Promoting The Expansion And Function Of Human Corneal Endothelial Cells And Therapy Of Corneal Endothelial Decompensation

Corneal disease is a major cause of blindness in the world,in which the corneal endothelial decompensation causes severe impairment of vision.The only solution is keratoplasty.However,the treatment is hampered by a worldwide shortage of donor corneas.In China There were about 300,000 patients that needed corneal transplantation each year,but the patients treated with surgeries were no more than 1%cases due to are performed every year because of the severe insufficient donor corneas.Tissue engineering is the science based on the principle of cell biology and engineering.Tissue cells with specific biological activity were combined with biological materials in vitro or in vivo building tissue and organs,in order to maintain,improve or repair damaged tissue and organ.The basic principle of tissue engineering is to organize cells(or stem cells)attach to the materials with good biocompatibility;Then the cell-biomaterial complex was implanted in specific parts of the body,or placed in a specific environment,forming a new organization with a specific structure and function step by step.Regenerative medicine,using the theory of biology and engineering,creates tissues or organs that are damaged so as to restore normal function of the organization and tissues.In recent years great progress has been made in regenerative medicine and tissue engineering.It is possible to construct corneal substitutes that have similar structure and function with the natural cornea by inoculating the cultured corneal cells on the scaffold material.New therapies may replace the conventional donor corneal transplantation along with the developments of regenerative medicine and tissue engineering.But sufficient functional corneal endothelial cells(CECs)are essential.The aim of this study was to promote the expansion and function of human corneal endothelial cells(HCECs)in vitro and in vivo.OASCs are derived from neural crest cells of the neuroectoderm.They possess a powerful proliferative capacity and multi-lineage differentiation potential as do many other stem cells.OASCs can be subcultured several times while maintaining the characteristic of stem cells.In addition,OASCs share the same embryonic origin with CECs.We hypothesized that human OASCs could promote the expansion and function of HCECs while maintaining their phenotype.Besides,orbital adipose tissues are easily accessible and they provide huge numbers of stem cells.We cultured human orbital adipose-derived stem cells and prepared conditioned medium by orbital adipose-derived stem cells.HCECs were isolated and cultured by using the conditioned medium in vitro.The cultivated HCECs could maintain fairly good proliferative and cell-based therapy capacity over]0 passages in this study.In animal experiments,corneal transparence was achieved after the injection of HCECs which demonstrated the good repair capacity of the cells.Our findings demonstrated that cultured HCECs with OASC-CM might be a promising source for research and clinical treatment.Objective1.Prepare conditioned medium by using orbital adipose-derived stem cells(OASCs).2.Investigate whether human adipose-derived stem cell-conditioned medium(CM)promote the proliferation and repair capacity of HCECs.3.Further test the proliferation and repair ability of cultured HCECs,and evaluate the feasibility of the cells in the treatment of corneal endothelial decompensation by animal experiments.MethodsPart I:Preparation of orbital adipose-derived stem cell-conditioned medium1.Isolation and culture of human orbital adipose-derived stem cellsThe orbital adipose tissues were repeatedly washed with PBS.Then the tissues were fragmented with surgical scissors and suspended in collagenase at 37℃ with gentle stirring.After 2-4h digestion,fragmented tissues were re suspended in Dulbecco’s modified Eagle’s medium low glucose(DMEM-LG)containing 10%fetal bovine serum for 5min at room temperature and then filtered through a 70p.m strainer.The fluid was washed with PBS and centrifuged twice for 5 min at 1200 rpm at RT.Cell suspensions were plated in T-25 flask in DMEM-LG supplemented with 10%FBS,10%penicillin-streptomycin and incubated at 37 ℃ in 5%CO2.The phenotypes of human orbital adipose-derived stem cells(OASCs)were detected by Flow cytometry and Immunofluorescence.2.Preparation of conditioned mediumOASCs were washed 3 times with PBS and the medium was replaced with basal growth medium.The OASCs were kept for cultivate.Then the medium was collected and filtered(0.22μm)and stored at-80 ℃ to preserve the biological activity.Part II:Experimental study on promoting the proliferation and function of human corneal endothelial cells by conditioned medium1.Isolation and culture of human corneal endothelial cellsAfter corneas were washed with M199,the Descemet’s membranes(DM)containing HCECs were stripped and digested with collagenase.The HCECs were resuspended and seeded in 1 well of a 12-well plate coated with FNC Coating Mix.The cells were cultured in OASC-CM(CM-HCECs)as the experimental group.The BM was composed of Opti-MEM-I,8%FBS,5 ng/mL human epidermal growth factor(hEGF),20μg/mL ascorbic acid,200 mg/L calcium chloride,0.08%chondroitin sulfate,and 50μg/mL penicillin-streptomycin.2.To detect the proliferation and repair ability of cultured human HCECsCEC relative markers such as zonula occludens-1(ZO-1),Na+/K+ ATPase and N-cadherin were detected by Real-time reverse transcription-polymerase chain reaction(RT-PCR),Western Blot and immunofluorescence.Cell Counting Kit-8(CCK-8)assay and Wound Healing assay were performed to evaluate the