Limbal Stem Cells in Health and Disease

Stem cells are present in all self-reviewing tissues and have unique properties. The ocular surface is made up of two distinct types of epithelial cells, constituting the conjunctival and the corneal epithelia. These epithelia are stratified, squamous and non-keratinized. Although anatomically continuous with each other at the corneoscleral limbus, the two cell phenotypes represent quite distinct subpopulations. The stem cells for the cornea are located at the limbus. The microenvironment of the limbus is considered to be important in maintaining stemness of the stem cells. They also act as a barrier to conjunctival epithelial cells and prevent them from migrating on to the corneal surface. In certain pathologic conditions, however, the limbal stem cells may be destroyed partially or completely resulting in varying degrees of stem cell deficiency with its characteristic clinical features. These include conjunctivalization of the cornea with vascularization, appearance of goblet cells, and an irregular and unstable epithelium. The stem cell deficiency can be managed with auto or allotransplantation of these cells. With the latter option, systemic immunosuppression is required. The stem cells can be expanded ex vivo on a processed human amniotic membrane and transplanted back to ocular surface with stem cell deficiency without the need of immunosuppression.     

In search of markers for the stem cells of the corneal epithelium

The anterior one-fifth of the human eye is called the cornea. It consists of several specialized cell types that work together to give the cornea its unique optical properties. As a result of its smooth surface and clarity, light entering the cornea focuses on the neural retina allowing images to come into focus in the optical centres of the brain. When the cornea is not smooth or clear, vision is impaired. The surface of the cornea consists of a stratified squamous epithelium that must be continuously renewed. The cells that make up this outer covering come from an adult stem cell population located at the corneal periphery at a site called the corneal limbus. While engaging in the search for surface markers for corneal epithelial stem cells, vision scientists have obtained a better understanding of the healthy ocular surface. In this review, we summarize the current state of knowledge of the ocular surface and its adult stem cells, and analyse data as they now exist regarding putative corneal epithelial stem cell markers.     

Potential localization of putative stem/progenitor cells in human bulbar conjunctival epithelium

Although the conjunctival fornix appears to contain the greatest proportion of stem cells, it is likely that pockets of conjunctival epithelial stem cells may also exist throughout the conjunctival epithelium. This study was to investigate the potential localization of putative stem/progenitor cells in the human bulbar conjunctival epithelium by evaluating 6 keratins and 13 molecules that have been previously proposed stem cell associated or differentiation markers. We found that cornea specific cytokeratin (CK) 3 was not expressed by the bulbar conjunctival epithelial cells. In contrast, CK4 and CK7 were expressed by the superficial cells of bulbar conjunctival epithelium. CK14 and CK15 were confined to the basal cell layer. CK19 was strongly expressed by all layers of the bulbar conjunctival epithelium. The expression patterns of molecular markers in the basal cells of human bulbar conjunctival epithelium were found to be similar to the corneal epithelium. Basal conjunctival epithelial cells strongly expressed stem cell associated markers, including ABCG2, p63, nerve growth factor (NGF) with its receptors tyrosine kinase receptor A (TrkA) and neurotrophin low‐affinity receptor p75NTR, glial cell‐derived neurotrophic factor (GDNF) with its receptor GDNF family receptor alpha 1 (GFRα‐1), integrin β1, α‐enolase, and epidermal growth factor receptor (EGFR). The differentiation associated markers nestin, E‐cadherin and involucrin were not expressed by these cells. These findings indicate that the basal cells of bulbar conjunctival epithelium shares a similar expression pattern of stem cell associated markers to the corneal epithelium, but has a unique pattern of differentiation associated cytokeratin expression. J. Cell. Physiol. 225: 180–185, 2010. © 2010 Wiley‐Liss, Inc.     

