Laboratory Science JAMES V. J ESTER, PHD, SECTION EDITOR

The Corneal Epithelial Niche

MARY ANN STEPP, PHD1 AND JAMES D. ZIESKE, PHD2

ABSTRACT In recent years, it has become generally accepted thelium. Stem cells are most simply defined as cells that that the corneal epithelial stem cells are localized in the basal have the capacity to self-renew as well as the ability to gener- cell layer of the limbal epithelium. However, a number of ques- ate differentiated cells.2-6 When adult stem cells are consid- tions remain regarding the number, markers, generation, and ered, a further criterion is added requiring that the cells maintenance of the corneal epithelial stem cells. One of the must self-renew throughout the life span of the animal. key questions concerns what makes up the microenvironment Stem cell research has recently become one of the most or niche that is responsible for allowing the stem cells to re- active areas in biology research; however, to date, no mo- main and function throughout the life of the tissue. This review lecular markers have been recognized that definitively iden- will consider the unique aspects of the limbus and compare tify stem cells. Therefore, stem cells are usually defined by these to what is known about other stem cell niches. a group of common characteristics.2-6 First, stem cells are KEY WORDS , limbal epithelium, the ultimate precursors for all other cells in a tissue or stem cell, niche, transient amplifying cell, α9 integrin, even in the entire organism. Second, stem cells maintain α-enolase, microenvironment their own population. They are capable of asymmetric cell division, giving rise to one daughter cell that remains a stem cell and a second daughter cell that will go on to I. INTRODUCTION: differentiate. Third, stem cells make up only a small per- WHAT IS A STEM CELL? centage of the total cells in the tissue, ranging from as low lthough it had been speculated for a number of as 0.01% of the cells in bone marrow to 2-4% of the cells years that corneal epithelial cells are repopulated in the small intestine. Fourth, stem cells are relatively un- A by centripetal migration of cells from the limbus differentiated. Fifth, stem cells divide infrequently in vivo, or conjunctiva, it was not until 1986 that Schermer et al1 Sixth, stem cells have progeny that can rapidly replicate in proposed that stem cells localized in the limbus were re- vitro when placed into cell culture. Many recent defini- sponsible for generating and maintaining the corneal epi- tions of stem cells also require that the cells must be multipotent, i.e., they can differentiate into more than one cell type. These definitions state that cells that give rise to Accepted for publication November 2004 only one cell type are termed progenitor cells. According to From the 1Department of Anatomy and Cell Biology, George Washington this strict definition, corneal epithelial stem cells, which University Medical Center, Washington, DC, USA, and 2Schepens Eye Re- search Institute and Department of Ophthalmology, Harvard Medical School, have been reported to give rise only to corneal epithelial Boston, MA, USA cells, would be termed progenitor cells. However, we will Supported by NIH/NEI Grant No. R01 EY13559-03 to MAS and R01 use the less restrictive definition in this review and refer to EY05665 to JDZ. the corneal epithelial precursor cells as stem cells. The authors have no proprietary interest in any concept or product dis- A second classification of cells to be defined are the cussed in this article. progeny of non-stem daughter cells that result from the Single copy reprint requests to Mary Ann Stepp, PhD (address below). asymmetric cell division of a stem cell. These cells are more Corresponding authors: Mary Ann Stepp, PhD, Department of Anatomy committed to differentiation than the stem cell and also and Cell Biology, George Washington University Medical Center, 2300 I Street NW, Washington, DC 20037; Tel: 202-994-0557; Fax 202-994-8885; have a more limited ability to proliferate. They have a range E-mail: [email protected]; and, James D. Zieske, PhD, Schepens Eye Re- of proliferative capacities. The cell that is the immediate search Institute, 20 Staniford Street, Boston, MA 02114; Tel: 617-912-0282; progeny of stem cell division can undergo more rounds of Fax 617-912-0107; E-mail: [email protected] proliferation than the cell that arises after several cell divi- Abbreviations are printed in boldface where they first appear with their sions. Regardless, the primary purpose of these cells is to definitions and are listed in Table 1. increase the number of cells resulting from each stem cell division ultimately resulting in a tissue. These cells have ©2005 Ethis Communications, Inc. The Ocular Surface ISSN: 1542- 0124. Stepp MA, Zieske JD. The corneal epithelial stem cell niche. been traditionally referred to as transient amplifying cell 2005;3(1):15-26. (TAC).1 TACs are also sometimes termed transit cells4 and transit amplifying cells.3

THE OCULAR SURFACE / JANUARY 2005, VOL. 3, NO. 1 / www.theocularsurface.com 15 THE CORNEAL EPITHELIAL STEM CELL NICHE / Stepp and Zieske

