In sickness and in health: Corneal epithelial stem cell biology, pathology and therapy

Our window to the world is provided by the cornea on the front surface of the eye. The integrity and functionality of the outermost corneal epithelium is essential for vision. A population of limbal epithelial stem cells (LESCs) are responsible for maintaining the epithelium throughout life by providing a constant supply of daughter cells that replenish those constantly lost from the ocular surface during normal wear and tear and following injury. LESC deficiency leads to corneal opacification, inflammation, vascularization and discomfort (Daniels et al., 2001, 2007). Cultured LESC delivery is one of several examples of successful adult stem cell therapy in patients. The clinical precedence for use of stem cell therapy and the accessibility of the transparent stem cell niche make the cornea a unique model for the study of adult stem cells in physiological conditions as well as in disease.     

Contemporary limbal stem cell transplantation – a review

Conjunctivalization of the cornea is the hallmark of limbal stem cell deficiency (LSCD). This is often associated with persistent corneal epithelial defects and a fibrovascular pannus. LSCD can be unilateral or bilateral and partial or total. In partial LSCD involving the visual axis sequential sector conjunctival epitheliectomy (SSCE) is a useful option. In total LSCD, transplantation of limbal tissue or of ex vivo expanded sheets is the mainstay. In unilateral cases autolimbal transplant is the procedure of choice. In bilateral cases living (related) and cadaver donors are considered. The former has the advantage of being fresh and can be human leucocyte antigen matched. Procedures for harvesting limbal tissue from living donors are identical. Different strategies are required for harvesting tissue from cadaver whole globes or sclero‐corneal rims. Recipient eye preparation requires removal of the fibrovascular tissue. Donor explants are generally sutured directly on the denuded recipient surface without the preparation of a ‘bed’ to fit the explant. It is imperative that inflammation is meticulously controlled before limbal transplantation especially if tissue from living donors is used. Limbal transplantation, with the exception of a corneal graft, should be the last surgical intervention planned. Meticulous postoperative care and treatment with antibiotics, steroids, artificial tears and autologous serum are required. With allografts long‐term immunosuppression is necessary. Limbal transplantation is contraindicated in the presence of severe dry eye. Despite its complexities limbal transplantation does significantly improve vision related quality of life. Autografts give the best results and living related donor grafts are next best. Majority of cadaver grafts fail in 5 years.     

羊膜移植联合自体角膜缘干细胞移植治疗眼部烧伤

目的：探究羊膜移植联合自体角膜缘干细胞移植治疗对眼部烧伤患者眼表修复时间、修复率及并发症的影响。

方法：回顾性分析我院行自体角膜缘干细胞移植治疗(对照组)和行羊膜移植联合自体角膜缘干细胞移植治疗(观察组)的各61例61眼眼部烧伤患者临床资料。记录两组术后眼表修复率、修复时间、术后12wk视力情况及并发症发生情况(睑球粘连、角膜新生血管、角膜溶解溃疡),并比较两组术前及术后2wk细胞因子\〖血管内皮生长因子(VEGF)、转化生长因子-β1(TGF-β1)、胰岛素样生长因子-1(IGF-1)\〗水平差异。

结果：观察组术后眼表修复率高于对照组(P<0.05),且修复时间低于对照组(P<0.05)。术后12wk时,观察组视力情况明显优于对照组(P<0.05)。观察组术后并发症总发生率明显低于对照组(P<0.05)。术后2wk时,两组血清VEGF、TGF-β1、IGF-1水平均较术前升高(P<0.05),且对照组高于观察组(P<0.05)。

结论：羊膜移植联合自体角膜缘干细胞移植对眼部烧伤患者治疗效果显著,可促进患者眼表修复,改善术后视力,调节患者角膜血管新生情况,降低术后并发症风险。     

Ex vivo expansion of limbal epithelial stem cells: amniotic membrane serving as a stem cell niche

