Department of Medicine, Washington University School of Medicine, Saint Louis, Missouri 63110
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ABSTRACT |
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Highly regulated programs for airway epithelial cell proliferation and differentiation during development and repair are often disrupted in disease. These processes have been studied in mouse models; however, it is difficult to isolate and identify epithelial cell-specific responses in vivo. To investigate these processes in vitro, we characterized a model for primary culture of mouse tracheal epithelial cells. Small numbers of cells seeded at low density (7.5 × 104 cells/cm2) rapidly proliferated and became polarized. Subsequently, supplemented media and air-liquid interface conditions resulted in development of highly differentiated epithelia composed of ciliated and nonciliated cells with gene expression characteristic of native airways. Genetically altered or injured mouse tracheal epithelial cells also reflected in vivo patterns of airway epithelial cell gene expression. Passage of cells resulted in continued proliferation but limited differentiation after the first passage, suggesting that transit-amplifying cell populations were present but with independent programs for proliferation and differentiation. This approach provides a high-fidelity in vitro model for evaluation of gene regulation and expression in mouse airway epithelial cells.
airway; cilia; Clara cell; progenitor cell
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INTRODUCTION |
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AIRWAY EPITHELIAL CELL proliferation and differentiation during lung development and repair are highly regulated, complex processes (6, 24). In lung disease, these processes are influenced by growth factors, cytokines, or proteases released by mesenchymal and inflammatory cells (11, 24). To determine epithelial cell-specific function in diseases such as asthma and cystic fibrosis, primary human airway epithelial cell culture has been valuable (11, 21, 28), although defined mutations and phenotypes are limited and analysis is complicated by genetic variation between individuals.
To characterize the role of a single gene in airway epithelial cell function during lung development or disease, isogenic animals have been generated with deficient or augmented gene expression. Like human disease, these mice often have complex responses involving airway epithelial cells (11, 30). To identify a cell-specific response in airway epithelial cells from genetically defined mice, it would be desirable to culture and manipulate these cells in vitro. However, in contrast to human and other species, there are few approaches for primary culture of differentiated mouse airway epithelial cells (5, 7, 15). Available primary culture protocols for mouse tracheal epithelial cells (MTEC) require large numbers of cells and result in minimal cell differentiation (5, 7, 15). Problems in primary culture include the inherent low cell yield from each mouse trachea and possible differences in growth factor requirements for amplifying progenitor cell populations and induction of specific cell phenotypes.
We have defined conditions for primary culture of MTEC that result in rapid proliferation and generation of a highly differentiated epithelium reflecting wild-type and mutant phenotypes. In vitro passage and subsequent regeneration of differentiated epithelial cell populations suggest the presence of progenitor-like cells located in the proximal trachea.
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MATERIALS AND METHODS |
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Culture media and supplements. Media formulations were modified from previously described methods (14, 17, 19, 26-28). Supplements were from Sigma-Aldrich (St. Louis, MO) unless indicated. "Hams F-12 pen-strep" is Ham's F-12 media with 100 U/ml penicillin and 100 µg/ml streptomycin. "MTEC Basic" media is DMEM-Ham's F-12 (1:1 vol/vol), 15 mM HEPES, 3.6 mM sodium bicarbonate, 4 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml fungizone. "MTEC/Plus" is MTEC basic media supplemented with 10 µg/ml insulin, 5 µg/ml transferrin, 0.1 µg/ml cholera toxin, 25 ng/ml epidermal growth factor (Becton-Dickinson, Bedford, MA), 30 µg/ml bovine pituitary extract (17), 5% FBS, and freshly added 0.01 µM retinoic acid. "MTEC/NS" is MTEC basic media supplemented with 2% NuSerum (Becton-Dickinson) and freshly added 0.01 µM retinoic acid. "MTEC/SF" (serum-free) is MTEC basic media supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, 0.025 µg/ml cholera toxin, 5 ng/ml epidermal growth factor, 30 µg/ml bovine pituitary extract, 1 mg/ml BSA, and freshly added 0.01 µM retinoic acid.
