Isolation and Characterization of Bone Marrow-Derived Mesenchymal Stem Cells

The BM-MSCs were isolated, cultured, and characterized as described earlier [3 (link)]. Briefly, mononuclear cells obtained from the BM aspirates by density gradient centrifugation were cultured in 25 cm² flasks (BD Biosciences, USA) at 37°C in 5% CO₂ using 5 ml of complete culture media consisting of α-MEM, 1% GlutaMAX, 16.5% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Gibco and Thermo Fisher Scientific, USA). After 48 hours, nonadherent cells were removed and medium was replaced. When culture reached 70–80% confluency, adherent cells were harvested using 0.05% trypsin (Gibco) and replated for further expansion. The cells of the 3^rd passage were used in all the experiments.
For immunophenotypic characterization, the BM-MSCs were stained by incubating the cells for 30 minutes with the following preconjugated antibodies: CD73 (PE), CD90 (PE), CD105 (PE), CD166 (PE), CD34 (FITC), CD45 (FITC), HLA-DR (FITC), and CD13 (PE) (all from Serotec, (http://www.abdserotec.com)). Cells stained with isotype-matched antibodies served as controls. After washing, the cells were acquired on a BD FACS-Canto flow cytometer (BD Biosciences, San Jose, CA, USA) and the data analysis was done using the FACS express software.