Derivation and Maintenance of Epiblast Stem Cells

E6.0–6.5 embryos were dissected in FHM medium (Millipore) and the epiblast was isolated as described52 (link). Briefly, the embryonic portions were collected separately and grown in CDM (F12 Nutrient Mix/IDMEM, 5 mg/ml BSA, 450 μM Monothioglycerol, 7 μg/ml Insulin (Sigma), 15 μg/ml Transferrin (Roche), 100 IU/ml Pen/Strep, 1% Chemically Defined Lipid (Life Technologies), 12 ng/ml bFGF (R&D Systems), 20 ng/ml Activin A (Peprotech), 10 μM ROCK Inhibitor (Y27632, Tocris) at 37 °C and 6% CO₂. 3–4 days after plating individual epiblast outgrowths were partially dissociated into smaller pieces using a scalpel and transferred into freshly gelatin/DMEM/15% FCS-coated wells. After one week EpiSC colonies became visible, they were picked individually and processed as above. From the second passage onward the colonies were separated by 1mg/ml Collagenase IV (Invitrogen) treatment for two minutes at RT. After washing they were transferred into new wells, with special care to maintain big cell clusters. The ROCK Inhibitor improved proper attachment of the colonies to the plates53 (link). After long-term passaging the established EpiSCs stabilized in a more homogenous cell population.