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Published ahead of print on December 2, 2004, doi:10.1165/rcmb.2004-0079RC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 32, pp. 87-92, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0079RC

Rapid Communication

Embryonic Stem Cells Generate Airway Epithelial Tissue

Christelle Coraux, Béatrice Nawrocki-Raby, Jocelyne Hinnrasky, Claire Kileztky, Dominique Gaillard, Christian Dani and Edith Puchelle

INSERM UMR S 514, Reims; and CNRS UMR 6543, Centre de Biochimie, Nice, France

Correspondence and requests for reprints should be addressed to Dr. E. Puchelle, INSERM UMR S 514, CHU Maison Blanche, 45 rue Cognacq-Jay, 51092 Reims Cedex, France. E-mail: edith.puchelle{at}univ-reims.fr


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Embryonic stem (ES) cells are self-renewable and pluripotent cells derived from the inner cell mass of a blastocyst-stage embryo. ES cell pluripotency is being investigated increasingly to obtain specific cell lineages for therapeutic treatments and tissue engineering. Type II alveolar epithelial cells have been derived from murine ES cells, but the capacity of the latter to generate differentiated airway epithelial tissue has never been reported. Herein, we show by RT-PCR and immunocytochemistry that murine ES cells are able to differentiate into nonciliated secretory Clara cells, and that type I collagen induces this commitment. Moreover, when cultured at the air–liquid interface, ES cells give rise to a fully differentiated airway epithelium. By quantitative histologic examination, immunohistochemistry, and scanning electron microscopy, we show that the bioengineered epithelium is composed of basal, ciliated, intermediate, and Clara cells, similar to those of native tracheobronchial airway epithelium. Transmission electron microscopy and Western blotting reveal that the generated epithelium also exhibits the ultrastructural features and secretory functions characteristic of airway epithelial tissue. These results open new perspectives for cell therapy of injured epithelium in airway diseases, such as bronchopulmonary dysplasia, cystic fibrosis, or bronchiolitis obliterans.

Key Words: stem cells • airway epithelium • Clara cells


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage embryos (1, 2) that can replicate indefinitely in culture when maintained in an undifferentiated state by addition of leukemia inhibitory factor (LIF) to the culture medium (3). These undifferentiated cells have the characteristic to spontaneously differentiate into a broad spectrum of derivatives of all three germ layers (1, 2, 4, 5) when cultured as tridimensional structures called embryoid bodies (EB) (6). Available data demonstrate that ES cells can be induced to differentiate into specific cell lineages (7–12) by action of matrix components and/or growth factors. This pluripotentiality is being investigated increasingly for tissue engineering and recent results provide convincing results of skin reconstitution (11). The derivation from ES cells of cell types from endoderm origin has been poorly documented: studies reported the differentiation of hepatocytes (13), peancreatic islets (14), and more recently, of type II pneumocytes (15), but the capacity of ES cells to generate differentiated airway epithelial tissue has never been reported.

The aim of the present study was to test the ability of murine ES cells to differentiate into airway epithelial cells and to give rise to a well differentiated airway epithelium, composed of basal cells, ciliated cells, intermediate cells, nonciliated Clara cells, and similar to the epithelium covering human bronchioles (16). Clara cells are the most characteristic murine airway epithelial cell-type, producing Clara cell 10-kD protein (CC10) (17), a homodimeric protein considered as a specific marker for these cells (18), as well as surfactant proteins (SP) such as SP-B (19) and SP-D (20). In this study, we showed by investigating the expression of CC10 and SP-D that murine ES cells are able to differentiate into Clara cells, and we tested matrix substrates and growth factors to determine culture conditions supporting this derivation. We also demonstrated that murine ES cells can generate a functional tracheobronchial surface epithelium similar to the native mouse airway epithelium.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
ES Cell Cultures
The undifferentiated mouse ES cell line CGR8 (21) was routinely cultured in dishes coated with 0.1% gelatin (Sigma Aldrich, Saint-Quentin Fallavier, France) in 0.1 M, pH 7.2 PBS (Gibco BRL, Paisley, UK). The culture medium was composed of Glasgow minimum essential medium (GMEM; Gibco BRL) supplemented with 10% FCS (Gibco BRL), 1% nonessential amino acids (Gibco BRL), 1 mM sodium pyruvate (Gibco BRL), 0.1 mM 2-mercaptoethanol (Sigma Aldrich), penicillin/streptomycin (100 U/ml and 100 µg/ml, respectively; Gibco BRL) and 103 units/ml of LIF (ESGRO; Chemicon, Temecula, CA), which keeps ES cells in an undifferentiated state.

