Clinical Trial of Autologous Cultivated Limbal Epithelial Cell Sheet Transplantation for Patients with Limbal Stem Cell Deficiency.

PURPOSE: To confirm the efficacy and safety of Good Manufacturing Practice (GMP)-compliant autologous cultivated limbal epithelial cell sheets in government-controlled clinical trials that adhered to Good Clinical Practice stipulations for patients with unilateral limbal stem cell deficiency (LSCD). DESIGN: A prospective, multicenter, open-label, uncontrolled, single-arm clinical trial. PARTICIPANTS: Ten consecutive eyes of 10 patients with unilateral LSCD were followed for 2 years after surgery. Preoperative LSCD stage was IIB in 4 eyes and III in 6 eyes. METHODS: A limbal tissue biopsy was obtained from the healthy eye, after which limbal stem cells were dissociated and cultivated on temperature-responsive culture surfaces. All cell sheets were fabricated in a GMP-grade facility under established standard operating procedures. Cell sheets were evaluated using defined shipment criteria before transplantation, and only those that met the criteria were used. The cell sheet was transplanted onto each of the patients' diseased eye after removing the conjunctival scar tissue that covered the corneal surface. The severity of LSCD was determined according to a staging method agreed on by global consensus, with eyes evaluated as being in stages IA-C representing successful corneal epithelial reconstruction. Diagnosis and staging of LSCD were determined by the trial's Eligibility Judgment Committee and Effect Assessment Committee using slit-lamp photographs including fluorescein staining. Both committees comprised 2 or 3 third-party cornea specialists, who were provided with information anonymously and randomly. MAIN OUTCOME MEASURE: Corneal epithelial reconstruction rate was the primary end point. RESULTS: Corneal epithelial reconstruction was successful in 6 of 10 eyes (60%) 1 year postoperatively and was significantly higher than the 15% clinically significant efficacy rate achieved by allogeneic limbal transplantation. The reconstruction rate was 70% of eyes 2 years postoperatively. Additionally, improvements in visual acuity were noted in 50% and 60% of eyes at 1 and 2 years, respectively. No clinically significant transplantation-related adverse events were observed. CONCLUSIONS: The efficacy and safety of cultivated limbal epithelial cell sheet transplantation were thus confirmed, and the cell sheet, named "Nepic," is now approved as a cellular and tissue-based product in Japan. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Chondroitin Sulphate/Dermatan Sulphate Proteoglycans: Potential Regulators of Corneal Stem/Progenitor Cell Phenotype In Vitro.

Chondroitin sulphate (CS) proteoglycans with variable sulphation-motifs along their glycosaminoglycan (GAG) chains are closely associated with the stem cell niche of articular cartilage, where they are believed to influence the characteristics of the resident stem cells. Here, we investigated the immunohistochemical distribution of hybrid CS/dermatan sulphate (DS) GAGs in the periphery of the adult chicken cornea, which is the location of the cornea's stem cell niche in a number of species, using a monoclonal antibody, 6C3, that recognises a sulphation motif-specific CS/DS GAG epitope. This revealed positive labelling that was restricted to the subepithelial corneal stroma, as well as nearby bony structures within the sclera, called ossicles. When cultivated on cell culture dishes coated with 6C3-rich CS/DS, corneal stromal cells (keratocytes) that had been isolated from embryonic chicken corneas formed circular colonies, which took several days to reach confluency. A flow cytometric analysis of these keratocytes revealed changes in their expression levels of the indicative stem cell markers, Connexin 43 (Cx43), Paired Box 6 (PAX6), B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1), and C-X-C Chemokine Receptor 4 (CXCR4) suggestive of a less-differentiated phenotype compared with expression levels in cells not exposed to CS/DS. These findings support the view that CS/DS promotes the retention of a stem cell phenotype in corneal cells, much as it has been proposed to do in other connective tissues.

Barium-induced toxic anterior segment syndrome.

Toxic anterior segment syndrome (TASS) is a rapid-onset inflammation of the eye following uneventful ocular surgery. We report a case of TASS following Baerveldt glaucoma implant (BGI) surgery. Inductively coupled plasma-mass spectrometry (ICP-MS) identified barium in the eye and in the eluate from the bleb of the BGI. We attribute TASS in our patient to the dissolution of barium from the BGI and its entry into the eye, where it causes severe inflammation.

