Ординатура / Офтальмология / Английские материалы / Ocular Periphery and Disorders_Dartt, Bex, Amore_2011

calmodulin, whereas one type of channel in this domain is activated as a direct consequence of emptying of intracellular Ca2+ stores. This channel is formed by a tetrameric configuration of transient receptor potential (TRP) protein subunits. Each of the different types of TRP channels identified in the corneal epithelium is activated by different stimuli. Currently, three different types are expressed and studied in corneal epithelial cells. Even though their activation leads in all cases to increases in plasma membrane Ca2+ influx, the types of responses elicited are very different from one another. The four identified types are TRPC4, TRPV1, TRPV4, and TRPM8. In human cornea epithelial cells, TRPC4 is activated during exposure to EGF. TRPV1 is stimulated by an agonist contained in an extract of red peppers (capsaicin) leading to increases in proinflammatory cytokine expression. TRPV4 is activated by a decreased bathing-solution osmolarity and elicits a RVD response. On the other hand, TRPM8 is temperature sensitive. The Ca2+ signaling role attributable to activation of each of these three different TRP isoforms is dependent on the ability of endoplasmic reticulum Ca2+ transporters to rapidly take up into intracellular stores the Ca2+ that has flowed in through these different plasma membrane TRP channels. Otherwise, a sustained rise in intracellular Ca2+ concentration may be cytotoxic. The importance of Ca2+ signaling in mediating receptor control of corneal epithelial renewal and transparency suggests that its modulation by drugs is of potential value in a clinical setting.

K+, Cl–, and Na+ Channels

Modulation of K+ channel activity is essential for mediating different responses associated with corneal epithelial function. For example, changes on K+ channel activity mediate EGF-induced mitogenic responses, ultraviolet (UV) lightinduced apoptosis, and adrenoceptor-induced increases in net Cl transport. In these cells, there is expression of Ca2+-dependent and inwardly rectifying K+ channels. Even though activation of these different K+ channels types induces intracellular K+ losses, the responses are very different from one another. Additional studies are required to understand how their activation leads to such disparate responses.

Chloride channel activity modulation occurs in response to adrenergic receptor-induced increases intracellular Ca2+ concentration and rises in intracellular cyclic adenosine monophosphate (cAMP) levels. Corneal epithelial cells express cystic fibrosis transmembrane regulator (CFTR) channels that are stimulated by rises in cAMP and underlie stimulation of net Cl transport. They also express a Cl channel designated as ClC-3 and their activation is suggested to underlie a regulatory volume response mediating shrinkage during exposure to a hypotonic challenge.

There is some evidence for the expression of tetrodotoxin-blockable Na+ channels in corneal epithelial cells. The functional importance of such activity is unclear since their activation only occurs at membrane voltages far less negative than those described in corneal epithelial cells.

Summary

Ion transporter and membrane permeability regulation are essential to the maintenance of corneal epithelial health. Such control is essential for mediating responses that are required for corneal epithelial renewal and the maintenance of corneal transparency. The corneal epithelial cells express both primary and secondary ionic transport mechanisms whose regulation occurs through receptor activation. These ion transporters in concert with changes in membrane permeability underlie: (1) osmotically coupled fluid flow; (2) cell-volume regulation; (3) intracellular pH regulation; and (4) Ca2+ signaling. Such control assures that epithelial renewal maintains barrier function and the finetuning contribution capability of the corneal epithelium to elicit adequate fluid egress from the cornea for sustaining tissue transparency. Additional studies are warranted to identify novel drug targets in the signaling pathways mediating receptor control of ion transporter and channels. This endeavor will possibly identify improved techniques for restoring corneal epithelial function subsequent to injury.

See also: Corneal Epithelium: Response to Infection; Dry Eye: An Immune-Based Inflammation; Stem Cells of the Ocular Surface; The Surgical Treatment for Corneal Epithelial Stem Cell Deficiency, Corneal Epithelial Defect, and Peripheral Corneal Ulcer.

Further Reading

Al-Nakkash, L., Iserovich, P., Coca-Prados, M., Yang, H., and Reinach, P. S. (2004). Functional and molecular characterization of a volume-activated chloride channel in rabbit corneal epithelial cells.

Journal of Membrane Biology 201: 41–49.

Al-Nakkash, L. and Reinach, P. S. (2001). Activation of a CFTRmediated chloride current in a rabbit corneal epithelial cell line.

Investigative Ophthalmology and Vision Science 42: 2364–2370. Bildin, V. N., Yang, H., Crook, R. B., Fischbarg, J., and Reinach, P. S. (2000). Adaptation by corneal epithelial cells to chronic hypertonic

stress depends on upregulation of Na:K:2Cl cotransporter gene and protein expression and ion transport activity. Journal of Membrane Biology 177: 41–50.

