To better characterize human retinal pigment epithelial (RPE) cells, their maturation was studied by time-lapse observation and immunostaining of the tight junction protein ZO-1. During subconfluency with active migration, the cells had an elongated shape. During cell division to reach continency, RPE cells became small and tight, exhibiting cobblestone-like morphology. In addition, RPE maturation at the peripheral region of the culture vessel was delayed when compared with the central region, demonstrating local heterogeneity during maturation. To correlate cellular migration and maturation, we compared frequencies of migration rate and number of ZO-1-positive cells at the central and peripheral regions. Cells having migration rates less than 5.0 mu m/h in the central region were 1.4-fold higher than in the peripheral region at day 5. Regardless of locational differences in the culture vessel, the frequency of cells having migration rates less than 5.0 mu m/h showed 90% agreement with the frequency of ZO-1-positive cells. To inhibit cell migration, RPE cells were exposed to medium containing 50 mu g/ml Rac1 inhibitor at day 5. Frequencies of ZO-1-positive cells and cells having migration rates less than 5.0 mu m/h at the peripheral region were similar to those at the central region. The results show that migration is an important factor affecting maturation, and demonstrate that location heterogeneity during maturation is caused by different migratory behaviors in the culture vessel. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.