Tissue engineered corneal epithelium derived from clinical-grade human embryonic stem cells.

PURPOSE: To construct tissue engineered corneal epithelium from a clinical-grade human embryonic stem cells (hESCs) and investigate the dynamic gene profile and phenotypic transition in the process of differentiation. METHODS: A stepwise protocol was applied to induce differentiation of clinical-grade hESCs Q-CTS-hESC-1 and construct tissue engineered corneal epithelium. Single cell RNA sequencing (scRNA-seq) analysis was performed to monitor gene expression and phenotypic changes at different differentiation stages. Immunostaining, real-time quantitative PCR and Western blot analysis were conducted to detect gene and protein expressions. After subcutaneous transplantation into nude mice to test the biosafety, the epithelial construct was transplanted in a rabbit corneal limbal stem cell deficiency (LSCD) model and followed up for eight weeks. RESULTS: The hESCs were successfully induced into epithelial cells. scRNA-seq analysis revealed upregulation of ocular surface epithelial cell lineage related genes such as TP63, Pax6, KRT14, and activation of Wnt, Notch, Hippo, and Hedgehog signaling pathways during the differentiation process. Tissue engineered epithelial cell sheet derived from hESCs showed stratified structure and normal corneal epithelial phenotype with presence of clonogenic progenitor cells. Eight weeks after grafting the cell sheet onto the ocular surface of LSCD rabbit model, a full-thickness continuous corneal epithelium developed to fully cover the damaged areas with normal limbal and corneal epithelial phenotype. CONCLUSION: The tissue engineered corneal epithelium generated from a clinical-grade hESCs may be feasible in the treatment of limbal stem cell deficiency.

Assessment of a self-assembling peptide gel, SPG-178, in providing a clear operative field for trabeculectomy surgery for glaucoma in an animal model.

The presence of blood during ophthalmic surgery is problematic, as it can obstruct a surgeon's view of the operative field. This is particularly true when performing trabeculectomy surgery to enhance ocular fluid outflow and reduce intraocular pressure as a treatment for glaucoma, one of the most common vision loss conditions worldwide. In this study, we investigated the performance of a transparent, self-assembling peptide gel (SPG-178) and its ability to maintain visibility during trabeculectomy surgery. Unlike the hyaluronic acid gel commonly used in ophthalmic surgery, SPG-178 did not permit the ingress of blood into the gel itself. Rather, it forced blood to flow peripherally to the gel. Moreover, if bleeding occurred under the SPG-178 gel, perfusion with saline was able to effectively flush the blood away along the interface between the SPG-178 and the ocular tissue (in this case scleral) to clear the surgical field of view. In experimental trabeculectomy surgeries with mitomycin C used as an adjuvant, there were no differences in the postoperative recovery of intraocular pressure or bleb morphology with or without the use of SPG-178. SPG-178, therefore, when used in a gel formulation, represents a new material for use in intraocular surgery to ensure a clear operative field with likely beneficial treatment outcomes.

Cell jamming, stratification and p63 expression in cultivated human corneal epithelial cell sheets.

Corneal limbal epithelial stem cell transplantation using cultivated human corneal epithelial cell sheets has been used successfully to treat limbal stem cell deficiencies. Here we report an investigation into the quality of cultivated human corneal epithelial cell sheets using time-lapse imaging of the cell culture process every 20 minutes over 14 days to ascertain the level of cell jamming, a phenomenon in which cells become smaller, more rounded and less actively expansive. In parallel, we also assessed the expression of p63, an important corneal epithelial stem cell marker. The occurrence of cell jamming was variable and transient, but was invariably associated with a thickening and stratification of the cell sheet. p63 was present in all expanding cell sheets in the first 9 days of culture, but it's presence did not always correlate with stratification of the cell sheet. Nor did p63 expression necessarily persist in stratified cell sheets. An assessment of cell jamming, therefore, can shed significant light on the quality and regenerative potential of cultivated human corneal epithelial cell sheets.

High-fat diet induces dry eye-like ocular surface damages in murine.