Candia, O. A. and Alvarez, L. J. (2008). Fluid transport phenomena in ocular epithelia. Progress in Retinal and Eye Research 27: 197–212.

Capo´-Aponte, J. E., Wang, Z., Bildin, V. N., Pokorny, K. S., and Reinach, P. S. (2007). Fate of hypertonicity-stressed corneal epithelial cells depends on differential MAPK activation and p38MAPK/Na-K-2Cl cotransporter1 interaction. Experimental Eye Research 84: 361–372.

Lu, L. (2006). Stress-induced corneal epithelial apoptosis mediated by K+ channel activation. Progress in Retinal Eye Research

25: 515–538.

Lu, L., Reinach, P. S., and Kao, W. W. (2001). Corneal epithelial wound healing. Experimental Biology and Medicine 226: 653–664.

Pan, Z., Yang, H., Mergler, S., et al. (2008). Dependence of regulatory volume decrease on transient receptor potential vanilloid 4 (TRPV4) expression in human corneal epithelial cells. Cell Calcium 44: 374–385.

Reinach, P. S., Capo´-Aponte, J. E., Mergler, S., and Pokorny, K. S. (2008). Roles of corneal epithelial ion transport mechanisms in mediating responses to cytokines and osmotic stress.

In: Tombran-Tink, J. and Barnstable, C. J. (eds.) Ocular Transporters: In Ophthalmic Diseases and Drug Delivery, Series Ophthalmology Research, pp. 17–46. Totowa, NJ: Humana Press.

Reinach, P. S., Holmberg, N., and Chiesa, R. (1991). Identification of calmodulin-sensitive Ca2+-transporting ATPase in the plasma membrane of bovine corneal epithelial cell. Biochimica et Biophysica Acta 1068: 1–8.

Wolosin, J. M. and Candia, O. A. (1987). Cl– secretagogues increase basolateral K+ conductance of frog corneal epithelium. American Journal of Physiology 253: C555–C560.

Wu, X., Yang, H., Iserovich, P., Fischbarg, J., and Reinach, P. S. (1997). Regulatory volume decrease by SV40-transformed rabbit corneal epithelial cells requires ryanodine-sensitive Ca2+-induced Ca2+ release. Journal of Membrane Biology 158: 127–136.

Yang, H., Mergler, S., Sun, X., et al. (2005). TRPC4 knockdown suppresses epidermal growth factor-induced store-operated channel activation and growth in human corneal epithelial cells.

Journal of Biological Chemistry 280: 32230–32237.

Yang, H., Sun, X., Wang, Z., et al. (2003). EGF stimulates growth by enhancing capacitative calcium entry in corneal epithelial cells.

Journal of Membrane Biology 194: 47–58.

Zhang, F., Yang, H., Wang, Z., et al. (2007). Transient receptor potential vanilloid 1 activation induces inflammatory cytokine release in corneal epithelium through MAPK signaling. Journal of Cellular Physiology 213: 730–739.

Glossary

Basement membrane – Every epithelial sheet elaborates a noncellular dual-layered membrane of collagen and proteoglycans to which the cells are anchored. In addition to anchoring the cells, basement membranes (also called basal laminae) serve to maintain epithelial differentiation and to provide a biological barrier preventing contact between the epithelial cells and the underlying mesenchymal tissue.

Cell therapy – Stem cells are injected directly into pathological tissues to restore tissue function. Conjunctiva – The squamous epithelium overlaying the sclera and inner eyelid.

Corneal stroma – This tough connective tissue provides 90% of the corneal thickness and consists mostly of collagen, proteoglycans, and water. Cells make up 4% of the stromal tissue.

Crypts – These structures, originally named in the intestine, represent small dead-end tubes that protrude from the surface of an epithelial layer into the surrounding mesenchymal tissue. Cytokeratin – Hair and the epidermis consist primarily of the fibrous proteins, keratins. Moist

epithelia such as intestine and cornea were originally thought not to be ‘keratinized’ but the outer layers cells in these tissues express intracellular keratin proteins as they differentiate. Due to the large number of keratin genes, keratin expression is highly tissue specific.

Endothelium – Corneal endothelium is a single epithelial layer of cells on the posterior (inner) side of the cornea which provides a hydrodynamic pumping function to maintain corneal hydration. It is unrelated to vascular endothelium.

