Integrins in Slow-Cycling Corneal Epithelial Cells at the Limbus in the Mouse - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:07 |只看该作者 |倒序浏览 |打印

作者：Ahdeah Pajoohesh-Ganjia,b, Sonali Pal-Ghosha, Samuel J. Simmensc, Mary Ann Steppa,d : N. X. I4 A$ {$ z8 v0 W
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      【摘要】
   Adult corneal epithelial stem cells (CESCs) have been shown to reside at the periphery of the cornea at a site called the corneoscleral junction or limbus. Although studies have shown that these cells are slow cycling, their molecular characteristics are not well understood. Using a whole-mount procedure, we show that whereas 9-integrin is present in a subset of the basal cells at the corneal limbus and absent in the central cornea, &szlig;1-, &szlig;4-, 3-, and 6-integrins are more highly expressed overall in central corneal basal cells. To characterize CESCs based on their slow-cycling nature, we simultaneously evaluated 5-bromo-2-deoxyuridine (BrdU) label-retaining cells (LRCs) and integrin expression (9, &szlig;1, and &szlig;4) in a total of 1,889 cells at the limbus of adult mice that had been injected as neonates with BrdU. Whereas the LRCs were usually observed adjacent to 9-integrin-positive cells, most LRCs were 9-integrin¨Cnegative and expressed high levels of &szlig;1- and &szlig;4-integrin. In addition, we observed more BrdU-positive LRCs at the superior and inferior quadrants of adult mouse corneas than at the nasal and temporal quadrants, and determined that 0.94 to 3.6% of the limbal basal cells were slow cycling. We conclude from these data that the slow-cycling LRCs in the adult mouse cornea are enriched in cells that express high levels of &szlig;1- and &szlig;4-integrin and little 9-integrin.
      【关键词】 Adhesion receptors Integrins Mouse3 b! w/ F2 [0 a3 ?
   INTRODUCTION  ^: N+ b5 ]$ ?! Q! \

Corneal epithelial stem cells (CESCs), also referred to as limbal stem cells, reside at the corneoscleral junction or limbus . The important roles integrins play in mediating cell adhesion and signaling provide the rationale for our hypothesis that integrins are involved in both retaining CESCs within their microenvironment at the niche and allowing them to retain their stem-like properties over the life of the organism.
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In the present study, we use the slow-cycling property of stem cells to determine if the LRCs have distinct integrin expression profiles. Numerous investigators have used the slow-cycling property of adult epithelial stem cells as a tool to identify their location within tissues , we evaluated corneal LRCs for expression of these integrins as well." q0 u- d# U9 s, V$ r

Our results show that the LRCs of the mouse ocular surface represent 0.94 to 3.6% of limbal basal cells. Those cells that retained the most label were also shown to express the most ß1-and ß4-integrin and the least 9-integrin. As label retention decreased within the LRCs, levels of ß1- and ß4-integrin also decreased, but 9-integrin increased. Although these studies rule out an epithelial stem cell marker function for 9-integrin, they show that the LRCs are found in close proximity (within 10¨C20 µm) to 9-integrin¨Cpositive cells. In addition, our results show that there were more LRCs in the superior and inferior regions of the mouse cornea than in the temporal and nasal regions.

MATERIALS AND METHODS( x, w0 S, O4 O, z& Z6 v
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Animals
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All experiments were carried out in accordance with the guidelines and the approval of The George Washington University Institutional Animal Use and Care Committee as well as the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. BALB/c pregnant mice were obtained from Hilltop Lab Animals, Inc. (Scottdale, PA, http://www.hilltoplabs.com) at 14 days gestation. After a week in quarantine, they were moved to the animal room for delivery and long-term maintenance. The pups were then injected with the appropriate amount of 5-bromo-2-deoxyuridine (BrdU) solution (see below).

