Corneal epithelial stem cells and their niche at a glance

The cornea is the transparent region of the ocular surface and is essential for maintaining vision, as it enables light to enter the eye and stimulate the photoreceptor cells of the retina (Notara et al., 2010a). It also acts as a physical barrier between the internal structures of the eye and the outside world, thus protecting the eye from environmental damage (Notara et al., 2010a).

Structurally, the cornea consists of an avascular, collagen-rich stromal tissue that is lined by a self-renewing, stratified, non-keratinisng squamous epithelium (Daniels et al., 2001) (see poster). The transparent nature of the cornea is largely due to specific features of the corneal stroma. Particularly important characteristics in this respect include the absence of blood vessels, the distinct organisation of collagen fibres and the low numbers of stromal cells (Xuan et al., 2016). The corneal epithelium lines the external surface of the stroma and protects it from environmental insults. It is therefore essential for the maintenance of the attributes of the stroma that enable transparency. Furthermore, unlike keratinising epithelia such as the epidermis, in which the outermost cell layers replace their cytoplasm with keratin proteins, the corneal epithelium maintains living cells at the surface, further aiding transparency.

Anatomically, the corneal epithelium is continuous with the epithelium that lines the conjunctiva (Dziasko and Daniels, 2016). However, they are separated by a junctional zone known as the limbus and are phenotypically distinct, as they express distinct cytokeratins (cytokeratin 3 and 12 in the cornea, cytokeratin 13 in the conjunctiva) (Notara et al., 2010a) (see poster). In addition, the conjunctiva contains mucin-producing goblet cells, which are absent from the cornea (Notara et al., 2010a). The unique attributes of the corneal epithelium are essential in allowing it to function as an effective barrier, while simultaneously remaining fully transparent.

Given the barrier function that the corneal epithelium performs and the range of insults it is exposed to, its long-term maintenance is critical and is mediated by epithelial stem cells that reside within the tissue. Presently, our understanding of how corneal epithelial stem cells (CESCs) are regulated during homeostasis, repair and disease remains incomplete, and further elucidation of the cellular and molecular mechanisms that control CESC function will have important clinical implications. In particular, this will advance our knowledge of a variety of disorders of the ocular surface that can cause blindness and will potentially lead to new therapeutic strategies that can restore the corneal epithelium and thus visual acuity. This Cell Science at a Glance article will summarise our current understanding of the biology of CESCs, with a particular focus on the role the stromal microenvironment, or niche, plays in regulating stem cell function.

The location of CESCs has been intensively investigated for a number of years and remains a highly active and somewhat controversial area of research. The prevailing and widely accepted model is that CESCs reside exclusively at the limbus, which is at the junction between the cornea and the conjunctiva (see poster). The evidence supporting this is based on a variety of studies. Firstly, epithelial cells within the basal layer of the limbal epithelium exhibit characteristics of immature, undifferentiated cells that are consistent with the presence of stem cells (Daniels et al., 2001; Dziasko and Daniels, 2016; Notara et al., 2010a). Specifically, epithelial cells within this location lack expression of cytokeratin 3 and 12, which are expressed by mature, differentiated corneal epithelial cells, while they retain the expression of cytokeratin 14, which is expressed by immature stem or progenitor cells in the basal layer of a variety of stratified epithelia (Daniels et al., 2001; Dziasko and Daniels, 2016; Notara et al., 2010a). In addition, many cells within the limbus also express putative stem cell markers. These include the ΔN isoform of p63 (also known as TP63), which is expressed by proliferative stem or progenitor cells in several stratified epithelia, and the transporter protein ABCG2, which confers the so-called ‘side-population’ phenotype and is often considered to be a universal stem cell marker (Daniels et al., 2001; Dziasko and Daniels, 2016; Notara et al., 2010a). Other putative stem cell markers expressed by cells within this region include N-cadherin and Fzd7 (Dziasko and Daniels, 2016). Furthermore, it has been demonstrated that the limbal epithelium contains a high proportion of quiescent cells that rarely divide, a property that is exhibited by long-lived stem cells in a variety of other tissues (Cotsarelis et al., 1989). Although the expression pattern of these markers is generally consistent with the presence of stem cells, it is important to emphasise that a definitive phenotype for corneal epithelial stem cells, which correlates with bona fide stem cell activity, remains to be determined.

In light of this, the most convincing evidence supporting the presence of stem cells in the limbus is the demonstration that cells isolated from this region can readily generate long-term proliferative clones in vitro (holoclones) and can reconstitute the cornea upon grafting (Daniels et al., 2001, 2007). Indeed, the utility of stem cells that have been isolated from the limbus is powerfully demonstrated by their use in the clinic, as they can be used to regenerate the cornea in patients who have suffered severe damage to the ocular surface following injury or disease (Daniels et al., 2001, 2007).

