Establishment and characterization of a telomerase-immortalized porcine bronchial epithelial cell line

2.1 Experimental animals

One snatch-farrowed, porcine-colostrum-deprived large Yorkshire piglet (7-week-old male) and nine BALB/c nu/nu mice (4-week-old females) were purchased from Zhoubang Biological Technology Company of Nanjing and Animal Experiment Center of Yangzhou University, respectively. The piglet had neither sign of cardiopulmonary disorders nor respiratory symptoms at clinical examination. In addition, the piglet was not under any drug treatment or vaccinated. Before the animal experiment, antibody detection test results based on serologic testing demonstrated that the following pathogens were free, including M. hyopneumoniae, swine fever virus, porcine respiratory and reproductive syndrome virus, pig o-type foot-and-mouth disease, PCV, porcine pseudorabies and SIV.

2.2 Ethics statement

All animal experiments were performed according to animal welfare standards and were approved by the Ethical Committee for Animal Experiments of Jiangsu Academy of Agricultural Sciences, China.

2.3 Isolation and culture of primary PBECs

The primary PBECs were isolated according to the method for mouse tracheobronchial epithelial cells (H. C. Lam et al., 2011) and human epithelial cells from lungs (Ramirez et al., 2004; Vos et al., 2005) with some modifications. The piglet was euthanized with an overdose of 20% sodium pentobarbital (2 ml/kg; Cat. #P3761; Sigma-Aldrich, St. Louis, MO) through forelimb intravenous injection to ensure it suffered minimal pain. Directly after euthanasia, 75% alcohol was used to disinfect the epidermal tissue of the thoracic and abdominal skin of the piglet. The skin was cut along the sternum from the throat to the last rib until the rib cage was removed to expose the chest. Another set of surgical scissors and scalpel were used to remove most of the skin around the tracheal in case of throat contamination. Subsequently, the trachea was lifted after connective tissue was removed. The trachea distal to the lung was cut immediately after the upper trachea was fastened with a hemostatic clamp. The remaining trachea together with the intact lung tissue were removed from the chest and placed in a sterilized tray covered with ice-cold phosphate-buffered saline (PBS) before being transferred to the biosafety cabinet. For further bronchial separation, the lung tissue was divided into single lobes with flattened tweezers. Subsequently, a surgical dissecting mirror, microsurgical forceps and scissors were used to remove the excess lung tissue surrounding the trachea, eventually leading to the isolation of bronchial tissue.

The isolated bronchial tissues were washed with ice-cold D-Hank’s balanced salt solution (DBSS; Ca2⁺/Mg2⁺ free; Gibco, Carlsbad, CA) and manually cut into approximately 1 mm³ pieces with microscopic scissors. The minced bronchial tissue pieces were digested in a total of 20 ml of digestion solution for 30 min at 37°C in a 5% CO₂ atmosphere. The digestion solution consisted of Dulbecco’s modified Eagle's media–nutrient mixture F-12 (DMEM–F12), containing 4 ml of ethylenediaminetetraacetic acid (0.0005 M), 8 ml of collagenase I (0.5 mg/ml; Worthington, Lakewood, NJ), and 8 ml of 0.25% trypsin (Gibco, Carlsbad, CA). Twenty microliters of the solution was removed to observe the digestion condition under microscopy every 20 min. Ice-cold DMEM–F12 medium containing 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA) was added to stop the digestion immediately when the cells become round and scattered. The crude cell-containing suspension was filtered through a 200-mesh filter after gentle pipetting. The filtrate was collected after the filter was rinsed twice with ice-cold DBSS. The supernatant was discarded after centrifugation of the suspension at 1200 rpm for 10 min, and the precipitation was resuspended with DMEM–F12 containing 10% FBS before being placed into four 10 cm dishes (Thermo Fisher Scientific, Waltham, MA) and two wells of 12-well cell plate (Corning, Tewksbury, MA). Cultures were grown in DMEM–F12 medium containing 100 U/ml penicillin (HyClone, Marlborough, MA), 0.1 mg/ml streptomycin (HyClone, Marlborough, MA), 15 μg/ml enrofloxacin (Aladdin, Shanghai, China), 0.1 mg/ml gentamicin, and 5 μg/ml amphotericin (Dingguo, Beijing, China). To get the primary PBECs, the medium in four dishes was changed to DMEM–F12 containing 2% FBS after 12 hr to restrict the outgrowth of nonepithelial cells, such as smooth muscle cells. In addition, PBEC cultures were supplemented with antibiotics and regulation factors of epithelial cells (Cat. #CC-4175; LONZA, Walkersville, MD), including 26 µg/ml bovine pituitary extract, 15.5 ng/ml human recombinant epidermal growth factor (hEGF), 5 µg/ml human recombinant insulin, 1.4 µM hydrocortisone, 2.7 µM epinephrine, 9.7 nM tri-iodo-l-thyronine, 0.3 nM retinoic acid, and 10 µg/ml transferrin. Culture medium was changed every day, and an additional 10 ng/ml hEGF and 0.1 ng/ml retinoic acid (Sigma-Aldrich, St. Louis, MO) were added each day. To get the primary smooth muscle cells used as control, the supernatant of cells in 12-well cell plate was changed to DMEM–F12 medium containing 10% FBS and without adding epithelial cells growth factors after smooth muscle cells demonstrated adherent growth. Then, the growth of primary PBECs could be restricted and primary smooth muscle cells will gradually become the predominant cells. Morphological features of the primary cultures were evaluated every day by light microscopy (Nikon, Chiyoda-ku, Tokyo, Japan). The cells were passaged upon reaching 80% confluence after approximately 3 days in culture by digestion with 0.125% trypsin (Gibco, Carlsbad, CA) diluted in D-Hank’s medium.