OUTLINE Table 1. Abbreviations

I. Introduction: What is a stem cell? BMP bone morphogenetic protein II. Evidence for the limbal localization of corneal epithelial stem cells CESC corneal epithelial stem cell III. The niche Dpp Decapentaplegic A. The germinal niche of the fly: Role of cell:cell EGF epidermal growth factor contact via cadherins B. Stem cell niches in non-ocular human tissues ESC epidermal stem cell 1. The epidermal stem cell niche: the bulge FGF fibroblast growth factor 2. The gastrointestinal niche: the crypt base GFAP glial fibrillary acidic protein 3. The germinal niche in the adult brain: the subventricular zone and the subgranular zone GISC gastrointestinal stem cell 4. The niche: the role of GSC germ line stem cell the osteoblast HSC hematopoietic stem cell C. The limbal stem cell niche: a unique microenvironment supporting quiescent corneal IGF insulin-like growth factor epithelial stem cells ISEMF intestinal subepithelial myofibroblast LSC limbal stem cell II. EVIDENCE FOR THE LIMBAL LOCALIZATION LSCD limbal stem cell deficiency OF CORNEAL EPITHELIAL STEM CELLS mRNA messenger RNA Over the past 20 years, research from many laboratories has led to the conclusion that the corneal epithelial stem cells NSC neuronal stem cell are localized in the limbus.7-12 These cells have been termed SGZ subgranular zone both corneal epithelial stem cells (CESC), based on their func- SVZ subventricular zone tion, and limbal stem cells (LSC), based on their localization. We will use the term CESC in this review. TAC transient amplifying cell The first suggestion that the limbus is the source of CESC TGF transforming growth factor was made in 1971 by Davenger and Evensen,13 who showed that, occasionally, pigmented cells were observed to migrate Tn-C Tenacin-C from the limbus into the central . It should be noted, VEGF vascular endothelial growth factor however, that the term stem cell is never used in their report. Centripetal migration of cells from the limbus to central cor- nea has subsequently been documented in animal models.14,15 Schermer et al,1 who demonstrated that the keratin K3 was an expression pattern similar to that of K3. In contrast, sev- localized in all corneal and limbal epithelial cells except the eral proteins are expressed preferentially in the limbal basal limbal basal cells, presented a second line of evidence that cells. These proteins have been scrutinized for their potential the stem cells are localized in the limbus. Coupled with cell for being CESC markers. Included in this group of potential culture studies, Schermer’s report documented that the limbal markers are: 1) metabolic enzymes—α-enolase,26,27 cyto- basal cells were less differentiated than the other corneal epi- chrome oxidase,28 carbonic anhydrase,29 and glucose trans- thelial cells. This report was the first to speculate that the porter I30; 2) growth factor receptors—epithelial growth fac- CESC were localized in the limbus. A third line of evidence is tor receptor,31 TGF-β receptors I and II,32,33 and TrkA34; 3) that limbal basal cells proliferate less frequently than the rest cytoskeleton components—keratin 1935 and vimentin36; 4) of the corneal epithelium. Lavker and coworkers16 demon- cell cycle components—cyclins D and E,37 and p10738; and strated this property by injecting mice with 3H-thymidine 5) others—α9 integrin,39 melanin,40 p63,41 and ATP-bind- and finding that only limbal basal cells retained the label. ing cassette transporter G2.12,42 Fourth, only limbal basal cells are clonogenic in cell culture Most of the proteins considered as potential markers experiments.17-19 Finally, perhaps the strongest evidence, are expressed in numerous basal cells at the limbus; an studies have shown that transplantation of limbal tissue or example is seen in Figure 1, which shows the co-localiza- even cultured limbal basal cells can result in healing of cor- tion of α-enolase and α9-integrin in the adult rat limbus. neal epithelium.20-22 The distribution of these two proteins is partially overlap- As stated above, one piece of evidence that the CESC are ping with some limbal basal cells that express both pro- localized in the limbus is the lack of expression of the differ- teins and others that express more or less of one or the entiation marker K3 in these cells.1 Subsequent to this find- other. This distribution emphasizes the heterogeneity of ing, other proteins, including K12,23 aldehyde dehydroge- cellular phenotypes present at the limbus and is some- nase class 3,24 and connexin-4325 have been shown to have what at odds with the findings that only a small popula-

