Limbal Epithelial Stem Cells Research Articles

Emerging Therapeutic Strategies for Limbal Stem Cell Deficiency.

Identification and characterization of the limbal epithelial stem cells (LESCs) has proven to be a major accomplishment in anterior ocular surface biology. These cells have been shown to be a subpopulation of limbal epithelial basal cells, which serve as the progenitor population of the corneal epithelium. LESCs have been demonstrated to play an important role in maintaining corneal epithelium homeostasis. Many ocular surface diseases, including intrinsic (e.g., Sjogren's syndrome) or extrinsic (e.g., alkali or thermal burns) insults, which impair LESCs, can lead to limbal stem cell deficiency (LSCD). LSCD is characterized by an overgrowth of conjunctival-derived epithelial cells, corneal neovascularization, and chronic inflammation, eventually leading to blindness. Treatment of LSCD has been challenging, especially in bilateral total LSCD. Recently, advances in LESC research have led to novel therapeutic approaches for treating LSCD, such as transplantation of the cultured limbal epithelium. These novel therapeutic approaches have demonstrated efficacy for ocular surface reconstruction and restoration of vision in patients with LSCD. However, they all have their own limitations. Here, we describe the current status of LSCD treatment and discuss the advantages and disadvantages of the available therapeutic modalities.

Current approaches to the problem of carrier selection for limbal stem cells cultivation in the treatment of limbal stem cell deficiency

Diseases and damages of the ocular surface are one of the common causes of decreased vision and blindness. Dysfunction or death of limbal epithelial stem cells (LESC) plays an important role in the development of pathological processes in these conditions, which leads to the development of the limbal stem cell deficiency (LSCD). Currently, one of the methods to treat LSCD is a transplantation of cultured ex vivo LESC. The most common carriers for the cultivation of LESC in the world is the amniotic membrane (AM). However, the presence of certain disadvantages in using AM for the cultivation of LESC compels to search new types of carriers made from biological or synthetic materials. In this review, we have analyzed various types of carriers: collagen, fibrin, chitosan with gelatin, silk fibroin, keratin, contact lenses, polylactide-co-glycolide, polycaprolactone, and the possibility of their application as carriers for the LESC cultivation followed by transplantation on the ocular surface is considered.

Protection of Corneal Limbus from Riboflavin Prevents Epithelial Stem Cell Loss after Collagen Cross-Linking.

Purpose To investigate whether the protection of corneal limbus from riboflavin exposure during collagen cross-linking (CXL) prevents limbal epithelial stem cell (LESC) loss. Methods Ten New Zealand white rabbits received an epithelium-off CXL using an accelerated protocol. Seven days before procedure, 5-bromo-2-deoxyuridine (BrdU) was intraperitoneally injected. During procedure, riboflavin was applied to the corneal surface within a 9 mm diameter retention ring in 5 rabbits, thereby preventing the limbus from riboflavin exposure. In other 5 rabbits, riboflavin was instilled every 2 min, allowing the spillover to the limbus. One day after UVA irradiation, corneas were subjected to histological and molecular assays. Results There were no differences in corneal thickness and epithelial healing between the groups. The numbers of BrdU-labelled and p63+ limbal epithelial cells were markedly reduced in the group without a ring, but significantly increased when a ring was used. Robust expression of CK3/12 was observed in the limbal epithelium in the group with a ring. The mRNA levels of ABCG2, FGF2, IL-1β, and IL-6 were significantly increased in the corneas with a ring. Conclusions Protection of limbus from riboflavin during CXL was effective in preserving LESCs. However, inflammation was increased in the cornea treated with riboflavin using a ring.

Computer simulation of neutral drift among limbal epithelial stem cells of mosaic mice

The use of mice that are mosaic for reporter gene expression underlies many lineage-tracing studies in stem cell biology. For example, using mosaic LacZ reporter mice, it was shown that limbal epithelial stem cells (LESCs) around the periphery of the cornea maintain radial sectors of the corneal epithelium and that radial stripe numbers declined with age. Originally, the corneal results were interpreted as progressive, age-related loss or irreversible inactivation of some LESC clones. In this study we used computer simulations to show that these results could also be explained by stochastic replacement of LESCs by neighbouring LESCs, leading to neutral drift of LESC populations. This was shown to reduce the number of coherent clones of LESCs and hence would coarsen the mosaic pattern in the corneal epithelium without reducing the absolute number of LESCs. Simulations also showed that corrected stripe numbers declined more slowly when LESCs were grouped non-randomly and that mosaicism was rarely lost unless simulated LESC numbers were unrealistically low. Possible reasons why age-related changes differ between mosaic corneal epithelia and other systems, such as adrenal cortices and intestinal crypts, are discussed.

