Noriko Koizumi

Cultivated corneal epithelial stem cell transplantation in ocular surface disorders

PURPOSE To investigate the outcome of cultivated corneal epithelial transplantation for severe stem cell deficiencies using denuded amniotic membrane (AM) as a carrier. DESIGN Retrospective, noncomparative case series. PARTICIPANTS Thirteen eyes of 11 patients were studied. These consisted of five eyes with acute Stevens-Johnson syndrome (SJS), two with chronic SJS, one with an acute chemical injury, two with chronic chemical injuries, two with ocular cicatricial pemphigoid, and one with drug-induced pseudopemphigoid. All of these eyes had total stem cell deficiencies. MAIN OUTCOME MEASURES Adaptation of the cultivated corneal epithelium onto the host corneal surface was confirmed 48 hours after surgery. The reconstruction of the ocular surface and visual acuity were measured. METHODS Corneal limbal epithelium from donor corneas was cultivated for 4 weeks on a denuded AM carrier, with 3T3 fibroblast coculture and air lifting. The cultivated corneal epithelium showed four to five layers of stratification and was well differentiated. After conjunctival tissue removal from the cornea up to 3 mm outside the limbus and subconjunctival tissue treatment with 0.04% mitomycin C, cultivated allocorneal epithelium, including the AM carrier, was transplanted onto the corneal surface up to the limbus. Lamellar keratoplasty, using preserved donor graft without epithelium, was performed simultaneously for five chronic-phase patients showing corneal stromal scarring. Systemic immunosuppression was used to prevent allograft rejection. RESULTS In all 13 eyes, the entire corneal surface, on which cultivated allocorneal epithelium had been placed, was free from epithelial defects 48 hours after surgery, indicating complete survival of the transplanted corneal epithelium. Visual acuity improved in all eyes after surgery, and 10 of the 13 eyes were restored to good vision (postoperative visual acuity improved two or more lines) 6 months after the operation. During the follow-up period (mean +/- standard deviation, 11.2 +/- 1.3 months), the corneal surfaces were clear, although three eyes experienced epithelial rejection. CONCLUSIONS Cultivated corneal epithelial transplantation using denuded AM as a carrier can be used for severe stem cell deficiencies.

Amniotic Membrane as a Substrate for Cultivating Limbal Corneal Epithelial Cells for Autologous Transplantation in Rabbits.

Purpose. To examine the viability of using human amniotic membrane as substrate for culturing corneal epithelial cells and transplanting them onto severely injured rabbit eyes. Methods. An ocular-surface injury was created in the right eye of eight rabbits by a lamellar keratectomy extending 5 mm outside the limbus. Next, from the limbal region of the uninjured left eyes of five of these animals, a small biopsy of corneal epithelial cells was taken and cultured on acellular human amniotic membrane. One month later, the invading conjunctiva that covered the corneal surface of all eight injured eyes was surgically removed. Five of the eyes then received grafts of amniotic membrane containing autologous cultured epithelial cells, whereas the other three received grafts of acellular amniotic membrane alone. Results. A confluent primary culture of limbal corneal epithelial cells was established on acellular human amniotic membrane after 14 days. Cells were partially stratified and fairly well attached to the underlying amniotic membrane, although a fully formed basement membrane was not evident. The three rabbits that received amniotic membrane transplantation alone all had total epithelial defects on the graft in the early postoperative period. Eyes that were grafted with amniotic membrane that contained cultivated epithelial cells, however, were all successfully epithelialized up to 5 days after surgery. Conclusion. Autologous transplantation of cultivated corneal epithelium is feasible by using acellular amniotic membrane as a carrier.

Enhancement on primate corneal endothelial cell survival in vitro by a ROCK inhibitor.

