Basal Cells of the Human Adult Airway Surface Epithelium Retain Transit-Amplifyi - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 01:00 |只看该作者 |倒序浏览 |打印

作者：Rodolphe Hajja, Thomas Baranekb, Richard Le Naourb, Pierre Lesimplea, Edith Puchellea, Christelle Corauxa作者单位：aInstitut National de Sant et de Recherche Mdicale Unit , Centre Hospitalier Universitaire Maison Blanche, Reims, France;bEquipe Associe , Institut Fdratif de Recherche , Reims, France
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      【摘要】
   In numerous airway diseases, such as cystic fibrosis, the epithelium is severely damaged and must regenerate to restore its defense functions. Although the human airway epithelial stem cells have not been identified yet, we have suggested recently that epithelial stem/progenitor cells exist among both human fetal basal and suprabasal cell subsets in the tracheal epithelium. In this study, we analyzed the capacity of human adult basal cells isolated from human adult airway tissues to restore a well-differentiated and functional airway epithelium. To this end, we used the human-specific basal cell markers tetraspanin CD151 and tissue factor (TF) to separate positive basal cells from negative columnar cells with a FACSAria cell sorter. Sorted epithelial cells were seeded into epithelium-denuded rat tracheae that were grafted subcutaneously in nude mice and on collagen-coated porous membranes, where they were grown at the air-liquid interface. Sorted basal and columnar populations were also analyzed for their telomerase activity, a specific transit-amplifying cell marker, by the telomeric repeat amplification protocol assay. After cell sorting, the pure and viable CD151/TF-positive basal cell population proliferated on plastic and adhered on epithelium-denuded rat tracheae, as well as on collagen-coated porous membranes, where it was able to restore a fully differentiated mucociliary and functional airway epithelium, whereas viable columnar negative cells did not. Telomerase activity was detected in the CD151/TF-positive basal cell population, but not in CD151/TF-negative columnar cells. These results demonstrate that human adult basal cells are at least airway surface transit-amplifying epithelial cells. 4 I9 A/ e5 J" W+ Z" M$ R
      【关键词】 Human airway epithelium Basal cells Transit-amplifying cells CD Tissue factor
   INTRODUCTION" q* @! i5 J7 W: t1 w- e$ a

In numerous airway diseases, such as asthma, bronchiolitis obliterans, chronic obstructive pulmonary disease, and cystic fibrosis, the pseudostratified airway surface epithelium composed of basal, secretory, and ciliated cells is severely damaged and must regenerate to restore its defense functions. This regeneration process involves stem cells and/or transit-amplifying cells (TACs). Despite the slow cell turnover observed in lung epithelia, much evidence supports the presence of stem cells/TACs in the airways that are activated after injury. When mature or immature human fetal tracheal cells .: r: ?; C1 ]3 R# N- `
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Of the three main airway surface epithelial cell types, basal . Despite all these findings, the nature of human airway epithelial stem cells/TACs is still unclear.$ a4 a! ?% `9 z" |; C" F
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It is currently thought that stem cells and TACs express specific surface and intracellular markers that distinguish them from other somatic cell types. A TAC derives from a stem cell and is committed to differentiate into one or more lineages by active and limited divisions. Initially, it may not be committed and may retain self-renewal properties . Thus, a detectable telomerase activity in a cell population would predict the presence of TACs./ `! H; q, ?: L5 N

The aim of our study was to identify the population of the human adult airways that retains TAC properties capable of restoring a fully differentiated mucociliary and functional epithelium. In this perspective, we focused our work on human airway basal cells previously described as the stem cell/TAC population in animals. We used the tetraspanin CD151 and tissue factor (TF) to separate basal cells from secretory and ciliated cells by FACS. CD151 is a major component of prehemidesmosomal structures and plays a role in the formation and the stability of hemidesmosomes . Using an in vivo humanized nude mouse xenograft model and an in vitro air-liquid interface culture model, we studied the capacity of sorted basal and columnar cells to reconstitute a differentiated functional epithelium and analyzed their telomerase activity.) q* ?1 C% R7 g