BrdU Injection

On the third day after birth, BALB/c mouse pups were injected twice a day (8 a.m. and 3 p.m.) for 4 days with 25 µl of a 1-mg/ml solution of BrdU per gram body weight. (BrdU crystals were dissolved in water using a 37¡ãC water bath.). The pups were allowed to mature and were sacrificed using a lethal injection of sodium pentobarbital (approximately 12 µl/g body weight). Eyes were sutured for orientation purposes, and left and right eyes were stored separately. This enabled us to orient the eyes for analysis of LRCs and integrin expression by quadrant (superior, nasal, inferior, and temporal).; x6 f2 P- F2 h

Antibodies and Chemicals7 Q8 \5 O% g, @$ ]+ n( |) B2 Q. N
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Polyclonal antibodies against 3-, 9-, and ß1-integrin were characterized previously , and antibodies against 6-integrin (catalog no. CD49f) and ß4-integrin (catalog no. CD104) were purchased from BD Pharmingen (San Diego, http://www.bdbiosciences.com/pharmingen). All primary antibodies were diluted in blocking buffer at 1:100, except for 6-integrin, which was diluted at 1:200. Alexa secondary antibodies¡ªgoat anti-rabbit, anti-rat, and anti-mouse 488 (catalog nos. A-110012, A-11006, and A-11001, respectively)¡ªwere purchased from Molecular Probes, Inc. (Eugene, OR, http://probes.invitrogen.com) and diluted at 1:500 in blocking buffer. Cy5 goat anti-mouse (catalog no. 115-175-166) was purchased from Jackson Immunoresearch Labs (West Grove, PA, http://www.jacksonimmuno.com) and was used at a dilution of 1:75. Finally, the anti-BrdU kit (catalog no. 1296736) and the BrdU crystals (catalog no. 280879) were purchased from Roche Diagnostics (Indianapolis, http://www.roche-applied-science.com).
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The following were purchased from Fisher Scientific (Pitts-burg, PA, http://www.fisherscientific.com): slides (catalog no. 12-544-7), cover slip (catalog no. 12-548-5P), horse serum (catalog no. ICN2921249), HCl (catalog no. SA49), KCl (catalog no. BP-366-500), 100% methanol (catalog no. A454-4), Trypsin 2.5% (catalog no. NC9254859), and Tween 20 (catalog no. BP337-500). The following were purchased from Sigma-Aldrich (St. Louis, http://www.sigmaaldrich.com): CaCl2 (catalog no. C-3881), 100% ethanol (catalog no. E-702-3), glycine (catalog no. G7403), KH2HPO4 (catalog no. P-5379), Na2HPO4 (catalog no. S-3264), NaCl (catalog no. S-9888), and Triton X-100 (catalog no. T8532). The ProLong mounting media (catalog no. P-7481) and propidium iodide (PI) (catalog no. 1304) were obtained from Molecular Probes, and the Fluoromount-G mounting media (catalog no. 17984-25) was from Electron Microscopy Science (Ft. Washington, PA, http://www.emsdiasum.com).

Immunofluoresence  R0 l* r! C! [# \1 L7 Z$ J8 M

After sacrifice, the eyes were enucleated, washed in 1x phosphate-buffered solution (PBS) (1x PBS is herein after referred to as PBS; 10x PBS was made as follows: 14.4 g Na2HPO4, 2.4 g KH2HPO4, 2 g KCl, 80 g NaCl, in a total volume of 1 liter of water, pH 7.4), and fixed for 2 hours in a 4:1 dilution of methanol:DMSO prechilled at ¨C20¡ãC. They were then incubated in 100% methanol at ¨C20¡ãC for long-term storage. For 9-integrin, all eyes were transferred from 100% methanol into PBS in a graded methanol series (70, 50, and 30% methanol in PBS, 30 minutes each). For other integrins, the eyes were transferred from 100% methanol into a graded methanol and Triton X-100 series (70/30, 50/50, 30/70% methanol/Triton X-100; 5 minutes each). Eyes were then washed in PBS twice for 30 minutes each. All washes and incubations were done at room temperature (RT) with gentle shaking, unless specified otherwise. Next, they were incubated with blocking buffer (100 ml PBS with 1 g bovine serum albumin added and stirred for 30 minutes; 1 ml of horse serum added and stirred for an additional minute) for 2 hours, followed by primary antibody incubation overnight at 4¡ãC. The next day, eyes were washed three times with PBS and 0.02% Tween 20 (PBST) for 1 hour each, blocked for 2 hours, then incubated with the appropriate secondary antibodies overnight at 4¡ãC. The following day, the eyes were washed three times with PBST for 1 hour each. To visualize the integrins alone, at this step, the eyes were incubated in PI, for 5 minutes followed by three washes with Millipure water, 5 minutes each. To achieve the best result in flattening the eyes, four incisions were made, and the eyes were put on a black filter before adding the ProLong or Fluoromount-G mounting media and cover slipping.

