Nectin-3 expression is elevated in limbal epithelial side population cells with strongly expressed stem cell markers

Corneal epithelial stem cells (CESCs) are essential for maintaining the ocular surface. However, the lack of surface markers for CESCs remains a serious obstacle in the identification of CESCs. Previously, we showed that rabbit limbal epithelial side population (rLE-SP) cells exhibited stem cell phenotypes including increased expression of CD61, a marker for mouse hematopoietic stem cells. Here, we demonstrate that nectin-3, an immunoglobulin-like cell-cell adhesion molecule, is highly expressed in rLE-SP cells. Additionally, nectin-3⁺ cells were significantly enriched among CD61⁺rLE-SP cells as compared to CD61⁻rLE-SP cells. In mouse bone marrow side population cells, a correlation between expression of nectin-3 and CD61 was also observed. These data strongly suggest that nectin-3 may contribute to the identification of CESCs.     

PFN1 and integrin‐β1/mTOR axis involvement in cornea differentiation of fibroblast limbal stem cells

Ex vivo limbal stem cell transplantation is the main therapeutic approach to address a complete and functional re‐epithelialization in corneal blindness, the second most common eye disorder. Although important key points were defined, the molecular mechanisms involved in the epithelial phenotype determination are unclear. Our previous studies have demonstrated the pluripotency and immune‐modulatory of fibroblast limbal stem cells (f‐LSCs), isolated from the corneal limbus. We defined a proteomic profile especially enriched in wound healing and cytoskeleton‐remodelling proteins, including Profilin‐1 (PFN1). In this study we postulate that pfn‐1 knock down promotes epithelial lineage by inhibiting the integrin‐β1(CD29)/mTOR pathway and subsequent NANOG down‐expression. We showed that it is possible modulate pfn1 expression levels by treating f‐LSCs with Resveratrol (RSV), a natural compound: pfn1 decline is accompanied with up‐regulation of the specific differentiation epithelial genes pax6 (paired‐box 6), sox17 (sex determining region Y‐box 17) and ΔNp63‐α (p63 splice variant), consistent with drop‐down of the principle stem gene levels. These results contribute to understand the molecular biology of corneal epithelium development and suggest that pfn1 is a potential molecular target for the treatment of corneal blindness based on epithelial cell dysfunction.     

Profile of biological characterizations and clinical application of corneal stem/progenitor cells

Corneal stem/progenitor cells are typical adult stem/progenitor cells. The human cornea covers the front of the eyeball, which protects the eye from the outside environment while allowing vision. The location and function demand the cornea to maintain its transparency and to continuously renew its epithelial surface by replacing injured or aged cells through a rapid turnover process in which corneal stem/progenitor cells play an important role. Corneal stem/progenitor cells include mainly corneal epithelial stem cells, corneal endothelial cell progenitors and corneal stromal stem cells. Since the discovery of corneal epithelial stem cells (also known as limbal stem cells) in 1971, an increasing number of markers for corneal stem/progenitor cells have been proposed, but there is no consensus regarding the definitive markers for them. Therefore, the identification, isolation and cultivation of these cells remain challenging without a unified approach. In this review, we systematically introduce the profile of biological characterizations, such as anatomy, characteristics, isolation, cultivation and molecular markers, and clinical applications of the three categories of corneal stem/progenitor cells.     

Critical appraisal of ex vivo expansion of human limbal epithelial stem cells

The stem cells (SCs) of the corneal epithelium located in the limbal basal layer are the ultimate source to maintain corneal epithelial homeostasis. Like other adult tissue-specific SCs, self renewal and fate decision of limbal SCs are regulated by a specialized in vivo microenvironment, termed "niche". Loss of limbal SCs or dysfunction of the limbal niche renders corneas with a unique clinical disease labeled limbal stem cell deficiency (LSCD). Besides transplantation of autologous or allogeneic limbal SCs or amniotic membrane, a new strategy of treating LSCD is to transplant a bio-engineered graft by expanding limbal SCs ex vivo. Herein, we conduct a critical appraisal of six protocols that have successfully been practiced in treating human patients with LSCD, and identify issues whether niche regulation has been disrupted or maintained during isolation and expansion. Consequently, we propose a future direction that may circumvent the potential pitfalls existing in these conventional protocols by preserving the interaction between limbal SCs and their native niche cells during isolation and expansion. Such an approach may one day help realize considerable promise held by adult SCs in treating a number of diseases.     