16 THE OCULAR SURFACE / JANUARY 2005, VOL. 3, NO. 1 / www.theocularsurface.com THE CORNEAL EPITHELIAL STEM CELL NICHE / Stepp and Zieske tion of the limbal basal cells (perhaps as few as 100 cells/ tions called adherens junctions; it is also a transcription fac- limbus) are true CESCs.15 One possible explanation for tor that can regulate the expression of a number of genes these observations involves the niche hypothesis. important in development, including several that control According to the niche hypothesis, originally proposed cell polarity and early segmentation in the embryo. When by Schofield,43 cells are influenced by their microenviron- free in the cytoplasm, β-catenin will bind to a complex ment to become and remain stem cells. Thus, it may be pos- that forms after activation of the Wnt signaling pathway. sible that the entire limbus provides a microenvironment that Wnt’s are ligands that activate Wnt receptors and induce allows the limbal basal cells to maintain stem-like character- proteolytic breakdown of β-catenin. Their name is derived istics, and it is not until the cells leave the niche that they from the wingless phenotype that mutations in these pro- differentiate so that they can be readily distinguishable from teins cause in developing flies. While the niche in the fly CESCs by expression of α9 integrin or α-enolase. ovary has provided insight into the role of Wnt-mediated In this review, we will examine the niche concept, dis- cell:cell adhesion in the maintenance of the GSCs in the cussing what is known about niches in other tissues and ovary and in the role of morphogens in the initial specifi- the microenvironment that may make up the CESC niche. cation of the GSCs, we still do not know the roles played by extracellular matrix proteins within the niche in the fly. III. THE ADULT STEM CELL NICHE Adult stem cells lie within a protected area termed the B. Stem cell niches in non-ocular human tissues niche. The niche is made up of three components: the stem 1. The epidermal stem cell niche: the bulge cells themselves, supporting cells within the mesenchyme, Remarkable progress in understanding the epidermal stem and the extracellular matrix produced by both stem and cell (ESC) niche has been fueled by new methods for creat- support cells. Progress in understanding the molecular and ing transgenic mice that can be used for lineage analysis. The cell biology of several of the adult stem cell niches in mam- work of Tumbar and colleagues51 and of Morris and col- mals has been significant over the past several years. Here, leagues52 has demonstrated that the cells in the bulge region after a brief consideration of what we have learned from of the hair follicle are, in fact, pluripotent stem cells that can drosophila, we will discuss the stem cell niche, as it is give rise to epidermis, hair, and the epithelial cells that make currently conceived in mammalian skin, intestine, brain, up the sebaceous glands. Molecular profiles of ESCs isolated and in the bone marrow. by both groups using two different techniques have yielded similar results and show, indeed, that stem cells are largely A. The germinal niche of the fly—role of cell:cell quiescent and produce distinctive combinations of messen- contact via cadherins ger RNAs (mRNAs), copies of the information carried by a In drosophila, the ovary is an excellent model for the study gene. ESCs are CD34+ and express high levels of α6 integrin of support cells and growth factors in the niche. Both appear as well as specific matrix molecules, including the α1 chain to be necessary for the maintenance of the germ line stem for collagen type VI and tenascin-C. As in fly stem cells, growth cells (GSCs) and somatic stem cells that are housed in the fly factors are crucial for maintaining ESCs. mRNAs encoding ovarioles.44,45 Lying adjacent to the GSCs are the cap cells; molecules mediating Wnt-signaling are generally these are mesenchymal cells that form a major part of the downregulated in the ESCs, but are upregulated when ESCs germinal niche in the ovary. When one GSC divides, one of are stimulated to divide. ESC survival seems to be associated its two daughters remains associated with a cap cell and the with upregulation of the transforming growth factor β (TGFβ) other ends up one cell away from the cap cell.46 The cell in and fibroblast growth factor (FGF) pathways and down- contact with the cap cell remains a GSC, whereas the other regulation of bone morphogenetic protein (BMP)-mediated cell becomes committed to forming a new egg (oogenesis). signaling. Regardless of which of these specific signaling path- The cap cells express growth factors required for the mainte- ways are most critical, it is clear that these important cells posses nance of the GSCs, including Decapentaplegic (Dpp), a fly mRNA profiles distinct from their non-stem cell neighbors. homologue of bone morphogenetic proteins (BMPs) 2 and 4, The identity of the support cells in the mesenchyme Wingless, and Hedgehog. Loss of Dpp or any of the proteins that are in contact with the ESCs within the niche in the downstream of Dpp in its signaling cascade results in loss of GSCs. bulge of the hair follicle is not totally clear. One possibil- Conversely, overexpression of Dpp results in an expansion of ity is that they are mesenchymal cells of the dermal pa- the GSCs.46-48 These data indicate that gradients of Dpp and pilla. These cells are clearly important in initiating a new other growth factors regulate the numbers of GSCs. wave of hair follicle growth; however, they are not in di- Studies of fly development have also shown that the rect contact with the bulge during all stages in the growth precise arrangement of cells within the niche is regulated of the hair follicle. A second potential source of support in part by the cell:cell adhesion molecule DE-cadherin.49,50 cells is the arrector pili muscle. The basement membrane DE-cadherin and Armadillo, the fly homologue of β- beneath the ESC at the bulge is produced by both the ESC catenin, are concentrated at the interface between cap cells themselves and the arrector pili muscle cells. and the GSCs and are required for the initial localization The elegant transmission electron microscopy (TEM) of the GSCs within the ovariole. β-catenin not only inter- studies by Akiyama and colleagues53 show the basement acts with cadherins in forming the epithelial cell:cell junc- membrane underlying cells of the bulge region adjacent to

THE OCULAR SURFACE / JANUARY 2005, VOL. 3, NO. 1 / www.theocularsurface.com 17 THE CORNEAL EPITHELIAL STEM CELL NICHE / Stepp and Zieske the cell membrane of a smooth muscle cell of the arrector pili meostasis and more rapidly after tissue damage.55 muscle. Bulge epithelial cells have adhesion complexes simi- Cheng and Leblond56 first proposed the Unitarian lar in appearance to hemidesmosomes along their underly- theory of epithelial cell formation in the small intestine ing basement membrane zone; immediately beneath their over 40 years ago. This theory states that all of the cell basement membrane is the cell membrane of the smooth types of the small intestine arise from a single precursor muscle cell. Given the fact that α6 integrin, a component of cell. Since those original studies, others have shown that the hemidesmosomes, keeps appearing in molecular profiles the Unitarian theory holds true for the entire surface of as a possible marker for stem cells, perhaps it is the interac- the GI tract. The details are best worked out in the small tion between the basal lamina of the ESC and the smooth intestine, where the stem cells are known to be located muscle cell membrane via specialized hemidesmosomes that just superior to the Paneth cells at the base of the crypts of keeps these cells within their niche during quiescence. Lieberkuhn. The possible involvement of muscle in maintaining stem An important tool for the study of epithelial lining cells cells is supported by additional, albeit circumstantial, evi- in the GI tract is the microcolony assay, first developed dence. Tension in the form of mechanical stress has been over 30 years ago.57 Animals are given a cytotoxic dose of shown to mediate lineage commitment decisions in mesen- radiation. Within 4 days, the GISCs within the crypts un- chymal cell populations, including conversion of a stem cell dergo apoptosis, but their niche can still be identified by into adipocyte or bone54; it is possible that tension applied to the location of the Paneth cells that are resistant to the the ESCs via their direct attachment to the smooth muscle effects of irradiation. After all the GISCs have died due to cells of the arrector pili muscle mediates, via integrins, the irradiation, the empty crypts have been termed crypt ghosts. transmission of survival signals to the ESC. Further, addi- These types of studies have shown that crypts are clonal tional evidence from the study of the gastrointestinal niche and that survival of one cell within a crypt will allow for show clearly that myofibroblasts play central roles in the regeneration of the entire crypt and overlying villus. Ad- maintenance of the stem cell niche in the intestine.55 ditional studies using mouse embryo aggregation assays In summary, the ESC is a pluripotent cell capable of and transgenic and mutant mice have confirmed these repopulating the skin, hair, and sebaceous gland compart- data.55,58 The stem cell niche within the crypt is produced ments. Commitment to form one of its three differenti- by a fenestrated sheet of intestinal subepithelial ated lineages involves activation of Wnt signaling, and the myofibroblasts (ISEMFs).59 These cells are unique in that maintenance of quiescence likely involves cell:cell and/or they form a syncytium that extends throughout the entire cell substrate adhesion and signaling pathways involving lamina propia and, thus, is in close contact with blood TGFβ and/or FGF. The unique matrix of the ECS niche is vessels. This niche remains within the crypt ghosts after created by the ESCs themselves and surrounding cells in the majority of the stem cells have died following radia- the mesenchyme, including permanent resident smooth tion treatment. The ISEMFs make a variety of growth fac- muscles cells that make up the arrector pili muscle as well tors that are thought to regulate the proliferation and dif- as the cells of the dermal papilla. ferentiation of the overlying GISCs. Although the GISCs have been studied for many years, 2. The gastrointestinal niche: the crypt base no markers exist, and progress toward identification of The gastrointestinal (GI) tract includes the small and large their molecular characteristics has been slow. An exciting intestine and stomach and is comprised of four distinct cell report was derived from studies of a mouse that expresses types: columnar cells, mucin-secreting or goblet cells, endo- a transgene for the diphtheria toxin A fragment in Paneth crine cells, and, in the small intestine, Paneth cells, which cells of the small intestine; as a result of this mutation, the secrete lysozyme and the antibacterial cryptins. The colum- mice lack Paneth cells. Despite the loss of Paneth cells at nar cells are most abundant; they possess apical microvilli their crypt base, the GISC cell population expands in these and are termed enterocytes in the small intestine and mice.60 Using laser capture microdissection to isolate pro- colonocytes in the colon. The goblet cells produce mucin to genitor cells from normal crypts and from mutant crypts facilitate the passage of food along the intestine, and various lacking Paneth cells, Stappenbeck and colleagues60 were endocrine cells are present that produce enzymes and pep- able to generate molecular profiles of gut progenitor cells. tide hormones. The four distinct cell types that make up the Similar studies using isolated stomach progenitor cells re- lining of the GI tract are organized into small crypts, which vealed significant overlaps in gene expression profiles be- come together to form larger structures called villi. Paneth tween stomach and intestine. These profiles showed that c- cells and the neuroendocrine cells are found at the base of myc signal transduction pathways were activated in the pro- the crypts, where the gastrointestinal stem cells (GISCs) are genitor cells; this likely reflects the high rate of cell differen- located. tiation taking place in the gut and represents a major differ- In the stomach, the tissues are organized differently, ence between gut stem cells and those of the skin. and the epithelial lining takes the form of long tubular Progress has also been made in evaluating the roles of gland-like structures with foveolar, isthmus, neck, and base growth factors, including Wnts, in mediating the prolifera- regions within each gland. Turnover of the gut lining is tion and differentiation of cells within the GI tract. Interest in very rapid, occurring over a 2-7-day period during ho- this area is largely due to the large numbers of cancers that

