Cells culture of MSCs from transgenic pigs
This study was carried out in strict accordance to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal cells were retrieved under the approved animal protocols by the Institutional Animal Care and Use Committee of National Taiwan University (NTU107-EL-00128).
Cells in the study were divided into three groups: Group 1, enhanced green fluorescent protein pig MSCs (eGFP-pMSCs) from transgenic pigs (n=6); Group 2, in vitro eGFP plasmid transfected pig MSCs (n=6); and Group 3, pig MSCs without transfection (n=6).
In Group 1, passage-12 eGFP-pMSCs from the Department of Animal Science and Technology in National Taiwan University were cultured in minimum essential medium alpha (MEM-α) (Gibco) supplemented with 10% fetal bovine serum (Hyclone) and antibiotic solutions (100 U/mL penicillin, 100 μg/mL streptomycin, 50 μg/mL gentamicin and 250 ng/mL fungizone) (Gibco). The cells were maintained in a humidified 37℃ incubator supplied with 5% CO2.
Isolation and culture of pig mesenchymal stem cells
In group 2 and group 3, bone marrow aspirates of pig mesenchymal stem cell (pMSCs) were obtained from adult domestic pigs (age 15 months; weight 120 kg) from the Department of Animal Science, National Taiwan University (Taipei, Taiwan). Anesthesia was induced with ketamine (10 mg/kg of BW, Sigma-Aldrich, St. Louis, MO) and maintained with inhalation of anesthetic halothane (Sigma-Aldrich, St. Louis, MO). The tibial area was prepared and sterilized, and approximately 5 mL of bone marrow was aspirated into a syringe containing 6,000 U of heparin. Bone marrow mononuclear cells were obtained by negative immunodepletion of CD3+, CD14+, CD19+, CD38+, CD66b+, and glycophorin A+ cells using a commercially available kit (RosetteSep, Stem Cell Technologies, Vancouver, British Columbia, Canada), according to the manufacturer’s instructions. After a 20-minute incubation at room temperature, the cell-antibody mixture was diluted with twice the volume of phosphate- buffered saline (PBS) supplemented with 2% fetal bovine serum (FBS; Hyclone, Logan, UT) and 1 mM EDTA (Invitrogen), layered over an equal volume of Ficoll-Paque medium (1.077 g/cm 3; Amersham Bioscience), and centrifuged at 300 × g for 30 minutes at room temperature. Enriched cells were harvested from the buffer coat and washed twice with control medium, consisting of minimum essential medium (MEM) α (Sigma-Aldrich) supplemented with 20% FBS (Hyclone), 2 mM l-glutamine (Invitrogen), 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Invitrogen). The cells were maintained in a humidified 37 ℃ incubator supplied with 5% CO2 as group 1.
Quantitative assessment of cell viability after transfection
In group 2, plasmid enhanced green fluorescent protein gene (pEGFP-N1) was
transfected into pMSCs with TransIT®-2020 (Mirus, Madison, WI, USA) according
to the manufacturer’s instructions. For the cell viability experiments, we prepared
TransIT®-2020/pEGFP pMSCs in 6-well plates, removed the growth medium and
washed them with one time PBS. On day 1, the cells were transfected with
different ratios of transIT-2020: pEGFP-N1 in 1:1, 2:1, 3:1 and 4:1. Either 1 μg or 2.5
μg of pEGFP-N1 were compared. On days 2, 3 and 4, 800 μL of growth medium and
20 μL of cell counting kit-8 (CCK-8; Enzo Life Sciences, East Farmingdale, NY, USA)
solution was added to each well of the plate. The plate was then incubated for 1 hour
37 °C in 5% CO2. Absorbance was measured at 450 nm using a microplate reader
(TECAN-Infinite 200 PRO, New Taipei City, Taiwan).
In vitro transfection eﬃciency of pMSCs
The pMSCs were seeded onto 6-well plates at a density of 1×106 cells/well and cultivated in 2 mL of DMEM with 10% FBS on day 0. On day 1, the cells were treated with different ratios of TransIT®-2020 and pEGFP-N1. After days 2, 3 and 4, the pMSCs were harvested and mixed with 200 μL of 4% paraformaldehyde on ice for 15 minutes. The pMSCs were then centrifuged at 1500 rpm for 5 minutes at 4°C and re-suspended with 300 μL of PBS in round bottom tubes (Corning Inc. Kennebunk, ME, USA). The expression of GFP in the cells were examined using a fluorescence-activated cell sorting (FACS) Calibur flow cytometer (Becton Dickinson, Heidelberg, Germany). The transfection rate was evaluated using Becton Dickinson Cell Quest software. Different proportions of reagent and plasmid were tested to obtain the highest transfection concentration.