PURPOSE The transplantation of cultivated corneal endothelial cells (CECs) has gained attention recently for the treatment of patients with corneal endothelial dysfunction. However, an efficient culturing technique for human (H)CECs has yet to be properly established. The present study was conducted to investigate the applicability of the Rho kinase (ROCK) inhibitor Y-27632 in promoting cultivation of cynomolgus monkey (M)CECs. METHODS MCECs of cynomolgus monkeys were cultured in a medium containing 10 microM Y-27632. The number of viable cells adherent to culture plates were monitored by a luminescent cell-viability assay and colony growth was detected by toluidine blue staining. Proliferating cells were detected by Ki67 expression using flow cytometry and a BrdU-labeling assay for immunocytochemistry. Annexin V-positive apoptotic cells were analyzed by flow cytometry. RESULTS The number of viable cultivated MCECs was enhanced by Y-27632 addition after 24 hours in culture. The colony area of the culture in the presence of Y-27632 was higher than in the absence of Y-27632 on day 10. In Y-27632-treated cultures, the number of Ki67-positive cells was significantly increased at 24 and 48 hours, and the number of proliferating BrdU-positive cells was increased at 48 hours. The number of Annexin V-positive apoptotic cells was decreased at 24 hours. CONCLUSIONS The inhibition of Rho/ROCK signaling by specific ROCK inhibitor Y-27632 promoted the adhesion of MCECs, inhibited apoptosis, and increased the number of proliferating cells. These results suggest that the ROCK inhibitor may serve as a new tool for cultivating HCECs for transplantation.

Immunosuppressive properties of human amniotic membrane for mixed lymphocyte reaction

The combination of allograft limbal transplantation (ALT) and amniotic membrane transplantation (AMT) has been applied in the treatment of severe ocular surface diseases. The beneficial effect of this combination has been thought to result from possible immunosuppressive ability of amniotic membrane (AM). However, the mechanisms of any such ability remain unknown. In this study, we investigated whether human AM has the ability to suppress allo‐reactive T cell responses in vitro. For mixed lymphocyte reaction (MLR), lymphocytes isolated from lymph nodes of C57BL/6 mice (Mls1b, Vβ6+) were cultured with irradiated splenocytes from DBA/2 mice (Mls1a, Vβ6−) with or without human AM. For carboxyfluorescein diacetate succinimidyl ester (CFSE) experiments, responder lymph node cells were labelled with a stable intracellular fluorescent dye and cultured with irradiated stimulator cells. The ratio of responder Vβ6+ T cells was then determined by FACS analysis, and the division profiles of responder Vβ6+ T cells were analysed by CFSE content. Furthermore, Th1 and Th2 cytokine synthesis by allo‐reactive T cells in MLR culture supernatants was determined by enzyme‐linked immunosorbent assay (ELISA). Addition of AM to the MLR culture resulted in the significant inhibition of thymidine incorporation compared with control culture lacking AM. The population of responder CD4+Vβ6+ T cells was significantly reduced in the AM‐treated culture in comparison to control. CFSE analysis revealed less division and lower proliferation of responder CD4+Vβ6+ T cells in cultures with AM than without. In addition, allo‐rective T cell synthesis of both Th1 (IL‐2 and IFNγ) and Th2 (IL‐6 and IL‐10) type cytokine was significantly decreased in the presence of AM. These results indicate that human AM has the ability to suppress allo‐reactive T cells in vitro. This inhibitory effect likely contributes to the success of the ALT‐AMT combination.

ROCK Inhibitor Converts Corneal Endothelial Cells into a Phenotype Capable of Regenerating In Vivo Endothelial Tissue

Corneal endothelial dysfunction accompanied by visual disturbance is a primary indication for corneal transplantation. We previously reported that the adhesion of corneal endothelial cells (CECs) to a substrate was enhanced by the selective ROCK inhibitor Y-27632. It is hypothesized that the inhibition of ROCK signaling may manipulate cell adhesion properties, thus enabling the transplantation of cultivated CECs as a form of regenerative medicine. In the present study, using a rabbit corneal endothelial dysfunction model, the transplantation of CECs in combination with Y-27632 successfully achieved the recovery of corneal transparency. Complications related to cell injection therapy, such as the abnormal deposition of the injected cells as well as the elevation of intraocular pressure, were not observed. Reconstructed corneal endothelium with Y-27632 exhibited a monolayer hexagonal cell shape with a normal expression of function-related markers, such as ZO-1, and Na(+)/K(+)-ATPase, whereas reconstruction without Y-27632 exhibited a stratified fibroblastic phenotype without the expression of markers. Moreover, transplantation of CECs in primates in the presence of the ROCK inhibitor also achieved the recovery of long-term corneal transparency with a monolayer hexagonal cell phenotype at a high cell density. Taken together, these results suggest that the selective ROCK inhibitor Y-27632 enables cultivated CEC-based therapy and that the modulation of Rho-ROCK signaling activity serves to enhance cell engraftment for cell-based regenerative medicine.