MATERIALS AND METHODS

Antibodies- ^6 {0 Y4 U8 z7 D) i! t( K
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All antibodies used were from mouse species. Purified TF and TF fluorescein isothiocyanate (FITC)-conjugated anti-human monoclonal antibodies (IgG1; 1:75 and 3 µl/106 cells, respectively) were purchased from American Diagnostica (Neuville sur Oise, France, http://www.americandiagnostica.com). Purified CD151 and phycoerythrin (PE)-conjugated anti-human monoclonal antibodies (IgG1, clone 14A2.H1; 1:50 and 6 µl/106 cells, respectively), as well as PE-conjugated nonimmune IgG1 isotype control (clone MOPC-21; 6 µl/106 cells), were from BD Biosciences (San Diego, http://www.bdbiosciences.com). Anti-cytokeratin (CK)-13 (IgG1, clone KS-1A3; 1:1,000), anti-CK18 (IgG1, clone CY-90; 1:1,000) monoclonal antibodies, nonimmune IgG (dilution adjusted to the primary antibody concentration), 4',6'-diamino-2-phenylindole (DAPI) (200 ng/ml), and Griffonia simplicifolia isolectin B4-FITC (GSI-B4; 4 µg/ml) came from Sigma-Aldrich (St. Louis, http://www.sigmaaldrich.com). Rabbit anti-aquaporin 3 polyclonal antibody (AQP3; 1:100) was from Euromedex (Souffelweyersheim, France, http://www.euromedex.com). Anti-MUC5AC monoclonal antibody (IgG1, clone CLH2; 1:50) was from Novocastra Ltd. (Newcastle upon Tyne, U.K., http://www.novocastra.co.uk). Anti-human ß-tubulin monoclonal antibody (IgG2b, clone KMX-1; 1:15,000) was from Chemicon (Temecula, CA, http://www.chemicon.com). Anti-zonula occludens (ZO)-1 monoclonal antibody (IgG1, clone ZO1¨C1A12; 1:20) was from Zymed Laboratories (San Francisco, http://www.clinisciences.com). Anti-human cystic fibrosis transmembrane conductance regulator (CFTR) monoclonal antibody (IgG2a, clone 24¨C1; 1:20) was from R&D Systems Inc. (Minneapolis, http://www.rndsystems.com). Alexa Fluor 488 and Alexa Fluor 594 goat anti-mouse (IgG H L; 1:200) secondary antibodies were from Molecular Probes Inc. (Eugene, OR, http://probes.invitrogen.com). FITC-conjugated goat anti-mouse secondary polyclonal antibody (F(ab')2; 1:500) and nonimmune IgG1 isotype control (clone DAK-GO1; 1:500) were from DakoCytomation (Carpinteria, http://www.dakocytomation.com). The blocking goat anti-mouse IgG Fab fragments (H L; 1:50), as well as FITC-conjugated nonimmune IgG1 isotype control (3 µl/106 cells), came from Jackson Immunoresearch Laboratories (West Grove, PA, http://www.jacksonimmuno.com).
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Human Airway Tissues

The use of human tissues was authorized by the bioethical law 94-654 of the Public Health Code of France, with a written consent from the patients. Human airway tissues were collected after nasal polypectomy of nine patients who did not suffer from any other disease. Fresh human tissues were immediately transferred to the laboratory in RPMI 1640 medium supplemented with 25 mM HEPES, 200 U/ml penicillin, and 200 µg/ml streptomycin (Gibco-BRL, Paisley, U.K., http://www.gibcobrl.com) and washed with cold RPMI 1640. A portion of each tissue was embedded in optimum cutting temperature (O.C.T.) compound (Tissue-Tek, Zoeterwoude, The Netherlands, http://www.emsdiasum.com), frozen in liquid nitrogen and stored at ¨C80¡ãC until used for histology and immunohistochemistry.
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Epithelial Cell Isolation and FACS
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Human airway surface epithelial cells were isolated from nasal tissues by incubation with 0.1% type XIV collagenase (Sigma-Aldrich) in RPMI 1640 medium supplemented with 200 U/ml penicillin and 200 µg/ml streptomycin overnight at 4¡ãC. Cell-denuded tissues were assessed by histology and showed a denuded basal lamina with intact glandular and duct cells. Isolated epithelial cells were washed, suspended in RPMI 1640 culture medium, and counted. Cells were sieved with a 70-µm nylon BD Falcon cell strainer (BD Biosciences) and washed with sterile phosphate-buffered saline (PBS) (Gibco-BRL) without Ca2  and Mg2  supplemented with 2 mM EDTA (Sigma-Aldrich). Cells were centrifuged and suspended in PBS-EDTA containing 5% of bovine serum albumin (BSA) (Sigma-Aldrich) for 30 minutes.
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For CK13 analyses, 2 x 106 cells were treated with saponin 0.1% (Sigma-Aldrich) in PBS for 10 minutes at 4¡ãC, incubated with anti-CK13 antibody for 25 minutes at 4¡ãC, and then incubated with FITC-conjugated anti-mouse secondary antibody for 25 minutes at 4¡ãC. Nonimmune mouse IgG1 was used as isotype control. Three x 104 events were recorded, and CK13 analyses were performed on FACSCalibur (BD Biosciences) to distinguish marked basal cells from the unmarked columnar cell population (secretory and ciliated cells).7 X- Y$ P- F$ f
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For cell sorting, 22.5 ¡À 5.8 x 106 cells (range 12¨C34 x 106) were treated in each experiment. Cells were incubated with both anti-CD151-PE and anti-TF-FITC antibodies diluted in PBS for 20 minutes at 4¡ãC. Nonimmune IgG1-PE and IgG1-FITC diluted in PBS were used as isotype controls. Cells were then washed twice and suspended in PBS-EDTA. DAPI was added to stain dead cells. Cell sorting was performed with a FACSAria cell sorter (BD Biosciences) equipped with blue, red, and violet lasers. Dead cells were excluded from basal and columnar cells by DAPI versus forward scatter (FSC) dot plots. Doublets were excluded from the basal cell population by side scatter (SSC)-W versus SSC-H and FSC-W versus FSC-H dot plots. Finally, bright basal cells (CD151 /TF ) and negative columnar cells (CD151¨C/TF¨C) were selected and sorted using the cell sorter's purity option at a rate of 5,000 events per second. Sorted populations were reanalyzed (3 x 104 events recorded) for purity and viability, and 1 x 105 cells of each population were cytospun on gelatin-coated slides for CK13 and MUC5AC staining cell count performed as described below. Nonsorted control cells that followed the same procedure described above, without any antibody addition, were stained with DAPI to assess for dead cells. Viable nonsorted cells were then seeded on plastic, on porous membranes, and into xenografts as described below.