Streaming of labelled cells in the conjunctival epithelium

This study examines epithelial cell streaming and turnover in normal rat bulbar conjunctiva. Twenty seven male adult random-bred Hebrew rats weighing between 250–300 g, were injected i.p. with [³H]-thymidine. Three rats were killed at various times, thereafter from 1 h to 28 days. The enucleated eyes were fixed in formalin, cut into 5 μ thick sections, dipped into liquid emulsion, exposed for three weeks and stained with haematoxylin and eosin. Conjunctival epithelium was scanned from the limbus and outward, using an ocular micrometer grid with 10 x 10 divisions. In each consecutive field the grid was positioned along the basement membrane which was defined as the x-axis. The y-axis extended from the basement membrane outward. The x, y coordinate of each nucleus with three grains or more and its grain content were recorded along the entire epithelium. Conjunctival epithelium is divided into two cell kinetic compartments: a progenitor (P), along the basal and supra basal layer, in which cells proliferate, and a non proliferating Q-compartment, in the layers above. One hour after labelling most of the labelled cells were in the basal and supra basal layers. From then onward labelled cells streamed along both axes. Their x-velocity was 10·5±2·4 μ/day and the y-velocity 9·3 ± 5·4 μ/day. Cells are eliminated at the epithelial surface which is the outer Q-compartment boundary. Basal cell turnover was estimated from grain count dilution curves. The time it takes for the grains in a cell to reach half of their initial value was 8·3 days. It is closely related to the cell's generation time. The present study demonstrates that conjunctival epithelium in the rat streams along two axes, x, and y: 1 The x-axis extends along the basal layer, from the limbus and outward. 2 The y-axis extends from the basal layer into the layers above it. Cells first stream along the x-direction and then turn y-ward. Since cells are ultimately exfoliated from the conjunctival surface, and since the conjunctiva maintains steady state, we propose that stem cells located in the limbus generate transitional cells that stream along the two axes. Macroscopically the limbus is circular, and the stem cells are situated around the cornea. Each stem cell and its streaming progeny can be viewed as a conjunctival epithelial unit. We propose that conjunctival and corneal epithelium, are the descendants of an uncommitted stem cell that generates two differentiation pathways, a corneal and a conjunctival.     

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     

The spectrum of cytokeratins expressed in the adult human cornea, limbus and perilimbal conjunctiva

The aim of this study was to detect a spectrum of cytokeratins (CK) present in the adult human cornea, limbus and perilimbal conjunctiva. Cryosections from seven corneo-scleral discs were fixed, and indirect immunofluorescent staining was performed using antibodies directed against CK1-CK10 and CK13-CK20. The percentage of positive cells was calculated in the epithelium of the cornea, limbus and perilimbal conjunctiva. Quantitative real time RT-PCR (qRT-PCR) was used to detect CK6 and CK18 expression in the corneal and conjunctival epithelium. The most intense staining present throughout the cornea was observed for CK3, CK5 and CK14; CK19 was found at the corneal periphery only. CK4 and CK10/13 revealed mild to moderate positivity mostly in the superficial layers of the cornea. The suprabasal cell layers of all examined areas showed a strong positivity for CK16. A heterogeneous staining pattern with a centrifugal decrease in the signal was observed for CK8 and CK18. CK5/6, CK14 and CK19 were present in the limbus, where a positive signal for CK3 was observed in the suprabasal and superficial cells only. In contrast to the cornea, CK15 appeared in the basal and suprabasal layers of the limbus. The perilimbal conjunctiva showed strong immunostaining for CK10/13, CK14 and CK19. A moderate signal for CK7 was detected in the superficial layers of the conjunctiva. qRT-PCR confirmed CK6 and CK18 expression in the corneal and conjunctival epithelium. The detailed characterization of the corneal, limbal and perilimbal conjunctival epithelium under normal circumstances may be useful for characterizing the changes occurring under pathological conditions.     