PURPOSE: A high-fat diet leads to dysfunction in multiple systems of the body. Herein we investigate the effects of a high-fat diet on the ocular surface using a murine model. METHODS: Four-week-old male C57BL/6 mice were fed with a standard-fat diet (10 kcal% fat, SFD) or a high-fat diet (60 kcal% fat, HFD) for 1 or 3 months. Phenol red thread test was used to detect tear production, oregon green dextran (OGD) staining was performed to assess corneal epithelial permeability, and PAS staining was conducted to ascertain the presence of conjunctival goblet cells. Squamous metaplasia in the ocular surface and corneal epithelial barrier function were detected by immunofluorescent staining, zymography and Western blot analysis. Oxidative stress related protein expression was evaluated by immunostaining and Western blot analysis. Corneal and conjunctival cell apoptosis was determined by TUNEL assay and caspase-3 expression. RESULTS: A HFD induced obvious ocular surface damages, including decreased tear production, notable OGD staining and distinct goblet cell loss. It also resulted in corneal epithelial barrier dysfunction and significant squamous metaplasia of the corneal and conjunctival epithelia. The HFD also upregulated key factors that regulate oxidative stress in the ocular surface, and upregulated cell apoptosis in ocular surface epithelial cells. CONCLUSIONS: A HFD induces dry eye-like ocular surface damages in mice via the activation of oxidative stress and an induction of apoptosis in the cells of the ocular surface.

Hyperlipidemia Affects Tight Junctions and Pump Function in the Corneal Endothelium.

Hyperlipidemia impacts on various diseases, such as atherosclerosis, hypertension, and diabetes mellitus. However, its influence, if any, on ocular tissues is largely unknown. Herein, we developed hyperlipidemic murine models by feeding 4-week-old male wild-type mice with a high-fat diet and apolipoprotein E knockout (ApoE-/-) mice with a high-fat diet or standard diet to investigate the corneal endothelial change under hyperlipidemic conditions. Oil Red O staining showed an accumulation of lipid droplets in corneal endothelial cells (CECs) of hyperlipidemic mice. Other manifestations included a reduced cell density and distorted cell morphology, a disruption of the endothelial cell tight junctions and adhesion junctions, a reduced number of surface microvilli, down-regulation of Na+-K+-ATPase expression and function, activation of oxidative stress, changes in mitochondrial ultrastructure, and increased apoptosis. CEC recovery after injury, moreover, was diminished in hyperlipidemic mice; and high palmitate levels were found in the aqueous humor. In vitro hyperlipemia model, moreover, was found to be associated with dose-dependent CEC cytotoxicity, altered cell morphology, reduced pump function, and an induction of oxidative stress, leading to functional and pathologic changes in the corneal endothelium.

Recovering vision in corneal epithelial stem cell deficient eyes.

A healthy corneal epithelium, which is essential for proper vision and protection from external pathogens, is continuously replenished throughout life by stem cells located at the limbus. In diseased or injured eyes, however, in which stem cells are deficient, severe ocular problems manifest themselves. These are notoriously difficult to manage and as a result the last 20 or so years has seen a number of therapeutic strategies emerge that aim to recover the ocular surface and restore vision in limbal stem cell deficient eyes. The dominant concept involves the generation of laboratory cultivated epithelial cell sheets expanded from small biopsies of the epithelial limbus (for patient or donors) or another non-corneal epithelial tissue such as the oral mucosa. Typically, cells are grown on sterilised human amniotic membrane as a substrate, which then forms part of the graft, or specially formulated plastic culture dishes from which cells sheets can be released by lowering the temperature, and thus the adherence of the plastic to the cells. Overall, clinical results are promising, as is discussed, with new cultivation methodologies and different cell lineages currently being investigated to augment the treatment options for visual disturbance caused by a corneal epithelial limbal stem cell deficiency.

CD200 facilitates the isolation of corneal epithelial cells derived from human pluripotent stem cells.

The in vitro induction of corneal epithelial cells (CECs) from human induced pluripotent stem cells (iPSCs) represents a new strategy for obtaining CE stem/progenitor cells for the surgical reconstruction of a diseased or injured ocular surface. The clinical promise of this strategy is considerable, but if the approaches' potential is to be realised, robust methods for the purification of iPSC-derived CE lineage cells need to be developed to avoid contamination with other cells that may carry the risk of unwanted side effects, such as tumorigenesis. Experiments conducted here revealed that during CEC isolation, CD200-negative selection using a cell sorter considerably reduced the contamination of the cell population with various non-CECs compared with what could be achieved using TRA-1-60, a conventional negative marker for CECs. Furthermore, CD200-negative sorting did not affect the yield of CECs nor that of their stem/progenitor cells. Single-cell gene expression analysis for CEC sheets obtained using CD200-negative sorting showed that all analysed cells were CE-lineage cells, expressing PAX6, delta-N p63, and E-cadherin. Non-CECs, on the other hand, expressed non-CEC genes such as FGFR1 and RPE65. CD200, thus, represents a robust negative marker for purification of induced CE lineage cells, which is expressed by undifferentiated iPSCs and non-CECs, including iPSC-derived neural and retinal cells.