Cell Culture

Nonsorted control cells, as well as sorted CD151 /TF  and CD151¨C/TF¨C populations, were seeded on plastic in six-well BD Falcons (BD Biosciences) for cell proliferation at a density of 1.5 x 105 cells per square centimeter in the proliferation medium Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 medium (Gibco-BRL; 3/1 vol/vol) supplemented with 0.87 µM bovine insulin, 65 nM human transferrin, 1.6 nM recombinant human epidermal growth factor (EGF), 1.38 µM hydrocortisone, 30 nM retinyl acetate, 9.7 µM 3,3',5-triiodo-L-thyronin, 2.7 µM (¨C)epinephrine, 35 µg/ml bovine pituitary extract, 5 µM ethanolamine, 5 µM O-phosphorylethanolamine, 30 nM sodium selenite, 1 nM manganese chloride tetrahydrate, 0.5 µM sodium metasilicate nonahydrate, 1 nM ammonium molybdate tetrahydrate, 5 nM ammonium vanadate, 1 nM nickel sulfate hexahydrate, 0.5 nM stannous chloride dihydrate (Sigma-Aldrich), 100 U/ml penicillin, and 100 µg/ml streptomycin. To improve cell adhesion, 10% fetal calf serum (Gibco-BRL) was added for the first 24 hours.

For cell differentiation, sorted populations were seeded on collagen IV-coated (Sigma-Aldrich) 12-mm Costar Transwell porous polyester membranes (0.4-µm pore size) (Corning Costar, New York, http://www.corning.com/lifesciences) at a density of 1.5 x 105 cells per cm2 and cultured up to confluence in proliferation medium. Then, the medium was definitively removed from the upper compartment and replaced in the basal one by bronchial epithelial basal medium (Cambrex, Walkersville, MD, http://www.cambrex.com)/DMEM high glucose (Gibco-BRL) medium (1/1 vol/vol) (supplemented with 0.5 ng/ml EGF, 5 µg/ml insulin, 0.5 µg/ml hydrocortisone, 10 µg/ml transferrin, 0.5 µg/ml epinephrine, 6.5 µg/ml triiodothyronin, 0.13 mg/ml bovine pituitary extract , 100 U/ml penicillin, and 100 µg/ml streptomycin), and cells were grown at the air-liquid interface. All cultures were incubated at 37¡ãC in a 100% humidified incubator in the presence of 5% CO2. Membranes were processed for histological and immunohistochemical analyses when homogenous cilia beating was observed after 25¨C35 days. Six human airway tissues were used for cell proliferation studies, and three of these were used for air-liquid interface cultures. All experiments were performed in triplicate.
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Airway Epithelial Secretion Collection from Air-Liquid Interface Cultures

When homogenous cilia beating was observed in air-liquid interface cultures, surface epithelia were washed with PBS, and then 100 µl of PBS was added to the apical surface of regenerated epithelia to collect airway epithelial secretions. After incubation for 7 hours at 37¡ãC, supernatants were collected and stored at ¨C80¡ãC until use.
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Airway Xenografts. o$ T) @; u6 V* R3 w
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All experiments and procedures were performed in compliance with the French Ministry of Agriculture regulations for animal experimentation. Humanized xenografts were prepared as described previously . Briefly, tracheae of adult Wistar rats (Charles River Laboratories, Saint-Aubin-L¨¨s-Elbeuf, France, http://www.criver.com) were frozen at ¨C80¡ãC and thawed to remove the rat surface epithelium. Rat tracheae were then tied aseptically at their ends to sterile polyethylene tubing and stored at ¨C80¡ãC until use. Nonsorted control cells, as well as sorted CD151 /TF  and CD151¨C/TF¨C human airway populations (5 x 105 cells per 80 µl in proliferation medium), were seeded into epithelium-denuded rat tracheae that were grafted subcutaneously into the flanks of 7 week-old female nude mice anesthetized with an intraperitoneal injection of pentobarbital sodium (40 mg/kg) (Centravet, Gondreville, France, http://www.centravet.fr). Nude mice were housed under pathogen-free conditions. Tracheal xenografts were flushed twice a week with serum-free DMEM/Ham's F-12 medium supplemented with 200 U/ml penicillin, 200 µg/ml streptomycin, 50 µg/ml gentamicin, 2.5 µg/ml amphotericin, and 420 U/ml colimycin to wash and remove dead cells and accumulated mucus from the lumen. Animals were sacrificed with an overdose of pentobarbital sodium after 35 days of engraftment, and tracheal xenografts were removed for histological and immunohistochemical analyses. Three human airway tissue samples were used for xenograft experiments that were performed in triplicate.$ ]5 ~4 p" ~: Q1 t* n6 K3 w1 G
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Histology
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Human airway tissues, air-liquid interface membranes, and xenografts were embedded in O.C.T. and frozen in liquid nitrogen. Five-micrometer frozen sections were cut with a microtome, collected on gelatin-coated slides, and either stained with hematoxylin-eosin using Rapid-Chrome Frozen Sections Staining Kit (Thermo Shandon Inc., Pittsburgh, http://www.thermo.com) and mounted in Eukitt (Electron Microscopy Sciences, Hartfield, PA, http://www.emsdiasum.com) for histology or frozen at ¨C20¡ãC for immunohistochemistry. Images of plastic cultures were acquired on a Nikon microscope (Champigny-Sur-Marne, France, http://www.nikon.com), and images of airway tissue, air-liquid interface membrane, and xenograft sections were acquired on an Axiophot microscope (Carl Zeiss, Oberkochen, Germany, http://www.zeiss.com).; h9 f0 O- D7 j4 U+ A3 W
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Immunohistochemistry

