Stephen J. Tuft

Characterization of the Limbal Epithelial Stem Cell Niche: Novel Imaging Techniques Permit In Vivo Observation and Targeted Biopsy of Limbal Epithelial Stem Cells

It is anticipated that stem cell (SC) therapy will enable the regeneration of diseased tissues and organs. Understanding SC niches is an essential step toward realizing this goal. By virtue of its optical transparency and physical separation of SC and transient amplifying cell compartments, the human cornea provides a unique opportunity to visualize and observe a population of adult stem cells, limbal epithelial stem cells (LESCs), in their niche environment. To date, the characteristics of the LESC niche have remained unclear. State‐of‐the‐art imaging techniques were used to construct a three‐dimensional (3D) view of the entire human corneal limbus and identify the structural characteristics of the LESC niche. Two distinct candidate LESC niche structures were identified. Cells within these structures express high levels of the putative limbal stem cell markers p63α and ABCG2; however, current methods cannot identify for certain which exact cells within this cell population are truly LESCs. These structures could be located and observed in vivo in normal human subjects, but not in patients with clinically diagnosed corneal LESC deficiency. The distribution of these structures around the corneal circumference is not uniform. Biopsies targeted to limbal regions rich in LESC niche structures yielded significantly higher numbers of LESCs in culture. Our findings demonstrate how adult stem cell niches can be identified and observed in vivo in humans and provide new biological insight into the importance of LESC niche structures in maintaining normal LESC function. Finally, the concept of targeted biopsy of adult SC niches improves stem cell yield and may prove to be essential for the successful development of novel adult stem cell therapies.

Prognostic Factors for the Progression of Keratoconus

PURPOSE The progression of keratoconus to a stage where penetrating keratoplasty (PK) is required for visual rehabilitation has considerable implications for affected patients. To assist with counselling, the authors have attempted to identify which factors measurable early in the course of the disease may indicate the likelihood of subsequent surgery. METHODS The authors reviewed the records of all patients who attended a single center over a 7-year period for contact lens management of their keratoconus. The influence of clinical variables on the time taken for the worst eye to progress to PK was evaluated by actuarial methods and multivariate analysis. RESULTS Included in the study were 2723 patients with a mean follow-up for unoperated eyes from the first visit of 4.5 years (range, 3 months to 28 years). Data were available for multivariate analysis in 2363 patients. At the end of the study period, 757 eyes (21.6% of all patients) had been grafted. The number of eyes progressing to PK was independently related to both the maximum and minimum keratometry, a corneal cylinder of more than 1.9 mm, the Snellen acuity, the racial group (P < 0.0001), and the age at presentation (P = 0.0006). Sex, laterality, systemic atopic disease, maternal or paternal age at birth, joint hypermobility, and a family history of keratoconus were not statistically related to outcome. Progression to PK in one eye increased the risk of progression in the contralateral eye (P < 0.0001) and a linear model of disease progression is proposed. CONCLUSIONS Several clinical variables can be measured in patients at the presentation of keratoconus that influence the probability of a subsequent PK.

Corneal stem cells in review

The cornea provides the eye with protection and the refractive properties essential for visual acuity. The transparent epithelium is highly specialized with basal and stratified squamous cells that are renewed throughout life from a stem cell population. The stem cells are thought to reside at the corneal limbus and may be maintained by a variety of intrinsic and extrinsic factors such as the local environment, survival factors, and cytokines. A number of markers have been localized to the limbus in an attempt to identify stem cells; however, definite stem cell identification remains elusive. During homeostasis and following injury to the corneal epithelium, the limbal stem cells divide to produce daughter transient amplifying cells that proliferate, migrate, and differentiate to replace lost cells. However, this cannot occur if the stem cell population is depleted. Limbal stem cell deficiency then results in corneal re‐epithelialization by the neighboring conjunctiva, causing pain, poor vision, and even blindness. This review will focus on corneal epithelial stem cells in ocular surface repair and regeneration. The current knowledge of stem cell biology in the corneal epithelium, clinical consequences of stem cell deficiency, and therapeutic strategies aimed at reversing stem cell deficiency will be discussed.

Clinical Features of Atopic Keratoconjunctivitis

The clinical spectrum of ocular disease in 37 patients with atopic keratoconjunctivitis (AKC) is described. Patients typically had a severe blepharoconjunctivitis. Associated corneal scarring, suppurative keratitis, or keratoconus were the major causes of visual loss. Serum and tear samples from these patients were analyzed to quantify total and specific IgE antibodies. The results were compared as a case control study with results from samples from 55 patients with other forms of atopic disease and 16 nonatopic volunteers. Although the mean values for total and specific IgEs in the serum of patients with atopic disease were markedly higher than the values from nonatopic controls (P less than 0.00002), a difference between the disease groups could not be demonstrated (P greater than 0.05). There were also differences between both the total IgE (P = 0.0002) and pollen-specific IgE (P = 0.015) in tears from patients with atopic disease and nonatopic controls, but not for house dust mite or cat dander-specific IgEs. These results suggest that clinical differences between groups of patients with chronic allergic external eye disease are not associated with specific patterns of IgE production.

