Pd0325901

E14 (obtained from the American Type Culture Collection) and F1 (M. musculus¹²⁹ × M. castaneus obtained from Barbara Panning) mouse ES cells were cultured on 0.1% gelatin-coated plates in ES medium (DMEM containing 15% FBS, 0.1 mM MEM nonessential amino acids, 1 mM sodium pyruvate, 2 mM GlutaMAX, 0.1 mM 2-mercaptoethanol, 1000 U/mL LIF, 3 μM CHIR99021 [GSK3β inhibitor, Biovision], 1 μM PD0325901 [MEK inhibitor, Invivogen]) and maintained in a pluripotent state in the absence of a feeder layer (Mlynarczyk-Evans et al. 2006 (link); Ying et al. 2008 (link)). MEFs were isolated from embryonic day 12.5 (E12.5) C57Bl/6 mouse embryos and cultured in DMEM containing 10% FBS and 2 mM GlutaMAX. F1 TS cells (M. musculus^C57BL/6 × M. castaneus, obtained from Susannah Varmuza) were maintained as described in Tanaka (2006) (link) (Miri et al. 2013 (link)). EBs were formed by the hanging drop method; to initiate differentiation, 1000 cells were suspended in 20-μL droplets of ES medium without LIF and 2i (Ohnuki and Kurosawa 2013 (link)). EBs were transferred to 0.1% gelatin on day 4 to facilitate further growth. EBs were collected at daily intervals up to day 12, and RNA was isolated for gene expression analysis by RT-qPCR. All animal experiments were approved by the University Animal Care Committee (UACC) at the University of Toronto and the Bioscience Local Animal Care Committee (LACC).

Mouse ESCs (E14TG2a; ATCC [CRL-1821]), were maintained in feeder-free conditions on 0.1% gelatin in DMEM supplemented with 15% (v/v) fetal bovine serum (FBS), 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 2 mM GlutaMAX, 0.1 mM 2-mercaptoethanol, 1,000 U/mL LIF, 3 μM CHIR99021 (GSK3β inhibitor, Biovision) and 1 μM PD0325901 (MEK inhibitor, Invivogen); referred to as LIF/2i medium. The differentiation medium contained the same components with the exception of LIF/2i. For FGFR inhibition, the MEK inhibitor was replaced with 25 nM PD173074 (Selleckchem). For TPA (NEB) treatment, cells were cultured overnight in low serum (0.2%) ESC medium, followed by treatment with 200 nM TPA in the absence of LIF/2i. For ESCs maintained in LIF alone, cells were cultured in ESC medium containing LIF without 2i for 8–10 passages before removal of LIF to induce differentiation. For evaluation of AP activity, cells were seeded at 250 cells/well in a 12-well plate and allowed to differentiate for 5 days. AP staining (Millipore) was performed following the manufacturer’s instructions. EBs were formed by the hanging-drop method; 1,000 cells were suspended in 20-μL droplets of ESC medium without LIF/2i (Ohnuki and Kurosawa, 2013 (link)). EBs were transferred to 0.1% gelatin on day 4, and collected until day 12; RNA was isolated for gene expression analysis by qRT-PCR.