2.4 Characterization of primary cell cultures

To determine whether the isolated cells were bronchial epithelial cells, cytokeratin 18 immunostaining was performed according to previous studies (Schierack et al., 2006; Wang et al., 2014; Xie et al., 2015). Specifically, primary PBECs at passage 2 and primary smooth muscle cells containing few primary PBECs at passage 2 (negative control) were grown in a 24-well plate before fixation with 4% paraformaldehyde in PBS for 10 min at room temperature (RT). Subsequently, cells were treated with 0.1% Triton X-100 in PBS for 5 min at RT. Cells were blocked with an immunostaining blocking buffer containing 1% bovine serum albumin (Beyotime, Shanghai, China) for 30 min and then incubated with a 1:600 dilution of primary monoclonal antibody against cytokeratin 18 (Cat. #ab668; RRID: AB_305647; Abcam, Cambridge, MA) for 1.5 hr at 37°C. After three PBS washes, cells were incubated with fluorescein isothiocyanate (FITC)–conjugated goat anti-mouse immunoglobulin G (IgG; H + L; Cat. #A0568; Beyotime, Shanghai, China) at a 1:1,000 dilution for 1 hr at 37°C. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 15 min before cells were observed using fluorescence microscopy (Zeiss, Tokyo, Japan). To assess the percentage of PBECs in isolated primary cells, six microscopic fields at ×100 magnification were randomly selected for each of three wells in a 24-well plate to manually count the cell number using the same cutoff values and the same exposure settings.

2.5 Establishment of immortalized hTERT-PBECs

After culturing for two passages, primary PBECs were plated into 24-well plates at a density of 2 × 10⁵ cells per well and cultured in DMEM–F12 medium containing 2% FBS plus the regulation factors and antibiotics described as above. Before transfection, the medium was changed to fresh medium with FBS and antibiotics. The primary cells were transfected with the pEGFP-hTERT recombinant vector constructed in our previous study (Xie et al., 2015) when the cells reached 80–85% confluence. In addition, the pIRES2-EGFP vector was also transfected as a control using Lipofectamine^® 3000 (Invitrogen, Waltham, MA) according to the manufacturer’s instructions. The cells were observed using fluorescence microscopy (Zeiss, Tokyo, Japan) after being in stationary culture for more than 24 hr. pEGFP-hTERT contains green fluorescent protein (EGFP), which was used as a marker to verify the transfection success rate of the recombinant plasmid into primary PBECs. Because pEGFP-hTERT contains the neo gene, which confers resistance to the G418 antibiotic, positive cell clones were selected with 400 µg/ml G418 (diluted in 1 mol/L HEPES; Sigma-Aldrich, St. Louis, MO) in complete culture medium after 24 hr, and monoclonal cells appeared after 6 days of selection. Stable hTERT overexpression cell lines were established from large and healthy G418-resistant colonies. After 24 hr of incubation, the medium was replaced, and the positive cells were selected by treatment with 400 mg/ml G418 antibiotics, and cells were selected a second time using the same procedure. Eventually, positive cell clones were obtained after three rounds of selection. After confirmed as mycoplasma negative, the newly established cell line, hTERT-PBECs, at passage 30 was sent to China Center for Type Culture Collection (CCTCC) for preservation (CCTCC no: 201749).