Limbal transplantation in the management of chronic contact-lens-associated epitheliopathy

We describe the clinical management of 6 patients who developed a chronic corneal epitheliopathy 1–18 years after commencing soft contact lens wear. All had a history of exposure to thiomersal in contact lens fluids. The corneal changes were characterised by epithelial haze and superficial stromal vascularisation which extended from the limbus towards the visual axis. Five patients were observed for a minimum of 18 months after stopping contact lens wear before undergoing limbal transplantation. A good result was obtained in 1 patient who had worn a contact lens in one eye only. Recurrent epithelial changes were observed on the recipient eyes of the remaining patients who had previously worn contact lenses bilaterally, and in 1 patient epithelial haze also developed adjacent to the donor site in the previously clinically normal donor eye. All 5 patients experienced an improvement in symptoms post-operatively but in 2 patients the visual acuity later deteriorated because of epithelial irregularity. The sixth patient has not had surgery. We conclude that limbal stem cell dysfunction in chronic contact-lens-associated epitheliopathy may be subclinical and that autograft transplantation in bilaterally exposed patients may fail to restore the epithelial phenotype of the host eye whilst jeopardising the epithelial integrity of the donor eye by depleting its stem cell reserve.

Ex Vivo Expansion and Transplantation of Limbal Epithelial Stem Cells

OBJECTIVE To determine, using objective measures, the outcome of ex vivo cultured limbal epithelial stem cell (LESC) transplantation performed in compliance with good manufacturing practice using a novel culture system without 3T3 feeder cells. DESIGN Prospective, noncomparative, interventional case series. PARTICIPANTS Ten eyes of 10 patients with profound LESC deficiency arising from chemical injury (4 eyes), aniridia (3 eyes), ectodermal dysplasia (1 eye), Reigers anomaly with Pax6 haploinsufficiency (1 eye), and unknown cause (1 eye). METHODS Allogeneic (7 eyes) or autologous (3 eyes) corneal LESCs were cultured on human amniotic membrane. Tissue was transplanted to the recipient eye after superficial keratectomy. Impression cytology and confocal microscopy were performed 6 months after surgery with clinical follow-up to 13 months. Success was defined as an improvement in the defined clinical parameters of LESC deficiency, an improvement in visual acuity, the restoration of a more normal corneal phenotype on impression cytology, and the appearance of a regular hexagonal basal layer of cells on corneal confocal microscopy. MAIN OUTCOME MEASURES Clinical parameters of LESC deficiency (loss of epithelial transparency, superficial corneal vascularization, epithelial irregularity, and epithelial breakdown), visual acuity, impression cytology and cytokeratin expression profiles, and in vivo confocal corneal confocal microscopy. RESULTS The success rate using this technique was 60% (autografts 33%, allografts 71%). All patients with a successful outcome experienced an improvement in visual acuity of >/=2 lines Snellen acuity. Preoperatively, CK3+ and CK19+ cells accounted for 12+/-2.4% (mean +/- standard error of the mean) and 80+/-2.15% of cells, respectively, whereas postoperatively these accounted for 69+/-6.43% (P<0.0001) and 30+/-6.34% (P<0.0001) of cells, respectively. Goblet cells accounted for 8+/-1.19% of cells preoperatively and 1+/-0.35% of cells postoperatively (P<0.0001). CONCLUSIONS These data demonstrate that it is possible to culture LESCs ex vivo in compliance with good manufacturing practice regulations. A set of objective outcome measures that confirm the efficiency of this technique in treating LESC deficiency is described. The widespread use of such standardized and objective outcome measures would facilitate a comparison between the different culture methods in use.

Surgically induced necrotising sclerokeratitis (SINS)--precipitating factors and response to treatment.

The clinical features, treatment, and visual outcome of 52 eyes from 43 patients who developed scleritis following surgery were reviewed. In all patients the scleral inflammation developed adjacent to a surgical wound. Ninety six per cent had necrotising disease and 23% also had evidence of secondary posterior scleritis. Many different types of ocular surgery were implicated and the majority (75%) of the patients had two or more surgical procedures before the onset of the scleritis. Although cataract extraction through a limbal incision resulted in the largest subgroup, scleritis also followed glaucoma, strabismus, and retinal detachment surgery. The latent period between surgery and the appearance of inflammation was short (mean 9 months) except for a small group in whom scleritis occurred many years after squint surgery. Sixty three per cent of patients had evidence of a systemic disease. Early diagnosis and aggressive medical treatment significantly improved the visual outcome. The precipitating factors, pathogenesis, and course of this condition are discussed.