18 THE OCULAR SURFACE / JANUARY 2005, VOL. 3, NO. 1 / www.theocularsurface.com THE CORNEAL EPITHELIAL STEM CELL NICHE / Stepp and Zieske arise from these highly proliferative cells. It has been shown cell types via their long processes. The B cells give rise to that Wnt/β-catenin signaling controls epithelial homeostasis; both type C cells that act as transit amplifying cells, as well as mutations in molecules involved in Wnt-signaling such as type A cells that are a committed population of migratory APC, Tcf-4, and Fkh-6, lead to tumor formation. Delta is the neuroblasts that will migrate to the olfactory bulb to become name given to a family of transmembrane receptors that are interneurons. All of these cells lie adjacent to the lumen of expressed on cell surfaces that bind to ligands on adjacent the ventricle and sit in close contact with ependymal cells cells called Notch receptors. The interaction of Delta and Notch and are surrounded by a basement membrane arising from has been shown in flies and worms to induce changes in cell endothelial cells of adjacent blood vessels. fate in the cells expressing Notch receptors. Data from the NSCs isolated from the mouse SVZ and SGZ can be study of GISCs also show that Notch/Delta signaling path- grown in culture as neurospheres; these are round cell:cell ways regulate the differentiation of goblet, Paneth, and aggregates of neural progenitor cells that grow in a rela- enteroendocrine cells.61,62 tively undifferentiated state in culture under defined con- In summary, in the GI tract, the GISCs are located at ditions. Methods for differentiating these cells into spe- the base of the intestinal crypts and a single GISC can give cific neural and glial lineages have been developed; these rise to all the cells of the crypt. Despite the availability of methods involve plating neurospheres on adhesive sub- good animal models to study this niche, the rapid turn- strates and giving the cells various combinations of growth over of the epithelial lining in the gut has made it hard to factors, including FGF2 and EGF. The development of these isolate and identify markers for these cells in vivo or in in vitro methods for manipulating NSCs has led to many vitro. While it is clear that crypts are clonal in vivo and important discoveries, including the elucidation of growth that myofibroblasts provide the stromal support for these factors affecting neurogenesis, e.g., bFGF, insulin-like cells, the direct molecular level interactions that take place growth factor 1 (IGF1), TGFα, vascular endothelial growth within the niche in the GI tract remain elusive. Since the gut factor (VEGF), Eph/ephrin, Shh, and others.63 lining turns over more rapidly than do the epithelial cells As an example, Garcion and colleagues65 have shown making up the skin or cornea, it is likely that important dif- that tenascin-C is an important regulator of stem cell re- ferences at the molecular level will be found between the sponses to growth factors. Using neurosphere cultures from GISCs and other more slowly regenerating epithelia. wild type and tenascin-C knockout mice, these investiga- tors showed that Tenacin-C (Tn-C)- mediated the conver- 3. The germinal niche in the adult brain: the sion of the NSCs from an FGF2 responsive state to an epi- subventricular zone and the subgranular zone dermal growth factor (EGF) responsive state. In addition, Only within the past several years have we begun to BMP4 inhibited NSCs from acquiring the expression of think of the adult brain as having stem cells for the pro- the EGF receptor. Tn-C knockout NSCs fail to acquire the duction of new neurons. The brain is formed during de- EGF receptor unless they are provided with an exogenous velopment from neuroepithelial stem cells in the ventricular source of Tn-C; the failure of the Tn-C knockout cells to zone that gives rise to both neurons and glial cells. Glia acquire EGF receptors is associated with changes in the are largely considered to be support cells; two important specification of differentiated cell types. The Tn-C null stem classes of glia in the central nervous system are astrocytes cells generate more neurons and fewer glial cells than do and oligodendrocytes. Oligodendrocytes function to normal cells that express Tn-C. Thus, Tn-C is implicated myelinate the axons of neurons, whereas astrocytes have as an important niche component in the SVZ and SGZ in diverse functions. Astrocytes have a small central cell body vitro; in vivo in the normal mammalian brain, it is present with numerous star-like extensions rich in intermediate at both sites. filament proteins, including glial fibrillary acidic protein Other niche components in the SVZ and SGZ are base- (GFAP), that make contact with neurons and endothelial ment membrane proteins and growth factors that are present cells of adjacent blood vessels. around the neural progenitors at the niche. The endothelial The two sites for continued generation of neurons from cells that form the adjacent blood vessels produce many of progenitor cells in the adult brain are the subventricular these, including VEGF. It has been shown that the NSCs ex- zone (SVZ) of the lateral ventricle and the dentate gyrus press VEGF receptor 2 (VEGFR2), implicating VEGF signal- of the subgranular zone (SGZ) of the hippocampus.63 ing in the maintenance of the stem cell phenotype in the Unlike the other adult tissue specific stem cells character- brain.66 The ependymal cells release the morphogen noggin, ized to date, the adult neuronal stem cells (NSCs) have which antagonizes BMP signaling. BMPs cause the NSCs to the morphology of differentiated astrocytes, and they are differentiate along the glial lineage, and, thus, noggin helps intermixed at the NSC niche with other astrocytes and keep the stem cells multipotent. with the endothelial cells that make up the vasculature in The basement membrane surrounding the niche con- the SVZ and SGZ. Within these specific sites, astrocytes tains collagens, laminins, and heparan sulfate and chon- appear to serve as both niche and stem cell.64 droitin sulfate proteoglycans in addition to Tn-C.67,68 It In the SVZ, the lineages of the neural progenitors have acts as a reservoir to allow the formation of morphogen been determined. The NSCs themselves are called type B cells; gradients along which differentiating neuronal and glial these cells are in direct cell:cell contact with all of the other cells migrate and differentiate. In support of this concept,

