Cell Markers and the Side Population Phenotype in Ocular Surface Epithelial Stem Cell Characterization and Isolation

Laboratory Science JAMES V. JESTER, PHD, SECTION EDITOR

Cell Markers and the Side Population Phenotype in Ocular Surface Epithelial Stem Cell Characterization and Isolation J. MARIO WOLOSIN, PHD ABSTRACT The ocular surface is covered by two rapidly renewing and embryologically-related linings, the corneal and conjunctival epithelia. The long-term survival of these tissues is ultimately dependent on their respective resident stem cells. In the corneal epithelium, the stem cells and their early precursors are exclusively circumscribed to the narrow vascularized limbal rim that provides epithelial precursor cells to the critically transparent central cornea. Limbal damage causes an interruption of this essential cell supply and allows the invasion of the corneal surface by the conjunctival epithelium, an event that ultimately leads to corneal scarring. The limited supply of immunocompatible tissue is a major hindrance to efforts to develop effective procedures for ocular surface reconstruction. This review describes some of the current work and strategies being developed to achieve the isolation of the limbal stem cell and define its genetic, biochemical, and functional make-up. The study of isolated ocular surface stem cells will foster basic understanding of the environmental requisites for their survival and proliferation in a self-replicative mode, leading eventually to advances in therapeutic approaches. KEY WORDS conjunctiva; connexin43, p63, ABCG2; cornea; epithelia; limbus; side population; stem cells

Accepted for publication November 2005 From the Department of Ophthalmology and Black Family Stem Cell Institute, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY, USA Supported by: EY 014878, EY 015132 and Core Center Grant EY 01867 The author has no commercial interest in any concept or product discussed in this article. Single copy reprint requests to: J. Mario Wolosin, PhD(address below). Corresponding author: J. Mario Wolosin, PhD, Department of Ophthalmology and Black Family Stem Cell Institute, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA. Tel : 212241-8980. Fax: 212-289-5945. Email: [email protected] Abbreviations are printed in boldface where they first appear with their definitions. ©2006 Ethis Communications, Inc. The Ocular Surface ISSN: 15420124. Wolosin M. Cell markers and the side population phenotype in ocular surface epithelial stem cell characterization and isolation. 2006;4(1):10-23.

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I. BACKGROUND A. The Ocular Surface Epithelia and the Stem Cellbased Renewal Plan

he vertebrate ocular surface is lined with two specialized, stratified, constantly renewing epithelia, the limbal/corneal and conjunctival lineages. These tissues originate from the initial ectodermal-optic vesicle interaction that defines oculogenesis. The close range interaction between PAX-6-positive head ectoderm and the optic vesicle leads to the development of the lens placode and, subsequently, through its invagination, the lens vesicle. The small number of PAX-6-positive cells remaining on the ectodermal surface expands in the subsequent days and, through two distinct pathways, become the two ocular surface epithelia.1-3 PAX-6 expression becomes a permanent, functionally significant component of these epithelia, which distinguishes them from the PAX6 negative epidermis.4-8 The two ocular surface epithelia undergo continuous daily wear, which, in the absence of keratinization, results in rapid constitutive shedding of isolated cells and continuous cell replacement. In its most basic features, their self-renewal is similar to that described for many continuously renewing tissues,9-11 including the epidermis, the endodermally derived gastrointestinal epithelia, and the blood system (Figure 1). Briefly, under steady state, a very small number of stem cells residing in specialized environments or niches divide or “cycle” infrequently. Proliferation may result in the generation of two stem cells, two slightly differentiated progenitors or, through asymmetric cell division,12 one stem cell and one slightly differentiated daughter cell. Because DNA is most vulnerable to mutation during synthesis, this slow cycling property is critical to protect these cells from dangerous transformations.13 This property is experimentally revealed by the ability of stem cells to retain, for a substantial amount of post-labeling time, radiolabeled thymidine or BrdU that was incorporated into their DNA in an earlier proliferation round.10,14,15 Additionally, the progeny of stem cells undergo gradual differentiation. The differentiation involves a marked increase

T

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CELL MARKERS AND SIDE POPULATION PHENOTYPE / Wolosin OUTLINE I. Background A. The ocular surface epithelia and the stem cell-based renewal plan B. Limbal stem cell segregation: consequences and solutions II. Limbal stem cell markers A. Cx43 exclusion B. p63 isoforms C. ABCG2 III. Hoechst side populations in ocular epithelia IV. Stem cell properties in the SP and LSSC cohorts A. In vivo slow cycling B. Clonogenicity and resistance to PMA C. Colony formation efficiency V. Concluding remarks

in the proliferation rate. The proliferation spurt implies that under homoeostatic conditions, or immediately after a drastic cell loss, the primary replacement of lost cells is accomplished by cell proliferation of the stem cell progeny. However, after a number of replication rounds, these transient amplifier (TA) cells undergo mitotic arrest and

Figure 1. The stem cell-based tissue renewal plan and the limbal/ corneal epithelial precursor cell segregation. A stem cell residing on a specialized niche divides every few weeks or months to generate a new stem cell and a slightly differentiated progeny that will go on to yield rapidly dividing but ultimately terminally differentiating cells. These latter transient amplifier cells provide tissues with the immediate cell replacement needs. The long-term survival of the tissue is dependent on the small pool of stem cells. A question mark in the sketch suggests that precursor cells in the very early stages of differentiation may be able to recover the stem cell phenotype when provided with the right ‘niche.’ In the limbal/corneal epithelium all stem and early progeny cells are localized in the basal layer of the vascular limbus, the only zone comprising cells not expressing the corneal differentiation cytokeratins K3 and K12.

