Enhanced generation of retinal progenitor cells from human retinal pigment epithelial cells induced by amniotic fluid

Retinal progenitor cells are a convenient source of cell replacement therapy in retinal degenerative disorders. The purpose of this study was to evaluate the expression patterns of the homeobox genes PAX6 and CHX10 (retinal progenitor markers) during treatment of human retinal pigment epithelium (RPE) cells with amniotic fluid (AF), RPE cells harvested from neonatal cadaver globes were cultured in a mixture of DMEM and Ham's F12 supplemented with 10% FBS. At different passages, cells were trypsinized and co-cultured with 30% AF obtained from normal fetuses of 1416 weeks gestational age.

Compared to FBS-treated controls, AF-treated cultures exhibited special morphological changes in culture, including appearance of spheroid colonies, improved initial cell adhesion and ordered cell alignment. Cell proliferation assays indicated a remarkable increase in the proliferation rate of RPE cells cultivated in 30% AF-supplemented medium, compared with those grown in the absence of AF. Immunocytochemical analyses exhibited nuclear localization of retinal progenitor markers at a ratio of 33% and 27% for CHX10 and PAX6, respectively. This indicated a 3-fold increase in retinal progenitor markers in AF-treated cultures compared to FBS-treated controls. Real-time PCR data of retinal progenitor genes (PAX6, CHX10 and VSX-1) confirmed these results and demonstrated AF's capacity for promoting retinal progenitor cell generation.

Taken together, the results suggest that AF significantly promotes the rate of retinal progenitor cell generation, indicating that AF can be used as an enriched supplement for serum-free media used for the in vitro propagation of human progenitor cells.