Keratocyte – These are dendritic, quiescent cells of the corneal stroma. They produce the complex extracellular matrix of the corneal stroma but, during wound healing, also produce an opaque scar tissue. Limbus – In the eye, the border between the transparent cornea and the opaque sclera is known as the limbus. The limbal region of the overlying epithelia is continuous with no obvious boundary, but the conjunctival and corneal cells can be distinguished by the types of cytokeratins that are expressed.

Neural crest – In early embryonic development a small population of cells between the ectoderm and

the neural tube migrates out into the embryo, differentiating into a wide variety of tissues including autonomic nerves, cartilage, smooth muscles, and melanocytes. A number of ocular tissues (corneal stroma and iris) are derived from neural crest. Niche – A stem cell niche is a phrase loosely used to describe the microenvironment in which stem cells are found, which interacts with stem cells to regulate stem cell fate.

Squamous epithelium – Multilayered epithelia show differences between the basal cells attached to the basal lamina and the more superficial cells of the layer. The apical cells flatten and express tissuespecific keratins. These outer cells die by apoptosis and leave the layer (known as desquamation) to be replaced by underlying cells. Single-layer epithelia such as the corneal endothelium do not undergo the process of desquamation and renewal.

The Ocular Surface

Anatomy

Beneath the protective tear film, the human ocular surface is covered with a contiguous layer of moist squamous epithelium, consisting of the bulbar conjunctival epithelium over the sclera and the transparent corneal epithelium of the cornea (Figure 1). The bulbar conjunctival epithelium rests on a loose, vascularized connective tissue which allows the movement of the eyelid over the sclera and maintains the limbal vascular supply. This epithelium is contiguous with the corneal epithelium and with the conjunctiva of the fornix located in the folded region between the sclera and the eyelid. The conjunctival epithelium contains goblet cells – the source of soluble mucins in the tear film. These cells are an essential element in maintenance of the integrity of the ocular surface. The cornea is covered with a phenotypically unique epithelial sheet 5–11-cell layers deep which is transparent to light and devoid of goblet cells. The flattened cells of the corneal surface overlie wing-shaped cells – which, in turn, rest on a cuboidal basal epithelium. Unlike the conjunctival epithelium, the corneal epithelium is firmly connected to rigid tissue of the stroma via an anchoring complex involving keratin and collagen fibrils extending into the acellular anterior collagenous layer of the stroma. The interface between the corneal and conjunctival epithelium is known as the

corneoscleral limbus (Figure 1(a)). This region features a network of physical folds in the surface of stromal tissue known as the palisades of Vogt (Figure 1). As described below, the palisades harbor a small population of cells identified as limbal stem cells (LSC). The limbal region also contains pigmented cells and a population of immune cells related to the Langerhans’ cells of the dermis.

Underlying the corneal epithelium is the corneal stroma, a tough, transparent connective tissue making up 90% of the thickness of the cornea (Figure 1(c)). The stroma contains layers (lamellae) of aligned collagen fibrils sandwiching quiescent keratocytes, flattened mesenchymal cells. The specialized arrangement of the collagenous ultrastructure in the stroma is responsible for the remarkable tensile strength of this tissue as well as its unique light transparency. On the posterior side of the stroma a single cell layer – the corneal endothelium – is separated from the stromal tissue by Descemet’s membrane. The three tissue layers of the cornea each make an important physiological contribution to maintenance of corneal transparency. In addition, the cornea provides 75% of the refractive power required to focus the light on the retina and acts as an effective biological barrier.

Development

Formation of the human cornea begins at approximately 5–6 weeks of gestation. After the lens vesicle pinches off from the surface ectoderm of the head, the overlying

ectoderm transforms into a layer of cuboidal epithelial cells, which continue to develop into the corneal epithelium. At 6–7 weeks, neural crest cells migrate between this epithelium and the lens, forming the corneal endothelium. Shortly afterward, a second wave of cell migration from neural crest forms the stroma – which then begins to generate collagenous matrix in the 8th week.

Homeostasis and Repair

The cellular layers of the cornea and conjunctiva differ markedly in several important characteristics of homeostasis and repair. In the corneal epithelium, the superficial cells are lost by exfoliation and replaced by the underlying wing cells. These, in turn, are continually replaced by mitotically active basal cells. In addition to the basal to superficial migration of corneal epithelial cells, there is a well-documented migration of epithelial cells from the periphery to the center of the cornea, measured at 2–3 mm per day. The dynamics of the corneal epithelium have been characterized as the X,Y,Z equation, in which the exfoliation rate (X ) equals the rate of production of new cells from the basal cells (Z) plus cells added by the centripetal migration of cells (Y ). When injured, the corneal epithelium closes wounds first by migration of the intact sheet over the denuded area, followed by a delay before increased cell division is observed. Bulbar conjunctival epithelium is contiguous with the corneal epithelium without any obvious physical barrier; however,

these cells express different cytokeratin markers and are found not to migrate in the unwounded eye. Cell division by basal cells and exfoliation is observed throughout the conjunctiva.

