 1 x 105 cells from each sorted fraction (AQP3– and AQP3+) were inoculated into denuded human fetal tracheas, which were in turn transplanted subcutaneously into SCID mice [27]. Four to 20 weeks after implantation, grafts were harvested and analyzed by histology. Regardless of the duration of development, no control host graft inoculated with culture medium alone supported epithelial regeneration; the airway lumen became occluded by fibroblastic cells and showed no evidence of re-epithelialization. All donor cell-seeded grafts, by contrast, were successfully repopulated with epithelial cells. The whole inner surface of the graft was eventually covered by a typical pseudostratified human airway epithelium, containing normally distributed basal, ciliated, and secretory cells. After short-term development, grafts inoculated with either AQP3+ or AQP3– cell fractions exhibited marked histologic differences. At 4–6 weeks after engraftment, inoculation of AQP3– cells had already resulted in the reconstitution of a mature epithelial lining including basal, ciliated, and secretory cells (Fig. 4AGo, Table 1Go). Grafts seeded with AQP3+ cells only, by contrast, exhibited a double layer of undifferentiated epithelial cells after 4 and 6 weeks (Fig. 4BGo, Table 1Go). Twenty weeks after seeding, AQP3+ cell–derived epithelium was well differentiated and pseudostratified (Fig. 4DGo, Table 1Go). The mature epithelium generated by AQP3– cells after 4 weeks was maintained after 20 weeks (Fig. 4CGo, Table 1Go). At that stage, no differences were observed between AQP3+ and AQP3– cell–derived structures, and both contained numerous submucosal glands within the mesenchyme.



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  		Figure 4. Histological sections from grafts reconstituted with AQP3+ or AQP3– cells and developed in the SCID mouse. (A): Four weeks after engraftment, a graft seeded with sorted apical, AQP3– cells is lined with a containing basal, ciliated and secretory cells. (B): In contrast, basal, AQP3+ cells transplanted in the same conditions have given rise to a bilayered structure constituted of undifferentiated epithelial cuboidal cells. After 20 weeks in the SCID mouse, both AQP3+ (C) and AQP3– (D) cells have generated a mature, well-differentiated, mucociliary pseudostratified epithelium. Scale bars = 50 µm. Abbreviations: AQP3, aquaporin-3; SCID, severe combined immunodeficiency.

 
After 20 weeks of development, the surface epithelium developed from either sorted AQP3+ or AQP3– cells expressed the expected markers of the human airway mucosa. The AQP3 water channel was restricted to basal cells (Fig. 5BGo), and the CFTR protein was located at the apical domain of ciliated cells, as expected (Fig. 5CGo). The tight junction protein ZO-1 was present in the subapical membranes of apical cells (Fig. 5DGo). Interestingly, each sorted cell subpopulation generated numerous submucosal glands within the mesenchyme. These newly developed glandular structures appeared functional. Serous cells were detected using the lysozyme marker (Fig. 6BGo), whereas mucous cells expressed mucins such as MUC5AC (Fig. 6DGo), MUC4 (Fig. 6FGo), and the IgA secretory component (Fig. 6HGo).



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  		Figure 5. Epithelial surface cell markers in a human airway mucosa reconstituted from AQP3+ cells and developed for 20 weeks in the SCID mouse. The newly developed pseudostratified epithelium, seen in (A) in Nomarski interference microscope, expresses the AQP3 water channel solely on basal cells (B) and the CFTR protein on the apical aspect of ciliated cells (arrows) (C). (D): ZO-1, the junctional protein linked to CFTR by the actin cytoskeleton, is present in apical membranes of apical cells (arrows). Scale bars = 25 µm. Abbreviations: AQP3, aquaporin-3; CFTR, cystic fibrosis transmembrane conductance regulator; SCID, severe combined immunodeficiency.

 


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  		Figure 6. Epithelial glandular cell markers in the human airway mucosa reconstituted from AQP3+ cells and developed for 20 weeks in the SCID mouse. The glands newly formed by AQP3+ cells are composed by both serous cells which express the lysozyme marker (B), and mucous cells positive for MUC5AC (D), IgA secretory component (sc) (F), and MUC4 (H). (A, C, E, G): Nomarski interference microscopic views of B, D, F, and H, respectively. Scale bars = 50 µm. Abbreviations: AQP3, aquaporin-3; SCID, severe combined immunodeficiency.

