Limbal Epithelial Crypt Research Articles

In situ distribution of markers for stemness, quiescence, proliferation and differentiation in the human cornea and limbus

Aims/Purpose: Identify the distribution of the most commonly used markers for stemness, quiescence, proliferation, and differentiation in limbal compartments such as human limbal epithelial crypts, posterior and anterior limbus, as well as the central and posterior cornea.Methods: Corneal‐limbal biopsies from cadaver human donors (n = 3) embedded in paraffin were proceeded for immunohistochemistry fluorescence and examined for stemness/progenitor markers (p63α, SOX9, ABCG2, N‐cadherin), proliferation markers (ki‐67), quiescence marker (CEBPD), differentiation markers (CX43, KRT3, KRT12, KRT13), and epithelial marker (E‐cadherin). Images were obtained by Zeiss Axio Observer. Fluorescent images were obtained using the Zeiss Axio Imager M1 fluorescence microscope (ZEISS, Oberkochen, Germany).Results: The stemness/progenitor markers p63α and SOX9 were found in basal and suprabasal cells of limbal epithelial crypts and limbal epithelium, whereas the ABCG2 and N‐cadherin were exclusive for limbal basal epithelium in all limbal structures. Proliferation marker Ki‐67 was most abundant in suprabasal layers of the limbus and rarely found in the posterior cornea, while absent in anterior cornea. Quiescence marker CEBPD was positive in the basal cells of both, limbus and cornea. Differentiation markers KRT3 and KRT12 were positive in the whole cornea and intermediate and superficial layers in anterior and posterior limbus, while rarely present in limbal epithelial crypts. However, CX43 was staining almost all suprabasal layers in the limbus as well as basal and suprabasal layers in the cornea. KRT13 was exclusive for superficial layer of the anterior and posterior limbus layers staining conjunctival cells.Conclusions: Here we identify the localization of the most common markers used to determine the stemness, quiescence, proliferation, and differentiation status to unravel the in situ distribution of these markers in the limbal epithelial crypts, posterior and anterior limbus, as well as the central and posterior cornea. The study accumulated new knowledge about the distribution of these markers and the corneal‐limbal organization.

A Novel Technique of Amniotic Membrane Preparation Mimicking Limbal Epithelial Crypts Enhances the Number of Progenitor Cells upon Expansion.

We aimed to investigate whether a novel technique of human amniotic membrane (HAM) preparation that mimics the crypts in the limbus enhances the number of progenitor cells cultured ex vivo. The HAMs were sutured on polyester membrane (1) standardly, to obtain a flat HAM surface, or (2) loosely, achieving the radial folding to mimic crypts in the limbus. Immunohistochemistry was used to demonstrate a higher number of cells positive for progenitor markers p63α (37.56 ± 3.34% vs. 62.53 ± 3.32%, p = 0.01) and SOX9 (35.53 ± 0.96% vs. 43.23 ± 2.32%, p = 0.04), proliferation marker Ki-67 (8.43 ± 0.38 % vs. 22.38 ± 1.95 %, p = 0.002) in the crypt-like HAMs vs. flat HAMs, while no difference was found for the quiescence marker CEBPD (22.99 ± 2.96% vs. 30.49 ± 3.33 %, p = 0.17). Most of the cells stained negative for the corneal epithelial differentiation marker KRT3/12, and some were positive for N-cadherin in the crypt-like structures, but there was no difference in staining for E-cadherin and CX43 in crypt-like HAMs vs. flat HAMs. This novel HAM preparation method enhanced the number of progenitor cells expanded in the crypt-like HAM compared to cultures on the conventional flat HAM.

