Study On The Corneal Endothelial Cells Substituted With Transplantation Of Bone Marrow-derived Mesenchymal Stem Cells Of Rhesus Monkey

Purpose: Achieve and cultural rhesus monkey bone marrow mesenchymal stem cells (BMSCs) in vitro, then, transplant it into the endothelial surface of corneal whose Descemet's membrane was splited. Observe the change in cell morphology and function, to examine the feasibility of the corneal endothelial cells substituted with transplantation of bone marrow-derived mesenchymal stem cells.Methods: The experimented monkeys are divided into 2 groups: experimental group (3 monkeys) and control group (1 monkey). Mesenchymal stem cells (MSCs) were isolated from bone marrow by density gradient centrifugation combined with adhering means. The cultured cells were identification was perform by flow cytometry and detect its ability of differentiation by differentiate into adipocytes in vitro, and the differentiated cells were determined by oil Red O. After labeled by 5-bromodeoxyuridine (Brdu), cultured cells were transplanted onto the endothelial surface of corneal whose Descemet's membrane was splited by centrifugation method. Post-operation, the transparency of corneas were observed, corneal graft were remove after 1 month, 2 month, 3 month respectively for measurement purpose. Suchas pathology detection, anti Brdu monoclonal antibody staining, electron microscopy observation of the distribution and the connection between transplanted cells and the changes of their morphology.Result: Density gradient centrifugation combined with adhering method can yield high purity MSCs. A small amount of cells attached to the wall after 24 hours of cultured in vitro, 48 hours later, attaching rate reach into 5%, and after 12 to 16 days, cells were confluence. Cells show a spindle shape and arranged in parallel or swirling. By flow cytometry analysis, cultured cells were positive in CD29 (94.26%) when compared with the low positive rates for CD34 (7.51%), and CD45 (4.02%). After 3 weeks of differentiation, MSCs were differentiated into adipocytes, and shows a orange-red staining in the cytoplasm when dyed by Oil Red O, meanwhile, nuclei stained blue. Both corneal grafts of experimental and control groups became edema after 2 weeks of transplantation. The corneal grafts of experimental group maintain a certain transparency after 7-8 weeks transplantation. But, the corneal graft of control group run severe edema, and bullous keratopathy occurred. Scanning electron microscopy showed that the transplanted cells attached loosely to the endothelial surface of the corneal graft, gave a spindle shape after one month transplantation, cells contact to each other, and showed spindle and polygonal shape, after 2 months surgery, cells contact to each other closely after 3 months surgery. Cells contained Brdu were stained brown in the nuclei by immunohistochemical method.Conclusion: Density gradient centrifugation combined with adhering method can yield high purity MSCs. Cells differentiated into corneal endothelial like cells when transplanted onto endothelial side of the cornea.