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Figure 2. The corneal epithelial stem cell (CESC) niche. The CESCs are in a unique position to receive cellular signals from numerous sources, as indicated by the arrows. Conjunctival and corneal epi- thelial cells, conjunctival and corneal-derived stromal fibroblasts, as well as vascular endothelial and associated smooth muscle cells, all are potentially important for generation and maintenance of the stem cell niche. In addition to factors provided by these resident tissue cells, cytokines and matrix molecules may also impact the niche through leakage from the adjacent blood vessels. These fac- tors are proposed to form cytokine gradients within the niche that maintain the CESCs in a quiescent, undifferentiated phenotype for the life of the organism. The basement membrane beneath the CESCs is distinct in its composition from that underlying the con- junctiva and corneal epithelial cells. It is likely that proteoglycans and matrix molecules within the CESC basement membrane and underlying stroma bind to and release cytokines to direct cell divi- sion and differentiation. By mediating attachment to the basement membrane and integrating signal transduction events from the ex- tracellular environment, integrins play key roles in maintaining stem cells in their undifferentiated state.

In summary, the niche at the SVZ and SGZ contains NSCs that give rise to neurons in the adult mammalian brain. NSCs have the morphological appearance of astro- cytes and express some markers (GFAP) previously asso- Figure 1. Micrographs demonstrating the immunolocalization of ciated with differentiated glial cell types. This niche is com- (A) α-enolase and (B) α9-integrin in adult rat corneal limbal cells. prised of the NSCs called type B cells and their progeny the Colocalization is seen in (C). Several points should be noted. First, the localization of α-enolase is representative of many markers that type C and A cells, ependymal cells, and endothelial cells are expressed in the majority of the limbal basal cells. Second, α9- from adjacent blood vessels. The basal lamina surrounding integrin expression is more restricted. Third, colocalization of the the vascular endothelium extends and surrounds these clus- two proteins suggests that there is heterogeneity in the limbal basal cell population with a subset of α9-integrin bright cells (arrows). ters of NSC and support cells, bringing growth factors and matrix molecules to which the stem cells respond. when pieces of SVZ containing NSCs are transplanted to 4. The hematopoietic stem cell niche: the role of the another region within the SVZ or SGZ, the cells retain their osteoblast ability to produce neurons. However, when placed at other In many ways, we know more about the stem cells sites in the brain distal from the SVZ or SGZ, the ability of that give rise to the formed elements of the blood than we this tissue to generate new neurons is lost. When NSCs do about all the other adult stem cells combined. This is derived from the SGZ are grown briefly in culture and trans- because of their use clinically in bone marrow transplants planted back into the SGZ, they retain the ability to make to restore the immune system in patients with metastatic interneurons; placed in non-neuronal sites, they become glial diseases of the circulating cells of the blood, including leu- cells exclusively.69,70 Thus, the microenvironment at these kemias. In bone marrow transplants, patients are two sites not only maintains the stem cell compartment, but myeloablated; the cells within their own bone marrow are it also can direct the differentiation of those stem cells. killed, usually by a high dose of radiation. This kills not