To induce their spontaneous differentiation, ES cells were grown as EB (22). After LIF removal and trypsin detachment (0.025% trypsin [Gibco BRL]/1 mM EDTA [Sigma Aldrich] in PBS–1% chicken serum [Gibco BRL]), 103 undifferentiated cells in 20-µl drops of medium containing 20% FCS were deposited on nonadherent bacterial grade Petri dish covers, then inverted to form hanging drops (Day 0 of the culture). After 2 d, EB were collected, washed, and cultivated for another 3 d in suspension in new nonadherent bacterial grade Petri dishes in medium containing 10% FCS. EB were then seeded on plastic culture dishes for RNA extraction, or on glass coverslips for immunocytochemistry, for 3, 10, 16, or 23 d (i.e., Days 8, 15, 21, or 28).

To induce ES cell differentiation without EB formation, after LIF removal and trypsin detachment, undifferentiated ES cells (2 x 103/cm2) were seeded, on either uncoated plastic dishes or on type I collagen (250 µg/ml [Sigma Aldrich] in 0.1% acetic acid)–coated dishes, gelatin (0.1% in PBS)-coated dishes, type IV collagen (250 µg/ml [Sigma Aldrich] in PBS)–coated dishes, or type VI collagen (200 µg/ml [Sigma Aldrich] in 0.05% acetic acid)–coated dishes for 8 or 15 d in medium containing 10% FCS. To assess their effects on ES cell cultures, keratinocyte growth factor (KGF, 5 µM; R&D Systems, Minneapolis, MN) or retinoic acid (RA, 33 nM; Sigma Aldrich) was added to the culture medium on Day 0 or Day 8 of the culture.

Airway Epithelium Generation Procedure
After 8 d of culture on uncoated plastic dishes or on type I collagen–coated dishes, ES cells were detached by incubation with 0.5% dispase solution (Boehringer Mannheim, Mannheim, Germany) in PBS. ES cells (2 x 105/cm2) were then seeded on Millicell-HA porous membranes (Millipore, Bedford, MA) on the same uncoated or coated surface as before and cultured for 8 more d, followed by 15 d at the air–liquid interface, or at liquid–liquid interface. Cultures were then prepared for histology, immunohistochemistry, reverse transcriptase–polymerase chain reaction (RT-PCR), transmission electron microscopy (TEM), or scanning electron microscopy (SEM).

RT-PCR Analysis
Total RNA was extracted with the High Pure RNA Isolation kit as recommended by the manufacturer (Roche Diagnostics, GmbH, Mannheim, Germany). RT-PCR was performed with 10 ng of total RNA using the GeneAmp Thermostable RNA PCR kit (Perkin Elmer, Foster City, CA) and two pairs of oligonucleotides (Eurogentec, Seraing, Belgium). The following forward and reverse primers, respectively, were designed for mouse CC10: 5'-CGCCATCACAATCACTGTGGTCA-3', 5'-GAGGGTATCCACCAGTCTCTTCA-3' and mouse 28S: 5'-GTTCACCCACTAATAGGGAACGTGA-3', 5'-GGATTCTGACTTAGAGGCGTTCAGT-3'. RT-PCR products were separated by acrylamide gel electrophoresis, stained with SYBR Gold (Molecular Probes, Eugene, OR) and visualized by fluorimetric scanning (Fuji, LAS-1000, Raytest, France). The expected sizes of the RT-PCR products of CC10 and 28S are 198 and 212 bp, respectively.

Immunocytochemistry and Immunohistochemistry
EB grown on glass coverslips were fixed in acetone precooled to –20°C for 10 min, air dried, and rehydrated in PBS. Air–liquid interface cultures were embedded in Optimal Cutting Temperature compound (OCT; Tissue-Tek, Sakura Finetek, Zoeterwoude, The Netherlands). Sections (5 µm thick) were fixed in methanol precooled to –20°C for 10 min and rinsed in PBS.

