Kenta Yamasaki

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.

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.

Gene expression and immunolocalisation of a calcium-activated chloride channel during the stratification of cultivated and developing corneal epithelium

The spatial and temporal localisation of a calcium-activated chloride channel (CLCA) and its mRNA was investigated, during the in vivo and in vitro development of stratified epithelia, by fluorescence immunohistochemistry and quantitative polymerase chain reaction in embryonic chicken corneas and the expansion of excised human corneal stem cells on amniotic membrane. Single-layered human epithelial cultures on amniotic membrane and early day embryonic chicken corneas expressed relatively little human CLCA2 or its chicken homologue. However, as the epithelium in both models matured and the number of cell-layers increased, the gene expression level and protein staining intensity increased, primarily within the basal cells of both the cultured and embryonic tissues. These results demonstrate that human CLCA2 protein and mRNA expression are elevated during epithelial stratification, suggesting that this protein plays a role in the growth of multi-layered corneal epithelia during both natural development and tissue cultivation.

Calcium-activated Chloride Channel-2 in Human Epithelia

Calcium-activated chloride channels (CLCAs) are a family of multifunctional proteins that are widely distributed in tissues. To investigate the distribution of human CLCA-2 (hCLCA2) in human epithelia at the light and electron microscopic levels, we raised a primary antibody against a synthetic polypeptide sequence from natural hCLCA2. Corneal, skin, vaginal, esophageal, and laryngeal epithelia were immunopositive for hCLCA2 at the cytosolic aspect of the basal cells adjacent to the basement membrane. Epithelia of stomach and small intestine showed no hCLCA2 immunoreactivity. This study reports the cellular distribution of hCLCA2 in human epithelia and suggests its possible involvement in epithelial stratification and cell-substrate adhesion.

Clinical Outcomes of Phototherapeutic Keratectomy in Eyes With Thiel-Behnke Corneal Dystrophy

PURPOSE To investigate the functional and morphologic midterm outcome of phototherapeutic keratectomy (PTK) for Thiel-Behnke corneal dystrophy diagnosed by gene-mutation analysis. DESIGN Retrospective, single-center clinical study. METHODS Between July 2001 and May 2010, 10 consecutive PTKs were performed in 10 eyes of 5 patients (2 male, 3 female; mean age: 55 ± 13 years) with superficially accentuated opacities caused by Thiel-Behnke corneal dystrophy and were followed up for at least 12 months (range: 12-108 months). Main outcome measures included (1) best-corrected visual acuity (BCVA), (2) uncorrected visual acuity (UCVA), (3) spherical equivalent, and (4) recurrence rate. The probability of recurrence of Thiel-Behnke corneal dystrophy after PTK was calculated using the Kaplan-Meier method for survival analysis. RESULTS The p.Arg555Gln mutation was found within the TGFBI gene in all 5 patients. Average logarithm of minimal angle of resolution (logMAR) BCVA change was -0.55 ± 0.26. Average logarithm UCVA change was -0.54 ± 0.31. In 5 of the 10 eyes, recurrence of central superficial opacification was clinically identified during the follow-up periods, and in 4 of those 5 eyes, the level of the recurrence was so significant that the visual acuity was reduced more than 2 lines. The maximum follow-up period of the 1 eye without significant post-PTK recurrence was 108 months. CONCLUSIONS PTK is a successful therapy for Thiel-Behnke corneal dystrophy, and results in midterm stable visual acuity and corneal transparency. Unlike in Reis-Bücklers corneal dystrophy cases, PTK delays the need for more invasive surgical intervention in Thiel-Behnke corneal dystrophy.

Lattice Corneal Dystrophy Type IV (p.Leu527Arg) Is Caused by a Founder Mutation of the TGFBI Gene in a Single Japanese Ancestor

PURPOSE Lattice corneal dystrophy (LCD) type IV (LCD4) is a late-onset corneal dystrophy with amyloid deposition at the deep stromal layer of cornea. As with other corneal dystrophies, this LCD subtype is also caused by a mutation (p. Leu527Arg) of the transforming growth factor, beta-induced (TGFBI) gene. Although LCD type I has been reported worldwide, LCD4 has been reported only in the Japanese population. In the present study, a haplotype analysis was performed to investigate whether this LCD subtype is caused by a founder mutation. METHODS Genomic DNA samples were extracted from 13 unrelated patients with LCD4. As a control, genomic DNA samples from 96 normal volunteers were also analyzed. For the haplotype analysis, the samples were amplified by polymerase chain reaction (PCR), TA-cloned, isothermally amplified, and subjected to a 1-base primer extension assay against a mutation site (c.1580T>G) and six known single-nucleotide polymorphisms (SNPs; rs4669, rs2072239, rs7727725, rs17689879, rs6871571, and rs3792900), which are located adjacent to the mutation site. RESULTS The haplotype analysis revealed that all the disease-carrying alleles from the 13 LCD4 patients shared an identical haplotype, whereas non-disease-carrying alleles from the normal volunteers and the LCD4 patients exhibited four haplotypes. There was a statistically significant difference in the haplotype distribution between the disease-carrying and the non-disease-carrying alleles. CONCLUSIONS The findings of this study strongly indicate that LCD4 was caused by a founder mutation of the TGFBI gene that occurred in a single Japanese ancestor.

