Hdaci

Transfection was carried out in 6-well plate format. Plasmid complex, at a total volume of 20 μL/cm², consist of 250 ng/cm² DNA, 1.1 µL/cm² Polyethylenimine MAX (Polyscience; 1 mg/mL) and DPBS, was incubated at room temperature for 15 min. The plasmid complex was then added dropwise into MSCs (150000 cells/cm² in 6-well plates) supplemented with 500 ng/mL Lipofectamine^™ 2000 Transfection Reagent (ThermoFisher Scientific) and 0.5 μM Vorinostat (Histone deacetylase inhibitor; HDACi, BioVision) in complete medium. The culture media was replaced with fresh media at 24 h post-transfection. Then, cells were further incubated for 24 h before analysis. Cell images were taken with EVOS FL Cell Imaging System (Thermo Fisher Scientific) equipped with a GFP (Ex470/Em510) fluorescent light cube. For flow cytometric analyses, cells were washed by DPBS, trypsinized using TrypLE Express. Percentage of fluorescent positive cells was quantified by Attune NxT Flow Cytometer system (ThermoFisher Scientific), and the raw data was analysed using non-modified MSCs as the negative control at < 0.8%, using Invitrogen Attune NxT software (Version 3.1.2, ThermoFisher Scientific).

AD-MSCs were kept at passages 3–7 for transfection. AD-MSCs were seeded at 50,000 per well in 24-well plate format and incubated for 24 h before transfection. Polyethylenimine MAX (PEI, Polyscience) was added to PBS at 1 μg of plasmid DNA to 3 μL of PEI (1 mg/mL). The mixture, at a total volume of 50 μL, was incubated at room temperature for 15 min. The complex was then added to the cell culture in a dropwise manner. Transfection Enhancer were supplemented to complete media before the addition of transfection mixture into the culture. The Enhancer consist of DOPE/CHEMS (9:2 M ratio Fusogenic lipid, Polar Avanti Lipid) and 1.25 μM Vorinostat (Histone deacetylase inhibitor; HDACi, Bio Vision) [43 (link), 44 (link)]. The culture media were replaced with fresh media at 24 h post-transfection. Then, cells were further incubated for at least 24 h before analysis.