unless mentioned otherwise. iPSCs were cultured on irradiated mouse embryonic fibroblasts (MEFs, MTI-GlobalStem, Gaithersburg, MD) in DMEM/F12 media supplemented with knockout serum replacement (KSR, 20% v/v), non-essential amino acids (NEAA-1X), GlutaMAX (1X), beta-Fibroblast Growth Factor (FGF2, PeproTech, Inc, Rocky Hill, NJ) and 2-mercaptoethanol (0.1 mM). The quality of cells was monitored daily and differentiated cells were mechanically removed under a light microscope in a biosafety hood. iPSCs were culture up to 80% confluence and dissociated into single cell suspension after treated with Accutase (diluted in PBS (1.5:1) containing Rock inhibitor (Y-27632 dihydrochloride, Tocris Biosciences, Minneapolis, MN) to improve cell survival. The MEFs and iPSCs were separated by plating the single cell suspension onto 0.1% gelatin (0.1%, EMD Millipore, Billerica, MA) coated dishes for 45 min, after which time the MEFs attach to the substrate and the non-adherent iPSCs were collected.
