| Background：The degeneration, necrosis and the final loss of RPE cells usually cause the damage ofphotoceptors and vision loss. Till now, there is no effective therapy to treat RPEdegeneration. The major issue in the cell therapy in the retina degeneration is how to getenough RPE cells because the cells are low immune rejection and tumorigenicity. Thedevelopment of stem cell technology provides a novel choice for the treatment of retinaldiseases. Especially, embryonic stem cells (ESCs) are considered to be important seed cellsfor the treatment of retinal degenerative diseases. However, ESCs usually have the risks ofimmune rejection and tumor formation. Compared with the clinical application of ESCs,the adult stem cells, autologous and non-ethical advantage, can avoid immune rejection andthe risk of tumor formation mesenchymal stem cells (MSCs) can be induced to differentiateinto RPE-like cells and may become the promising cells for the treatment of retinaldegenerative diseases. However, the biological characteristics and functions of the MSCsderived from RPE cells are still fully understood.Methods：1. Isolation and identification of hMSCs, porcine RPE cells1.1Isolation of hMSCs, porcine RPE cells1.2. FACS and identification of differention properties of hMSCs; Identification ofporcine RPE cells by immunofluorescence2.Inducing RPE differentiation of hMSCs2.1. co-culturing with RPE using transwell and RPE conditioned medium inducedhMSCs to differentiate to RPE like cells 2.2. Identification of co-culture induced hMSCs to differentiate to RPE like cells：cellmorphology, intracellular pigment content quantitative analysis, transmission electronmicroscopy detection, Real-time PCR, immunofluorescence staining, phagocytosis and celltomography experiments, Western Blot, verify MERTK protein in inducing cell-specificphagocytosis role and induce cell secretion of neurotrophic factors testing.3.Transplantation of hMSC-RPE in acute and chronic retinal degeneration model3.1. Building sodium iodide treated acute retinal degeneration rat model; identificationof the model: HE staining and F-ERG testing.3.2. Subretinal transplatation of hMSC and hMSC derived RPE cell in the acutesubretinal transplantation: cell morphology, immunofluorescence staining hMSCs derivedRPE cells, phagocytic assay and immunofluorescence staining, F-ERG and Western Blotafter transplantation.3.3. subretinal transplantation in RCS rats model: measuring the thickness of the outernuclear layer, immunofluorescence staining, F-ERG and phagocytosis assay aftertransplantation.Results：1. Isolation and identification of hMSCs, porcine RPE cells1.1. hMSCs morphology formed mostly spindle fibroblast-like appearance andvortex-like arrangement. Porcine RPE cells are polygonal in shape, and filled withbrown-black pigment granules.1.2. Flow cytometry analysis revealed that hMSCs were strongly positive for CD73,CD90and CD105, but negative for CD14, CD45and CD34. After osteogenic induction,hMSCs stained by alizarin red exhibited matrix mineralization formation after21-days inculture. hMSCs were also able to differentiate towards an adipose phenotype after culturingfor21days with adipogenic medium. In addition, the cells could also be induced to be achondrogenic phenotype after four weeks in culture following the formation of ahigh-density pellet and a serum-free chondrogenic medium with TGF-β3, positive alcianblue staining. Porcine RPE cells were positive staining for CRALBP, RPE65, ZO-1.2. Inducing RPE differentiation of hMSCs2.1. hMSCs derived RPE-like cells can be observed some pigment granulesintracellular, the content of melanin pigment is closer to the adult porcine RPE cells, some of which origins from the pigment granules secreted into the extracellular RPE cells wereinduced phagocytosis into the cell.2.2. The pigment content is of no significant difference between hMSCs derivedRPE-like cells induced by co-culture and native RPE cells. The pigment content is ofsignificant difference between hMSCs derived RPE-like cells induced conditioned mediumand native RPE cells.2.3. Electron microscopy also revealed the presence of mature pigment granules andfurthermore, microvilli was clearly present in hMSC-derived RPE cells after14-dayco-culture2.4. Various RPE-specific markers in hMSC-derived RPE-like cells were determined.These genes were significantly up-regulated; MITF, OTX2, tyrosinase, bestrophin,PMEL17, RPE65, PEDF,PAX6and CRALBP after being co-cultured with adult pig RPEcells