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. Author manuscript; available in PMC: 2021 Sep 1.
Published in final edited form as: Exp Eye Res. 2020 Jul 19;198:108094. doi: 10.1016/j.exer.2020.108094

CORNEAL EPITHELIAL BIOLOGY: LESSONS STEMMING FROM OLD TO NEW

Robert M Lavker 1,*, Nihal Kaplan 1, Junyi Wang 1,2, Han Peng 1
PMCID: PMC7508907  NIHMSID: NIHMS1617072  PMID: 32697979

The anterior surface of the eye functions as a barrier to the external environment and protects the delicate underlying tissues from injury. Central to this protection are the corneal, limbal and conjunctival epithelia. The corneal epithelium is a self-renewing stratified squamous epithelium that protects the underlying delicate structures of the eye, supports a tear film and maintains transparency so that light can be transmitted to the interior of the eye (Basu et al., 2014; Cotsarelis et al., 1989; Funderburgh et al., 2016; Lehrer et al., 1998; Pajoohesh-Ganji and Stepp, 2005; Parfitt et al., 2015; Peng et al., 2012b; Stepp and Zieske, 2005). In this review, dedicated to James Funderburgh and his contributions to visual science, in particular the limbal niche, corneal stroma and corneal stromal stem cells, we will focus on recent data on the identification of novel regulators in corneal epithelial cell biology, their roles in stem cell homeostasis, wound healing, limbal/corneal boundary maintenance and the utility of single cell RNA sequencing (scRNA-seq) in vision biology studies.

1. What stem cells have taught us about corneal epithelial biology

Over 30 years ago, a series of seminal papers from the Tung-tien Sun and Robert Lavker laboratories fundamentally altered the manner in which the vision field regarded how corneal epithelial homeostasis was maintained. The initial data coming from the Sun laboratory in 1986 (Schermer et al., 1986), formed the foundation for the concept that stem cells of the corneal epithelium were preferentially located in the limbal epithelial basal layer. Such a conclusion was drawn from keratin expression data demonstrating that keratin 3, a marker for an advanced stage of differentiation was located in the corneal epithelial basal layer but was absent in the limbal epithelial basal layer. This suggested that limbal epithelial basal cells were in a more “primitive”, less differentiated state, than the corneal epithelial basal cells and gave rise to the following model, where the stem cells of the corneal epithelium resided in the limbal basal layer and their progeny (transit amplifying; TA cells) moved centripetally into the corneal epithelium. The second paper published in 1989 from the Lavker laboratory (Cotsarelis et al., 1989), in collaboration with the Sun laboratory, confirmed and extended this concept. Using a technique to localize cells that rarely cycled (label-retaining cells; LRCs), a fundamental characteristic of stem cells, these investigators demonstrated that LRCs were a subpopulation of basal cells preferentially located in the limbal epithelium. Furthermore, following a central corneal epithelial wound, these rarely cycling cells were shown to rapidly enter the proliferative population to aid in the reepithelialization of the corneal epithelium. The third paper in this series, published in 1998 (Lehrer et al., 1998), used a double-labeling technique that enabled investigators to trace the movement of the limbal-located LRC’s (stem cells) that had divided to become TA cells. This technique revealed that the limbal epithelial basal cells were a heterogenous population consisting of both stem and TA cells (Fig. 1). The double-labeled TA cells were observed to migrate into the peripheral corneal epithelium. TA cells were also proposed to be either “early or young” TA cells with considerable proliferative capacity, or “late or mature” TA cells with limited (1-2) rounds of division. These findings also established the concept that, under resting conditions, a TA cell did not have to use all of its replicative capacity prior to becoming a post-mitotic differentiated cell. However, upon stress (e.g., wounding) TA cells used all of their amplification divisions to repair the defect. Collectively, these three papers made major contributions to our current understanding of ocular anterior segmental epithelial homeostasis.

Figure 1. Label-retaining cells represent the limbal epithelial stem cells.

Figure 1.

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Label-retaining cells (red-stained nuclei; referred to as label-retaining cells; LRCs) are located in the basal layer of the limbal epithelium. The TA cells (silver grains in nuclei) are primarily located in the corneal epithelium (Lehrer et al., 1998). An occasional cell with a red (BrdU+ stained nucleus plus silver (tritiated-thymidine) grains is noted, which represents a LRC that has divided. Such a cell would be an early TA cell.

