Day (0): Cells were gently collected in to 50 ml conical tube (Corning) using 10-ml Stripette® and centrifuged at 300 × g for 6 minutes at room temperature (all media changes will require this step using these parameters.). Media was aspirated to waste, and media was changed to basal medium complete with FGF2 (50 ng/ml), BMP4 (50 ng/ml), Activin-A (12.5 ng/ml), RI (1 μm) and LiCl (2mM) at 2 ml per well in a 6-well plate, Cells were then placed in humidified tri-gas incubator under hypoxic cell culture conditions (5%O2, 5%CO2, 37°C).
