V A Shanmuganathan

Limbal epithelial crypts: a novel anatomical structure and a putative limbal stem cell niche

Background/aims: There is substantial evidence that mammalian epithelial stem cells are located within well defined niches. Although the corneoscleral limbus is acknowledged as the site of corneal epithelial stem cells no anatomical niche for such cells has yet been described. The authors undertook to re-evaluate the microanatomy of the limbus in order to identify possible sites that may represent a stem cell niche. Methods: Systematic serial 5–7 μm sections of human corneoscleral segments obtained from cadaver donors, were examined. The sections were stained with haematoxylin and eosin or toludine blue. Sections with specific areas of interest were further examined immunohistologically for the corneal epithelial marker cytokeratin 14 and the “stem cell” marker ABCG2 transporter protein. Results: Distinct anatomical extensions from the peripheral aspect of the limbal palisades were identified. These consist of a solid cord of cells extending peripherally or circumferentially. The cells stained positive for CK14 and ABCG2. Conclusions: A novel anatomical structure has been identified at the human limbus, which demonstrates characteristics of being a stem cell niche. The authors have termed this structure the limbal epithelial crypt.

Stem cell differentiation and the effects of deficiency

AbstractStem cells have several unique attributes, the key features being their potency and plasticity. They have the ability to give rise to multiple cell lineages and to transdifferentiate into totally different cell type(s) when relocated to a novel stem cell niche. Most self-renewing tissues are served by stem cells. At the ocular surface, the corneo-scleral limbus is believed to provide the niche for corneal epithelial stem cells. A large body of circumstantial evidence, both clinical and basic, supports this view. However, specific identification of limbal stem cells has proved elusive. Cytokeratin markers, vimentin, epidermal growth factor receptors, p63, and others have been used to identify epithelial cell populations at the limbus, which could harbour putative stem cells. In contrast, none of the known haematopoietic stem cell markers namely, CD34 and CD133, stain any specific subset of corneal or limbal epithelial cells. Singly or collectively, none of these markers point to any unique cell(s) that could be regarded as stem cells, supporting the notion that the corneal epithelium is served by ‘committed progenitors’ rather than by stem cells. Disease or destruction of the corneo-scleral limbus is associated with consequential events that eventually lead to visual impairment or blindness. Conjunctivalisation and vascularisation of the corneal surface and persistent or recurring epithelial defects are hallmarks of limbal deficiency.

Morphological characteristics of the limbal epithelial crypt

Aim: In 2005 we reported the discovery of a novel anatomical structure at the limbus, which we termed the limbal epithelial crypt (LEC). The purpose of this study was to further evaluate the distribution, immunophenotypical, and ultra structural characteristics of the LEC as a putative niche of stem cells. Methods: Sequential histological sections of human corneo-scleral limbal rims were examined for the presence and distribution of the LEC. Immunophenotypical characterisation of the LEC cells using a panel of antibodies of interest was undertaken. Transmission electron microscopy of the LEC was used to examine the ultra structural and morphometric features of cells within the LEC and adjacent limbus. Results: A total of 74 LECs were identified in eight corneo-scleral rims. These varied in number, size and distribution within rims. Cells within the crypt demonstrated the following phenotype: CK3−/CK19+/CD 34−/Vimentin+/p63+/Connexin 43+/MIB1 (Ki67)−. Presence of Cx43 was also demonstrated in the rete pegs adjacent to the LEC. Basal cells of the LEC were significantly smaller than basal cells found in adjacent rete pegs and also smaller than suprabasal limbal and central corneal epithelial cells (p<0.05). Morphologically they had a high nuclear:cytoplasmic ratio and were adherent to the underlying basement membrane by means of complex convolutions of cytoplasmic processes. Conclusions: LECs are sparse but a consistent finding in the human corneo-scleral limbus. The LEC contains a unique sub-population of cells expressing several characteristics that are consistent with it representing a putative stem cell niche.

