PURPOSE: To examine the characteristics of retinal pigment epithelial (RPE) cells cultured on amniotic membrane (AM). The present study examined how AM modulates RPE cell differentiation. METHODS: Human RPE cells were cultured on the basement membrane side of dispase treated AM. After one week of cellular confluence, cultures were terminated, conditioned medium was collected, and total RNA was extracted. The expression pattern of several genes considered to participate in the function of differentiated RPE was evaluated. Ultrastructural changes were evaluated by transmission electron microscopy. RESULTS: Morphologically, RPE cells cultured on AM exhibited ultrastructural epithelial features such as microvilli of the apical membrane and intercellular junctions. Gene expression of RPE65, CRALBP, bestrophin, and tyrosinase related protein (TRP)-2 was upregulated in RPE cells cultured on AM compared to cells cultured on plastic. In addition, protein production of vascular endothelial growth factor, thrombospondin-1, and pigment epithelium derived factor was markedly increased in cells cultivated on AM. Gene expression of cathepsin D, brain derived neurotrophic factor, and basic fibroblast growth factor, however, did not differ between RPE cells cultured on plastic or AM. CONCLUSIONS: RPE cells cultured on AM demonstrated an epithelial phenotype morphologically and several growth factors important for maintaining retinal homeostasis were upregulated. AM might be a useful matrix substrate to retain the differentiated and epithelial phenotype of RPE for subretinal transplantation.
PURPOSE: To examine the characteristics of retinal pigment epithelial (RPE) cells cultured on amniotic membrane (AM). The present study examined how AM modulates RPE cell differentiation. METHODS:Human RPE cells were cultured on the basement membrane side of dispase treated AM. After one week of cellular confluence, cultures were terminated, conditioned medium was collected, and total RNA was extracted. The expression pattern of several genes considered to participate in the function of differentiated RPE was evaluated. Ultrastructural changes were evaluated by transmission electron microscopy. RESULTS: Morphologically, RPE cells cultured on AM exhibited ultrastructural epithelial features such as microvilli of the apical membrane and intercellular junctions. Gene expression of RPE65, CRALBP, bestrophin, and tyrosinase related protein (TRP)-2 was upregulated in RPE cells cultured on AM compared to cells cultured on plastic. In addition, protein production of vascular endothelial growth factor, thrombospondin-1, and pigment epithelium derived factor was markedly increased in cells cultivated on AM. Gene expression of cathepsin D, brain derived neurotrophic factor, and basic fibroblast growth factor, however, did not differ between RPE cells cultured on plastic or AM. CONCLUSIONS: RPE cells cultured on AM demonstrated an epithelial phenotype morphologically and several growth factors important for maintaining retinal homeostasis were upregulated. AM might be a useful matrix substrate to retain the differentiated and epithelial phenotype of RPE for subretinal transplantation.