For immunofluorescence stainings, nonspecific binding was blocked with PBS containing 3% BSA (Sigma Aldrich) for 30 min at room temperature (RT). Coverslips were incubated with goat polyclonal antibody to CC10 (Santa Cruz Biotechnology, Santa Cruz, CA), and culture sections with mouse monoclonal antibody to tubulin ß (Sigma Aldrich) or biotinylated Griffonia Simplicifolia Isolectin B4 (GSI-B4; Sigma Aldrich) for 60 min at RT. Coverslips and culture sections were then washed in PBS, incubated with relevant biotinylated antibodies (Amersham, Aylesbury, UK) in 3% BSA-PBS, or 3% BSA-PBS alone in the case of GS-I-B4, for 60 min at RT, washed in PBS, incubated with Alexa Fluor 488–conjugated streptavidin (Molecular Probes) in PBS for 30 min at RT and washed in PBS.

For immunoperoxidase stainings, sections of air–liquid cultures were fixed in methanol, rinsed in PBS, and then treated with 0.3% hydrogen peroxide for 5 min at RT to quench endogenous peroxidase activity. Nonspecific binding was blocked with 3% BSA-PBS for 30 min at RT. Slides were incubated with goat polyclonal antibody to CC10 (Santa Cruz Biotechnology) or goat polyclonal antibody to SP-D (Santa Cruz Biotechnology) in 3% BSA-PBS for 60 min at RT. After three 5-min washes in PBS, subsequent steps were performed with the peroxidase LSAB+kit (labeled-streptavidin biotin method; Dako Cytomation, Glostrup, Denmark). Peroxidase activity was revealed with AEC (3-amino-9-ethylcarbazole) chromogen, which generated a red-brown product.

All coverslips and slides were counterstained with Harris' hematoxylin for 10 s, then mounted with Gel Mount anti-fading solution (Biomeda, Foster City, CA). Slides were examined under an Axiophot fluorescence microscope (Zeiss, Le Pecq, France). Negative controls omitted the primary antibody.

Western Blotting
To detect the presence of the Clara cell–secreted protein CC10, we collected the products secreted at the apical side of the ES cell cultures at the air–liquid interface. We used mouse trachea and lung proteins as controls. Mouse tissues were homogenized in RIPA buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1% Igepal (vol/vol), 1% sodium deoxycholate (wt/vol), 5 mM iodoacetamide, 0.1% SDS (wt/vol)] containing a complete protease-inhibitor cocktail (Roche Diagnostics), left on ice for 15 min, and centrifuged at 5,000 x g for 10 min at 4°C. Proteins (50 µg for ES cell secretory products and 5 µg for mouse tissues, quantitated by BC Assay Protein Quantitation kit; Interchim, Montluçon, France) were mixed with Laemmli buffer (BioRad, Hercules, CA) containing 5% ß-mercaptoethanol (vol/vol), boiled for 5 min, separated on a 12% SDS-PAGE gel and transferred onto a 0.2-µm pore size nitrocellulose membrane (NEN, Buckinghamshire, UK). The membrane was then blocked with 5% (wt/vol) nonfat dry milk in PBS containing 0.1% Tween 20 (wt/vol) for 120 min at RT, before incubation with anti-CC10 antibody (Santa Cruz Biotechnology) overnight at 4°C. The blot was then incubated with a horseradish peroxidase–conjugated swine anti-goat antibody (Dako Cytomation) for 60 min at RT. Signals were detected with an Enhanced Chemiluminescence (ECL+) kit (Amersham Pharmacia Biotech, Buckinghamshire, UK). Western blot bands were visualized by chemiluminescent scanning (LAS-1000; Fuji).

TEM
To study the ultrastructure of the bioengineered airway epithelium using TEM, air–liquid interface cultures were rinsed in PBS, fixed in 2.5% glutaraldehyde-PBS (Sigma Aldrich) for 60 min at 4°C, postfixed in 1% osmium tetraoxide-H2O (Merck, Darmstadt, Germany) for 120 min at 4°C, dehydrated through graded concentrations of ethanol at RT, and then embedded in agar resin 100 (Agar Scientific, Orsay, France). Semithin sections were stained with 1% toluidine blue in 13.1 mM Na2B4O7 buffer, and observed under a Zeiss Axiophot microscope. Ultrathin sections were stained with uranyl acetate and lead citrate, and observed using a Hitachi 300 transmission electron microscope (Elexience, Verrières Les Buisson, France) operating at 75 kV.

