Locational heterogeneity of maturation by changes in migratory behaviors of human retinal pigment epithelial cells in culture

Abstract

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 confluency, 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 μm/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 μm/h showed 90% agreement with the frequency of ZO-1-positive cells. To inhibit cell migration, RPE cells were exposed to medium containing 50 μg/ml Rac1 inhibitor at day 5. Frequencies of ZO-1-positive cells and cells having migration rates less than 5.0 μm/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.

Facilitation of uniform maturation of human retinal pigment epithelial cells through collective movement in culture

“…Migratory behavior is known to change in response to cellular phenotype, and the migratory behavior of individual RPE cells has been shown to be closely related to tight junction formation associated with their maturation (21). In the present study, time-lapse observation and immunostaining realized retrospective analysis of maturation of RPE cells.…”

“…0F3292; Lonza, Walkersville, MD, USA) were seeded at 5.0 Â 10 4 cells/cm 2 on laminin-coated 48-well plates (culture area in each vessel: 0.95 cm 2 ; diameter: 5.5 mm; Corning Constar, Cambridge, MA, USA) according to a method described previously (21). The laminin-coated surface was prepared by applying a solution of laminin-1 (SigmaeAldrich, St. Louis, MO, USA) on the bottom surface of 48-well plates at 2 mg/cm 2 .…”

“…1). The positional centroids (x i , y i ) of each cell in the ROI were determined by using the image processing software (LabVIEW software; National Instruments, Austin, TX, USA) as described previously (21). The migration rate, V, of individual cells was determined from the displacement of positional centroids for 12 h. As shown in Fig.…”

“…To assess tight junction formation, ZO-1 and cell nuclei were immunostained according to a previously described method (21). In brief, cells from the culture were washed, with PBS were fixed with 4% paraformaldehyde for 15 min at 4 C, and permeabilized by incubation for 5 min in 0.2% Triton X-100.…”

“…In addition, RhoA activity is an antagonistic toward Rac1 activity (18e20). In a previous study, the confluent cultures were conducted to understand the profiles of RPE cells in the initial state of maturation process and the migration affected spatial heterogeneity of tight junction formation in the vessel (21). In the present study, the spatio-temporal analysis for migratory behavior was further conducted to clarify the initiation of maturation of the cells in the vessel, and the uniform maturation was attempted.…”