Human stem cell based corneal tissue mimicking structures using laser-assisted 3D bioprinting and functional bioinks

There is a high demand for developing methods to produce more native-like 3D corneal structures. In the present study, we produced 3D cornea-mimicking tissues using human stem cells and laser-assisted bioprinting (LaBP). Human embryonic stem cell derived limbal epithelial stem cells (hESC-LESC) were used as a cell source for printing epithelium-mimicking structures, whereas human adipose tissue derived stem cells (hASCs) were used for constructing layered stroma-mimicking structures.The development and optimization of functional bioinks was a crucial step towards successful bioprinting of 3D corneal structures. Recombinant human laminin and human sourced collagen I served as the bases for the functional bioinks. We used two previously established LaBP setups based on laser induced forward transfer, with different laser wavelengths and appropriate absorption layers. We bioprinted three types of corneal structures: stratified corneal epithelium using hESC-LESCs, lamellar corneal stroma using alternating acellular layers of bioink and layers with hASCs, and finally structures with both a stromal and epithelial part. The printed constructs were evaluated for their microstructure, cell viability and proliferation, and key protein expression (Ki67, p63α, p40, CK3, CK15, collagen type I, VWF). The 3D printed stromal constructs were also implanted into porcine corneal organ cultures.Both cell types maintained good viability after printing. Laser-printed hESC-LESCs showed epithelial cell morphology, expression of Ki67 proliferation marker and co-expression of corneal progenitor markers p63α and p40. Importantly, the printed hESC-LESCs formed a stratified epithelium with apical expression of CK3 and basal expression of the progenitor markers. The structure of the 3D bioprinted stroma demonstrated that the hASCs had organized horizontally as in the native corneal stroma and showed positive labeling for collagen I. After 7 days in porcine organ cultures, the 3D bioprinted stromal structures attached to the host tissue with signs of hASCs migration from the printed structure. This is the first study to demonstrate the feasibility of 3D LaBP for corneal applications using human stem cells and successful fabrication of layered 3D bioprinted tissues mimicking the structure of the native corneal tissue.

Iatrogenic limbal stem cell deficiency following drainage surgery for glaucoma

ObjectiveTo describe conjunctival epithelial overgrowth of the cornea after surgery for glaucoma. MethodsThis is a retrospective case series (setting: Moorfields Eye Hospital). Fourteen eyes of 13 patients with suspected limbal stem cell deficiency (LSCD) and corneal conjunctivalization after glaucoma drainage surgery. Conjunctivalization was defined as corneal epithelium that demonstrated late stain after topical application of fluorescein. Patient demographics, clinical features, potential risk factors, treatment, and final visual acuity were recorded. Main outcome measures were potential risk factors for conjunctivalization, complications, and response to treatment. ResultsEleven eyes had multiple procedures involving the limbus, and in 11 eyes mitomycin C (MMC) or 5 fluorouracil had been used as an adjunct to reduce fibrosis. Affected eyes typically had a segment of late stain with fluorescein based at the site of previous glaucoma surgery, but in one eye there was total loss of the corneal epithelial phenotype. All eyes previously had topical treatment for their glaucoma but only 2 had an ocular surface disease associated with LSCD. Most cases were asymptomatic, but in 3 eyes there was visual loss when the abnormal phenotype crossed the visual axis. In these 3 eyes there was recurrent epithelial breakdown, often at the interface between the 2 epithelial phenotypes. In one individual, these symptoms resolved after limbal epithelial transfer from the unaffected contralateral eye. ConclusionsGlaucoma drainage surgery can damage the adjacent corneal limbal epithelial stem cell population. This can be associated with recurrent epithelial breakdown and reduced vision. If there is visual loss, limbal epithelial transplantation is a potential treatment option.