Identification, maintenance, and expansion of stem cells for subsequent transplantation has become a new strategy for treating many diseases in most medical subspecialties. The stem cells of the corneal epithelium are located in the limbal basal layer and are the ultimate source for constant corneal epithelial renewal. Like those in other tissues, limbal stem cells are supported by a unique stromal microenvironment called the stem cell niche, which consists of certain extracellular matrix components, cell membrane-associated molecules, and cytokine dialogues. Destructive loss of limbal stem cells or dysfunction of their stromal environment renders many corneas with a clinical entity called limbal stem cell deficiency, which is characterized by variable extents of conjunctival ingrowth depending on the severity of limbal damage. A new strategy of treating limbal stem cell deficiency is to transplant a bio-engineered graft by expanding limbal epithelial stem cells ex vivo on amniotic membrane. This review summarizes the published literature data collectively explaining how amniotic membrane is an ideal biological substrate that can help maintain and support the expansion of limbal epithelial stem cells.     

Coculture of autologous limbal and conjunctival epithelial cells to treat severe ocular surface disorders: Long-term survival analysis

Background:

Cultivated limbal epithelium for reconstruction of corneal surface is a well-established procedure; however, it is not adequate for damage which also extensively involves the conjunctiva. In severe cases of ocular surface damage that warrant additional conjunctival transplantation apart from cultivated limbal stem cell transplantation, we describe the long-term survival of a novel method of cocultivating autologous limbal and conjunctival epithelium on a single substrate.

Materials and Methods:

Forty eyes of 39 patients with severe limbal stem cell deficiency and conjunctival scarring or symblepharon underwent transplantation of autologous cocultivated epithelium on human amniotic membrane. A ring barrier was used to segregate the central limbal and peripheral conjunctival epithelia in vitro. Patients were followed up at regular intervals to assess stability of the ocular surface, defined by absence of conjunctivalization into the central 4 mm of the cornea and absence of diffuse fluorescein staining. Penetrating keratoplasty (PKP) was subsequently performed, where indicated, in patients with surface stability.

Results:

The cumulative survival probability was 60% at 1 year and 45% at 4 years by Kaplan–Meier analysis (mean follow-up duration: 33 ± 29 months, range: 1–87 months). Best-corrected visual acuity improved to greater than 20/200 in 38% eyes at the last follow-up, compared with 5% eyes before surgery. Immunohistochemistry in five of the corneal buttons excised for PKP showed an epithelial phenotype similar to cornea in all five.

Conclusions:

Synchronous use of cultured limbal and conjunctival epithelium offers a feasible alternative and a simpler one-step surgical approach to treat severe ocular surface disorders involving limbus and conjunctiva.     

改良带角膜缘干细胞的游离结膜瓣移植术治疗翼状胬肉

目的:探讨改良带角膜缘干细胞的游离结膜瓣移植术治疗翼状胬肉的临床疗效及对泪膜功能的影响。方法:病例对照研究。将我院2017-03/2021-03收治的60例60眼翼状胬肉患者,按随机数字表法分为对照组和观察组,各30例30眼,对照组给予翼状胬肉切除联合羊膜移植术治疗,观察组给予翼状胬肉切除联合改良带角膜缘干细胞的游离结膜瓣移植术治疗,治疗周期均为21d。比较两组患者临床疗效、手术时间、角膜创面修复时间,术前和术后1、3mo的干眼评分、泪膜破裂时间值和基础泪液分泌试验值以及不良反应发生情况。结果:观察组临床治疗总有效率高于对照组(χ²=5.963,P=0.015),观察组手术时间长于对照组(t=-2.643,P<0.05),角膜创面修复时间短于对照组(t=2.182,P<0.05)。两组患者在术后1、3mo,干眼评分与术前比较均下降(均P<0.05),且观察组与对照组比较有差异(t=2.082、3.956,均P<0.05)。术后1、3mo泪膜破裂时间与术前比较均升高(均P<0.05),且观察组与对照组比较有差异(t=4.245、2.07...     

