Cells preparation was performed as previously described with minor modifications . Adipose tissue (approximately 15-20 grams) was collected from intra-abdominal fat deposit or supragluteal subcutaneous region of adult horses at the abattoir immediately after slaughtering. Tissue was minced into small pieces under sterile conditions and washed several times with phosphate buffer saline solution (PBS) containing penicillin (1,000 IU/ml) and streptomycin (100 mg/ml) (Sigma-Aldrich Corp., ST. Louis, MO, USA). The tissue was then digested with 7.5 mg/ml type I collagenase (Sigma-Aldrich) in Dulbecco's modified Eagle's medium low glucose (DMEM) (Lonza, Inc. Rockland, ME, USA) medium under mild shaking for 1 hour at 37°C. After digestion, the remaining tissue fragments were removed, cells suspension was filtered through a 50 μm nylon mesh and centrifuged at 250 × g for 10 minutes. The supernatant was removed and the cell pellet resuspended in DMEM containing 10% v/v fetal bovine serum (FBS) (Lonza), 1,000 IU/ml penicillin and 100 mg/ml streptomycin. Finally cells were diluted to 5.0 ×104 cells/cm2 in culture medium, seeded in 25 cm2 tissue culture flasks and incubated at 37°C in 5% CO2. After 48 h the medium was changed with fresh medium to remove non adherent cells; thereafter, medium was renewed every 3 days. The first subculture was performed after 7-10 days. Subsequent subcultures were performed when cells reached about 80% confluence.
Constructs design and generation
pHSV-TK luciferase, pCMV-luciferase, pSV40-luciferase, pPGK-luciferase and pTK-renilla were obtained from Promega (Promega Corp., Madison, WI, USA). pBoHV-4 IE2 was constructed by sub-cloning a PCR fragment, corresponding to the BoHV-4 Immediate Early 2 (IE2) promoter in front of the luciferase reporter gene contained into the pGL3-basic vector as previously described . To obtain pEGFP-IRES-neo, the EGFP ORF was cut with NheI and BamHI and legated into the NheI/BamHI sites of pIRESneo 2 (Clontech Laboratories Inc., Mountain View, CA, USA). Whereas, to get pCMV-EGFP/neo, neo ORF was amplified with a couple of primers (neo/KpnI/sense, 5'-cgg ggt acc atg att gaa caa gat gga ttg-3' and neo/KpnI/antisense, 5'-cgg ggt acc tca gaa gaa ctc gtc aag aag-3') and the resulting amplicon sub-cloned in frame to the carboxy-terminal of EGFP into the pEGFP-C1 vector (Clontech Laboratories). Whereas, pSecE2 was obtained sub-cloning the Bovine Viral diarrhea Virus glycoprotein E2 into the pSecTag2 vector (Invitrogen Corp., Carlsbad, CA, USA) as previously described .
Dual luciferase reporter assay
Confluent EADSCs in twenty-four well plates were co-transfected with 0.5 μg of reporter construct or 0.5 μg pGL3 empty vector, as a negative control and 0.05 μg of pTK-Renilla to normalize the efficiency of transfection, using LTX transfection reagent (Invitrogen) as suggested by the manufacturer. Transfection mixture was prepared in DMEM without serum and antibiotics and left on the cells for 6 h at 37°C, 5% CO2 in air, in a humidified incubator. After 6 h, the transfection mixture was replaced with complete medium (RPMI-1640 (Lonza), 10% FBS, 50 IU/ml of penicillin, 50 μg/ml of streptomycin and 2.5 μg/ml of Amphotericin B (Sigma-Aldrich)) and left to recover for 18 h at 37°C, 5% CO2 in air, in a humidified incubator. Twenty-four hours post transfection, cells were analyzed for Luciferase expression.
Luciferase reporter assay was performed with a Dual Luciferase Reporter Assay System kit (Promega) with minor modifications. Following treatments, cells were washed with PBS, lysed with 100 μl of lysis passive buffer by freeze-thawing at -80°C. 10 μl of the cell lysate were added to 50 μl of LAR and Luciferase activity was determined with a PerkinElmer Victor3 Multilabel Counter (PerkinElmer, Waltham, MA, USA), according to the manufacturer's specifications. Individual assays were normalized for Renilla activity with a second reading, adding 50 μl of Stop & Glo substrate (Promega). Experiments were performed with 4 replicates at each time point and each experiment repeated three times. Statistical differences were tested by ANOVA.