A novel technique of amniotic membrane suturing for ex vivo cultivated limbal epithelial stem cell expansion increases the number of progenitor cells

AbstractPurpose: Human limbal epithelial stem cells (HLESCs) reside in limbal epithelial crypts and continuously compensate for the loss of cells in the corneal epithelium. The lack or malfunction of HLESCs leads to a severe ocular surface disease called limbal epithelial stem cell deficiency (LSCD). Cultivated limbal epithelial stem cell transplantation (CLET) of expanded autologous limbal tissue on a human amniotic membrane (HAM) carrier is one of the most common methods for monocular LSCD. We hereby present a novel suturing technique of HAM prior to limbal biopsy expansion and cultivation that mimics natural limbal crypts.Methods: HLESCs derived from limbal biopsies were cultured ex vivo in complex media (COM) on flat or crypt‐like sutured HAMs for 3 weeks. The samples were then processed for immunohistochemistry (IHC) and analysed for progenitor markers: p63α and SOX9; markers for quiescence: CEBPD; proliferation: Ki‐67; differentiation: CX43 and CK3/12; transmembrane markers: E‐cadherin and N‐cadherin. The positivity of markers was counted and compared in cells cultivated on flat vs. crypt‐like sutured HAMs.Results: HLESCs cultured on folded, crypt‐like sutured HAMs had significantly more positivity for p63α, SOX9 (progenitor markers) and Ki‐67 (proliferation marker) compared to those cultured on flat HAMs. Fewer cells were positive for CK3/12 (differentiation marker) in cultures on folded compared to flat HAMs. The number of cells positive for CEBPD (quiescence marker), CX43 and E‐cadherin (differentiation markers) were equal in both groups (flat vs. folded HAMs).Conclusions: A novel technique of HAM suturing that forms crypt‐like undulations mimicking limbal epithelial crypts increases the number of early progenitor cells and could hence increase the quality of the ex vivo expanded limbal epithelial tissue and success for transplantation.

Bioinspired Precision Engineering of Three-Dimensional Epithelial Stem Cell Microniches.

Maintenance of the epithelium relies on stem cells residing within specialized microenvironments, known as epithelial crypts. Two-photon polymerization (2PP) is a valuable tool for fabricating 3D micro/nanostructures for stem cell niche engineering applications. Herein, biomimetic gelatin methacrylate-based constructs, replicating the precise geometry of the limbal epithelial crypt structures (limbal stem cell "microniches") as an exemplar epithelial niche, are fabricated using 2PP. Human limbal epithelial stem cells (hLESCs) are seeded within the microniches in xeno-free conditions to investigate their ability to repopulate the crypts and the expression of various differentiation markers. Cell proliferation and a zonation in cell phenotype along the z-axis are observed without the use of exogenous signaling molecules. Significant differences in cell phenotype between cells located at the base of the microniche and those situated towards the rim are observed, demonstrating that stem cell fate is strongly influenced by its location within a niche and the geometrical details of where it resides. This study provides insight into the influence of the niche's spatial geometry on hLESCs and demonstrates a flexible approach for the fabrication of biomimetic crypt-like structures in epithelial tissues. This has significant implications for regenerative medicine applications and can ultimately lead to implantable synthetic "niche-based" treatments.

Columnar keratopathy: An early manifestation of limbal stem cell deficiency

# Background/Purpose Stem cells of the corneal epithelium reside in the limbal palisades and associated limbal epithelial crypts. The limbus constitutes a barrier between the conjunctival and corneal phenotype of cells. When the limbal barrier is breached, stem cell deficiency (LSCD) results with conjunctivalisation of the cornea. The purpose of this study was to document an early manifestation of LSCD which is consistent and distinct across several causes of LSCD. # Methods Seventeen eyes of 11 consecutive patients with diverse known causes of LSCD were included. Patients were examined clinically with the slit lamp and fluorescein staining, and by in vivo confocal microscopy. All corneas were photographed with a slit lamp camera. Seven patients underwent in vivo confocal microscopy examination. # Results There were 6 males and 5 females. Six cases had bilateral involvement and the other 5 were unilateral. All patients showed predominantly superior loss of limbal anatomy with effacement of the palisades of Vogt, superficial vascularization encroaching on the peripheral cornea and alternating columns, 6–20 in number, of late fluorescein staining conjunctivalised and normal epithelium extending for a variable distance towards the corneal centre. These were related to prolonged contact lens wear, atopic keratoconjunctivitis, superficial ocular surface burns, ocular cicatricial pemphigoid, rosacea keratoconjunctivitis, old trachoma and neurotrophic keratopathy. In vivo confocal microscopy confirmed features of LSCD. # Conclusion Alternating columns of conjunctivalized and normal epithelia encroaching on the cornea from the superior limbus is a distinct manifestation of early LSCD across a diverse range of causes. The reason for this manifestation is unclear but could be related to the random dysfunction or loss of the palisade architecture.