Мodification of Mechanical Limbal Stem Cell Deficiency Model

Introduction. Ocular surface diseases related with limbal epithelial stem cells dysfunction were united in term “limbal stem cell deficiency” (LSCD). For experimental study of the regenerative processes and evaluation of the success of new LSCD treating methods LSCD model is required. Various LSCD models were proposed in the experiment to study: mechanical, thermal, chemical, medicamental. The main lack of these models were the relative high cost and complexity of execution. The mechanical model allows for the guaranteed removal of tissues containing LESCs, and therefore seems to be the most acceptable. We offered a modification of the mechanical LSCD model in rabbits. Purpose to create a standardized modification of the mechanical limbal stem cell deficiency model in the experiment. Material and methods. The experimental study was performed in 10 mature Chinchilla rabbits (20 eyes) with an average weight 2.5–3.5 kg. With the local anesthesia, after a 40-second application of the filter paper impregnated with 20% ethanol, the corneal epithelium was removed. With microsurgical diamond blade we metered limb portion of 4 mm width, 0.2 mm deep, and it was removed along the 360° rim. Results. On the 30th day we discovered corneal opacity and neovascularization with conjunctival pannus extending to the optical zone of the cornea. Histological examination revealed tissue edema, inflammatory infiltration, and newly formed vessels. In some places, thinning of epithelium to one row of flattened cells was observed. The Bowman membrane was deformed and practically not detected. Histological examination and impression cytology confirmed the presence of goblet cells in the corneal epithelium. Conclusions. Our modification of the mechanical limbal stem cell deficiency model is devoid of the main lacks of previous models, such as the high cost and complexity of execution, provides intraoperative limbal tissue resection depth control and excludes the possibility of the eye perforation. It is effective, universal, standardized and available for widespread use.

Outcomes of Penetrating Keratoplasty Following Autologous Cultivated Limbal Epithelial Stem Cell Transplantation.

The purpose of this study is to investigate the outcomes of penetrating keratoplasty (PKP) following autologous cultivated limbal epithelial stem cell transplantation (CLET). A prospective, single center, interventional cohort study investigating patients with unilateral total limbal stem cell deficiency (LSCD) treated with CLET who underwent PKP. Patients with confirmed corneal re-epithelialization > 6 months post-CLET, and with best-corrected visual acuity (BCVA) <0.3 logMAR were offered PKP. CLET survival assessed by slit lamp, corneal impression cytology (CIC), and in vivo confocal microscopy. Confirmation of corneal re-epithelialization by histological and immunocytochemical (ICC) examination of trephined corneal buttons. Mean change in best-corrected visual acuity (logMAR) following PKP and PKP survival at 12 months were calculated. Twenty patients underwent PKP. Mean time of PKP was 19 months (range 11-41 months, SD 7.26) post-CLET. Median follow-up time post-PKP was 15 months (range 1-32, SD 10.2). CIC and ICC of all corneas confirmed corneal re-epithelialization before PKP. Mean pre-PKP BCVA was 1.46 (range 0.3-2.7, SD 0.94) improving to a mean post-PKP BCVA of 0.74 (range 0-2.7, SD 0.87); mean improvement in BCVA post-PKP of 36 letters (95% CI 15.0-57.1, p = .002). Kaplan-Meier mean graft survival was 90.9% (95% CI 50.8-98.7) at 12 months. We recommend a two-stage approach with CLET followed by PKP >12 months later. Patients experienced a significant improvement in BCVA following PKP. PKP did not have a detrimental effect on CLET survival. PKP survival post-CLET is better than that reported for high risk PKP. Stem Cells 2018;36:925-931.

Generation and characterisation of decellularised human corneal limbus.

Limbal epithelial stem cells (LESC) reside in a niche in the corneo-scleral transition zone. Deficiency leads to pain, corneal opacity, and eventually blindness. LESC transplantation of ex-vivo expanded human LESC on a carrier such as human amniotic membrane is a current treatment option. We evaluated decellularised human limbus (DHL) as a potential carrier matrix for the transplantation of LESC. Human corneas were obtained from the local eye bank. The limbal tissue was decellularised by sodium desoxychelate and DNase solution and sterilised by γ-irradiation. Native limbus- and DHL-surface structures were assessed by scanning electron microscopy and collagen ultrastructure using transmission electron microscopy. Presence and preservation of limbal basement membrane proteins in native limbus and DHL were analysed immunohistochemically. Absence of DNA after decellularisation was assessed by Feulgen staining and DNA quantification. Presence of immune cells was explored by CD45 staining, and potential cytotoxicity was tested using a cell viability assay. In the DHL, the DNA content was reduced from 1.5 ± 0.3μg/mg to 0.15 ± 0.01μg/mg; the three-dimensional structure and the arrangement of the collagen fibrils were preserved. Main basement membrane proteins such as collagen IV, laminin, and fibronectin were still present after decellularisation and γ-irradiation. CD45-expressing cells were evident neither in the native limbus nor in the DHL. DHL did not convey cytotoxicity. The extracellular matrix (ECM) of the limbus provides a tissue specific morphology and three-dimensionality consisting of particular ECM proteins. It therefore represents a substantial component of the stem cell niche. The DHL provides a specific limbal niche surrounding, and might serve as an easily producible carrier matrix for LESC transplantation.

