Hidetoshi Tanioka

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.

Cultivated corneal endothelial transplantation in a primate: possible future clinical application in corneal endothelial regenerative medicine.

Purpose: To review our attempt to devise a method of cultivated corneal endothelial transplantation using primates in which corneal endothelium, like that of humans, has low proliferative ability. Methods: Monkey corneal endothelial cells (MCECs) were cultivated, with subcultures grown on collagen type I carriers. The corneal endothelia of 6 eyes of 6 monkeys were scraped intensively, after which cultivated MCEC sheets were inserted into the anterior chamber of 4 eyes. As controls, a collagen sheet without MCECs was transplanted in 1 eye of a monkey, and a suspension of cultivated MCECs was injected into the anterior chamber of 1 eye of another monkey. Results: MCECs produced a confluent monolayer of closely attached hexagonal cells, which expressed both ZO-1 and Na+-K+ adenosine triphosphatase. Early postoperative period MCEC sheets were attached to Descemet membrane, and corneal clarity was recovered. Six months after transplantation, MCEC-transplanted corneas remained clear, and closely attached hexagonal cells were observed. In 1 animal with longer observation, polygonal cells were observed by in vivo specular microscopy at a density of >2000 cells/mm2 and remained >1600 cells/mm2 for ≤2 years. Control eyes showed irreversible bullous keratopathy throughout the observation period. Conclusions: Cultivated MCECs become attached to the transplanted eye and maintain a clear cornea ≤2 years postoperatively, suggesting that corneal endothelial cells of primates might have proliferative ability in vivo once they have been cultured and proliferated in vitro. Our monkey model constitutes an important step forward for regenerative medicine with possible future application in patients with corneal endothelial dysfunction.

The Role of Interleukin-33 in Chronic Allergic Conjunctivitis

PURPOSE The authors discovered a genetic association between the ST2L gene and atopy. The ST2L gene encodes a membrane-bound functional marker for Th2 cells. Recently, a novel Th2 cytokine, interleukin-33 (IL-33), was discovered to be a specific ligand for ST2L. The authors investigated the role of IL-33 in chronic allergic conjunctivitis. METHODS Immunohistochemical analysis was carried out using giant papillae samples obtained from patients with atopic keratoconjunctivitis. The authors used proinflammatory stimuli to clarify IL-33 mRNA/protein-inducing signals with cultured human conjunctival epithelial cells, fibroblasts, human umbilical vascular endothelial cells, and mast cells. These cells were also used to examine the expression of ST2L (IL-33R). Finally, cultured mast cells were stimulated with recombinant IL-33 (rIL-33) to examine the downstream signals. RESULTS The authors found IL-33 protein expression in human vascular endothelial cells in the giant papillae and in the control conjunctivae. IL-33 expression was also observed in conjunctival epithelium of the giant papillae but not in the control conjunctivae. IL-1 beta stimulation upregulated IL-33 mRNA expression in conjunctival fibroblasts. The authors also confirmed mature IL-33 protein expression in ocular resident cells by Western blot analysis. Preferential ST2L expression was observed in human mast cells, and phosphorylation of p38 MAPK and IL-13 mRNA induction was observed in human cultured mast cells after rIL-33 stimulation. Phosphorylation of p38 MAPK was inhibited by soluble ST2 protein. CONCLUSIONS The IL-33-ST2 signaling cascade plays some roles in the pathophysiology of chronic allergic conjunctivitis through the activation of mast cells.

Cultivated human conjunctival epithelial transplantation for total limbal stem cell deficiency.

PURPOSE To determine the feasibility of cultivated conjunctiva as a viable epithelial sheet for transplantation and corneal resurfacing in eyes with limbal stem cell deficiency (LSCD). METHODS Human corneal epithelial (HCE) and human conjunctival epithelial (HCjE) cells were cultivated on human amniotic membrane (AM) to confluence and then air lifted to allow further stratification and differentiation. Denuded AM and cultivated HCE and cultivated HCjE cells were then transplanted into 18 eyes of rabbits with induced LSCD. The cultivated and engrafted epithelia were examined by transmission electron microscopy (TEM) and immunohistochemistry. Two weeks after transplantation, the eyes were examined by slit lamp biomicroscopy and scored on epithelial integrity, corneal haze, and corneal neovascularization. RESULTS Both cultivated and engrafted HCjE sheets demonstrated confluent epithelial sheets with five to six layers of well-stratified epithelium. TEM examination of engrafted HCjE revealed numerous microvilli, desmosomes, and hemidesmosomes, identical with in vivo corneal epithelium. Immunohistochemical analysis of both HCjE and HCE cells showed the presence of CK3, CK4, and CK12, with absence of Muc5AC. Clinical outcomes for eyes receiving HCjE transplants and HCE transplants were comparable, with most having transparent, smooth corneas, free of epithelial defects. CONCLUSIONS The study showed that microscopically, HCjE cells have features similar to HCE cells, with clinically equivalent outcomes. The ex vivo cultivation of conjunctiva to form transplantable epithelial sheets for corneal replacement is a promising new treatment modality in patients with LSCD.

