| ObjectiveCorneal endothelial dysfunction which caused by trauma,surgery,endophthalmitis and other pathological factors,mainly manifested as corneal endothelial cell damage or loss.Because human corneal endothelial cells can not regenerate,if the loss of endothelial cells exceeds its critical value,it will cause corneal edema,blurred vision and eye irritation.In severe cases,secondary corneal ulcer will occur.At present,the treatment of corneal endothelial dysfunction mainly depends on corneal transplantation,but the lack of donor cornea seriously limits the clinical treatment.With the continuous development of tissue engineering and cell engineering technology,the research of tissue engineering cornea has made some progress.Constructing tissue engineering corneal endothelium for transplantation is the current research hotspot.Human embryonic stem cells are the ideal source of seed cells,t.However,the reported researchs have some shortcomings,such as long induction time,low induction efficiency,the demand of high surgical technic,poor in vivo verification effect,short in vivo survival time after cell transplantation or failure to maintain stability.In this study,the method of corneal endothelial cell transplantation was improved by anterior chamber injection of corneal endothelial mini sheets.Human embryonic stem cells(hESC)were induced by small molecules of RA,Y-27632 and cytokine bFGF into corneal endothelial cell-like cells in vitro to establish a method of directional differentiation of hESC-derived corneal endothelial cells.Combined with anterior chamber injection transplantation of mini sheets,New Zealand white rabbits and Cynomolgus monkey corneal endothelial dysfunction model were established to verify the therapeutic function of hESC-derived endothelial cell-like cells in vivo,and to provide a new strategy and scheme for clinical treatment of corneal endothelial dysfunction.Methods1.Preparing and transplantation of corneal endothelial mini sheetsPrimary rabbit corneal endothelial cells(RCECs)transplantation was performed by anterior chamber injection single cells and Descemet's Stripping Endothelial Keratoplasty(DSEK).The effect was observed and compared by slit lamp microscopy.Rabbit primary corneal endothelial cells were isolated and cultured by different cell digestive enzymes(Accutase,trypsin-EDTA and Dispase).The preparation conditions of mini sheets were determined by comparing the size and cell viability of the sheets.The in vitro adhesion ability,in vivo adhesion ability,in vivo intercellular junction formation ability,corneal transparency and thickness were compared between mini sheets and single cells.2.Inducing hESC to differentiate into corneal endothelial cell-like cells in vitrohESC were cultured with mTeSRTM1;hESC-derived crest cells were differentiated with UM medium supplemented with RA+bFGF,and identified by immunofluorescence staining and flow cytometry;corneal endothelial cells were differentiated with DP medium supplemented with Y-27632 and identified by immunofluorescence staining,flow cytometry and PCR.The safety of hESC-derived corneal endothelial cells were identified by NOD/SCID immunodeficient mice transplantation and HE staining.3.Identification of function of hESC-derived corneal endothelial cell-like cells in vivoThe induced corneal endothelial cell-like cells were prepared into mini sheets and transplanted into the right eye of New Zealand white rabbits by anterior chamber injection.The control group was the experimental rabbits who only scraped corneal endothelial cells.The cornea was observed by slit lamp microscopy after operation,corneal thickness was measured by corneal pachymeter,and the recovery of corneal transparency was compared between the transplanted cell group and the control group.The morphology of endothelial cells was observed by scanning the central corneal area with in vivo confocal corneal microscopy at different time points.Immunofluorescence staining was used to observe the connection of transplanted cells and endothelial pump function.Digoxin labeled cells were used to observe the survival of transplanted cells.In addition,the model of corneal endothelial dysfunction in cynomolgus monkeys was transplanted by anterior chamber injection of mini sheets.The cornea was observed by slit lamp microscopy after operation,and the recovery of corneal transparency was observed.Corneal thickness was measured by corneal pachymeter,and the changes of corneal thickness were observed.The morphology of transplanted endothelial cells was observed by in vivo confocal corneal microscopy scanning in the central corneal area.Immunofluorescence staining was used to observe