Nerve terminals at the human corneoscleral limbus

AimsTo demonstrate and characterise distinct subepithelial compact nerve endings (CNE) at the human corneoscleral limbus.MethodsTen fresh human donor corneoscleral discs (mean age, 67 years) and 26 organ-cultured corneoscleral rims (mean...

Diagnosis of corneal limbal stem cell deficiency.

A state of limbal stem cell deficiency (LSCD) can be secondary to a number of etiologies, resulting in either a reduction in the total number of limbal stem cells or an abnormality in stem cell function. Initially, the epithelium becomes irregular and hazy; however, this condition may progress to persistent corneal epithelial defects, stromal scarring, ulceration, and even perforation. Since LSCD secondary to a variety of etiologies may be reversible, and various factors are prognostic of disease progression, timely diagnosis is important. This review will describe current knowledge of diagnostic techniques for LSCD and understanding of epithelial stem cell function. Conjunctivalization, regarded as the most reliable clinical finding diagnostically, can be identified as late staining of epithelium with fluorescein. While identifying loss of the palisades of Vogt by slit-lamp examination, can provide a high suspicion of LSCD, but this is not diagnostic. Impression cytology is a simple, noninvasive technique that aids in the diagnosis of LSCD, but a negative result also cannot rule out the diagnosis. Recent findings have also shown that imaging techniques including in-vivo confocal microscope and optical coherent tomography can also aid in diagnosing LSCD; however, several challenges remain before these techniques become standard diagnostic methods in clinical practice. Meanwhile, determination of the absence of limbal epithelial crypts and focal stromal projections using image reconstruction techniques may assist in the diagnosis of LSCD. Furthermore, histologic markers may help not only to improve sensitivity and specificity of conventional techniques in diagnosis of LSCD, but also to identify human limbal stem cells and determine their number and function in LSCD. Efforts to develop and improve techniques for diagnosing LSCD are ongoing. Increased knowledge of limbal stem cells and components of their niches may not only help in understanding the pathogenesis of LSCD but may improve its diagnosis, thereby ameliorating the prognosis of patients with this devastating disease.

Optimised laser microdissection of the human ocular surface epithelial regions for microarray studies