Mouse tracheal cell isolation. Tracheal cells from wild-type C57Bl/6, SV129/J, C57Bl/6-SV129/J hybrid, Friend Virus B-Type (FVB), Clara cell secretory protein (CCSP)-deficient (kindly provided by B. Stripp, University of Pittsburgh), and Foxj1-deficient mice were evaluated (3, 22). For most studies, wild-type C57Bl/6 or hybrid mice, 3-6 wk of age were used. MTEC harvest was modified from previously described methods (7, 19, 28). Mice were killed and then briefly immersed in 70% ethanol (avoiding airway submersion). With the use of a sterile technique, tracheas were resected from the larynx to the bronchial main branches and collected in ice-cold Ham's F-12 pen-strep. In a tissue culture hood, muscle and vascular tissues were dissected from tracheas in cold media. Tracheas were washed with media, opened longitudinally, and then incubated in Ham's F-12 pen-strep containing 1.5 mg/ml pronase (Roche Molecular Biochemicals, Indianapolis, IN) for 18 h at 4°C. The tube was then put on ice, and FBS was added to a final concentration of 10%. The tracheas were inverted 12 times, transferred to another tube of Ham's F-12 pen-strep with 10% FBS, inverted again, placed in one-third tube of media, inverted to further release cells, and then discarded. Contents of the three tubes were pooled and collected by centrifugation at 400 g for 10 min at 4°C. Cells were resuspended in 200 µl/trachea of Ham's F-12 pen-strep containing 0.5 mg/ml crude pancreatic DNase I (Sigma-Aldrich) and 10 mg/ml BSA. The cells were then incubated on ice for 5 min, centrifuged at 400 g for 5 min at 4°C, and resuspended in MTEC basic media with 10% FBS. After incubation in tissue culture plates (Primera; Becton-Dickinson Labware, Franklin Lakes, NJ) for 3-4 h in 5% CO2 at 37°C to adhere fibroblasts, nonadherent cells were collected by centrifugation, resuspended in 100-200 µl MTEC/Plus per trachea, and counted. No attempt was made to achieve a single cell suspension from cell clumps. The average yield of tracheal cells was 1.81 × 105 cells/trachea [±0.58 × 105 (SD)] from 34 preparations obtained from 162 mice weighing 10-20 g. Cell viability determined by trypan blue exclusion was >90%. Cytocentrifuge preparations of these cells revealed that ~99% expressed cytokeratin, as determined by pan-cytokeratin antibody staining.
In vitro culture of mouse tracheal cells.
Supported polycarbonate and polyester porous (0.4 µM pores) membranes
(Transwell and Transwell Clear; Corning-Costar, Corning, NY) were
coated with filter-sterilized 50 µg/ml type I rat tail collagen
(Becton-Dickinson) in 0.02 N acetic acid using 1.0 ml/cm2
membrane for 18 h at 25°C. Membranes were seeded with 7.5 × 104 cells/cm2 and incubated with MTEC/Plus
filling upper and lower chambers in 5% CO2 at 37°C.
Media were changed every 2 days until the transmembrane resistance
(Rt) was >1,000 · cm2,
as measured by an epithelial Ohm-voltmeter (World Precision Instruments, Sarasota, FL). Media were then removed from the upper chamber to establish an air-liquid interface (ALI), and lower chambers
only were provided fresh MTEC/NS or MTEC/SF media every 2 days. Cells
on Transwell Clear membranes were monitored by inverted-phase microscopy. To remove epithelial cells from membranes for enumeration or passage, cells were incubated in Cell Dissociation Solution (Sigma-Aldrich) supplemented with 0.25% trypsin and 2.7 mM EDTA at
37°C for 15 min and resuspended in Ham's F-12 pen-strep with 10%
FBS. Cells were collected by centrifugation and resuspended in
MTEC/Plus for reseeding.
Immunofluorescent labeling and analysis.
