iPS cells were cultured and expanded by growth on a layer of irradiated mouse embryonic fibroblasts (MEFs) using human embryonic stem (hES) cell media containing Dulbecco’s Modified Eagle Medium with F12 (DMEM/F12, 1:1 ratio) (Invitrogen) supplemented with 20% Knockout Serum Replacer (Invitrogen), 1× non-essential amino acids (Invitrogen), 1mM L-glutamine solution (Invitrogen), 0.1mM β-mercaptoethanol (MP Biomedicals, Solon, OH, USA), and 4ng/mL basic fibroblast growth factor (bFGF) (Millipore, Billerica, CA, USA). Colonies were observed on a daily basis, and any colonies exhibiting spontaneous differentiation were removed. iPS media was replaced on a daily basis. iPS cells were passed every 7 days onto irradiated MEFs using a 1mg/mL dispase (Invitrogen) in DMEM/F12.