Slides were air-dried, and sections were fixed with cold acetone at ¨C20¡ãC for 10 minutes, washed with PBS, and blocked with PBS-BSA 3% for 30 minutes at room temperature (RT) to prevent unspecific bindings. Sections were then incubated with the primary antibody diluted in PBS-BSA 1% for 1 hour at RT, washed with PBS, incubated with the Alexa Fluor 488 secondary antibody diluted in PBS-BSA 1% for 1 hour at RT, washed with PBS, incubated with DAPI diluted in PBS for 10 minutes, washed with PBS, counterstained with Harris hematoxylin (Shandon) for 5 seconds, washed with PBS, and finally mounted in Aquapolymount solution (Polysciences Inc., Warrington, PA, http://www.polysciences.com). Green, red, or violet fluorescence was observed, and images acquired were on an AxioImager Z1 microscope (Carl Zeiss). Negative controls were performed by replacing primary antibodies with mouse nonimmune IgG fractions and with PBS-BSA 1% alone. For dual staining, sections were incubated with primary antibody and Alexa Fluor 488 secondary antibody as described above. After being washed with PBS, sections were exposed to goat anti-mouse Fab (H L) fragments for 1 hour to block free sites of the mouse primary antibody. Then, sections were washed with PBS and proceeded for another staining following the same procedure described above using the Alexa Fluor 594 secondary antibody. Three samples were used for each staining.
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Antibacterial Activity of Airway Epithelial Secretions4 g  O/ o2 b8 V2 v8 J0 Y

Bactericidal activity of airway epithelial secretions collected from air-liquid interface cultures was performed as described previously . Briefly, wild-type laboratory Staphylococcus aureus strain 8325-4 (generous gift from Dr. T.J. Foster, Trinity College, Dublin, Ireland) was cultured in trypticase soy broth (bioM¨¦rieux, Marcy l'Etoile, France, http://www.biomerieux.fr) for 2 hours at 37¡ãC under agitation (120 rpm) to obtain log-phase bacterial cultures. Then, bacteria were washed twice in sterile PBS and water, respectively, and diluted in sterile water to a final concentration of 250 colony-forming units (CFUs)/µl. To assess antibacterial activity, 2 µl of bacterial suspension (500 CFUs) was mixed with 30 µl of collected airway epithelial secretions, incubated for 2 hours 30 minutes at 37¡ãC under low agitation (70 rpm), and seeded on trypticase soy agar plates (bioM¨¦rieux). As control, 500 CFUs of S. aureus were incubated with 30 µl of sterile PBS and seeded on agar. After overnight incubation at 37¡ãC, surviving bacteria were counted and expressed as percentage of PBS-grown control bacteria referenced as 100%. Logarithmic scale was used to represent the percentage axis. Epithelial secretions of the three air-liquid interface cultures realized above, from nonsorted cells and CD151 /TF  sorted cells, were used and experiments were performed in duplicate.- w" ]! Q, K" g! D7 _