Ocular surface epithelial and stem cell development

Phenotypic features and developmental events involved in the genesis of the limbo-corneal and conjunctival epithelia are described. Together, these two epithelia define the ocular surface. They derive from a small cohort of optic vesicle-induced PAX6+ head ectodermal cells that remain on the surface following lens vesicle formation by the main PAX6+ cell cohort. Both epithelia are stratified, and display wet, non-keratinizing phenotypes. The most significant spatial feature of the limbo-corneal epithelium is the segregation of its supporting stem and early precursor cells to the limbus, the outer vascularized rim separating the cornea from the conjunctiva. These stem cells express ABCG2, a xenobiotic transporter present in stem cells from other organs. ABCG2 transport activity excludes the DNA dye Hoechst 33342, allowing the isolation of the ocular stem cells by flow cytometry, as a unique cohort known as a side 'side population'. Limbal stem cells do not form gap junctions and exist as metabolically isolated entities. Tracking of expression changes in Cx43, the main gap junction protein expressed in both the pre-epithelial ectoderm and in the mature central corneal epithelium, indicates that a limbal stem cell phenotype starts developing very soon after lens vesicle invagination, in advance of the appearance of any recognizable anatomical sub-epithelial limbal feature. Differences in Cx43 expression also reveal the very early nature of the divergence in limbo-corneal and conjunctival lineages. The putative involvement of several early genes, including gradients of PAX6 and differences in expression patterns for members of the Id or msh gene expression regulators are reviewed.     

Rat limbal epithelial side population cells exhibit a distinct expression of stem cell markers that are lacking in side population cells from the central cornea

The side population (SP) phenotype is shared by stem cells in various tissues and species. Here we demonstrate SP cells with Hoechst dye efflux were surprisingly collected from the epithelia of both the rat limbus and central cornea, unlike in human and rabbit eyes. Our results show that rat limbal SP cells have a significantly higher expression of the stem cell markers ABCG2, nestin, and notch 1, compared to central corneal SP cells. Immunohistochemistry also revealed that ABCG2 and the epithelial stem/progenitor cell marker p63 were expressed only in basal limbal epithelial cells. These results demonstrate that ABCG2 expression is closely linked to the stem cell phenotype of SP cells.     

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.     

Cultured human corneal epithelial stem/progenitor cells derived from the corneal limbus

Stem/progenitor cells of the human corneal epithelium are present in the human corneal limbus, and several corneal epithelial stem/progenitor cell markers have been reported. Recently, the neurotrophin family receptors were reported to be useful markers of corneal epithelial stem/progenitor cells. Therefore, we examined an enzymatic separation method for obtaining corneal epithelial stem/progenitor cells and measuring the change in the expression of low-affinity neurotrophin receptor p75 (p75^NTR), a receptor belonging to the neurotrophin family. As a result, it was found that our separation method preserved cell viability. Furthermore, p75^NTR was mainly observed in epithelial basal cells as were the corneal epithelial stem/progenitor markers p63 and integrin β1. p75^NTR was also observed in the cultured cells, but its frequency decreased with passage. In conclusion, we propose that our culture method will enable the culture of corneal stem cells and that it is a useful tool for elucidating the molecular basis of the niche that is necessary for the maintenance of epithelial stem cells in the corneal limbus. Furthermore, we conclude that p75^NTR is a useful cell marker for evaluating the characteristics of stem/progenitor cells in culture.     

Amnion and ocular surface problems

The amniotic membrane, the most internal placental membrane, has various properties useful in ophthalmology. Collected on delivery by elective Caesarean section, the amnion is prepared under sterile conditions, and, usually, cryopreserved until its use as a biological bandage or as a substrate for epithelial growth in the management of various ocular surface conditions. Specifically, the amnion is used to : (1) limit formation of adhesive bands between eyelids and eyeball (symblepharon) or the progression of a fibrovascular outgrowth towards the cornea (pterygium) or to (2) facilitate the healing of corneal ulcers, bullous keratopathy, and corneal stem cell deficiency. In this last condition, either hereditary or acquired after a thermal or a chemical burn, corneal stem cells, located at a transitional zone between the cornea and conjunctiva, are lost. These cells are essential for renewal of corneal epithelium in normal and in diseased states. The loss of these cells leaves the corneal surface free for invasion by conjunctival epithelium. Not only, does conjunctival epithelium support the development of vascularisation on the normally avascular cornea, but some conjunctival cells differentiate into mucus secreting goblet cells. Such a change in phenotype leads to loss of corneal transparency and visual disability. The removal of this fibro-vascular outgrowth in combination with transplantation of both amniotic membrane and corneal stem cells are used to treat this condition. The amnion stimulates the proliferation of less differentiated cells which have the potential to reconstruct the cornea. This potential is at the origin of the hypothesis that the amnion may provide an alternative niche for limbal stem cells of the corneal epithelium. It abounds in cytokines and has antalgic, anti-bacterial, anti-inflammatory and anti-immunogenic properties, in addition to allowing, like fetal skin does, wound healing with minimal scar formation. These desirable properties are responsible for the increasing use of amniotic membrane in ophthalmology. The complete understanding of the mechanisms of action of amniotic membrane for ocular surface diseases has yet to be understood. Once revealed by research, they may provide new pharmacological avenues to treat ocular surface diseases.     