Successful regrafting of cultivated corneal epithelium using amniotic membrane as a carrier in severe ocular surface disease

Purpose. Our group performed cultivated allogeneic corneal epithelial transplantation in 13 eyes from 11 patients with severe ocular surface disorders. After the clinical application of this new surgical treatment, some patients experienced epithelial and subepithelial opacities. We applied our procedure again in these patients to achieve successful ocular surface reconstruction. Methods. The corneal limbal epithelial cells from donor corneas were cultivated for 4 weeks on denuded amniotic membrane (AM) carrier, with 3T3 fibroblast coculture and airlifting. The study subjects consisted of 3 patients. At 3 and 12 months after the first operation, the failed epithelial graft with AM was replaced with new allogeneic corneal epithelium cultivated on AM. Results. At 48 hours after transplantation, the corneal surfaces of the 3 eyes were clear and smooth; the entire corneal surfaces were evenly covered with the transplanted cultivated corneal epithelium, which did not stain with fluorescein. The ocular surface epithelia of these patients are all stable without epithelial defects. Conclusions. We have shown that, in cases where the initially transplanted cultivated epithelium becomes opaque, it is possible to repeat the transplantation process with new cultivated epithelium on AM.

Comparison of ultrastructure, tight junction-related protein expression and barrier function of human corneal epithelial cells cultivated on amniotic membrane with and without air-lifting

PURPOSE To evaluate the usefulness of the air-lifting technique for culturing corneal limbal epithelial cells on amniotic membrane (AM) for use in ocular surface reconstruction. A cultured sheet that has a good barrier function should be better for this purpose. In corneal epithelium, tight junctions (TJ) play a vital role in the barrier function. The TJ complex includes the integral transmembrane proteins occludin and the claudins, and some membrane-associated proteins such as ZO-1. In this paper, we investigated the barrier function and the expression of TJ related proteins. METHODS Corneal limbal epithelium obtained from donor corneas and cultivated on acellular AM was divided into two groups. These were the non-air-lifting (Non-AL) group, which was continuously submerged in medium, and the air-lifting (AL) group, which was submerged in medium for 3 weeks, then exposed to air by lowering the medium level. Morphology and the permeability to horseradish peroxidase (HRP) were determined by electron microscopy. Tight junction (TJ)-related protein and mRNA expression changes were assessed by immunoblotting and reverse transcription-polymerase chain reaction. RESULTS The cultures of both groups formed 4-5-layer-thick, well-stratified epithelium. The AL cultures had tightly packed epithelial cells with all the HRP/diaminobenzidine (DAB) reaction product accumulated on the apical surface of the superficial cells. The Non-AL culture, by contrast, had more loosely packed epithelial cells with larger intercellular spaces. The HRP/DAB reaction product penetrated the intercellular space to a depth of 3-4 cell layers. Statistically, there was a significant difference in intercellular spaces and desmosome count in the superficial cells between the groups. With AL, TJ-related proteins localized at the apical portion of the lateral membrane. TJ-related protein and mRNA amounts were not changed by AL while claudin subtype expression became more consistent and closer to that of in vivo corneal epithelium. CONCLUSIONS The AL technique reduces intercellular spaces in the superficial cells and promotes the formation of the barrier function. It is useful in culturing corneal epithelial cells for use in ocular surface reconstruction.

Comparison of intact and denuded amniotic membrane as a substrate for cell-suspension culture of human limbal epithelial cells

BackgroundWe have previously developed a limbal epithelial culture system using a cell-suspension method on denuded amniotic membrane (AM). However, other workers reported that intact AM is advantageous for limbal epithelial culture in that it preserves stem cell characteristics. In this study, we cultivated human limbal epithelial cell-suspensions on both intact and denuded AM and compared the morphology and adhesion of the limbal epithelial cells on these two substrates.MethodsHuman limbal epithelial cells were dissociated from donor eyes using dispase and gentle pipetting and then seeded onto intact and denuded AM as cell suspension. Limbal epithelial cells on AM were co-cultured with a MMC-treated 3T3 fibroblast feeder layer and epithelial differentiation was promoted by air lifting. Cultures were examined by light, scanning and transmission electron microscopy and differences in cellular attachments and intercellular spacing were quantified. Basement membrane complexes were examined by indirect immunofluorescence.ResultsLimbal cells grown on denuded AM were well stratified and differentiated. Cells were well attached to each other and to the basement membrane. In contrast, limbal cells cultured on intact AM failed to stratify and in places formed a monolayer.The culture on denuded AM had significantly (P<0.001) more desmosomal junctions as well as significantly (P<0.001) more junctional attachments to the carrier than the intact culture. In addition, the intercellular spaces between cells cultivated on denuded AM were significantly (P<0.001) smaller than those between cells grown on the intact substrate. In cultures on both denuded and intact AM, the basement membrane zone displayed a positive staining for collagen VII, integrins alpha-6 and beta-4 and laminin 5.ConclusionsWe successfully cultivated well-stratified and -differentiated limbal cells on denuded AM, while on the intact AM limbal cells failed to stratify and in places formed only a monolayer of cells. The limbal cells cultivated on denuded AM were well attached to the AM stroma and were morphologically superior to the limbal epithelium cultivated on intact AM. We conclude that for purposes of transplantation of differentiated epithelial sheets, denuded AM is probably the more practical carrier for human limbal epithelial cell cultures when using our cell-suspension culture system.