Figure 2. Density-coded scatter plots of total human limbal or corneal epithelia (black color is highest density, blue color is lowest). The difference in light-scattering values is consistent with a substantial increase in average cell size and intracellular complexity in the cell’s transition from the limbal to the corneal domain.

terminally differentiate. It is possible that this transition from stem cells to TA cells involves intermediate states in which cells retain some of the properties of the stem cell and even the ability to re-acquire the stem cell status under favorable conditions.11 B. Limbal Stem Cell Segregation: Consequences and Solutions

Schermer et al discovered that basal cell movement from the limbus across the limbo-corneal boundary is associated with de novo, sudden expression of the tissuespecific differentiation cytokeratins.16 This finding led them to propose that in the limbal/corneal lineage, stem cells are exquisitely restricted to the limbus, the vascularized outer rim separating the conjunctiva from the avascular cornea. Other studies based on label-retaining cell localization14 and the effect on corneal health of limbal conjunctival autografts or keratolimbal allograft transplants aimed

Figure 3. Loss of the limbal epithelium allows corneal surface colonization by the conjunctival epithelium. The top panel shows the normal ocular surface. The corneal, limbal and conjunctival epithelia are colored in blue, lilac and green, respectively. The limbal zone has been magnified to depict the hypothetical distribution of stem (red) and other precursor cells (see Figure 1). The bottom panel summarily depicts the effect of limbal failure. The conjunctival epithelium, including its stem cells (red), has colonized the corneal surface.

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at restoring limbal epithelial stem cell17,18 have confirmed the initial hypothesis of Schermer et al. The phenotypic changes of basal cells at the limbal/ corneal demarcation are not subtle and are likely to involve multiple other severe changes associated with differentiation. Globally, this is revealed by a comparison of either cell sizes in humans using confocal microscopy, or the two light-scattering properties of cells analyzed by flow cytometry.19 Forward light scatter (FSC) is generated by particles that are relatively large with respect to the wavelength of the incident light (0.488 μm). Thus, since cells are about 20 times larger than the analyzing argon laser beam, this forward light scatter reflects the size of a cell. Light side scatter (SSC) is generated by particles smaller than the length of the incident light; its magnitude is proportional to the cytosolic complexity of the cells, a feature typically referred to as cell granularity. The epithelial cells of the central cornea show markedly higher averages than the limbal cells in both scatter parameters, i.e., they not only change their cytokeratin expression, but exhibit a large increase in size and complexity (Figure 2). This stem cell segregation to the limbus and the drastic differentiation of the limbal/corneal basal cell at the demarcation between the two domains has critical clinical consequences. Full circle or regional damage to the limbus due to chemical /thermal injuries, microbial infection, or autoimmune reactions results in the loss of precursors for the corneal epithelium.20 Secondarily, the limbal cell loss allows corneal surface colonization by the conjunctival epithelium (Figure 3). The latter tissue’s cellular properties and pro-angiogenic capacity lead to the inflammatory condition known as limbal stem cell deficiency. This condition may involve neovascularization and extensive corneal scarring. The formal adoption of the concept of stem cell segregation in the limbal/corneal epithelium has allowed a new perspective on certain pathological conditions of the cornea and led to the development of limbal transplantation methods for the treatment of limbal deficiencies in human patients.21,22 Currently, there is a very limited supply of cells available for clinical procedures using autologous tissue transplantation. Therefore, there is a great interest in finding ways to attain ex vivo preservation and numerical expansion of the stem cell population prior to surgical restoration. Toward this end, some degree of in vitro stem cell expansion has been reported in the blood system.23 The use of specialized substrata, such as preserved amniotic membrane, may provide a niche-like environment for ocular surface epithelial cells.24-26 Full preservation of the stem cell phenotype, though, may be a difficult task in vitro, due to the need for asymmetric cell division. Such division events are at the core of the nature of the stem cell. It is dependent on the exquisitely tuned and yet-to-be understood cues present in the site of stem cell residence or niche. Stepp and Zieske have recently reviewed the limbal stem cell niche in this journal.27 The existence of a physically limiting niche implies that, during division, the cell 12

closer to the niche environment somehow derives a polarity advantage that allows it to preserve more of the cytosolic components that stabilize the stem cell phenotype.12,28,29 Thus, under the standard substratum-symmetric conditions used to foster proliferation in culture for the generic tissue cell, loss of the stem cell phenotype may be difficult to avoid. In terms of clinical utility of expanded cells in culture, full preservation of stemness may not be a requisite. As initially proposed by Schoffield,11 it is possible that cells in the early stages of differentiation may be able to re-acquire the full stem cell status when transferred to the natural niche environment. This needs to be weighted as a factor in the interpretation of experimental and clinical transplantation studies that achieve restoration of the stem cell stock following expansion of limbal epithelial cell populations in cell culture. II. LIMBAL STEM CELL MARKERS The ability to isolate viable somatic stem cells from any given tissue is certain to open new means to manipulate them to human benefit. When unique epitopes or surface markers are identified on the stem cell plasma membrane, they can be used as a tether to isolate the intact cells by techniques such as cell panning or fluorescence-activated cell sorting. This was first demonstrated in the hematopoietic system with the identification of CD34 as a bone marrow stem cell surface marker.30,31 The purification of hematopoietic stem cells has led to extraordinary advances in both basic research and clinical practice in hematology. Similar advances in the epithelial field are still to materialize, limited in part by the absence of such biochemical landmarks for epithelial cells in general and for limbal and or conjunctival epithelial stem cells, in particular. In the limbal/corneal epithelium, earlier studies aimed at identifying stem cells relied on limbal-to-corneal expression comparisons. The underlying logic of such studies was that, given the absence of stem cells in the corneal domain, marker expressed only within the limbus may reflect a stem cell component whose expression extends to some or all of the early stem cell progeny, disappearing only upon substantial cell differentiation at the limbal/corneal demarcation.32,33 The intrinsic weakness of this approach is that expression, or lack of it, may solely be a function of environmental cues. Soluble factors derived from the circulation within the limbus, major differences in the cellular complexity of the underlying stroma, and differences in basement membrane composition may be a major determinant of the expressed phenotype without representing a direct relationship of the actual differentiation state of the cell. More recent studies have tried to identify limbal stem cell markers by searching for components displaying a limbal basal cell distribution consistent with the notion that only a few isolated cells can actually be stem cells, as inferred from the distribution of slow-cycling cells in vivo.14 Several reviews on the subject of limbal stem cell markers have been recently published elsewhere.34,35 In their extensive, well-