To visualize the integrins and BrdU simultaneously, eyes of BrdU-injected mice were obtained following the protocol previously stated. After appropriate primary and secondary antibody staining for 9-, ß1-, or ß4-integrin, eyes were fixed in ethanol (prechilled 70% ethanol in 50 mM glycine buffer, pH 2) for 20 minutes at ¨C20¡ãC followed by two washes in PBS for 30 minutes each and incubated with 2.5% Trypsin in 0.1% CaCl2 in PBS, for 10 minutes. They were then briefly washed in PBS and transferred to a solution of 4 M HCl for 10 minutes. Eyes were washed twice in PBS, for 20 minutes each and then incubated in blocking buffer for 2 hours followed by anti-BrdU antibody overnight incubation at 4¡ãC. The next day, they were washed three times with PBST for 1 hour, blocked for 2 hours, and incubated with anti-mouse secondary antibody overnight at 4¡ãC. The following day, the eyes were washed three times with PBST for 1 hour each, incubated in PI for 5 minutes, washed three times with Millipure water for 5 minutes each, and flattened on a black filter.# z$ {% d5 ~% T& |6 y! Y
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Confocal Microscopy

The appearance of a flat-mounted mouse cornea prepared for analysis as described above is schematically represented in Figure 1A. Based on the location of a suture placed in the temporal sclera prior to enucleation, incisions were made to divide the cornea into four quadrants anatomically; within each of these quadrants, confocal images from three nonoverlapping regions were acquired. All images were captured with a confocal microscope at the Center for Microscopy and Image Analysis (CMIA) at the George Washington University Medical Center. A Bio-Rad MRC 1024 confocal laser scanning microscope (Bio-Rad, Hercules, CA, http://www.bio-rad.com) equipped with krypton-argon laser and an Olympus IX-70 inverted microscope (Olympus, Tokyo, http://www.olympus-global.com) were used to image the localization of Alexa Fluor 488 (488 nm laser line excitation; 522/35 emission filter), Alexa Fluor 594 or propidium iodide (PI) (568 nm excitation; 605/32 emission filter), and Cy5 (647 nm excitation; 680/32 emission filter). Optical sections of confocal epifluorescence images were acquired sequentially at 1-µm intervals using a x20 objective lens (numerical aperture , 0.7) or at 0.5-µm intervals using a x60 oil objective (NA, 1.40), with Bio-Rad LaserSharp v3.2 software. Images were acquired from the apical aspect of the epithelium into the underlying stroma; this typically required capturing approximately 25 optical sections. Thirty-six confocal areas were imaged at x60 magnification per integrin; nine areas of 167 x 167 x 0.5 µm were imaged from each of the four quadrants: superior, nasal, inferior, and temporal. No fewer than three eyes were used for each integrin analyzed.* G  u1 F! r# a8 G9 ]8 U4 r
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Figure 1. Whole-mounted mouse corneas: orientation of eyes and regional localization of 9-integrin. (A): A suture was placed in the temporal sclera of the left and right eyes prior to enucleation. Four incisions were placed as shown to flatten and orient the cornea. Red boxes indicate that for each quadrant images from three regions were taken. (B): En face images were taken with a x60 oil objective of a confocal microscope (shown from the basal aspect) from the adult mouse cornea at the limbus. 9-Integrin (green) is regionally distributed around the circumference of the mouse eye with the most intense expression at the nasal region; nuclei are stained with propidium iodide (red).: j$ T2 U6 p0 r8 ~
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Image Analysis$ V- j, w/ B: b- s/ P
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Adobe Photoshop v7.0 software with Bio-Rad plugins was used both to convert images from Bio-Rad PIC into TIFF files and to merge images to create montages. Image Pro Plus v5.1 software (Media Cybernetics, Crofton, MD, http://www.mediacy.com) was used to merge stacks of laser confocal images and to render three-dimensional (3D) images via an Image Pro Plus 3D Constructor v5.1 module./ r* E9 l8 j5 r9 o: Z0 U7 \* x
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Statistical Analyses