20 THE OCULAR SURFACE / JANUARY 2005, VOL. 3, NO. 1 / www.theocularsurface.com THE CORNEAL EPITHELIAL STEM CELL NICHE / Stepp and Zieske only the hematopoietic stem cells (HSCs) that are located in the bone marrow, but it also kills their prog- eny, including all the cells of the eryth- roid and myeloid lineages. The patient is then transfused with cells isolated from a matched donor bone marrow and enriched for the HSCs. The HSCs go into the bone marrow from the cir- culation, a process called homing, ad- here to the marrow niche within the marrow stroma, and initiate hemato- poiesis. Bone marrow transplants have been performed for over 100 years, and it has been through at- tempts to improve their efficiency and reduce mortality that advances in the basic understanding of the HSCs have emerged. The HSCs are CD34+/Lin–/Sca1+/ Figure 3. Micrographs demonstrate the immunolocalization of α9 and β1 integrin in adult Kit1+ and can be enriched from whole mouse . Note that heterogeneity exists in the limbal basal cells with respect to localization of integrin and extracellular matrix. Adult mouse corneas were used for whole bone marrow aspirates by cell sort- mount experiments, as described by Pajoohesh-Ganji, et al.87 The top row shows co-local- ing procedures so that the transfused ization of α9 and β1 integrins; the bottom row shows co-localization of α9 integrin and one cells contain primarily cells that will of its ligands, tenascin-C (Tn-C). 3-D reconstructions were assembled and projected from the apical to basal aspect through the limbal basal cells. The upper right corners in each be able to engraft successfully. The image are oriented toward the central cornea, and the lower left corners are oriented to- HSCs can be grown and maintained ward the conjunctiva. Along the limbal rim, α9 integrin is expressed in a region-specific in culture. Transgenic and knockout manner as shown in the two different regions presented. β1 integrin is much more abun- dant than α9 integrin and many cells that have high levels of β1 integrin expression have mice can be used for bone marrow no α9 integrin. Tn-C is present in a ring around the limbus lying just beneath the limbal transplant studies, and recently, two basal cells; some but not all of those cells express α9 integrin. groups of investigators were able to report the repopulation at high efficiency of the entire bone Calvi and colleagues75 studied bone formation and he- marrow from a single transplanted HSC.71,72 matopoiesis in another mouse model that was overexpressing It had been shown early on that the more primitive of the an activated receptor for parathyroid hormone under the con- hematopoietic cells first appear after transplant on the en- trol of the promoter for collagen α(1). They confirmed the dosteal surfaces of the trabecular bone. The cells that HSCs association between HSCs and osteoblasts and went on to initially interact with were thought at different times to be show that Notch-1 was expressed by HSCs and that osteo- stromal fibroblasts, myofibroblasts, and/or endothelial cells. blasts expressed the Notch1-ligand, Jagged 1, a protein that The other cell known to be present at the same site, the os- is member of the Delta family of Notch ligands. Notch-1 sig- teoblast, was largely ignored by those studying hematopoie- naling was confirmed to play a role in HSC survival in that sis until about 10 years ago when Taichman and Emerson73 addition of Notch-1 inhibitors suppressed HSC proliferation. showed that osteoblasts supported hematopoiesis and HSC Yet another study has emerged that adds to our under- survival. Recent studies have confirmed those early results, standing of the complexity of the signaling between os- using independent techniques. Zhang and colleagues74 and teoblast and HSC. Arai and colleagues76 believe that the Calvi and colleagues75 both show that the HSCs bind to os- osteoblast:HSC interaction is critical for the induction of teoblasts directly. survival signals that maintain HSCs in a quiescent state Zhang’s studies of a mouse with a conditional ablation within the niche. The quiescent state protects stem cells of BMP receptor 1A (BMPR1A) revealed increased depo- from depletion in response to proliferative signals. This sition of trabecular bone, increased endosteal surface, and group showed that HSCs express the tyrosine kinase re- decreased marrow cavity. Surprisingly, these mice had twice ceptor Tie2 and that they interact with osteoblasts that as many HSCs in their marrow as are present in mice with- express the Tie2 ligand angiopoietin-1 (Ang-1). Tie2:Ang- out the conditional ablation of BMPR1A. Detailed analy- 1 interaction induced HSCs to become quiescent. ses showed that there were more osteoblasts in these mice While these results clear up many questions, they also and, as a result, there were more HSCs bound within the leave many questions about the HSC niche unanswered. marrow. These investigators went on to show that the in- The exact mechanism of homing is still unclear, but integrin teraction between the HSC and the osteoblasts was in part α4β1 is clearly implicated in mediating the interaction mediated by N-cadherin-mediated adhesion between os- between the HSC and the niche. Conditionally ablated mice teoblasts and HSCs. lacking α4β1 in the HSCs accumulate HSCs in the pe-