1.1. Limbal location of corneal epithelial stem cells helps explain ocular surface diseases

Based on the work from numerous investigators the original limbal epithelial stem cell (LESC) concept has been continuously modified (Corradini et al., 2012; Li et al., 2007; Pajoohesh-Ganji and Stepp, 2005; Park et al., 2019; Pellegrini et al., 1999; Stepp and Zieske, 2005; Sun et al., 2010; Zieske, 1994). For example, it is now recognized that in addition to quiescence and extensive proliferative capacity, which are wellaccepted general properties of ‘‘sternness”, LESCs express putative stem cell markers (e.g., N-cadherin, ABCB5, ABCG2, Lrig1, keratin 15 (K15), p63). Another important amendment to the stem cell concept was that early TA cells, which represent immediate stem cell progeny, reside in the limbal epithelial basal layer and that LESCs and early TA cells are phenotypically and functionally indistinguishable. Mature or late TA cells are enriched in the central corneal epithelial basal layer (Budak et al., 2005; de Paiva et al., 2005; Ksander et al., 2014; Pellegrini et al., 2001; Richardson et al., 2018).

The impact that the LESC concept has had on the clinical treatment of corneal aberrations cannot be understated. Prior to the late 1980’s corneal transplants were routinely performed using a portion of healthy corneal epithelium to replace the damaged tissue. While this method had a good success rate of restored vision, there were times when persistent corneal epithelial defect occurred and vison became compromised. Current treatments of limbal injury are aimed at replacing the damaged corneal epithelium with either a portion of healthy limbal epithelium or a sheet of cells enriched in limbal progenitor cells. With the realization that stem cells were exclusively located in the limbal epithelium, Kenyon and Tseng (Kenyon and Tseng, 1989) pioneered limbal autograft transplantation for patients with damaged ocular surface epithelium, which delivered a piece of limbus to the damaged area. This dramatically improved the transplant success rate; however, there was an inherent risk of compromising the remaining valuable limbal epithelium. To overcome this potential problem, novel techniques such as ex vivo limbal epithelial stem cell expansion on carriers (e.g., fibrin (Fasolo et al., 2017; Rama et al., 2001; Rama et al., 2010), amniotic membrane (Grueterich et al., 2002; Kim and Tseng, 1995; Le and Deng, 2019; Lee and Tseng, 1997; Shortt et al., 2009; Tsai et al., 2000)) were developed for transplantation to reduce the damage of the healthy eye. Specifically, clinical and experimental evidence showed that cultured limbal epithelial transplantation is effective to maintain a healthy ocular surface in patients with a success rate of 70-80% (Baylis et al., 2011; Shortt et al., 2007b; Utheim, 2013). Transplantation with a high proportion of limbal epithelial stem cells with the capacity of self-renewal as well as carriers that impart anti-inflammatory and anti-angiogenesis properties is the key for graft success. Rama et al (Rama et al., 2010) found that the graft success was associated with the percentage of p63-bright holoclone-forming (stem cell-enriched) cells. One of the problems that still exists is a lack of definitive markers to unequivocally identify limbal epithelial stem cells. Recently, ABCB5 (Ksander et al., 2014) and other markers (Budak et al., 2005; Daniels et al., 2006; de Paiva et al., 2005; Morita et al., 2015; Pajoohesh-Ganji et al., 2004; Sartaj et al., 2017; Watanabe et al., 2004) have been employed to isolate populations of limbal epithelial cells that have certain stem cell properties with the potential for ex vivo expansion.