Niche regulation of corneal epithelial stem cells at the limbus

Among all adult somatic stem cells,those of the corneal epithelium are unique in their exclusive location in a definedlimbai structure termed Palisades of Vogt.As a result,surgical engraftment oflimbal epithelial stem cells with or withoutex vivo expansion has long been practiced to restore sights in patients inflicted with limbal stem cell deficiency.Neverthe-less,compared to other stem cell examples,relatively little is known about the limbal niche,which is believed to play apivotal role in regulating self-renewal and fate decision of limbal epithelial stem cells.This review summarizes relevantliterature and formulates several key questions to guide future research into better understanding of the pathogenesis oflimbal stem cell deficiency and further improvement of the tissue engineering of the corneal epithelium by focusing onthe limbal niche.     

Enrichment of corneal epithelial stem/progenitor cells using cell surface markers, integrin alpha6 and CD71

Corneal epithelial stem cells are believed to reside in the basal layer of the limbal epithelium, but no definitive cell surface markers have been identified. For keratinocytes, stem/progenitor cells are known to be enriched by cell surface markers, integrin α₆ and CD71, as a minor subpopulation which shows high integrin α₆ and low CD71 expressions (α₆^bri/CD71^dim). In the present study, we investigated the possibility that corneal epithelial stem cells can be enriched by integrin α₆ and CD71. The α₆^bri/CD71^dim cells were separated by fluorescence-activated cell sorting, as a minor subpopulation of the limbal epithelial cells. They were enriched for relatively small cells, showing a higher clonogenic capacity and expression of stem cell markers, but a lower expression of differentiation markers, compared to other cell populations. The cells were localized immunohistochemically in the basal region of the limbal epithelium. These results indicate that the α₆^bri/CD71^dim subpopulation enriched corneal epithelial stem cells.     

Understanding cornea epithelial stem cells and stem cell deficiency: Lessons learned using vertebrate model systems

Animal models have contributed greatly to our understanding of human diseases. Here, we focus on cornea epithelial stem cell (CESC) deficiency (commonly called limbal stem cell deficiency, LSCD). Corneal development, homeostasis and wound healing are supported by specific stem cells, that include the CESCs. Damage to or loss of these cells results in blindness and other debilitating ocular conditions. Here we describe the contributions from several vertebrate models toward understanding CESCs and LSCD treatments. These include both mammalian models, as well as two aquatic models, Zebrafish and the amphibian, Xenopus. Pioneering developments have been made using stem cell transplants to restore normal vision in patients with LSCD, but questions still remain about the basic biology of CESCs, including their precise cell lineages and behavior in the cornea. We describe various cell lineage tracing studies to follow their patterns of division, and the fates of their progeny during development, homeostasis, and wound healing. In addition, we present some preliminary results using the Xenopus model system. Ultimately, a more thorough understanding of these cornea cells will advance our knowledge of stem cell biology and lead to better cornea disease therapeutics.     

Evaluating alternative stem cell hypotheses for adult corneal epithelial maintenance

In this review we evaluate evidence for three different hypotheses that explain how the corneal epithelium is maintained. The limbal epithelial stem cell(LESC)hypothesis is most widely accepted. This proposes that stem cells in the basal layer of the limbal epithelium, at the periphery of the cornea, maintain themselves and also produce transient(or transit) amplifying cells(TACs). TACs then move centripetally to the centre of the cornea in the basal layer of the corneal epithelium and also replenish cells in the overlying suprabasal layers. The LESCs maintain the corneal epithelium during normal homeostasis and become more active to repair significant wounds. Second, the corneal epithelial stem cell(CESC) hypothesis postulates that, during normal homeostasis, stem cells distributed throughout the basal corneal epithelium, maintain the tissue. According to this hypothesis, LESCs are present in the limbus but are only active during wound healing. We also consider a third possibility, that the corneal epithelium is maintained during normal homeostasis by proliferation of basal corneal epithelial cells without any input from stem cells. After reviewing the published evidence, we conclude that the LESC and CESC hypotheses are consistent with more of the evidence than the third hypothesis, so we do not consider this further. The LESC and CESC hypotheses each have difficulty accounting for one main type of evidence so we evaluate the two key lines of evidence that discriminate between them. Finally, we discuss how lineage-tracing experiments have begun to resolve the debate in favour of the LESC hypothesis. Nevertheless, it also seems likely that some basal corneal epithelial cells can act as long-term progenitors if limbal stem cell function is compromised. Thus, this aspect of the CESC hypothesis may have a lasting impact on our understanding of corneal epithelial maintenance, even if it is eventually shown that stem cells are restricted to the limbus as proposed by the LESC hypothesis.     

