Bone marrow-derived MSC (BM-MSC) were isolated using anti-Stro-1 antibody (R&D Systems, Minneapolis, MN) and magnetic cell sorting (Miltenyi Biotec, Auburn, CA) as previously described.44 (link) Cell cultures were maintained in gelatin (Sigma-Aldrich, St Louis, MO)-coated flasks and MSCGM (Mesenchymal Stem Cell Growth Medium BulletKit, Lonza, Atlanta, GA) supplemented with penicillin/streptomycin (100U/ml) (Gibco-Life Technologies, Carlsbad, CA) at 37 °C and 5% CO2 in a humidified atmosphere. MSC were passaged when they reached 70% confluence using trypsin solution (Lonza) for 7 minutes at 37 °C. Cell number and viability were determined using the Trypan Blue (Gibco-Life Technologies) exclusion method, and cells were replated at 3,000 cells/cm2. Phenotypic and differentiative characterization of Stro-1+ BM-MSC has been previously reported.44 (link) MSC were found to be positive (>95%) for CD29, CD73, CD90, and CD105; and negative (<0.05%) for CD34, CD45, CD14, CD19, and HLA-DR. Furthermore, upon culture in appropriate media, these cells differentiated into bone, cartilage, and adipocytes.
All experiments were performed using cells from four different donors (n = 4), at passages 6–9. After genetic modification with HLA-G1, independently of the method used, cells are designated MSC-HLA-G1.
All experiments were performed using cells from four different donors (n = 4), at passages 6–9. After genetic modification with HLA-G1, independently of the method used, cells are designated MSC-HLA-G1.