BackgroundThe most important challenge of performing insitu transcriptional profiling of the human ocular surface epithelial regions is obtaining samples in sufficient amounts, without contamination from adjacent tissue, as the region of interest is microscopic and closely apposed to other tissues regions. We have effectively collected ocular surface (OS) epithelial tissue samples from the Limbal Epithelial Crypt (LEC), limbus, cornea and conjunctiva of post-mortem cadaver eyes with laser microdissection (LMD) technique for gene expression studies with spotted oligonucleotide microarrays and Gene 1.0 ST arrays.MethodsHuman donor eyes (4 pairs for spotted oligonucleotide microarrays, 3 pairs for Gene 1.0 ST arrays) consented for research were included in this study with due ethical approval of the Nottingham Research Ethics Committee. Eye retrieval was performed within 36 hours of post-mortem period. The dissected corneoscleral buttons were immersed in OCT media and frozen in liquid nitrogen and stored at −80°C till further use. Microscopic tissue sections of interest were taken on PALM slides and stained with Toluidine Blue for laser microdissection with PALM microbeam systems. Optimisation of the laser microdissection technique was crucial for efficient and cost effective sample collection.ResultsThe starting concentration of RNA as stipulated by the protocol of microarray platforms was taken as the cut-off concentration of RNA samples in our studies. The area of LMD tissue processed for spotted oligonucleotide microarray study ranged from 86,253 μm2 in LEC to 392,887 μm2 in LEC stroma. The RNA concentration of the LMD samples ranged from 22 to 92 pg/μl. The recommended starting concentration of the RNA samples used for Gene 1.0 ST arrays was 6 ng/5 μl. To achieve the desired RNA concentration the area of ocular surface epithelial tissue sample processed for the Gene 1.0 ST array experiments was approximately 100,0000 μm2 to 130,0000 μm2. RNA concentration of these samples ranged from 10.88 ng/12 μl to 25.8 ng/12 μl, with the RNA integrity numbers (RIN) for these samples from 3.3 to 7.9. RNA samples with RIN values below 2, that had failed to amplify satisfactorily were discarded.ConclusionsThe optimised protocol for sample collection and laser microdissection improved the RNA yield of the insitu ocular surface epithelial regions for effective microarray studies on spotted oligonucleotide and affymetrix platforms.

Interactive 3D computer model of the human corneolimbal region: crypts, projections and stem cells

This study aims to clarify the existence of and to map the localization of different proposed stem cell niches in the corneal limbal region. One human eye was cut into 2200 consecutive sections. Every other section was stained with haematoxylin and eosin, digitized at low and high magnification, aligned, 3D reconstructed and visualized using interactive 3D visualization software. The visualization software has interactive tools that make free rotations in all directions possible and makes it possible to create virtual sections independent of the original cutting plan. In all, one low-magnification and 24 high-magnification interactive 3D models were created. Immunohistochemistry against stem cell markers p63 and ΔNp63α was performed as a supplement to the 3D models. Using the interactive 3D models, we identified three types of stem cell niches in the limbal region: limbal epithelial crypts (LECs), limbal crypts (LCs) and focal stromal projections (FSPs). In all, eight LECs, 25 LCs and 105 FSPs were identified in the limbal region. The LECs, LCs and FSPs were predominantly located in the superior limbal region with seven LECs, 19 LCs and 93 FSPs in the superior limbal region and one LEC, six LCs and 12 FSPs in the inferior limbal region. Only few LECs, LCs and FSPs were localized nasally and temporally. Interactive 3D models are a powerful tool that may help to shed more light on the existence and spatial localization of the different stem cell niches (LECs, LCs and FSPs) in the corneal limbal region.

The impact of age on the physical and cellular properties of the human limbal stem cell niche.

The limbal niche in the corneoscleral junction of the eye, habitat of the limbal epithelial stem cells (LESC), facilitates corneal epithelial regeneration by providing physical support and chemical signalling. Anatomical structures within the limbus, namely, limbal epithelial crypts and focal stromal projections, are believed to function as a putative niche for LESCs. In this study, the impact of age on the topography of this niche was investigated. Also, the relationship between niche topography and limbal epithelial cell phenotype was assessed. Ex vivo imaging of the limbus in cadaveric tissue of donors aged from infancy to 90 years was carried out using electron and confocal microscopy. The data suggested that the area occupied by the crypts was sharply reduced after the age of 60 years. The niche microstructures also became smoother with donor age. The phenotypic assessment of cultured limbal epithelial cells harvested from donors of different ages showed that the levels of putative stem cell markers as well as telomerase activity and telomere length remained unchanged, regardless of niche topography. However, the colony forming efficiency of the cultures was significantly reduced with age (p < 0.05). This is the first comprehensive study of the effect of age on the structural and phenotypic characteristics of the human limbal niche. The results have a significant biological value as they suggest a correlation of limbal architecture with decline of re-epithelialisation rate in older patients. Overall, the data also suggest that LESCs harvested from younger donors may be more suitable for cultured LESC therapy production.

