ASCs were isolated from lipoaspirate obtained from eight healthy donors of which one was male and seven were female (age between 28–57 years; mean age 43.4). BMSCs were obtained from bone marrow aspirate from eight healthy donors, two males and six females (age 20–46 years; mean age 27.8). The use of ASCs and BMSCs from healthy volunteers was approved by the National Ethical Committee protocol no. H-3-2009-119. All donors agreed to and signed the informed consent. After isolation and cultivation, ASCs and BMSCs were both stimulated with VEGF and compared with untreated control.
Bone marrow preparation and BMSC isolation
50 ml bone marrow aspirate was obtained from the iliac crest by needle aspiration under local anesthesia. The sample was diluted 1:2 with phosphate-buffered saline pH 7.4 (PBS, −Ca2+ -Mg2+, Gibco, Invitrogen, Denmark, cat.no. 10010–015). Mononuclear cells (MNCs) were harvested by gradient centrifugation on Lymphoprep (1077 g/cm3, Medinor, Denmark, cat.no. 1114547), washed with PBS and counted using NucleoCounter® NC-100™ (Chemometec, Denmark) according to manufacturer’s instructions. Primary cell cultures of MNCs were established by seeding 2 × 107 cells/T75-flask (Nunc, Thermo Scientific, Denmark, cat.no. 156494) in complete medium containing Dulbecco’s Modified Eagle Medium, low glucose (1 g/l) (DMEM) supplemented with 25 mM HEPES and L-Glutamin, (PAA Laboratories, Austria, cat.no. E15-808), 10% Fetal Bovine Serum Farma grade (FBS, PAA Laboratories, cat.no. A11-512) and 1% Penicillin/Streptomycin (Gibco, cat.no. 15140–122). The cells were incubated in standard conditions at 37°C in humid air with 5% CO2. The medium was changed 5 days after initial seeding, and subsequently every 3–4 days.
Lipoaspirate preparation and ASC isolation
Approximately 100 ml lipoaspirate was obtained from liposuctions of subcutaneous abdominal fat performed under local anesthesia. The lipoaspirate was washed twice with PBS to remove residual blood. The adipose tissue was digested by incubation with collagenase (Collagenase NB4 (0.6 PZ U/ml, Serva GmbH, Germany) dissolved in HBSS (+CaCl2 + MgCl2, GIBCO, cat.no. 14065–049) diluted to a concentration of 2 mM Ca2+) at 37°C for 45 min. under constant rotation. The collagenase was neutralized with complete medium and the suspension was filtered through a 100 μl mesh (Cell Strainer, BD Falcon, cat.no. 352360); centrifuged at 1200 g for 10 min. at room temperature, re-suspended and counted. MNCs were seeded at a density of 4.5x106 cells/T75-flask in complete medium and incubated at standard conditions. After two days in culture, cells were washed with PBS to remove non‒adhering leukocytes, and complete medium added anew with subsequent change of media every three-four days.
When the culture reached a confluence level of approximately 90%, cells were washed with PBS, detached with 3 ml TrypLE® (TrypLE® Select, Gibco, cat.no. 12563–029) for 10 min. at 37°C and neutralized with 7 ml complete medium. The suspension was centrifuged at 300 g for 5 min. at room temperature, counted, and seeded at 3.5x105 cells/T75-flask for the experimental setup. The cells exhibited stem cell characteristics, by adhering to plastic, expressing stem cell markers and being able to differentiate [22, 29–31].
Stimulation with VEGF
BMSCs and ASCs were cultured in complete medium until their confluence was estimated to be 80%. They were then either kept in complete medium (control), or changed to serum‒deprived medium (DMEM + 2%FBS + 1%P/S) added 50 ng/ml recombinant human VEGF‒A165 (rhVEGFA165, R&D Systems, USA, cat.no. 293-VE-CF). All media was renewed every two-three days and cells were cultured for one week after which they were harvested for further processing.
Nucleic acid extraction
Total RNA was extracted using Qiagen RNeasy® Mini Kit (Qiagen Hamburg GmbH, Hamburg, Germany, cat.no. 74106) according to the manufactures protocol. 1 ml syringe (Omnific‒F 1 ml, B.Braun Melsunger AG, Germany, cat.no. 300013) was used to homogenize the lysed cells before applying the Qiagen protocol. RNA purity was measured using NanoDrop® 1000 Spectrophotometer (Thermo Scientific, MA, USA), and the eluate was stored at −80°C. RNA purity and integrity were confirmed using RNA Nano Chips (Agilent, Santa Clara, CA, cat.no. 5067–1521) and the Agilent 2100 Bioanalyzer by following instructions of the Agilent RNA 6000 Nano Kit.
