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Differentiation Potential of a Basal Epithelial Cell Line Established From Human Bronchial Explant

November 13, 2007

By Halldorsson, Skarphedinn Asgrimsson, Valthor; Axelsson, Ivar; Gudmundsson, Gudmundur Hrafn; Steinarsdottir, Margret; Baldursson, Olafur; Gudjonsson, Thorarinn

Abstract Due to the cellular complexity of the airway epithelium, it is important to carefully define bronchial cell lines that capture the phenotypic traits of a particular cell type. We describe the characterization of a human bronchial epithelial cell line, VA10. It was established by transfection of primary bronchial epithelial cells with retroviral constructs containing the E6 and E7 oncogenes from HPV16. The cell line has been cultured for over 2 yr, a total of 60 passages. Although prolonged culture resulted in increased chromosomal instability, no major phenotypic drift in marker expression was observed. The cells expressed cytokeratins 5, 13, 14, and 17 suggesting a basal-like phenotype. This was further supported by the expression of alpha6beta4 integrins and the basal cell-associated transcription factor p63. The VA10 cell line generated high trans-epithelial electrical resistance in suspended and air-liquid interface culture, indicating functionally active tight junction (TJ) complexes. Immunocytochemistry showed the typical reticular structures of occludin and TJ-associated F-actin. VA10 produced pseudostratified layer in air-liquid interface culture with expression of p63 restricted to the basal layer. Furthermore, VA10 produced round colonies when cultured in laminin-rich reconstituted basement membrane, and immunostaining of claudin-1 and the basolateral marker beta4 integrin revealed colonies that generated polarization as expected in vivo. These data indicate that VA10 epithelia have the potential to model the bronchial epithelium in vivo and may be useful to study epithelial regeneration and repair and the effect of chemicals and potential drug candidates on TJ molecules in airway epithelia. Keywords Bronchial epithelia * Basal cells * Differentiation * Tight junctions * p63

Introduction

The respiratory tract is typically divided into proximal conducting airways and distal alveolar regions (Karp et al. 2002; Knight and Holgate 2003; Rawlins and Hogan 2006). The bronchial epithelium plays a key role in lung defense against inhaled particles, microbes, and toxic chemicals (Whitsett 2002). Studies also suggest that bronchial epithelial function and repair are important in the pathogenesis of asthma, chronic obstructive pulmonary disease, cystic fibrosis, and lung cancer (Knight and Holgate 2003; Cookson 2004). However, the study of the bronchial epithelium in vivo is limited by complex methodological and ethical issues and requires invasive procedures that affect epithelial structure and function. Such issues are circumvented by the use of primary cultures that have proven useful to study airway inflammation, differentiation, and carcinogen es is (Gruenert et al. 1995). The major advantage of this approach is that the cells do indeed represent the tissue of origin. Transformation of primary cells is made less likely by a relatively short period of time in culture (Fridriksdottir et al. 2005). The disadvantages, however, include limited access to biopsy material and a finite life span of the explanted cells. In addition, primary cells may display interbiopsy variations, affecting the reproducibility of results. In contrast, established cell lines provide the necessary supply of cells with similar genotype and phenotype, allowing them to be used for complex continuous long-term studies (Fridriksdottir et al. 2005).

The bronchial epithelium is composed of various cell types including columnar ciliated epithelial cells, goblet cells, serous cells, neuroendocrine cells, clara cells, and basal cells (Knight and Holgate 2003; Rawlins and Hogan 2006). Due to the cellular complexity of the bronchial epithelium, it is important to establish cell lines that capture specific phenotypic traits of their in vivo counterparts. Applying well-defined cell lines will allow careful modeling in vitro of structure and function of bronchial epithelium which is the key to the studies of epithelial differentiation, pathophysiology of lung diseases, and response to novel drug candidates.

Basal cells are ubiquitous in the conducting epithelium and are the only cell type that is firmly attached to the basement membrane via alpha6beta4 integrins (Knight and Holgate 2003). They are known to express low molecular weight cytokeratins (CKs) such as CK 5, 13, 14, and 17 and the basal cell-associated transcription factor p63 (Daniely et al. 2004). Similar to basal cells in other organs such as the skin (Watt 1998), breast (Gudjonsson et al. 2002), and prostate (Hudson et al. 2001), basal cells in lungs are thought to contain a subpopulation of cells with stem cell/ progenitor characteristics giving rise to a more differentiated progeny. In the lung, the basal cells are believed to be responsible for the generation of mucous and ciliated epithelial cells (Hong et al. 2004; Knight and Holgate 2003).

