EFFECTS OF RETINAL-PIGMENT EPITHELIAL CELL-SECRETED FACTORS ON NEONATAL RAT RETINAL EXPLANT PROGENITOR CELLS

This study demonstrates the effects of conditioned media from transfor med neonatal rat retinal pigment epithelial cells (tnrRPE-CM) in a cul ture system consisting of neonatal rat retinal explants. For this stud y, retinal explants from postnatal day 2 (PN2) normal rats were cultur ed for over 3 weeks on a poly-D-L-ornithine-coated surface in RPE-CM o nly, 10% serum, or a serum-free defined media, and then examined by ph ase-contrast and scanning electron microscopy and immunocytochemistry. After 2 days in vitro, long ganglion cell-like neurites projected fro m retinal explants grown in tnrRPE-CM. These neurites increased in num ber and length with prolonged time in culture. In addition, by 5 days, round cells were observed adjacent to neonatal explants grown in tnrR PE-CM. By day 10, these round cells had increased in number and were s een along the neurites, in massive clusters immediately adjacent to th ese explants and dispersed throughout the culture-plate surface. Media conditioned by primary cultures of normal neonatal rat RPE cells caus ed a similar, but less robust, cellular response in retinal explants w hen compared to tnrRPE-CM. At 10 days, retinal explants grown in 10% s erum showed only a few short processes, but no round cells, while thos e explants grown in defined media appeared to be degenerating. The rou nd migrating cells are classified as retinal progenitor cells since th ey immunostained for opsin and interphotoreceptor retinoid-binding pro tein (IRBP), two photoreceptor cell markers, and a few for cellular re tinaldehyde binding protein (CRALBP), a Muller cell marker. Neurite ou tgrowth and retinal progenitor cell production from explants were elim inated when the tnrRPE-CM was subjected to trypsin or heat treatment, indicating that the factor(s) responsible for promoting these cellular events was most likely proteinaceous. Growth factors, including basic fibroblast growth factor, were unable to generate long neurite outgro wth or progenitor cell production as observed in RPE-CM-supplemented e xplant cultures. We report that CM from cultures of primary and transf ormed neonatal rat RPE cells promoted ganglion cell-like neurites and the production of migrating retinal progenitor cells that primarily ex pressed photoreceptor-specific markers, from neonatal rat retinal expl ants. This evidence further confirms the important role of RPE in reti nal development. The production of large numbers of progenitor cells b y an RPE-secreted factor(s) may have important implications for possib le therapeutic approaches to help correct retinal disease states by re placing lost cells through transplantation technology. (C) 1996 Wiley- Liss, Inc.