Matrigel was used as a substrate to support the expansion of hPSCs. At least 1 day before the experiment, a bottle of Matrigel was thawed overnight at 4 °C. The bottle was transferred on ice to a tissue culture hood and kept cold during its preparation as Matrigel solidifies at RT. The Matrigel was diluted 1:50 with DMEM by repetitively pipetting in cold DMEM taken directly from the refrigerator. Immediately prior to passaging the hPSCs, a 10 cm plate was coated with 1–2 ml of diluted Matrigel and shaken well to cover the entire surface. After incubation at RT for at least 30 min, the Matrigel was aspirated from the plate.