Another outcome from the LESC theory is the appreciation that several diseases of the ocular surface can be classified as limbal epithelial stem cell deficiencies (LSCD). LSCD is characterized by overgrowth of conjunctival-derived epithelial cells, corneal neovascularization, chronic inflammation and opacification with severe visual loss, eventually leading to blindness (Pellegrini et al., 2013; Puangsricharern and Tseng, 1995; Rama et al., 2010). Many ocular surface diseases, including intrinsic (e.g., Sjogren’s syndrome) or extrinsic (e.g., alkali or thermal burns) insults, which either impair LESCs or the limbal microenvironment/niche for LESCs survival, can lead to LSCD (Dua et al., 2000; Kenyon and Tseng, 1989; Puangsricharern and Tseng, 1995; Singh et al., 2013). Recurrent erosion and persistent corneal epithelial defects can be found in LSCD, presenting with pain, photophobia, and decreased vision (Dong et al., 2018; Liang et al., 2009; Pauklin et al., 2009). In addition to restoring LESCs, the rebuilding of the limbal niche is also crucial for the treatment of LSCD. Limbal niche has unique gene expression patterns and extracellular matrix protein profiles as well as niche cells that are specifically suited to the maintenance and function of LESCs (Grueterich et al., 2003; Li et al., 2007; Notara et al., 2010; Shortt et al., 2007a; Stepp and Zieske, 2005; Yazdanpanah et al., 2019a; Yazdanpanah et al., 2019b). In LSCD, such a niche is frequently damaged by acute/chronic inflammation (Liang et al., 2009; Tseng et al., 2016). LESCs transplanted to an injured ocular surface with a damaged niche, are likely to be lost over time (Tseng et al., 2016; Yazdanpanah et al., 2019a). Strategies for reconstructing the limbal niche are focused on decreasing inflammation and restoration of proper function of the niche cells (Li et al., 2007; Yazdanpanah et al., 2019a). Funderburgh and others showed that mesenchymal stem cells (MSCs) are a group of fibroblast-like multipotent mesenchymal stromal cells and can be isolated from a wide variety of tissues including bone marrow, nervous tissue, adipose tissue, placental tissue and also limbal stroma (Basu et al., 2014; Funderburgh et al., 2016; Ma et al., 2014). Corneal stromal stem cells are a type of MSC, isolated from the limbal stroma and have the capacity to suppress early neutrophil infiltration and T cell proliferation in response to corneal trauma and block fibrotic scarring (Hertsenberg and Funderburgh, 2015). MSCs have also been tested in the treatment of LSCD (Funderburgh et al., 2016; Hertsenberg and Funderburgh, 2015; Massie et al., 2015; Navas et al., 2018; Pinnamaneni and Funderburgh, 2012; Shojaati et al., 2019; Shukla et al., 2020; Zhang et al., 2015) . Transplantation of MSCs shows a similar therapeutic effects in the experimental LSCD model compared with LESC transplantation (Holan et al., 2015). Some studies suggested the differentiation of MSCs into corneal epithelial cells (Gu et al., 2009; Reinshagen et al., 2011). However, most of the studies suggested that MSCs produce and secrete growth factors, microRNAs and immunomodulatory factors through paracrine signaling, and exosomes to improve the corneal microenvironment that can support the activation of the residual LESCs (Lan et al., 2012; Li and Zhao, 2014; Shojaati et al., 2019; Ye et al., 2006; Zhang et al., 2015). Studies have shown that MSCs can produce immunomodulatory factors, which can attenuate the expression of TNFa, INFr and increase the anti-inflammatory cytokines (TGFb and IL1 Ra) as well as improve the limbal niche (Lan et al., 2012; Wen et al., 2014). Except for the soluble factors, MSCs also release extracellular vesicles which can effectively promote regenerative phenotype in an miRNA-dependent manner (Shojaati et al., 2019).

2-. Recent overtures to tackle limbal stem cells

2-1-. Single-Cell RNA Sequencing Helps Redefine the Limbal/Corneal Epithelial Stem/Early Transit Amplifying Cell Population:

By definition, stem cells are a rare population comprising less than 1-2% of the proliferating cells (Becker et al., 1963; Till and Mc, 1961). In most tissues, stem cells and their immediate progeny, the early TA cells are in close apposition to each other, making it difficult to obtain discrete populations for analyses (Cotsarelis et al., 1990; Fuchs, 2018). One of the newest techniques to help circumvent this problem is scRNA-seq. scRNA-seq enables the interrogation of cellular heterogeneity at the resolution of individual cells (Der et al., 2017; Kaplan et al., 2019; Tang et al., 2009; Wen and Tang, 2016; Wu et al., 2018). It is also an ideal means of studying the function of an individual cell or group of similar cells in the context of their microenvironment. To date, there has been little use of scRNA-seq technology to investigate the corneal/limbal epithelial stem and TA cell populations.

