Pd0325901

ESCs were derived from preimplantation blastocysts of CBA/J-mated CBA/J female
mice by applying the reported ESC propagation method^{20 (link)} with slight modifications. Briefly, the embryos were flushed out from the
decidua of a pregnant CBA/J mouse at 3.5 days post-coitum. The collected embryos
were cultured overnight in a 35-mm dish with KSOM medium at 37°C in 5%
CO₂ to obtain the blastocyst-stage embryos. The next day, embryos
that normally developed to the blastocyst stage were selected. Each of the
selected embryos was put on an MEF feeder cell-coated well of a 96-well plate in
serum-free ESC culture medium (NDiff227, Takara Biochemicals, Japan)
supplemented with leukemia inhibitory factor (10 ⁶ u/ml: Wako,
Japan) and two kinase chemical inhibitors (PD0325901, 1 µM: ReproCELL, Japan;
CHIR99021, 3 µM: Wako, Japan). After a few days, the blastocysts were attached
on the well, and cells grew from them. The expanded cells were passaged by
dissociation with Accutase (Funakoshi, Japan). We define this passage event as
the passage (p)1. Until p3, the expanded cells were maintained on MEF feeders
with the same ESC culture medium. At p4, the cells were passaged on a laminin
(iMatrix-511 silk, 1.25 µg/ml; Takara Biochemicals, Japan)-coated well and,
after this, maintained under the feeder-free condition with the same ESC culture
medium.