Prior to passaging, the cells were inspected under an inverted microscope. In the rare case that any differentiating colony was detected, those colonies were marked and aspirated before passaging. The medium from the hPSCs was aspirated. The plate was washed twice with 1–2 ml of PBS (without Ca++ and Mg++) to remove the remaining magnesium and calcium from the medium. When washing, PBS was added to the wall of the plate slowly to avoid washing the colonies off the plate. After two washes, the cells were incubated with 1 ml of EDTA at RT for 5 min within the hood. After 5 min, 1–2 ml of KSR medium (20% knockout serum replacement, DMEM, 2 mM l-Glutamine, 10 µM β-Mercaptoethanol and penicillin–streptomycin) with ROCK inhibitor (Y-27632, 5 µM) was added to neutralize the EDTA. The cells were resuspended with a P1000 pipette to lift the colonies off the plate and collect at the bottom of the plate, which was tilted at a 30° angle. A cell lifter was also used to detach the remaining cells by gentle mechanical dislodgement. The collected