The simplest approach to examining the association between integrin and BrdU levels is through Pearson correlation. However, because of the pooling of data across eyes and sampled regions within eyes, the assumption of statistical independence is likely to be violated. Therefore a "mixed model"  was used to estimate and test these associations. In a mixed model, an independent variable is considered a "random effect" if its values can be considered analogous to being randomly sampled from a population, but would be considered a "fixed effect" if the interest lies in differences among specific chosen (fixed) values of the variable. For this study, eye and regions within the eye were considered random effects, whereas the quadrant was considered a fixed effect. Mixed models were estimated using the default estimation (restricted maximum likelihood) and testing options of the Proc Mixed module of SAS 8.2 (SAS Institute, Inc., Cary NC, http://www.sas.com). Where the effects of eye, region, and quadrant are small, the test of the Pearson correlation, ignoring eye, region, and quadrant effects, will give a result very similar to that of the corresponding mixed model. In those instances, we present these simpler Pearson correlation analyses. Spearman rank-order correlations were also calculated, but are not reported because they differed trivially from the Pearson correlations.& e, ]) b5 p* l' @6 H+ a
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RESULTS
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9-Integrin¨Cpositive basal cells are regionally distributed around the circumference of the adult BALB/c mouse eye (6¨C8 weeks old) at the limbus, as shown in Figure 1B. The number of 9-integrin¨Cpositive cells varies from one quadrant of the eye to the next, such that, at the nasal region, 9-integrin¨Cpositive cells can make up as many as 70% of the basal cells, whereas, at the inferior region, fewer than 15% of the cells were 9-integrin positive. The restriction of 9-integrin to the limbus occurs over time during eye development, and the width of the limbal epithelium containing the 9-integrin¨Cpositive cells varies by quadrant from a minimum of 150 to a maximum of 300 µm at the inferior and nasal regions, respectively . In the current study, after first investigating the pattern of expression of ß1-, 3-, ß4-, and 6-integrins, we identified the 9-, ß1-, and ß4-integrin expression profiles of the slow-cycling LRCs on the mouse ocular surface.
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Unlike 9-Integrin, ß1-, ß4-, 3-, and 6-Integrin Are More Abundant in the Central Cornea
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Each panel in Figure 2 shows a stack of images 10-µm thick and viewed from the basal to apical aspect using the 3D Constructor module of Image Pro Plus software. Each stack was then rotated 90¡ã to show the cross section (CS), indicated by the white dashed lines, presented below each image. Figure 2A and 2B shows the staining for ß1-integrin and PI in the limbal basal cells and in the central corneal epithelial basal cells, respectively. The cross-sectional view shows that ß1-integrin was present at the basolateral and apical aspects of the basal cells and appeared more abundant in the central cornea than in the limbus. It is interesting to note that ß1-integrin was not present immediately under the nuclei of the basal cells. We could confirm that antibody penetration was not responsible for the lack of staining beneath the nuclei because we observed integrin staining around cells of the vasculature deep in the stroma (supplemental online movie).$ `5 T9 W+ I' T, F