The cells of the corneal stroma – the stromal keratocytes – show little cell division in normal adults. The keratocytes undergo rapid cell division after localization in the cornea in late embryogenesis, but, following birth, the keratocyte cell number stabilizes and little or no mitosis can be detected throughout life. In the case of inflammation or wounding, however, the stromal keratocytes become activated and mitotic. The activated keratocytes adopt a fibroblastic phenotype and, later, display characteristics of myofibroblasts. Connective tissue secreted by these cells during wound healing is not transparent but becomes opaque scar tissue. Following healing, the cells become quiescent, but human corneal scars are very slow to resolve; it is not clear if the resident cells ever return to a fully keratocytic phenotype. These properties suggest a limited process of tissue renewal in the corneal stroma.

Renewal of corneal endothelial cells is even more limited than that of the keratocytes. Following childhood, human corneal endothelial cells do not divide. Compensation for endothelial damage is accomplished by flattening of the remaining cells to cover the posterior surface of the cornea. In vitro as well, human corneal endothelial cells show only limited ability to divide following infancy.

These characteristics have led to a conventional view that, while the corneal epithelium is maintained by a stem cell population, the stroma and the endothelium – with limited ability for self-renewal – are not maintained by mitotically active, tissue-resident stem cells.

Properties of Stem Cells

Stem cells, by definition, undergo asymmetric cell division; that is, they undergo self-renewal while giving rise to differentiated daughter cells. Embryonic stem cells derived from the inner cell mass of the blastocyst are pluripotent, giving rise to most cells of the body. In culture, embryonic stem cells can be propagated indefinitely in an undifferentiated state. Although they can be isolated from umbilical cord blood, fully pluripotent stem cells are thought to be scarce or absent in adult tissues. Stem cells in adult organisms have long been associated with self-renewing tissues, such as the hematopoietic system, dermis, and intestine. The resident stem cells in these tissues are generally capable of generating only one type of cell, making them unipotent. In recent years, understanding of a new class of stem cells has emerged: known as mesenchymal stem cells (MSC), these cells appear to be present in small numbers in many somatic tissues and can also be isolated from bone marrow. Studies

show these MSC to participate in injury repair, and – when expanded in culture – the MSC exhibit potential to differentiate into a number of lineages, and thus can be considered as being multipotent. Considerable effort has gone into identifying common characteristics of adult stem cells, but in spite of such efforts there appears to a dearth of global phenotypic markers for such cells. In vitro, clonal growth, extended life span, and the ability to express phenotypic markers of multiple differentiated cell types have been useful in stem cell identification, but cell surface markers for stem cells – as well as properties enabling isolation of these populations – often need to be defined for individual tissues.

In self-renewing tissues, stem cells appear to be localized in an anatomical niche, a restricted microenvironment which interacts with stem cells to regulate stem cell fate. The niche provides physical and chemical factors that both control the replication and maintain the differentiation potential of the stem cells. Typically, the niche is near a vascular bed, usually containing a population of unrelated cells that serve as feeder cells.

Stem Cells in the Corneal Epithelium

Characteristics of Limbal Stem Cells

Abundant research supports the idea that a stem cell population is localized in the corneoscleral limbal region. These cells – termed limbal stem cells (LSC) – share a number of features with the stem cells of other selfrenewing tissues. They have small cell size and high nuclear-to-cytoplasmic ratio. They lack expression of differentiation markers expressed by corneal epithelial cells, specifically cytokeratins (CK) 3 and 12. Like stem cells in other self-renewing tissues, the LSC divide very infrequently (slow cycling), and, therefore, DNA-labeling agents – such as bromodeoxyuridine – are retained in these cells for months. Label retention has often been used in identifying stem cell candidates in intact tissues. Cells from the limbal region also grow clonally in large colonies known as holoclones, whereas clones from the central cornea are less abundant and grow through fewer population doublings. Furthermore, unlike central corneal cells, LSC proliferation is resistant to inhibition by phorbol esters. LSC express several genes linked to stem cell self-renewal, including CEBPD, Bmi1, and Notch1. Cornea-specific ablation of Notch1 resulted in differentiation of LSC into hyperplastic, keratinized, epi- dermal-like cells, indicating the probability that Notch1 expression is essential in maintenance of the LSC stem potential. LSC also express a transporter protein, ABCG2, responsible for efflux of fluorescent dye Hoechst 33342. Expression of ABCG2 enables isolation of cells using fluorescence-activated cell sorting (FACS). Cells isolated using this dye-efflux assay are known as a side population,

based on the distribution pattern of the fluorescent cells following FACS. Side-population cells from the corneal epithelium are present exclusively in the limbus; following sorting, they exhibit slow cycling and clonal growth properties consistent with their identification as stem cells. Ability to isolate LSC has allowed a detailed comparison of the differences between this population and the other cells of the corneal epithelium. A summary of these differences is given in Table 1.