The cDNA synthesis reaction was prepared using AffinityScript (Stratagene, Denmark, cat.no. 600559) in an eight‒tube strip (0.1 mL, MicroAmp™, Appied Biosystems®, Invitrogen, cat.no. 4358293) on ice. The total reaction volume was 20 μl with 0.5 μg RNA, 10 μl cDNA synthesis master mix, 3 μl Oligo (dT) primer, 1 μl AffinityScript RT RNase block enzyme mixture, and RNAse-DNAse free water to 20 μl total volume. Tubes were closed with caps (Applied Biosystems, cat.no. N801-0535) and the reactions were performed with an initial stage of 25°C for 5 min., 42°C for 45 min. and 95°C for 5 min. (Veriti 96 well fast thermal cycler, Applied Biosystems model no. 9901). Following synthesis, the cDNA was stored in aliquots at −20°C.
Quantiative real-time PCR
qPCR was performed in triplicate per donor for each group within the same qPCR-run. A calibration curve was run simultaneously on the RG candidate tested on the donors. Only data obtained from runs fulfilling the same criteria for efficiency and correlation coefficient as the primer verification was included for analysis. The Cq threshold was set to the value of 0.1 for all qPCR runs. Brilliant II SYBR®green QPCR Low ROX master mix (Agilent, cat.no. 600806) was used with a total reaction volume of 25 μl in 96-well optical reaction plates (Agilent, cat.no. 401333) with 5 μl of diluted cDNA. The plate was sealed with optical plastic caps (Agilent, cat.no. 401425). qPCR was performed using Mx3000 (Stratagene) and the results were collected using Mx3000 version 4.0 software for Windows (Stratagene). The reaction was initiated by heating to 95°C for 10 min., followed by 40 cycles elongation at 60°C for 1 min. and denaturation at 95°C for 30 sec.
To verify the chosen RGs, a normalization experiment was set up. vWF was used as target gene, and normalized to different combinations of RG candidates. The level and the standard deviations of the fold changes between VEGF treated cells and controls were compared for normalization to different RG combinations. The fold changes in vWF expression between VEGF treatment and controls were calculated with the 2-∆∆Cq-method.
A 3% 3–1 NuSieve agarose gel (Lonza, Switzerland, cat.no. 50090) was made according to the laboratory protocol with 1x TAE buffer diluted from 50x TAE buffer (Qiagen, cat.no. 129237). For visualization of qPCR products, 10% non-toxic GelStar Nucleic Acid Gel Stain (Lonza, cat.no. 50535) was added to the melted agarose before pouring the solution into a plastic well with combs. After gel solidification the combs were removed and the solid gel transferred to an electrophoresis tank (BioRad, CA, USA) and covered with 1 x TAE buffer. For every 8 μl PCR product, 2 μl Gelpilot 5x loading dye (Qiagen, cat.no. 1037650) was added and the 10 μl sample is loaded by directly injecting into the wells. A negative control was included to show no contamination of the product. Gelpilot 1 kb ladder (Qiagen, cat.no. SM0318) were used. The gel was run between 70 and 100 volt in 45 min.– 60 min. and visualized under ultraviolet light.
Step 1. Cq values were plotted in Microsoft Excel and assessed visually for outliers in the lower end.
For further analysis, we used the GenEx software (MultiD Analyses AB, Sweden), and more specifically its algorithms Normfinder and geNorm. In short, Normfinder selects the most suitable RG based on the standard deviations (SDs) for intragroup and intergroup variation, while geNorm compares the RGs pair-wise and performs sequential elimination resulting in the best correlated RGs. Average Cq-values of the technical triplicates were used for analysis.
Step 2. For each cell type, the mean Cq-values of all RG candidates from all donors were entered in GenEx for the treatment and control groups.
Step 3. The function Normfinder was performed taking group classification of treatment into account, in order to verify that the candidate RGs were stably expressed and not regulated by treatment. The RG candidates with SDs reaching or exceeding 0.2 were removed from the panel by inactivation in the Data Manager of GenEx before proceeding to the next step. Normfinder was run again, without taking groups into account to give the best estimate of the genes’ stabilities and an assessment of the optimal number of RGs. Lastly, geNorm was run to determine the best correlated RGs. The ∆∆Cq-method was used when assessing the effect of VEGF treatment on the expression of vWF normalized to various RGs, with subsequent use of paired t-test and Pearson’s Correlation in IBM SPSS. Results were considered significant at p-values below 0.05.