THE OCULAR SURFACE / JANUARY 2005, VOL. 3, NO. 1 / www.theocularsurface.com 21 THE CORNEAL EPITHELIAL STEM CELL NICHE / Stepp and Zieske ripheral circulation; these data and other data from stud- and 2) conjunctival epithelia; the 3) corneal and 4) con- ies in which normal mice were transplanted with HSCs junctival stromal fibroblasts; the 5) vascular endothelial lacking α4β1 confirmed that α4β1 integrin plays an im- cells; and 6) associated smooth muscle cells of the epis- portant role in lymphocyte homing.77 One α4β1 ligand is cleral blood vessels. All of these cells could secrete growth, Tn-C. Tn-C is abundant within the bone marrow stroma,78 differentiation, and/or survival factors that accumulate in and Tn-C null mice have suppressed hematopoiesis that the limbal stroma and overlying epithelial basement mem- can be restored by the exogenous addition of Tn-C.79 brane. Distinct from these factors that are the products of Whether osteoblasts or other stromal cells produce Tn-C resident cells, there is also the additional influence of pro- in the bone marrow is not clear. teins from serum that could be released at the limbal vas- Taken together with previous studies, data from several culature. In response to this unique combination of over- groups of investigators shows that the osteoblast is an impor- lapping signals, we propose that a subpopulation of cor- tant part of the HSC niche. Cell:cell adhesion, mediated in neal epithelial cells are induced during early eye develop- part by cadherins, and transmission of cell:cell signals via ment into maintaining a quiescent phenotype and func- Notch-1:Jagged-1 and Tie2:Ang-1 interactions are important tioning as CESCs. We believe that the same combination in maintaining HSCs within their niche and regulating their of factors that induce corneal epithelial stem cell quies- proliferation and differentiation. While the ability of HSCs to cence establishes and maintains the limbal stem cell niche. home to the bone marrow niche is impaired in mice lacking When the CESCs are deprived of their niche, they re- α4β1 integrin, additional molecules on the HSCs themselves spond to growth and differentiation signals in ways simi- and in the stromal matrix are no doubt involved in the regu- lar to corneal epithelial cells, and they act as early tran- lation of HSC homing to the bone marrow. Understanding siently amplifying cells (eTACs), dividing rapidly for sev- how HSCs home to the marrow is important, as tumor me- eral population doublings to generate large numbers of tastasis to peripheral organs is, in essence, a type of homing. progeny. This is what happens when human CESCs are In summary, studies on other niches suggest certain com- placed into cell culture.80,81 Similarly, if the niche is dam- mon threads in the generation and maintenance of adult stem aged by severe trauma, changes in the availability of cell niches. First, the support cells in the niche often appear growth/differentiation/survival factors along with changes to be myofibroblasts, smooth muscle cells, or endothelial cells. in the environment itself induce the remaining CESCs to Second, a gradient of growth factors produced by these cells differentiate into eTACs, resulting in a reduction of CESCs appears to be crucial in the maintenance of the stem cells. at the site of trauma. This is how trauma to the limbus can Third, stem cells frequently appear to adhere to a specialized result in LSCD. In addition, we know that wounds to the basement membrane secreted by the stem cells and the sup- central cornea that spare the cells at the limbus, but re- port cells. In the remainder of this review, we will examine quire proliferation during active cell migration, place the these commonalities in the limbal stem cell niche. CESCs at risk.82 The limbal stem cell niche can be thought of as having C. The limbal stem cell niche: a unique two distinct functional sites within it: a quiescent site and microenvironment supporting quiescent a proliferative site. In homeostatic conditions in adult eyes, corneal epithelial stem cells the balance of signals is shifted toward maintaining the The CESCs within the limbal stem cell niche are local- stem cell in its nondifferentiated state, and survival is fa- ized there due to a combination of anatomical and bio- vored over cell division and cells are quiescent. CESC cell chemical events first set up during development. The niche division is considered to be an asymmetric cell division is maintained by a combination of factors intrinsic to the because it gives rise to one cell that is an eTAC and one CESCs themselves as adult tissue-specific stem cells, as cell that remains a CESC. During homeostasis, CESCs pro- well as by factors present in the environment. These ex- liferate, but do so infrequently to replace corneal epithe- trinsic factors include molecules produced by nearby vas- lial cells lost in the tear film. Events or diseases that inter- cular endothelial cells, smooth muscle cells that surround fere with either the establishment or the maintenance of those blood vessels, and/or molecules released from those gradients of growth and survival factors in the basement blood vessels. In addition, factors released by the adjacent membrane and stroma that make up the niche would lead conjunctival cells and the TACs intermixed with the CESCs to limbal stem cell deficiency (LSCD). In this view, the also likely play a role in the maintenance of the CESC popu- CESCs are formed from corneal epithelial cells that hap- lation. A diagram highlighting the roles of intrinsic versus pen to find themselves in the right place exposed to the extrinsic factors in specification and maintenance of the right factors at the right time. As long as the corneal epi- CESCs is shown in Figure 2. In this section, we present a thelial cell has not committed to withdraw from the cell model for the CESC niche that is based upon research done cycle, we believe that it could be induced to revert to a on the limbus and also draws upon the research done on CESC if it were placed within the appropriate site. The other adult stem cell niches. conjunctival cells and/or their underlying stromal fibro- The stroma that underlies the limbal epithelium is blasts are proposed to secrete factors that inhibit the im- unique in that it is positioned to receive signals from at migration of corneal epithelial cells into the conjunctiva. least six distinct cell populations: the overlying 1) corneal CESCs, corneal epithelial basal cells, and their underlying