Bone Morphogenetic Protein 4 (BMP4) Enhances the Differentiation of Human Induced Pluripotent Stem Cells into Limbal Progenitor Cells

Corneal epithelium maintains visual acuity and is regenerated by the proliferation and differentiation of limbal progenitor cells. Transplantation of human limbal progenitor cells could restore the integrity and functionality of the corneal surface in patients with limbal stem cell deficiency. However, multiple protocols are employed to differentiate human induced pluripotent stem (iPS) cells into corneal epithelium or limbal progenitor cells. The aim of this study was to optimize a protocol that uses bone morphogenetic protein 4 (BMP4) and limbal cell-specific medium. Human dermal fibroblast-derived iPS cells were differentiated into limbal progenitor cells using limbal cell-specific (PI) medium and varying doses (1, 10, and 50 ng/mL) and durations (1, 3, and 10 days) of BMP4 treatment. Differentiated human iPS cells were analyzed by real-time polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemical studies at 2 or 4 weeks after BMP4 treatment. Culturing human dermal fibroblast-derived iPS cells in limbal cell-specific medium and BMP4 gave rise to limbal progenitor and corneal epithelial-like cells. The optimal protocol of 10 ng/mL and three days of BMP4 treatment elicited significantly higher limbal progenitor marker (ABCG2, ∆Np63α) expression and less corneal epithelial cell marker (CK3, CK12) expression than the other combinations of BMP4 dose and duration. In conclusion, this study identified a successful reprogramming strategy to induce limbal progenitor cells from human iPS cells using limbal cell-specific medium and BMP4. Additionally, our experiments indicate that the optimal BMP4 dose and duration favor limbal progenitor cell differentiation over corneal epithelial cells and maintain the phenotype of limbal stem cells. These findings contribute to the development of therapies for limbal stem cell deficiency disorders.     

Corneal stem cells and tissue engineering: Current advances and future perspectives

Major advances are currently being made in regenerative medicine for cornea. Stem cell-based therapies represent a novel strategy that may substitute conventional corneal transplantation, albeit there are many challenges ahead given the singularities of each cellular layer of the cornea. This review recapitulates the current data on corneal epithelial stem cells, corneal stromal stem cells and corneal endothelial cell progenitors. Corneal limbal autografts containing epithelial stem cells have been transplanted in humans for more than 20 years with great successful rates, and researchers now focus on ex vivo cultures and other cell lineages to transplant to the ocular surface. A small population of cells in the corneal endothelium was recently reported to have self-renewal capacity, although they do not proliferate in vivo. Two main obstacles have hindered endothelial cell transplantation to date: culture protocols and cell delivery methods to the posterior cornea in vivo. Human corneal stromal stem cells have been identified shortly after the recognition of precursors of endothelial cells. Stromal stem cells may have the potential to provide a direct cell-based therapeutic approach when injected to corneal scars. Furthermore, they exhibit the ability to deposit organized connective tissue in vitro and may be useful in corneal stroma engineering in the future. Recent advances and future perspectives in the field are discussed.     

Tooth-derived stem cells: Update and perspectives

Tissue engineering is an emerging field of science that focuses on creating suitable conditions for the regeneration of tissues. The basic components for tissue engineering involve an interactive triad of scaffolds, signaling molecules, and cells. In this context,stem cells(SCs) present the characteristics of selfrenewal and differentiation capacity, which make them promising candidates for tissue engineering. Although they present some common markers, such as cluster of differentiation(CD)105, CD146 and STRO-1, SCs derived from various tissues have different patterns in relation to proliferation, clonogenicity, and differentiation abilities in vitro and in vivo. Tooth-derived tissues have been proposed as an accessible source to obtain SCs with limited morbidity, and various tooth-derived SCs(TDSCs) have been isolated and characterized, such as dental pulp SCs, SCs from human exfoliated deciduous teeth, periodontal ligament SCs, dental follicle progenitor cells, SCs from apical papilla, and periodontal ligament of deciduous teeth SCs. However, heterogeneity among these populations has been observed, and the best method to select the most appropriate TDSCs for regeneration approaches has not yet been established. The objective of this review is to outline the current knowledge concerning the various types of TDSCs, and discuss the perspectives for their use in regenerative approaches.     

