As previously described (Lieberman et al. 2012), iPSCs were cultured on a feeder layer of irradiated mouse embryo fibroblasts using human embryonic stem cell media containing DMEM with F12 (DMEM/F12, 1:1 ratio, Thermo Fisher Scientific) supplemented with 20% Knockout Serum Replacer (Thermo Fisher Scientific), 1x non-essential amino acids, 1 mM L-glutamine (Thermo Fisher Scientific), 0.1 mM β-mercaptoethanol (MP Biomedicals), and 4 ng/mL of basic fibroblast growth factor (bFGF, Millipore). iPSCs were monitored daily and colonies exhibiting spontaneous differentiation were manually removed. Media was fully replaced daily and cells were cultured for 7 days, or to confluency, before being passaged using 1 mg/mL Dispase (Thermo Fisher Scientific) in DMEM/F12.
