Rapid differentiation of human ESCs into RPE cells by defined xeno-free culture system

Abstract

Background Subretinal transplantation of retinal pigment epithelium (RPE) cells for the treatment of age-related macular degeneration (AMD) have accelerated the drive to develop xeno-free cultivation system that support the rapid differentiation of human embryonic stem cells (hESCs) into ES-RPE cells. Objective This study was to report a modified xeno-free culture system and method for accelerating derivation of hESCs to differentiate into RPE cells. Methods This study was approved by Ethic Committee of Zhongshan Ophthalmic Center.HESC H1 line was cloned and cultured in Vitronectin XF™-coated 6-well dish with xenogenetic-free medium.Cells were cultured in 50 ng/ml noggin, 10 ng/ml DKK-1 and 10 ng/ml insulin like growth factor-1 (IGF-1) medium for 2 days, and then the concentration of noggin was decreased to 10 ng/ml and 5 ng/ml basic fibroblast growth factor (bFGF) and cultured for the following 2 days.Sequentially, noggin and bFGF were removed and cultured for 2 days.Finally, 1 μmol/L CHIR99021 was added in medium for 6 days.Morphological changes in the progress of ESCs differentiation into RPE were observed by Living Cell Imaging System.The expression of Mitf and RPE65, RPE cells-specific markers, in the cells were detected by immunofluorescence technique, and the relative expression levels of RPE cells-specific marker mRNA were assayed using real time fluorescent quantitation PCR. Results Polygonal-shape monolayer cells which contained pigments were initially observed at day 14 after cultured with the cobblestone- like arrangement.Mitf and RPE65 were strongly expressed in the hES-derived RPE cells 35 days after induced, showing red fluorescence, and the cells presented hexagonal shape at cultured day 60 with numerous pigment granules in cytoplasm.Compared with before differentiation, the expression levels of Mitf mRNA in hES-RPE cells increased by (3.43±2.77) folds and (8.91±2.83) folds, and the expression levels of RPE65 mRNA increased by (14.60±3.94) folds and (87.16±9.32) folds at day 7 and day 14 after differentiation, respectively (all at P<0.05). Conclusions A defined xeno-free culture system is successfully established by adding niacinamide, DKK-1, noggin, IGF-1 and CHIR99021 in xeno-free medium, and this system can accelerate the derivation and differentiation of hESCs into RPE-like cells. Key words: Embryonic stem cells/drug effect; Cell culture techniques/methods; Cell differentiation; Retinal pigment epithelium; Culture media/drug effects; Cell line; Humans; Xeno-free