Epithelial stem cells of the eye surface

OBJECTIVES: Epithelial stem cells of the eye surface, of the cornea and of the conjunctiva, have the ability to give rise to self renewal and progeny production of differentiated cells with no apparent limit. The two epithelia are separated from each other by the transition zone of the limbus. The mechanisms adopted by stem cells of the two epithelia to accomplish their different characteristics, and how their survival, replacement and unequal division that generates differentiated progeny formation are controlled, are complex and still poorly understood. They can be learned only by understanding how stem cells/progenitors are regulated by their neighbouring cells, that may themselves be differently unspecialised, forming particular microenvironments, known as 'niches'. Stem cells operate by signals and a variety of intercellular interactions and extracellular substrates with adjacent cells in the niche. Technical advances are now making it possible to identify zones in the corneal limbus and conjunctiva that can house stem cells, to isolate and expand them ex vivo and to control their behaviour creating optimal niche conditions. With improvements in biotechnology, regenerative cornea and conjunctiva transplantation using adult epithelial stem cells becomes now a reality. RESULTS AND CONCLUSIONS: Here we review our current understanding of stem cell niches and illustrate recent significant progress for identification and characterization of adult epithelial stem cells/progenitors at cellular, molecular and mechanistic levels, improvement in cell culture techniques for their selective expansion ex vivo and prospects for a variety of therapeutic applications.     

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.     

Adult corneal epithelium basal cells possess the capacity to activate epidermal, pilosebaceous and sweat gland genetic programs in response to embryonic dermal stimuli

Recent work has shown remarkable plasticity between neural and hematopoeitic, as well as between hematopoeitic and muscle stem cells, depending on environmental stimuli (Fuchs, E. and Segre, J. A. (2000) Cell 100, 143-155). Stem cells give rise to a proliferative transient amplifying population (TA), which is generally considered to be irreversibly committed. Corneal epithelium provides a particularly useful system for studying the ability of TA cells to activate different genetic programs in response to a change in their fibroblast environment. Indeed, corneal stem and TA cells occupy different localities - stem cells at the periphery, and TA cells more central (Lehrer, M. S., Sun, T. T. and Lavker, R. M. (1998) J. Cell Sci. 111, 2867-2875) - and thus can be discretely dissected from each other. It is well known that pluristratified epithelia of cornea and skin display distinct programs of differentiation: corneal keratinocytes express keratin pair K3/K12 and epidermal keratinocytes keratin pair K1-2/K10; moreover, the epidermis forms cutaneous appendages, which express their own set of keratins. In our experiments, central adult rabbit corneal epithelium was thus associated either with a mouse embryonic dorsal, upper-lip or plantar dermis before grafting onto nude mice. Complementary experiments were performed using adult mouse corneal epithelium from the Rosa 26 strain. The origin of the differentiated structures were identified in the first case by Hoechst staining and in the second by the detection of beta-galactosidase activity. The results show that adult central corneal cells are able to respond to specific information originating from embryonic dermis. They give rise first to a new basal stratum, which does not express anymore corneal-type keratins, then to pilosebaceous units, or sweat glands, depending of the dermis, and finally to upper layers expressing epidermal-type keratins. Our results provide the first evidence that a distinct TA cell population can be reprogrammed.     

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.     

Letter from the Guest Editor

Understanding the mechanisms of stem cell proliferation, self-renewal and differentiation is fundamental for stem cell biology. Stem cells proliferate by either symmetric division or asymmetric division. Through asymmetric division, stem cells self-renew and differentiate to mature cells. Stem cells could also divide symmetrically to give rise to differentiated cells. Besides intrinsic cues, proliferation and self-renewal of most stem cell types also rely on extrinsic signals from niche or surrounding cells. Failure in any of these factors may result in disturbed stem cell proliferation, self-renewal or differentiation and/or generate cancer stem cells that drive cancer development.     

MicroRNA-145 regulates human corneal epithelial differentiation

Background

Epigenetic factors, such as microRNAs, are important regulators in the self-renewal and differentiation of stem cells and progenies. Here we investigated the microRNAs expressed in human limbal-peripheral corneal (LPC) epithelia containing corneal epithelial progenitor cells (CEPCs) and early transit amplifying cells, and their role in corneal epithelium.

