The effects of continual passaging on cellular morphology, colony shape and EB formation. (A) > 95% of colonies displayed spontaneous differentiation and loss of GFP expression in peripherally located cells of low-passage GG3.1 and miPS-25 cell clusters. Serial passaging results in morphological stability and uniform GFP expression in > 85% of cell colonies in miPS-25 cultures. (B) EB diameter increased after multiple passages (iPSC LP represents cells at P20-30) compared to early-passage cultures (iPSC EP represents cells at P7-9). ESC-derived EB diameter also differs significantly from EP-derived EB (n = 3 for each group). Scale bars indicate 100 μm. Values are mean ± SD. *P < 0.01.