All cytokines selleck chemicals llc were purchased from Peprotech EC (Paris, France), nicotinamide and dexamethasone were obtained from Sigma-Aldrich (St Louis, MO) and ITS from BD Pharmigen (Franklin Lakes, NJ, USA). Nuclear and cytoplasmic proteins and RNA were collected at 7, 14 and 21 days of culture. Flow Cytometry Analysis For immunophenotype studies, basal and differentiated human MSC were detached and stained with fluorescein- or phycoerythrin-coupled antibodies and analyzed with a FACSCalibur flow cytometer (Becton, Dickinson). Anti-CD34-FITC, anti-CD45-PE and anti-CD133 were purchased from Miltenyi Biotec (Berlin, Germany), anti-CD73-PE, anti-CD90- PE and anti-CD166 were from BD Pharmigen (Franklin Lakes, NJ), anti-CD13-FITC, anti-CD44-FITC and anti-CD49e-FITC were from Beckman Coulter, Inc (CA, USA), anti-CD105-FITC was from R&D Systems (MN, USA), and anti-CD29-FITC, anti-CD184-PE and VEGFR2 were from eBioscience, Ltd (London, UK).
Quantitative real time RT- PCR analysis Total RNA was extracted following a modification of Chomezynski and Sacchi’s protocol with Trizol reagent Sigma-Aldrich (St Louis, MO). Total RNA was quantified by spectrophotometry (ND-1000, Nanodrop Tecnologies, DE, USA). One ��g of total RNA was treated with DNAse (DNAse kit, Sigma-Aldrich, St Louis, MO) and complementary DNA was amplified using the QuantiTect Reverse Transcripction kit (Qiagen, Hilden, Germany). Primers were designated with the free Oligo 7 software and their sequences are listed in Table 3. Quantitative real-time PCR was performed in a Light cycler 480 (Roche Diagnostics, Basel, Switzerland).
Table 3 Primers used for quantitative RT-PCR analyses. Immunocytochemical analysis Human MSCs were cultured on chamber slides (Nunc, Rochester, NY, USA) for 21 days and then were fixed and treated during 20 min with 0.01 M citrate buffer pH 6. Cells were incubated for 1 h at room temperature with: anti-PCNA (175 dilution, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), anti-albumin (DakoCytomation Glostrup, Denmark, 12000 dilution), anti-��-fetoprotein (R&D Systems, Minneapolis, MN, USA, 10 ��g/ml), anti-cytokeratin-19 (R&D Systems, Minneapolis, MN, USA, 10 ��g/ml), or anti-��-1-antitrypsin (DakoCytomation Glostrup, Denmark, 1800 dilution) primary antibodies. HRP-labelled polymer conjugated to secondary antibodies was used for 30 minutes at room temperature and diaminobenzidine was added to detect positive staining.
Finally, cells were counterstained with hematoxylin (DakoCytomation Glostrup, Denmark). During all the procedure three washes with PBS were performed after each step. Confocal microscopy analysis Undifferentiated and differentiated human Batimastat MSCs were cultured on chamber slides and, after the corresponding treatments; they were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 15 minutes at room temperature.