Telomerase Activity
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Telomerase activity was assessed by the telomeric repeat amplification protocol assay (TRAP). Sorted CD151 /TF  and CD151¨C/TF¨C cells were centrifuged, and pellets were stored at ¨C80¡ãC until use. Proteins were extracted in ice-cold CHAPS 1x buffer (1% CHAPS, 1 mM MgCl2, 1 mM EGTA, 10 mM Tris-HCl, pH 7.5, 0.1 mM benzamidine, 10% glycerol, 5 mM ß-mercaptoethanol) (Sigma-Aldrich) and quantified with the Quick Start Bio-Rad assay kit (Bio-Rad, Hercules, CA, http://www.bio-rad.com) following the manufacturers' instructions. Absorbance was read on a Xenius microplate spectrophotometer (SAFAS, Monte Carlo, Monaco, http://www.safas.com). Polymerase chain reaction (PCR) was performed in a final 25-µl reaction volume composed of 5 µl of distilled water containing 800 ng of CD151 /TF  or CD151¨C/TF¨C protein extract and 20 µl of the reaction mixture containing 20 mM Tris, pH 8.0, 1.5 mM MgCl2, 63 mM KCl, 1 mM EGTA, 0.005% Tween 20, 20 µg/ml BSA (Sigma-Aldrich), 50 µM dNTP (Takara, Shiga, Japan, http://www.takara.co.jp), 9 pmol of TS primer (5'-AATCCGTCGAGCAGAGTT-3'), 6.2 pmol of reverse primer of elongated TS (5'-GTGCCCTTACCCTTACCCTTACCCTAA-3'), 4.6 pmol of nontelomeric reverse primer of TSNT (5'-ATCGCTTCTCGGCCTTTT-3'), 0.5 yoctomole of TSNT internal control (5'-AATCCGTCGAGCAGAGTTAAAAGGCCGAGAA-GCGAT-3') (Eurogentec, Seraing, Belgium, http://www.eurogentec.be), and 1 unit of Taq DNA polymerase (Takara). Five µl of distilled water was added to the reaction mixture without or with Taq polymerase for negative controls. Two-hundred nanograms of A549 cell extract diluted in 5 µl of distilled water constituted the positive control. Tubes were incubated in the thermocycler for 20 minutes at 30¡ãC and then 1 minute at 90¡ãC followed by 30 cycles of 30 seconds/92¡ãC, 30 seconds/52¡ãC, and 30 seconds/72¡ãC. After amplification, PCR products were separated on a 12% nondenaturing acrylamide gel at 200 V for 55 minutes and then stained with SYBR Gold (Molecular Probes). Bands were acquired under UV on a LAS-1000 equipped by a Fuji camera (Stamford, http://www.raytest.fr).
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Statistical Analyses" e9 I& D5 y) A2 y) t2 Z  q

Values are expressed as the mean ¡À SD. The nonparametric Mann-Whitney test was used for statistical analyses that were performed on StatView software (SAS Institute, Cary, NC, http://www.statview.com). Significant differences were defined for p : _$ K0 D7 P) U0 O' r, {
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RESULTS7 h; E1 Y0 O1 A; E3 n/ B3 y

CD151 and TF Are Basal Cell-Specific Markers( t. ^) f5 k3 G# {# E: l( F2 i6 v$ S3 I

