*4.8. Cell Culture*

KITD816V-TSCs were derived from E3.5 blastocysts, which were obtained after mating of R26-LSL-KITD816V mice with Deleter-Cre mice [33]. Cre recombinase expression is controlled by human CMV minimal promoter [54]. Cells were cultured on tissue culture plastic in humidified

incubators (Heracell 240i, Thermo Scientific, Waltham, MA, USA) with 7.5% CO<sup>2</sup> at 37 ◦C. TSC were grown in a trophoblast stem (TS) cell medium supplemented with 70% mouse embryonic fibroblast (MEF) conditioned medium (CM) [45]. TS medium was supplemented with 25 ng/mL human recombinant FGF4 (Reliatech, Wolfenbüttel, Lower Saxony, Germany; #100-017L) and 1 µg/mL heparin (Sigma-Aldrich, St. Louis, MO, USA; #H3149-10KU).