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Figure 4. Connexin43 in the human limbal/ corneal epithelium. A. Nomarski interference contrast micrograph incorporating the corneal periphery (b) and the Palisade of Vogt zone of the limbus (c). B and C. Immunofluorescence micrographs of b and c, respectively. In the corneal periphery, connexin expression is vigorous and extends to multiple suprabasal cell layers. In contrast, in the Palisades, connexin43 stain is scant and appears to be completely absent in numerous basal cells. Bar is approx. 50 μm for B and C.

documented review, Schlotzer-Schrehardt and Kruse described the expression of integrin alpha9, vimentin, KGFR, and the multiple drug-resistance transporter ABCG2 as limited to the basal cells of the limbal zone. Only the latter protein, though, displays a clustered expression that is consistent with intralimbal stem cell distribution. A large set of additional potential protein candidates, including integrins and growth- and differentiation-associated receptors, were found not to comply with a corneal/limbal and or intralimbal distribution consistent with stem cells. Thus, this review will explicitly focus on three proteins that, at least superficially, comply with the stem cell distribution criteria. A. Cx43 Exclusion

Connexin 43, a gap junction component, was the first protein whose distribution conformed to the criterium of a discrete limbal distribution, albeit by absence rather than by over-expression. Gap junctions allow cells to exchange and equilibrate ions and small solutes and, thus, contribute to coordinated responses within the cells of a tissue. The corneal epithelium of all vertebrate species examined displays a very high density of connexin43 in gap junctions. Within the limbus, this connexin is weakly expressed. The change from weak expression to high expression occurs suddenly at the limbal/corneal junction, essentially in conjunction with the conversion of basal cells from tissue-specific cytokeratin-negative to -positive status.33 In addition, notwithstanding differences from species to species and differences between different quadrants of the limbus, careful examination of intralimbal Cx43 stain in adult mammalian tissues revealed the presence of connexin43negative basal cells, as single cells or in patches, interspaced within the faintly expressing congeners. In rabbit neonates,

where the percentile of stem cells is expected to be higher than in the adult animal, entire patches of connexin43negative cells were easily distinguishable. Likewise, fully negative areas within some areas of the human Palisades of Vogt (Figure 4) or in the equivalent zone in the bovine36 can be found. These features and physiological measurements of solute transmissibility from cell to cell, indicating the absence of cytosol sharing within the limbal zone, led to the proposition that the stem cells of the limbal/corneal epithelium and stem cells in certain other epithelial cells, may be distinguished by the complete absence of connexin gap junctions. Teleologically, metabolic isolation makes sense: stem cells need to protect an expected unique intracellular milieu from the effects of solutes transmitted from rapidly cycling neighbors. Schlotzer-Schrehardt and Kruse35 identified an equivalent exclusion pattern for several proteins related to cell-cell adhesion and cell contact, including P-cadherin and the integrins alpha2, alpha3, alpha6 and beta4, suggesting that communication deficits in the stem cells may extend to other mechanisms of cell-cell interactions. The ectodermal cells, from which the limbal/corneal epithelia derive are connexin43-positive. Hence, it stands to reason that de-expression of connexin43 must occur at some point during the genesis of the limbal stem cell. Accordingly, Wolosin et al. tracked the expression of connexin43 from the earliest ocular developmental stages in the rat 37 The study provided potentially important clues regarding limbal genesis. On embryonic day (E) 12.5— soon after establishment of the embryonic corneal epithelium, at a stage that precedes the start of expression of the tissue specific cytokeratins in the corneal zone or the development of any anatomical feature associated with a limbal structure— individual connexin43-negative corneal epithelial cells were observed directly adjacent to the edges of the developing retina. These few connexin43-negative cells multiplied during the subsequent days to form a fully negative zone that could be tracked throughout embryonic development and by E20 was localized at the morphologically identifiable limbus (Figure 5). If down-regulation of connexin43 ’marks‘ the establishment of the limbal stem cell, it is possible that this embryonic-stage limbal stem cell influences in some interactive manner the formation of the adult limbal stem cell niche, rather than being an independently formed niche that causes the stem cell to emerge at a later stage from a generic embryonic corneal epithelial cell at the limbal location. Connexin43 is also expressed in the conjunctival epithelium, but the expression in this epithelium is much weaker than in the corneal epithelium. This fact, and the intrinsic complexity of this epithelium, makes it very difficult to determine whether the absence of connexin43 in this tissue is related to stemness. B. p63 Isoforms

The p63 protein, a member of the p53 superfamily, is an essential cell-fate determinant for stratified epithelium,38,39

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Figure 5. Expression of Cx43 and K12 at the rat limbal/ corneal demarcation on embryonic day 20. A. Thumbnail view of section used for the immunostaining in B and C. This section was poststained with H&E after collection of immunostaining images. B. Framed area in A immunostained for connexin43. Corneal (Co), limbal (Li); bulbar (bCn) and palpebral (pCn) conjunctiva zones and the eyelid (ey) are indicated. Arrows mark the putative limits of the limbus, as inferred from connexin43 expression. C. Higher magnification of area of B. D. Cryosection adjacent to section used for B/ C stained for the corneal specific keratin 12. Note that along the limbal/corneal differentiation axis (left to right), enhanced expression of connexin43 occurs a few cells ahead (bracket with arrowheads) of the de novo expression of K12. Bar is approx. 50 μm for B and 20 μm for C and D.