Recently, we conducted a scRNA-seq study to define the stem/early TA cell population in normal, resting limbal and corneal epithelia (Kaplan et al., 2019). Unbiased clustering detected 10 distinct clusters (Figure 2). In three mesenchymal cell populations, a subcluster was identified as corneal mesenchymal stem cells based on their (1) low levels of keratocan, (2) expression of Six2 and Scf, and (3) high levels of Fhl 1 and Cd90 (Thy1). These corneal mesenchymal stem cells have anti-fibrotic properties during corneal wound healing (Hertsenberg et al., 2017; Karamichos et al., 2014; Morgan et al., 2016). The remaining seven clusters expressed high levels of Pax6, adherens, and tight junctional as well as keratin genes, all markers of epithelial cells (Kaplan et al., 2019). Gap junctional genes were also preferentially expressed in epithelial cell populations. We identified three limbal/corneal epithelial cell subpopulations designated as stem/early TA, mature TA, and differentiated corneal epithelial cells. In these three corneal/limbal epithelial cell populations, our study established a comprehensive atlas of gene expression. Thus, novel stem cell regulators were identified including Thioredoxin-interacting protein (Txnip) and PDZ-binding kinase (Pbk). TXNIP functions to maintain stem/early TA cell quiescence through G0/G1 cell cycle arrest via p27kip1. This makes excellent biological sense, as quiescence is one of the features of limbal epithelial stem cells and Txnip may be a limbal epithelial-preferred gene that contributes, in part, to stem cell maintenance. We determined that PBK arrested corneal epithelial cells in G2/M phase of the cell cycle contributing to a rarely cycling phenotype (Kaplan et al., 2019). Collectively, our study forms the foundation for further dissecting the roles of stem/TA cell populations in maintaining epithelial homeostasis. Therefore we expanded our scRNA-seq analysis to study the contribution of autophagy to stem cell maintenance, as we have demonstrated that autophagy plays an important role in maintaining the proliferative capacity of limbal epithelial stem cells (Park et al., 2016).

Figure 2. Single-cell RNA sequencing identifies stem/early TA, late TA and differentiated corneal epithelial cell populations.

Figure 2.

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Single-cell RNA sequencing identifies 10 clusters of cells from wild type mouse limbal/corneal tissues. t-SNE plot of 2,513 cells are visualized. Cells are colored by the clusters. Three of 7 clusters represent epithelia: Conjunctival epithelium (1 and 10) stem/early TA (3), late TA (8), differentiated corneal epithelial cell (2, 4, 5) populations (Kaplan et al., 2019).

2-2-. Autophagy contributes to limbal epithelial stem cell homeostasis:

Autophagy plays critical roles in maintenance of tissue homeostasis and is an important protective factor in tissues governed by stem cells. This self-cannibalistic process begins with the induction of double-membranes (initiation). Initiation starts with a stress signal, which releases the inhibition of mTOR on the UNC51-like kinase (ULK1). The later recruits and phosphorylates Atg13, Fip200, and Atg101. Such a ULK1 complex activates Beclin1, the key factor in the PI3K complex that is essential for phagophore formation. The PI3K complex recruits WIPI via PtdIns3P leading to the formation of a nascent phagophore. The last stage is autophagosome elongation and closure involving an Atg12 / Atg5 / Atg7 cascade catalyzing a ubiquitin like conjugation. This cascade results in the lipidation of LC3I to LC3II onto forming double-membrane autophagosomes. Followed by maturation, autophagosomes fuse with lysosomes to form autolysosomes, where degradation is conducted. End-stage autophagy is when autolysosomes are recycled to reform lysosomes via autophagic lysosome reformation (ALR; Figure 3A) (Chen and Yu, 2013; Peng et al., 2017a, b; Rong et al., 2012; Rong et al., 2011; Yazdanpanah et al., 2019a).

Figure 3. Autophagy positively affects epithelial proliferative capacity.

Figure 3.

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(A) Scheme of autophagic flux (Peng et al., 2017a). (B) Immunohistochemical staining of BrdU+ (brown) cell in central corneal epithelia of Beclin-1+/− mice and littermate control (Beclin-1+/+), 24 h after wounding (Park et al., 2016). (C) Model proposing how autophagy regulates epithelial proliferation via increased PBK and H2AX expression and decreased ATF3 expression, resulting in stem/TA cell activation (Kaplan et al., 2019).