New Graft Insertion Device for Descemet Stripping Automated Endothelial Keratoplasty.

PURPOSE: Corneal endothelial dysfunction is a major indication for corneal graft surgery worldwide, and although surgical intervention through a range of posterior lamellar surgeries has proven to be hugely beneficial, challenges remain. This is especially so where the anterior chamber is relatively shallow, as is often the case in the Asian population, though not exclusively so. In this study, we introduce a new insertion device to deliver endothelial graft tissue for Descemet stripping automated endothelial keratoplasty (DSAEK). METHODS: A new surgical tool was designed and manufactured so as to enable a 1-step insertion of corneal graft tissue into the anterior chamber based on a pressure-flow concept, rather than the a pull-through one. This was tested ex vivo to assess endothelial cell damage, and then performed in 12 first-in-human surgeries. RESULTS: Precut DSAEK lenticules implanted in donor corneas ex vivo through the new technique showed that less endothelial cell damage occurs compared with a pull-through technique. Grafts were successful in all patients receiving the new surgery, with no cases of primary graft failure. CONCLUSIONS: The newly developed DSAEK inserter is a simple and useful tool for endothelial graft delivery, lessening intraoperative mechanical stress on the graft tissue.

A Surgical Cryoprobe for Targeted Transcorneal Freezing and Endothelial Cell Removal.

PURPOSE: To examine the effects of transcorneal freezing using a new cryoprobe designed for corneal endothelial surgery. METHODS: A freezing console employing nitrous oxide as a cryogen was used to cool a series of different cryoprobe tip designs made of silver for high thermal conductivity. In vitro studies were conducted on 426 porcine corneas, followed by preliminary in vivo investigations on three rabbit corneas. RESULTS: The corneal epithelium was destroyed by transcorneal freezing, as expected; however, the epithelial basement membrane remained intact. Reproducible endothelial damage was optimally achieved using a 3.4 mm diameter cryoprobe with a concave tip profile. Stromal edema was seen in the pre-Descemet's area 24 hrs postfreeze injury, but this had been resolved by 10 days postfreeze. A normal collagen fibril structure was seen 1 month postfreeze, concurrent with endothelial cell repopulation. CONCLUSIONS: Transcorneal freezing induces transient posterior stromal edema and some residual deep stromal haze but leaves the epithelial basement membrane intact, which is likely to be important for corneal re-epithelialization. Localized destruction of the endothelial monolayer was achieved in a consistent manner with a 3.4 mm diameter/concave profile cryoprobe and represents a potentially useful approach to remove dysfunctional corneal endothelial cells from corneas with endothelial dysfunction.

Coordinated generation of multiple ocular-like cell lineages and fabrication of functional corneal epithelial cell sheets from human iPS cells.

We describe a protocol for the generation of a functional and transplantable corneal epithelium derived from human induced pluripotent stem (iPS) cells. When this protocol is followed, a proportion of iPS cells spontaneously form circular colonies, each of which is composed of four concentric zones. Cells in these zones have different morphologies and immunostaining characteristics, resembling neuroectoderm, neural crest, ocular-surface ectoderm, or surface ectoderm. We have named this 2D colony a 'SEAM' (self-formed ectodermal autonomous multizone), and previously demonstrated that cells within the SEAM have the potential to give rise to anlages of different ocular lineages, including retinal cells, lens cells, and ocular-surface ectoderm. To investigate the translational potential of the SEAM, cells within it that resemble ocular-surface epithelia can be isolated by pipetting and FACS sorting into a population of corneal epithelial-like progenitor cells. These can be expanded and differentiated to form an epithelial layer expressing K12 and PAX6, and able to recover function in an animal model of corneal epithelial dysfunction after surgical transplantation. The whole protocol, encompassing human iPS cell preparation, autonomous differentiation, purification, and subsequent differentiation, takes between 100 and 120 d, and is of potential use to researchers with an interest in eye development and/or ocular-surface regeneration. Experience with human iPS cell culture and sorting via FACS will be of benefit for researchers performing this protocol.