Mosaic analysis of stem cell function and wound healing in the mouse corneal epithelium

Background  

The mouse corneal epithelium is a continuously renewing 5–6 cell thick protective layer covering the corneal surface, which regenerates rapidly when injured. It is maintained by peripherally located limbal stem cells (LSCs) that produce transient amplifying cells (TACs) which proliferate, migrate centripetally, differentiate and are eventually shed from the epithelial surface. LSC activity is required both for normal tissue maintenance and wound healing. Mosaic analysis can provide insights into LSC function, cell movement and cell mixing during tissue maintenance and repair. The present study investigates cell streaming during corneal maintenance and repair and changes in LSC function with age.     

Limbal stem cells: Central concepts of corneal epithelial homeostasis

A strong cohort of evidence exists that supports the localisation of corneal stem cells at the limbus. The distinguishing characteristics of limbal cells as stem cells include slow cycling properties, high proliferative potential when required, clonogenicity, absence of differentiation marker expression coupled with positive expression of progenitor markers, multipotency, centripetal migration, requirement for a distinct niche environment and the ability of transplanted limbal cells to regenerate the entire corneal epithelium. The existence of limbal stem cells supports the prevailing theory of corneal homeostasis, known as the XYZ hypothesis where X represents proliferation and stratification of limbal basal cells, Y centripetal migration of basal cells and Z desquamation of superficial cells. To maintain the mass of cornea, the sum of X and Y must equal Z and very elegant cell tracking experiments provide strong evidence in support of this theory. However, several recent stud-ies have suggested the existence of oligopotent stem cells capable of corneal maintenance outside of the limbus. This review presents a summary of data which led to the current concepts of corneal epithelial homeostasis and discusses areas of controversy surrounding the existence of a secondary stem cell reservoir on the corneal surface     

Rare corneal clones in mice suggest an age-related decrease of stem cell activity and support the limbal epithelial stem cell hypothesis

The anterior ocular surface comprises the cornea, conjunctiva and a narrow intermediate region called the limbus. It is widely accepted that the corneal epithelium is maintained by stem cells but different hypotheses propose that the stem cells that maintain the mouse corneal epithelium during normal homeostasis are located either in the basal limbal epithelium or throughout the basal corneal epithelium. There are no specific markers to help test these alternatives and new methods are required to distinguish between them. We observed that KRT5^LacZ/− transgenic mice produced rare β-galactosidase (β-gal)-positive radial stripes in the corneal epithelium. These stripes are likely to be clonal lineages of cells derived from stem cells, so they provide a lineage marker for actively proliferating stem cells. The distributions of the β-gal-positive radial stripes suggested they extended centripetally from the limbus, supporting the limbal epithelial stem cell (LESC) hypothesis. Stripe frequency declined between 15 and 30 weeks, which predicts a reduction in stem cell function with age. Pax6^+/−, KRT5^LacZ/− corneas had small patches rather than stripes, which confirms that corneal maintenance is abnormal in Pax6^+/− mice.     

Reconstruction of corneal epithelium with cryopreserved corneal limbal stem cells in a goat model

We describe a procedure to construct an artificial corneal epithelium from cryopreserved limbal stem cells (LSCs) for corneal transplantation. The LSCs were separated from limbal tissue of male goats. The primary LSCs were identified by flow cytometry and were expanded. They were examined for stem cell-relevant properties and cryopreserved in liquid nitrogen. Cryopreserved LSCs were thawed and then transplanted onto human amniotic membrane, framed on a nitrocellulose sheet, to construct corneal epithelium sheets. The artificial corneal epithelium was transplanted into the right eye of pathological models of total limbal stem cell deficiency (LSCD). Then, the effects of reconstruction were evaluated by clinical observation and histological examination. Polymerase chain reaction analysis was used to detect the SRY gene. The data showed that transplantation of cryopreserved LSCs, like fresh LSCs, successfully reconstructed damaged goat corneal surface gradually, but the SRY gene expression from male goat cells could only be detected in the first 2 months after transplantation. The therapeutic effect of the transplantation may be associated with the inhibition of inflammation-related angiogenesis after transplantation of cryopreserved LSCs. This study provides the first line of evidence that cryopreserved LSCs can be used for reconstruction of damaged corneas, presenting a remarkable potential source for transplantation in the treatment of corneal disorders.     