Tumor-Associated Calcium Signal Transducer 2 Is Required for the Proper Subcellular Localization of Claudin 1 and 7 : Implications in the Pathogenesis of Gelatinous Drop-Like Corneal Dystrophy

Gelatinous drop-like dystrophy (GDLD) is a rare autosomal recessive form of corneal dystrophy characterized by subepithelial amyloid depositions on the cornea. Previous clinical and laboratory observations have strongly suggested that epithelial barrier function is significantly decreased in GDLD. Despite the decade-old identification of the tumor-associated calcium signal transducer 2 (TACSTD2) gene as a causative gene for GDLD, the mechanism by which the loss of function of this causative gene leads to the pathological consequence of this disease remains unknown. In this study, we investigated the functional relationship between the TACSTD2 gene and epithelial barrier function. Through the use of immunoprecipitation and a proximity ligation assay, we obtained evidence that the TACSTD2 protein directly binds to claudin 1 and 7 proteins. In addition, the loss of function of the TACSTD2 gene leads to decreased expression and change in the subcellular localization of tight junction-related proteins, including claudin 1, 4, 7, and ZO1 and occludin, both in diseased cornea and cultured corneal epithelial cells. These results indicate that loss of function of the TACSTD2 gene impairs epithelial barrier function through decreased expression and altered subcellular localization of tight junction-related proteins in GDLD corneas.

Functional Role of Thymic Stromal Lymphopoietin in Chronic Allergic Keratoconjunctivitis

PURPOSE Previous reports have shown that thymic stromal lymphopoietin (TSLP) plays a role in atopic diseases. This study was undertaken to investigate the expression of TSLP in the giant papillae obtained from patients with vernal keratoconjunctivitis (VKC) or atopic keratoconjunctivitis (AKC), and its functional roles were analyzed. METHODS TSLP mRNA expression was examined in resected conjunctival samples obtained from four patients with VKC/AKC and three control subjects by reverse transcription-polymerase chain reaction. Anti-TSLP, anti-dendritic cell-limbic system-associated membrane protein (anti-DC-LAMP), and anti-tryptase immunohistochemical staining was performed with 10 resected giant papillae. Human conjunctival epithelial (HCJE) cells were stimulated with poly I:C, with and without endosomal inhibitor, to examine TSLP mRNA expression. Cultured human mast cells were stimulated with recombinant (r)TSLP to analyze the downstream effect of TSLP. RESULTS All four VKC/AKC samples showed TSLP mRNA expression; however, no TSLP mRNA expression was found in the control conjunctivae. Anti-TSLP immunohistochemical staining showed preferential expression in the epithelial cells and some infiltrated cells of the giant papillae, but not in the control conjunctivae. Double immunohistochemical staining with TSLP and DC-LAMP or tryptase showed the existence of activated dendritic cells and mast cells near TSLP-positive cells in the giant papillae. Real-time PCR analysis showed that poly I:C induced TSLP mRNA expression in HCJEs in an endosomal-function-dependent manner and that rTSLP could induce IL-13 mRNA expression in the mast cells synergistically with IL-33. CONCLUSIONS The TSLP protein produced in conjunctival epithelial cells plays a role in severe ocular allergy through the activation of dendritic cells and mast cells in synergy with other cytokines.

Genomic Aberrations and Cellular Heterogeneity in SV40-Immortalized Human Corneal Epithelial Cells