Lypofection: was performed using LTX transfection reagent (Invitrogen) as suggested by manufacturer and described above, the only difference was that reagents were scaled-up to 25 cm2 flasks. Calcium phosphate precipitation: was performed as previously described by others . Electroporation: EADSCs were sub cultured to a fresh 25 cm2 flask when growth reached 90% confluence and were incubated at 37°C in a humidified atmosphere of 95% air-5% CO2. Plasmid DNA (5 μg) in 600 μl DMEM without serum was electroporated (Equibio apparatus, Wolf Laboratories Limit., York, UK), 186 V, 960 μF, 4-mm gap cuvettes (Wolf Laboratories)) into EADSCs. Electroporated cells were returned to a new flask containing complete medium.
EADSCs were electroporated as described above. Electroporated cells were returned to the flask, fed the next day with complete medium supplemented with 400 μg/ml of G418 (Sigma-Aldrich) and split 1:2 when they reached confluence at 2 days post electroporation. Cells were grown till complete selection.
BoHV-4-based vector preparation, infection and recovery assay
Infection of EADSCs was performed with 1 MOI (multiplicity of infection) of BoHV-4EGFPDTK, which was propagated as previously described . A BT cell line (bovine turbinate cells, ATCC, CRL-1390, LGC Standards S.r.l., Milano, Italy) was used to propagate the virus, because of its high sensitivity towards infection of BoHV-4. BT cells were cultured in DMEM containing 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin, and incubated at 37°C in a humidified atmosphere containing 5% CO2. BoHV-4EGFPDTK was propagated by infecting confluent monolayers with 1 of 50% tissue culture infective dose (TCID50) per 5 cells. After 4 days, cultures were frozen and thawed three times to release the virus. After removal of cell debris by low-speed centrifugation, the TCID50 was determined by limiting dilution. Virus stock thus obtained was stored at -80°C.
Cell cultures in six-well plates were infected with virus at a MOI of 10 TCID50/cell for 1 h. After infection, the inactivation of extracellular virus was carried out by low-pH treatment . Briefly, the medium was removed and the plates were washed once with PBS, and then incubated for 2 min with either PBS (control plates) or a buffer (pH 3) containing 40 mM citric acid (Sigma-Aldrich), 10 mM KCl (Sigma-Aldrich), and 135 mM NaCl (Sigma-Aldrich). This procedure completely inactivated the absorbed non-penetrated infectious particles. Cultures were washed with medium and cultured till CPE completely destroyed the cell monolayer, after which 1 ml of the medium was removed from each well and centrifuged for 5 min at 1000 × g in a bench-top centrifuge to remove any cellular debris. TCID50 were determined on MDBK cells by limiting dilution.
Lentiviral vector preparation and transduction of EADSC
Third generation of lentiviral vector were prepared as previously described . Seventy-five cm2 flasks of 293T cells were transfected by calcium-phosphate precipitation based method. Two hours before transfection, growing media (DMEM, 10% FBS, 50 IU/ml of penicillin, 50 μg/ml of streptomycin and 2.5 μg/ml of Amphotericin B) were changed with the fresh ones and four plasmids DNA transfection mix were prepared by adding 9 μg of envelope plasmid (pMD2-VSVG), 12.25 μg of core packaging plasmid (pMDLg/pRRE), 6.25 μg of REV plasmid (pRSV-REV) and 25 μg of transfer vector (pCCLsin18.PPT.Prom.GFP.Wpre). The plasmid solution was made up to a final volume of 500 μl with 450 μl of H2O and 50 μl of 2.5 M CaCl2 (Sigma-Aldrich). The precipitate was formed by drop wise addition of 500 μl of the 2× HBS solution (280 mM NaCl (Sigma-Aldrich), 100 mM HEPES (Sigma-Aldrich), 1.5 mM Na2HPO4 (Sigma-Aldrich), pH 7.12) to the 500 μl plasmid DNA-CaCl2 mix and finally added to the 293T cells. The Ca-Phosphate-DNA precipitate was allowed to stay on the cells for 14-16 h, after which the medium was replaced with 10 ml of fresh medium. Forty-eight hours after changing the media, the cell supernatant containing viral particles was collected, centrifuged at 500 × g for 5 minutes, filtered through a 0. 22 μm nitrocellulose filter, aliquoted and stored at -80°C. Transducing units on ml (TU/ml), were determined by limiting dilution on 293T cells.