In this study, we established a new human bronchial epithelial cell line. It is characterized by the expression of basal-cell markers. When induced to differentiate, this cell line formed high transepithelial electrical resistance (TER), with well-defined expression of tight junction (TJ) molecules, and in vivo-like polarization in air-liquid and three-dimensional culture. This cell line will therefore be particularly suitable to study transepithelial transport, TJ proteins, and cell differentiation and repair in bronchial epithelia.

Materials and Methods

Cell culture. Primary bronchial epithelial cells (a gift from Prof. Michael J. Welsh, University of Iowa, Iowa City, IA, USA) were cultured on collagen-coated (Vitrogen-100 Cohesion, Palo Alto, CA, USA) plastic flasks T-25 flasks (Nunc, Roskilde, Denmark) in serum- and antibiotic-free bronchial epithelial growth medium with supplements (BEGM+S; Cambrex, East Rutherford, NJ, USA). Supplements consisted of 2 ml Bovine Pituitary Extract; 0.5 ml insulin, 0.5 ml hydrocortisone, 0.5 ml gentamycin (GA-1000), 0.5 ml retinoic acid, 0.5 ml transferrin, 0.5 ml triiodothyronin, 0.5 ml epinephrine and 0.5 ml human epidermal growth factor in 500 ml medium. For immunocytochemical experiments, cells were grown on Lab-Tek glass Chamber Slides (Nalge Nunc, Naperville, IL, USA) coated with collagen. For TER experiments, cells were seeded in the upper chamber of Transwell permeable support filter system with a pore size of 0.4 [mu]m (Coming Costar Corporation, Acton, MA, USA) and cultured in 50:50 DMEM-Ham’s F-12 medium (Gibco, Burlington, Canada) supplemented with 5% fetal bovine serum (Gibco). On the day after seeding, the cells were cultured and maintained in 50:50 DMEM-Ham’s F-12 medium supplemented with 2% Ultroser G (Biosepra, Cergy-Saint- Christophe, France). When cultured at the air-liquid interface, medium was removed from the apical side of the epithelium after reaching confluence. Cultures were rinsed with phosphate-buffered saline (PBS) every 2-3 d. For three-dimensional cultures, cells were seeded into 300 [mu]l of growth factor reduced Matrigel (Becton Dickinson Labware, Bedford, MA, USA) and cultured for 14 d in BEGM+S. After culture period, gels were frozen in ice-cold n-Hexan and mounted in tissue-freezing medium for sectioning. Gels were sectioned in a cryostat at 7-[mu]m intervals, fixed in methanol, and stained using an immunofluorescence protocol. Single-cell cloning was done in 96-well plate.

Establishment of a human bronchial epithelial cell line. Transfection of normal human bronchial epithelial cells was performed with sterile filtered retrovirus supernatant from the PA317 LXSN packaging cell lines, containing retroviral construct with human papilloma virus-16 E6 and E7, and the neomycin resistance gene (CRL-2203, American Type Culture Collection, Rockville, MD, USA). Transfection was done in the presence of 8 [mu]g/ml polybrene (Sigma-Aldrich). Transfected cells were selected by cultivation in the presence of 500 [mu]g/ml neomycin (Life Technologies, Gaithersburg, MD, USA).

Soft agar assay. To test for anchorage independent growth, VAlO cells were cultured in agar solution (USB, Cleveland, OH, USA). As a positive control, we used A549 lung adenocarcinoma cell line. About 7.000 cells were mixed with 1.5 ml agar (0.4 and 0.5%) and plated into six-well culture dish and cultured for 30 d. Each experiment was conducted in triplicate.

Karyotype analysis. Karyotype analysis was performed at the Chromosome Laboratory of the Department of Pathology, Landspitali- University Hospital using standard cytogenetic procedures. Briefly, cells were incubated with Metaphase Arresting solution (Genial Genetic Solutions, Runcorn, UK) for 3 h, followed by hypotonic treatment (0.0075M KCl) for 20 min at 37[degrees] C, fixed with methanol/ acetic acid (one third) and G-banded with trypsin solution and Leishman’s stain. At passage 13, ten VA-10 cells were analyzed and 12 cells at passage 35. Karyotypes were described following an international system for human cytogenetic nomenclature (ISCN) recommendations.

Growth curve. Analysis of cell growth was performed using a standard protocol. Cells were plated onto 24-well plates and cultured at 37[degrees] C in a humidified 5% CO2 atmosphere. After 24 h, three wells of each culture were trypsinized and counted by using a hemocytometer. This was repeated daily for 7 d and the results plotted as a growth curve. Measurement of transepithelial electrical resistance. A Millicell-ERS voltohmmeter (Millipore, Billerica, MA USA) was used to measure the TER value of confluent filters. All measurements were done in triplicate and TER values normalized for the area of the filter after background subtraction. Statistical analysis was performed using Student’s t test. The data are presented as mean+-standard error of the mean (SEM).

