Es cell fbs

mESCs used for this study were maintained in feeder-free conditions in standard ESC Complete media containing DMEM high glucose (Sigma-Aldrich) supplemented with 10% serum (ES Cell FBS; Gibco), LIF (Esgro-LIF; Millipore), Glutamax (Life Technologies), and MEM Nonessential Amino Acid Solution (ATCC; protocols were adapted from Faunes et al., 2013 (link)). Healthy cells were maintained with daily media changes and regular passaging every 48–72 h at a proportion of 1:10 on 0.1% gelatin-coated (Millipore) plates after dissociation using TrypLE Express (Life Technologies). For differentiation using RA (Sigma-Aldrich), cells were plated in Complete medium with 0.5 µM RA in the absence of LIF for 48 h. For long-term cultures, cells were plated in limiting dilutions in 6- or 96-well plates for multiple passages (14 d) in stem conditions (serum plus LIF) with DMSO or iCRT3, with daily media changes. AP staining was performed for every passage to monitor the relative pluripotency levels. Small molecules used include 10 µM iCRT3 (ChemDiv) and 1 µM XAV939 (Sigma-Aldrich), which were diluted with DMSO. L-Wnt3a and control L cells were gifts from R.T. Moon (University of Washington, Seattle, WA).

E14 mouse embryonic stem cells (mESCs) were cultured using mouse ES medium prepared in DMEM high glucose (Hyclone) supplemented with 15% ES cell FBS (Gibco), 2 mM l-glutamine (Gibco), 1X Pen-Strep (Gibco), 100 μM MEM non-essential amino acids (Gibco), 100 μM β-mercaptoethanol (Gibco), and 1000 U/ml leukemia inhibitory factor (LIF; ESGRO, Millipore). The mouse ES line was cultured on plates pre-coated with 0.1% gelatine (porcine). Mouse embryonic fibroblasts (mEFs) and 293T cells were cultured directly on non-coated, treated culture plates. The media for mEF cells was prepared using DMEM high glucose (Hyclone), along with 10% heat inactivated (HI) FBS, 2 mM l-glutamine (Gibco), 1X Pen-Strep (Gibco) and 100 μM MEM non-essential amino acids. All cultures were maintained at 37°C with 5% CO₂.