Aquaporin-3 Expression in Human Fetal Airway Epithelial Progenitor Cells - 《stemcells》2004-2005文献干细胞之家



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a INSERM U506, H?pital Paul Brousse, Villejuif, France;
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b INSERM UMR514, IFR 53, Universit谷 de Reims, Reims, France;
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c Rangos Research Center, Children’s Hospital, Pittsburgh, Pennsylvania, USA
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Key Words. Aquaporin ? Airway ? SCID mouse ? Epithelium8 V) h) i* j1 J+ S& Y
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Correspondence: Bruno P谷ault, 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@chp.edu( Z: g& I. B! p, [8 q* m+ f1 J% |
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ABSTRACT

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 . Similar to other permanently renewing epithelial tissues such as the intestine and epidermis , renewal in the steady state and repair ability suggest the existence of stem cells in the airways . 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 ., h& t$ t! @2 `
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Several approaches have been developed to identify progenitor cells in the normal or injured airway epithelium such as 3H-thymidine radiolabeling , in vitro or in vivo development of sorted subsets of cells , and cell-lineage tracking with recombinant retroviruses . Studies of cell turnover in the normal respiratory epithelium designate basal cells as potential stem cells . However, the strong proliferation of secretory cells after chemical or physical injury  suggests that these may also represent candidate progenitor cells. Cells constituting the airway mucosa have been separated on differences in density , or by flow cytometry on the basis of cell size  or surface-antigen expression . 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 . The analysis of gland development in that model suggested that only a subset of basal cells could form both surface epithelium and glands . The existence of multipotent epithelial stem cells in a niche located within submucosal gland ducts has been retrospectively suggested . 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 . 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 . 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.+ B+ h8 s3 {/ C

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 . 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.$ z9 u+ m3 b9 Q$ W

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.% L8 e" t+ p0 w4 R  O6 r& r

MATERIALS AND METHODS: _$ w8 _5 N: t  H2 f3 ^' L/ K

Antigenic Discrimination of Epithelial Cell Subsets in Human Airway Xenografts% V3 U9 R9 O- {$ [8 y2 @8 }
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As described previously, human airway grafts in SCID mice were eventually terminally differentiated and lined entirely with a ciliated and secretory pseudostratified epithelium . 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.
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We first examined the binding to graft sections of lectins described as markers in the rat airway epithelium . 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. 1A), whereas CK 13 was restricted to the basal cell layer (Fig. 1B). 6 and ?4 integrin subunits showed a very restricted distribution on the basal membrane of basal cells (Figs. 1C, 1D). Among adhesion molecules, the hyaluronate receptor CD44 was located on all basal cell membranes (Fig. 1E), and CD166, expressed otherwise by embryonic neuroblasts and by hematopoietic stem cells and stromal cells , was found on the apical and basolateral membranes of ciliated and secretory cells (Fig. 1F). Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein was, as expected, restricted to the apical membrane of ciliated cells (Fig. 1G). 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. 1H). Of note, AQP3 was not detected at all in epithelial cells lining gland ducts and within submucosal glandular cells.

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 6 (C) and ?4 (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." I4 L+ \! P/ m. u( i$ v9 C9 s

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.( ?3 ^- e' ]3 n$ d7 o
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Dissociation and Characterization by Flow Cytometry of Human Airway Epithelial Cells
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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 . 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.
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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. 2A). 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.! I5 o* y8 \/ w- C  Z) ^% j6 v
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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./ V  T4 @# H: k* R1 [
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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.2C), 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. 2D).

Ability of Sorted Cells to Restore the Human Airway Epithelium in SCID Mice& p" n! n/ N* `% g+ A

Next, we used flow cytometry to sort suprabasal and basal cells from dissociated epithelium. We performed four sorting experiments based on AQP3 expression (Fig. 3, Table 1). 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 3B. 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, 3D). 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.

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
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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 . 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. 4A, Table 1). Grafts seeded with AQP3  cells only, by contrast, exhibited a double layer of undifferentiated epithelial cells after 4 and 6 weeks (Fig. 4B, Table 1). Twenty weeks after seeding, AQP3  cell–derived epithelium was well differentiated and pseudostratified (Fig. 4D, Table 1). The mature epithelium generated by AQP3– cells after 4 weeks was maintained after 20 weeks (Fig. 4C, Table 1). At that stage, no differences were observed between AQP3  and AQP3– cell–derived structures, and both contained numerous submucosal glands within the mesenchyme.' s; a/ z; h2 _$ Z! G- N1 g
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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.1 I5 ?7 [9 {6 C; R3 }# Z

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. 5B), and the CFTR protein was located at the apical domain of ciliated cells, as expected (Fig. 5C). The tight junction protein ZO-1 was present in the subapical membranes of apical cells (Fig. 5D). 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. 6B), whereas mucous cells expressed mucins such as MUC5AC (Fig. 6D), MUC4 (Fig. 6F), and the IgA secretory component (Fig. 6H).) N1 [$ a# o- |* }! B" N6 Z

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.
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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).) a' u0 o( Z5 B% `0 V! O* l

DISCUSSION( i8 A5 k! g% u
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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 . 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., S4 R4 K7 p, a9 ~7 Q6 ?% p
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