The autophagic process in the limbal/corneal epithelia has been largely ignored. Using a GFP-LC3 transgenic mouse, we observed a high degree of autophagy in the stem cell-enriched limbal epithelial basal layer compared with more differentiated corneal epithelial basal cells (Park et al., 2016). Active autophagy in the stem cell-enriched limbal epithelium is consistent with autophagy being a stem cell protective factor (Boya et al., 2018). Thus, it is not surprising that scRNA-seq revealed less stem cells in Beclin1 deficient mice, which is required for the early stages of autophagy (Kaplan et al., 2019). Such a reduction in stem cells and their immediate progeny is consistent with the marked reduction in holoclone colonies from human limbal epithelial cells with compromised autophagy (Park et al., 2016). A decrease in expression of Ki67 was seen in the Beclin+/− mice versus wild-type in both the stem/early TA cell populations (Kaplan et al., 2019). Consistent with an apparent compromise in limbal epithelial stem cell behavior, corneal epithelial wound-induced proliferative response of mice deficient in Beclin 1 (Beclin1+/−; Figure 3B) was reduced (Park et al., 2016). Specifically, we noted a significant reduction in BrdU-labelled cells in these mice compared with littermate controls. This suggests that autophagy may be a necessary component for activation of the limbal epithelial stem cell/TA cell populations. It is also possible that such reduced activation in limbal epithelium can be due to a reduced number of stem cells in Beclin1 deficient limbal epithelium (Kaplan et al., 2019), setting a precedent for future investigations.

These observations indicate a critical role for autophagy in maintaining stem cell numbers, which may occur via ensuring proper stem cell self-renewal. Interestingly, we observed dysregulation of ATF3 and PBK, genes associated with the cell cycle and stress response, which may provide valuable insight into how autophagy functions as a positive regulator of proliferation in the stem/early TA cells (Kaplan et al., 2019). Wild-type mice had high expression of PBK in the corneal epithelium. We suggest that such a reduction in PBK might also help explain the compromised wound-healing response in the Beclin 1+/− mice. We propose that in the context of re-epithelialization following wounding, one of the functions of autophagy is to ensure a timely upregulation of epithelial proliferation. To accomplish this, PBK expression is promoted, whereas ATF3 expression is attenuated, resulting in stem/TA cell activation (Figure 3C). Future studies are required to explore how these genes are regulated.

2-3-. microRNAs regulate limbal epithelial stem cell homeostasis

miRNAs are small (~22 nucleotides in length), “noncoding” or “non-messenger” RNAs that are part of the RNAi silencing machinery (for reviews see (Ambros, 2004; Berezikov, 2011; Friedman et al., 2009; Lavker et al., 2009; Lewis et al., 2005; Ying and Lin, 2004; Zamore and Haley, 2005)). A “target-centric” approach is common to miRNA investigations using miRNAs as a discovery tool to identify proteins that were not previously associated with either the corneal or limbal epithelia. For example, in the corneal epithelium, miR-205 targets the lipid phosphatase SHIP2 (Yu et al., 2008). SHIP2 was not formerly considered in the context of stratified squamous epithelia, but is now known to regulate the actin cytoskeleton and migration, as well as cell survival in corneal epithelial cells (Li et al., 2010; Yu et al., 2010; Yu et al., 2008). The corneal epithelial-preferred miR-31 (Ryan et al., 2006), targets factor inhibiting hypoxia-inducible factor-1 (FIH-1) (Peng et al., 2012a). FIH-1, a previously unknown component of the ocular surface epithelium is now recognized as a pleiotropic hydroxylase that negatively regulates corneal epithelial differentiation via Notch signaling (Peng et al., 2012b) and glycogen stores via a c-kit/Akt/GSK-3β signaling pathway (Peng et al., 2012a; Peng et al., 2013), and positively regulates proliferation via p63 signaling (Kaplan et al., 2020; Peng et al., 2012b). More recently, we found that miR-184, the most corneal epithelial-preferred miRNA (Ryan et al., 2006), directly targets and represses the proangiogenic factors, friend of Gata 2 (FOG2), platelet-derived growth factor (PDGF-β), which is essential for the recruitment of pericytes by PDGF-βR–expressing endothelial cells, and phosphatidic acid phosphatase 2b (PPAP2B) to maintain corneal avascularity (Park et al., 2017). A mutation of the seed region of miR-184 is also reported to be responsible for many ocular anterior abnormalities, e.g. severe familiar keratoconus with early-onset anterior polar cataract (Hughes et al., 2011) as well as endothelial dystrophy, iris hypoplasia, congenital cataract, and stroma thining (EDICT syndrome) (Iliff et al., 2012). miR-184 is also expressed in corneal endothelial cells (Frausto et al., 2014; Zhao et al., 2013), which may contribute to the secretion of anti-angiogenic factors.