2.6 Cell growth kinetics and replicative lifespan determination

Unless otherwise stated, primary PBECs and immortalized hTERT-PBECs used in all subsequent experiments are primary PBECs at passage 4 and hTERT-PBECs at passage 60, respectively. The hTERT-PBECs and primary PBECs were seeded in 24-well plates at 2 × 10⁴ cells per well in 500 µl of culture medium. The medium was changed every two days. On each day, cells in three wells were harvested by trypsinization, and cell numbers were counted according to a previous study (Hong, Zhang, Xu, Su, & Sun, 2007; Moffatt-Jauregui et al., 2013). Each test was performed in triplicate.

To determine the replicative lifespan, primary PBECs and hTERT-PBECs were trypsinized and collected after centrifugation. Cells were fixed overnight with 10 ml of 75% ice-cold ethanol at 4°C and then washed three times using cold PBS. After centrifugation, the supernatant was discarded, and cells were washed twice with cold PBS and treated with 0.4 ml propidium iodide (Cat. #550825; BD Biosciences, San Jose, CA) for 15 min in the dark at RT. For proper cell cycle analysis, the count of cells should be more than 1 × 10⁶. The percentages of gated cells in the G1/G0, G2/M, and S phases were calculated. Cell cycle quantification was examined by flow cytometry (FCM) on a BD Accuri C6 (BD Biosciences, San Jose, CA), and data were processed to determine the cell cycle distribution using FlowJo software (Su et al., 2013; Wang et al., 2014).

2.7 Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of hTERT

Total RNA was extracted from primary PBECs, hTERT-PBECs, and human epithelial type 2 (HEp-2) cells (positive control; CLS Cat. #300397/p694_Hep-2; RRID: CVCL_1906) using a Total RNA Extraction Kit (Cat. #R6834; Omega, Norcross, GA) according to the manufacturer’s instructions. RNA was extracted from approximately 1 × 10⁶ cells mentioned, and complementary DNA (cDNA) was synthesized using an oligo(dT) primer and HiScript^® Reverse Transcriptase (Cat. #R101; Vazyme, Nanjing, China). The transcriptional level of hTERT was analyzed by RT-PCR, and β-actin was used as an internal control. The primer sequences of hTERT (forward: 5′-CCGTGGTTTCTGTGTGGT-3′; reverse: 5′-GAAGCGGCGTTCGTTGT-3′) and β-actin (forward: 5′-CACGCCATCCTGCGTCTGGA-3′; reverse: 5′-AGCACCGTGTTGGCGTAGAG-3′) were designed referring to the human and porcine messenger RNA (mRNA) homologous sequence. The amplified PCR products of hTERT and β-actin were 661 and 100 base pairs, respectively.

2.8 Western blot analysis

As previously described (Su et al., 2013), no less than 5 × 10⁶ primary PBECs, hTERT-PBECs, and HEp-2 cells (positive control) were washed twice with PBS and collected before using a total protein extraction kit (Cat. #E211; Vazyme, Nanjing, China). Cell proteins were rapidly frozen in liquid nitrogen and stored at −70°C until protein concentrations were measured. Approximately 15 µg of total protein was resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane (Millipore). The membranes were washed three times with PBS and then blocked with 5% nonfat milk (Yili Industrial Group Co., Ltd., Hohhot, China) in Tris-buffered saline (TBS) containing 0.5% Tween 20 (TBST) at 37°C for 2 hr with shaking. Subsequently, the membranes were incubated with anti-hTERT (Cat. #sc-377511; RRID: AB_11150127; Santa Cruz Biotechnology, Dallas, TX) or anti-β-actin (Cat. #BS6007MH; Bioworld, St. Louis, MN) at 1:600 in blocking solution at 37°C for 1.5 hr. After three washes using TBST, the membranes were incubated with secondary antibody (horseradish peroxidase–labeled goat anti-mouse IgG (H + L); Dingguo, Beijing, China) at 1:10,000 in blocking solution at 37°C for 1.5 hr followed by incubation with chromogenic reagents (Tiangen, Beijing, China).