Permeability to Lanthanum Nitrate and TEM
The functionality of the bioengineered airway epithelium tight junctions was examined by visualizing the diffusion of a low-molecular-weight tracer, lanthanum nitrate, into the intercellular spaces, according to the method described by Revel and Karnovsky (23). Air–liquid interface cultures were rinsed in PBS and fixed for 60 min at 4°C in 2.5% glutaraldehyde-PBS. After several washes with PBS and S-collidin (Sigma)-HCl buffer, pH 7.8, the cultures were apically postfixed for 120 min at RT with a 1:1 (vol:vol) mixture of 0.2 M lanthanum nitrate (Sigma) in S-collidin-HCl buffer and 1% osmium tetraoxide-H2O, dehydrated through graded concentrations of ethanol at RT, and then embedded in agar resin 100. Ultrathin sections were stained with uranyl acetate and lead citrate, and observed using a Hitachi 300 transmission electron microscope operating at 75 kV.

SEM
To examine the ES cell cultures using SEM, air–liquid interface cultures were prepared as for TEM through dehydration, then critical-point-dried with CO2, affixed to stubs, coated with 12-nm-diameter gold-palladium, and observed using a Philips XL30 scanning electron microscope (FEI Company, Limeil-Brévannes, France) operating at 10 kV.

Quantification by SEM of the Surface Areas of the Cultures Covered with Well Differentiated Ciliated Epithelium
The surface areas of the cultures covered with ciliated epithelium were quantified using a computer-assisted scanning electron microscope. Twenty-eight fields, in three different cultures, were analyzed at a constant magnification of 665. Software developed in our lab determined, for each field, the total surface area (µm2) and that covered by ciliated epithelium: then the percentage of the culture covered by ciliated epithelium was calculated. Data are presented as means ± SD.

Quantification by Light Microscopy of Cell Types Constituting the Differentiated Airway Epithelium Derived from ES Cells
The percentages of each cell type (ciliated cell, basal cell, intermediate cell, and Clara cell) in native mouse tracheas and the airway epithelium generated from ES cells were quantified by light microscopy (magnification x 63) of toluidine blue–stained semithin sections: 13 fields in two tracheas and 18 fields in three different cultures, containing 298 and 283 cells, respectively. Then, the percentages of each cell type in the generated airway epithelium and mouse trachea were calculated and are presented as means ± SD.

Statistical Analysis
The mean percentages of each cell type in the mouse trachea and in the airway epithelium generated from ES cells were compared using an unpaired Student's t test. A P value < 0.05 was considered to be significant.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
ES Cells Spontaneously Differentiate into Clara Cells
We assessed the potential of ES cells grown as EB to differentiate into airway epithelial cells, by analyzing the expression of the mRNA encoding the Clara cell–specific marker CC10 at the successive times during culture. As shown in Figure 1A, after 35 cycles of PCR amplification, CC10 mRNA was detected in ES cells cultivated as EB as of Day 15 of culture with sustained expression thereafter, but not in undifferentiated ES cells. EB cultures were also processed for immunocytochemistry to evaluate the expression of the CC10 protein. Immunolabeling of CC10 was detected in the cytoplasm of EB outgrowth cells of 21-d-old cultures (Figure 1B). We never observed ciliated cells in EB cultures by inverted light microscopy (data not shown).



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  		Figure 1. ES cell differentiation into Clara cells through EB. (A) Electrophoresis of RT-PCR products of CC10 mRNA extracted from control mouse trachea (lane 1), undifferentiated ES cells (lane 2), and ES cells grown as EB for 8 d (lane 3), 15 d (lane 4), 21 d (lane 5), and 28 d (lane 6). The expected 198-bp transcript is detected as of Day 15 in ES EB cultures. The 28S housekeeping gene gives rise to the expected 212-bp transcript. (B) Immunofluorescence labeling of the Clara cell marker CC10 in the cytoplasm of some cells in EB cultures on Day 21 (a). (b) Corresponding phase-contrast micrograph. Bars = 30 µm.

 
Type I Collagen Induces Clara Cell Differentiation of ES Cells
We assessed the ability of ES cells to differentiate into airway epithelial cells without EB formation, by culturing them on different substrates and with or without growth factor supplementation. The RT-PCR analysis demonstrated that ES cells grown on type I collagen expressed CC10 mRNA as early as Day 8 of culture, whereas ES cells grown either on plastic (Figure 2A) or on gelatin, type IV collagen, or type VI collagen did not (data not shown). However, after 15 d, CC10 mRNA expression was observed in ES cell cultures on type I collagen, on plastic (Figure 2B), and on gelatin, type IV collagen or type VI collagen (data not shown). Influence of growth factors on Clara cell differentiation from ES cells was also investigated: KGF addition to the culture medium did not induce CC10 mRNA expression, regardless of the substrate (data not shown). Similarly, RA did not induce more CC10 mRNA expression than type I collagen alone (data not shown). We never observed ciliated cells by inverted light microscopy in ES cell cultures, whatever the substrate used with or without KGF or RA, and regardless of the culture duration (data not shown).