Figure 2. Whole-mount staining for ß1-, ß4-, and 3-integrin and propidium iodide (PI) on adult mouse cornea projected from the basal aspect. The corneas of 6-week-old mice were stained for ß1-, ß4-, or 3-integrin (green) and propidium iodide (red). There is more ß1-, ß4-, and 3-integrin present at the central cornea than in the limbus. Note the absence of these integrins from right beneath the nuclei. Abbreviation: CS, cross section.
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Figure 2C and 2D shows staining for ß4-integrin and PI in the limbus and the central cornea, respectively. ß4-integrin forms heterodimers with the 6 subunit, and the staining obtained for 6-integrin was identical to that of ß4-integrin (data not shown). Viewing the tissues from their basal aspect showed that, whereas ß4-integrin was present at the basement membrane, there was also variability in the amount and intensity of this integrin among individual basal cells. This variability was not easily observed in the cross-sectional view, where the staining seemed to be linear and uniform. ß4-integrin was present primarily in the basolateral, apical, and basal aspects of the basal cells, and, viewing cross sections, appeared to be more abundant in the central cornea than at the limbus.- f: A6 u  X$ L" P- {" f  Q
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We then assessed the distribution of 3-integrin, the primary ß1 family integrin on the mouse ocular surface (Fig. 2E, 2F). We found that the localization of 3-integrin was similar to that observed for ß1-integrin; it was primarily present at the lateral aspect of the basal cells and appeared to be more abundant in the cornea than in the limbus. From these data, we concluded that ß1-, ß4-, and 3-integrins are more abundant in the central and cornea, with their expression decreasing toward the limbus. Even though there was variability observed in the amount and intensity of these integrins within individual cells at the limbus, there were no differences in the regional distribution of these integrins in the manner shown previously for 9-integrin. ß1-, ß4-, 3-, and 6-integrins were distributed uniformly among the four quadrants of the mouse eye.% Z# l" ~6 w* ]/ L, U1 I
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Triple-Labeling Corneas for BrdU, Integrin Antibody, and PI Positively Identified LRCs and Their Integrin Profiles In Situ in the Limbal Epithelium* f( |9 q- |- E0 q" @- X
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En face images were taken from each of the four different quadrants of each eye with a x60 oil objective of a confocal microscope as described; each region acquired represented approximately 484 basal cells. Typical data for the 9-integrin and BrdU from two regions of the cornea are shown in images presented in Figure 3. Figure 3A and 3F shows images viewed basally, which were then rotated 90¡ã to show the cross section (Fig. 3B, 3G). Using the 3D Constructor module of Image Pro Plus software, the white rectangular region indicated in Figure 3B and 3G was digitally magnified and is presented in Figure 3C¨C3E and 3H¨C3J, respectively, showing the 9-integrin, BrdU, and PI triple-stained tissues in Figure 3C and 3H, the 9-integrin and BrdU double-stained tissues in Figure 3D and 3I, and 9-integrin alone stained tissues in Figure 3E and 3J.
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Figure 3. High-resolution imaging of triple-labeled corneas for 9-integrin, BrdU, and PI. Eyes from adult BrdU-injected mice were stained for 9-integrin (green), BrdU (blue), and PI (red). BrdU can be present in discrete or multiple patches in single cells, and label-retaining cells (LRCs) can be observed in clusters or alone. Also, whereas an occasional 9-integrin-positive cell is also label-retaining, most 9-integrin-positive cells are not LRCs. Abbreviations: BrdU, 5-bromo-2-deoxyuridine; PI, propidium iodide; CS, cross section.) L7 p. Y1 j7 ^- h/ g

The limbal region presented in Figure 3A¨C3E shows two 9-integrin¨Cpositive cells surrounded by several 9-integrin¨Cnegative basal cells. There are two BrdU-positive cells in the limbal basal cell layer, indicated by arrowheads, next to and behind one of the 9-integrin¨Cpositive cells. A second cell layer has one BrdU-positive cell as indicated by the arrow. To approximate the boundaries of the BrdU-positive cell nuclei, it was necessary to rotate and use handles, a tool in the Image Pro Plus software that allowed us to move through the stack of optical sections while viewing the PI-stained nuclei and BrdU staining simultaneously.

Although we often observed LRCs in clusters adjacent to patches of 9-integrin¨Cpositive cells (Fig. 3C¨C3E), this was not always the case. Figure 3I and 3J shows a single LRC isolated away from other LRCs; this LRC is 9-integrin¨Cpositive and is surrounded by several 9-integrin¨Cpositive cells that did not retain BrdU. This cell is shown closely apposed to BrdU-positive stromal fibroblasts (asterisk). To define the boundary between the epithelium and the corneal stroma and the BrdU-positive cells contained there, again, it was necessary to rotate the 3D images and move through the tissue from its apical to basal aspect and observe the abrupt change in organization and orientation of the nuclei of cells within the stroma. Whereas the nuclei of the epithelial basal cells were either cuboidal or columnar oriented perpendicular to the basal cell surface, those of the stromal fibroblasts were oriented parallel to the basal cell surface, as shown by the asterisk shown in Figure 3H (a movie showing how LRCs were identified in the mouse cornea is provided in the online supplemental data). Because of this intimate association between epithelial LRCs and underlying stromal fibroblasts, care had to be taken with each epithelial LRC cell evaluated to carefully assess the border between the epithelial LRCs and the stromal fibroblasts.2 D8 N4 k* ]; w7 |- F) R3 L