To separate human adult airway epithelial basal cells from airway columnar cells, we identified two membrane basal cell-specific markers. The tetraspanin molecule CD151 and TF were specific for all basal cells¡ªas confirmed by CK13 (a cytoplasm-specific basal cell marker) colabeling (Fig. 1A¨C1D, 1E¨C1H, respectively)¡ªon which they were colocalized as well (Fig. 1I¨C1L). CD151 and TF were also specifically expressed on human bronchial epithelial basal cells (data not shown). Moreover, CD151 and TF epitopes were still intact after enzymatic cell dissociation, as assessed by immunohistochemistry on cytospun cells (data not shown). In addition to CD151 and TF, we tested GSI-B4, which has been shown to be specific for basal cells in animals , recognized all surface epithelial cells in our human airway tissues (data not shown). Thus, these results encouraged us to use CD151 and TF as markers of all basal cells for epithelial cell sorting.
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Figure 1. CD151 and TF localization on human nasal epithelia. Sections were dual-stained with anti-CD151 and anti-CK13 (B¨CD), anti-TF and anti-CK13 (F¨CH), or anti-CD151 and anti-TF (J¨CL) antibodies. Staining of CD151 and TF was specific for basal cells (B, F, J, K, L) and colocalized with CK13 (D, H). DNA was visualized with DAPI (A, E, I). Scale bars = 25 µm. Abbreviations: DAPI, 4',6'-diamino-2-phenylindole; Lu, lumen; m, mesenchyme; TF, tissue factor.
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Identification of the Basal Cell Population in Isolated Cells by Flow Cytometry/ T6 H2 E& h1 _0 o
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Human airway epithelial cells were isolated from tissues, sieved, and stained with anti-CK13 antibody. When we proceeded to flow cytometry analyses, FSC versus SSC dot plot representation (Fig. 2A) suggested that cells gated on R1 should be basal cells, based on their physical shape. The CK13 analyses confirmed that suggestion, as shown in Figure 2C. Cells gated on R2 were negative to CK13 (Fig. 2E) and represented, therefore, the columnar cell population. Thus, the identification of the basal and columnar cell populations allowed us to proceed to cell sorting.* z. s5 B1 |1 b+ ?* a' j8 [
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Figure 2. Identification of the basal cell population by fluorescence-activated cell sorting. The whole population of epithelial cells (A) was exposed to the anti-CK13 antibody and then to the FITC secondary antibody. Staining was bright when cells were gated on R1 (C) (basal cell population) but negative when they were gated on R2 (E) (columnar cell population). (B, D): same isotype control adjusted either on R1 or on R2 gate. Abbreviations: FITC, fluorescein isothiocyanate, FSC-H, forward scatter height; PE, phycoerythrin; SSC-H, side scatter height.* G! n4 {4 ?2 N6 R
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Fluorescence-Activated Cell Sorting1 o" K% H4 s$ @& b! t2 r
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Cells were sieved and double-stained with anti-CD151-PE and anti-TF-FITC antibodies. In an FSC versus SSC dot plot, basal and columnar populations were circumscribed (Fig. 3A, a), and cells that were positive to DAPI were excluded. Cell sorting was performed as described in Materials and Methods by excluding doublets from basal cells and gating the bright positive CD151 /TF  basal cell population (Fig. 3A, d) and the negative CD151¨C/TF¨C columnar cell population (Fig. 3A, e). The numbers of collected cells were 5.4 ¡À 1.9 x 106 and 1.1 ¡À 0.6 x 106 cells for positive (CD151 /TF ) and negative (CD151¨C/TF¨C) populations, respectively. By reference to the original total isolated cell population, their percentages were 25% ¡À 9% and 4.8% ¡À 3.1%, respectively. The low percentage obtained for the CD151¨C/TF¨C population is likely due to the exclusion of dead cells and columnar cell aggregates (Fig. 3A, e). The reanalysis by DAPI of sorted populations for viability showed that 99.2% ¡À 0.4% of CD151 /TF  cells and 98.9% ¡À 0.6% of CD151¨C/TF¨C cells were viable. Purity was 99.5% ¡À 0.3% for CD151 /TF  sorted basal cells (Fig. 3B, a, b) and 97.9% ¡À 0.7% for CD151¨C/TF¨C sorted columnar cells (Fig. 3B, c, d). We further characterized sorted positive and negative populations, cytospun on gelatin-coated slides, by immunohistochemistry with MUC5AC and CK13 (data not shown). Positive CD151 /TF  population contained 0.04% ¡À 0.03% MUC5AC-positive contaminating secretory cells, and negative CD151¨C/TF¨C population contained 1.2% ¡À 0.2% CK13-positive contaminating basal cells, which was in agreement with purity results obtained by FACS analyses. Moreover, the negative CD151¨C/TF¨C population showed that 23.5% ¡À 6.1% of total cells were positive for MUC5AC, and that 60.5% ¡À 5.8% were ciliated cells, as judged by their morphology. Sorted CD151 /FT  basal cells were also all positive for CK13, except the 0.04% of contaminating cells positive for MUC5AC., J+ _1 Y+ ~; o7 z( x7 x% q5 m

Figure 3. Sorting of human airway epithelial cells. (A): The whole population (a) was double-stained with anti-CD151-PE and anti-TF-FITC antibodies. Viable CD151 /TF  basal cells (d) and CD151¨C/TF¨C columnar cells (e) were derived from P1 and P2, respectively (a), and sorted using the sorter's yield and purity options. FITC and PE isotypes were adjusted on both P1 (b) and P2 (c) gates. (B): Purity analyses of sorted CD151 /TF  cells (a, b) and CD151¨C/TF¨C cells (c, d). Abbreviations: FITC-A, fluorescein isothiocyanate-area; FSC-A, forward scatter area; P, population; PE-A, phycoerythrin area; SSC-A, side scatter area; TF, tissue factor.! i. T* i' L4 g' m) |# c% L* f% d