Methodology/Principal Findings

Human LPC epithelia was extracted for small RNAs or dissociated for CEPC culture. By Agilent Human microRNA Microarray V2 platform and GeneSpring GX11.0 analysis, we found differential expression of 18 microRNAs against central corneal (CC) epithelia, which were devoid of CEPCs. Among them, miR-184 was up-regulated in CC epithelia, similar to reported finding. Cluster miR-143/145 was expressed strongly in LPC but weakly in CC epithelia (P = 0.0004, Mann-Whitney U-test). This was validated by quantitative polymerase chain reaction (qPCR). Locked nucleic acid-based in situ hybridization on corneal rim cryosections showed miR-143/145 presence localized to the parabasal cells of limbal epithelium but negligible in basal and superficial epithelia. With holoclone forming ability, CEPCs transfected with lentiviral plasmid containing mature miR-145 sequence gave rise to defective epithelium in organotypic culture and had increased cytokeratin-3/12 and connexin-43 expressions and decreased ABCG2 and p63 compared with cells transfected with scrambled sequences. Global gene expression was analyzed using Agilent Whole Human Genome Oligo Microarray and GeneSpring GX11.0. With a 5-fold difference compared to cells with scrambled sequences, miR-145 up-regulated 324 genes (containing genes for immune response) and down-regulated 277 genes (containing genes for epithelial development and stem cell maintenance). As validated by qPCR and luciferase reporter assay, our results showed miR-145 suppressed integrin β8 (ITGB8) expression in both human corneal epithelial cells and primary CEPCs.

Conclusion/Significance

We found expression of miR-143/145 cluster in human corneal epithelium. Our results also showed that miR-145 regulated the corneal epithelium formation and maintenance of epithelial integrity, via ITGB8 targeting.     

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.     

Identification of epithelial label-retaining cells at the transition between the anal canal and the rectum in mice

In certain regions of the body, transition zones exist where stratified squamous epithelia directly abut against other types of epithelia. Certain transition zones are especially prone to tumorigenesis an example being the anorectal junction, although the reason for this is not known. One possibility is that the abrupt transition of the simple columnar epithelium of the colon to the stratified squamous epithelium of the proximal portion of the anal canal may contain a unique stem cell niche. We investigated whether the anorectal region contained cells with stem cell properties relative to the adjacent epithelium. We utilized a tetracycline-regulatable histone H2B-GFP transgenic mice model, previously used to identify hair follicle stem cells, to fluorescently label slow-cycling anal epithelial cells (e.g. prospective stem cells) in combination with a panel of putative stem cell markers. We identified a population of long-term GFP label-retaining cells concentrated at the junction between the anal canal and the rectum. These cells are BrdU-retaining cells and expressed the stem cell marker CD34. Moreover, tracking the fate of the anal label-retaining cells in vivo revealed that the slow-cycling cells only gave rise to progeny of the anal epithelium. In conclusion, we identified a unique population of cells at the anorectal junction which can be separated from the other basal anal epithelial cells based upon the expression of the stem cell marker CD34 and integrin a6, and thus represent a putative anal stem cell population.     

Identification of epithelial label-retaining cells at the transition between the anal canal and the rectum in mice

In certain regions of the body, transition zones exist where stratified squamous epithelia directly abut against other types of epithelia. Certain transition zones are especially prone to tumorigenesis an example being the anorectal junction, although the reason for this is not known. One possibility is that the abrupt transition of the simple columnar epithelium of the colon to the stratified squamous epithelium of the proximal portion of the anal canal may contain a unique stem cell niche. We investigated whether the anorectal region contained cells with stem cell properties relative to the adjacent epithelium. We utilized a tetracycline-regulatable histone H2B-GFP transgenic mice model, previously used to identify hair follicle stem cells, to fluorescently label slow-cycling anal epithelial cells (e.g., prospective stem cells) in combination with a panel of putative stem cell markers. We identified a population of long-term GFP label-retaining cells concentrated at the junction between the anal canal and the rectum. These cells are BrdU-retaining cells and expressed the stem cell marker CD34. Moreover, tracking the fate of the anal label-retaining cells in vivo revealed that the slow-cycling cells only gave rise to progeny of the anal epithelium. In conclusion, we identified a unique population of cells at the anorectal junction which can be separated from the other basal anal epithelial cells based upon the expression of the stem cell marker CD34 and integrin α6, and thus represent a putative anal stem cell population.Key words: stem cells, transitional epithelium, keratinocyte, slow-cycling, label retaining cell     

Stand by me: Fibroblasts regulation of the intestinal epithelium during development and homeostasis

The epithelium of the small intestine is composed of a single layer of cells that line two functionally distinct compartments, the villi that project into the lumen of the gut and the crypts that descend into the underlying connective tissue. Stem cells are located in crypts, where they divide and give rise to transit-amplifying cells that differentiate into secretory and absorptive epithelial cells. Most differentiated cells travel upwards from the crypt towards the villus tip, where they shed into the lumen. While some of these cell behaviors are an intrinsic property of the epithelium, it is becoming evident that tight coordination between the epithelium and the underlying fibroblasts plays a critical role in tissue morphogenesis, stem-cell niche maintenance and regionalized gene expression along the crypt-villus axis. Here, we will review the current literature describing the interaction between epithelium and fibroblasts during crypt-villus axis development and intestinal epithelium renewal during homeostasis.     