 
In parallel, four independent airway repopulation experiments were performed, using the same protocol and conditions described above with sorted CD166+ and CD166– airway epithelium cells. Three experiments were protracted for 4 weeks and one for 8 weeks. No epithelium repletion was ever observed with either donor cell subset (not shown).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
Epithelial tissues that make up the interface between organs and the outside environment are naturally subject to physical, chemical, or biologic aggressions but can compensate the ensuing erosion by a significant potential for regeneration. The intestinal crypt has long been a paradigmatic niche in which a small subset of epithelial stem cells orderly sustain the permanent renewal of the digestive mucosa [3]. A hierarchy of progenitors has been deciphered in the skin [33–35] at the top of which would stand multipotent stem cells located in hair follicles [36]. Significant progress has also been achieved in proving the existence of stem and progenitor cells in the animal airway epithelium, although the identification of such cells has been principally retrospective and their distribution and exact potential are still debated [25]. It is generally thought that of the three major cell types present in the airway epithelium, both secretory and basal cells retain the capacity to divide and differentiate. Ciliated cells, by contrast, are considered to be irreversibly differentiated and unable to divide [6, 7].

In this study, we aimed toward the identification and characterization of progenitor cells in the human airway mucosa. First, we used immunohistochemistry and flow cytometry to identify cell-surface markers, allowing us to delineate distinct epithelial cell subpopulations within the human fetal airway. We then investigated the developmental potential of the sorted cell subsets in SCID-hu mice [27].

The first conclusion we could draw from efficient pronase dissociation of the human fetal airway mucosa into single, viable cells was that no cell subset therein can be isolated by flow cytometry on physical criteria only. This is an important difference with the results of experiments performed in rats [13] and rabbits [16], in which basal and secretory cells could be distinguished and sorted on side- and forward-scatter characteristics. As for cell-surface molecular markers, {alpha}6ß4 integrin or CD44 was never detected on human airway epithelial cells dispersed by pronase digestion, thus preventing the discrimination of positive or negative subsets. We also confirmed that G. simplicifolia lectin, a marker for basal cells in the rat airway epithelium [29, 37], has no affinity for human airway epithelial cells [38, 39].

Two cell-surface molecules that resisted pronase digestion were detected on human airway epithelial cells. CD166, also known as HCA, is a homophilic adhesion molecule belonging to the immunoglobulin family and conserved from Drosophila to the human species. In humans, CD166 is present at the surface of embryonic neuroblasts during proliferation, neurite elongation and synaptogenesis [30], and also on the most primitive hematopoietic stem cells [31]. CD166 is, in addition, expressed at the surface of stromal cells in blood-forming tissues and is involved in stem cell adhesion to the stroma [32]. On sections of the airway mucosa, we detected CD166 at the surface of suprabasal, differentiated epithelial cells. This population could also be, for the first time, pictured by flow cytometry, accounting for approximately 60% of total dissociated cells, using the same marker. Thus CD166 could be used as a lineage marker for the positive selection of suprabasal cells in the human airway epithelium. However, no epithelial regeneration was observed in grafts seeded with positive or negative sorted cells, suggesting the blocking effect of the antibody and a low level of CD166 expression on basal progenitor cells.

Conversely, the AQP3 cell-surface water channel, the expression of which was first described within the rat mucosa [40, 41], was detected only on basal cells and possibly on a minor subset of intermediate cells of the human fetal airway surface epithelium, as confirmed by more recent observations [42, 43]. On FACS analysis, the whole AQP3+ cell population accounted for approximately 20% of total dissociated cells, which is in agreement with previous quantitative estimations for basal cells in the human airway epithelium [44, 45].

