DSEK, DSAEK and DEMK had brought new technologies for corneal transplantation in the treatment of corneal endothelial dysfunction in recent years [6, 23]. But these methods also have disadvantages such as technical difficulty, cell loss, lamellae dislocation, graft rejections and graft failure after surgery [24–27]. However, all types of corneal transplantation depend on donor cornea and healthy HCECs. Corneal substitutes are expected to completely solve the worldwide shortage of corneal donors with the rapid development of tissue engineering and regenerative medicine[15]. Among the three elements of seed cells, scaffold materials and three-dimensional culture, seed cells are the most critical factor. Recently researchers had studied the effect of pharmacological agents on CEC proliferation, such as rho-associated protein kinase inhibitor Y-27632 and inhibitors of the p38 mitogen-activated protein kinase (MAPK) [10, 28]. The former researchers used cell injection with Y-27632 to treat human bullous keratopathy and the experimental results were gratifying. However, excessive cell proliferation usually leads to EMT with loss of function [29]. Therefore, how to obtain a sufficient number of HCECs with therapeutic ability remains a knotty problem.
OASCs and CECs are both derived from neural crest cells of the neuroectoderm [30, 31]. OASCs possess a powerful proliferative capacity and multi-lineage differentiation potential [20]. In the previous study, we prepared a conditioned medium obtained from OASCs (OASC-CM) and cultivated HCECs with OASC-CM (CM-HCECs). The results of in vitro experiments showed that CM-HCECs could highly express CEC related markers (N-Cadherin, Na+/K+ ATPase and ZO-1) even after 10 passages. Meanwhile, the proliferation and repair ability of HCECs were significantly enhanced. In order to test the therapeutic ability of CM-HCECs in vivo, we carried out animal experiments of corneal endothelial dysfunction in rabbit and monkey models. The results of 10 months observation after surgery showed that the cornea could recover and remain transparent in a short time which preliminarily proved the therapeutic effect of CM-HCECs [18].
In this study, we conducted monkey experiments and observed 24 months after cell transplantation. Through the experimental results we discovered that the corneas of the TR2Y group recovered rapidly and remained transparent. The central corneal thickness (CCT) maintained at about 500µm 1-24 months after surgery. The average endothelial cell density was the lowest in the third month, which was considered to be related to postoperative immune reaction, microenvironment changes and apoptosis caused by inflammatory factors [32, 33]. After 3 months, the cell density gradually increased, indicating that the transplanted cells had adapted to the new microenvironment and remained stable. At 24 months after cell transplantation, the cell density in the TR2Y group remained at about 2500 cells/mm2, which was slightly lower than that of over 3000 cells/mm2 in normal monkeys before the operation. In addition, there were no pathological changes in eyes of the TR2Y group by a gonioscope, fundus photography and B-mode ultrasound. The result indicated that transplanted cells coexisted harmoniously with the host. Histological examination and Immunofluorescence also showed that the transplanted cells formed single cell layer on DM and played the function of tight junction and pump function.
RNA-sequencing was used to study the gene expression pattern of HCEC, CM-HCECs and TR2Y cells after cell transplantation. Venn analysis and correlation analysis showed that the proportion of co-expressed genes and the correlation coefficient in HCECs, CM-HCECs and TR2Y cells were quite high. The results of GO annotation analyses also showed that the three cells were similar in gene expression pattern and belonged to the same type of cell.
Venn analysis showed that the CM-HCECs and TR2Y cells had high proportion of co-expressed genes. Correlation analysis showed that there was a high correlation between CM-HCECs and TR2Y cells and the r value was 0.911. It showed that most cells maintained the original expression pattern and remained in stable status after the transplantation. Venn analysis showed that the proportion of co-expressed genes of HCECs and tr2y was 77.53% which was higher than that of HCECs and CM-HCECs (77.48%). And the correlation analysis showed that the r value was 0.809 in HCECs vs TR2Y cells and 0.712 in HCECs vs CM-HCECs. The result indicated the transplanted cells were closer to HCECs. It could be seen from the analysis of the expression difference between CM-HCECs and TR2Y groups that transplanted cells had obvious changes in extracellular matrix, cell adhesion and other related functions. The result indicates that the adhesion of TR2Y cells is enhanced after cell transplantation. We consider that the microenvironment in monkey anterior chamber is more conducive to cell survival and growth than that in vitro [34].
In this study, keratic precipitates (KP) and anterior chamber exudation occurred after the transplantation. This may due to the immune reaction caused by some transplanted cells falling onto the lens or into the anterior chamber. In addition, heterologous grafts and usage of serum containing medium could also cause immune or rejection reactions [18]. But the reactions were moderate and could be controlled by conventional therapy. This may benefit from that the cultured HCECs are endowed with the function of immunomodulatory by CM [35, 36]. On the other hand, transplantation through cell-injection also has limitations such as inaccurate location of cells, cells lost and the requirement for special body position after surgery. We provide long-term observation and analysis results on advanced primates although the number of experimental animals is limited.
In this preclinical research the general condition of the monkeys was good during 24 months of postoperative observation. There were no indications of abnormal intraocular pressure, lens opacity and changes of fundus. In the future research, we will improve experiment method and procedure and conduct clinical trials.