Despite its importance in maintaining corneal epithelial homeostasis, much less attention has been directed at the miRNA signature in the stem cell-enriched limbal epithelium. To address this deficiency, we used a combinatorial approach of laser capture microdissection to isolate relatively pure populations of limbal epithelial basal cells (stem cell-enriched) and corneal epithelial basal cells (TA-enriched) with miRNA expression profiling (Peng et al., 2015). Such profiling demonstrated that the miR-103/107 family are preferentially expressed in the basal cells of the stem cell-enriched limbal epithelium and target novel proteins involved in processes related to stem cells (Park et al., 2015; Peng et al., 2015) . We showed that miRs-103/107 target p90RSK2, a kinase that regulates G0/G1 progression and in this manner helps maintain a slowcycling phenotype. The proliferative capacity of human epidermal and limbal keratinocytes can be enhanced by ectopic expression of the miR-103/107 family, in part, by targeting Wnt3a. Furthermore, we showed that miRs-103/107 target mitogenactivated protein kinase kinase kinase 7 (MAP3K7) and thereby negatively regulated the p38/AP-1 pathway. The negative regulation of p38 by miRs-103/107 contributed to enhanced proliferative capacity, a hallmark of stem cells. Since miRs-103/107 also promoted increased holoclone colony formation by regulating JNK activation through non-canonical Wnt signaling, we believe that this microRNA family preserves “sternness” by mediating the crosstalk between the Wnt/JNK and MAP3K7/p38/AP-1 pathways (Park et al., 2015; Peng et al., 2015). By targeting NEDD9 (HEF1), miRs-103/107 insured maintenance of the essential stem cell niche molecule, E-cadherin (E-cad) in limbal keratinocytes. We also demonstrated that the tyrosine phosphatase PTPRM is a target of miRs-103/107 and thereby maintained low levels of Cx43, which is a feature of several stem cell-enriched epithelia (Park et al., 2015; Peng et al., 2015). Collectively, these findings indicate that miRs-103/107 play critical roles in orchestrating multiple aspects of the adult epithelial stem cell (Figure 4). One of the major challenges of ex vivo epidermal and corneal epithelial cell therapy is expansion and preservation of stemness from a small biopsy. Since ex vivo expansion of limbal-derived stem cells is a crucial step in treating corneal blindness, we believe that ectopic expression of miRs-103/107 may represent a novel means of expanding the proliferative capacity of the limbal-derived epithelial cells.

Figure 4. miR-103/107 regulates LESC homeostasis.

Figure 4.

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Limbal preferred miR-103/107 maintains limbal stemness by directly targeting NEDD9 to regulate adherens junctions, PTPRM to regulate Gap junctions, p90RSK2 to regulate cell cycle (Peng et al., 2015) and MAP3K7 to regulate proliferative capacity (Park et al., 2015).

Other miRNAs have been identified within the limbal/corneal epithelia. For example, Teng et al identified several limbal epithelial-enriched microRNAs (miR-10b, 126, 127, 139, 142-3p, 143, 145, 155, and 338) by microRNA array profiling. KEGG pathway analysis suggested that miR-10b, 126, 139-5p, 143, 145, 155 were strongly associated with stem cell regulation (Teng et al., 2015). Among them, overexpression of miR-10b significantly increased two putative LESC markers, KRT15 and KRT17, and the pluripotent stem cell marker, OCT4. Interestingly, DKK1, a Wnt signaling inhibitor, is a predicted direct target of miR-10b. In submerged cultures of HCECs and organ-cultured corneas, miR-10b negatively regulated DKK1 expression. This observation lead to the idea that miR-10b plays a positive role in maintaining LESC homeostasis via sustaining Wnt signaling (Kulkarni et al., 2017). miR-146a is another limbal epithelial-enriched microRNA (Kulkarni et al., 2017; Lee et al., 2011). Overexpression of miR-146a in HLECs resulted in an upregulation of Frizzled-7, and K15. Conversely, knockdown of miR-146a caused downregulation of these putative LESC markers (Winkler et al., 2014). Finally, microRNAs also have a role in the differentiation of stem cells. For example, miR-450b represses SOX2 and induces differentiation of limbal epithelial stem/progenitor cells (Bhattacharya et al., 2019). These findings further support the roles of microRNAs in limbal stem cell homeostasis and opens up a world of possibilities for treatment modalities in the corneal epithelial field.

