Deae dextran sulfate

All lentiviral supernatants were prepared by cotransfection of HEK-293T cells with one of the vector transfer constructs, murine ecotropic envelope vector (pCAG-Eco, Addgene Plasmid # 35617), and the packaging vectors pMDLg/pRRE and RSV-Rev. For gene transduction, bone marrow-derived macrophages were used on day 5 of differentiation. Two million cells were plated in 12-well plates, and viruses (MOI = 20) were added to each well in the presence of 6 μg/ml of DEAE-dextran sulfate (Sigma-Aldrich, St. Louis, MO).

Human BM-MSCs were seeded at density of 5 × 10³ cells/cm². The following day, the cells were incubated for ±20 h with LV-miR-148b-3p or LV-miR-148b-5p or LV-Ctrl in culture medium supplemented with 2.5 μg/ml DEAE-dextran sulfate (Sigma-Aldrich Chemie, Taufkirchen, Germany). The other day, cells were rinsed with PBS and the transfection medium was changed with osteogenic differentiation medium (ODM) containing DMEM-LG included 15% FBS, and 100 U/ml Pen/Strep supplemented with 10 mM ß-glycerophosphate, 50 μg/ml ascorbic acid, and 0.1 μM dexamethasone (all from Sigma-Aldrich Chemie, Taufkirchen, Germany). To check the transduction efficiency, direct EGFP fluorescence was evaluated at day 7 using an inverse fluorescence microscopy (Nikon Eclipse TE2000-U, Tokyo, Japan).

Ad-MSCs were plated at density of 5 × 10³ cells/cm². The next day, the cells were infected with LV-Tbx20 or LV-Ctrl particles in culture medium containing 2 µg/ml DEAE-dextran sulfate (Sigma-Aldrich Chemie, Taufkirchen, Germany) under antibiotic-free conditions. After 24 h, the cells were washed with PBS and the transfection medium was replaced with osteogenic differentiation medium. The transduction efficiency was checked by direct EGFP fluorescence microscopy. All experiments were performed in triplicate.