4.10. Differentiation Assay

The H9 hESC colonies were dissociated into small pieces for spontaneous differentiation assay and seeded as spheres on ultra-low attachment plates in homemade E8 medium for seven days to form embryoid bodies. The embryoid bodies were further differentiated for seven days in Knockout DMEM supplemented with 2 mM L-glutamine, 20% FBS, 1% penicillin–streptomycin, 0.1 mM non-essential amino acid, 0.1 mM 2-mercaptoethanol. Total RNA was isolated from embryoid bodies, and real-time PCR analysis was performed as described above.