Co-ordinated ocular development from human iPS cells and recovery of corneal function.

The eye is a complex organ with highly specialized constituent tissues derived from different primordial cell lineages. The retina, for example, develops from neuroectoderm via the optic vesicle, the corneal epithelium is descended from surface ectoderm, while the iris and collagen-rich stroma of the cornea have a neural crest origin. Recent work with pluripotent stem cells in culture has revealed a previously under-appreciated level of intrinsic cellular self-organization, with a focus on the retina and retinal cells. Moreover, we and others have demonstrated the in vitro induction of a corneal epithelial cell phenotype from pluripotent stem cells. These studies, however, have a single, tissue-specific focus and fail to reflect the complexity of whole eye development. Here we demonstrate the generation from human induced pluripotent stem cells of a self-formed ectodermal autonomous multi-zone (SEAM) of ocular cells. In some respects the concentric SEAM mimics whole-eye development because cell location within different zones is indicative of lineage, spanning the ocular surface ectoderm, lens, neuro-retina, and retinal pigment epithelium. It thus represents a promising resource for new and ongoing studies of ocular morphogenesis. The approach also has translational potential and to illustrate this we show that cells isolated from the ocular surface ectodermal zone of the SEAM can be sorted and expanded ex vivo to form a corneal epithelium that recovers function in an experimentally induced animal model of corneal blindness.

PAX6 Isoforms, along with Reprogramming Factors, Differentially Regulate the Induction of Cornea-specific Genes.

PAX6 is the key transcription factor involved in eye development in humans, but the differential functions of the two PAX6 isoforms, isoform-a and isoform-b, are largely unknown. To reveal their function in the corneal epithelium, PAX6 isoforms, along with reprogramming factors, were transduced into human non-ocular epithelial cells. Herein, we show that the two PAX6 isoforms differentially and cooperatively regulate the expression of genes specific to the structure and functions of the corneal epithelium, particularly keratin 3 (KRT3) and keratin 12 (KRT12). PAX6 isoform-a induced KRT3 expression by targeting its upstream region. KLF4 enhanced this induction. A combination of PAX6 isoform-b, KLF4, and OCT4 induced KRT12 expression. These new findings will contribute to furthering the understanding of the molecular basis of the corneal epithelium specific phenotype.

Mesenchymal-epithelial cell interactions and proteoglycan matrix composition in the presumptive stem cell niche of the rabbit corneal limbus.

PURPOSE: To investigate whether mesenchymal-epithelial cell interactions, similar to those described in the limbal stem cell niche in transplant-expired human eye bank corneas, exist in freshly enucleated rabbit eyes and to identify matrix molecules in the anterior limbal stroma that might have the potential to help maintain the stem cell niche. METHODS: Fresh limbal corneal tissue from adult Japanese white rabbits was obtained and examined in semithin resin sections with light microscopy, in ultrathin sections with transmission electron microscopy, and in three-dimensional (3D) reconstructions from data sets of up to 1,000 serial images from serial block face scanning electron microscopy. Immunofluorescence microscopy with five monoclonal antibodies was used to detect specific sulfation motifs on chondroitin sulfate glycosaminoglycans, previously identified in association with progenitor cells and their matrix in cartilage tissue. RESULTS: In the rabbit limbal cornea, while no palisades of Vogt were present, the basal epithelial cells stained differentially with Toluidine blue, and extended lobed protrusions proximally into the stoma, which were associated with interruptions of the basal lamina. Elongate processes of the mesenchymal cells in the superficial vascularized stroma formed direct contact with the basal lamina and basal epithelial cells. From a panel of antibodies that recognize native, sulfated chondroitin sulfate structures, one (6-C-3) gave a positive signal restricted to the region of the mesenchymal-epithelial cell associations. CONCLUSIONS: This study showed interactions between basal epithelial cells and subjacent mesenchymal cells in the rabbit corneal limbus, similar to those that have been observed in the human stem cell niche. A native sulfation epitope in chondroitin sulfate glycosaminoglycans exhibits a distribution specific to the connective tissue matrix of this putative stem/progenitor cell niche.

The effect of riboflavin/UVA collagen cross-linking therapy on the structure and hydrodynamic behaviour of the ungulate and rabbit corneal stroma.