EADSCs were plated on 25 cm2 flasks and grown till they reached 80% (~ 106 cells) of confluence. The lentiviral vector was added onto each flask at an MOI of 1. EADSCs were incubated overnight at 37°C and then the media was replaced with 5 ml of fresh media. After 2 days of incubation, cells were analyzed for EGFP expression under a fluorescence microscope or flow cytometry.
Transfected or transduced EADSCs, were then washed twice with PBS and 5 μl of 7-Amino-actinomycin D (7-AAD, BD Biosciences, San Jose, CA, USA) solution were added to asses cell death. Cells were acquired by FACSCalibur flow cytometer (BD Biosciences) equipped with a 15 mW Argon laser using 488 nm band pass filter, calibrated with CALIBRITE 3 beads (BD Biosciences) and FACSComp Software (BD Biosciences). Analysis were performed using Cell Quest software (BD Biosciences).
ADSCs osteogenic differentiation and specific staining
For inducing osteogenic differentiation mesenchymal stem cells were plated at a density of 5,000 cells/cm2 in a 25 cm2 culture flask. When cells reached about 80% of confluence they were treated with dexamethasone (Sigma-Aldrich) (0.1 μM), β-glicerophospate disodium (Sigma-Aldrich) (10 mM) and ascorbic acid (Sigma-Aldrich) (250 μM) in DMEM with 10% FBS. After 10-15 days, osteogenic differentiation was demonstrated by von Kossa silver reduction method and alizarin staining. The differentiated cultures were fixed 20 minutes at room temperature with a PBS 4% paraformaldehyde (Sigma-Aldrich) solution. Cells were washed twice with distilled water and then stained either with alizarin or von Kossa method. For the von Kossa staining, the von Kossa Bioptica kit (04-170801) (Bioptica s.p.a., Milano, Italy) was used, as described by the supplier. Briefly, cells were incubated subsequently in lithium carbonate solution, silver nitrate solution, reducing solution and sodium sulphite solution (provided by the kit) to determine the specific substitution of calcium ions by cationic silver. Nuclei were counterstained with Mayer's Carmalum (Bioptica).
For alizarin red staining, the StemPro Osteogenesis Differentiation kit (Invitrogen) was employed as described by the supplier. Fixed cells were washed twice with distilled water and incubated with 2% Alizarin Red S solution (pH 4.2), provided by the kit, for 2 to 3 minutes. Subsequently, stained cells were washed three times with distilled water and visualized under a light microscope and images were captured for qualitative analysis.
Protein free cell supernatants (20 μl) were electrophoresed through 10% SDS-polyacrylamide gels and transferred to nylon membranes by electro-blotting. Membranes were incubated with monoclonal anti-BVDV-gE2 (clone 157; VRMD, Inc., Pullman, USA), probed with horseradish peroxidase-labeled anti-mouse immunoglobulin antibody (Sigma-Aldrich), and visualized by enhanced chemiluminescence (ECL Kit; PIERCE, Rockford, IL, USA).
Serum neutralization tests were performed as follows. Twenty-five microliters of each serum sample were added to the first line of wells of 96-well plates. Twenty-five microliters of DMEM were added to each well and, for each serum tested, serial two-fold dilutions were made. Positive and negative serum controls were included. Twenty-five microliters of virus suspension containing 100 TCID50 of BVDV was added to each well. After 1 h of incubation at 37°C, 50 μl of a MDBK cell suspension were added to each well and the plates were incubated for 3 days at 37°C in a humidified atmosphere of 95% air-5% CO2. Expression of viral infectivity and serum neutralizing activity through CPE were detected by microscopy and or by crystal violet (Sigma-Aldrich) staining of the cell monolayer. The neutralization antibody titers were expressed as the reciprocal (log 2) of the final dilution of serum that completely inhibited viral infectivity.
Animal handling and care
Horses were cared for and used in accordance with Italian laws for animal experimentation. Horses were maintained at 24°C with a controlled light cycle (12 h of light, starting at 6:00 a.m.) and with food and water ad libitum. Blood samples were obtained and viral injections were performed via the auricular vein at scheduled intervals.