2.9 Telomerase activity assay

Telomerase activity analysis was performed according to a previous study with some modifications (He et al., 2009). Briefly, primary PBECs, hTERT-PBECs, and HEp-2 cells (positive control) were assayed with a TeloTAGGG Telomerase PCR ELISAPLUS Detection Kit (Cat. #11854666001; Roche, Pleasanton, CA) according to manufacturer’s instructions. In addition, HEp-2 cells were treated for 10 min at 85°C (named as H-HEp-2) to inactivate telomerase protein for use as a negative control. The concentrations of cells were adjusted to the same concentration, and cell extracts were separately prepared. The absorbance was determined at 450 nm with a reference wavelength of 690 nm. Each test was performed in triplicate. It was considered there was no telomerase activity when the absorbance was less than 0.2 according to a previous study (He et al., 2009).

2.10 Karyotype analysis

The number of chromosomes in hTERT-PBECs was determined following standard methods (Ha Thi et al., 2014; Wang et al., 2014). Briefly, cells were initially cultured in a T25 cell culture flask (Thermo Fisher Scientific, Waltham, MA) for more than 24 hr, and cells were then treated with colcemid (Aladdin, Shanghai, China) at a final concentration of 0.2 μg/ml at 37°C for 5 hr. Subsequently, cells were trypsinized, centrifuged, and incubated in 0.075 M KCI at 37°C for 20 min. Cells were then fixed with a freshly prepared ice-cold acetic acid–methanol (1:3, vol/vol) solution and subjected to blowing and careful mixing with straws before collection by centrifugation at 1,000 rpm. This step was repeated, and two or three drops of dispersed cell suspension were smeared on each ice-cold slide from a 0.5 m height and then air-dried. The chromosomes were stained with 4% Giemsa solution in Gurr’s buffer, and the number of chromosomes in metaphase (n = 100 cells) was observed microscopically.

2.11 Relative quantitative RT-PCR analysis

Total cellular RNA was isolated from both primary PBECs and hTERT-PBECs after direct cell lysis using the Total RNA Extraction Kit (Cat. #R6834; Omega, Norcross, GA). No less than 1 μg of total cellular RNA was reverse-transcribed in a 10-µl reaction volume using the HiScript II Q RT SuperMix for quantitative PCR (+gDNA wiper; Cat. #R223; Vazyme, Nanjing, China) before running on an ABI 7500 Real Time PCR System using the HiScript II One Step qRT-PCR SYBR^® Green Kit (Cat. #Q221; Vazyme, Nanjing, China). RT-PCR was performed using cDNA under specific conditions for a total of 16 genes. These selected genes are involved in cell cycle (sox2, sox17, ccna1, ccna2, ccnb1, and ccnb2; H. L. Chen, Chew, Packer, & Gallo, 2013; Ha Thi et al., 2014; Klajic et al., 2014; Lange, Keiser, Wells, Zorn, & Whitsett, 2009; Tompkins et al., 2011), innate immunity (IL-6, IL-8, CSF2, JNK1, CXCL2, and STAT3; Lamontagne, Mell, & Bouchard, 2016; Machugh et al., 2012; Nalpas et al., 2013), and oxidative stress response (DUOX1, DUOX2, SOD1, and GPX1; Brockmeier et al., 2017; X. Chen et al., 2017; Nicholson et al., 2012). Glyceraldehyde 3-phosphate dehydrogenase and β-actin were used as the internal controls. PCR primers used in the quantitative assays are listed in Supporting Information Table S1. The fold change of mRNA expression in primary PBECs and hTERT-PBECs was determined using the  method.