22 THE OCULAR SURFACE / JANUARY 2005, VOL. 3, NO. 1 / www.theocularsurface.com THE CORNEAL EPITHELIAL STEM CELL NICHE / Stepp and Zieske stromal fibroblasts also likely secrete factors that inhibit cornea, we see Tn-C beneath some but not all of the α9 the migration of conjunctival epithelial cells into the cen- integrin-positive limbal basal cells. A common theme tral cornea. emerges from Figures 1 and 3: the limbal niche consists of Survival of the CESCs in their nondifferentiated state a population of corneal epithelial cells each of which ex- is most likely regulated via mesenchymal cells in the press a unique molecular phenotype. stroma; these cells likely produce survival factors that Regardless of specifics, the model presented in Figure maintain the CESC as nondifferentiating cells. In the bone 2 is useful in predicting and testing specific hypotheses marrow, we know that for stem cell replacement therapy regarding causes for the development of limbal stem cell to work well, the niche must be empty; the patient is deficiency conditions. Data supporting the above model myeloablated. In procedures involving treatments of hu- comes from various sources, and we have built it on the man corneas with cultured CESCs, care should be taken strong foundation of research and insights provided by to remove the damaged or nonfunctional CESCs and/or our colleagues.10,12 We know from work on flies and in conjunctival cells present at that site, which could block skin that niches function in maintaining stem cell quies- engraftment. The limbal area needs to be free of scar tis- cence and proliferation. Our own studies of corneal wound sue and the adjacent conjunctival epithelium, and the healing support the idea that the niche itself is not altered grafted corneal epithelial cells must be in close approxi- in response to smaller corneal epithelial debridement mation to the underlying stromal fibroblasts. wounds, but is altered after larger wounds.82 The pres- During migration of corneal epithelial cells after small ence of a specific limbal stem cell niche that functions to wounds, there does not appear to be a loss of CESCs,82 maintain these cells in a quiescent state where they are suggesting that the CESC niche remains quiescent during communicating differently with their non-stem cell neigh- re-epithelialization after small wounds. Then, after migra- bors25 is supported by: 1) the recent work of Espana and tion is complete, the niche functions to enhance prolifera- colleagues83 showing that the limbal stroma enhances tion, and CESCs are signaled to divide until the corneal CESC survival; 2) past work showing that the basement epithelial cell layer is brought back to its normal thick- membrane beneath the limbal basal cells was distinct in ness. Wounds that are large are more of a challenge to the terms of matrix composition84-86; 3) and our work show- CESCs.82 The CESC niche is forced to remain function- ing that integrins were differentially expressed at the lim- ally proliferative for an extended time, and the majority of bus.87 Data from the brain and gastric niche suggest that the eTACs near the niche are also actively proliferating mesenchymal cells, including smooth muscle cells and and migrating away from the niche toward the central cor- myofibroblasts, secrete factors that are critical for sustain- nea. Because the corneal epithelial sheet is migrating at ing the niche both functionally and structurally. Extensive the same time the CESCs are proliferating, the proliferat- work on HSCs has contributed to our assumptions about ing CESCs could be swept into the migratory cell popula- the importance of niche-emptying for successful engraft- tion, where they eventually become post-mitotic and ter- ment and to the thinking that successful corneal epithelial minally differentiate. Conjunctival epithelial cells would cell engraftment likely involves a type of homing wherein then fill the empty stem cell niche. The lack of CESCs at the CESCs express specific integrins and adhere within this site would allow for the slow movement of conjuncti- the niche to specific ligands. val cells onto the cornea. Furthermore, the occupation of We have incorporated the findings of Ferrais and col- the niche by conjunctival cells physically prevents neigh- leagues88 and Pearton and colleagues89 into our model. boring eTACs from reoccupying that site after cell migation These studies show that central corneal epithelial cells from is complete. adult rabbit corneas can be reprogrammed to function as While we have used the term factors repeatedly to sig- ESCs and produce skin and sebaceous gland when grafted, nify cues that induce cell proliferation or cell survival, di- along with embryonic mouse dermal fibroblasts, back into rect cell:cell communication and cell:substrate adhesion adult mice. If adult central corneal epithelial cells can be to matrix proteins also play important roles as environ- made to adopt an ESC fate, surely they can be made to mental cues to direct cells toward specific fates. Some of adopt a CESC fate, if provided the right combination of the factors we refer to above are likely to be cell:cell adhe- factors. In the experiments by Ferrais et al,88 those factors sion molecules and/or matrix ligands rather than survival/ were provided by dermal fibroblasts isolated from neona- growth factors. Figure 3 shows evidence for the differen- tal mice. If we accept that the corneal TACs can be repro- tial localization of α9 and β1 integrins and Tn-C in the grammed, then, in order to reprogram them to become normal adult mouse limbus. β1 integrin is less abundant CESCs and not ESCs, it is necessary to identify the factor(s) at the limbus compared to the central cornea and con- in mesenchymal cells that are needed to accomplish this junctiva, whereas α9 integrin is present exclusively within transition. It is possible that these factors exist only in a subpopulation of the limbal basal cells. Despite the overall neonates and are produced by specific cell types at very reduction in the intensity of cells staining for β1 integrin specific times during development. However, we know that at the limbus, some cells at this site possess abundant β1 for some patients with LSCD, the cornea can be success- integrin and little α9 integrin. When the α9 integrin ligand fully treated with transplantation of cultures of corneal Tn-C is colocalized with α9 integrin in the adult mouse TACs.81,90,91 So, the CESCs can be functionally restored

THE OCULAR SURFACE / JANUARY 2005, VOL. 3, NO. 1 / www.theocularsurface.com 23 THE CORNEAL EPITHELIAL STEM CELL NICHE / Stepp and Zieske from TACs in adults. One challenge ahead is to determine cling limbal epithelial basal cells that can be preferentially how this happens and to try to increase its frequency of stimulated to proliferate: implications on epithelial stem cells. success. To do that, we need to continue looking carefully Cell 1989;57:201-9 at the limbal stem cell niche, both at the level of the CESCs 17. Ebato B, Friend J, Thoft RA. Comparison of limbal and pe- and at the stromal elements that are so important in creat- ripheral human corneal epithelium in tissue culture. Invest ing and maintaining it. We need to understand how it func- Ophthalmol Vis Sci 1988;29:1533-7 tions to maintain the ocular surface and what goes wrong 18. Lindberg K, Brown ME, Chaves HV, et al. In vitro propagation in LSCD. of human ocular surface epithelial cells for transplantation. Invest Ophthalmol Vis Sci 1993;34:2672-9 ACKNOWLEDGEMENTS 19. Pellegrini G, Golisano O, Paterna P, et al. Location and clonal We appreciate the excellent technical assistance of Ahdeah Pajoohesh- analysis of stem cells and their differentiated progeny in the Ganji, Sonali Pal Ghosh, and Audrey E.K. Hutcheon. Also, we would human ocular surface. J Cell Biol 1999;145:769-82 like to thank past members of the Stepp and Zieske laboratories 20. Kenyon KR, Tseng SC. Limbal autograft transplantation for who have contributed much of the data that forms the basis for the ocular surface disorders. Ophthalmology 1989;96:709-22; dis- ideas developed in this review. Finally, we owe a huge debt of grati- cussion 722-3 tude to our numerous colleagues who have listened to and fostered 21. Pellegrini G, Traverso CE, Franzi AT, et al. Long-term restora- our thinking and our science over the years. tion of damaged corneal surfaces with autologous cultivated . corneal epithelium. 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