Bone marrow-derived mesenchymal stem cells (National Institute of Neuroscience, Italy)

Bone marrow (BM) was obtained by aspiration from the posterior iliac crest of healthy donors, after informed consent. BM cells were layered over a 1.073 g/ml Percoll gradient. The cells in the interface were collected, washed twice in 0.1 M phosphate-buffered saline (PBS; pH 7.4), plated in MSC medium at 8 × 100,000 cells/cm2 in T flasks and maintained at 37 °C in an atmosphere of 5% CO2. After 3 days, the non-adherent cells were removed and replaced with fresh culture medium. Subsequent complete medium changes were performed every 4 days. After 15 days for the first passage and every week for the following passages, BM cells were detached by treatment with 0.25% trypsin containing 0.01% EDTA for 10 min at 37 °C. They were plated at a density of 8000/cm2 and expanded for several passages to prevent confluence.