fulfilling a number of functions in development and in mature organisms.40,41 The p63–/– mouse is born without any substantial epidermal component, indicating the significance of p63 for the survival of the epithelia derived from the ectoderm. The protein is expressed as multiple protein types with 2 subclasses, TA and ΔN, which arise as a result of alternative promoter usage.41 TA isoforms contain transcriptional activation domains at the N-terminus and can induce p53-target genes. ΔNp63 isoforms lack the N-terminus and cannot activate transcription. Instead, these isoforms seem to block transcriptional activation by p53 and TA isoforms of p63, hence providing an antiapoptotic effect in a dominant negative fashion. Using a pan-p63 antibody and a chromogenic staining method, Pellegrini et al42 observed that p63 was expressed in isolated human limbal basal cells not expressing the proliferation marker PCNA. This pattern, combined with the fact that cell clonogenicity correlated with p63 expression, led these investigators to propose p63 as the first positive marker of limbal stem cells. This proposal has generated great interest and a certain degree of controversy. One source of the controversy regards detection methods; when immunodetection methods more sensitive than the chromogenic method used by Pellegrini et al were used, most human and rabbit limbal basal cells, and even many adjacent suprabasal cells and basal cells 14

throughout the corneal zone, were confirmed to express p6343,44 (Figures 6 and 8). In spite of this apparent contradiction, the p63/stem cell relationship has been the subject of multiple investigations. Because of the complete absence of differentiation, epithelial stem cells are expected to be small and, thus, to have the largest nuclear-to-cytoplasm ratios.45,46 In accordance with this notion, Arpitha et al measured p63 expression as a function of nucleus-to-cytoplasm ratio in cyto-spins of human limbal cells.47 They established that the highest expression occurred in cells displaying the highest nuclear-to-cytoplasm ratio. These same cells were also negative for connexin43 and cytokeratin K3. Salehi-Had et al measured the levels of p63 in limbal epithelial cells grown in culture in sparse or dense conditions.48 They noted that essentially all adherent cells were p63-positive, negating an absolute stem cell/p63 correlation. Later, the loss of p63 in these cultures was found to be dependent on culture density. Since cell density is a well-established factor in the induction of differentiation, this result is consistent with the expression of p63 in a large fraction of the limbal/corneal basal cells in vivo and indicates that the loss of p63 expression is related to terminal differentiation. Nevertheless, the highly confluent differentiated cultures contained a small proportion of cells with strong p63 expression. This suggests that these cells may represent a subpopulation resilient to differentiation under conditions that cause differentiation of other cells, a property expected for stem cells. Epstein et al examined the relationship between p63 content and the onset of proliferation of rabbit limbal cells.49 The rationale for these studies is that, compared to the rapid proliferation status of TA cells, stem cells, having a slow-cycling phenotype in vivo, may exhibit a delay in their initial proliferation and/or longer cycling times after they are set in culture. This concept is depicted schematically in

Figure 6. Expression of p63 in the human limbus as determined with pan-p63 antibody. The histogram describes the intensity of nuclear stain for a continuous patch of 85 basal cells from the inserted micrograph. Notice that a) the majority of the basal cells are positive: b) 4-7 cells, or 5-9%, have distinctively higher p63 levels; and c) nuclei of some of the cells leaving the basal layer are also p63-positive.

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Figure 7. p63 staining in the rabbit limbus and in four colonies (C1-4) originating from single limbal epithelial cells cultured for 7 days over a feeder layer of 3T3 cells. Notice that larger colonies, i.e., those originating from faster- or earlier-proliferating precursors, have lower p63 stain.

Figure 8. The effect of delayed initial proliferation on the generation of cell duplets in limbal cultures. It is assumed that the higher the p63, the slower the cell cycling rate. The number of days included in the sketch corresponds to the experimental days reported in Table 1.

Figure 7. The results, detailed in Table 1, show that cell duplets present several days post-plating (i.e., those that result from a very recent cell division or from a division used by other researchers had any significant component that is proceeding slowly) contained higher p63 levels than of wounding at the time of collection, further studies are either duplets that formed soon after plating or those that clearly necessary to confirm this theory. developed into substantial colonies during this time (FigWith respect to the expression of distinct isoforms, ure 8). Thus, this ex vivo study establishes that cells with Kawasaki et al used laser capture microdissection to isothe highest level of p63 have a slower-cycling phenotype, late cells from the basal and suprabasal compartment from consistent with stem cells. In fact, p63high cells amount to human cornea, limbus, and conjunctiva. They then meaabout 5% of the basal cells of the limbus (Figure 6), a sured levels of isoform message RNA by reverse tranpercentile that is consistent with estimates of the percentscriptase-polymerase chain reaction (PCR).54 Using primers for most TA and ΔN sequences, they found that only age of stem cells derived from label-retaining studies. FiΔN isoforms are expressed and confirmed that ΔNp63α nally, in rat and mouse, the distribution of p63 does not was the dominant form in the limbus. However, they did seem to conform with that expected for a stem cell marker, find evidence for the two other ΔNp63 variants in the corbeing higher in the basal cells of the corneal periphery nea and in the suprabasal limbus. than in the limbus.50,51 The complex relationships between differentiation staIn the rabbit, Wang et al found expression of a TA tus and p63 may originate from the expression of more isoform. This isoform displayed a highly differentially than one p63 isoform. Using an isoform-specific antibody, limbal expression. In contrast, the expression of a ΔN Di Iorio et al examined the expression of the three isoforms isoform(s) is minimally abated with the transition from of the ΔNp63 family in the human.52 They found that only the limbal to the corneal domain.55 One can see from this the α isoform is expressed in intact tissue samples. Woundlatter study that, depending on the relative levels of exing, though, led to the expression of the two other ΔNp63 pression of different isoforms in different species and the forms, β and γ. Furthermore, using the new antibody spedifferences in expression between cells of a given domain, cific for the α type and a classification of colonies accordthe relationship between stemness and p63 expression may ing to the Barrandon and Green methodology, 53 they demonstrated that exTable 1 Intensity of p63 staining in clonal assays of rabbit limbal cells as a pression of this isoform occurs only function of number of cells in the colony at various post-seeding in holoclones, the clones presumably days. derived only from stem cells. Thus, they attribute the expression of p63 Day 2 Day 4 Day 7 in the large majority of the human # of cells/colony Mean ±SD Mean ±SD Mean ±SD limbal cells (e.g., as in Figure 6) and 1 71.3 4.4 78.7 16.1 129.4 6.6 its expression within cells of the corneal domain to be due to damaged 2 56.1 4.7 99.8 10.0 148.9 9.6 specimens in which the other isoforms became expressed as a result of 3-6 24.9 5.7 15.1 8.3 23.1 6.3 a wound healing/proliferative re7-12 23.4 7.6 sponse. Considering that there is no indication that the human corneas THE OCULAR SURFACE / JANUARY 2006, VOL. 4, NO. 1 / www.theocularsurface.com