Immunocytochemical analyses. Cells were fixed using either methanol at -20[degrees] C or 3.7% formaldehyde followed by 0.1% Triton X-100. For immunoperoxidase stainings, we used EnVision+ System-HRP (DAB) as instructed by the manufacturer (Dakocytomatation, Denmark). The following antibodies were used: Mouse anti-Cytokeratin 5/6, Rabbit anti-Claudin-1, Mouse anti- Occludin, Mouse anti-E-Cadherin, Rabbit anti-JAM-A (all from Zymed), Mouse anti-Cytokeratin 13 (Sigma), Mouse anti-Cytokeratin 17 (from DakoCytomation), Mouse anti-Cytokeratin 14, Mouse anti-p63 (Novocastra), Alexa Fluor 488 phalloidin (stain for F-actin, Molecular Probes), and Mouse anti-beta4 Integrin (Chemicon). Cells were counterstained with haematoxilin. Images were captured using a Leica DFC320 digital imaging system (Leica, Bensheim, Germany). For immunofluorescent stainings, isotype-specific Alexa Fluor secondary antibody conjugates were used and TO-PRO-3 nucleic acid stain, all from Molecular Probes.

Confocal microscopy. Immunofluorescent images were captured using a Zeiss LSM 5 Pascal Confocal Microscope system (Carl Zeiss AG, Munich, Germany) with Plan-Neofluar 40x and Plan-Apochromat 63 x oil-immersion objectives. Monolayers and transwell filters were mounted with coverslips and Fluoromount-G mounting medium (SouthernBiotech, Birmingham, AL, USA). Z scans were performed by taking a series of images at the same location with 0.40-[mu]m focal intervals.

Western blot. Equal amounts of proteins, as determined by Bradford method (Bradford 1976), were loaded and run on a NuPAGE 10% Bis-Tris gel (Invitrogen, Carlsbad, CA, USA) and transferred to a polyvinylidene difluoride membrane (Invitrogen). The blots were blocked in 5% nonfat milk and subsequently incubated with the primary antibody overnight followed by incubation with secondary antibodies, horseradish peroxidase-conjugated antimouse, or rabbit for l h (Amersham Biosciences UK Ltd., Little Chalfont, England). Protein bands were visualized using enhanced chemiluminescence system and Hyperfilm (Amersham Biosciences).

Results

A bronchial epithelial cell line with basal cell-likephenotype. Primary bronchial epithelial cells were transfected with a retroviral construct containing E6 and E7 oncogenes from human papilloma virus (HPV) 16 as well as the neomycin (G418) resistance gene. After transfection, cells were cultivated in BEGM medium with supplements (see “Material and Methods”) containing 500 [mu]g/ml G418 to select for the transfected cells. The resulting bronchial epithelial cell line referred to as VA10 showed a typical cobblestone epithelial phenotype in monolayer culture (Fig. 1a, b). Initial characterization was conducted in serum-free bronchial epithelial medium with supplements (see “Material and Methods”). Immunofluorescence stainings demonstrated expression of cytokeratins 5/6, 13, 14, and 17 suggesting that VA10 contains cells with a basal- like phenotype (Fig. 2a). The basal phenotype was further supported by the expression alpha6beta4 integrin (not shown) and the basal cell associated transcription factor p63 (Fig 2b). Interestingly, a subpopulation of p63-negative cells appears in culture upon induction of differentiation with 2% Ultroser G (Fig. 2b), indicating a generation of more differentiated cell population. Single-cell cloning of VA10 cells resulted in a subline that retained marker expression of the parental cell line.

Marker expression and nonmalignant phenotype in long term culture. The cell line has been cultured for more than 2 yr, over 65 passages with split ratio 1:6. There has been no major phenotypic drift in marker expression of VA10 cells in early (65) passages. At passage 13, cells retain a normal karyotype, but at passage 35, trisomy of chromosomes X, 5, 8, and 20 was noted (Fig. 3a). Despite the difference in karyotype at different passages, the cells had similar growth curves and contact inhibition at confluency independent of passage number. At higher passages, population doubling time was longer, but the saturation density was similar (Fig. 36). Immunocytochemistry of keratins, p63, and alpha6beta4 integrins showed no differences between early or late passages (not shown). To test for anchorage-independent growth, cells were cultured in 0.4 and 0.5% agar in the 17, 31, and 65 passages. As a positive control, we used A549 lung adenocarcinoma which forms large colonies in soft agar assay. After 30 d in culture, no colonies were formed, indicating that VA10 cells are not tumorigenic before passage 65 (data not shown).