While the above data supports the hypothesis that stem cells capable of long-term maintenance of the corneal epithelium are present in the limbus, whether or not CESCs reside exclusively in the limbus remains an open question. A recent investigation found evidence for stem cell activity throughout the ocular surface in a variety of mammalian species, including mouse, pig and rabbit (Majo et al., 2008). In this study, stem cells could be isolated from several regions of the cornea in addition to the limbus, although they were present in higher numbers within the limbal epithelium and peripheral cornea. Furthermore, a lineage-tracing experiment demonstrated that cells derived from the limbus only contribute to the cornea during repair, and that they remain dormant during homeostasis (Majo et al., 2008). These results therefore suggest that stem cells in the limbus do not significantly contribute to the homeostasis of the corneal epithelium, but do perform an important regenerative function following injury.

One possible explanation for these findings may be that the CESCs in the limbus are a dormant population that becomes activated only during wound healing, whereas CESCs present in the corneal epithelium perform the bulk of the task of routine homeostatic maintenance. The presence of multiple populations of stem cells is consistent with findings obtained from other self-renewing tissues, such as the epidermis, intestine and bone marrow (Arwert et al., 2012; Tian et al., 2011; Wilson et al., 2008). Interestingly, in these tissues, there is considerable evidence that different stem cell compartments contribute differentially to tissue maintenance depending on the circumstances. For example, in both the intestine and the bone marrow, dormant populations of stem cells have been identified, which only become active during wound healing, whereas a separate stem cell compartment mediates the homeostatic maintenance of the tissue. It is therefore possible that limbal stem cells are a dormant population of stem cells that function only in extreme circumstances, whereas maintenance during normal homeostasis is performed by other stem or progenitor cells that are distributed throughout the cornea. Further work is needed to definitively establish the relative contributions of limbal and non-limbal stem cells during both homeostasis and repair of the ocular surface. However, it is clear that the corneal epithelium contains stem cells that exhibit remarkable regenerative capacity.

The activity of stem cells in any system needs to be tightly regulated in order to ensure that a tissue remains in equilibrium. In this respect, the tissue microenvironment, or niche, in which stem cells reside plays a critical role in regulating stem cell fate decisions (Watt and Hogan, 2000). The stem cell niche thus represents a highly specialised, anatomically distinct region of a tissue that provides the appropriate microenvironmental cues required to maintain a population of cells that is capable of meeting the regenerative demands of a tissue at any given time. Examples of stem cell niches include the crypt base of the small intestine, in which intestinal epithelial stem cells reside (Sato et al., 2011), and the bulge region of hair follicles (see Box 1), where cutaneous epithelial stem cells are found (Arwert et al., 2012). Within these specialised microenvironments, a variety of signals are provided, which ensure appropriate stem cell activity. However, the precise cellular and molecular mechanisms by which the niche regulates stem cell behaviour is only beginning to be elucidated. Nevertheless, it is becoming clear that a variety of niche components, such as vasculature, mesenchymal cells and the extracellular matrix (ECM), play a critical role in providing a range of cues that influence stem cell fate decisions (Watt and Hogan, 2000; Watt and Huck, 2013). These include soluble biochemical factors, mechanical cues and metabolic factors, as well as cell-contact-dependent signals (Watt and Hogan, 2000).

With respect to the cornea, despite the uncertainty surrounding the location and identity of CESCs in the ocular surface, all of the available experimental evidence indicates that they are highly enriched in the limbal epithelium. The limbus is thus a region of considerable interest with regards to identifying the niche components that regulate CESCs (see poster).

In the human ocular surface, there are several features of the limbus that distinguish it from both the conjunctiva and the cornea. Perhaps most strikingly, the stromal tissue in the limbus forms papillae-like invaginations known as the ‘Palisades of Vogt’, in between which are limbal epithelial crypts (see poster) (Dziasko and Daniels, 2016). Within these crypt structures, a high proportion of basal epithelial cells express putative stem cell markers, such as Fzd7, N-cadherin and ABCG2, which is consistent with the notion that the limbus provides a specialised stromal environment that is capable of supporting CESCs (Dziasko and Daniels, 2016). In addition, the limbal stroma is heavily vascularised and contains distinct ECM components compared to the corneal stroma (α1 and α2 collagen IV, β2 laminin and vitronectin) (Dziasko and Daniels, 2016), all of which may be critical in maintaining CESCs. There is also evidence that direct physical interactions between mesenchymal cells in the limbal stroma and epithelial cells in the limbal crypts are important in maintaining the stem cell population (Dziasko and Daniels, 2016).