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or may not be apparent when pan-isoform antibodies are used. In conclusion, a stemness/P63high relationship appears to exist but may be obscured in many conditions or in different species by the co-expression of sequence variants unrelated to the stem cell phenotype that are also recognized by the antibody employed. Regardless of final verification of the status of p63 as an absolute marker of stemness, several studies have used the expression of p63 in cultures as an indication of preservation of the stem and/or precursor state. Hernandez-Galindo et al compared the effect of phorbol myristate acetate (PMA) in explant cultures of human limbal epithelial cells during ex vivo expansion on amniotic membrane or on a plastic cell culture surface.56 Addition of PMA to cultured epidermal keratinocytes or corneal epithelial cells induces the majority of cells to cease proliferating and commence differentiation. However, stem cells have a different response. In vivo, they are induced to proliferate, and, in culture, they withstand this “differentiative effect” of PMA.57,58 Under control conditions, little difference was found between the two substrata in terms of p63 expression and the lack of corneal connexin expression. However, upon introduction of PMA, cells grown over plastic rapidly lost p63 and gained connexin expression. These changes toward a more differentiated phenotype were prevented or ameliorated by the amniotic membrane, suggesting that this biological substratum stabilizes the stem or cell precursor phenotype. P63 has also been used in several attempts at stem cell purification, whereby the enrichment of presumptive stem cells is indicated by the higher level of p63 in that fraction relative to a non-stem cell control population. Li et al and de Paiva et al fractionated cultured human limbal stem cell populations according to either the rapidity of attachment to collagen IV substratum or cell size, respectively.59,60 They used a similar battery of recognized tests to assess the relative enrichment of stem cells including p63. The message RNA for this protein was found to be higher in the fraction containing the more rapidly attaching or smaller cells, respectively. The same fractions also displayed a number of other features, including higher colony formation efficiencies, consistent with enrichment in stem cells. Stained with the pan-p63 isoform antibody, the human

or rabbit conjunctival epithelium shows a basal cell antigen distribution similar to that of the limbus. At the gene level, the human conjunctiva also showed an isoform distribution resembling that seen in the limbus.54 C. ABCG2

Based on several converging observations, Zhou et al61 proposed that the protein ABCG2, a transporter belonging to the extended family of ATP- binding cassette (ABC) proteins, may be highly expressed in many stem cell types (see next section). Consequently, a number of researchers have examined its expression in the ocular surface.7,35,60,6265 These studies show that ABCG2 is expressed in the human limbal and conjunctival epithelium but not in the stem cell-free corneal epithelial domain.63 Reflecting the complexity of the human limbal zone and the possible variability between specimens and staining methods, the limbal staining for ABCG2 seems to display a variety of patterns. Nevertheless, it can be stated that ABCG2 expression occurs as discrete foci in both tissues, consistent with the stem cell criteria (Figure 9). Typically, each one of these foci includes a number of basal cells and, in some clusters of cells, adjacent suprabasal cells. Within the limbus, cell group size is smaller near the limbal/corneal junctions. Cluster size and frequency increase toward the conjunctival side of the tissue, in particular, in the Palisades of Vogt. In this site, long suspected be the main zone for the human limbal stem cells, ABCG2 is present in some specimens (but not in others) as multiple stratified layers, forming a near continuum of staining in which individual clusters cannot be easily distinguished (see double asterisk in Figure 9). While staining for ABCG2, Dua et al identified heretofore unrecognized, stunning-looking, epithelial crypts extending from the Palisades base, which are fully ABCG2positive.65 Probably, the deep incursion of these crypts into the limbal stroma and their narrow upper necks made it difficult to recognize these as epithelial cells in the past. Given the relationship of ABCG2 to stem cells, the authors proposed that the crypts were stem cell niches containing high numbers of the most primordial limbal stem cells. Fittingly, the ability of limbal epithelial cells to infiltrate the stromal matrix has been independently documented.66

Figure 9. Cluster distribution of ABCG2 in the limbus and conjunctiva. A1-4. Images from the human limbus. The corneal zone is located to the right of the image. In A1 asterisks are positioned above clusters of ABCG2-rich basal cells. In a Palisade of Vogt distal to the cornea (double asterisk), all basal and several layers of suprabasal cells copiously express ABCG2 antigen. The A2 and A3 micrographs depict cell clusters that include suprabasal cells. The A4 sample depicts a cluster of positive cells within a Palisade of Vogt, where most cells show no suprabasal stain. The dashed line outlines the base of the epithelial cells. B. Representative sample of an ABCG2+ cell cluster in human conjunctiva. C. Rat conjunctiva.

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Several intriguing observations can be derived from the ABCG2 limbal staining patterns. In the epidermis, the basal cell layer is viewed as an evenly distributed set of proliferative units, each emanating from one stem cell.67 The limbal ABCG2 staining fits this model if one assumes that only the cell (or cells) at the center of the cluster is a stem cell and that the surrounding cells, both basal and suprabasal, are cells that are directly derived from the last division cycle of this stem cell. Given the long period of time between one cell division and the next for any stem cell, this assumption implies that the adjacent ABCG2– positive cell remains immobile over this long period. Another possible explanation for the ABCG2-positive cell clusters is that the niche environment induces ABCG2 expression. Cluster size and preservation of ABCG2 expression in suprabasal cells may reflect quantitative differences in the niche-inductive abilities. In the Palisades of Vogt and in Dua’s crypts, larger niches may accommodate multiple stem cells side-by-side, leading to ABCG2 expression in all cells, irrespective of their differentiation status. In any event, given that ABCG2 expression reflects a degree of stemmess, the pattern of ABCG2 staining in the human limbus indicates a gradient of stem cells skewed toward the areas more distal to the corneal domain. This resembles the situation described above in respect to the exclusion of connexin43 in this area. ABCG2 in the human conjunctiva is quite high and, thus, visualization of a cluster arrangement is sometimes difficult. Antigen clusters are more easily observed in the rat, a second species recognized by one anti-ABCG2 antibody (Figure 9). For use as a tether for immunopanning, ABCG2 is the most attractive candidate of the three proteins discussed. Connexin43 is an integral membrane protein presenting an extracellular domain, and thus, it may provide the opportunity for stem cell purification by negative immunoselection methods. This protocol is uncertain, because connexins may undergo rapid internalization upon cell-cell dissociation, thereby generating a false-positive cell component. p63 is a nuclear protein and, as such, it is not useful for viable cell purification. The main problem with ABCG2, however, is that a large fraction of the ABCG2-positive cells may not be stem cells. Nevertheless, ABCG2 has been applied by de Paiva et al to obtain a stem cell-enriched population by flow cytometry, using an antibody directed against the exposed side of ABCG2.64 This immuno-isolated population complied, marker-wise, with the expected properties of limbal stem cells and displayed higher clonogenic capacity than an ABCG2negative population. III. HOECHST SIDE POPULATIONS IN OCULAR EPITHELIA