Retention of functionally intact tight junctions. We have shown that VA10 epithelia generate high TER in suspended culture that can be further enhanced by the macrolide antibiotic azithromycin (Asgrimsson et al. 2006). In the current study, we cultured the cell line at the air-liquid interrace and measured TER (Fig. 4a). After an initial lag period of 25 and 40 d, respectively, TER gradually increased in suspended culture and air-liquid interrace culture (Fig. 4a), suggesting the expression of functional TJ proteins. To define the spatial expression of TJ proteins, we used immunocytochemistry and confocal microscopy (Fig. 4b, c). Figure 4b shows the reticular pattern of occludin and the TJ associated F- actin filaments. Confocal Z scanning revealed the expected apical localization of occludin and TJ associated F-actin (Figure 4c).

Polarization and differentiation of VA10 epithelia. VA10 cells produce round colonies when cultured in reconstituted basement membrane (rBM; Fig. 5a). Immunostaining of the TJ marker claudin-1 and the basolateral marker beta4 integrin (Fig. 5b) suggests that the colonies polarize as expected in vivo. These data indicate that the cells have the potential to generate apical to basolateral polarity in three-dimensional culture, thereby representing a model to mimic the bronchial epithelium in vivo. Interestingly, cells maintained in 50:50 DMEM-Ham’s F-12 medium supplemented with 2% Ultroser G produced a dual layer of cells. The lower layer retained nuclear p63 expression while the upper layer did not (Fig. 6), suggesting that loss of p63 expression is a marker of differentiation and that the basal cells in VA10 may have progenitor cell characteristics.

Discussion

Access to representative cell culture models to study cellular composition and molecular signaling in bronchial epithelia is important to better understand lung development and the pathophysiology of lung diseases, in the present study, we established and characterized a bronchial epithelial cell line, VA10. Interestingly, the cell line expressed phenotypic traits of basal cells of the proximal conducting bronchial epithelium including cytokeratins 5, 13, 14, and 17, the hemidesmosomal integrin alpha6beta4, and the basal cell associated transcription factor p63. The cells showed contact inhibition in monolayer and generated a pseudostratified epithelium in air-liquid interface culture with basal expression of p63. The epithelium expressed functional TJ complexes as evidenced by high TER when cultured on permeable transwell filters in submerged culture and in an air- liquid interface culture. Furthermore, site-specific markers indicated that VA10 epithelia produced in vivo-like polarization in three-dimensional culture.

Bronchial epithelial cell lines. Immortalized bronchial epithelial cell lines are widely used to study the structure, function, and development of the respiratory tract (Karp et al. 2002; Knight and Holgate 2003). However, the cellular complexity of bronchial epithelia requires carefully defined cell lines that represent specific in vivo phenotypic traits. Interestingly, many available bronchial cell lines are not precisely defined with respect to their cellular origin and lack the characterization of critical markers for differentiated epithelial cells such as TJ complexes (Forbes and Ehrhardt 2005). The most cited lung epithelial cell line, A549, was initiated from a human bronchoalveolar cell carcinoma (lieber et al. 1976). This cell line represents type II alveolar cells; however, it has been widely used to study lung biology in general, although the structure and function of malignant alveolar epithelial cells is quite different from those of normal bronchial epithelial cells. The human bronchial cell lines 16HBE14o- , Calu-3, and BEAS-2B have been successfully applied to study drug transport and differentiation, drug metabolism, and drug delivery due to their ability to form TJs (reviewed in Forbes and Ehrhardt 2005). The cell line 16HBE14o- was developed by transformation with SV40 large T antigen of cultured human bronchial epithelial cells (Cozens et al. 1994). In contrast, the Calu-3 cell line was derived from bronchial adenocarcinoma (Shen et al. 1994) and the BEAS-2B line from normal human epithelial cells immortalized using the adenovirus 12-SV40 hybrid virus (Reddel et al. 1988). Calu-3 and 16HBE14o- cell lines have been identified as the best differentiated model available and have been used to study barrier function and the activity of TJ complexes (Forbes and Ehrhardt 2005).