Membranes were fixed with 4% paraformaldehyde in PBS, pH 7.4, for 10 min at 25°C, washed in PBS, cut from supports into two to six pieces,
and processed for immunodetection in 24- or 96-well dishes. Cell
sections (6 µm) were obtained after fixed membranes were submerged in
warm 2% agarose and then embedded in paraffin. Nonspecific antibody
binding was blocked using 5% donkey serum and 3% BSA in PBS for 30 min at 25°C. Samples were incubated for 18 h at 4°C with
isotype-matched control antibody or primary antibody in blocking
solution. Control or primary antibody binding was detected using FITC-
or indocarbocyanine-labeled secondary antibody (Jackson ImmunoResearch
Laboratories, West Grove, PA). No detectable staining was observed for
isotype-matched control antibodies. Membranes were mounted on slides
with Vectashield (Vector, Burlingame, CA) containing 4',6
diamidino-2-phenylindole to stain intracellular DNA. Photomicroscopy
was performed using an Olympus BX51 camera (Melville, NY) to acquire
images with a CCD (MagniFire; Olympus) interfaced with MagniFire
software. At least three fields of cells were counted directly or
analyzed using National Institutes of Health Image software (version
1.25; National Institutes of Health; http://rsb.info.nih.gov/nih-image). Primary antibodies and dilutions or concentrations were as follows: rabbit pan-cytokeratin (1:500; Biomedical Technology, Stoughton, MA), rabbit anti-Z0-1 (0.5 µg/ml; Zymed, San Francisco, CA), rabbit anti-TTF-1 (2.0 µg/ml;
Biopat, Caserta, Italy), rabbit anti-aquaporin 4 (2.5 µg/ml;
Chemicon, Temecula, CA), mouse anti-Muc5AC (0.4 µg/ml; Lab Vision,
Fremont, CA), mouse anti--tubulin-IV (30 µg/ml; BioGenex, San
Ramon, CA), and rabbit anti-mouse CCSP (1:500; kindly provided by F. DeMayo, Baylor College of Medicine, Houston, TX). Staining with
biotinylated lectin Bandeiraea (Griffonia) simplicifolia
BS-1 isolectin S4 (50 µg/ml; Sigma-Aldrich) detected by Texas
red-labeled avidin (Vector) was used to identify basal cells, as
described previously (20).
Cell proliferation assay. Cells were incubated in media containing 10 µM 5-bromo-2'-deoxyuridine (BrDU; Sigma-Aldrich) for 2 h, fixed as above, treated with 4 N hydrochloric acid for 15 min, and then neutralized in 0.1 M sodium borate, pH 8.5 for 20 min. Cells were incubated with rat anti-BrDU antibody (0.5 µg/ml; Accurate Chemical, Westbury, NY) in PBS containing 0.2% Triton X-100 for 30 min at 25°C, and primary antibody was detected using rabbit anti-rat FITC-labeled antibody (Jackson ImmunoResearch Laboratories). Cells expressing BrDU were counted as described above. To study responses after wounding, cells on membranes were embossed with the proximal end of an 8-mm glass Pasteur pipette to denude a 1-mm ring of cells before BrDU labeling.
Scanning electron microscopy. Tracheas and membranes were prepared for scanning electron microscopy (EM), as previously described (18). Briefly, samples were fixed with 2.5% glutaraldehyde, stained with 1.25% osmium tetroxide, critical point dried under liquid carbon dioxide, gold sputter coated, and visualized on a Hitachi S-450 microscope (Tokyo, Japan).
Statistical analysis. Rt and cell numbers were analyzed for statistical significance using the Student's t-test and a one-way ANOVA for a factorial experimental design. The two-sample and multicomparison significance level for one-way ANOVA was 0.05. If significance was achieved by one-way analysis, post-ANOVA comparison of means was performed using Scheffé's F-test (29).
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RESULTS |
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Formation of tight junctions and polarization of cultured mouse
tracheal cells.
MTEC seeded at low density on membranes developed a polarized cell
layer demonstrated by development of Rt,
exclusion of media from the upper chamber during ALI conditions, and
expression of tight-junction protein ZO-1 (Fig.
1). In the presence of supplemented, FBS-containing media (MTEC/Plus), Rt increased
over the first 5-6 days as cells became confluent (Fig.
1A). ALI was created for each preparation when
Rt of all membranes was >1,000
· cm2. At this time (ALI day 0),
media were removed from the upper chamber, and cells were cultured in
MTEC/NS or MTEC/SF media. Rt gradually decreased
after creation of ALI, as noted by others (7). Fewer than
4% of Transwell inserts never formed electrically tight junctions. To
correlate the increase in Rt with gene-specific expression, ZO-1, a component of tight-junction assembly, was evaluated
(13). ZO-1 was expressed in portions of the peripheral cell membranes (Fig. 1B) before development of high
Rt; however, after ALI was created, peripheral
membranes uniformly expressed ZO-1, consistent with the establishment
of a layer of polarized epithelial cells.