for14days compared with control hMSCs without co-culture induction. Furthermore,the expression levels of the above genes in hMSCs-derived RPE were comparable to thosein freshly isolated adult RPE.2.5. An immunofluorescence assay was used to assess the expression of typical RPEmarker proteins in order to confirm that the increased gene expression led to changes inprotein levels; these included CRALBP, RPE65and ZO-1.2.6. Green fluorescence was observed in both adult pig RPE cells and hMSCs-derivedRPE cells after24h in culture suggesting these cells had acquired the ability to phagocytoseextracellular elements of the POS. Green fluorescence was not observed in the non-inducedhMSCs. After14-day co-culture, the expression of MERTK in induced hMSCs wassignificantly enhanced as compared with hMSCs without co-culture induction. Meanwhile,the expression of αVβ5Integrin was also enhanced slightly. hMSCs-derived RPE cellsexposed to MERTK antibodies showed a significant reduction in the number of POSingested over a5h period compared to control cells.2.7. The results from the ELISA assay showed that there was significant enhancementof BDNF and GDNF in hMSC-derived RPE, relative to hMSCs without co-cultureinduction. However, these values were significantly less than the values seen for the porcineRPE samples. 3. Transplantation of hMSC-RPE in acute and chronic retinal degeneration model3.1. Hematoxylin and eosin staining of retinal section from NaIO3injected rat showsthat compared to the confluent RPE monolayer in the uninjured eyes, the NaIO3-treatedeyes demonstrate a paucity of RPE cells with only very occasional rounded RPE cellsremained on the underlying Bruch’ s membrane. Compared with normal rats the sodiumiodate rats display significantly lower F-ERG in A wave and B wave amplitude.3.2. Confocal images showed that hMSC-RPE cells could express mitochondrial andCRALBP after transplantation tested by immunofluorescence staining.3.3. F-ERG results show that there was signifincantly increased in A wave and B-waveamplitude after transplantation of hMSC-RPE cells compared with hMSC transplantationand sham operation.3.4. After transplantation in the model of acute retinal degeneration, Western Blotshowed that rhodopsin expression was higher in hMSC-RPE cell transplanted rats thanhMSC cell transplanted rats.3.5. RCS rat retinal outer nuclear layer thickness was significantly thicker after hMSCs-RPE cells transplantation.3.6. Confocal images showed that hMSCs-RPE cells could express mitochondrial andCRALBP after transplantation tested by immunofluorescence staining.3.7. F-ERG results show that there was signifincantly increased in A wave and B-waveamplitude4weeks after transplantation hMSCs-RPE cells in RCS rats model.3.8. Western Blot results show MERTK expression in the retina after transplantaionwere positive in4weeks after transplantaion hMSC-RPE cells and negative in4weeks aftertransplantaion hMSC-RPE cells and both sham operation.3.9. Confocal images showed that rhodopsin positive POS could be found in PRE65positive transplanted cells4weeks after transplantation, but could not be found8weeksafter transplantationConclusion：1. We successfully obtained hMSCs and pig RPE cells. The morphology andphenotype of these two cells are consistent with hMSCs and porcine RPE cellscharacteristic. 2. We used transwell system to establish hMSCs and RPE cells non-contact co-culturesystem in vitro to induce differentiation of hMSCs to RPE cells. The induced cells showedbiological properties and functions similar to native RPE cells.3. In the intravenous injection of sodium iodate rat model of acute retinal degeneration,the changes of organization and structure are consistent with acute retinal degenerationchanges.4. The rats with acute retinal degeneration induced by sodium iodate injury weretransplanted by RPE cells derived from hMSCs had potent protective effects on visualfunction and maintenance.5. In the RCS rat model of chronic retinal degeneration, followed by transplantation ofRPE cells derived from hMSCs, the rats had phagocytic ability of photoreceptor outersegment. However, after8weeks of transplantation, the number of viable cells wassignificantly decreased, and only a small amount of cells expressed markers of RPE cellsmature. In addition, we did not observe the transplanted cells with phagocytic ability ofphotoreceptor outer segments as well as the maintenance of visual function. |
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