Perinatal cell culture
The present study was approved by the Siriraj Institution Review Board (SIRB). Human UCs and PLs were collected from full-term deliveries and immersed in sterile phosphate-buffered saline (PBS) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin under sterile conditions. The WJ, PLs and UCs were stored and processed separately. The samples were washed twice using PBS prior to their preparation into 2–3-cm2 sections. Perinatal tissues were collected in 15 ml centrifugal tubes containing PBS and then washed twice under centrifugation at 984 × g for 5 min. The pellets from the washing procedure were digested using 0.5% trypsin-EDTA at 37°C for 30 min and placed in 25-cm2 tissue culture flasks in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. The tissues were incubated under 5% CO2 until MSC outgrowth occurred from the explant. The complete medium was changed twice a week. Subpassaging was performed using 0.5% trypsin-EDTA to expand the quantity of cells used in the experiment.
MSC characterization
Criteria
The cells from the BM and perinatal sources were characterized as MSCs based on the following criteria: plastic adherence, positive and negative specific cell surface markers and multi-potential differentiation (13).
MSC surface markers
The cell surface markers for the MSCs were examined using a flow cytometer (FACSCalibur™, Becton-Dickinson, Franklin Lakes, NJ, USA). The immunophenotype of the MSCs was characterized using the following mouse monoclonal antibodies: cluster of differentiation CD34-PE, CD45-FITC, CD73-PE (all purchased from BD Pharmingen, San Diago, CA, USA), CD90-FITC, CD105-FITC and CD106-PE (all purchased from AbD SeroTec, Raleigh, NC, USA). The MSCs between passages 2 and 5 were harvested using 0.5% trypsin-EDTA. A 5×105 cell suspension (50 μl) was incubated at 4°C for 30 min in the dark with mouse anti-human antibodies. Following incubation, the cells were washed twice with cold PBS and centrifuged at 984 × g for 5 min at 4°C. The cells were then fixed with 300 μl 1% paraformaldehyde prior to analysis by a flow cytometer (Becton-Dickinson).
Osteogenic and adipogenic differentiation of MSCs
The MSCs from passages 2–5 were cultured in Advance STEM Osteogenic Differentiation medium (OsteoDiff) for osteoblast differentiation (Hyclone Laboratories, Inc., Logan, UT, USA) and Advance STEM Adipogenic Differentiation medium (AdipoDiff; Hyclone Laboratories) for adipocyte differentiation. The MSC cells were seeded at a density of 1×105 cells/ml and maintained in each differentiation medium. The media were changed twice a week. The differentiated conditioned MSCs were maintained for 3 weeks, at which time their osteogenic and adipogenic properties were evaluated using immunocytochemical staining with alkaline phosphatase and Oil Red O (Sigma-Aldrich, St. Louis, MO, USA).
In vitro treatment with 5-azacytidine
The MSCs obtained from the BM, UC, PL and WJ during passages 2–5 were used in this experiment. The MSCs were seeded at densities of 1×105 cells in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin for 24 h. The treatment group was treated with 10 μM 5-azacytidine (Sigma-Aldrich) in complete medium for 24 h subsequent to which it was changed into complete medium for 3 weeks. The morphology, growth rate and viability were then observed.
RNA preparation and real-time RT-PCR
RNA was collected from the control and 5-azacytidine-treated groups at weeks 1 and 3. The total RNA was isolated from the cells using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and quantified by spectrophotometry at 260 nm. The total RNA (2 μg) was used for reverse transcription with the first-strand cDNA synthesis kit (Invitrogen Life Technologies). Real-time PCR was performed for 40 cycles using the ABI 7500 real-time PCR system (Applied Biosystems, Bedford, MA, USA) and analyses for cardiac- and myogenic-specific genes were performed using the following primers: α-cardiac actin (260 bp), sense 5′-TCTATGAGGGCTACGCTTTG-3′ and antisense 5′-GCCAATAGTGATGACTTGGC-3′; Troponin T (TnT; 225 bp), sense 5′-AGAGCGGAAAAGTGGGAAGA-3′ and antisense 5′-CTGGTTATCGTTGATCCTGT-3′; Nk×2.5 (136 bp), sense 5′-CTGCCGCCGCCAACAAC -3′ and antisense 5′-CGCGGGTCCCTTCCCTACCA-3′; and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 139 bp) sense 5′-GTCAACGGATTTGGTCGTATTG-3′ and antisense 5′-CATGGGTGGAATCATATTGGAA-3′. Each cycle consisted of denaturation at 95°C for 10 sec, annealing at 60°C for 10 sec and extension at 72°C for 40 sec. The products were quantified by the comparative Ct method. Samples were compared with the control group, normalized against GAPDH and presented as fold change increases.
Immunofluorescence staining
The MSCs from passages 2–5 were cultured on chamber slides (Lab-Tek, Thermo Fisher Scientific, Rochester, NY, USA) at 1×105 cells/well with DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. The 10 μM 5-azacytidine treatment and control groups from days 3 and 7 were used for immunofluorescence analysis. The cells were washed twice with PBS prior to fixation using 4% paraformaldehyde in PBS at 4°C for 30 min. Non-specific binding was blocked using 4% bovine serum albumin (BSA), followed by incubation with purified goat polyclonal TnT and mouse monoclonal GATA4 primary antibodies (both Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The samples were then washed twice with PBS and overlaid with rabbit anti-mouse FITC- (Millipore, Billerica, MA, USA) and donkey anti-goat PE-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc.) for 30 min at 4°C in the dark. The samples were washed twice, followed by DAPI counterstaining and mounting using ProLong Gold Antifade (Invitrogen Life Technologies).
Statistical analysis
The data were analyzed using SPSS version 11.5 (SPSS, Inc., Chicago, IL). Data are expressed as the mean ± SEM. The significance was analyzed using the Mann-Whitney U test. P<0.05 was considered to indicate a statistically significant difference.