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MTEC growth kinetics.
ZO-1 expression also suggested changes in cell size and number. We
found that cell number increased markedly within the first week of
culture, reflecting proliferation as detected by BrDU incorporation and
resulting in a multilayered epithelium (Fig. 2). Although 7.5 × 104/cm2 cells were seeded on the membrane,
after 3 days, total cell number was approximately one-half (40.6%),
indicating that only a fraction of seeded cells adhered to the
membrane, as described in tracheal cell culture from other species
(25). During a discrete proliferation phase in the first
7-10 days, cells grown in MTEC/SF resulted in a greater number of
cells than in MTEC/NS. After this phase, BrDU incorporation in MTEC was
0.1-0.2% in MTEC/NS and 1-2% in MTEC/SF. In either media,
multiple cell layers developed with tall ciliated and Clara cells
apparent at ALI day 5 (Fig. 2B). MTEC grown in
MTEC/NS typically had one to two layers, whereas MTEC in MTEC/SF had
two to three layers. The multicell layers at ALI day 14 resembled native trachea (Fig. 2B) and tracheal cell
cultures from other species (8, 25, 28).
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MTEC apical surface differentiation.
To further characterize changes in cell histology, epithelial cell
apical surface morphology was observed by scanning EM (Fig. 3). This showed large, flat, uniform
cells with microvilli at ALI day 0 (Fig. 3A).
From ALI day 0 to day 10, cells became smaller, more dome-like, and ciliated. Thus, after proliferation, there was a
distinct phase of epithelial cell differentiation. By ALI day
10, the surface of the cultured MTEC morphologically resembled mouse trachea (Fig. 3B).
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Expression of differentiation markers in MTEC.
Changes in histology and morphology correlated with known markers of
airway epithelial cell differentiation (Fig.
4). As observed in
vivo, airway epithelial cell transcription factor TTF-1 was expressed
in all MTEC evaluated at ALI days 0, 7, and
14 (6, 24). Lectin B. simplicifolia
isolectin B4 (BS-B4) staining was used as a marker of basal cells and
had an affinity for cells in the layer adjacent to the membrane but not
upper layers, also as observed in vivo (20). Similarly,
aquaporin 4 expression was detected in the basolateral domain of cells
after ALI day 0 (1). Unlike normal lung
(10, 25), Muc5AC was expressed in large numbers of cells
at ALI day 5 and in fewer cells by ALI day 14,
but typical mucus cells were not found in samples processed for
histological evaluation (Fig. 2B).
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MTEC proliferation and differentiation during injury and passage.
To determine if MTEC were capable of proliferation and
redifferentiation in vitro as required for wound healing, mature cells (ALI day 18) grown in MTEC/NS were wounded and observed for
proliferation and differentiation. Uninjured MTEC exhibited a low
percentage of BrDU incorporation (as shown in Fig. 2), but 1 day after
injury BrDU incorporation was increased markedly at the wound edge
(Fig. 5A). Later (3 days),
nearly the entire denuded region was replaced by large numbers of
ciliated cells, suggesting the presence of an MTEC population capable
of epithelial cell proliferation and differentiation, similar to in
vivo repair (16).
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DISCUSSION |
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We found that MTEC can be differentiated rapidly using defined
conditions to model native mouse trachea with several advantages. First, in the presence of a supplemented media, cells rapidly proliferate, permitting successful initiation of cultures from a small
number of cells harvested from trachea (7.5 × 104
cells/cm2; less than one-half a trachea). This allows
evaluation of MTEC when numbers of mice are limited and facilitated
analysis of MTEC from surviving Foxj1()/(
) mice that are runted and
die at <3 wk of age (3, 22). In contrast, prior MTEC
protocols used a seeding density of 1.2 × 106
cells/cm2 (6 adult mouse tracheas; see Ref.