PURPOSE: To examine the effect of riboflavin/UVA corneal crosslinking on stromal ultrastructure and hydrodynamic behaviour. METHODS: One hundred and seventeen enucleated ungulate eyes (112 pig and 5 sheep) and 3 pairs of rabbit eyes, with corneal epithelium removed, were divided into four treatment groups: Group 1 (28 pig, 2 sheep and 3 rabbits) were untreated; Group 2 (24 pig) were exposed to UVA light (3.04 mW/cm(2)) for 30 minutes and Group 3 (29 pig) and Group 4 (31 pig, 3 sheep and 3 rabbits) had riboflavin eye drops applied to the corneal surface every 5 minutes for 35 minutes. Five minutes after the initial riboflavin instillation, the corneas in Group 4 experienced a 30 minute exposure to UVA light (3.04 mW/cm(2)). X-ray scattering was used to obtain measurements of collagen interfibrillar spacing, spatial order, fibril diameter, D-periodicity and intermolecular spacing throughout the whole tissue thickness and as a function of tissue depth in the treated and untreated corneas. The effect of each treatment on the hydrodynamic behaviour of the cornea (its ability to swell in saline solution) and its resistance to enzymatic digestion were assessed using in vitro laboratory techniques. RESULTS: Corneal thickness decreased significantly following riboflavin application (p<0.01) and also to a lesser extent after UVA exposure (p<0.05). With the exception of the spatial order factor, which was higher in Group 4 than Group 1 (p<0.01), all other measured collagen parameters were unaltered by cross-linking, even within the most anterior 300 microns of the cornea. The cross-linking treatment had no effect on the hydrodynamic behaviour of the cornea but did cause a significant increase in its resistance to enzymatic digestion. CONCLUSIONS: It seems likely that cross-links formed during riboflavin/UVA therapy occur predominantly at the collagen fibril surface and in the protein network surrounding the collagen.

A study of host corneal endothelial cells after non-Descemet stripping automated endothelial keratoplasty.

PURPOSE: To determine the short-term fate of the host endothelium and Descemet membrane after non-Descemet stripping automated endothelial keratoplasty (nDSAEK). METHODS: Eight unilateral DSAEK (n = 4) or nDSAEK (n = 4) surgeries were performed in the right eyes of 8 rabbits. Corneal transparency and thickness were followed-up by slit-lamp microscopy, and 2 weeks postoperatively, corneas were evaluated by immunohistochemistry and transmission electron microscopy. RESULTS: Corneas remained clear after both DSAEK and nDSAEK. One week after DSAEK, the stroma-to-stroma surgical interface was identifiable as a zone of fibrotic tissue a few microns thick, whereas in the nDSAEK group, the recipient corneal endothelium and Descemet membrane were clearly visible at the graft-host interface. The retained endothelial cells were positive for Na/K-ATPase but assumed a markedly different morphology from healthy endothelial cells, with cell processes extending into the graft stroma or engulfing strands of irregularly dissected grafted stromal tissue where they occasionally appeared to compartmentalize the transplanted matrix and became detached from the underlying Descemet membrane. CONCLUSIONS: Host endothelial cells 2 weeks after nDSAEK express markers of pump function, but appear to be morphologically altered, occasionally detaching from the adjacent Descemet membrane, extending into the graft stroma or engulfing strands of the grafted stroma at the interface. The short-term persistence and subsequent phenotypical alternation of residual endothelial cells, aligned to structural changes to Descemet membrane, might influence graft adherence after nDSAEK.

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.

The persistence of transplanted amniotic membrane in corneal stroma.

PURPOSE: To characterize the long-term incorporation of transplanted amniotic membrane into corneal stroma. DESIGN: Experimental study. METHODS: Rabbit amniotic membrane, stained with a fluorescent dye (DTAF), was implanted unilaterally into the corneal stroma of four adult rabbits. Corneas were examined clinically and by transmission electron microscopy and fluorescent microscopy at 1, 3, 7, and 10 months after surgery. RESULTS: Visibility of the transplanted amniotic membrane, in situ, on slit-lamp examination decreased over time. However, fluorescent and electron microscopy clearly demonstrated that the amniotic membrane remained structurally unchanged and intact within the corneal stroma up to 10 months after implantation. CONCLUSIONS: Amniotic membrane allografts persist intact within an intracorneal space for many months postoperatively and are not quickly broken down or dissolved by the host tissue.