Limbal epithelial stem cells in corneal surface reconstruction

Cornea serves as the partial front barrier and major light reflection organ of the eye. The integrity of corneal surface is essential for ocular function. Injuries or congenital diseases could significantly destruct the homeostasis of the ocular surface, especially the microenvironment of limbal epithelial stem cells (LESCs), and will eventually cause dysfunction of corneal regeneration and diminish of LESCs. The loss of LESCs by different reasons are named limbal stem cell deficiency (LSCD), which is one of the leading cause of vision loss worldwide. To restore the corneal surface, LESC transplantation in the form of tissue or cell cultures is currently a viable and promising method to treat LSCD. In this review, we aim to introduce the characters and niche of LESCs, and discuss different aspects of its application in cornea surface reconstruction.

The Role of Limbal Epithelial Stem Cells in Regulating Corneal (Lymph)angiogenic Privilege and the Micromilieu of the Limbal Niche following UV Exposure.

The cornea is a clear structure, void of blood, and lymphatic vessels, functioning as our window to the world. Limbal epithelial stem cells, occupying the area between avascular cornea and vascularized conjunctiva, have been implicated in tissue border maintenance, preventing conjunctivalisation and propagation of blood and lymphatic vessels into the cornea. Defects in limbal epithelial stem cells are linked to corneal neovascularisation, including lymphangiogenesis, chronic inflammation, conjunctivalisation, epithelial abnormalities including the presence of goblet cells, breaks in Bowman's membrane, persistent epithelial defects and ulceration, ocular surface squamous neoplasia, lipid keratopathy, pain, discomfort, and compromised vision. It has been postulated that pterygium is an example of focal limbal deficiency. Previous reports showing changes occurring in limbal epithelium during pterygium pathogenesis suggest that there is a link to stem cell damage. In this light, pterygium can serve as a model disease of UV-induced stem cell damage also characterised by corneal blood and lymphangiogenesis. This review focuses on the role of corneal and limbal epithelial cells and the stem cell niche in maintaining corneal avascularity and corneal immune privilege and how this may be deregulated following UV exposure. We present an overview of the PUBMED literature in the field as well as recent work from our laboratories.

In Vivo Confocal Microscopic Observations of Vortex Keratopathy in Patients with Amiodarone-Induced Keratopathy and Fabry Disease.

Purpose To compare the morphology of two types of vortex keratopathy: amiodarone-induced keratopathy and the Fabry disease-associated keratopathy. Patients and Methods Eight patients who were receiving oral amiodarone therapy and 3 patients with Fabry disease, a mother and her 2 daughters, were examined by slit-lamp biomicroscopy and in vivo confocal microscopy (IVCM) regularly. Results Amiodarone-induced keratopathy developed in 7 of the 8 patients, and it was detected as early as 7 days by IVCM and 14 days by slit-lamp biomicroscopy. The in vivo confocal microscopic images showed a clustering of corneal epithelial cells with a highly reflective cytoplasm in both types of keratopathy. In the amiodarone-induced keratopathy, the highly reflective epithelial cells were first found at the center of the cornea and then spread to the periphery with increasing time on amiodarone. In Fabry disease, the highly reflective epithelial cells were consistently observed extending from the limbus to the central cornea. Conclusion These findings suggest that the corneal epithelial cells most likely endocytose amiodarone from the tear film in the amiodarone-induced keratopathy. In Fabry disease, globotriaosylceramide deposits are taken up by the lysosomes of the limbal epithelial stem cells, and they differentiate and migrate to the center of the cornea to form the whorl pattern.