Validation of Endogenous Control Genes for Gene Expression Studies on Human Ocular Surface Epithelium

PurposeTo evaluate a panel of ten known endogenous control genes (ECG) with quantitative reverse transcription PCR (qPCR), for identification of stably expressed endogenous control genes in the ocular surface (OS) epithelial regions including cornea, limbus, limbal epithelial crypt and conjunctiva to normalise the quantitative reverse transcription PCR data of genes of interest expressed in above-mentioned regions.MethodThe lasermicrodissected (LMD) OS epithelial regions of cryosectioned corneoscleral buttons from the cadaver eyes were processed for RNA extraction and cDNA synthesis to detect genes of interest with qPCR. Gene expression of 10 known ECG—glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta actin (ACTB), peptidylprolyl isomerase (PPIA), TATA-box binding protein (TBP1), hypoxanthine guanine phosphoribosyl transferase (HPRT1), beta glucuronidase (GUSB), Eucaryotic 18S ribosomal RNA (18S), phosphoglycerate kinase (PGK1), beta-2-microglobulin (B2M), ribosomal protein, large, P0 (RPLP0)—was measured in the OS epithelial regions by qPCR method and the data collected was further analysed using geNorm software.ResultsThe expression stability of ECGs in the OS epithelial regions in increasing order as determined with geNorm software is as follows: ACTB<18S<TBP<B2M<PGK1<HPRT1<GUSB<GAPDH<PPIA-RPLP0. In this study, geNorm analysis has shown the following ECGs pairs to be most stably expressed in individual OS epithelial regions: HPRT1-TBP in cornea, GUSB-PPIA in limbus, B2M-PPIA and RPLP0-TBP in LEC and conjunctiva respectively. However, across the entire ocular surface including all the regions mentioned above, PPIA-RPLP0 pair was shown to be most stable.ConclusionThis study has identified stably expressed ECGs on the OS epithelial regions for effective qPCR results in genes of interest. The results from this study are broadly applicable to quantitative reverse transcription PCR studies on human OS epithelium and provide evidence for the use of PPIA-RPLP0 ECGs pair in quantitative reverse transcription PCR across the OS epithelium.

The Porcine Limbal Epithelial Stem Cell Niche as a New Model for the Study of Transplanted Tissue-Engineered Human Limbal Epithelial Cells

Transparency of the human cornea is dependent upon the integrity of its epithelium and hence a population of limbal epithelial stem cells (LESCs). We have previously shown that LESCs reside in limbal epithelial crypts at the periphery of the human cornea. In this study the anatomy and functionality of the porcine limbus was evaluated for the first time as a novel model of the human limbus. Scanning electron microscopy, confocal microscopy, and histology revealed common structures in the porcine and human limbus in terms of the location and topography of palisades of Vogt and limbal epithelial crypts. Epithelial cells harvested from crypt regions achieved higher colony forming efficiency than cultures established from the noncrypt regions and central cornea. Also, expression of the putative SC markers p63α and integrin β1 brightness was higher in the basal layer of the crypt regions, as shown by immunocytochemistry. De-epithelialized porcine corneas were used as an in vitro organ culture model to study the fate of transplanted human epithelium cultured from the limbus. Multilayered epithelium was observed after ∼1 week. Subsequently, wounds were inflicted on the central corneal epithelium. The wounded tissue healed within 5-7 days, and multilayering of the central corneal epithelium was re-established. The transplanted epithelia were repeatedly wounded at least four times and the wounds healed by 1 week. Putative SC marker expression of the transplanted epithelia was confirmed using immunohistochemistry. These results demonstrate that the porcine limbus shares features with the human limbus and as such provides a suitable model for the study of cultured limbal epithelial cell transplantation. These data have significant clinical value as this model can provide information on LESC fate post-transplantation and their ability to respond to injury, which is not possible to study in patients.

