Culture, Differentiation, and Maintenance of Stem Cells

ESCs were maintained on laminin in Falcon® plates and cultured in Chemically Defined Medium (CDM) supplemented with 0.7 µM PD0325901 (AxonMedCHem), 2.5 µM CHIR99201 (AxonMedChem), and 700 U/ml LIF (Cell Guidance Systems). Passage of ESCs is performed every 3 days after Trypsin treatment. The conversion of ESCs into cEpiSCs was performed by switching medium to CDM supplemented with 20 ng/ml Activin A (Cell Guidance Systems) and 12 ng/ml FGF2 (Cell Guidance Systems)^{53 (link)}. cEpiSCs were seeded on serum-coated Falcon® plates and passed every 3 or 4 days after collagenase treatment. TSCs were cultured on serum-coated Falcon® plates according to Ohinata and Tsukiyama^{30 (link)} with some modifications: We used a serum-free medium i.e. CDM supplemented with 12 ng/ml FGF2, 20 ng/ml Activin A, 10 nM XAV939 (Sigma) and 5 nM Y27632 (Cell Guidance Systems) called FAXY medium and passed every 3 days. Immunostaining and RNA-FISH experiments were performed on cells cultured at least for 14 days or 10 days for cEpiSCs.