Limbal Stem Cell Niche

Centripetal migration of pigmented cells in healing epithelial wounds was first observed in the 1940s, and, in 1971, Davanger and Evenson proposed that the source of the migrating cells was the palisades of Vogt. These are a series of radially oriented fibrovascular ridges concentrated along the upper and lower corneoscleral limbus (Figure 1(a) and (b)) described originally in the early twentieth century. The morphology of these features has been elucidated by Daniels and by Dua, showing that they are present throughout the limbus but more concentrated in the superior and inferior regions. The ridges and valleys of the palisades are maintained by a specialized vascular system and form an interwoven network, often terminating in tunnels or ‘crypts’ under the surface of the stroma (Figure 1(b)). The bases of these crypts contain cells strongly expressing the several marker genes used in identifying LSC (Table 1), providing further evidence that these crypts represent the LSC niche. Recently, it was observed that pigmented melanocytes in the limbal crypts express N-cadherin, as do the LSC. During expansion in

Table 1 Expression markers for determining epithelial stem cell differentiation

Modified from Secker and Daniels (2008).

culture the LSC lose N-cadherin expression as they differentiate. N-cadherin-dependent cell–cell interactions with melanocytes may consequently represent a feature of the niche environment involved in maintaining the slow cycling and stem cell potential of the LSC.

The presence of label-retaining cells in the limbus and the centripetal migration of the more rapidly dividing cells in the corneal epithelium prompted Schermer and colleagues to suggest that the slow-cycling LSC are precursors to the mitotic basal cells throughout the epithelium. These mitotic daughter cells are termed transient amplifying (TA) cells. The TA cells are hypothesized to migrate from the limbus throughout the central cornea before ceasing cell division and differentiating to the superficial cells of the corneal surface. Labeling with both tritiated thymidine and bromodeoxyuridine followed by various chase periods provided kinetics showing that migrating basal cells are indeed mitotic, but are not label retaining – that is, cycle faster than the LSC. The LSC appear small and round compared to the TA cells, thus may be more primitive than the TA cells. Cloning studies also show the mitotic cells in the central stroma to be less able than LSC to generate the large holoclones indicative of high replicative potential.

TA cells are not fully committed to corneal epithelial differentiation. TA cells in adult central corneal epithelium – when transplanted to embryonic dermis – have demonstrated potential to differentiate into hair follicles and other dermal tissue types. Thus, these basal cells of the central epithelium maintain a clear stem cell potential. Are these central corneal stem cells progeny of the LSC? DNA-labeling kinetics are consistent with an interpretation that slow-cycling cells in the limbus are the origin of mitotic cells of the central cornea. Lamellar keratoplasty in male rabbits with corneal tissue from females led to the gradual replacement of the sex chromatin in the central corneal graft, suggesting that LSC from the host were the source of the cells that mature and exfoliate from the superficial central cornea. In spite of these and a number of related studies, however, there is still only indirect evidence that LSC are the progenitors of the mitotic basal TA cells in undisturbed corneal epithelium. In fact (as discussed below), recent studies suggest the possibility that the LSC may contribute to the central corneal epithelial cell populations only in healing wounds.

Limbal Stem Cell Deficiency

It is well documented that, following wounding, the LSC become more mitotically active and participate in centripetal migration. Cells from the limbus can, in fact, rapidly repair wounds involving the entire central corneal epithelium. However, injuries or conditions resulting in loss or inactivation of the LSC present a markedly

