Feeder-free And Serum-free Directed Differentiation Of Human Embryonic Stem Cells Into Retinal Pigmented Epithelium

Dysfunction and degeneration of retinal pigmented epithelium(RPE)can cause the deterioration of the environment in the retina and the apoptosis of photoreceptor and eventually leading to the occurrence of degenerative disease of the retina.This will make patients with hypopsia and blind.However,there is still no effective treatment for these diseases.Recent studies have shown that cell therapy to replenish the degenerating RPE cells may potentially halt disease progression.Human embryonic stem cells(hESCs)own the capacity of self-renew and differentiating into almost all the different cell types in the adult body.This leads to hESCs may serve as an unlimited donor source of RPE cells for transplantation.According to the results of animal model experiments and clinical trials,hESCs derived RPE(hESCs-RPE)is safe in vivo.And there is no tumorigenic and other side effect when hESCs-RPE transplanted into the eyes.Moreover,the vision of patients was partially recovered.However,the existing culture system of hESCs-RPE always contained ingredients of animal origin such as fetal bovine serum and feeder.This may bring potential risk of contamination and high cost.None-animal culture system will be good to study the mechanism of RPE culture and large-scale,commercial production of hESCs-RPE in the future.In this article,we developed a serum-free and feeder-free culture system was used to culture hESCs.Before differentiation,immunofluorescence staining,karyotype analysis and flow cytometry were used to detect the morphology and pluripotency of hESCs to ensure that subsequent differentiation go well.After 7 days culture of hESCs,cells completed the fusion,and the hESCs culture medium was replaced with RPE differentiation medium.To obtain pure hESCs-RPE,cells were passaged.Moreover,immunofluorescence staining,RT-PCR,scanning electron microscopic imaging and karyotype analysis were used to detect the structural characterization of hESCs-RPE.Phagocytosis of hESCs-RPE was detected to ensure the functional characterization.This none-animal origin culture system is match to the clinical requirement,this will provide a seed for the follow-up experiments.The hESCs-RPE obtained from feeder-free and serum-free system will provide reference for the researchers who do the related clinical researches.