Polybreme

HEK 293T cells were used for lentiviral production, followed by concentration by ultra-centrifugation. Briefly, a second generation packaging system composed of three plasmids (transfer vector with expression construct LV-GFP, the packaging plasmid psPAX2, and the envelope protein expression plasmid pMD2.G – pMD2.G, a gift from Didier Trono (Addgene plasmid # 12259¹; RRID: Addgene_12259), was mixed in a ratio of 2:1:1 in DMEM (Thermofisher Scientific) and Fugene6 transfection reagent (Roche) and 293T cells were transfected during 4 h to overnight. The supernatant from the transfected plate was collected every 24 h in serum-free conditions and concentrated by ultracentrifugation (Beckman XL-90) at 90.000 rpm for 2 h at 4°C. The concentrated viral solution was passed through a 0.45 μm low-protein binding filter and aliquots were used to transfect the hiPSCs lines. The cell lines F002.1A.13, Gibco^® iPSC6.2 and EMC24i/R2 (C6) were successfully transfected, by first incubating the cells in a 24-well plate during 30 min at 37°C with E8 medium, 10 μM ROCKi and 5 μg/mL of Polybreme (Sigma). Then, LV-GFP previously dissolved in E8 was added dropwise and the cells were incubated during 1 h30 min. Culture medium was then changed daily. After cell growth and expansion, FACS sorting of GFP⁺ cells was performed using BD FACSAria^TM III (BD Biosciences-US) in order to obtain pure populations.