Epithelial cell characteristics of cultured human limbal explants

Aim: To determine the immunohistochemical characteristics of putative corneal epithelial stem cells remaining on limbal explants maintained in culture. Methods: Human limbal explant cultures were generated from 25 residual corneoscleral donor rims following penetrating keratoplasty. Serial sections of these explants were studied using immunohistochemical techniques with a panel of antibodies, on day 0 and 1, 2, and 3 weeks. Results: The number of epithelial cells expressing cytokeratin 19 and vimentin increased with duration in culture, while the number of cells expressing cytokeratin 3 decreased. Connexin 43 expression was lost by 1 week in culture. p63 was expressed by cells that had migrated around the explant and the number of p63 positive cells decreased with longer duration in culture. The explants were initially negative for Ki67, but the epithelial cells were positive at 1 week, and expression of Ki67 was progressively lost with increasing duration in culture. The initial uniform staining of the epithelium for epidermal growth factor receptor and α enolase remained unchanged at 3 weeks. Conclusions: There is an expansion of less differentiated (cytokeratin 3 negative and CK19/vimentin positive) epithelial cells on corneoscleral explants maintained in culture for 3 weeks. The pattern of expression of p63 noted in this study does not support the suggestion that it is a marker of limbal stem cells. The decline in p63 and Ki67 expression among the epithelial cells of the cultured explant button implies that as the epithelial sheet outgrowing from the explant button reaches confluence, the proliferative status of the cells remaining on the explant button declines. These findings are of clinical relevance as explants of limbal tissue are used in limbal stem cell transplantation. There is no information available to date on the fate of epithelial cells on such explants. This study provides some insight into this and suggests that an expansion of the stem cell pool or its progeny may occur in limbal explants.

Optimization of Amniotic Membrane (AM) Denuding for Tissue Engineering

INTRODUCTION Amniotic membrane (AM) has gained increasing popularity as a useful carrier for ex vivo-expanded cells for tissue engineering, particularly in ocular surface reconstruction. However, current methods employed for denuding AM are highly variable, and the consequent effects on the structural and molecular composition of the AM basement membrane (BM) are ambiguous. We compare the effects of the main denuding procedures, and propose a highly effective standardized alternative. METHODS AMs preserved for transplantation were denuded using published ethylenediaminetetraacetic acid (EDTA)- and dispase-based methodologies and our novel thermolysin-based procedure. Scanning and transmission electron microscopy and immunohistochemistry, for BM components (collagens IV and VII, laminin 5, and integrins alpha6 and beta4), were used to assess effectiveness of denuding epithelium, whilst maintaining the integrity of the BM. RESULTS EDTA- and dispase-based denuding techniques resulted in the disaggregation and even destruction of the BM structure and molecular composition. Employing thermolysin effectively denuded epithelium whilst maintaining BM structural and molecular integrity. CONCLUSION Current procedures for preparing AM are variable and often ineffective, resulting in nonstandard membranes. Our novel thermolysin-based technique effectively denudes the AM whilst preserving an essentially intact and consistent BM. Therefore, we propose that this novel thermolysin procedure may potentially improve overall generation of tissue-engineered constructs using AM.

The efficacy of sirolimus in the treatment of patients with refractory uveitis.

Aims: To determine the efficacy of sirolimus in the treatment of patients with severe non-infectious uveitis. Methods: Eight patients with severe non-infectious uveitis were recruited to an open study. Inclusion criteria were limited to patients whose disease was not controlled with at least two or more separate steroid sparing immunosuppressants (either because of unacceptable side effects or ineffectiveness of the drug) or who required regular doses of corticosteroids either as high dose systemic or orbital floor injections in order to control their disease. Intraocular inflammation, visual acuity, symptoms, corticosteroid burden, drug toxicity, and side effects were monitored. Results: Sirolimus therapy was effective in five of the eight patients, all of whom had their dose of corticosteroids reduced or discontinued. Treatment in three patients was considered a failure as it caused intolerable side effects and/or failed to control the uveitis. Side effects were common and were typically gastrointestinal or cutaneous in nature. The severity of symptoms was dose dependent in most cases and occurred at trough blood levels above 25 ng/ml. Conclusion: Sirolimus is an effective and potent immunosuppressive treatment in the majority of patients with non-infectious uveitis and can reduce the need for long term supplementary corticosteroid therapy. Further studies are required to establish the long term efficacy and safety of sirolimus alone or in combination with other steroid sparing immunosupressants.