the adherence and survival of transplanted cells in the Descemet's membrane,cell junction and endothelial pump function after induced corneal endothelial cell transplantation.Results1.Comparison of corneal endothelial cell transplantation methods(1)Comparison of cell transplantation by anterior chamber injection and DSEKThere was no significant difference in the rate of corneal edema subsidence between the two groups.The corneal opacity was not obvious after corneal edema disappear in single cells anterior chamber injection group.The corneal grafts transplanted by DSEK method had poor adherence in the early stage after operation,with a small amount of interlamellar effusion and some of the matrix grafts were nubecula after corneal edema subsided.(2)Effects of different digestive enzymes on primary RCECsDispase had weak digestibility and the cells were mainly dissociated into large sheets of cells.Trypsin-EDTA had strong digestibility,cells rapidly dissociate into single cells.Accutase cells had moderate digestibility,and the cells were mainly dissociated into cell sheets containing 4-10 cells.With the prolongation of digestion time,the morphology of the grafts did not change much.Within 10 minutes Acutase or trypsin-EDTA has less digestive damage to isolated cells and the cell viability of dissociated cells was close to or over 90%,the cell viability of the two digestive enzymes dissociated cells decreased significantly after 15 minutes.(3)The effect of mini sheets transplantation in animal modelsThe number of cells attached to mini sheets is significantly more than that of single cells in 1 hour in vitro and 6 hours in vivo after transplantation in New Zealand white rabbits.The expression of ZO-1 and endothelial pump Na~+/K~+-ATPase in mini sheets group was higher than that in single cells group 48 hours after anterior chamber injection.In vivo confocal corneal microscopy showed that hexagonal cells were observed in corneal endothelial cells 7 days after mini sheets injection,and the connections between endothelial cells are more closely,while the cells were irregular between 7 days and 21days after single cells injection,and there was no normal structural connection between cells.Injecting mini sheets has higher cell density than injecting single cells.Slit lamp microscopy showed that corneal transparency recovered rapidly 7 days after mini sheets injection,and gradually 14 days after single cells injection.Corneal thickness measurements and analysis showed that the corneal thickness decreased rapidly and remained stable 7 days after mini sheets injection,while the corneal thickness decreased to nearly normal 14 days after single cells injection.2.To establish a method of directional differentiation of hESC-derived corneal endothelial cells(1)Differentiation of hESC into neural crest cells in vitroThe morphology of hESC changed gradually,which showed that the cell volume became larger,the ratio of nucleus to cytoplasm became smaller,and the cell morphology gradually became polygonal.Fluorescence staining showed that the induced cells expressed neural crest marker genes(HNK-1?P75?SOX10?PITX2 and AP-2?),and the positive rate of P75 and HNK-1 by flow cytometry was 84.733%and 72.993%.(2)Differentiation of neural crest cells into corneal endothelial cell-like cells in vitroThe morphology of the neurocrest cells changed.The cells were polygonal,small and arranged in a paving stone-like shape.Fluorescence staining showed that the cells expressed the marker genes of corneal endothelial cells(ZO-1,Na~+/K~+-ATPase,N-cadherin),but did not express the markers of vascular endothelial cells(vWF,CD31).Flow cytometry showed that 98.090%of the cells expressed functional protein gene Na~+-K~+-ATPase.(3)Tumorigenicity of induced cellsSubcutaneous mass growth was observed in immunodeficient mice inoculated with embryonic stem cells 5 to 6 weeks after inoculation,and paraffin section staining was performed to show morphology of polyembryonic histiocytes.Immunodeficient mice inoculated with induced corneal endothelial-like cells showed no tumor formation 8months after inoculation.3.The therapeutic function of hESC-derived corneal endothelial cell-like cells in animal models(1)The therapeutic function of hESC-derived corneal endothelial cell-like cells in the corneal endothelial dysfunction New Zealand White Rabbit ModelThe slit lamp microscopic showed that the corneal transparency of rabbits in the transplanted cells group began to recover gradually 7 days after operation.Corneal transparency was basically restored 2 weeks after operation.The corneal transparency remained stable for 4 to 8 weeks after operation.The cornea of the