Study Of Tissue Engineering Human Retinal Pigment Epithelium Complex

Background: Retinal pigment epithelium(RPE) is a monolayer of cells, which is located between the neural retina and Bruch’s membrane. RPE is involved in the formation of the blood-retinal barrier, can secrete nutrition factors and phagocytose photoreceptor outer segment(POS). As a result, RPE plays a great role in the maintenance of normal physiological function of the retina. Dysfunction of RPE usually causes sight-threaten diseases, including age-related macular degeneration(AMD), retinitis pigmentosa(RP) and Stargardt’s macular dystrophy. At present, no effective treatment is found to cure these retinal degeneration diseases. RPE transplantation is expected to replace the patients’ compromised RPE cells and to protect the photoreceptors, which is a promising approach.The source of RPE donor cells is a key constraint of the clinical translation of RPE transplantation. Embryonic stem cells(ESCs) hold the capacity of unlimited proliferation, self-renewal and pluripotent differentiation. Besides, the stable characteristics of ESCs make them suitable for clinical application to produce scale-up RPE donor cells. Currently, the hESC-derived RPE cells induced by spontaneous differentiation method(SD-RPE) have been used in clinical trials. Three-dimensional(3D) human ESCs culture is a new method of ESC differentiation. By 3D cultures, the ESCs were differentiated into optic-cup like structures in the previous studies. The RPE cells derived from 3D ESCs cultures(3D-RPE) are likely to be closer to in vivo counterpart due to the differentiation method of 3D cultures mimicking normal development of embryonic retinal tissue.The transplantation of RPE cells affects the survival and function of RPE cells in vivo. Currently, there are two ways of transplantation: cell suspension and cell sheet transplantation. Although the surgical procedure of cell suspension transplantation is easier, the grafted cells usually clump up and cannot adhere or grow on the Bruch’s membrane as a monolayer of polarized cells, which is harmful for the long-term survival and function of RPE donor cells. Cell sheet transplantation can maintain the monolayer and polarized structure of RPE cells in vivo to extend the effect of RPE transplantation. RPE cells were cultured on biomaterials in vitro to construct tissue engineering RPE cells complex, which is a polarized and functional monolayer of RPE cells. Then the RPE sheet with scaffold can be transplanted into the subretinal space. Nowadays, many biomaterials were used to construct tissue engineering RPE cells, imitating the RPE- Bruch membrane complex. However, the factors of biomaterials, such as biocompatibility, biodegradability, operability, the adhesion and growth of RPE cells should be taken into account to construct tissue engineering RPE cells.Object: To study the biological characteristics of RPE cells derived from three-dimensional human embryonic stem cell cultures. To explore the biocompatibility of biomaterials used for RPE cells culture. To prepare biomaterials with good biocompatibility and to construct tissue engineering RPE complex. To study the biological characteristics of tissue engineering RPE complex and the effect of rescuing retinal degeneration.Methods and Results: This study consists of three parts:Part One. The biological characteristics of RPE cells derived from three-dimensional human embryonic stem cells cultures1. We studied the morphology, ultrastructure and proliferation of 3D-RPE cells at different time points and found that 3D-RPE cells exhibited cobblestone-like morphology, which were similar with the native RPE cells. Microvilli were apparent at the apices of 3D-RPE, pigment granules were accumulated in the apical cytoplasm, and nuclei were located at the basal part. Caveolaes occurred at both the apex and the base of the cell membrane. Besides, all of the intercellular connections specific to RPE cells existed including tight junction, gap junction, adherences junction and desmosomes. Compared with the time-matched SD-RPE cells, 3D-RPE cells had better proliferation.2. We studied the genes expression profile of 3D-RPE cells at different time points and found that 3D-RPE-40 dhad the highest correlation with native hfRPE. As differentiation advancement, the genes associated with differentiation up-regulated. However, the level of genes in the 3D-RPE cells was lower than those in the SD-RPE cells.3. We studied the functions of phagocytosis and secretion, polarity, tight junctions and gap junctions in 3D-RPE cells. We found that 3D-RPE cells formed better tight junctions and earlier polarity than time-matched SD-RPE cells. However, the functions of phagocytosis and secretion in SD-RPE cells were better than time-matched 3D-RPE cells.Part Two. The tissue engineering human RPE cells complex based on PET membrane1. We studied the characteristics of 3D-RPE cells cultured on PET membrane and found that PET/RPE complex was polarized monolayer of RPE cells, which could phagocytose POS, secrete nutrition factors and form tight junction.2. We studied the effect of grafted PET/RPE complex in the subretinal space of RCS rats and found that PET/RPE complex could rescue the retinal degeneration of RCS rats. However, the effect had no significant difference with RPE suspension transplantation. Besides, PET/RPE complex transplantation induced inflammation.Part Three. The tissue engineering human RPE cells complex based on GO film1. We studied the cytotoxicology of different graphene to eye cells and cocultured different chemical modification, different surface charge and different oxidation of graphene with four eye cells(ESC-RPE cells, human conjunctiva epithelium cells, human corneal epithelium cells and human vascular epithelium cells) to detect the viability, apoptosis, oxidative stress and mitochondrial membrane potential. We found that polyethylene glycol(PEG) modification can significantly improve the biocompatibility of graphene oxide(GO). Surface charges have no significant effect to the cytotoxicology of GO. The degree of oxidation of GO was negatively correlated with the vitality of eye cells while that was positively correlated with the oxidative stress of eye cells.2. We studied the characteristics of 3D-RPE cells cultured on PEG-GO-S film and found that PEG-GO-S/RPE complex could secrete nutrition factors and form tight junctions.3. We studied the biocompatibility of grafted PEG-GO-S/RPE complexin the subretinal space of RCS rats and found that the grafted PEG-GO-S/RPE complex did not caused severe inflammatory response.Conclusions:1. 3D-RPE cells possess morphology, ultrastructure, polarity, gene expression profile, and functions of authentic RPE. 3D-RPE cells are a new donor for cell therapy of retinal degenerative diseases.2. As differentiation advancement, 3D-RPE cells could mature gradually with increasing pigment, decreasing proliferation and increasing functions. Compared with the time-matched SD-RPE cells, 3D-RPE cells mature slower.3. PET membrane could be used to construct polarized and functional RPE cell sheet. PET/RPE transplantation could rescue retinal degeneration of RCS rats even though it induced inflammation.4. The chemical modification and oxidation are the key factors to decide the cytotoxicology of graphene. However, the surface charges have no significant effect to the cytotoxicology of graphene. PEG-GO-S nanosheets exert low toxicity to eye cells and PEG-GO-S film can be used to construct functional monolayer of RPE cells.