Capacity of Sorted Cells to Proliferate and Reconstitute a Fully Differentiated Epithelium+ A, [5 q7 T, O% p- k; j- ?$ R
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The ability of sorted basal and columnar cell populations to proliferate and restore a pseudostratified mucociliary airway epithelium was assessed in vitro and in vivo. The reproducibility of the results was excellent, which means that seeded cells from all donors were able to repopulate air-liquid interface cultures and xenografts. Moreover, no variations between donor-tissue samples were noted. All highly pure and viable CD151 /TF  basal cells seeded on plastic were able to proliferate (Fig. 4D), as did nonsorted control cells (Fig. 4A), whereas seeded viable CD151¨C/TF¨C columnar cells did not adhere (Fig. 4G). In all air-liquid interface cultures and xenografts, CD151 /TF  basal cells were able to reconstitute a pseudostratified mucociliary epithelium composed of basal, ciliated and secretory cells (Fig. 4E, 4F) as did nonsorted control cells (Fig. 4B, 4C), whereas CD151¨C/TF¨C columnar cells were not able to (Fig. 4H, 4I). Regenerated epithelia in air-liquid interface cultures and in xenografts were further analyzed for markers generally expressed on well-differentiated airway surface epithelia in vivo. Regenerated epithelia from CD151 /TF  basal cells, as well as control regenerated epithelia, stained positively, in the same way, for CK13 (basal cells) (Fig. 5A¨C5D), CK18 (columnar cells) (Fig. 5E¨C5H), MUC5AC (secretory cells) (Fig. 5I¨C5L), ß-tubulin (ciliated cells) (Fig. 5M¨C5P), ZO-1 (a tight junction-associated protein that characterizes the epithelial barrier integrity) (Fig. 5Q¨C5T), and CFTR (a chloride channel expressed on the apical membrane of ciliated cells) (Fig. 5U¨C5X). Of note, MUC5AC stains all differentiated secretory cells, as described previously , who quantified the numbers of basal, secretory, and ciliated cells in the human large airways.5 K; R0 f- M& L0 a. C
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Figure 4. Histology of 2D cultures and reconstituted epithelia from sorted CD151 /TF  positive basal cells. (A¨CC): Control 2D cultures, air-liquid interface cultures, and xenografts, respectively. (D¨CF): CD151 /TF  basal cells adhered and proliferated on plastic (D) and reconstituted a fully differentiated epithelium in air-liquid interface cultures (E) and in xenografts (F). (G¨CI): CD151¨C/TF¨C columnar cells neither adhered (G) nor reconstituted airway epithelia in air-liquid interface cultures (H) and in xenografts (I). Original magnification, x400 (A, D, G). Scale bars, 25 µm. Abbreviations: 2D, two-dimensional; ac, apical compartment; bc, basal compartment; Lu, lumen; m, mesenchyme; TF, tissue factor.  r/ `6 @/ V5 f
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Figure 5. Characterization of regenerated epithelia from CD151 /TF  basal cells in air-liquid interface cultures and xenografts. Epithelial differentiation markers CK13 (A¨CD), CK18 (E¨CH), MUC5AC (I¨CL), ß-tubulin (M¨CP), ZO-1 (Q¨CT), and CFTR (U¨CX) were expressed similarly on CD151 /TF  and control regenerated epithelia. (A'¨CD'): CD151 (red) and TF (green) were colocalized (yellow) on basal cells in CD151 /TF  and control regenerated epithelia. Scale bars = 25 µm. Abbreviations: ac, apical compartment; bc, basal compartment; CFTR, cystic fibrosis transmembrane conductance regulator; Lu, lumen; m, mesenchyme; TF, tissue factor; ZO, zonula occludens.. T' w  N/ ~* K% E- o3 D+ y% D' `
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Table 1. Percentage of the different cell populations (cell type/total cells) using the differentiation markers CK13 (basal cells), MUC5AC (secretory cells), and ß-tubulin (ciliated cells) after complete epithelial regeneration from CD151 /TF  cells and nonsorted control cells in air-liquid interface cultures and xenografts
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Functionality of Regenerated Epithelia4 K; y3 c; ]; M4 I

After underlining the differentiated state of generated epithelia from CD151 /TF  basal cells and nonsorted control cells, we sought to determine whether these epithelia exhibited any functional activity. Therefore, the capacity of generated epithelia in air-liquid interface cultures to secrete factors that kill bacteria was tested. After incubation with S. aureus, we observed that culture-airway surface epithelial secretions significantly killed over 99% of bacteria. In fact, when mixtures of bacteria and epithelial secretions of control (nonsorted cells) and CD151 /TF  (sorted cells) regenerated epithelia were seeded on agar, bacterial recovery was 0.32% and 0.26%, respectively, compared with bacterial growth in PBS alone (Fig. 6). No statistical difference was found between bactericidal activities of secretions collected from control and those from CD151 /TF  regenerated epithelia.
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Figure 6. Effect of airway epithelial cell secretions collected from air-liquid interface well-differentiated cultures on S. aureus growth. Sterile PBS was used as control of bacterial growth and referenced as 100%. Of the incubated bacteria, 0.32% ¡À 0.2% and 0.26% ¡À 0.2% survived after their exposal to secretions of regenerated epithelia from NSC and SC, respectively. No statistical difference was found between antibacterial activities of the two types of secretions. Abbreviations: NSC, nonsorted cells; PBS, phosphate-buffered saline; SC, sorted CD151 /TF  cells.

Assessing for TAC Presence) k9 I4 \  B, K# n3 k' R

Sorted CD151 /TF  basal cells and CD151¨C/TF¨C columnar cells were subjected to telomerase activity using the TRAP assay. The telomere ladder located above the primer dimers in the positive control lane corresponds to the telomerase activity, which was detectable in the CD151 /TF  basal cell population but not in CD151¨C/TF¨C columnar cells (Fig. 7).

