Human Bone Marrow-Derived MSC Isolation

Human bone marrow-derived MSCs were isolated from human bone marrow aspirates of two different donors. Donor A was a 22-year old male and donor B was a 23-year old female. Mononuclear cells were isolated by density gradient centrifugation with a Biocoll gradient (Biochrom). Plastic-adherent cells were passaged before reaching confluence and were sub-cultured at a density of 2 x 10³ to 5 x 10³ cells/cm² in Dulbecco´s Modified Eagle´s Medium (DMEM, FG0415, Biochrom) supplemented with 10% fetal calf serum (FCS, Thermo Scientific, Hyclone, Waltham, USA), 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Life Technologies), 100 U/mL penicillin (Biochrom), 100 mg/mL streptomycin (Biochrom) and 2 ng/mL human recombinant FGF2 (Peprotech, Hamburg, Germany). For all experiments, cells were used in passage 6. MSCs characteristics were confirmed randomly by flow cytometric analysis of cell surface molecules and by in vitro differentiation as described elsewhere [38 (link),39 (link)].