Comparative transcriptional profiling of the limbal epithelial crypt demonstrates its putative stem cell niche characteristics

BackgroundThe Limbal epithelial crypt (LEC) is a solid cord of cells, approximately 120 microns long. It arises from the undersurface of interpalisade rete ridges of the limbal palisades of Vogt and extends deeper into the limbal stroma parallel or perpendicular to the palisade. There are up to 6 or 7 such LEC, variably distributed along the limbus in each human eye.Morphological and immunohistochemical studies on the limbal epithelial crypt (LEC) have demonstrated the presence of limbal stem cells in this region. The purpose of this microarray study was to characterise the transcriptional profile of the LEC and compare with other ocular surface epithelial regions to support our hypothesis that LEC preferentially harbours stem cells (SC).ResultsLEC was found to be enriched for SC related Gene Ontology (GO) terms including those identified in quiescent adult SC, however similar to cornea, limbus had significant GO terms related to proliferating SC, transient amplifying cells (TAC) and differentiated cells (DC). LEC and limbus were metabolically dormant with low protein synthesis and downregulated cell cycling. Cornea had upregulated genes for cell cycling and self renewal such as FZD7, BTG1, CCNG, and STAT3 which were identified from other SC populations. Upregulated gene expression for growth factors, cytokines, WNT, Notch, TGF-Beta pathways involved in cell proliferation and differentiation were noted in cornea. LEC had highest number of expressed sequence tags (ESTs), downregulated and unknown genes, compared to other regions. Genes expressed in LEC such as CDH1, SERPINF1, LEF1, FRZB1, KRT19, SOD2, EGR1 are known to be involved in SC maintenance. Genes of interest, in LEC belonging to the category of cell adhesion molecules, WNT and Notch signalling pathway were validated with real-time PCR and immunofluorescence.ConclusionsOur transcriptional profiling study identifies the LEC as a preferential site for limbal SC with some characteristics suggesting that it could function as a 'SC niche' supporting quiescent SC. It also strengthens the evidence for the presence of "transient cells" in the corneal epithelium. These cells are immediate progeny of SC with self-renewal capacity and could be responsible for maintaining epithelial turn over in normal healthy conditions of the ocular surface (OS). The limbus has mixed population of differentiated and undifferentiated cells.

Pathological changes of anatomical structure and markers of limbal stem cell niche due to inflammation

The corneoscleral limbus is the site of corneal epithelial stem cells (SC). The aim of this study is to evaluate the expression of different SC markers in the normal human limbus and to determine how this is affected by inflammation. Corneoscleral specimens from healthy and inflamed donor eyes were examined by immunohistochemistry/immunofluorescence for p63, vimentin, laminin 5, integrin α6, β1, β4, ABCG2, desmoglein 3, connexin 43, N-cadherin, and cytokeratins 12 and 15. The distribution and anatomic structure of the limbal crypts and the percentage of SC marker antigens in healthy donors were analyzed. In inflamed tissues, we evaluated the anatomic structure of the limbal epithelial crypt (LEC) and the positivity for SC markers. In normal limbus, the niche structures were distributed differently. The variability of their number correlated with the percentage of p63 positivity. Integrin β1 staining directly correlated with p63 positivity while the remaining proteins were variably and widely distributed. Double staining for p63 and vimentin did not reveal any co-localization. In inflamed eyes, the basal cells in the crypts were "stretched" and surrounded by inflammatory cells, and only a few SC markers were still present. Diseases involving the limbus may result in marked changes of expression of SC markers within the LEC and also alter the crypt structure.

