Illumina HiSeq 2500 sequencing; Transcriptomic analyses of adipocyte differentiation from human mesenchymal stromal-cells (MSC)
Transcriptomic analyses of adipocyte differentiation from human mesenchymal stromal-cells (MSC)
Human mesenchymal stromal-cells were obtained from bone marrows of three donors, recruited via the Bone-Marrow Transplant Program of the Hematology Service at Reina Sofia University Hospital (Cordoba, Spain). All of them signed written informed-consent. MSC isolation, characterization and storage were performed as we have previously described (Casado-Diaz et al., 2008) MSC were cultured in 75 cm2 flasks from Nunc (Kamstrupvej, Denmark), containing 12 ml Minimum Essential-Medium Alpha (MEM ) Eagle from Lonza (Basel, Switzerland), supplemented with 10% fetal bovine-serum (FBS) from Gibco - Life Technologies - Thermo Fisher Scientific (Waltham, MA, USA), 2 mM UltraGlutamine (Lonza), 1 ng basic fibroblast-growth factor (bFGF)/ml from Sigma-Aldrich (Saint Louis, MO, USA), 100 U ampicillin and 0.1 mg streptomycin/ml. Cells were grown in an incubator at 37 oC with 5% CO2 and humidity, until about 80 to 90% confluence. Then, cultures were maintained in such medium without bFGF (controls) or likewise but supplemented with 5x107 M dexamethasone, 0.5 mM isobutylmethylxanthine and 50 mM indomethacin (Sigma-Aldrich) inducing agents to trigger differentiation into adipocytes. Adipogenic differentiation at day 14 was determined by oil red-O staining of lipid droplets (LD). Then, cells were fixed with 3.7% formaldehyde for 10 min, washed with isopropanol (60% in water) and stained for 15-20 min with a mixture of 8.2 ml of 0.3% (w/v in isopropanol) of oil red-O plus 6.8 ml of distilled water. They were then washed with water, stained with hematoxylin and visualized under a light microscope (all chemicals from Sigma-Aldrich). Total RNA were isolated from both control and induced-to-differentiate cultures with mirVana miRNA Isolation Kit from Ambion - Life Technologies - Thermo Fisher Scientific, at days seven and 14 after adipogenesis induction. RNA were quantified using a ND-1000 spectrophotometer from NanoDrop - Thermo Fisher Scientific, and their integrity checked by agarose-gel electrophoresis (AGE). RNA samples were pooled from each time and donor, to generate two samples: control (RNA pooled from days seven and 14) and cells induced into adipocytes (RNA pooled from days seven and 14) for subsequent downstream analyses. ST-DGE genome-wide transcription profilings were performed with 5 g total RNA input, as we have previously described (Molina et al., 2011; Zawada et al., 2011), using TrueQuant technology from GenXPro (Frankfurt Main, Germany), in order to identify Polymerase Chain-Reaction (PCR)-derived copies in the dataset, as we have reported (Muller et al., 2014). Populations of small RNA (17 to 30 b) were size-selected from total RNA using flashPAGE Fractionator System from Ambion - Life Technologies - Thermo Fisher Scientific, for preparation of miRNA libraries.
Experimental Factor: 5x107 M dexamethasone, 0.5 mM isobutylmethylxanthine and 50 mM indomethacin
stimulus