Retinal pigment epithelium (RPE) transplantation is a particularly promising treatment of retinal degenerative diseases affecting the RPE-photoreceptor complex.Embryonic stem cells (ESCs) provide an abundant donor cell source for RPE transplantation.What's more, human ESC-derived RPE cells induced by spontaneous differentiation method (SD-RPE) have already been used in the clinical trials.To advance clinic translation and search for more ideal donor cells, we comprehensively studied the time-course characteristics of RPE cells derived from three-dimensional ESC cultures (3D-RPE);the results were compared with those of SD-RPE cells.We showed that both 3D-RPE and SD-RPE cells could mature gradually as differentiation proceeded, and 3D-RPE cells were more plastic and less mature than time-matched SD-RPE cells.Notably, global gene expression profile proved that 3D-RPE cells resembled native human fetal RPE (hfRPE) more closely than SD-RPE cells and transplanted 3D-RPE cells could rescue retinal degeneration caused by RPE dysfunction.This study showed that 3D-RPE cells might be an new donor cells for cell therapy.