A unique relationship of ABCG2 to stem cells has been established as a result of a series of discoveries that provide for an instructive narrative on scientific progression. The DNA binding dye Hoechst 33342 has long been used as a vital dye to identify cells in different stages of the cell cycle. Bound to DNA, under ultraviolet excitation, Hoechst 33342 emits both

blue and red fluorescence. The blue fluorescence is the primary emission of the dye monomer in solution. The red fluorescence originates from π +- π+ orbital interactions that become possible when this planar dye intercalates in the DNA. This interaction generates new intermediate quantum energy levels. Non-radiational energy decay from the main excited energy state to these new states followed by radiational decay from there to ground level results in low energy (red) emission. The more dye accumulated in DNA, the higher the transference of fluorescence yield from blue to red emission, a bathochromic shift. Because the interaction between the dye and DNA is a physical phenomenon quite independent of cell status, when stained cells are analyzed by flow cytometry, all the cells containing a single copy of DNA (G0/G1) localize in the blue/red emission plot around a single spot (Figure 10). Cells that are in G2/M stages maintain the same red/ blue ratio but emit at double the absolute intensity, because they contain double the amount of DNA per cell. Cells in the S phase display an intermediate intensity, proportional to the amounts of extra DNA already accumulated. Additionally, aging/terminally differentiating cells can be expected to produce deviant results, because their DNA “packing” is changed, allowing more Hoechst accumulation (leading to lower blue/red ratios). A decade ago, Goodell et al reported that, in addition to these expected spectral domains, populations of mouse bone marrow cells contained a population of cells located in blue/red Hoechst emission plots in a peculiarly shaped zone to the left (blue) of the main blue/red axis.68 These cells became known as side populations (SPs). They demonstrated that these cells were identified with the most primitive bone marrow precursors, as defined by the mouse hematopoietic cell markers and bone marrow repopulating capacity. The source of the SP was determined to be the presence of one or more multidrug resistance transporters capable of efficiently removing Hoechst before it can bind to nuclear DNA. These ATP binding cassette (ABC) efflux pumps remove xenobiotics from cells using ATP-derived energy, thereby protecting cells from a variety of cytotoxic insults.69,70 Thus, under proper kinetic conditions ABC transport-rich cells may display 1) an overall decrease in fluorescence, and 2) a spectral shift toward the blue, due to amelioration of the bathochromic shift resulting from less dye bound in the DNA (see Figure 10). Later, with expanded understanding and genetic identification of the large variety of ABC-type transporter families, Zhou et al published what now can be viewed as a seminal proposal, plainly stating that, “the ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype.“61 Thereafter, multiple studies linked the SP activity to cell stemness in pancreas, mammary gland, and lung,71,72,73 confirming the likelihood of Zhou’s proposal. However, it would be premature to jump to the conclusion that this is a universal feature of stemness rather than a frequent one. The stem cell status of SP cells in the epidermis

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has been questioned,74 and the protein may be expressed at substantial levels in differentiated cells, as in blood and in the colon. Several limbal studies have explored the expression of the SP phenotype.7,60,63,64,76,77 In our studies, we took into consideration the common developmental origin and functional closeness of the limbal/corneal and conjunctival epithelia, and we decided to simultaneously examine both limbal/ corneal and conjunctival epithelia SPs. The practical advantage of this approach is to overcome the extreme numerical limitation of working only with the small number of cells provided by the limbus. SP cohorts were identified in the

Figure 11. Side populations, ABCG2 effect, and correlation to scatter plots in human limbal epithelial cells. Top panels. The control plot shows a well-defined SP that is abolished by the ABCG2-specific inhibitor fumitremorgin C. Bottom panel. Correlations between Hoechst and light scatter shows that a substantial fraction of the SP cells display a remarkably low SSC (LSSC). Conversely a substantial fraction of the total LSSC population consists of SP cells.

Figure 10. Flow cytometry Hoechst plots in the absence (top panel) and presence (bottom panel) of ABCG2 transport. The close packing of intercalated dye in the DNA generates a bathochromic emission shift, from blue to red. Under these conditions all cells exhibit a constant blue/red emission ratio. The only other significant parameter is the amount of DNA/cell. Cells with more than one DNA copy (cells in S and G2-M stages) emit more total fluorescence. The presence of an efficient Hoechst 33342 efflux transporter (bottom panel) leads to decreased bound dye, and thus less absolute dye concentration and less bathochromic shift due to larger distances between intercalated monomers.