PURPOSE Simian virus (SV)40-immortalized human corneal epithelial (HCE-T) cells have been widely used as an in vitro model of human corneal epithelial cells. The nature of this cell line was assessed for genomic aberrations and cellular heterogeneity. METHODS For the quantitative measurement of genomic aberrations, array-based comparative genomic hybridization (CGH) analysis was performed. For identification of cellular heterogeneity, cell morphology, growth kinetics, transepithelial electrical resistance, and transfection/transcriptional efficiency were analyzed. Real-time PCR and chromosomal fluorescent in situ hybridization (cFISH) against some gained or lost loci were performed, to assess genomic heterogeneity. Expressed sequence tags (ESTs) for this cell line were collected to assess differences in the gene expression profiles between HCE-T cells and normal corneal epithelial cells. Southern blot analysis and inverse PCR analyses were used to determine the genomic integration site of the SV40 large T antigen gene (LTAG). RESULTS Array CGH analysis demonstrated that the genomic content of HCE-T cells is different from the normal healthy genome. The results from cellular functional assays, real-time PCR, and cFISH strongly indicated that HCE-T cells consist of a significant number of heterogeneous cell populations. The genomic integration site of the SV40 large T antigen was at p22.1 of chromosome 9. CONCLUSIONS The results indicate that HCE-T cells have an altered genomic content and that they are composed of heterogeneous cell populations. This should be considered when conducting experiments or interpreting the results of studies that use this cell line.

Assessment of epithelial integrity and cell viability in epithelial flaps prepared with the epi-LASIK procedure.

PURPOSE: To assess the histological integrity and cell viability in epithelial flaps prepared with epikeratomes. SETTING: Department of Ophthalmology, Kyoto Prefectural University of Medicine and The Baptist Eye Clinic, Kyoto, Japan. METHODS: Epithelial flaps were prepared by epi‐LASIK surgery. After immediate fixation, they were examined by light and electron microscopy. To assess cell viability in fresh epithelial flaps, biostaining experiments were performed using propidium iodide (PI), calcein‐AM, and Hoechst 33342 dye. In addition, some epithelial flaps were organ‐cultured for 24 hours. RESULTS: Light and electron microscopy showed that most of the inspected areas showed nuclei and cytoplasm at significantly reduced density and discontinuity of the basement membrane. Biostaining experiments showed that approximately 90% of the basal cells in epithelial flaps were PI‐positive dead cells; organ cultures showed detachment of basal cells from the epithelial flap after 24 hours of incubation. CONCLUSION: Most basal cells in epithelial flaps prepared with different epikeratome devices were dead.

Investigation of the Corneal Filament in Filamentary Keratitis

PURPOSE To date, no studies have elucidated the composition of the corneal filament in detail. In this study, an immunohistochemical technique was used to clarify the exact composition of the corneal filament in filamentary keratitis. In addition, the mechanisms responsible for filament formation were identified. METHODS Filaments were obtained from 13 patients with filamentary keratitis with a background of penetrating keratoplasty, aqueous tear deficiency, and severe ocular surface disorders, who were receiving treatment at an outpatient facility. From those tissues, transverse and longitudinal frozen sections were prepared and subjected to an indirect fluorescent immunohistochemical analysis with primary antibodies, including cytokeratins (CK1, -4, -6, -10, -12, and -13), mucins (MUC1, -4, -5AC, and -16), keratinization-related proteins (transglutaminase [TGase]-1 and filaggrin), cell proliferation marker Ki67, and markers of infiltration cells (HLA-DR and neutrophil-elastase). TUNEL staining was used for the detection of apoptosis. Fluorescent images of the sections were inspected with a fluorescence microscope. RESULTS The filaments were composed of CK12-positive cells and had a roll-formed central core. They were covered with MUC5AC- and -16-positive mucins including CK4- and -13-positive cells and neutrophil-elastase-positive cells. The filaments also included broken cells and DNA fiber-form postlesional nuclei that were positive for TUNEL staining. However, those areas stained weakly for CK6 and HLA-DR; faintly for CK1, CK10, MUC1, and MUC4; and not at all for Ki67, TGase-1, and filaggrin. CONCLUSIONS The results of this research have the potential to open new pathways toward understanding the mechanism that generates the filament in filamentary keratitis, as well as new treatments in the future.

Small-angle fibre diffraction studies of corneal matrix structure: a depth-profiled investigation of the human eye-bank cornea

In the cornea of the eye light transmission is facilitated by the regular arrangement and uniform diameter of collagen fibrils that constitute the bulk of the extracellular corneal matrix. Matrix architecture, in turn, is believed to be governed by interactions between collagen fibrils and proteoglycan molecules modified with sulfated glycosaminoglycan side chains. Here, we outline the contribution made by small-angle X-ray scattering studies of the cornea in understanding the role of sulfated glycosaminoglycans in the control of collagen architecture in cornea, and present new depth-profiled microbeam data from swollen human eye-bank corneas that indicate no significant change in collagen fibril diameter throughout the tissue, but a lower collagen interfibrillar spacing in the anterior-most stromal regions compared with the ultrastructure of the deeper cornea.