7). Second, selective media supplementation and ALI
conditions also rapidly led to a high degree of differentiation with
large numbers of ciliated, CCSP-, mucin-, and aquaporin-expressing
cells that have not been described previously in MTEC cultures. This
makes possible in vitro study of gene regulation using approaches not
feasible in vivo. Third, culture conditions allow proliferation and
redifferentiation in vitro for the study of airway repair in the
absence of immune cell mediators. Finally, primary culture of cells
from genetically deficient mice mirrored in vivo phenotypes in Foxj1
and CCSP null MTEC. This provides a potent alternative approach for in
vitro analysis of airway epithelial cells from isogenic mice with
deficient or augmented gene expression to characterize and manipulate
cell-specific responses.
Conditions used for MTEC proliferation and differentiation were similar to those used to culture tracheal cells from human, rat, and other species. Those studies showed that ALI, insulin, epidermal growth factor, and retinoic acid were important for cell proliferation, ciliogenesis, and mucus cell differentiation (4, 8, 17, 23, 27, 28). We also found that a simple media with a modified serum supplement (NuSerum; see Ref. 9) compared with Ultroser G (see Refs. 7 and 28) resulted in many more ciliated cells, increased cell layer number, and increased cell height (unpublished observation), likely because of differences in proprietary additives. Conditions we used favor ciliogenesis but were associated with a rise then fall in numbers of CCSP-expressing cells. This change in secretory cell populations was seen by others during human airway cell culture (8) and could be a result of undefined secreted factors or changes in matrix composition that support different cell types. Although we noted Muc5AC expression in many cells, we did not specifically create conditions found by others to favor mucus cell differentiation such as lowering epidermal growth factor concentrations or using a collagen gel substratum (10, 25). In mature cells, mucus was present on the apical aspect of cells, and electron-dense granules were found by transmission EM in nonciliated cells that may contain mucin or CCSP (data not shown), suggesting that functional secretory cells are present. Characterization of secretory cells and the effect of varying concentrations of retinoic acid, epidermal cell growth factor, or cytokines on changes in cell populations or gene expression may be addressed in future studies.
Passage, proliferation, and subsequent differentiation of the primary
culture MTEC suggest that populations of transit-amplifying cell
populations are present, similar to progenitor-like cells found in in
vivo (2, 12) and in vitro rat and human airway cell
cultures (27). We found that, after 3 days, only a small percentage of cells initially seeded on membranes became adherent and
proliferated, indicating a subpopulation of progenitor-like cells
survived in the culture conditions (27). Molecular markers of airway progenitor cells are not defined; however, at ALI days 3, 0, and 3, these cells expressed
fundamental airway epithelial cell transcription factors TTF-1 and
hepatocyte nuclear factor-3
(data not shown) but not CCSP or
-tubulin-IV. B. simplicifolia BS-1 isolectin S4
(BS-B4), a putative basal cell marker associated with repairing
epithelial cells (20), was detected in over one-half of
the cells at this time. However, BS-B4 also stained many cells at ALI
days 7 and 14 when proliferation was low,
suggesting that BS-B4 may not be a specific marker of
transit-amplifying cells. Consistent with the observations of others,
we also found that, under the growth conditions provided by our in
vitro culture system, cells most capable of proliferation and
differentiation appeared to reside in the proximal trachea, possibly in
or around tracheal glands (2, 7); however, we did not
evaluate gland cell marker expression. Finally, these findings show
that programs for proliferation are not directly linked to
differentiation, since, despite the presence of a transit-amplifying
population that permitted proliferation, differentiation was limited
after the first passage. It is possible that an additional cell
population capable of proliferation and differentiation has limited
survival within the conditions established.
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ACKNOWLEDGEMENTS |
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We thank Kwong Kim, Phil Karp, Reen Wu, and Bob Senior for helpful discussions, Mike Veith for assistance with electron microscopy, Barry Stripp for Clara cell secretory protein (CCSP)-deficient mice, Francesco deMayo for CCSP antibody, and the Digestive Disease Research Core for tissue processing.
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FOOTNOTES |
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This study was supported by National Institutes of Health awards RO1-HL-56244 and RO1-HL-63988 (to S. L. Brody) and P30 DK-52574.
Address for reprint requests and other correspondence: S. L. Brody, Washington Univ. School of Medicine, Campus Box 8052, 660 S. Euclid Ave., St. Louis, MO 63110 (E-mail: brodys{at}msnotes.wustl.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
August 9, 2002;10.1152/ajplung.00169.2002
Received 3 June 2002; accepted in final form 5 August 2002.
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