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 pool of colonies was dispersed and broken into smaller clusters by triturating the cells with the plate continuously tilted. Vigorous mixing or pipetting was avoided to prevent the cells from shearing. The homogeneous cell suspension was plated at about 2.5 × 106 cells per Matrigel-coated 10 cm plate with E8 medium. Rock inhibitor (Y-27632, 5 µM) was added on the first day of passaging to prevent dissociation-induced death of hPSCs [25]. The plate was immediately shaken back and forth and side to side to evenly distribute the cells and prevent the cells from aggregating densely in one area. The plate was then placed in the incubator to allow the cells to attach and expand. Human PSCs were maintained in E8 medium (Fig. 1a), which was changed every day.