Inducing dry eye disease using a custom engineered desiccation system: Impact on the ocular surface including keratin-14-positive limbal epithelial stem cells

PurposeDry eye disease (DED) is characterized by loss of tear film stability that becomes self-sustaining in a vicious cycle of pathophysiological events. Currently, desiccation stress (DS) is the dominant procedure for inducing DED in mice, however its’ effect on limbal epithelial stem cells (LESCs) has been overlooked. This study aimed to establish a DS model via the use of a novel hardware to investigate the impact on the ocular surface including LESCs.MethodsA mouse transporter unit was customized to generate a dehumidified environment. C57BL/6J mice were exposed to mild DS and injected with scopolamine hydrobromide (SH) or remained untreated (UT) under standard vivarium conditions for 10 consecutive days (n = 28/group). Clinical assessments included phenol red tear-thread test, fluorescein staining and optical coherence tomography assessments. Histopathological and immunofluorescence was used to evaluate tissue architecture, goblet cell (GC) status, lacrimal gland (LG) inflammation and epithelial phenotype on the ocular surface. Whole flat-mounted corneas were immunostained for keratin-14 (K14), then imaged by confocal microscopy and analyzed computationally to investigate the effect of DS on LESCs.ResultsCustom modifications made to the animal transporter unit resulted in dehumidified cage relative humidity (RH) of 43.5 ± 4.79% compared to the vivarium 53.9 ± 1.8% (p = 0.0243). Under these conditions, aqueous tear production in mice was suppressed whilst corneal permeability and corneal irregularity significantly increased. H&E staining indicated stressed corneal basal epithelial cells and increased desquamation. DS-exposed mice had reduced GC density (41.0 ± 5.10 GC/mm vs 46.9 ± 3.88 GC/mm, p = 0.0482) and LGs from these mice exhibited elevated CD4⁺ cell infiltration compared to controls. DS elicited K14⁺ epithelial cell displacement, as indicated by increased fluorescence signal at a distance of 50–100 μm radially inwards from the limbus [0.63 ± 0.053% (DS) vs 0.54 ± 0.060% (UT), p = 0.0317].ConclusionsApplication of mild DS using customized hardware and SH injections generated features of DED in mice. Following DS, ocular surface epithelial cell health decreased and LESCs appeared stressed. This suggested that potential downstream effects of DS on corneal homeostasis are present, a phenomenon that is currently under-investigated. The method used to induce DED in this study enables the development of a chronic model which more closely resembles disease seen in the clinic.     

Moving epithelia: Tracking the fate of mammalian limbal epithelial stem cells

Lineage tracing allows the destiny of a stem cell (SC) and its progeny to be followed through time. In order to track their long-term fate, SC must be permanently marked to discern their distribution, division, displacement and differentiation. This information is essential for unravelling the mysteries that govern their replenishing activity while they remain anchored within their niche microenvironment. Modern-day lineage tracing uses inducible genetic recombination to illuminate cells within embryonic, newborn and adult tissues, and the advent of powerful high-resolution microscopy has enabled the behaviour of labelled cells to be monitored in real-time in a living organism. The simple structural organization of the mammalian cornea, including its accessibility and transparency, renders it the ideal tissue to study SC fate using lineage tracing assisted by non-invasive intravital microscopy. Despite more than a century of research devoted to understanding how this tissue is maintained and repaired, many limitations and controversies continue to plague the field, including uncertainties about the specificity of current SC markers, the number of SC within the cornea, their mode of division, their location, and importantly the signals that dictate cell migration. This communication will highlight historical discoveries as well as recent developments in the corneal SC field; more specifically how the progeny of these cells are mobilised to replenish this dynamic tissue during steady-state, disease and transplantation. Also discussed is how insights gleaned from animal studies can be used to advance our knowledge of the fundamental mechanisms that govern modelling and remodelling of the human cornea in health and disease.