In vivo, both AQP3+ basal cells and AQP3– suprabasal cells sorted from a donor human fetal trachea and injected intraluminally into a denuded host SCID-hu airway, had regenerated in 5 months, in all grafts analyzed, a normal, pseudostratified, and ciliated airway epithelium as well as submucosal secretory glands. Yet, epithelium regrowth from the AQP3– cell subset proceeded differently from that mediated by AQP3+ basal cells. As early as 4 weeks post-transplant, airway grafts seeded with AQP3– cells were already lined with a pseudostratified epithelium, whereas reconstitution from AQP3+ cells proceeded through the development of a bilayered, poorly differentiated epithelium. This sequence recapitulates the normal ontogeny of the airway mucosa. Besides the fact that differences may exist between mammalian species, our results are in agreement with those of previous studies in the rat and rabbit, which showed that, in the short term, sorted basal cells generated a poorly differentiated surface epithelium whereas sorted secretory cells yielded a well-differentiated epithelium [13, 17, 18]. The authors concluded that progenitor cell activity was detected within airway epithelium secretory cells, and that basal cells had only limited progenitor capacity. However, our analysis performed on both short and long terms shows that, similarly to suprabasal cells, basal epithelial cells have after 5 months also regenerated a well-differentiated, pseudostratified, and ciliated airway epithelium as well as secretory glands. Our observations are now in agreement with the conclusions of several recent reports in which cre/lox-mediated cell tracking in transgenic mice has shown the residence of stem cells within the basal layer of the tracheal epithelium [27–29]. It will be important to try and correlate AQP3 expression with that of the keratin isoforms used to typify candidate airway stem cells in these studies.

It is noteworthy that airway epithelium regenerated from purified AQP3+ or AQP3– cells contained a normally distributed population of AQP3+ basal cells, CFTR+ suprabasal cells, and secretory submucosal glands. Lysozyme is a marker of serous glandular cells [46], and MUC5AC and MUC4 are expressed in mucous secretory cells of the surface and glandular epithelium [47, 48]. The secretory component of the secreted immunoglobulin A is also a good marker of a polarized, well-differentiated glandular epithelium. Our data demonstrate that each population was renewed in our transplantation setting and suggest the existence of progenitor cells for surface and glandular airway epithelium among both AQP3+ and AQP3– epithelial cells. Still, AQP3– cells would renew the airway epithelium much faster than their positive counterparts.

It will be important to determine whether the AQP3– epithelial progenitors are secretory cells, inasmuch as these cells have been shown in the rat to repopulate the surface epithelium in 4 weeks [13, 17].


   CONCLUSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
Direct stem cell identification with cell-surface markers was pioneered two decades ago in the hematopoietic system. Since then, the blood cell hierarchy has been precisely deciphered using this approach, and the possibility of sorting human blood cell progenitors has been widely exploited therapeutically. Lack of usable markers and appropriate assay systems, as well as technical difficulties in the processing of more compact tissues, has delayed the identification of stem cells in other, notably epithelial, tissues. The present work is therefore a proof of concept that human respiratory epithelium progenitors can be prospectively purified, although much remains to be done to delineate an organized genealogy of stem and precursor cells within this tissue. This could eventually be of therapeutic significance since, besides permanent renewal in the steady state, the airway mucosa can repair itself when injured in diseases such as asthma and cystic fibrosis. Finally, we also want to stress that the same general prospective approach is now also being used to identify cancer stem cells [49]. We foresee that the strategy and tactics we followed in the present study will also apply to the investigation of the development of bronchial and lung cancers.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
We are grateful to Drs. M. Catala, A.-L. Delezoide, F. Menez, and J. Martinovic for providing human fetal tissues. We also acknowledge the expert assistance of Z. Mishal with flow cytometry, Bin Sun and Mihaela Crisan with immunohistochemistry, and Kristen Dering with figure editing. We thank Dr. C. Coraux for helpful comments on the manuscript. This work was supported by grants from the European Community, Association Vaincre la Mucoviscidose (Paris), Ministère de la Recherche (Paris), and INSERM/Association Française contre les Myopathies (Paris).


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 Results
 Discussion
 Conclusion
 References
 

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Received August 13, 2004; accepted for publication March 25, 2005.



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