The molecular mechanisms by which the various components of the limbal stroma may regulate the CESC population require further elucidation. However, the expression of specific biochemical factors by limbal stromal cells has been implicated in stem cell maintenance. Examples include Wnt ligands (Dziasko and Daniels, 2016; Nakatsu et al., 2011; Ouyang et al., 2014), which are important in other stem cell niches such as the intestinal crypt (Clevers et al., 2014), and cytokines and chemokines such as IL-6 (Notara et al., 2010b). Cell-contact-dependent pathways, such as the Notch signalling cascade, as well as direct interactions with ECM components and vasculature, may also be important (Dziasko and Daniels, 2016).

An additional feature that has been shown to be important in regulating stem cell function is the mechanical properties of the surrounding tissue. Factors, such as elasticity and topography, have been shown to influence how a cell responds to other microenvironmental cues, such as growth factors and/or cytokines (Aragona et al., 2013). In this regard, stem cell niches often exhibit a distinct topography and are composed of specific ECM components, each of which will endow the niche with particular mechanical traits (Hsu et al., 2014; Watt and Huck, 2013). In this regard, the dome-like shape of the cornea is likely to impose distinct mechanical forces at different regions of the tissue (Majo et al., 2008), which may favour stem cell maintenance at specific locations. Furthermore, the specific ECM composition of the limbal stroma may also confer distinct mechanical properties. Consistent with this, there is some evidence that the limbus is slightly stiffer than the central cornea (Nowell et al., 2016). The niche also maintains appropriate metabolic conditions for stem cell maintenance, including nutrient availability and oxygen tension, each of which can have a profound effect on stem cell function (Rovida et al., 2014).

Thus, the distinct characteristics of the limbal stroma, such as ECM composition, vascularisation and growth factor expression, are likely to play an important role in maintaining a functional population of CESCs. Insight into the cellular and molecular mechanisms by which niche components regulate CESCs may be provided by examining how cutaneous epithelial stem cells are regulated by their microenvironment (see Box 1). In this tissue, biochemical factors secreted by mesenchymal cells located within the stem cell niche have been shown to regulate processes such as stem cell quiescence and activation. For example, expression of bone morphogenetic proteins (BMPs) by mesenchymal niche components promotes quiescence of cutaneous epithelial stem cells (Plikus et al., 2008), whereas expression of fibroblast growth factors (FGFs), TGF-β and the BMP inhibitor noggin promote their activation and proliferation (Greco et al., 2009; Oshimori and Fuchs, 2012). Functional studies in mice have also indicated that the vasculature plays an important role in the activation of cutaneous epithelial stem cells, although the mechanisms remain to be elucidated (Hsu et al., 2014). Sonic hedgehog (SHH) secretion by other epithelial components of the skin has also been shown to promote cell cycle entry in quiescent cutaneous epithelial stem cells (Hsu et al., 2014). Furthermore, other components present within the cutaneous stem cell niche, such as peripheral nerves, immune cells and ECM components have also been implicated in regulating stem cell activity (Hsu et al., 2014). It will be interesting to determine whether similar cellular and molecular mechanisms operate in the cornea to control the function of CESCs. In addition, as referred to above, there are multiple stem cell compartments in the skin that each have their own distinct niche. Therefore, given that stem cell activity has been demonstrated at multiple locations on the ocular surface, it will be important to establish whether distinct niches are also present to support this organ.

There are several ocular surface disorders in which aberrant function of CESCs results in pathological changes in the corneal epithelium. One of the most common manifestations is conjunctivalisation of the cornea, whereby the corneal epithelium is replaced by conjunctival epithelial cells (Notara et al., 2010a). This is thought to occur due to depletion of CESCs in the limbus (limbal stem cell deficiency), resulting in migration of the conjunctiva onto the corneal surface (Notara et al., 2010a). Because of the significant phenotypic differences between the conjunctival and corneal epithelium, a range of abnormalities ensue, including opacification, neovascularisation and increased susceptibility to injury, all of which cause considerable pain and can result in corneal blindness (Notara et al., 2010a). Conditions that cause a depletion of corneal epithelial stem cells include severe chemical or thermal injury, chronic limbitis (inflammation of the limbus), contact lens keratopathy and Stevens–Johnson syndrome (an acute inflammatory disease) (Puangsricharern and Tseng, 1995). In each case, the depletion in stem cells is thought to arise from either the primary injury or the resulting inflammatory response.