The effect of lectins on the attachment and invasion of Enteromyxum scophthalmi (Myxozoa) in turbot (Psetta maxima L.) intestinal epithelium in vitro

The involvement of the lectin/carbohydrate interaction in the invasion of the turbot intestinal epithelium by Enteromyxum scophthalmi was studied in vitro using explants of turbot intestine and pre-treatment of parasite stages with the plant lectins of Canavalia ensiformis (Con A) and Glycine max (SBA). Both lectins inhibited the attachment and invasion of E. scophthalmi stages to the intestinal epithelium, though the inhibitory effect was higher for SBA than for Con A. Such results point to the involvement of N-acetyl-galactosamine (GalNAc) and galactose (Gal) residues and also of mannose/glucose residues in the E. scophthalmi-intestinal epithelium interaction. The inhibitory effect of both lectins on the parasite adhesion and penetration points to the interest of further studies to confirm the presence of putative lectins recognising GalNAc-Gal and mannose/glucose residues in turbot intestine. The obtained results demonstrated also the adequacy of turbot intestinal explants as an in vitro model to study the interaction with E. scophthalmi.     

Therapeutic use of limbal stem cells

Stem cells are defined as relatively undifferentiated cells that have the capacity to generate more differentiated daughter cells. Limbal stem cells are responsible for epithelial tissue repair and regeneration throughout the life. Limbal stem cells have been localized to the Palisades of Vogt in the limbal region. Limbal stem cells have a higher proliferative potential compared to the cells of peripheral and central cornea. Limbal stem cells have the capacity to maintain normal corneal homeostasis. However, in some pathological states, such as chemical and thermal burns, Stevens-Johnson syndrome, and ocular pemphigoid limbal stem cells fail to maintain the corneal epithelial integrity. In such situations, limbal stem cell transplantation has been required as a therapeutic option. In unilateral disorders, the usual source of stem cells is the contralateral eyes, but if the disease is bilateral stem cell allografts have to be dissected from family members or cadaver eyes. The advent of ex vivo expansion of limbal stem cells from a small biopsy specimen has reduced the risk of limbal deficiency in the donor eye. Concomitant immunosuppressive therapy promotes donor-derived epithelial cell viability, but some evidences suggest that donor-derived epithelial stem cell viability is not sustained indefinitely. Thus, long-term follow-up studies are required to ascertain whether donor limbal stem cell survival or promotion of recolonization by resident recipient stem cells occurs in restored recipient epithelium. However, this is not an easy task since a definitive limbal stem cell marker has not been identified yet. This review will discuss the therapeutic usage of limbal stem cells in the corneal epithelial disorders.     

Biological principals and clinical potentials of limbal epithelial stem cells

In this review, we describe a population of adult stem cells that are currently being successfully used in the clinic to treat blinding ocular surface disease, namely limbal epithelial stem cells (LESC). The function and characteristics of LESC and the challenges faced in making use of their therapeutic potential will be examined. The cornea on the front surface of the eye provides our window on the world. The consistency and functionality of the outer-most corneal epithelium is essential for vision. A population of LESC are responsible for replenishing the epithelium throughout life by providing a constant supply of daughter cells that replace those constantly removed from the ocular surface during normal wear and tear and following injury. LESC deficiency results in corneal inflammation, opacification, vascularisation and severe discomfort. The transplantation of cultured LESC is one of only a few examples of the successful use of adult stem cell therapy in patients. The clinical precedence for the use of stem cell therapy and the ready accessibility of a transparent stem cell niche make the cornea a unique model for the study of adult stem cells in health and disease. The authors thank the Special Trustees of Moorfields Eye Hospital (J.T.D.) and the BBSRC (M.N.) for financial support.     

Single-cell RNA sequencing in cornea research: Insights into limbal stem cells and their niche regulation

The corneal epithelium is composed of stratified squamous epithelial cells on the outer surface of the eye, which acts as a protective barrier and is critical for clear and stable vision. Its continuous renewal or wound healing depends on the proliferation and differentiation of limbal stem cells (LSCs), a cell population that resides at the limbus in a highly regulated niche. Dysfunction of LSCs or their niche can cause limbal stem cell deficiency, a disease that is manifested by failed epithelial wound healing or even blindness. Nevertheless, compared to stem cells in other tissues, little is known about the LSCs and their niche. With the advent of single-cell RNA sequencing, our understanding of LSC characteristics and their microenvironment has grown considerably. In this review, we summarized the current findings from single-cell studies in the field of cornea research and focused on important advancements driven by this technology, including the heterogeneity of the LSC population, novel LSC markers and regulation of the LSC niche, which will provide a reference for clinical issues such as corneal epithelial wound healing, ocular surface reconstruction and interventions for related diseases.