different outcome. After loss of the limbal cells, cells from the conjunctiva – including goblet cells – migrate across the corneal surface. This conjunctivalization results in inflammation, neovascularization, and growth of a fibrovascular pannus which severely reduces visual acuity. This phenomenon, known as limbal stem cell deficiency, can result from either hereditary or acquired causes. Transplantation of central corneal tissue without the accompanying LSC provides only temporary amelioration of this condition. Successful treatment, however, can be affected by autologous transplantation of limbal tissue from a contralateral, unaffected eye to the conjunctivalized eye. Reestablishing the LSC population can restore a stable and transparent corneal epithelium both in experimental animal studies and in human patients. Allografts can serve the same purpose but require immune suppression to prevent rejections. Expansion of LSC in culture has also been used to prepare epithelial sheets that restore long-term human epithelial function as well. It is worth noting that autologous, cultured epithelial sheets from buccal epithelial cells – as well as from conjunctival cells cultured under conditions in which goblet cells do not form – can also restore epithelial function. The biological implications of this use of nonlimbal cells to restore corneal epithelial function have not been fully explored. It is not clear, for example, if stem cells in these cultured layers colonize the limbal crypts and become a kind of ectopic, but functional, LSC population. The conclusions of the studies on limbal stem cell deficiency and its therapy are that the LSC are effective in replacing the corneal epithelial layer in a wound healing situation, and that the LSC create a biological barrier preventing conjunctival migration onto the corneal surface.

Understanding the Role for LSC in Corneal Homeostasis

Several studies have suggested the possibility that during normal corneal homeostasis the LSC may not contribute cells to the corneal epithelium. Studies of the destrinknockout mouse showed that these mice lack the centripetal migration of corneal epithelium. Destrin is an actin-binding protein involved in cytoskeletal dynamics and thus may be involved directly in such motility. The corneal epithelium forms correctly in these mice, but, later in life, the tissue becomes vascularized and hyperplastic. The presence of mitotic basal cells in the absence of migration suggests that the basal cells are not TA progeny of the LSC but arise from a distributed stem cell population in these mice. Thus, the LSC may not be necessary for establishing a fully differentiated corneal epithelium. In a later study using mice, limbal tissue expressing a b-galactosidase marker gene was transplanted to the limbus of non-b-Gal- expressing nude mice. Over a period of months, b-Gal was not found in the central cornea of unwounded mice, but

b-Gal cells rapidly migrated into the central cornea after wounding. b-Gal-expressing cells from central cornea were able to reconstitute a full epithelial sheet following transplantation, and furthermore, the reconstitution after transplantation could be carried out serially. These results indicate the existence of a nonlimbal population of corneal epithelial stem cells in mice that can give rise to a functional corneal epithelium. Such studies indicate LSC may not be necessary for formation or normal homeostasis of mouse cornea but support the principle that the LSC does participate in restoration of the tissue after wounding. There are no experimental data extending these results to humans, but until such data are presented, the role of the LSC in normal tissue homeostasis of the central corneal epithelium should be considered as unresolved.

Conjunctival Stem Cells

The bulbar conjunctiva contains basal mitotic cells that migrate to the surface and become quiescent as they differentiate; however, the cell layer does not undergo a lateral migration similar to that of the cornea. Differentiated conjunctival epithelium also differs from corneal epithelium in the expression of cytokeratins. Whereas cornea is positive for CK3 and CK12, conjunctiva lacks these keratins while expressing CK19, an antigen absent in cornea. Label-retaining cells can be observed in the fornix region, and clonal culture of these gives rise to both epithelial cells and goblet cells. Thus, the fornix appears to contain bipotent stem cells. Similarly, cells in the fornix are more strongly stimulated to proliferate by phorbol ester than those in the bulbar epithelium. These studies support the idea of a stem cell population in a fornicial niche. Clonogenicity studies, however, support the idea that cells with extended replicative potential (e.g., stem cells) are present both in the fornix and the bulbar surface. The lack of lateral migration of the cells of the bulbar conjunctiva, however, strongly supports the idea that during normal tissue homeostasis the tissue is maintained by a distributed population of resident stem cells.

Careful examination of stem-like cells in the bulbar conjunctiva has identified rare clusters of cells expressing the CK3/12 keratins characteristic of the cornea. Furthermore, transplantation of limbal b-galactosidase-expressing tissue into the limbal region of nude mice showed that, following wounding, some of these cells migrate into the conjunctiva and differentiate to both conjunctival epithelial and goblet cell phenotypes. These studies suggest that some of the stem cells distributed in the bulbar conjunctiva may derive from the LSC. Conversely, cells cultured from the bulbar region can be used to reconstitute the corneal epithelium in transplantation studies. Following transplantation to the cornea, these conjunctival-derived cells express the cornea-specific keratins CK3/12. The idea of

conjunctival/corneal transdifferentiation, once popular, is currently out of favor; however, these experiments support the possibility that stem cells in both the conjunctiva and cornea can – and do, in fact – produce differentiated cells in the neighboring tissue. The extent and the conditions under which this phenomenon occurs require further elucidation.