Pathologic epithelial and anterior corneal nerve morphology in congenital aniridic keratopathy

Abstract Purpose To document corneal morphology in Swedish families with congenital aniridia. Methods Detailed ophthalmic examinations were conducted in a number of affected and unaffected members. Digital slit lamp photography, anterior segment optical coherence tomography (ASOCT) and in‐vivo confocal microscopy (IVCM) examinations were performed bilaterally to document corneal morphology. Results Affected family members presented with different stages of aniridic keratopathy, with a corneal appearance varying from totally transparent to opaque and highly vascularised. Increased corneal thickness in affected members, particularly those with severe keratopathy, was noted by ASOCT. By IVCM, opaque corneas were characterized by active vessels and dense inflammatory cell infiltration. In corneas with milder keratopathy, pathologic epithelial findings included epithelial pleomorphism, focal epithelial opacities, and an absence of limbal epithelial crypts and focal stromal projections at the limbus. Nerves of the anterior cornea exhibited several distinct features, including an unusually close association of subbasal nerves with epithelial cells, an unusually high subbasal nerve density with highly branched nerves, and a prominent whorl region. Additionally, abnormally dense and tortuous anterior stromal nerves, attached to stromal keratocytes, were noted in unaffected members. Conclusion Altered epithelial morphology and a vigorous innervation of the anterior cornea were the most pronounced corneal findings in family members with milder forms of aniridic keratopathy. Further findings confirmed the known increase in corneal thickness and limbal stem cell abnormality in aniridia.

Corneal surface regeneration: epithelial sheets to epithelial crypts

AbstractThe corneal surface is the most important 123 square millimetres of the body’s surface. Its health is paramount for sight. Due to its exposed nature it is vulnerable to environmental insults causing minor to excessive epithelial cell loss. Large epithelial defects, with an intact limbus heal by the formation of 3 to 6 convex sheets that migrate centripetally, meet adjacent sheets and undergo contact inhibition to form geometric shapes. These finally close by the formation of ‘Y’ shaped contact lines (Rule 1). When the limbus is partially involved, there is a preferential healing of the limbus by circumferential migration of tongue shaped sheets from the remaining intact limbus. These meet to cover the limbus (Rule 2) and subsequent healing occurs as in rule 1. On occasions conjunctival epithelium migrates across the limbus resulting in partial limbal deficiency. When the entire limbus is affected, total conjunctivalisation of the cornea occurs (Rule 3). Within sheets, individual cells migrate in a predominantly clockwise whorl pattern (Rule 4). Physiological and wound healing related cell turnover is believed to occur from stem cells (SC) located at the limbus. Specialised anatomical structures, termed Limbal Epithelial Crypts (LEC) are associated with some limbal palisades and present all the hallmarks of a SC niche. SC characteristics are also seen in the peripheral cornea adjacent to a palisade with a LEC. Recent evidence suggests that the limbal SC play a critical role in wound healing but may have only a marginal role physiological corneal epithelial homeostasis.

Existence of limbal epithelial crypt within the infant limbus

Abstract Purpose The Limbal Epithelial Crypt (LEC) has been suggested to be the limbal epithelial stem cell niche and the limbus has been shown to demonstrate regional variations in association with the LEC. Our aims were to 1) to determine the existence of LEC in the infant age group and 2) compare and contrast the corneo‐scleral tissue between infant and the adult by use of immunofluorescent imaging. Methods Donor eyes were processed and snap frozen in liquid nitrogen. Using a Leica cryostat machine, corneo‐scleral sections were prepared and examined under microscopy for presence of LEC structures. Limbal sections were fixed, and stained with a range of current postulated markers of stem cells and cell differentiation by standard immunofluorescent imaging techniques. Results Immunostaining in the infant group demonstrates that both limbal and corneal epithelium consist of cells that are characteristic of cells with less differentiated nature. No classical adult LEC was discernable in corneo‐scleral rims of a 2 year old or a 4‐month old infant. However, adult LEC immunofluorescence, demonstrate similar characteristics to the infant limbus. Conclusion We hypothesize that the absence of classical adult LEC in infants suggests that LEC may arise during the natural human growth, and may act as a repository for limbal epithelial stem cells in the human adult.