Differentiation potential of epithelial cells immortalized with E6 and E7 oncogenes. VA10 was successfully established from normal primary bronchial epithelial cells by retroviral induction of the HPV-16 oncogenes E6 and E7. The major concern using retroviral constructs containing E6 and E7 to transfect cells is that immortality is achieved by inactivation of p53 and retinoblastoma protein. These may not be the only affected molecules, and other cellular functions may also be affected (Garbe et al. 1999; Zwerschke and Jansen-Durr 2000). Earlier studies suggest, however, that human cells derived from E6/E7 immortalization retain much of their original phenotype (Gudjonsson et al. 2004). In organotypic cultures of HPV-16 infected endocervical cells, the cells appeared normal, with ordinary stratification and production of a cornified layer (Halbert et al. 1992). In addition, normal adult human pancreatic epithelial cells transfected with E6/E7 remained polarized on collagen gels and did not grow in soft agar (Furukawa et al. 1996). Furthermore, E6 and E7 immortalized breast epithelial progenitor cells form branching structures in three-dimensional culture and retain the stem cell characteristics (Gudjonsson et al. 2002). Several studies describe transfection of bronchial epithelial cells with E6 and E7. Willey et al. (1991) established normal human bronchial epithelial cell line (BEP3) with cloned fulllength HPV- 16. This cell line was nontumorigenic after 100 population doublings in vitro but had diminished confluence-induced squamous differentiation. De Suva et al. (1994) concluded that HPV-16 E6 and E7 transfection were insufficient for immortalization of bronchial epithelial cells. Viallet et al. (1994) established a bronchial epithelial cell line, HBE-E6/E7, that resembles morphologically the basal cells of the normal human bronchial epithelium. This cell line is nontumorigenic and forms tubules in three-dimensional culture. The HBE4-E6/E7 cell line has been used to study the release of chemotactic factors from bronchial epithelium (Little et al. 2001) and in asthma (Burkart et al. 2006). Coursen et al. (1997) showed that transfection of bronchial epithelial cells gave rise to a cell population with increased life span before entering crisis. A subpopulation of these cells escaped crisis and achieved further population doublings, suggesting immortalization.

Modelling bronchial histology in vitro. The investigation of the human lung in vivo is appropriately limited by ethical issues. Obtaining lung tissue is also complicated by the physiological importance of pulmonary function. In addition, various methods such as bronchoscopic biopsies, bronchoalveolar lavage, and the sampling of airway surface liquid are known to distort the tissue architecture to be studied. Animal studies are similarly limited by ethical issues, sampling bias, differences between species, and high cost.

In summary, the VA10 cell line presented here combines several advantages. It is derived from normal human lung, maintains intact karyotype at low passages, generates polarized epithelia in three- dimensional culture, and expresses basal cell markers and functional TJ proteins as evidenced by high TER. High TER and well-defined expression of TJ proteins are useful characteristics to study transepithelial transport and the response of the TJ complex to pathogens, irritants and potential therapeutic agents (Asgrimsson et al. 2006). In addition, the expression of basal cell markers and in vivo-like polarization allow investigation of cell differentiation and repair that are central to the pathogenesis of common diseases such as bronchial carcinoma, chronic obstructive pulmonary disease, and asthma.

Acknowledgments Grant support was provided by The Icelandic Research Council, Landspitali University Hospital Research Fund, University of Iceland Research Fund, and Science and Technology Policy Council-Thematic program in postgenomic biomedicine. We thank Prof. Michael J. Welsh at the University of Iowa for providing primary bronchial cells.

Received: 9 January 2007 /Accepted: 14 June 2007 / Published online: 18 September 2007 / Editor J. Denry Sato

(c) The Society for In Vitro Biology 2007

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SH an VA contributed equally to this paper.

S. Halldorsson * G. H. Gudmundsson

Biology Institute, University of Iceland,

Reykjavik, Iceland

O. Baldursson

Faculty of Pharmacy, University of Iceland,

Reykjavik, Iceland

V. Asgrimsson * I. Axelsson * T. Gudjonsson

Faculty of Medicine, University of Iceland,

Reykjavik, Iceland

M. Steinarsdottir

Chromosome Laboratory, Department of Genetics

and Molecular Medicine, Landspitali University Hospital,

Reykjavik, Iceland

O. Baldursson

Division of Pulmonary Medicine, Landspitali University Hospital,

Reykjavik, Iceland

I. Axelsson * T. Gudjonsson

Experimental Cell Biology Research Unit,

Department of Hematology, Landspitali University Hospital,

(K-building), 101,

Reykjavik, Iceland

e-mail: thorarin@landspitali.is

Copyright Society for In Vitro Biology Sep/Oct 2007

(c) 2007 In Vitro Cellular & Developmental Biology; Animal. Provided by ProQuest Information and Learning. All rights Reserved.




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