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  		Figure 2. ES cell differentiation into Clara cells without EB formation. Electrophoresis of RT-PCR products of CC10 mRNA extracted from control mouse trachea (lanes 1), ES cells grown on plastic (lanes 2) and ES cells grown on type I collagen (lanes 3) only shows the expected 198-bp transcript in ES cultures grown on type I collagen after 8 d (A), whereas this transcript is present in cultures grown on plastic or type I collagen after 15 d (B). The housekeeping gene 28S generates the expected 212-bp transcript.

 
ES Cells Can Generate Functional Airway Epithelial Tissue Similar to Native Airway Epithelium
To test the potential of Clara cells derived from ES cells to generate a pseudostratified surface airway epithelium composed of multiple cell types, we used the air–liquid interface culture model. Briefly, after inducing ES cells to differentiate into Clara cells by culture on type I collagen for 8 d, all cells were harvested and seeded on acellular type I collagen–coated porous membranes for an additional week to allow the formation of confluent cultures, followed by 2 wk of culture at the air–liquid interface. Under these culture conditions, we could observe the generation of an airway epithelium composed of Clara cells, easily identifiable as bulging nonciliated cells, and basal, intermediate, and ciliated cells (Figure 3Ab) similar to those present in native mouse airway epithelium (Figure 3Aa). This ES-derived airway epithelium was detected at the surface of the cultures with an underlying tissue resembling to a mesenchyme in which we could observe blood vessels, and cartilaginous structures (data not shown). To better define the phenotype of the cells constituting the ES-derived bioengineered airway epithelium, we used cell-type specific markers. As expected, basal cells were bound by the GS-I-B4 (Figure 3Bb) and ciliated cells were positively immunostained with an antibody directed against the tubulin ß (Figure 3Bd). Clara cells expressed CC10 (Figure 3Be) and SP-D proteins (Figure 3Bf). By TEM, Clara cells in the ES-derived bioengineered airway epithelium revealed all the ultrastructural cytoplasmic components characteristic of the Clara cells found in native murine airway epithelium: an abundant rough endoplasmic reticulum (Figure 3Ca), numerous mitochondria (Figure 3Cb) and electron-dense secretory granules (Figure 4B). ES cells grown on plastic, then seeded on uncoated acellular porous membranes and cultured at the air–liquid interface, gave rise to a few ciliated cells, whereas ES cells grown on type I collagen, then seeded on type I collagen–coated membranes and cultured at the liquid–liquid interface never gave rise to ciliated cells (data not shown).



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  		Figure 3. Bioengineered airway epithelium from ES cells. (A) Histologic aspect of in vivo mouse tracheal epithelium (a) and in vitro ES-derived airway epithelium (b). Toluidine blue staining of semithin sections shows the presence of Clara cells (black arrowheads), ciliated cells (black arrows), basal cells (white arrow) and intermediate cells (white arrowhead) in both in vivo and in vitro epithelia. Bars = 30 µm. (B) Immunodetection of specific cell type markers in the ES-derived airway epithelium. The basal cells are bound by the GS-I-B4 (b) and the ciliated cells are positively immunostained with antibody directed against the tubulin ß (d). (a and c) Corresponding phase-contrast micrographs. The Clara cells express both CC10 (e) and SP-D (f) proteins. Bars = 30 µm. (C) Study by TEM of Clara cells in the ES-derived bioengineered airway epithelium. The cytoplasm of Clara cells exhibits (a) an abundant rough endoplasmic reticulum (white arrowheads), and (b) numerous mitochondria (white arrows). Bars = 40 nm.