Microarray study of limbal epithelial crypt demonstrates its putative limbal stem cell niche characteristics

Abstract Purpose We have discovered a novel anatomical structure at the limbus, termed the Limbal Epithelial crypt (LEC). Previous anatomical and immunohistological studies suggest its potential as the limbal stem cell niche. We aim to characterise the differential gene expression of the LEC by microarrays with a view to finding putative stem cell marker(s). Methods Frozen tissue blocks of corneoscleral buttons dissected from cadaver eyes were cryosectioned. These tissue sections from different ocular surface regions were laser microdissected. Extracted RNA was amplified &amp; hybridized to microarray chips. Raw data obtained with Genepix Pro6 software was filtered, normalized &amp; analysed on BASE, TMeV &amp; Jexpresspro software. Unpaired T‐Test, Significance Analysis of Microarrays and k‐means clustering were performed on the data. Quantitative gene expression analysis (qPCR) was performed on the genes of interest. Results 150 differentially expressed genes in the LEC were obtained(p ≤ 0.01). These genes were clustered according to stem cell related functions. qPCR was performed on the Integrin Beta 1 binding protein gene (ITGB1BP1). The protein expressed by this gene specifically binds to Beta 1 Integrin (ITGB1) which is a cell adhesion molecule, is predominantly an inhibitor of cell proliferation and has a role in maintaining stem cells in their niche. These genes were significantly expressed in the LEC (P=0.03). Conclusion The constituents of the LEC are stem cells with neighbouring niche cells, hence representing a Limbal Epithelial Stem Cell niche.

Limbal Epithelial Crypt

To determine the distribution of cell membrane proteins and extracellular matrix proteins around the limbal epithelial crypt (LEC) compared with adjacent limbus and corneal epithelium. Serial histological sections of human corneoscleral limbus rims were stained with antibodies of interest by standard immunohistochemistry. Superficial cells of the limbus were desmoglein 3 positive, compared with the negative basal cells of the limbus that correspond to cells with more stemlike properties. The LEC had a much lower proportion of desmoglein 3 staining in comparison. Tenascin C staining demonstrated regional variations of the limbus depending on their association with the LEC. Limbus that was associated with or adjacent to the LEC had a greater tenascin C expression compared with normal limbus, whereas the LEC demonstrated the greatest tenascin C expression. Based on these and similar results previously reported for connexin 43, we propose a novel model on the mechanism of corneal surface epithelium maintenance involving 3 different limbal regions: zone 1, limbus including the LEC; zone 2, limbus associated with the LEC; and zone 3, limbus distant to the LEC. The noted limbal variations may influence the selection of the donor site for limbal grafts in the future.

Evolving concepts on limbal stem cells and their niche

As a concentrated location for adult somatic stem cells, the niche plays a crucial role on maintaining the high proliferative ability and the inducing the particular differentiation of the stem cells by providing the special microenvironment containing definite extracellular matrix and niche cells. Nowadays limbal stem cells (LSCs) are the acknowledged adult somatic stem cells of corneal epithelium. Rapid progress has been accomplished in the theory and clinical practice on LSCs. However, the understanding on its niche has obviously fallen behind. This review summarized and analyzed the status quo of the study on niche of LSCs, mainly through the following aspects: the precise location of the niche was described as limbal epithelial crypts; the blood-derived and stroma-derived cytokine could regulate the proliferation, differentiation and apoptosis of LSCs by triggering particular intracellular information pathway; the survival and function of LSCs could be influenced by damaged niche microenvironment, while the differentiation of LSCs could be veered by another type of niche; in ex vivo LSCs expansion, reconstruction of the niche is needed and could be achieved by 3T3 cells or using amniotic membrane as the carrier.