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Figure 12. BrdU retention in rabbit conjunctival and limbal epithelium and in sorted cell populations following a 17-day BrdU loading and 5 weeks BrdU chase. All cells have been counterstained with PI (red). BrdU retention is revealed by the green color of the FITC conjugated anti-BrdU antibody

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limbal and conjunctival epithelia of three species, human (Figure 11), rabbit, and pig, but were never present in the stem cell-free corneal epithelial population. Two experiments directly linked the cohorts to the transport activity of ABCG2. First, fumitremorgin C, an ABCG2-specific transporter, as well as reserpine, a general ABC-type transporter inhibitor, markedly reduced or abolished SPs in both limbal and conjunctival cells.63 Second, the dependence of the SP phenotype, as studied in rabbit conjunctiva, complied with the kinetics of a transport phenomenon. At low Hoechst concentrations, an SP is easily identified, but increasing Hoechst above 5 μg/ml overwhelms the efflux capacity of the cell pump, causing the SP to disappear into the general G0/G1 population. Other studies have shown that SP limbal cells contain high levels of ABCG2 message, relative to their non-SP counterpart.60,64,76,77 Unfortunately, the biochemical characterization of these cells has, so far, been of a limited scope. This is understandable, given that a human or animal limbus yields typically about 1,000 SP cells. Such cell numbers are consistent with estimates of 100 stem cells per mouse limbus, obtained using clonal patches of LacZ-expressing cells in chimeric animals.78 Correlation analysis between the Hoechst emission and light-scatter properties shows several important features. About 50% of the SP cells have a very low forward light scatter, indicating, as expected, small cell size (Figure 11). However, the most significant feature in this correlation between SP type and physical properties is found in the side scatter features. The small cells displayed extremely low SSC values, to the extent that in the majority of the experiments, a large fraction of the SP cells can be distinguished in the scatter plots as a distinct area separated from the main cell cluster. This is true for both the limbus and conjunctival populations. We refer to this cluster as the SSClow or LSSC cohort. SP cells, not included within the LSSC cohort, have a FSC and SSC distribution similar to that of the general, non-SP cell population. Processing of the Hoechst/light scatter correlation analysis in the reverse direction shows that only a fraction of the LSSC cells belong to the SP population, leading to the definition of two LSSC subsets, LSSCSP+ and LSSCSP- cells. Anatomically, under phase contrast illumination, and consistent with the scatter features, the SP comprises a mix of small and large cells, vis-a-vis the size of the majority of cells isolated from the center of the G0/G1 main cell cluster. The LSSC consists of a population of small cells only. In contrast, the stem cell-free corneal epithelium contains no SP or LSSC cells. This later result suggests that the LSSC population may be directly related to cell stemness. IV. STEM CELL PROPERTIES IN THE SP AND LSSC COHORTS A. In Vivo Slow Cycling

The in vivo “slow cycling” feature is the most unique testable feature of adult stem cells of renewing epithelia. In the stratified epithelia derived from the ectoderm and

the columnar and glandular gastrointestinal epithelia, stem cells may cycle as seldom as once every few months, while most other proliferative cells reproduce as often as once a day. Hence, to identify label-retaining cells, tritiated thymidine or the thymidine analog BrdU are infused for several days into a neonate or young animal undergoing rapid growth. At this early developmental time, all or most proliferation-competent cells, including the stem cells, undergo cell division. Label administration is then stopped, and the animal is left undisturbed for several weeks. During this period, most cells continue to undergo rounds of cell division, and the acquired label becomes dramatically diluted. On the other hand, stem cells enter a near-quiescent state and divide rarely or not at all, retaining detectable levels of the label. Accordingly, to determine whether ocular surface epithelial SP cells possess the slow cycling phenotype, we implanted in young rabbits osmotic pumps containing BrdU. Following 8-10 days of continuous BrdU infusion, SP and/or LSSC limbal and conjunctival cells were sorted, attached to cover-slips by incubation or cyto-spinning and stained with an anti-BrdU antibody. Consistent with the substantial degree of epithelial labeling observed in tissue sections, 60-90% of the non-SP cells, representing the majority of the cells in the population, contained BrdU. In contrast, cells belonging to the SP subpopulation cells or the related LSSC fraction, displayed much lower percentiles of BrdU+, suggesting that most cells in this population did not divide over the 10-day interval. Next, we performed experiments conforming to the classical protocol used to identify label-retaining cells. BrdU was infused for a prolonged period of time (17 days), the pumps were removed, and the rabbits were subjected to a 5-6 week BrdU chase period. After this chase period, BrdU+ basal cells were scarce in both the limbus and conjunctiva (Figure 12). Cells displaying various levels of staining were also observed within the suprabasal strata near the epithelial surface, indicating that label disappearance during the BrdU chase interval occurs not only by dilution through multiple cell divisions, but also through cell exfoliation. Consistent with the tissue immunostaining patterns, the conjunctival epithelial G0/G1 fraction, accounting for the majority of the cell population, contained very few BrdU+ cells. In contrast, a substantial fraction of the cells in the conjunctival SP sample and limbal LSSC sample were BrdU-rich. (The multiple steps involved in Hoechst analysis leads to substantial cell attrition; thus, given the high degree of overlap between SP and LSSC cohorts, it was prudent to sort limbal cells according to light scattering into LSSC and non-LSSC cohorts, thereby avoiding the Hoechst loading step.) In summary, these cycling studies confirmed that a substantial fraction of the cells collected by flow cytometry according to the Hoechst exclusion (SP) or the LSSC feature conform to the classical definition of the stem cell in rapidly renewing tissues. Because the actual cycling rates for these cells are not known, we cannot ascertain whether

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the non-labeled cells in the SP or LSSC populations are slow cycling cells that did not happen to divide during the BrdU labeling interval or plainly, non-cycling cells of some type. B. Clonogenicity and Resistance to PMA