 


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  		Figure 4. SEM of generated airway epithelium (Aa) showing ciliated cells (white arrowheads) and cells exhibiting a nonciliated bulging apical membrane, characteristic of Clara cells (white arrows). Ultrastructural features of in vitro ES-derived bioengineered airway epithelium observed by TEM (Ab, Ac, Ad, Ae, and Af). This epithelium exhibits active ciliogenesis demonstrated by centriole migration (black arrows) toward the apical plasma membrane (b) leading to the formation of mature cilia composed of nine tubule-pairs at the periphery and a tubule-doublet in the center of the cilia (c). The bioengineered epithelium exhibits tight junctions (black arrow) at the apical part of lateral plasma membranes (d), desmosomes (black arrow) at the lateral plasma membranes (e) and hemidesmosomes (black arrows) connecting the basal plasma membranes to the underlying basement membrane (BM) (f). Bars = 10 µm (a); 150 nm (b), 30 nm (c), 75 nm (d), 295 nm (e), and 50 nm (f). (B) Functionality of the tight junctions analyzed using the lanthanum nitrate diffusion technique and visualized by TEM. Ultrathin sections show that lanthanum nitrate remains located at the apical surface of the ES-derived bioengineered airway epithelium (black arrows) and does not penetrate through the tight junctions (black arrowheads) between electron-dense secretory granule (SG)-containing Clara cells. Bar = 50 nm. (C) Electrophoresis of RT-PCR products of CC10 mRNA extracted from control mouse trachea (lane 1), undifferentiated ES cells (lane 2) and ES-derived airway epithelium (lane 3) shows the expected 198-bp transcript in bioengineered airway epithelium. 28S housekeeping gene generates the expected 212-bp transcript. (D) Western blot analysis of the CC10 protein in the secretions covering the air–liquid interface cultures reveal a 5-kD protein corresponding to the CC10 protein (lane 3) also observed in native mouse lung (lane 1) and tracheal (lane 2) tissues.

 
Using computer-assisted SEM, we examined the total surface of ES cultures at the air–liquid interface and quantified the area covered by well differentiated ciliated cells, which represent the most frequent cell phenotype in airway surface epithelium and the most easily detectable cell type. We calculated that 47.7 ± 10.1% of the surface of the ES cultures were covered by ciliated patches often containing bulging Clara cells (Figure 4Aa) and intermediate cells, with the remaining areas being covered by Clara cells, intermediate cells and undifferentiated epithelial cells. Comparison of the cell-type composition of the ES-derived bioengineered airway epithelium and mouse airway epithelium on culture and tissue sections, determined by light microscopy, showed: 56.6 ± 11.9% versus 52.6 ± 4.4% ciliated cells, 12.2 ± 8.4% versus 16.4 ± 3.4% Clara cells, 14.4 ± 6.6% versus 17.1 ± 3.7% basal cells, and 16.8 ± 7.1% versus 13.9 ± 3.4% intermediate cells, respectively. No significant difference could be observed in the percentage of each cell type between the native and ES-derived murine airway epithelium.

We investigated ES-derived bioengineered airway epithelium function, in terms of ciliogenesis, junction formation, and secretion. Ultrastructural analysis by TEM revealed active ciliogenesis demonstrated by centriole migration toward the apical plasma membrane of cells (Figure 4Ab). Mature cilia, composed of nine tubule-pairs at the periphery and a tubule-doublet in the center of the cilia, were observed (Figure 4Ac). Cellular junctions were seen in the generated airway epithelium: tight junctions at the apical part of the lateral plasma membranes of the cells (Figure 4Ad), desmosomes between neighboring cells (Figure 4Ae) and hemidesmosomes connecting the basal plasma membrane of cells to the underlying basement membrane (Figure 4Af). To confirm the functionality of the tight junctions observed, the diffusion of a low-molecular-weight tracer, lanthanum nitrate was analyzed by TEM. In the ES-derived bioengineered airway epithelium, lanthanum nitrate remained located apically and did not penetrate through the intercellular spaces (Figure 4B). We assessed CC10 expression by the generated airway epithelium: RT-PCR amplification of the mRNA encoding the CC10 protein showed expression of this Clara cell marker in the bioengineered airway epithelium (Figure 4C). Western blotting of ES-derived airway epithelial secretions in the fluid coating the air–liquid interface cultures detected the presence of the CC10 protein. Incubation with a specific anti-CC10 antibody revealed the presence of a 5-kD protein (Figure 4D).