Amniotic membrane transplantation for ocular surface reconstruction: indications and outcomes

Background:  The amniotic membrane is a useful tool in the management of several ocular surface diseases. Despite numerous studies, standardization of the use of the membrane and of outcome measures is lacking. Herein, the authors’ results of amniotic membrane transplantation (AMT) in ocular surface reconstruction against defined outcome measures are reported.

Tacrolimus immunosuppression in high-risk corneal grafts.

Background: Unlike the immune privilege enjoyed by low-risk corneal grafts, high-risk corneal grafts experience rejection rates comparable to liver and kidney transplants. Systemic immunosuppression reduces the risk of rejection in high-risk corneal grafts. Methods: Systemic tacrolimus, a specific T cell inhibitor, was used at a mean daily dose of 2.5 mg to immunosuppress 43 patients undergoing high-risk corneal transplantation. Immunosuppression was continued for a period of 18–24 months after the high-risk corneal graft. Results: During a mean follow-up period of 33.7 months, clarity of the graft was maintained in 65% of patients. Eight patients experienced rejection episodes while on tacrolimus, and this led to graft failure in five patients. Conclusion: Tacrolimus is relatively safe and effective in reducing rejection and prolonging graft survival in patients with high-risk keratoplasty compared with other series where similar immunosuppression was not used.

Argon laser iridotomy‐induced bullous keratopathy—a growing problem in Japan

Aims: To describe the long-term risk of bullous keratopathy following argon laser iridotomy (ALI) in Japan and to compare it with other centres in the world. Methods: We retrospectively reviewed the case records of all patients with ALI-induced bullous keratopathy that underwent penetrating keratoplasty at Kyoto Prefectural University of Medicine (KPUM) from January 2001 to December 2004. The results were compared with the other representative centres in Singapore and the UK. Results: Thirty-nine eyes of 33 patients were included in the study. The mean age of patients was 73.3±6.9 years (range, 58 to 87 years). Patients developed bullous keratopathy at a mean duration of 6.9±4.9 years (range, 0.2 to 16 years) after the laser iridotomy procedure. The majority of eyes that developed bullous keratopathy (59.0%) occurred following prophylactic ALI. KPUM had the highest percentage of ALI-induced bullous keratopathy cases that underwent penetrating keratoplasties, as compared with other centres in Singapore and the UK (20.0%, 1.8% and 0%, respectively). Conclusion: Bullous keratopathy may arise many years following ALI, and is a growing problem in Asian countries. This condition is a major cause of ocular morbidity in Japan, which has seen a worrying increase in the number of cases in recent years.

Epithelial proliferative potential of organ cultured corneoscleral rims; implications for allo-limbal transplantation and eye banking

Aims: To determine the epithelial proliferative capacity of organ cultured limbal tissue and correlate this with various donor and eye banking factors. Methods: 24 corneoscleral limbal (CSL) rims left over from penetrating keratoplasty were split in half and set up as in vitro explant cultures. Corneal epithelial proliferative potential (CEPP) was assessed by the number of “cycles” of growth achieved before explants underwent exhaustion and failure to generate an epithelium to subconfluence. The dependence of CEPP on the age of the donor, time of death to enucleation, time of enucleation to organ culture, and time in organ culture in the eye bank was determined. Results: CSL rims were capable of up to four cycles of culture with a wide variation between tissue samples. Of the various factors examined, death to enucleation time was the only statistically significant factor affecting the CEPP (regression coefficient: −0.062 (cycles/hour), CI −0.119 to −0.004, p  = 0.037). Time in organ culture had little effect on CEPP. Conclusions: Preselected organ cultured CSL rims from eye banks may offer a viable alternative tissue source for use in allo-limbal transplantation.