cells-proliferation ability.Part I:II In vivo evaluation of human corneal endothelial cell repair ability for corneal endothelial decompensation1.Establishment of corneal endothelial decompensation model for animals(New Zealand white rabbits and rhesus monkeys)To induce endothelial decompensation 3-mm limbal-corneal incisions were made,and the corneal endothelia were mechanically scraped with a modified irrigator needle.2.Transplantation of HCECs into the corneal endothelial decompensation modelThe cells were labeled by CFSE.Before cell injection,1100ul aqueous humor was extracted from the anterior chamber of the rabbit corneal endothelial decompensation models.Then CFSE-labeled HCECs were suspended in 100u1 Opti-MEM-I and injected into the anterior chamber of the eyes by an insulin needle.A peribulbar injection of Triamcinolone Acetonide and a subconjuctival injection of dexamethasone were given.After the injection,the operation eyes were kept in the face-down position for 5 hours under general anesthesia.3.Postoperative observationThe corneal transparency and thickness were observed and photographed using slit-lamp microscopy and optical coherent tomography.Part of the cornea was used to detect the CFSE signal and had immunofluorescent staining in the frozen section,and the other part was subjected to H&E staining.ResultsPart I:Preparation of orbital adipose-derived stem cell-conditioned mediumIn the study human OASCs were plastic adherent,spindle-shaped,fibroblast-like cells.Surface immuno-phenotype characterized by flow cytometry revealed that OASC’s highly expressed CD29,CD 105,CD49e and CD 166,suggesting their endothelial and stem cell origin.Immunofluorescence staining showed that OASCs strongly expressed vimentin in the cell plasma which is a kind of intermediate filament protein found in normal HCECs.Cultured OASCs demonstrated similar morphology and phenotype before passages 10.Part II:Experimental study on promoting the proliferation and function of human corneal endothelial cells by conditioned mediumCEC relative markers zonula occludens-1(ZO-1),Na+/K+ ATPase,N-cadherin,Col8a2 and SLC4A4 expressed in HCECs cultured by OASC-CM.The HCECs could maintain polygonal cell morphology and good proliferative capacity.Part III:In vivo evaluation of human corneal endothelial cell repair ability for corneal endothelial decompensationResults showed that CM-HCECs were successful in recovering transparency of the endothelium damaged corneas within 7 days although anterior chamber exudation could be seen.The corneal thickness rapidly decreased after injection of CM-HCECs.The changes of the central corneal thickness(CCT)were compared among different groups.Confocal microscope images and alizarin red S staining confirmed coverage of polygonal cells on the DM in the CM-HCEC group.Histological examination was done after the euthanasia of rabbits.The CFSE-labeled HCECs were detected by fluorescent microscope examination in the CM-HCEC group.H&E staining showed that the HCECs tightly adhered to the DM of the cornea in a monolayer.2.Animal experiments of rhesus monkeysSlit-lamp microscopy and OCT showed that the cells made the cornea transparent about 7 days after the injection.Rejection reactions such as keratic precipitates(KP)and anterior chamber exudation were noticed in the tenth to the fourteenth day after the injection,but almost a normal cornea and anterior chamber could be obtained after the postoperative treatment.One month after the surgery,the corneas of the CM-HCEC group became more transparent and thinner.The CCT of the CM-HCEC group was much thinner than that of the control group after surgery and almost the same as that in normal corneas.Intraocular pressures(IOP)were found to be no difference between the CM-HCEC group and normal group.The monkey models were observed for 10 months to evaluate the long-term effects of the HCECs injection.The corneas of the CM-HCEC group were still as transparent as normal cornea while corneal opacity and stromal edema were obvious in the control group.A gonioscope,fundus photography and B-mode ultrasound showed no pathological changes of the CM-HCEC group eyes.Immunofluorescent staining showed CFSE-positive signals of HCECs and positive fluorescent staining of the nuclei 2 months after the cell injection,which demonstrated that the injected HCECs regenerated the corneal endothelium.Na+/K+ ATPase and Zo-1 were also expressed in HCECs,indicating the persistent pump and barrier function.H&E staining showed that the HCECs formed a monolayer on the DM.3.Long-term postoperative observation in rhesus monkeysThe monkey models were observed for more than 1 year to evaluate the long-term effects of the HCECs Transplantation,The corneas of the CM-HCEC group were still as transparent as normal cornea.A gonioscope,fundus photography and B-mode ultrasound showed no pathological changes of the CM-HCEC group eyes.Conclusion1.The cultured human OASCs highly expressed stem cell relative markers.They are similar to other adipose-derived mesenchymal stem cells.2.The cells proliferation abilities were significantly enhanced and related functional markers were strongly positive while HCECs morphology was maintained by using OASC-CM.HCECs obtained some stem-cell-like properties.3.The cells proliferation abilities were significantly enhanced and related functional markers were strongly positive while HCECs morphology was maintained by using OASC-CM.We produced rabbit and monkey corneal endothelial decompensation models and successfully treated them through cell injection.With this approach many more functional HCECs could be available for research and cell-based therapy for corneal endothelial decompensation.