Mesenchymal stem cells were harvested from bone marrow.
Autologous MSCs were harvested from the iliac bone of each patient and expanded in culture for 4 weeks. Mononuclear cells were separated from the bone marrow by density gradient centrifugation, then washed with PBS, resuspended in low-glucose DMEM containing FBS (10%) and penicillin/streptomycin (100 mg/mL each), and plated at a density of 2-3x105 cells/cm2 in 75-cm2 flasks. The cultures were maintained at 37C in a humidified 5% (vol/vol) CO2 atmosphere for 5-7 days, after which nonadherent cells were removed by replacing the medium, and adherent cells were cultured for an additional 2-3 days. When the cultures approached confluence (70%-80%), adherent cells were detached by treatment with a trypsin/EDTA solution and replated at a density of 4-5x103 cells/cm2 in 175-cm2 flasks. Cells for infusion were serially subcultured up to passage P5. During culture, some cells of P1 or P2 were harvested and cryopreserved in DMSO (10%) and FBS (90%) for use in future infusions. P5 cells were used in all the treatment procedures.