| Purposes: Retinal Degeneration(RD)is a common kind of blinding eye disease,leading to a progressive apoptosis of photoreceptors caused by degeneration of primary retinal pigment epithelium(RPE)cells and/or photoreceptor cells,including age-related Macular diseases(AMD),retinitis Pigmentosa(RP),Stargardt disease and so on.So far,there is no effective treatment for these fundus diseases in clinic.The structure and function of the RPE cells are critical to visual formation and maintenance.Therefore,RPE cell transplantation is a promising treatment for RD.Human embryonic stem cells(h ESCs)and human induced pluripotent stem cells(hi PSCs)are important sources of RPE cells.Current studies support the feasibility and short-term safety of h ESC-RPE and hi PSC-RPE cell transplantation for the treatment of RD,with clinical trials ongoing.However,in this process,there are a lot of new problems and challenges to be solved: how to efficiently and stably increase production of ideal cells for transplantation,and how to intervene the epithelial-mesenchymal transition(EMT)of h ESC-RPE cells.To solve these problems,we are going to study in depth from two aspects:(1)to establish an efficient and stable method for the expansion of h ESC-RPE cells and explore its mechanism;(2)to intervene the epithelial-mesenchymal transition(EMT)of h ESC-RPE cells in transplantation and explore its mechanism.Methods and Results:This study consisits of two parts:Part Ⅰ: based on previous work of our laboratory,we established a method for the acquirement of h ESC-RPE cells differentiated from h ESCs by human retinal organoids(h Ret Os)culture,providing "seed" cells for large-scale,efficient and stable production under good manufacturing practices(GMP)standards in the future.The ultrastructure and expression of related proteins of h ESC-RPE cells were detected by transmission electron microscopy(TEM)and immunofluorescence(IF)staining,and the phagocytosis and barrier functions of h ESC-RPE cells were tested.These results indicated that h ESC-RPE cells,with typical RPE characteristics and functions,could be used for cell transplantation in clinic and subsequent experimental studies.Next,nicotinamide boosting promoted efficient and stable expansion of h ESC-RPE cells in vitro,enhangcing cell production for clinical transplantation in the future.Then we detected the mechanisms.We demonstrated that nicotiamide inhibits the senescence of h ESC-RPE cells and maintains epithelial phenotype by senescence staining and Western Blot(WB),which is dependent on improving mitochondrial dysfunction and promoting mitophagy to maintain mitochondrial quality control.These were confirmed by transepithelial electrical resistance(TER)detection,IF staining and quantitative detection of key metabolites such as NAD and ATP.Finally,the biological functions of h ESC-RPE cells in expansion were evaluated by pigment formation,TER detection and photoreceptor outer segment(POS)phagocytosis,so as to satisfy the requirements for cell transplantation.Part Ⅱ: We established with a h ESC-RPE cell EMT model with a cell density-and time-dependent manner,under the absence of cell-cell junctions.Through 2D/3D cell culture,RT-PCR and IF staining,the combination of Rep Sox and Y27632,that were screened from a small molecule library,could effectively reverse the middle and late EMT of h ESC-RPE cells.Combination of Rep Sox and Y27632 promoted proliferation and inhibited migration of h ESC-RPE cells by flow cytometry and wound healing assay.In vivo,combination of Rep Sox and Y27632 inhibited EMT of RPE cells and apoptosis of photoreceptor cells,successfully reversed the dispase-induced reduction in visual function.These were comfirmed by electrophysiological(ERG)examination and tissue immunofluorescence staining in subretinal fibrosis model rats.Conclusion:In this study,we successfully established a method for the acquirement of h ESC-RPE cells differentiated from h ESCs by human retinal organoids(h Ret Os)culture.Nicotinamide boosting promoted efficient and stable expansion of h ESC-RPE cells in vitro for cell transplantation in cilinic.Combination of Rep Sox and Y27632 intervened the EMT of h ESC-RPE cells.This study provides new ideas and strategies for mass production of HESC-RPE cells under GMP standards in the future and effective promoting h ESC-RPE cell transplantation in the treatment of RD in clinic. |
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