Embryoid bodies (EBs) were produced from adherent colonies that were enzymatically detached using dispase (0.5 mg ml−1) for 15–45 min, collected and then maintained in suspension using ultra low attachment plates (corning) in the presence of EB medium containing DMEM/F12 supplemented with 15% fetal bovine serum, 2 mM L-glutamine, 0.1 mM non-essential amino acids, and 1% penicillin/streptomycin. After 3–4 days, EBs were transferred to 0.1% gelatin-coated polystyrene chamber slides and cultured in differentiation medium (DMEM supplemented with 20% fetal bovine serum, 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acids and 1% penicillin/streptomycin) for 2–3 weeks to allow spontaneous endoderm formation. The medium was changed every other day. For mesoderm differentiation, EBs were maintained on gelatin-coated plate in differentiation medium supplemented with 100 μM ascorbic acid (Sigma). For ectoderm differentiation, EBs were cultured on Matrigel-coated plates in 1% N2 and 0.5% B27 (Invitrogen) medium supplemented with 1 μM retinoic acid for 2–3 weeks.