Corneal Stromal Stem Cells

The corneal stroma is a mesenchymal connective tissue making up 90% of the corneal thickness, with physical properties that provide the cornea its essential character. The stroma is formed during late embryogenesis by a population of neural crest cells migrating from the periocular mesenchyme. Chicken keratocytes from late embryogenesis retain neural crest progenitor properties even after transplantation into a new environment along cranial neural crest migratory passageways. In adult mammals, however, numerous in vitro experiments show that keratocytes rapidly lose their characteristic phenotype following several population doublings. Such a loss of phenotype occurs in healing wounds in vivo as well as in vitro. Recently, the authors found that the stroma of bovine corneas contain a small population of cells exhibiting self-renewal ability for an extended number of population doublings in culture. These corneal stromal stem-like cells were clonogenic and proliferated in vitro for over 100 doublings. A similar population of stem cells was isolated from human corneas as a side population using FACS. These stromal stem cells demonstrated potential for differentiation into several noncorneal cell types – a characteristic similar to that found in adult stem cells from other mesenchymal tissues. These cells also expressed several genes clearly designating them as tis- sue-specific mesenchymal stem cells, including ABCG2, Notch1, BMI1, SIX2, and KIT. ABCG2-expressing cells were localized in the limbal stroma subjacent to the epithelial basement membrane in the regions near the LSC niche.

Human corneal stromal cells remained viable for months after injection into mouse corneal stroma and were able to increase the transparency of lumican-knockout mouse corneas. In serum-free monolayer culture, keratocytes express stroma-specific gene products, but do not accumulate or organize extracellular matrix resembling that of the corneal stroma. However, when human stromal stem cells were cultured in attachment-free conditions they produced a stromal-like tissue including the stroma-specific keratan sulfate proteoglycan keratocan and generated parallel layers of collagen fibers resembling those in the stroma. These results suggest the potential use of stromal stem cells in bioengineering of corneal stroma or in direct stem cellbased therapy for corneal scars or corneal dystrophies.

Corneal Endothelial Stem Cells

The corneal endothelium is a single layer of flat hexagonal cells forming a boundary between the corneal stroma and the anterior chamber. This layer of cells functions as a pump to regulate stromal hydration. Although – like the keratocytes – the corneal endothelium is derived from neural crest, the endothelial cell characteristics are different from those of keratocytes. The human corneal endothelial cells are arrested in G1 phase in vivo and do not normally replicate to replace dead or injured cells. This lack of cell division results in a physiological reduction of cell density of about 0.3–0.5% per year. Scattered evidence, however, suggests the potential for some mitotic events in human corneal endothelium. Mitotic figures were observed in vivo by specular microscopy study following a rejection reaction on a corneal graft. Clusters of cells smaller than surrounding cells suggested that, at least under some circumstances, mitosis occurs in the endothelium of the adult human cornea. Recently, Yokoo and coworkers identified cells in the human corneal endothelium able to form cell-spheres in attachmentindependent culture. Cells in these spheres, formed under conditions similar to those used for isolation of neural stem cells, can be expanded and generate daughter cells expressing neuronal and mesenchymal molecular markers. These properties suggest a stem cell origin for the cells forming the spheres. The sphere-forming cells also adapted the polygonal morphology characteristic of endothelial cells, suggesting the presence of endothelial progenitors. These precursors were effective in vivo in restoring endothelial function in an animal model of corneal endothelial deficiency. Both peripheral and central rabbit corneal endothelia contain a significant number of precursors, but the peripheral endothelium contains more precursors and has a stronger self-renewal capacity than the central region by sphereforming assay. As the long-term culture of human endothelial cells has not been carried out and because of a lack of both stem cell and endothelial markers, positive identification of the proposed endothelial stem cells in situ has yet to be accomplished.

Conclusions

The ocular surface contains multiple populations of cells with stem cell-like properties. Some of these are localized to a distinctive niche in the corneoscleral limbus but other populations are more dispersed. Stem cells also are present in corneal stroma and endothelium. Stem cells support selfrenewal of the corneal and conjunctival epithelia and the limbal stem cells prevent conjunctivalization of the corneal surface; however, relationships among the different stem cell populations in normal tissue homeostasis – and in

response to wounding – are not yet fully characterized. Ocular-surface stem cells have a high potential for use in cell-based therapy for a variety of ocular pathologies, and in tissue engineering. Continuation of the characterization of ocular stem cells is, therefore, a high priority both for understanding the biology of the ocular surface and for development of sight-saving therapies.

Acknowledgments

The authors wish to thank Kira Lathrop for the excellent illustration and Martha Funderburgh for proofreading the manuscript. This work was supported by NIH Grant EY016415 and Research to Prevent Blindness Inc.