Acute Corneal Hydrops in Keratoconus

PURPOSE To determine the clinical factors associated with the development of acute corneal hydrops and its subsequent outcome. METHODS The authors identified 147 eyes (124 patients) with acute hydrops from a database of 5242 eyes (2723 patients) with keratoconus. They compared the clinical features of patients in whom hydrops developed with unaffected patients and assessed the progression to penetrating keratoplasty by actuarial methods. RESULTS Patients in whom acute hydrops developed tended to be younger males who had advanced corneal ectasia and a poor corrected Snellen visual acuity at the diagnosis of their keratoconus (P < 0.001). Acute hydrops also was more common in the presence of severe allergic eye disease (P < 0.001). The development of hydrops was a very significant risk factor for subsequently receiving a penetrating keratoplasty (P < 0.00001) and at the end of the study period 87 (59%) of the 147 eyes had surgery for visual rehabilitation. These eyes had a greater rate of graft rejection than eyes grafted without hydrops (P = 0.02). After resolution of the hydrops, 46 of the 60 unoperated eyes had been refitted with contact lenses, of which 28 (61%) achieved a Snellen visual acuity of 20/40 or better, although a better visual acuity often was present in the contralateral eye. Microbial keratitis developed in two of these eyes after refitting. CONCLUSIONS Although a penetrating keratoplasty often is indicated for visual rehabilitation after acute corneal hydrops, there is an increased rate of rejection. Only a minority of eyes were re-established in contact lenses after resolution of hydrops, but some patients achieved a functional level of visual acuity such that the procedure could be delayed or avoided.

The effect of amniotic membrane preparation method on its ability to serve as a substrate for the ex-vivo expansion of limbal epithelial cells.

Human amniotic membrane (HAM) is employed as a substrate for the ex-vivo expansion of limbal epithelial cells (LECs) used to treat corneal epithelial stem cell deficiency in humans. The optimal method of HAM preparation for this purpose is unknown. This study evaluated the ability of different preparations of stored HAM to serve as substrates for LEC expansion ex-vivo. The effect of removing the amniotic epithelial cells (decellularisation) from HAM prior to seeding of LECs, the effect of glycerol cryopreservation and the effect of peracetic acid (PAA) sterilization and antibiotic disinfection were evaluated using different HAM test groups. Human LECs were cultured on each preparation and the following outcomes were assessed: confluence of growth, cell density, cell morphology and expression of the putative LESC markers deltaN-p63alpha and ABCG2. Removing amniotic epithelial cells prior to seeding of LECs resulted in a higher percentage of confluence but a lower cell density than intact HAM suggesting that decellularisation does not increase proliferation, but rather that it facilitates migration of LECs resulting in larger cells. Decellularisation did not affect the percentage of cells expressing the putative LESC markers deltaN-p63alpha (< or =4% in both intact and acellular groups) and ABCG2 (< or =3% in both intact and acellular groups). Glycerol cryopreservation of HAM resulted in poor morphology and a low proportion of cells expressing deltaN-p63alpha (< or =6%) and ABCG2 (< or =8%). HAM frozen at -80 degrees C in Hanks Balanced Salt Solution (HBSS) was superior, demonstrating excellent morphology of cultured LECs and high levels of deltaN-p63alpha (< or =68%) and ABCG2 (< or =62%) expression (p<0.001). The use of PAA or antibiotics to decontaminate HAM does not appear to affect this function. The variables affecting the ability of HAM to serve as a substrate for LEC expansion ex-vivo are poorly understood. The use of glycerol as a cryoprotectant impairs this ability whereas simple frozen HAM appears to work extremely well for this purpose.

Visual outcome following posterior capsule rupture during cataract surgery

AIM To determine the relative risk of a poor visual outcome following posterior capsule rupture during cataract surgery. METHODS Prospective data were collected on consecutive eyes undergoing cataract extraction. The patients age, preoperative visual acuity, ocular comorbidity, grade of surgeon, and operative complications were documented. The best spectacle corrected visual acuity was recorded at discharge from the hospital service. RESULTS From a total of 1533 cases, 1420 (92.6%) eyes had complete follow up data. Posterior capsule rupture occurred in 59 (4.1%) cases. Eyes with posterior capsule rupture were 3.8 times more likely to have a final best spectacle corrected visual acuity less than 6/12. CONCLUSIONS Eyes having posterior capsule rupture during cataract surgery have a significant risk of reduced visual acuity.