Evolving concepts on the pathogenic mechanisms of aniridia related keratopathy

Heterozygosity for PAX6 deficiency (PAX6+/-) results in aniridia. Corneal changes in aniridia-related keratopathy (ARK) include corneal vascular pannus formation, conjunctival invasion of the corneal surface, corneal epithelial erosions and epithelial abnormalities, which eventually result in corneal opacity and contribute to visual loss. Corneal changes in aniridia have been attributed to congenital deficiency of corneal limbal stem cells. The aim of this paper is to review the potential mechanisms that may underlie the pathogenesis of aniridia related keratopathy. Current evidence, based on clinical observations and an animal model of aniridia suggest that the proliferative potential of the corneal limbal stem cells may not primarily be impaired. The corneal changes in aniridia may be related to an abnormality within the limbal stem cell niche. The mechanisms underlying progressive corneal pathology in aniridia appear multi-factorial and include: (1) abnormal corneal healing responses secondary to anomalous extracellular matrix metabolism; (2) abnormal corneal epithelial differentiation leading to fragility of epithelial cells; (3) reduction in cell adhesion molecules in the PAX6 heterozygous state, rendering the cells susceptible to natural shearing forces; and (4) conjunctival and corneal changes leading to the presence of cells derived from conjunctiva on the corneal surface.     

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.     

上皮肿瘤始于干细胞

人类肿瘤大约90％以上源自于上皮。上皮干细胞是唯一长期存在于上皮组织的细胞,可积累多次突变生成肿瘤。所以,人们认为肿瘤多起源于正常干细胞。然而,目前研究中关于上皮肿瘤干细胞及其与正常上皮干细胞的关系了解甚少。现综述近年来有关上皮干细胞和肿瘤发生的关系及其调控机制方面的研究进展。     

Stem Cell Niche Structure as an Inherent Cause of Undulating Epithelial Morphologies

The spatial organization of stem cells into a niche is a key factor for growth and continual tissue renewal during development, sustenance, and regeneration. Stratified epithelia serve as a great context to study the spatial aspects of the stem cell niche and cell lineages by organizing into layers of different cell types. Several types of stratified epithelia develop morphologies with advantageous, protruding structures where stem cells reside, such as rete pegs and palisades of Vogt. Here, multistage, spatial cell lineage models for epithelial stratification are used to study how the stem cell niche influences epithelial morphologies. When the stem cell niche forms along a rigid basal lamina, relatively regular morphologies are maintained. In contrast, stem cell niche formation along a free-moving basal lamina may prompt distorted epithelial morphologies with stem cells accumulating at the tips of fingerlike structures that form. The correspondence between our simulated morphologies and developmental stages of the human epidermis is also explored. Overall, our work provides an understanding of how stratified epithelia may attain distorted morphologies and sheds light on the importance of the spatial aspects of the stem cell niche.     

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     

CLED: a calcium-linked protein associated with early epithelial differentiation

Although it has been well established that Ca(2+) plays a key role in triggering keratinocyte differentiation, relatively little is known about the molecules that mediate this signaling process. By analyzing a bovine corneal epithelial subtraction cDNA library, we have identified a novel gene that we named CLED (calcium-linked epithelial differentiation), which encodes a messenger RNA present in all stratified squamous epithelia, hair follicle, the bladder transitional epithelium, and small intestinal epithelium. The deduced amino acid sequence of CLED, based on a bovine partial cDNA and its full-length, human and mouse homologues that have been described only as ESTs, contains 2 EF-hand Ca(2+)-binding domains, a myristoylation motif, and several potential protein kinase phosphorylation sites; the CLED protein is therefore related to the S100 protein family. In all stratified squamous epithelia, the CLED message is associated with the intermediate cell layers. Similar CLED association with cells that are above the proliferative compartment but below the terminally differentiated compartment is seen in hair follicle, bladder, and small intestinal epithelia. The only exception is corneal epithelium, where CLED is expressed in both basal and intermediate cells. The presence of CLED in corneal epithelial basal cells, but not in the adjacent limbal basal (stem) cells, provides additional, strong evidence for the unique lateral heterogeneity of the limbal/corneal epithelium. These results suggest that CLED, via Ca(2+)-related mechanisms, may play a role in the epithelial cell's commitment to undergo early differentiation, and that its down-regulation is required before the cells can undergo the final stages of terminal differentiation.     