3-. EphA2/EFNA1 and Limbal/Corneal epithelial boundary: keeping the stem cells in place

Physical and functional boundaries are present throughout living systems, and they are dynamic in nature to accommodate the functions of a specific tissue. Boundaries within these tissues are formed between populations of cells that do not identify each other as the same (Taylor et al., 2017; Ventrella et al., 2017). A similar boundary exists within the anterior segmental epithelium where more differentiated corneal epithelial basal cells are clearly segregated from their limbal epithelial basal cells enriched in progenitor cells (stem/early TA) in the limbus (Kaplan et al., 2018; Lavker et al., 2004; Pajoohesh-Ganji and Stepp, 2005; Schermer et al., 1986; Stepp and Zieske, 2005; Tseng et al., 2016). Expression patterns of several factors show compartmentalization at the limbal/corneal junction. For instance, calcium-linked, epithelial differentiation protein (CLED), early epithelial differentiation protein (EEDA) as well as miR-184 expression are compartmentalized in the corneal epithelium and their expression sharply stops at the limbal epithelium (Ryan et al., 2006; Sun et al., 2006; Sun et al., 2000). Conversely, α9-integrin, ABCG2 and enolase, are specifically expressed in limbal epithelial basal cells (Chung et al., 1995; Morita et al., 2015; Pajoohesh-Ganji and Stepp, 2005; Watanabe et al., 2004) (Figure 5).

Figure 5. Model of limbal/corneal epithelial boundary.

Figure 5.

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Schematic representation of human cornea (adapted from Li et al., 2007) and the reciprocal expression patterns of several signaling molecules, structural proteins and microRNAs across the limbal/corneal epithelia boundary. The reciprocal distribution of ephrin-A1 and EphA2 that overlaps in the peripheral corneal epithelium may help set the boundary between the limbus and cornea to support centripetal migration of epithelial cells and to maintain progenitor cell homeostasis in the limbus (Kaplan et al., 2018). LESC, limbal epithelial stem cells; e-TA, early transit amplifying cells; l-TA, late transit amplifying cells.

There is a constant need for regeneration of the corneal epithelium so that homeostasis can be maintained against environmental insults that disrupt the steadystate, self-renewing feature of this epithelium. This suggests the presence of a fluid boundary between epithelial cells that are in a constant state of flux at the limbal /corneal epithelial junction. Such a boundary is capable of altering its characteristics depending on changes in the surrounding environment. The signaling pathways that regulate the boundary between limbal and corneal epithelia have been the focus of numerous investigators (Blanco-Mezquita et al., 2011; Blanco-Mezquita et al., 2013; Cotsarelis et al., 1989; Lavker et al., 2004; Park et al., 2017; Ryan et al., 2006). Complete knowledge of what regulates a functionally integrated boundary between limbal/corneal epithelial cells and the molecular basis for organizing limbal and corneal epithelial tissue compartments will have implications for diseases where the limbal/corneal boundary is compromised such as LSCD, chronic inflammation and diabetes. Eph/ephrin-mediated cell-cell communication has been implicated in: (i) maintaining such boundaries via mediating transitions from proliferation to differentiation; (ii) regulating epithelial cell migration during tissue repair; and (iii) tissue patterning in general by differentially modulating cell adhesion and repulsion (Genander, 2012; Genander and Frisen, 2010; Kaplan et al., 2012; Kaplan et al., 2018; Lin et al., 2012; Perez White and Getsios, 2014; Perez White et al., 2017; Ventrella et al., 2017). Our previous work in this area, strongly suggests the contribution of Eph/ephrin signaling to limbal/corneal fluidity (Figure 5).

Eph receptors are a large family of receptor tyrosine kinases that mediate cell-cell communication when engaged by ephrin ligands found on the surface of neighboring cells. The unique juxtracrine positioning of Eph receptors and their ligands in opposing cells make them ideal signaling molecules for tissue patterning and boundary formation and they are abundantly expressed in adult epithelia (Lin et al., 2012; Miao et al., 2009). EphA and EphB receptors and their ligands have also been found in the anterior ocular surface epithelia (Kaplan et al., 2018; Kojima et al., 2007; Narayanan et al., 2005; Swamynathan et al., 2008). Expression of EphA1, EphA2, EphA3, ephrin-A1, ephrin-A2 have been detected in mouse corneal epithelium (Kojima et al., 2007).