control group was cloudy after operation,and the cornea remained nubecula 8 weeks after operation.Corneal thickness in the transplanted cells group began to decrease gradually 7 days after operation and returned to normal thickness within 2-3 weeks,while the corneal thickness of the control group did not decrease significantly after operation.The corneal thickness of the transplanted cells group remained around 1000?m for a long time.In vivo Confocal corneal microscopy scanning showed that the cells attached to the Descemet's membrane in the central cornea of the transplanted cells group were significantly smaller than those of the normal corneal endothelial cells 2 weeks after operation,with high cell density,no obvious hexagonal morphology and no obvious junction between the cells.At 5 weeks after operation,the cells were slightly larger than those of normal corneal endothelial cells,some of which had hexagonal morphology,and the junctions between the cells could be seen.The morphology and size of cells were similar to those of 5 weeks after operation,and the morphology of cells remained stable.Immunofluorescence staining showed that anti-human antigen was expressed in the elastic layer attached cells and ZO-1 and Na~+/K~+-ATPase were expressed in the transplanted cells at 1 week after operation.4 weeks after operation,a large amount of red fluorescence was observed in the central cornea by fluorescence microscopy.(2)The therapeutic function of hESC-derived corneal endothelial cell-like cells in the corneal endothelial dysfunction Cynomolgus Monkey ModelSlit lamp microscopy showed that corneal transparency began to recover gradually at7 days after transplantation,and clear pupil contour could be seen.The cornea was basically transparent and iris texture was clearly visible at 2 weeks after operation.Corneal thickness gradually decreased to normal within 14 days after transplantation.Tn vivo confocal corneal microscopy showed that at 11 days after operation the cells attached to the elastic layer were significantly smaller than those of the normal corneal endothelial cells.The cell density was higher.There was no obvious hexagonal shape and no obvious connection between the cells.The morphology of cells at 25 days was similar to that of 11 days,but the morphology of cells tended to be regular gradually.The size of cells was similar to that of normal corneal endothelial cells at 43 days,and some cells were similar to hexagonal shape.Connections between cells were observed.The morphology and size of cells were similar to that of 43 days after operation at 60 days,and the morphology of cells remained stable.3 days after operation,immunofluorescence staining showed that the anti-human antigen positive cells adhered to the Descemet's membrane of cornea,and some cells expressed Na~+/K~+-ATPase and SLC4A11.Conclusions1.Accctase was used to prepare mini sheets of corneal endothelial cells.Mini sheets anterior chamber injection could promote the rapid adherence of donor cells,accelerate the formation of tight junction of transplanted cells,and then play a barrier function,and promote the function of endothelial pump of transplanted cells.2.Using small molecules RA,Y-27632 and cytokine bFGF,hESC can be induced to differentiate into crest cells and then into corneal endothelial-like cells by two-step method.This method simulates the development of corneal endothelial cells in vivo.3.hESC-derived corneal endothelial cell-like cells can promote the regression of corneal edema and the recovery of corneal transparency in corneal endothelial dysfunction New Zealand white rabbits and cynomolgus monkeys,transplanted cells can survive for a long time in vivo and play endothelial pump function.Innovation and Significance1.Combining the advantages of traditional corneal endothelial transplantation and anterior chamber cell injection and preparing mini sheets for anterior chamber injection to transplant corneal endothelial cells,promoting the adherence and function of transplanted cells,improving the method of corneal endothelial cell transplantation.2.For the first time small molecules of RA,Y-27632 and cytokine bFGF were used to induce hESC to differentiate into corneal endothelial-like cells simulating the development of corneal endothelial cells in vivo rapidly and effectively which providing a new method for obtaining tissue-engineered corneal endothelial cells.3.For the first time the function of hESC-induced corneal endothelial-like cells were verified in large animal model of cynomolgus monkeys providing a new method for clinical treatment of corneal endothelial dysfunction. |
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