Limbal stem cells, a review of their identification and culture for clinical use

The surface of the eye is covered by two distinct epithelial populations, the conjunctival and corneal epithelia. The stem cell population for the corneal epithelia has been found to be located at the area known as the limbus. This is a narrow ring of tissue at the transitional zone between the cornea and conjunctiva. This stem cell population is responsible for generating transient amplifying cells which are responsible for renewing the cornea epithelia. There are currently no definitive markers for the stem cell population in the limbus. Instead using morphological features, such as small cells with a high nucleus-to-cytoplasm ratio, in conjunction with the presence of certain markers e.g. ΔNP63α and the absence of others, e.g. the cytokeratin pair 3 & 12, are taken as being indicative of the stem cell population. Damage can occur to the corneal epithelium due to a number of causes including, Steven-Johnson syndrome, and chemical or thermal burns. This results in invasion of the cornea by the conjunctival epithelium resulting in impaired vision. In 1997 Pellegrini et al. (Lancet 349, 990) successfully used cells sheets from cultured limbal cells to successfully treat patients with corneal damage. Since then several other groups, have successfully treated patients, using similar methods.     

Matrix-cytoskeletal interactions in the developing eye

The embryonic avian corneal epithelium in vitro responds to extracellular matrix (ECM) molecules in either soluble or polymerized form by flattening its basal surface, organizing the basal cortical actin cytoskeleton, and stepping up its production of corneal stroma twofold. Embryonic corneal epithelia, like hepatocytes and mammary gland cells, seem to contain heparan sulfate proteoglycan (HSPG) in their plasmalemma, which may interact with actin on the one hand or underlying collagen on the other. Work on the corneal epithelium suggests that, in addition to HSPG, specific glycoprotein receptors for laminin and collagen exist in the basal plasmalemma and play the critical role in actually organizing the basal epithelial cytoskeleton. As yet, uncharacterized proteins may link such receptors to actin. We suggest that ECM-dependent organization of the cytoskeleton is responsible for ECM enhancement of corneal epithelial differentiation. Cell shape and exogenous ECM also affect mesenchymal cell differentiation. In the case of the corneal fibroblast migrating in collagen gels, an actin cortex present around the elongate cell seems to interact with myosin in the cytosol to bring about pseudopodial extension. Both microtubules and actin microfilaments are involved in fibroblast elongation in collagen gels. It follows from the rules presented in this review that the mesenchymal cell surface is quite different from the epithelial cell surface in its organization. Nevertheless, epithelial cell surface-ECM interaction can be modified in the embryo at particular times to permit predesignated epithelial-mesenchymal transformations, as for example at the primitive streak. Though basal surfaces of definitive, nonmalignant epithelia adhere rather strictly to the rules of epithelium-ECM interaction and do not invade underlying ECM, the environment can be manipulated in vitro to cause these epithelia to send out pseudopodia and give rise aberrantly to mesenchymal cells in collagen gels. Further study of this phenomenon should cast light on the manner in which epithelial and mesenchymal cells organize receptors for matrix molecules on their cell surfaces and develop appropriate cytoskeletal responses to the extracellular matrix.     

Stem cell niches

The nature of the stem cell niche and its interaction with stem cells is one of fundamental problems in the biology of stem cells. Stem cell niches are formed during ontogeny. A niche can remain vacant and exist independently of stem cells; however, stem cell self-renewal cannot be maintained for long periods outside of the niche except for particular conditions, e.g., in vitro. A vacant niche can be occupied by excessive or transplanted stem cells and can provide for their functioning. A niche size allows a definite number of stem cells to be maintained. Excessive stem cells either differentiate in the presence of specific signal(s) or undergo apoptosis in the absence of such signal. Thus, the niches control the number of stem cells in the body and protect it from excessive stem cell proliferation. Under particular conditions, stem cells can leave and return to their niches. Stem cells are retained in the niche by cell-to-cell interactions and adhesion to the extracellular matrix. Both the niches and stem cells arise at a particular ontogenetic stage and are capable of long self-renewal. The development can be described in terms of the formation of stem cells and their niches.