Investigation of K14/K5 as a stem cell marker in the limbal region of the bovine cornea

Background

Identification of stem cells from a corneal epithelial cell population by specific molecular markers has been investigated previously. Expressions of P63, ABCG2 and K14/K5 have all been linked to mammalian corneal epithelial stem cells. Here we report on the limitations of K14/K5 as a limbal stem cell marker.

Methodology/Principal Findings

K14/K5 expression was measured by immunohistochemistry, Western blotting and Real time PCR and compared between bovine epithelial cells in the limbus and central cornea. A functional study was also included to investigate changes in K5/14 expression within cultured limbal epithelial cells undergoing forced differentiation. K14 expression (or its partner K5) was detected in quiescent epithelial cells from both the limbal area and central cornea. K14 was localized predominantly to basal epithelial cells in the limbus and suprabasal epithelial cells in the central cornea. Western blotting revealed K14 expression in both limbus and central cornea (higher levels in the limbus). Similarly, quantitative real time PCR found K5, partner to K14, to be expressed in both the central cornea and limbus. Following forced differentiation in culture the limbal epithelial cells revealed an increase in K5/14 gene/protein expression levels in concert with a predictable rise in a known differentiation marker.

Conclusions/Significance

K14 and its partner K5 are limited not only to the limbus but also to the central bovine cornea epithelial cells suggesting K14/K5 is not limbal specific in situ. Furthermore K14/K5 expression levels were not lowered (in fact they increased) within a limbal epithelial cell culture undergoing forced differentiation suggesting K14/K5 is an unreliable maker for undifferentiated cells ex vivo.     

Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells

In this paper we present keratin expression data that lend strong support to a model of corneal epithelial maturation in which the stem cells are located in the limbus, the transitional zone between cornea and conjunctiva. Using a new monoclonal antibody, AE5, which is highly specific for a 64,000-mol-wt corneal keratin, designated RK3, we demonstrate that this keratin is localized in all cell layers of rabbit corneal epithelium, but only in the suprabasal layers of the limbal epithelium. Analysis of cultured corneal keratinocytes showed that they express sequentially three major keratin pairs. Early cultures consisting of a monolayer of "basal" cells express mainly the 50/58K keratins, exponentially growing cells synthesize additional 48/56K keratins, and postconfluent, heavily stratified cultures begin to express the 55/64K corneal keratins. Cell separation experiments showed that basal cells isolated from postconfluent cultures contain predominantly the 50/58K pair, whereas suprabasal cells contain additional 55/64K and 48/56K pairs. Basal cells of the older, postconfluent cultures, however, can become AE5 positive, indicating that suprabasal location is not a prerequisite for the expression of the 64K keratin. Taken together, these results suggest that the acidic 55K and basic 64K keratins represent markers for an advanced stage of corneal epithelial differentiation. The fact that epithelial basal cells of central cornea but not those of the limbus possess the 64K keratin therefore indicates that corneal basal cells are in a more differentiated state than limbal basal cells. These findings, coupled with the known centripetal migration of corneal epithelial cells, strongly suggest that corneal epithelial stem cells are located in the limbus, and that corneal basal cells correspond to "transient amplifying cells" in the scheme of "stem cells----transient amplifying cells----terminally differentiated cells."     