Interestingly, ephrin-A1 ligand is concentrated in the limbal epithelium whereas the EphA2 receptor is more abundant in the corneal epithelium (Kaplan et al., 2018) (Figure 5). The reciprocal expression of these signaling molecules between limbus and cornea suggested a correlation between corneal epithelial homeostasis and EphA2/ephrin-A1 expression patterns in the anterior segmental epithelium. EphA2/ephrin-A1 signaling complexes regulate corneal epithelial cell migration, and an increase in ephrin-A1 expression could be involved in the delayed wound healing observed in diabetes (Kaplan et al., 2012). The reciprocal expression pattern of this receptor/ligand pair creates a driving force during recovery from an injury in the cornea while modifying signaling pathways that organize cell-cell adhesion and repulsion. Similarly, after an injury to the cornea, the expression levels of both total and unligated EphA2 (S897-EphA2) are increased following a decrease in ephrin-A1 levels in mouse cornea, which supports an increased migratory response (Kaplan et al., 2018; Miao et al., 2009).

We have shown that EphA2/ephrin-A1-induced repulsion of epithelial cells towards the injured area was dependent on increased EGFR signaling (Kaplan et al., 2018) via ADAM10 cleavage of EGFR ligands. EphA2 recruitment of ADAM10 to the confrontation site could be important for the shedding of EGFR ligands to mobilize corneal epithelial cells, which are dependent on EGFR signaling, similar to an ADAM17 dependent EGFR ligand shedding (Yin and Yu, 2009) in order to activate the migratory wound repair response. Our findings support the notion that the balance between EphA2 and ephrin-A1 defines a complex mechanism controlling steady state homeostasis of a fluid boundary at the limbal-corneal junction. Another functionally segregated boundary occurs between limbus and conjunctiva. The EphA2/ephrin-A1 expression pattern of conjunctiva is similar to its expression pattern in limbal epithelium in the human eye; however, there is a sharp boundary of ephrin-A1 between limbal and conjunctival epithelia in mouse eye. Despite these differences, there is little known about the role of Eph/ephrins in the conjunctiva. In disease conditions such as pterygium, the restricted expression pattern of ephrin-A1 is disrupted (John-Aryankalayil et al., 2006), further supporting a correlation between corneal epithelial homeostasis and EphA2/ephrin-A1 expression patterns in the cornea.

4. Conclusions and future perspectives

A healthy limbal/corneal epithelium requires coordinated actions of several signaling molecules and pathways intrinsic to these cells. In this review, we have discussed recent findings on the relationships between stem and early TA cells within the limbal epithelium and novel molecules, including miRNAs and the process of autophagy that regulates this important proliferative population. Finally, we have underscored the importance of the precise boundary between the limbal and corneal epithelia and provide information on how such a boundary is achieved and maintained. Our scRNA-seq data opens up a large amount of information for defining the boundary in the cornea. Compared with the traditional profiling of bulk populations, scRNA-seq methodologies enable the interrogation of relatively rare cell populations (e.g., stem and early TA cells, corneal stromal stem cells, inflammatory cell populations) (Kaplan et al., 2019). We believe that by dissecting out the unique gene signatures of these cell populations, we can understand the steady state limbal corneal boundary, as well as what happens when there is a detrimental situation such as burns and LESC deficiency.

HIGHLIGHTS.

  • Limbal/corneal epithelial stem cell biology

  • Application of single cell RNAseq to limbal epithelial stem cells

  • The role of autophagy in stem cell maintenance

  • How microRNAs contribute to limbal epithelial stem cell homeostasis

  • Eph/ephrins help to regulate the limbal/corneal epithelial boundary

Acknowledgements:

This research is supported by National Institutes of Health Grants EY06769, EY017539 and EY019463 (to R.M.L.); a Dermatology Foundation research grant and Career Development Award (to H.P.); an Eversight research grant (to H.P.), and the International Postdoctoral Exchange Fellowship Program 20180087 (to J. Wang).

Footnotes

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