Stemsure serum replacement

Fibroblasts were isolated from p63^fl/fl mouse embryos (E12.5) according to standard protocols [31 (link)] and applied to iPS cell generation using concentrated vesicular stomatitis virus-G-retroviral supernatant as described previously [32 (link), 33 (link)]. p63^fl/fl iPS cells were cultured on mitomycin C-inactivated p63^fl/fl mouse embryonic fibroblasts as feeder cells at 37°C in a humidified atmosphere with 5% CO₂ in ES medium [StemSure D-MEM (High Glucose) with Phenol Red and Sodium Pyruvate (Wako, Osaka, Japan), 15% StemSure Serum Replacement (Wako), 2 mM L-glutamine (Thermo Fisher Scientific), 1% (vol/vol) nonessential amino acids (Thermo Fisher Scientific), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich), 50 U/mL penicillin (Sigma-Aldrich), 50 mg/mL streptomycin (Sigma-Aldrich), and 1,000 U/mL leukaemia inhibitory factor (Wako)].

Embryoid bodies were generated by CTK medium treatment of day-7 hiPSC cultures to remove colonies from the culture dishes. Colonies were grown in differentiation medium in a suspension culture using EZ-BindShut II dishes (Iwaki, Japan). hiPSCs were induced to spontaneously differentiate in a medium composed of StemSure DMEM (Wako) supplemented with 20% StemSure serum replacement (Wako), 2 mM l-alanyl-l-glutamine (Wako), 1% MEM non-essential amino acids (Wako), and 0.1 mM 2-mercaptoethanol (Thermo Fisher Scientific). EBs were grown in suspension culture for 8 days, then plated onto gelatin-coated plates and allowed to differentiate for an additional 8 days in DMEM + 10% FCS.