Fluorescence evaluated by image and FACS after osteo-induction in monolayers
To promote cell differentiation, all groups received 0.5 mL/well of osteogenic induction medium (OIM). The OIM medium was composed of complete MEM-α medium enriched with 10−7 M dexamethasone (Sigma), 10 mM β-glycerophosphate (Sigma) and 50 μg/mL ascorbic acid (Sigma).(19)(20) The OIM medium was replenished every two or three days for a total of 7 days.
Fluorescent imaging and quantification in monolayers
Three groups of pMSCs were seeded at a density of 1×105 cells/well onto 48-well plates, and cultured in 2 mL of DMEM with 10% FBS. After treatment with OIM for a total of 7 days, fluorescent microscopy was acquired on a BioRad MRC 600 microscope from day 7 to day 28. Dishes were washed twice with PBS, ﬁxed in 4% v/v formaldehyde (methanol-free; Polyscience) for 15 minutes, permeabilized with 0.1% v/v Triton X-100 for 5 minutes, and incubated in 10 mg/mL bovine serum albumin and 100 g/mL RNAse for 45 minutes at room temperature. F-actin ﬁlaments were stained with Alexaﬂuor-conjugated phalloidin (Molecular Probes) for 20 minutes and nuclei were counter-stained with 10 g/mL propidium iodide (Sigma) for 10 minutes. Finally, samples were washed with PBS and mounted in Vectashield®. The quantitative distribution of the green fluorescent cells was analyzed using NIH Image J software (National Institutes of Health, USA) and presented as the modified integrated density (Modified IntDen). The modified integrated density was calculated based on the following equation: [Selected Integrated Density - (Area of Selected Cell × Mean Fluorescence of Background Beadings])(19). The images merged form light microscope and fluorescent microscope samples were also investigated.
Flow cytometry analysis of pMSCs in monolayer culture
After treatment with OIM for a total of 7 days, all staining procedures were conducted in non-adherent, round-bottom 48-well plates. Cells in the staining buffer were transferred to each well for corresponding antibodies and for controls including an unstained sample and an isotype control antibody. Samples were transferred to polystyrene FACS tubes (Thermo Fischer) and washed twice by centrifuging at 350g for 5 min and resuspending in 4 mL of PBS (Gibco). Cells were finally resuspended in 250 mL of PBS and retained on ice with protection from light until flow cytometry analysis. Flow cytometry was performed on a FACS Calibur instrument (BD Bioscience, Cambridge, UK).
Implantation of pMSCs into 3-D scaffolds
A hemostatic gelatin sponge, SpongostanTM (Ferrosan Medical Device, MS0003, thickness 0.1 cm), was used as the 3D scaffold.(19) To perform transplantation, the scaffolds were cut into disks with a diameter of 0.8 cm, sterilized by 75% (v/v) ethanol and washed three times with phosphate-buffered saline. The sterile scaffold disks were then immersed in Opti-MEM medium (Gibco) before use. The sterile scaffold disks were individually placed onto the wells of a 48 well-plate. 5×104 cells/disk in 50 μL of medium were seeded throughout the surface of the scaffold disk and incubated for 3 hours to allow cell attachment before the addition of medium.
Cell distribution and morphology analysis after osteogenic differentiation
The above three groups then received 0.5 mL/well of osteogenic induction medium (OIM) to promote cell differentiation. The OIM medium was composed of complete MEM-α medium enriched with 10-7 M dexamethasone (Sigma), 10 mM β- glycerophosphate (Sigma) and 50 μg/mL ascorbic acid (Sigma).(19) The OIM medium was replenished every two or three days for a total of 7 days. The constructs in each time point were washed twice with phosphate buﬀered saline (PBS; GIBCO™, Invitrogen Corp., Carlsbad, CA) and ﬁxed in 1.5% v/v glutaraldehyde in 0.14M sodium cacodylate (pH 7.4) for 30 mins at room temperature. Dehydration was performed by sequential immersion in serial diluted ethanol solutions of 50, 60, 70, 80, 90, and 100% v/v. The samples were then transferred to hexamethyldisilazane and air-dried at room temperature overnight. The cell distribution and morphology were analyzed by scanning electron microscopy (SEM) (Hitachi, SU8220).