2.12 In vitro soft agar assay

The soft agar assay was performed as previously described (He et al., 2009). Briefly, a bottom layer of 0.5% agar gel with 1.5 ml of culture media was prepared in a 24-well plate and stored overnight at 4°C to solidify the gel. Subsequently, hTERT-PBECs were trypsinized and re-suspended at concentrations of 5 × 10³, 1 × 10⁴, and 2 × 10⁴ cells/ml in DMEM–F12 medium containing 2% FBS. One milliliter of cell suspension (final concentration: 0.33% agar) was added into 1.5 ml of 0.5% agar. Cells were overlaid onto the solidified lower layer and incubated at 37°C in a humidified atmosphere with 5% CO₂ for 4 weeks. HEp-2 cells served as positive controls, and colonies in the plate were observed under a microscope for over 1 month. The presence of even a single colony indicated that cells were capable of anchorage-independent growth.

2.13 In vivo nude mice tumorigenicity assay

To evaluate the functional tumorigenicity of immortalized cells in vivo, a tumorigenicity assay was performed by subcutaneously injecting hTERT-PBECs into the left flank of 4-week-old nude mice (one site per mouse at 1.5 × 10⁶ cells per mouse for a total of three mice). As a positive control, HEp-2 cells at the same concentration were injected into the flanks of another three nude mice. Animals were housed under specific pathogen-free conditions and were observed weekly for tumor appearance for 2 months before they were killed.

2.14 Indirect immunofluorescence assay

To test the susceptibility of immortalized cells to swine respiratory-related viruses, SIV of H3N2 subtype and PCV2 were selected. Mardin Darby Canine Kidney (MDCK) cells and hTERT-PBECs were both infected and mock-infected with influenza A/swine/Shandong/3/2005(H3N2) at the multiplicity of infection (MOI) of 0.1, and Porcine Kidney 15 cells and hTERT-PBECs were infected and mock-infected with PCV2 isolate Haian (Genbank accession number FJ712216.1) at the MOI of 0.1. The procedure of indirect immunofluorescence assay was performed as previously described (Ding et al., 2017). Briefly, cells mentioned above were plated into 24-well plates at a density of 5 × 10⁴ cells per well in prior night before virus infection. After infected with influenza and PCV for 24 and 48 hr, respectively, cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min at RT. Cells were permeabilized with 0.2% Triton-X100 in PBS for 2 min at RT. After blocking with 5% bovine serum albumin for 2 hr at 37℃, the cells were exposed to positive pig serums for whole virus against influenza and PCV2 produced in our lab at a 1:500 dilution for 1 hr at 37°C. After three times washing with PBS, cells were incubated with FITC-conjugated goat anti-pig IgG (H + L; Cat. #ab6911; Abcam, Cambridge, MA) at a 1:1,000 dilution for 1 hr at 37°C. Cell nuclei were stained with DAPI for 5 min at RT before cells were visualized using fluorescence microscopy (Zeiss, Tokyo, Japan).

2.15 Statistical analysis

Data from quantitative real-time PCR analysis by using ABI 7500 Real Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA) and cell growth curves were analyzed by SPSS Statics software v20.0 developed by (IBM, LLC, Armonk, NY; http://www-01.ibm.com/software/uk/analytics/spss/; RRID: SCR_002865), and cell cycle analysis was analyzed by FlowJo software v7.6 developed by (FlowJo, LLC, Ashland, OR; https://www.flowjo.com/solutions/flowjo; RRID: SCR_008520). Statistical differences were assessed via analysis of variance, and p < 0.01 was considered to be significant.