Age-related expressions of p63 and other keratinocyte stem cell markers in rat cornea

In this study, we examined the postnatal expression patterns of p63 and other keratinocyte stem cell markers in the rat cornea in an attempt to determine the markers that best represent characteristics of corneal keratinocyte stem cells. We show that the expression of p63 in the rat cornea is unique and differs from that observed in humans. It changes with age, from central cornea-positive, peripheral cornea-positive, and limbus-positive, to central cornea-positive, peripheral cornea-positive, and limbus-negative, and finally to central cornea-negative, peripheral cornea-positive, and limbus-negative, as examined by immunohistochemical staining. However, when a more sensitive staining method was used, the limbus was also shown to be positive for p63, indicating a lower level of expression than that of the peripheral cornea. The basal layer of the rat limbal epithelium is the site where -catenin+, K14+, PCNA-, and K3- cells reside. This cell layer is also the site where slow-cycling cells are located. In contrast with observations made in humans, our results clearly indicate that p63 is expressed in stem cells and young transient amplifying cells of the rat cornea, with higher levels of expression in the latter.     

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.     

The corneal epithelial stem cell

The aim of this paper was to develop a GFP-expressing transgenic mouse model for the keratoepithelioplasty and to use this to follow the outcome of this form of graft, when placed on an inflamed corneal surface. Further aims were to characterize both the graft and the epithelial surface of the mouse and rat cornea using putative stem cell markers (P63 and Telomerase) and marker of cell differentiation (14-3-3 sigma). Keratepithelioplasty was carried out using a GFP transgenic mouse cornea as donor tissue. Fluorescent epithelial outgrowth from each keratepithelioplasty was scored and quantified. Donor corneal graft tissue was obtained from the paracentral region or the anatomical limbal region of murine corneas. Paracentral donor grafts (n = 20) consistently demonstrated a significant increase in proliferative potential compared to grafts obtained from the anatomical limbal region of the mouse cornea (n = 25) (P = 0.000, Mann-Whitney U). Correspondingly, P63 expression was maximal in the paracentral region of the mouse cornea, in keeping with the demonstrated increased proliferative potential of donor grafts harvested from this region of the cornea. The murine corneal epithelium demonstrated decreased rather than increased cellular layers at the limbal region, in contrast to that of the rat or human epithelium. In addition, as a general finding in all species tested, there was an apparent increase noted in P63 expression in basal corneal epithelial cells in regions that had increased cellular layers (limbus in humans and rats and the paracentral corneal region in the mouse). Epithelium, which had migrated from donor grafts onto recipient corneas, retained P63 expression for the period of time examined (up to 3 days postengraftment). In addition, the conjunctival surface of an injured conjunctivalized displayed an abnormal pattern of P63 expression. Telomerase expression was widespread throughout many layers of both the murine and rat corneal epithelium. In the mouse and rat corneal epithelium P63 expression was maximal in areas of increased proliferative potential. Its expression, however, was not confined to stem cells alone. Migrating cells from transplanted keratoepithelial grafts retained P63 expression at least in the early stages post-transplantation. Finally, damaged conjunctivalized corneas displayed an abnormal P63 expression pattern when compared to either normal conjunctiva or normal cornea.     

Circadian variation in the mitotic rate of the rat corneal epithelium. Cell divisions and migration are analyzed by a mathematical model

A stathmokinetic method was used to study the diurnal variation in the mitotic rate (MR) of the rat corneal epithelium, and in the adjacent conjunctival epithelium. A prominent circadian variation in cell proliferation was observed in both epithelia, both showing almost the same pattern, which may indicate that both tissues are submitted to the same regulatory mechanisms. The average rate of cell renewal during a 24 h period indicated a mean cell renewal time of 12.3 days. This is longer than previously assumed. The MR declined toward the central cornea. Based on the above observations and the known centripetal migration of cells in the corneal epithelium, we have developed a mathematical model showing isomorphism with the renewal of the corneal epithelium.     

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.