Stem cells and the less differentiated TA progeny proliferate in an isolated state (clonogenic growth) when supplied with the proper conditions. In the specific case of stratified epithelia, a test for stem cell status relies Figure 13. Effects of PMA in colony formation by rabbit limbal (Li) and corneal periphery on the selective proliferative response to (Co) cells and rabbit limbal SP and non-SP (Go/G1) cells. PMA (100 μM) was applied in the 4-72 hr post-plating interval. Small arrows in the Li-SP panel point to microcolonies (mc). PKC activating PMA.57,58,79,80 In vitro, Control samples were fixed and stained on day 12. PMA treated samples were fixed and this differential response to PMA is stained on day 14. Notice that the percentile of PMA-resilient clonogenic cells is much manifested in the ability of a fraction larger in the Li-SP than in the Li-nonSP. of limbal or conjunctival cells to “survive” exposure to PMA and go on to clonogenic assay in revealing stemness within an epitheestablish colonies. For instance, when young rabbit limbal lial population. and corneal periphery epithelial cells are set side-by-side Recognizing this important conundrum, Umamoto et in clonogenic assays, both cell populations yield apparal proposed that the limbal SP cells may be, in fact, a very ently similar colonies (Figure 13). However, if PMA is inprimitive population of highly quiescent cells, which, in cluded in the cultures in the 4-72 hours post-plating, the vivo, generate mitotically slow-cycling stem cells with high corneal population loses clonogenicity, whereas some proliferative potential.77 It is these cells, excluded from limbal cells survive the “differentiative effect” of PMA and the SP cohort, that have a slow cell cycle and colony-formform colonies, consistent with the notion that only the ing abilities. A second possibility is that, in fact, SP cells limbus contains stem cells. When the limbal rabbit cell are stem cells that cycle at a very slow rate in vivo and, population was sorted into SP and G0/G1 (i.e., non-SP) consequently, are not able to reach proliferative status in cells, PMA resilience was found to be concentrated in the vitro, causing the initiation of apoptotic mechanisms. In SP cohort. The PMA-treated SP dishes contained, in addithis context, it should be noted that, even though the pertion, a large number of microcolonies deriving from cells centile of PMA-resilient cells is much larger in the SP than that started to divide and make colonies after several days in the non-SP fractions, when the small size of the SP coin culture (as per microscopy monitoring). When the larger hort (<1%) is taken into account, there are probably more colonies were wiped out, these microcolonies continued PMA-resilient cells in the non-SP fraction. to grow, becoming large colonies by day 26.63 D. Colony Formation Efficiency

The flip side of these encouraging results with PMA treatment resides in the actual colony formation efficiency (CFE) of the SP cells. The CFE of the rabbit SP is much lower than one would expect from a population highly enriched in stem cells, ranging from less than 1% to no more than 3%. Probably more significant than the actual percentiles obtained under particular conditions is the fact that in side-by-side comparisons, the SP CFE is always lower than the CFE of the non-SP cells,63,77 even though the latter cohort is likely to contain a substantial proportion of non-basal, post-proliferative cells. This result is in clear contradiction to current operational definitions for the relationship between stemness and clonogenic ability.53 In-so-far as clonogenic capacity is a major recognized feature of stem cells, this low CFE either challenges the assumption that SP cells are stem cells or, conversely, introduces a question as to the absolute value of the 20

V. CONCLUDING REMARKS A body of evidence is accumulating to help define the cell marker and phenotypic character of the ocular stem cell types. Further studies, however, are needed to unravel the intrinsic features of these cells. First, a substantial fraction of the SP cells display a light scatter phenotype consistent with being small and lacking intracellular complexity. This LSSC phenotype is shared by another fraction of cells that do not exhibit the SP phenotype. The fact that cells with LSSC features are not found in the stem-cell free corneal domain provides a strong incentive to consider the possibility that the entire LSSC cohort consists of precursor cells. Intriguingly, Pavlovitch et al isolated a (similarly) small cell fraction (<8 μm) from mouse epidermis and found that, as with the rabbit limbal and conjunctival SP and LSSC cells, the mouse fraction also displayed extremely low colony-formation efficiency.46 Nevertheless, further exploration of

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this issue is needed to confirm this assumption—in particular, because the limbal and conjunctival epithelia incorporate non-epithelial cells, including melanocytes, dendritic cells, and most notably, peripheral T lymphocytes,81 a cell that is known to display very low SSCs82 and multiple ABC-type transporters.83 Second, it will be highly desirable to establish the degree of overlap between the various proposed stem cell markers or phenotypes by simultaneously staining for two or three of them and linking these results to the slow-cycling, label-retaining cells revealed by tritiated thymidine or BrdU pulse and chase. Presently, the completion of this task is more difficult than it would appear. ABCG2 staining is primarily limited to human tissue (due to antibody specificity and effectiveness), whereas label retaining cells can only be identified in animals. In their studies in human limbus, SchlotzerSchrehardt and Kruse report substantial overlap of connexin43 exclusion and ABCG2 expression.34 Yet, there are no guarantees that the stem cell pool displays a homogenous expression pattern, and, thus, a strict analysis of marker overlap may not lead to unambiguous interpretations. Third, there are apparent discrepancies between the results obtained by different stem cell purification methods that await resolution. Cells isolated according to the rapidity of attachment to collage type IV59 or via the ABCG2 external epitope60,64 are reported to display increased clonogenic capacity, whereas the cells isolated according to the functional feature conferred by the ABCG2 transporter, the SP phenotype,63,77 display very low clonogenic ability. The cell percentiles gathered by the first two methods are substantially larger than the size of the SP populations. Therefore, it is possible that the rapidattaching or the ABCG2-positive populations are either different from the SP population and/or encompass, in addition to SP cells, cells with high initial clonogenic capacity. It will be interesting to see what fraction of the cells isolated using the ABCG2 antibody are indeed SP cells. While these discrepancies are resolvable, clearly the main scientific challenge is to find the conditions that allow SP cells to survive and proliferate in vitro. Resolution of this quandary will open doors for the study of new methods to attain stem cell expansion in vitro and, ultimately, improve stem cell restoration in the clinical setting. REFERENCES 1. Davis JA, Reed RR. Role of Olf-1 and Pax-6 transcription factors in neurodevelopment. J Neurosci 1996;16:5082-94 2. Koroma BM, Yang JM, Sundin OH. The Pax-6 homeobox gene is expressed throughout the corneal and conjunctival epithelia. Invest Ophthalmol Vis Sci 1997;38:108-20 3. Nishina S, Shinichi K, Yamaguchi Y, et al. PAX6 expression in the developing human eye. Br J Ophthalmol 1999;83:723–7 4. Collinson JM, Quinn JC, Hill RE, West JD. The roles of Pax6 in the cornea, retina, and olfactory epithelium of the developing mouse embryo. Dev Biol 2003;255:303-12 5. Collinson JM, Chanas SA, Hill RE, West JD. Corneal develop-

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