3.1 Isolation, culture, and characteristics of primary PBECs

After the piglet was euthanized and disinfected with 75% alcohol, blood was drained from the abdominal incision, and a throat incision was made to expose the trachea to avoid contaminating the lung tissue. As shown in Figure 1a, all of the larynx and trachea tissues connecting the intact lung tissue were obtained. Isolated tissue was put in a tray before transferring to biosafety cabinet for further bronchus isolation. Figure 1b shows the isolated bronchus in a sterilized glass dish after gentle removal of the entire lung tissue around the trachea with microsurgical forceps and scissors. The isolated bronchial tissues were digested and cultured in medium with 10% FBS, and the supernatant was changed to medium with 2% FBS together with supplemental regulatory epithelial factors. Finally, few colonies of primary PBECs at passage 1 were grown to cell confluency greater than 80%. As shown in Figure 1c, primary cells displayed the cuboidal phenotype typical of mammary epithelial cells and grew in close contact to each other. Cytokeratin 18 immunostaining was performed after passage 1 primary cells were passaged to confirm that the primary cells isolated were indeed bronchial epithelial cells rather than nonepithelial cells, such as smooth muscle cells. The primary PBECs were stained green, and all cell nuclei were stained in blue using DAPI (Figure 1d), and the purity of primary PBECs was 92.6 ± 5.6%. For the negative control, only very limited cells (primary PBECs) were stained in green, whereas the majority cells (primary smooth muscle cell) were only blue-stained within cell nuclei.

3.2 Establishment of stable hTERT-PBECs

The primary PBECs could not be cultured for longer than six passages due to their reduced and short proliferative potential. Therefore, primary PBECs were transfected with the pEGFP-hTERT recombinant vector to overexpress hTERT and ultimately establish the stable hTERT-overexpressing cell line, hTERT-PBEC, which could be maintained for more than 60 passages. Because the recombinant plasmid encodes EGFP, it was used as a marker to verify successful transfection of recombinant plasmid by observation with fluorescence microscopy. Fluorescence intensity and quantity of hTERT-PBECs are shown in Figure 2a. Moreover, hTERT-PBECs were also immunostained for the cytokeratin 18 epithelial marker as described above. The hTERT-PBECs retained the characteristics of epithelial cells (Figure 2b), and the purity of hTERT-PBECs at passage 60 was 95.9 ± 3.2%.

3.3 Extended replicative lifespan of hTERT-PBECs

The proliferation potential of immortalized hTERT-PBECs was assessed based on their growth, morphological characteristics, and cell cycle analysis. Growth curves of both primary PBECs and hTERT-PBECs are shown in Figure 3a. Both cell types showed a rapid increase in cell proliferation 48 hr after passage. However, the concentration of primary PBECs was higher than the immortalized hTERT-PBECs from Days 3 to 5 (without significant difference), and the primary cells reached a maximal growth rate at Day 5 before declining. In contrast, hTERT-PBECs reached their peak at Day 7, and the concentration of the hTERT-PBECs was higher than that of primary PBECs from Days 7 to 9 (p < 0.01). However, no obvious differences were found between the morphological characteristics of the primary PBECs (Figure 3b) and the immortalized hTERT-PBECs (Figure 3c). To ascertain whether hTERT expression affected cell replicative lifespan or cell cycle progression, cell cycle distribution was assessed by FCM. Several differences were observed in the cell cycle phases between primary PBECs (Figure 3d) and hTERT-PBECs (Figure 3e) under the same culture conditions. The percentage of hTERT-PBECs in phase S was 11.55% higher than that of primary PBECs, whereas the percentage of hTERT-PBECs in phase G1 was lower than that of primary PBECs. All the above results indicated that the hTERT-PBECs have an extended replicative lifespan and display enhanced proliferative activity compared to primary PBECs.

3.4 Increased telomerase activity of hTERT-PBECs

As demonstrated in Figure 4a, hTERT was expressed in hTERT-PBECs and HEp-2 cells (positive control), but hTERT was not expressed in primary PBECs. Whole-cell extracts of primary PBECs, hTERT-PBECs, and HEp-2 cells were collected and subjected to western blot analysis. hTERT was detected in hTERT-PBECs and HEp-2 cells but was not observed in primary PBECs (Figure 4b). To determine the telomerase activity of primary PBECs and hTERT-PBECs, telomeric replication amplification protocol (TRAP) was performed. As depicted in Figure 4c, hTERT-PBECs showed significantly higher telomerase activity than that of primary PBECs. The absorbance of immortalized hTERT-PBECs was 0.85, whereas the absorbance primary PBECs was 0.15 (<0.2). The absorbance of the H-HEp-2 heat-inactivated negative control sample was 0.03, while the absorbance of the HEp-2 positive control was 0.89. These results suggested that primary PBECs do not express telomerase activity and that the telomerase activity of hTERT-PBECs was relatively higher.