   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
In addition to demonstrating the derivation of additional endoderm-derived cell types—the airway epithelial cells—from ES cells, our study provides, for the first time, evidence of the ability to generate a fully mature airway epithelial tissue. Our culture model allows derivation, from the pluripotent ES cells, of the four principal cell types composing the airway epithelium, which reorganized themselves to give rise to a well differentiated epithelium. However, we first show that murine ES cells are able to differentiate into Clara cells by using EB cultures, currently described to induce spontaneous differentiation of ES cells into multiple cell lineages (6), but this technique does not permit the obtaining of ciliated cell derivation. Inasmuch as it has been clearly shown that growth factors (7, 11) or matrix components (9, 11) could induce the ES cell commitment to differentiate into particular cell lineages, various culture conditions were tested using different culture substrates—among them type I collagen, a substrate known to promote airway epithelial cells differentiation (24), alone or with KGF, described to efficiently induce the Clara cell proliferation (25), or RA, routinely used to convert ES cells into numerous cell lineages (8, 12) and reported to stimulate the airway epithelial cell differentiation (26). Our results demonstrate that only ES cells grown on type I collagen differentiate into Clara cells as early as Day 8 of culture, whereas after 15 d, Clara cell differentiation is obtained on all the culture substrates tested. These observations suggest an inductive role of the type I collagen in the Clara cell commitment of ES cells. The appearance of Clara cells after 15 d of culture on all the culture substrates tested may be explained by the spontaneous differentiation from ES cells of cell lineages able to intrinsically produce type I collagen, which would induce the differentiation into airway epithelial cells. The same phenomenon could explain the Clara cell derivation by Day 15 of cultured EB, which are tridimensional structures known to allow intercellular interactions between ES-derived ectoderm, mesoderm, and endoderm cells. As for EB cultures, even when Clara cell differentiation is obtained, no ciliated cells are observed in ES cell cultures whatever the substrate with or without KGF or RA. The culture conditions used are then sufficient to induce Clara cell differentiation but not ciliated cell derivation, which is only obtained after air–liquid interface culture of type I collagen–induced ES cells. Thus, the air–liquid interface culture model, known to allow differentiation of airway epithelial cells (26), seems to directly induce ciliated cell differentiation from ES cells, or to lead to derivation of ciliated cells from airway epithelial progenitor cells, described to be Clara cells or basal cells (27–30).

Remarkably, the four principal cell types composing the surface airway epithelium are present and undergo organogenesis, but also, quantification of the cell components of this latter and of native mouse airway epithelium shows that they contain similar percentages of each cell type. In a more remarkable way, the ES-derived bioengineered airway epithelium exhibits some functions of a native epithelium. It shows active ciliogenesis as described by Lemullois and coworkers (31), that gives rise to mature cilia. One of the specialized functions of the airway epithelium is to form a tight barrier to prevent entry of inhaled material into the interstitial spaces. This function is ensured by cellular junctions, observed in our ES-generated airway epithelium, in particular by tight junctions whose impermeability to low-molecular-weight lanthanum diffusion through the intercellular spaces is demonstrated. Finally, we assessed the secretory function of the bioengineered airway epithelium by detecting the CC10 protein in the fluid coating the air–liquid interface cultures, and we show the presence of a 5-kD protein on immunoblots, consistent with the early reported CC10 molecular weight (32) and attesting to the ability of the ES-derived Clara cells to secrete the CC10 protein found in human bronchoalveolar lavages (33).

In summary, the results of this study demonstrate that ES cells have the capacity to generate a fully differentiated and functional tracheobronchial airway epithelium. In light of their potential to proliferate indefinitely, ES cells could theoretically provide an unlimited supply of cells and tissues for transplantation. Their great promise provides hope that airway epithelial tissue derivatives of ES cells could be grafted to reconstitute the airway epithelium in a variety of airway diseases, including bronchopulmonary dysplasia, cystic fibrosis, or bronchiolitis obliterans.


   Acknowledgments
 
The authors thank Jérôme Cutrona for the elaboration of the software used in this study.


   Footnotes
 
This work was supported by an Action Thématique Concertée (ATC) "Cellules Souches à Finalité Thérapeutique" grant from the Ministère de la Recherche, Institut National de la Santé et de la Recherche Médicale (INSERM), Association Française contre les Myopathies (AFM) and the Association Vaincre la Mucoviscidose (VLM).

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Conflict of Interest Statement: C.C. has no declared conflicts of interest; B.N-R. has no declared conflicts of interest; J.H. has no declared conflicts of interest; C.K. has no declared conflicts of interest; D.G. has no declared conflicts of interest; C.D. has no declared conflicts of interest; and E.P. has no declared conflicts of interest.

Received in original form March 1, 2004

Received in final form November 2, 2004


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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