Quantification of fluorescent in 3-D scaffolds
All samples were washed twice with PBS, ﬁxed in 4% v/v formaldehyde (methanol-free; Polyscience) for 15 min, permeabilized with 0.1% v/v Triton X-100 for 5 minutes, and incubated in 10 mg/mL bovine serum albumin and 100 g/mL RNase for 45 minutes at room temperature. F-actin ﬁlaments were stained with Alexa Fluor-conjugated phalloidin (Molecular Probes) for 20 minutes, and nuclei were counterstained with 10 g/mL propidium iodide (Sigma) for 10 minutes. Finally, the samples were washed with PBS and mounted in Vectashield®. Fluorescent images were recorded by confocal laser scanning microscopy (CLSM) (Leica, TCS SP8X). The quantitative distribution of fluorescent cells was analyzed with the same method illustrated in the monolayer section.
Osteogenic profiles in 3-D scaffolds
Alkaline phosphatase (ALP) staining and quantification
For staining on days 7 to 28, cells on the scaffold were washed twice with PBS and fixed for 5 min using 60% (v/v) citrate solution comprising 0.6 mL citrate concentrate solution (Sigma) in 29.4 mL deionized water and 20 mL acetone. The scaffolds were rinsed with deionized water and overnight stained with alkaline phosphatase staining solution. Alkaline phosphatase staining solution was a mixture of one part of naphthol AS-MX alkaline solution (Sigma) and 24 parts of fast violet stain solution (Sigma). On the next day, the staining solution was removed, and the scaffolds were washed five times with deionized water. Images of the stained discs were captured with a digital camera (Canon, PowerShot SX50 SH). The osteogenic quantification is assessed using an ALP assay kit (BioVision K412-500) after 7 to 28 days. The cells were washed with ice-cold PBS twice, and lysed using 300 μL RIPA (Radioimmunoprecipitation assay) lysis buffer (Sigma-Aldrich Corp., St Louis, MO, USA) for 5 min on ice. The cells were then rapidly scraped from the plate, and the cell lysates/RIPA buffer were transferred to a 1.5-mL microcentrifuge tube on ice for 20 min, followed by centrifugation at 8000 × g for 10 min at 4°C. The supernatant was then added to a new 1.5-mL microcentrifuge tube and stored at -20 °C., and then the volume was brought to 80 µL. Next, 50 µL of the 5 mM p-Nitrophenyl Phosphate (pNPP) solution was added to each well containing the test samples. The aliquots were incubated for 60 min at 25°C while protected from light. Subsequently, 20 µL of the stop solution was added to terminate the ALP activity in the sample. The absorbance at 405 nm was measured with a spectrophotometer (UV-Vis 8500). Then, the ALP activity was calculated using the following formula: [(Optical Density – Mean Optical Density of the Control Wells) × Total Volume × Dilution)/(18.45 × Sample Volume)].
Alizarin red S (ARS) staining and quantification
At days 7, 14, 21 and 28, cells cultured on the scaffolds were washed twice with PBS and fixed with 4% paraformaldehyde for 15 min. The fixative was removed and the discs with cells were rinsed with deionized water. The discs were stained with 2% ARS staining kit (Sigma-Aldrich Corp., St Louis, MO, USA) for 5 min to stain the calcium deposition on the discs. The staining solution was discarded, and the discs were carefully washed with deionized water until the excess stain was removed. The staining results were captured with a digital camera. To quantify the calcific stain on each scaffold disc, 1 mL/disc of 10 wt% cetylpyridinium chloride (Sigma-Aldrich Corp., St Louis, MO, USA) was added to the discs. The discs were left on an orbital shaker at 60 rpm for 1 h to completely resolve the dye from the discs. Finally, 100 μL of the dissolved solution from each disc was placed into a well of a 96-well plate and its absorbance measured at 540 nm with ELISA reader.
Immunohistochemistry (IHC) of osteogenic related markers
Histological slides of all three groups in different time points were incubated with citrate buﬀer (Dako, Glostrup, Denmark) at 60°C for heat-induced epitope retrieval and blocked with 1% hydrogen peroxide/methanol (Sigma-Aldrich, St Louis, MO, USA) for 30 min at room temperature. Subsequently, they were incubated overnight at 4°C with primary antibodies: collagen type I (Col-I) (1:1000, sc-59772, Santa Cruz Biotechnology, CA, USA) and osteocalcin (OC) (1:1000, sc-365797, Santa Cruz Biotechnology, CA, USA). The color reaction was developed with ready-to-use 3, 3’-diaminobenzidine (Dako Liquid DAB) color solution. The slides were counterstained with hematoxylin and visualized by a light microscope (Nikon Eclipse E600, Tokyo, Japan). The negative control used PBS instead of the primary antibody, and counterstaining was performed with hematoxylin.
All statistical analysis was performed using Student’s t-test and two-way ANOVA. Analysis of variance (ANOVA) was performed by using GraphPad Prism 5 software. Differences with a p value of < 0.05 were considered statistically significant.