3.5 Stable chromosomes of immortalized hTERT-PBECs

To analyze the karyotype stability of the immortalized hTERT-PBECs, actively proliferating hTERT-PBEC cultures at passage 60 were selected. As depicted in Figure 5a, hTERT-PBECs showed a typical and representative karyotype as well as a normal porcine diploid chromosome number (2n = 38). The chromosomal rearrangements are shown in Figure 5b, including 18 pairs of autosomes and one pair of sex chromosomes, which was consistent with the male sex of the piglet.

3.6 Gene expression analysis in primary PBECs and hTERT-PBECs

To investigate if hTERT introduction affected gene expression, relative quantitative RT-PCR was performed in both primary PBECs and immortalized hTERT-PBECs. Figure 6 shows the expression levels of 16 functional genes. The expression levels of the genes involved in the cell cycle, namely, sox2, ccna1, ccna2, ccnb1, and ccnb2, were significantly upregulated in immortalized hTERT-PBECs compared with primary cells, except for sox17, which was upregulated by only a 0.5-fold change. In contrast, the expression levels of genes involved in immune response and oxidative stress response remained unchanged as the relative mRNA levels ranged from 0.56 to 1.64 indicating less than a two-fold change, which is often used as a criterion for distinguishing significantly differentially expressed genes (Lamontagne et al., 2016; Nalpas et al., 2013).

3.7 Immortalized hTERT-PBECs are not transformed in vitro and do not exhibit a malignant phenotype in vivo

To confirm if immortalized hTERT-PBECs could lead to cellular anchorage-independent growth in vitro, a soft agar colony formation assay was performed. Several colonies were observed in the wells of positive control HEp-2 cells even at the lowest cell density of 5 × 10³ cells per well (Figure 7a), but no colonies were observed in the wells of hTERT-PBECs even after 4 weeks (Figure 7b). To determine whether immortalized hTERT-PBECs are tumourigenic in vivo, a definitive functional assay for tumorigenicity was performed by subcutaneously injecting hTERT-PBECs, primary PBECs, or tumourigenic HEp-2 cells into nude mice. Two months later, tumors were observed in all the three mice injected with HEp-2 cells (positive control; Figure 7c). In contrast, none of the mice injected with hTERT-PBECs (Figure 7d) or primary PBECs (negative control; data not shown) developed tumors. Samples were collected from the injection sites for histopathological examination, which confirmed the above observation results. Normal muscle tissue structures were observed at the site of injection of primary cells (data not shown) or immortalized hTERT-PBECs (Figure 7f,h), whereas pyogenic lesions were formed surrounding the tumor tissue. A large number of infiltrating dense inflammatory cells was observed at the HEp-2 injection site (Figure 7e,g). All these results indicated that immortalized hTERT-PBECs are not transformed and do not have tumourigenic potential.

3.8 Susceptibility of hTERT-PBECs to swine respiratory-related viruses

To test the susceptibility of hTERT-PBECs to SIV of H3N2 subtype and PCV2, indirect immunofluorescence assay was performed. As depicted in Figure 8, microscopic examination showed that immortalized hTERT-PBECs infected with SIV exhibited cytopathic effect (CPE) in some degree such as scattered growth and cell rounding. Similarly, in MDCK-SIV-infected group (positive control), CPE including cell rounding, detachment, cell apoptosis, and death was observed. Cells in SIV mock-infected groups showed normal cell morphology with no obvious CPE. Strong green fluorescent signals were observed in both MDCK cells and hTERT-PBECs infected with SIV, while cells in SIV mock-infected groups demonstrated no specific immunofluorescent signals. For cells infected with PCV2, no CPE was observed in PK15 cells (positive control), while hTERT-PBECs showed kind of CPE including cell rounding and death. However, typical circular green fluorescence signals could be observed in both PCV2-infected PK15 cells and hTERT-PBECs, while no specific green fluorescence signal was observed in PCV2 mock-infected groups. These results suggested that hTERT-PBECs were susceptible to swine respiratory-related viruses such as SIV and PCV2.