Figure 7. Telomerase activity in sorted CD151 /TF  and CD151¨C/TF¨C cells. CD151 /TF  and CD151¨C/TF¨C cell extracts (800 ng) were added to the reaction tubes. Telomerase activity, which corresponds to the telomere ladder located above the primer dimers, was positive in CD151 /TF  basal cells but negative in CD151¨C/TF¨C columnar cells. Internal controls correspond to the internal control fragment amplification added to all tubes. No protein extracts were added to the negative controls without (¨C) or with ( ) Taq polymerase. A549 protein (200 ng) extract was added to positive control tubes. Abbreviations: TF, tissue factor; TS, telomerase substrate.
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DISCUSSION4 y' [' A3 h; P7 p& P
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In this study, we demonstrate that human adult airway surface epithelial basal cells expressing CD151 and TF were capable of reconstituting a fully differentiated mucociliary and functional airway epithelium in vivo in xenografts and in vitro in air-liquid interface cultures. Moreover, the telomerase activity indicates the presence of TACs in sorted basal cells.
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CD151 has been shown to be involved in oncogenesis, in the control of metastasis, and in the modulation of cell motility both in vitro and in vivo . Moreover, we noticed that the antibodies used against CD151 and TF did not block cell adhesion to plastic and collagen. Therefore, we used these two protease-resistant markers to separate human adult CD151 /TF  basal cells from CD151¨C/TF¨C columnar cells.  K5 p. ]( |! v1 z

Contradictory results have been published on whether basal or secretory cells are stem cells/TACs of the airway surface epithelium. In addition, all the studies were performed on animals, such as rabbits and rats, using separation techniques based on cell density (elutriation) , thus validating the use of FT and CD151 as pan-basal cell markers.

When sorted basal cells were seeded, they adhered on plastic and had the capacity to proliferate and to restore a fully differentiated epithelium in air-liquid interface cultures and xenografts, whereas columnar cells did not have this ability. Moreover, differentiated epithelia reconstituted from CD151 /TF  basal cells were functional, as assessed by the capacity of their secretions to kill bacteria. When the functionality of the airway epithelium is altered, which is the case in several diseases, such as cystic fibrosis (pathology due to mutations in the cftr gene encoding the CFTR chloride channel), there is inactivation of some secreted antibacterial factors, such as ß-defensins .

Pure CD151 /TF  basal cells were able to divide and generate other basal cells as well as secretory and ciliated cells, which strongly suggests their self-renewal potential, as it has been emphasized that the capacity for self-renewal decreases as committed TACs differentiate . It would be interesting, as a first attempt, to test whether the described markers could be used to isolate purified basal cells from the distal airways.: `/ @3 o3 a9 J6 V, T& q
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In this study, we used the xenograft model in which we have previously described the regeneration dynamics of the human airway epithelium and showed the generation of glandular structures as well . A more focused work, studying the dynamics of gland development in grafts seeded with pure basal cells, would be very interesting.
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In each experiment, the same number of sorted basal and columnar cells was seeded in vitro and in vivo, but the negative columnar population never adhered. Because the sorted CD151¨C/TF¨C population contained 24% of secretory cells, we further seeded four times the amount of the original number of columnar cells on collagen-coated porous membranes to equalize the number of basal and secretory cells. Despite the elevated number of seeded secretory cells, they did not adhere. This could be related to the fact that secretory cells may lose their capacity to adhere to extracellular matrix molecules when isolated. In addition, the attachment of columnar cells to the basal lamina occurs mainly via desmosomal attachment to basal cells in situ . In our study, ciliated cells adhered neither in vitro nor in vivo.
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To demonstrate the existence of a potential TAC population among human basal cells, we analyzed sorted CD151 /TF  and CD151¨C/TF¨C cells for their telomerase activity. Telomerase is highly active in human embryonic tissues and cells .  j* x9 O5 M& {1 k5 q
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CONCLUSION5 Y$ B! T% K/ h3 d$ K5 m0 S: M
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Our data strongly support the concept that human adult airway basal cells retain TAC properties, as they are capable of proliferating and reconstituting a fully differentiated mucociliary and functional epithelium. Additional studies, such as clonogenicity tests, will define whether subpopulations of stem cells are likely to exist within human airway basal cells. In addition, the markers defined in this study would be of interest to isolate basal cells of high purity allowing their phenotyping (e.g., transcriptome and proteomic studies) to define "stemness" markers that will help for the identification of airway epithelial stem cells. Another interesting perspective of this work would be the transduction of isolated basal cells with a reporter gene that allows identifying new subpopulations within basal cells that retain stem cell properties.
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DISCLOSURES

The authors indicate no potential conflicts of interest.

ACKNOWLEDGMENTS& q9 ?7 w4 @9 P+ h# Y: l

We thank all the ENT and surgeons who provided us human airway tissues: Prof. J.-M. Klossek (Hôpital Jean Bernard, Poitiers, France), Dr. C. Ruaux (Clinique Mutualiste de la Sagesse, Rennes, France), Dr. P. Corlieu (Hôpital Tenon, Paris, France), and Dr. I. Durieu (Centre Hospitalier Lyon Sud, Lyon, France). We are also grateful to Dr. J-F. Riou (IFR53, Reims, France) for providing us the TRAP procedure. This work was supported by the French Association Vaincre la Mucoviscidose, R¨¦gion Champagne-Ardenne, and Adult Stem Cells Thematic Concerted Action (Institut National de Sant¨¦ et de Recherche M¨¦dicale, Association Française Contre les Myopathies, and the French Ministry of Research).: Z. A2 M% n3 d. W
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