Dmem with 4.5 g l glucose

For viral transduction of hAFSCs with Ub-mCherry virus, cells at 70% of confluence and passage 2 were rinsed once with phosphate buffered saline (PBS) 1× (Lonza) and detached from the plate (Accutase®; Innovative Cell Technologies, Inc.). The pellet was suspended in DMEM with 4.5 g/L glucose (Lonza) and the cell suspension was incubated with Ub-mCherry virus (20 μl/ml) and polybrene (1 μl/ml) for 1 hr at 37°C within a 5% CO₂ atmosphere. After the first incubation, DMEM with 4.5 g/L glucose (Lonza) and polybrene (1 μl/ml) were added and the cell suspension was plated in a plastic dish and kept for 3 hr under the previous conditions. Then, new medium was added and the cells were maintained for 48 hr at 37°C in a humidified atmosphere containing 5% CO₂. The lentiviral expression in transduced hAFSCs was checked and analyzed with fluorescence microscopy using Zeiss Oberver Z.1 inverted microscope to capture both red fluorescence and bright-field images. Cell sorting was performed using a FACSAria II (BD Biosciences). Voltages and gates were set based on unstained sample from untransduced hAFSCs. FACS Diva 8 (BD Biosciences) software was used for cell analysis. Human AF cherry positive cells were then plated and expanded in complete DMEM with 4.5 g/L glucose (Lonza) added with FGF (Invitrogen) (5 ng/ml) for further experiments.

DMEM with 4.5 g/L glucose and L-glutamine was purchased from Lonza. FBS and HEK293T cells were purchased from Life Technologies and ThermoScientific, respectively. Gibson Assembly Master Mix and required restriction enzymes were purchased from NEB. All other reagents were purchased from Sigma-Aldrich, unless otherwise noted.