Establishment of a Human Corneal Epithelial Cell Line Lacking the Functional TACSTD2 Gene as an In Vitro Model for Gelatinous Drop-Like Dystrophy

PURPOSE Gelatinous drop-like corneal dystrophy (GDLD) is characterized by subepithelial amyloid deposition that engenders severe vision loss. The exact mechanism of this disease has yet to be elucidated. No fundamental treatment exists. This study was conducted to establish an immortalized corneal epithelial cell line to be used as a GDLD disease model. METHODS A corneal tissue specimen was obtained from a GDLD patient during surgery. Corneal epithelial cells were enzymatically separated from the cornea and were dissociated further into single cells. The epithelial cells were immortalized by the lentiviral transduction of the simian virus 40 (SV40) large T antigen and human telomerase reverse transcriptase (hTERT) genes. For the immortalized cells, proliferative kinetics, gene expressions, and functional analyses were performed. RESULTS The immortalized corneal epithelial cells continued to proliferate despite cumulative population doubling that exceeded 100. The cells showed almost no sign of senescence and displayed strong colony-forming activity. The cells exhibited a low epithelial barrier function as well as decreased expression of tight-junction-related proteins claudin 1 and 7. Using the immortalized corneal epithelial cells derived from a GDLD patient, we tested the possibility of gene therapy. CONCLUSIONS We established an immortalized corneal epithelial cell line from a GDLD patient. The immortalized cells exhibited cellular phenotypes similar to those of in vivo GDLD. The immortalized cells are thought to be useful for the development of new therapies for treating GDLD corneas and for elucidation of the pathophysiology of GDLD.

A novel mutation of the TGFBI gene causing a lattice corneal dystrophy with deep stromal involvement

Lattice corneal dystrophy (LCD) type I is one of the five dominant TGFBI (transformimg growth factor β induced; formerly designated as bigh3 or keratoepithelin)-related corneal dystrophies with characteristic lattice-like refractile lines in the corneal stroma.1 Other than this common-type LCD, there have also been reported several minor-type LCDs caused by different mutations of the TGFBI gene.2 An 85-year-old man presented with complaints of bilateral blurred vision. His best-corrected visual acuity was 0.1 in OD and HM/30 cm in OS. He had bilateral corneal haze and cataract. The corneal haze contained many isolated or fused refractile opacities, most of them being dot-like, and some being lattice-like (figure 1A). The opacities were found at all depths of the corneal stroma, but mainly involved the deep stromal layer. The degree of corneal haze was severe in his left eye, but relatively mild in his right eye. His wife and two sons did not show any corneal opacity in their eyes. Cataract surgery was performed on his left eye, but his best-corrected visual acuity in that eye was improved …

Cytopathological Features of a Severe Type of Corneal Intraepithelial Neoplasia

Purpose: To report the cytopathological features of corneal intraepithelial neoplasia (CIN) through the investigation of cytokeratin expression pattern, keratinization, cell proliferation, apoptosis, and epithelial mesenchymal transition. Patient and Methods: Corneal tissue excised from a CIN patient was examined in this study. Cryosections of the excised CIN epithelial tissue were examined by immunostaining analysis using antibodies against cytokeratins, keratinization-related proteins, Ki-67, human telomerase reverse transcriptase (hTERT), and epithelial mesenchymal transition (EMT)-related proteins. Subcellular localization of F-actin was also analyzed using phalloidin. For the detection of apoptotic cells, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed. Real-time polymerase chain reaction was performed to quantify the expression level of hTERT in the CIN epithelium. Results: The CIN epithelium exhibited a significantly altered cytokeratin expression pattern compared to normal corneas with an upregulated expression of keratinization-related proteins. The CIN epithelium also demonstrated an increased number of Ki-67-positive cells with an upregulated expression of hTERT, while exhibiting an increased number of apoptotic cells. EMT did not occur in the CIN epithelium. Conclusion: CIN epithelium seems to be slightly dedifferentiated from the corneal epithelial lineage. The status of cell proliferation and apoptosis in the CIN epithelium was significantly altered from that of normal corneal epithelium, but its malignancy level does not appear to be as high as that of metastasis-competent malignant cancers.