Curcumin Promoting Directional Induction Efficiency From Human Embryonic Stem Cells To RPE

ObjectiveThis study was conducted to investigate the effects of Curcumin on human embryonic stem cells (hESCs) differentiation into Retinal pigment epithelial cells (RPE) in vitro, and to explore the Gene expression level of Wnt/beta-catenin signaling pathway during the process. In order to further improve the efficiency of directional induced hESC to RPE cells, so as to provide theoretical basis for the transformation of clinical treatment.Methods(1) Human embryonic stem cells were bought from Stem cell company, US. The hESCs were cultured in mTeSRTM medium; Curcumin was added on the15th day of self-differentiation. The hESCs cells were devided into experimental groups and control group, the details are as follows:①Experiment set up6treated concentration of Curcumin:Oμmol/L(control)、1μmol/L、2μmol/L、5μmol/L、10μmol/L and20μmol/L. Curcumin was removed after24hour. Total RNA were extracted at day22and day36of differentiation. RT-PCR was performed to examine the expression of RPE marker genes. The group which expresses highest level of RPE marker gene will had been chosen for further experiment.②Experiment set up5treated time of Curcumin:0hour(control)、24hour、3days、 week and2week. Total RNA were extracted at day22and day36of differentiation. RT-PCR was performed to detect the expression of RPE marker genes.(2) Human retinal pigment epithelial (hRPE) were immediately isolated from ocular trauma eye of healthy adults after removed. We scrape off RPE cell gently after pancreatic enzyme digestion. Collect cell suspension, cells were grown in hRPE medium in vitro. Half of the medium was Changed every2days. Western-blot was performed to examine the expression of CRALBP、RPE65and MITF. To detect cells phagocytosis, cells were treated with polystyrene microspheres.(3) The induced retinal pigment epithelial (iRPE) were isolated from curcumin treated group (1μmol/L,24h). Western-blot was performed to examine the expression of CRALBP、RPE65and MITF. Immunofluorescence were performed to examine the expression of Zo-1、MITF and PAX6. To detect cells phagocytosis, cells were treated with polystyrene microspheres.(4) To detect the gene expression change of Wnt/β-catenin signaling pathway during hESCs differentiation into RPE after treated with curcumin, total RNA were extracted at day22and day36of differentiation. RT-PCR was performed to examine the expression of LEF1、TCF7and MYC genes.Results(1) After24h of curcumin treat, group1μM and2μM show the highest degree in cytochrome. Followed with group5μM, group10μM and20μM show low degree of cytochrome. Results of RT-PCR revealed that the expressesion level of RPE marker gene was higher at curcumin treated group, especially at group1μM and2μM. With the increasing of curcumin concentration, the expressesion level of PAX6、CRALBP and RPE65was reducing.(2) After curcumin treatment, group24h and3d shows the highest degree in cytochrome. Followed with group1W, group2W shows low degree of cytochrome. Results of RT-PCR reveal that the expressesion level of RPE marker gene was higher at curcumin treated group, especially at group24h and3d. With the increasing of curcumin treatment time, the expressesion level of PAX6、CRALBP and RPE65was reducing.(3) hRPE cells have an irregular polygon morphology when cultured in vitro. With cells adherent growth, a large number of pigment particles gathered in the cytoplasm. Results of Western-blot revealed that CRALBP、RPE65and MITF protein express in the hRPE; Results of cells phagocytosis experiment show that hRPE possess phagocytosis.(4) Results of Western-blot reveal that CRALBP、RPE65and MITF protein express in the iRPE; Results of immunofluorescence reveal that Zo-1、MITF and PAX6protein express in the iRPE; Results of cells phagocytosis experiment show that iRPE possess phagocytosis.(5) Results of RT-PCR reveal that the expression level of LEF1、TCF7、MYC gene was higher at curcumin treated group, and there is compare to control group statistically significant difference (P<0.05).ConclusionThe experimental group treated with1μmol/L Curcumin for24h showed much higher grade of pigmentation and higher expression of RPE related genes. All the iRPE cells isolated and purified from Curcumin group showed definite expression of RPE related proteins with similar profile to that of human RPE cells. In Cucumin treated group, significant up-regulation was observed with Wnt target genes LEF1, TCF7and MYC. Curcumin treatment significantly enhanced the differentiation efficiency of human embryonic stem cells (hESCs) into retinal pigment epithelial cells (RPE). Stimulation of Wnt signaling by Curcumin may serve as an important mechanism underpins the efficient induction of RPE cells.