Diagnosis and Treatment of Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis with Ocular Complications

PURPOSE To present a detailed clarification of the symptoms at disease onset of Stevens-Johnson syndrome (SJS) and its more severe variant, toxic epidermal necrolysis (TEN), with ocular complications and to clarify the relationship between topical steroid use and visual prognosis. DESIGN Cross-sectional study. PARTICIPANTS Ninety-four patients with SJS and TEN with ocular complications. METHODS A structured interview, examination of the patient medical records, or both addressing clinical manifestations at disease onset were conducted for 94 patients seen at Kyoto Prefectural University of Medicine. Any topical steroid use during the first week at the acute stage also was investigated. MAIN OUTCOME MEASURES The incidence and the details of prodromal symptoms and the mucosal involvements and the relationship between topical steroid use and visual outcomes. RESULTS Common cold-like symptoms (general malaise, fever, sore throat, etc.) preceded skin eruptions in 75 cases, and extremely high fever accompanied disease onset in 86 cases. Acute conjunctivitis and oral and nail involvements were reported in all patients who remembered the details. Acute conjunctivitis occurred before the skin eruptions in 42 patients and simultaneously in 21 patients, whereas only 1 patient reported posteruption conjunctivitis. Visual outcomes were significantly better in the group receiving topical steroids compared with those of the no-treatment group (P<0.00001). CONCLUSIONS Acute conjunctivitis occurring before or simultaneously with skin eruptions accompanied by extremely high fever and oral and nail involvement indicate the initiation of SJS or TEN. Topical steroid treatment from disease onset seems to be important for the improvement of visual prognosis.

The ROCK Inhibitor Eye Drop Accelerates Corneal Endothelium Wound Healing

PURPOSE To evaluate the effect of Rho kinase (ROCK)-inhibitor eye drops on a corneal endothelial dysfunction primate model and human clinical case series of corneal endothelial dysfunction. METHODS As a corneal-endothelial partially injured model, the corneal endothelium of seven cynomolgus monkeys was damaged by transcorneal freezing; 10 mm of rock inhibitor Y-27632 was then applied topically 6 times daily. The phenotype of the reconstructed corneal endothelium was evaluated by immunohistochemical analysis and noncontact specular microscopy. For clinical study, the effect of Y-27632 eye drops after transcorneal freezing was evaluated in eight corneal endothelial dysfunction patients: four central corneal edema patients and four diffuse corneal edema patients. RESULTS Slit-lamp microscopy revealed that both Y-27632-treated and -nontreated corneas became hazy after transcorneal freezing, and then recovered their transparency within 4 weeks. ROCK inhibitor Y-27632 promoted recovery of corneal endothelial cell density and wound healing in terms of both morphology and function. The percentage of ZO-1 and Na(+)/K(+)-ATPase positive cells in the regenerated area in the Y-27632 group was significantly higher than in the controls. Noncontact specular microscopy revealed that corneal endothelial cell density was significantly higher in the Y-27632 group compared with the controls at 4 weeks; cell density reached approximately 3000 cells/mm(2), as opposed to 1500 cells/mm(2) in the control group. In addition to the animal study findings, the clinical study findings showed that Y-27632 eye drops effectively improved corneal edema of corneal endothelial dysfunction patients with central edema. CONCLUSIONS These findings show that rock inhibitor Y-27632 eye drops promote corneal endothelial wound healing in a primate animal model and suggest the possibility of Y-27632 as a novel therapeutic modality for certain forms of corneal endothelial dysfunction. (http://www.umin.ac.jp/ctr/ number, UMIN000003625.).