Primary genetic disorders may also induce CESC deficiency. For example, in the hereditary condition aniridia, haploinsufficiency of Pax6, which is essential for normal ocular development, results in a variety of ocular surface defects, including impaired corneal differentiation, conjunctavilisation and neovascularisation (Li et al., 2008). Studies using mice have suggested that the corneal defects observed in aniridia may result from a reduced activity of CESCs and thus represent a form of limbal stem cell deficiency (Collinson et al., 2004).

Another pathological condition of the corneal epithelium is squamous cell metaplasia, in which the normally non-keratinising corneal epithelium adopts a keratinised phenotype similar to that of the epidermis (Notara et al., 2010a). This condition frequently occurs following chronic inflammation of the ocular surface and also severely impairs visual acuity (Chen et al., 2009). The underlying cellular basis of this phenomenon is thought to be the transdifferentiaion of CESCs or their immediate progeny into a keratinising epithelia, a notion that is supported by several studies using mouse models (Mukhopadhyay et al., 2006; Vauclair et al., 2007). Indeed, it is likely that CESCs, like other epithelial stem cells, are multipotent (Bonfanti et al., 2010; Donati and Watt, 2015) and thus have the ability to generate a variety of epithelial lineages, in addition to the cornea. Interestingly, stem or progenitor cells isolated from the corneal epithelium have been shown to give rise to conjunctival-like cells in vitro (Majo et al., 2008), which could be relevant for corneal conjunctivalisation.

The loss of normal CESC function during disease may be a consequence of pathological changes that occur in the stem cell niche (see poster). As described above, many of the pathologies that ultimately result in stem cell deficiency or depletion involve abnormal immune or inflammatory responses. Such abnormal immune responses can induce profound changes in the microenvironment to which CESCs are exposed and thus impair their maintenance and/or survival. For example, immune-mediated pathology can damage niche components and induce conditions such as fibrosis in the sub-epithelial stroma (Aceves and Ackerman, 2009; Bonnans et al., 2014; Notara et al., 2010a). Significant metabolic changes in the niche can also occur (e.g. changes in oxygen tension and nutrient availability) (Rovida et al., 2014), and the expression of a variety of growth factors and cytokines can be induced in mesenchymal stromal cells (Bonnans et al., 2014; Grivennikov et al., 2010). Similar damage to the niche may also occur following chemical or thermal injury, either as a direct consequence of the insult or in response to the subsequent inflammatory response induced. Primary genetic disorders may also impair stem cell function through alterations to the niche. For example, in aniridia, there is evidence that Pax6 deficiency impairs the interaction between CESCs and mesenchymal cells in the limbal stroma (Ramaesh et al., 2005). Thus, the initial effect of ocular surface disease or injury might be the destruction or alteration of the niche, which subsequently results in the loss of microenvironmental cues that would normally maintain CESCs (see poster).

Abnormal function of corneal epithelial stem cells during disease or injury might also be a consequence of changes that occur in the niche. For example, squamous cell metaplasia of the ocular surface that occurs during chronic inflammation might be a result of niche remodelling. Indeed, our recent study has shown that chronic inflammation of the ocular surface induces increased ECM deposition in the corneal stroma, which subsequently leads to increased tissue stiffness (Nowell et al., 2016). This in turn elicits aberrant mechanotransduction in corneal epithelial stem and/or progenitor cells and promotes their conversion into a keratinising epithelium that resembles the epidermis (see poster). Thus, the abnormal differentiation of corneal epithelial stem and/or progenitor cells is an indirect consequence of microenvironmental changes induced by chronic inflammation.

In addition to improving our understanding of ocular surface disease, further elucidation of how exactly the niche changes during disease or injury, and how this subsequently affects CSCE function, will potentially improve the efficacy of CESC transplantation. The use of limbus-derived stem cells to restore the corneal surface following disease or injury is an important clinical application of CESCs. However, its success can be limited in patients in which the corneal stroma is severely damaged (Liang et al., 2009; Samson et al., 2002). Thus, an improved understanding of the factors that differentiate a ‘healthy’ niche from a diseased or dysfunctional niche may enable the development of clinical strategies that can improve the outcome of CESC transplantation.

CESCs, like other stem cells, are exquisitely sensitive to the microenvironment they are exposed to and inappropriate microenvironmental cues can result in their depletion and/or impaired function. Therefore, a more detailed understanding of how CESCs are regulated by the niche and how niche components change during disease or injury has the potential to result in improved therapeutic strategies for the treatment of a variety of ocular surface disorders. To this end, the identification of molecular markers that unambiguously identify CESCs will be a significant advance, as it will enable researchers to study the interactions between CESCs and their microenvironment in a more refined and comprehensive manner.