See also: Corneal Epithelium: Wound Healing Junctions, Attachment to Stroma Receptors, Matrix Metalloproteinases, Intracellular Communications; The Surgical Treatment for Corneal Epithelial Stem Cell Deficiency, Corneal Epithelial Defect, and Peripheral Corneal Ulcer.

Further Reading

Daniels, J. T., Dart, J. K., Tuft, S. J., and Khaw, P. T. (2001). Corneal stem cells in review. Wound Repair Regen 9(6): 483–494.

Daniels, J. T., Notara, M., Shortt, A. J., et al. (2007). Limbal epithelial stem cell therapy. Expert Opinion on Biological Therapy

7(1): 1–3.

Davanger, M. and Evensen, A. (1971). Role of the pericorneal papillary structure in renewal of corneal epithelium. Nature 229(5286): 560–561.

Du, Y., Funderburgh, M. L., Mann, M. M., SundarRaj, N., and Funderburgh, J. L. (2005). Multipotent stem cells in human corneal stroma. Stem Cells (Dayton, Ohio) 23(9): 1266–1275.

Lavker, R. M. and Sun, T. T. (2003). Epithelial stem cells: The eye provides a vision. Eye (London, England) 17(8): 937–942.

Lavker, R. M., Tseng, S. C., and Sun, T. T. (2004). Corneal epithelial stem cells at the limbus: Looking at some old problems from a new angle. Experimental Eye Research 78(3): 433–446.

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Glossary

Allogeneic – Taken from different individuals of the same species.

Aniridic keratopathy – The corneal opacification in congenital absence of the iris.

Atopic or vernal keratoconjunctivitis – The ocular conditions resulting from allergies. Type I and IV hypersensitivities have been demonstrated to play a role in the allergic response. Both disease processes manifest with classical symptoms of ocular allergy. Cicatrization – The formation of scar tissue. Corneal lenticules – Corneal disks, usually obtained from a donor.

Keratoepithelioplasty (KEP) – The transplantation of peripheral corneal lenticules harvested from donor tissue for the treatment of severe ocular surface diseases.

Keratolimbal allograft (KLAL) – A surgical procedure in which limbal tissue with peripheral cornea is obtained from donor eyes and transplanted to the recipient eyes. KLAL is performed to treat severe bilateral ocular surface disorders combined with limbal stem cell deficiencies.

Lamellar keratoplasty – An operation in which diseased corneal tissue is removed and replaced by lamellar corneal tissue from a donor. The procedure is performed either to improve vision (optical keratoplasty) or to provide structural support for the cornea (tectonic keratoplasty).

Limbal transplantation – The transplantation of limbal tissue including stem cells. Autografts and allografts of limbal transplantations were developed to improve the outcome of ocular surface reconstruction.

Mooren’s ulcer – A rapidly progressive, painful, ulcerative keratitis, which initially affects the peripheral cornea and may spread circumferentially and then centrally.

Ocular cicatricial pemphigoid (OPC) – A chronic disease that produces adhesions and progressive cicatrization and shrinkage of the conjunctival, oral, and vaginal mucous membranes.

Penetrating keratoplasty – The corneal transplant involving the replacement of all layers of the cornea, yet retaining the peripheral cornea.

Stevens–Johnson syndrome – A condition affecting the skin in which cell death causes the epidermis to separate from the dermis. The syndrome is thought to be a hypersensitivity complex affecting the skin and the mucous membranes.

Superficial keratectomy – The removal of corneal epithelium and anterior stroma.

Symblepharon – Adhesion of the eyeball to one or both eyelids.

The ex vivo expansion of corneal epithelial cells/ oral mucosal epithelial cells – A form of ocular surface reconstruction using cultivated corneal epithelial/oral mucosal epithelial cell sheets that are developed using tissue engineering techniques. Several types of cultivated epithelial sheets, with or without carrier materials, are used for the treatment of severe ocular surface diseases, such as Stevens–Johnson syndrome, ocular cicatricial pemphigoid, and severe chemical burns.

Introduction

The concept of an ocular surface has been widely accepted in the field of ophthalmology and investigations in this area have greatly improved our understanding of the important role that the ocular surface plays in the maintenance of vision and ocular health. The healthy ocular surface is composed of corneal and conjunctival epithelia, each of which has a distinct cellular phenotype. These two types of epithelia, with the presence of an intact tear film, maintain the ocular surface integrity. The corneal epithelium, especially, plays a critical role in maintaining corneal transparency and avascularity. On the basis of numerous investigations, it is now believed that corneal epithelial stem cells exist in the basal layer of the limbal regions where palisades of Vogt are seen in normal human subjects. Severe damage to the limbal