Comparison of culture media indicates a role for autologous serum in enhancing phenotypic preservation of rabbit limbal stem cells in explant culture.

In this study, we aimed to compare the effects of six different cell culture media and autologous serum (AS) on the phenotypic characteristics of rabbit limbal epithelial stem cells (LESC) cultivated on porous polyethylene terephthalate (PET) membranes. Limbal explants from rabbit corneas were grown on PET membrane inserts in five different media: DMEM-F12 with fetal bovine serum (FBS) (DMEM-F12-FBS), with pluripotin (DMEM-F12-pluripotin) and with autologous serum (DMEM-F12-AS), Epilife, Keratinocyte Serum Free Medium (KSFM) and Defined-Keratinocyte Serum Free Medium. The effects of different media were evaluated by total cell yield from explants, measuring the expression of proteins by immunofluorescence and gene expression by Real Time PCR. In all five media tested, most of the limbal epithelial cells (LEC) which proliferated from explants were positive for cytokeratin (CK) 14 (85-90%), indicating that all five media support the growth of LESC from explants. The expression of differentiation markers; CK 3 and 12 was highest in DMEM-F12-FBS (56%), was lower in Epilife and KSFM (26 and 19%, respectively), with the lowest values (13%) obtained in DMEM-F12-AS. Gene expression of limbal cultures on PET membrane inserts was compared to fresh limbal tissue. In DMEM-F12-FBS, DMEM-F12-pluripotin, and DMEM-F12-AS, expression of potential LESC markers CXCR4 and polycomb complex protein BMI-1 were similar to limbal tissue. DMEM-F12 with 10% AS maintained a higher percentage of potential stem cell marker genes and lower expression of genes involved in differentiation compared to Epilife or KSFM. Our study shows that rabbit LEC can be cultivated on PET inserts using DMEM-F12 with autologous serum without a requirement for amniotic membrane or feeder cells.

Xeno- and feeder-free differentiation of human pluripotent stem cells to two distinct ocular epithelial cell types using simple modifications of one method

BackgroundHuman pluripotent stem cells (hPSCs) provide a promising cell source for ocular cell replacement therapy, but often lack standardized and xenogeneic-free culture and differentiation protocols. We aimed to develop a xeno- and feeder cell-free culture system for undifferentiated hPSCs along with efficient methods to derive ocular therapy target cells: retinal pigment epithelial (RPE) cells and corneal limbal epithelial stem cells (LESCs).MethodsMultiple genetically distinct hPSC lines were adapted to a defined, xeno-, and feeder-free culture system of Essential 8™ medium and laminin-521 matrix. Thereafter, two-stage differentiation methods toward ocular epithelial cells were established utilizing xeno-free media and a combination of extracellular matrix proteins. Both differentiation methods shared the same basal elements, using only minor inductive modifications during early differentiation towards desired cell lineages. The resulting RPE cells and LESCs were characterized after several independent differentiation experiments and recovery after xeno-free cryopreservation.ResultsThe defined, xeno-, and feeder-free culture system provided a robust means to generate high-quality hPSCs with chromosomal stability limited to early passages. Inductive cues introduced during the first week of differentiation had a substantial effect on lineage specification, cell survival, and even mature RPE properties. Derivative RPE formed functional epithelial monolayers with mature tight junctions and expression of RPE genes and proteins, as well as phagocytosis and key growth factor secretion capacity after 9 weeks of maturation on inserts. Efficient LESC differentiation led to cell populations expressing LESC markers such as p40/p63α by day 24. Finally, we established xeno-free cryobanking protocols for pluripotent hPSCs, hPSC-RPE cells, and hPSC-LESCs, and demonstrated successful recovery after thawing.ConclusionsWe propose methods for efficient and scalable, directed differentiation of high-quality RPE cells and LESCs. The two clinically relevant cell types are generated with simple inductive modification of the same basal method, followed by adherent culture, passaging, and cryobanking.

