Cabd353003

Murine embryonic stem cells (mESCs) were cultured as described in Pucci et al. (2013) (link). Briefly, mESCs were seeded on 100 mm gelatin-coated plates (VWR, Mont-Royal QC, Canada, CABD353003 and Sigma, Oakville, ON, Canada, cat. # G1890) at a density of 0.9 × 10⁶ cells per plate and propagated in Glasgow Modified Eagle Medium (GMEM, Gibco, Invitrogen, acquired by Thermo-Fisher, Nepean, ON, Canada, cat. # 11710035) supplemented with 15% v/v fetal bovine serum (FBS; Wisent, St-Bruno, QC, Canada, cat. # 920–040), and 50 units/mL penicillin/streptomycin (Invitrogen, cat. # 15070–063), 0.1 mM non-essential amino acids (Life Technologies, acquired by Thermo-Fisher, Napean, ON, Canada, cat. # 11140050), 0.055 mM 2-mercaptoethanol (Life Technologies, cat. # 21985023) and 1000 U/mL of ESGRO LIF (1 × 10⁷ units, Millipore/Sigma, cat. # ESG1107). Cell cultures were maintained at 37°C with 5% v/v CO₂. Cells were split every two days, when they reached an approximate confluency of 80%. All cell lines used in this publication were regularly assessed for mycoplasma contamination and remained mycoplasma-free.

Differentiation analysis was performed as in Pucci et al. (2013) (link). Briefly, after 2 days of growth in LIF-containing media (as above), cells were plated at a density of approximately 0.2 × 10⁶ cells/100 mm plate on non-gelatin-coated dishes (VWR, cat. # CABD353003), in media without LIF but supplemented with 5 μM all-trans retinoic acid (ATRA, Sigma, R2625-50MG) for 6 days. At day 6, cells were trypsinized using 0.05% w/v trypsin-EDTA 0.5 mM (Invitrogen, cat. # 25300–054), transferred to gelatin-coated plates in GMEM media without ATRA and supplemented with LIF (as above) for 6 days or longer, as indicated. Cells were split to ensure they did not exceed 80% confluency After each step of the differentiation assay, and before reseeding the trypsinized cells, 0.2–0.5 × 10⁶ cells were used for flow cytometry analysis or 3 × 10⁶ cells for FACS. The pluripotent state was assessed by flow cytometry, using the Pou5f1-GFP reporter gene as a quantitative tool (see below). All experiments were repeated at least three times (n = 3 biological replicates), and typically included n = 3 technical replicates. A biological replicate represents a distinct mESC population (usually analyzed on a different day), and a technical replicate is the same biological replicate processed separately (usually on the same day).