Maintaining RNA Integrity for Transcriptomic Profiling of Ex Vivo Cultured Limbal Epithelial Stem Cells after Fluorescence-Activated Cell Sorting (FACS)

BackgroundTranscriptomic profiling of ex vivo cultured human limbal epithelial stem cells (hLESCs) will foster better understanding of corneal physiology and novel treatment paradigms to limbal stem cell deficiency (LSCD). However, currently such profiling studies are hampered due to difficulties with producing sufficient amounts of intact mRNA for deep RNA sequencing (RNA-seq) from subpopulations sorted on the basis of co-expression of membrane and intracellular antigens by fluorescence-activated cell sorting (FACS).MethodsTo address this problem, we systematically analyzed the critical steps, and found that ethanol fixation together with optimized downstream procedures provided a pipeline that yielded high quality total RNA in amounts to readily support the RNA-seq procedure, while still preserving good discrimination between the individual hLESC immunophenotypes.ResultsThe average RNA integrity number (RIN) was 7.7 ± 0.4, and the average yield was 4.6 ± 1.7 pg of RNA per cell. The sequencing analysis of the isolated RNA produced high quality data with more than 70% of read pairs mapping uniformly to the reference genome and 80% of bases with a Phred score of at least 30.ConclusionIn this study, we developed a reliable FACS-based procedure using ethanol as a fixative that would support accurate isolation of limbal epithelial progenitor subpopulations along with RNA yield and quality sufficient to enable deep transcriptomic profiling.

Transplant of Limbal Epithelial Stem Cells on Bioresorbable Scaffold

Transplant of Limbal Epithelial Stem Cells on Bioresorbable Scaffold

Easy xeno-free and feeder-free method for isolating and growing limbal stromal and epithelial stem cells of the human cornea.

Epithelial and stromal stem cells are required to maintain corneal transparency. The aim of the study was to develop a new method to isolate and grow both corneal stromal (SSC) and epithelial limbal (LSC) stem cells from small human limbal biopsies under culture conditions in accordance with safety requirements mandatory for clinical use in humans. Superficial limbal explants were retrieved from human donor corneo-scleral rims. Human limbal cells were dissociated by digestion with collagenase A, either after epithelial scraping or with no scraping. Isolated cells were cultured with Essential 8 medium (E8), E8 supplemented with EGF (E8+) or Green’s medium with 3T3 feeder-layers. Cells were characterized by immunostaining, RT-qPCR, colony forming efficiency, sphere formation, population doubling, second harmonic generation microscopy and differentiation potentials. LSC were obtained from unscraped explants in E8, E8+ and Green’s media and were characterized by colony formation and expression of PAX6, ΔNP63α, Bmi1, ABCG2, SOX9, CK14, CK15 and vimentin, with a few cells positive for CK3. LSC underwent 28 population doublings still forming colonies. SSC were obtained from both scraped and unscraped explants in E8 and E8+ media and were characterized by sphere formation, expression of PAX6, SOX2, BMI1, NESTIN, ABCG2, KERATOCAN, VIMENTIN, SOX9, SOX10 and HNK1, production of collagen fibrils and differentiation into keratocytes, fibroblasts, myofibroblasts, neurons, adipocytes, chondrocytes and osteocytes. SSC underwent 48 population doublings still forming spheres, Thus, this new method allows both SSC and LSC to be isolated from small superficial limbal biopsies and to be primary cultured in feeder-free and xeno-free conditions, which will be useful for clinical purposes.

Poly(ethylene glycol)-modified silk fibroin membrane as a carrier for limbal epithelial stem cell transplantation in a rabbit LSCD model

BackgroundLimbal epithelial stem cells (LESCs) play important roles in corneal epithelial homeostasis and regeneration, and damage to the limbus will lead to limbal stem cell deficiency (LSCD), with conjunctivalization and even visual impairment. Cultured LESCs have been used for ocular surface reconstruction, and silk fibroin (SF) membranes have shown potential as a substrate for LESC cultivation. Both culture methods and the carriers of LESCs affect outcomes following LESC transplantation.MethodsRabbit LESCs were cultured from tissue explant, single cell-suspension, and cell cluster culture methods. Ratios of p63α and/or ABCB5-positive LESCs, differentiated corneal epithelial cells (CK12 staining), and corneal tight junction formation (Claudin-1 staining) were examined to choose the most applicable LESC cultures. SF membranes were prepared and modified by 400-Da poly(ethylene glycol) (PEG). The characteristics of stem cells and normal corneal differentiation of LESCs cultured on PEG-modified SF membranes were further examined by immunofluorescence staining and flow cytometric analysis. LESCs cultured on PEG-modified SF membranes (LESC/SF grafts) and PEG-modified SF membranes (SF grafts) were transplanted onto rabbit corneas with total LSCD. New blood vessels, corneal epithelial defects, and cornea clarity were examined after transplantation. Furthermore, corneal epithelial thickness, stromal thickness, and the percentage area of CK12-positive corneal epithelium were quantified 4 months after transplantation.ResultsTissue explant and single cell-suspension cultures harvested more p63α and/or ABCB5-positive LESCs, generated more CK12-positive corneal epithelial cells, and formed more corneal tight junctions than cell cluster cultures. Prepared PEG-modified SF membranes were transparent, flexible, and sturdy enough for surgical manipulation. LESCs cultured on PEG-modified SF membranes maintained characteristics of stem cells and normal corneal differentiation. LESC/SF grafts inhibited new blood vessels and rescued corneal epithelial defects in the rabbit total LSCD model. In addition, LESC/SF grafts repopulated the limbus and increased corneal epithelial thickness, stromal thickness, and the area percentage of CK12-positive corneal epithelium.ConclusionsLESCs from tissue explant and single cell-suspension cultures were more applicable corneal epithelial cells for ocular surface reconstruction. LESC/SF grafts repaired corneal epithelial defects and reversed LSCD, and PEG-modified SF membranes were suitable to be a carrier for LESC transplantation.

Native and synthetic scaffolds for limbal epithelial stem cell transplantation.

The outcome of patients with corneal blindness that receive stem cell grafts to restore eye health and correct vision varies considerably and may be due to the different biological and synthetic scaffolds used to deliver these cells to the ocular surface. This review will highlight the positive attributes and limitations of the myriad of carriers developed for clinical use as well as those that are being trialled in pre-clinical models. The overall focus is on developing a standardized therapy for patients, however due to the multiple causes of corneal blindness, a personal regenerative medicine approach may be the best option.

Conjunctival-limbal autograft in total unilateral limbal stem cell deficiency

Background : Corneal epithelium is renewed by stem cells (SC) that reside at the corneal limbus. Reduced number of SC or their abnormal function lead to the ocular surface disease called limbal stem cell deficiency (LSCD), characterized by corneal conjunctivalization, vascularization, persistent epithelial defects, chronic inflammation, and loss of vision. In a case of total unilateral LSCD, autologous transplantation of limbal epithelial stem cells (LESC) from the healthy eye is needed. We describe the surgical technique of choice for autologous limbal transplantation, called conjunctival limbal autograft (CLAU) that we combined with amniotic membrane (AM) use. We present the results of CLAU in three patients with total unilateral LSCD due to chemical injury. Methods : Autologous limbal transplantation CLAU begins with the removal of fibrovascular pannus from the diseased corneal surface and the harvesting of two conjunctival-limbal grafts from the healthy eye. The grafts are then transplanted on to the limbal area of the recipient eye. AM is used as a patch to cover the denuded cornea and limbal grafts, as well as a barrier preventing the conjunctival epithelium from encroaching on to the temporal and nasal side of the corneal surface. In the donor eye, AM is used to cover the donor sites. CLAU with the use of AM was performed in 3 patients with unilateral LSCD due to chemical eye injury. In one patient limbal transplantation was combined with symblepharon lysis for entropium repair. In all cases AM was removed 3–6 days postoperatively to assess the growth of new epithelium from the limbal grafts. In all patients the ocular surface was covered with another AM until the cornea was completely epithelized and the new epithelium stable. In one patient the corneal regrafting and cataract removal was performed subsequently. Results : CLAU was successful in 2 patients and partially successful in 1 patient during the follow up. In all cases the growth of new epithelium from the limbal grafts was noted on day 3–6 after CLAU. The cornea was completely epithelized within 2 weeks in 2 patients and after 35 days in one patient. In two patients the corneal epithelium remained clear, smooth and stable during the follow up of 3.5 years and 4 months, respectively. In one patient, uneven epithelium probably representing a mosaic of corneal and conjunctival cells was noted in the central corneal region, where a small corneal ulcer developed 5 months after CLAU. In donor eyes no postoperative complications were noted, the donor sites epithelized within few days. Conclusions : Autologous limbal transplantation according to CLAU surgical technique combined with the use of AM is a successful and safe therapy for restoring corneal surface in total unilateral LSCD after chemical injury. It enables further surgical procedures for restoring the vision such as corneal transplantation and cataract surgery.