Images such as those shown in Figure 3 provide compelling evidence that most 9-integrin¨Cpositive cells are not slow-cycling LRCs. However, they also show that there were cells that were both BrdU- and 9-integrin¨Cpositive. Not all of the LRCs express the same repertoire of proteins, and some LRCs lose their label faster than others. To better assess the relationship between the LRCs and integrin expression, an experimental approach was required that would be suitable for statistical analysis allowing for assessment of sufficient numbers of cells so that we could get an idea of integrin profiles within populations of slow-cycling limbal basal cells.  v4 [3 F  b& a8 w8 i+ D

Assignment of Scores for Statistical Analyses
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As shown in Figure 3, BrdU could be present in either single or multiple discrete patches in the nucleus; only by careful analysis of 3D images and visual inspection of PI-stained nuclei could we approximate the boundaries of the nucleus for each BrdU-positive cell. Integrin staining was restricted to the cytoplasm and cell membranes of cells. Using confocal sections to assign regions of interest and determine pixel intensities was not possible since optimal BrdU and integrin staining were never found in the same confocal layer of these 3D images; rather, 3D images had to be rotated in different planes to obtain optimal staining for integrin and BrdU. For this reason and to allow us to obtain data on populations of cells, a scheme for assignment of scores by visual inspection was developed that minimized subjectivity and maximized the numbers of cells that could be assessed. To test for reliability in score assignments, data were analyzed statistically for consistency from region to region and eye to eye.

The scheme that was developed for assigning BrdU and integrin scores is shown in Figure 4. Each of the 36 regions obtained for each BrdU-integrin pair was analyzed using the 3D Constructor module of Image Pro Plus software, every BrdU-positive epithelial cell within each region was viewed individually, and a value was assigned to it based on both extent and intensity of its BrdU staining. Cells that had retained more BrdU were considered to be more stem-like (slower cycling) than those with less BrdU. BrdU was never found in the central corneal epithelial cells. After BrdU scores were assigned, integrin levels in each of the BrdU-positive cells were assigned a score. Scores assigned to BrdU levels were 0.5, 1, 2, or 3, and scores for integrins were 0, 1, 2, or 3., a# J$ B9 D9 [& ]
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Figure 4. Scores were assigned to both integrin- and BrdU-positive cells. The images shown are presented from the basal aspect of the cell to demonstrate how scores were assigned. (A): Typical cells scored for BrdU. The first image shows a cell stained for both BrdU (blue) and PI (red), and the second shows a cell stained only for BrdU. (B): Typical cells scored for the three different integrins studied. The first image shows a cell stained for both integrin (green) and PI (red), and the second shows a cell stained for only the integrin. Abbreviations: BrdU, 5-bromo-2-deoxyuridine; PI, propidium iodide.1 L  p4 N5 h" m7 h5 K1 m( O
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BrdU Retention Is Positively Correlated with ß1- and ß4-Integrin Expression but Negatively Correlated with 9-Integrin Expression
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Each eye was divided into four quadrants based on the location of sutures placed on the sclera of the eyes before enucleation. Three regions were randomly sampled from each quadrant (Fig. 1A); a total of 36 regions was assessed per integrin from a minimum of three eyes. Figure 5A¨C5C shows the number of BrdU-positive cells and their integrin expression profiles based on the assigned scores in eyes stained for 9-integrin (Fig. 5A), ß1-integrin (Fig. 5B), and ß4-integrin (Fig. 5C). To confirm that the assignment of BrdU scores was performed consistently, we analyzed the data for variations in the distribution of BrdU scores (0.5, 1, 2, 3) across eyes, regions, and quadrants, using the Mixed Linear Model as described. Based on the p-values obtained (eye, .054; region, .08; quadrant, .60), we concluded that the distribution of BrdU was not significantly different across eyes, regions, or quadrants. The consistency with which BrdU scores were assigned among the different eyes, regions, and quadrants analyzed provides further assurance that these data are as accurate as technically possible.$ @! _$ e+ K" c3 h# x1 [3 Y1 L) W- ?
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Figure 5. Mean integrin scores for BrdU-positive cells. The mean integrin scores were calculated for each BrdU value for each integrin analyzed for all three regions within each of the four quadrants evaluated and are presented in tabular form in (A¨CC); data are shown graphically in (D). The Pearson correlation coefficients indicate a significant positive correlation between ß1- and ß4-integrin and BrdU retention and a significant negative correlation between 9-integrin and BrdU retention. Abbreviation: BrdU, 5-bromo-2-deoxyuridine.( x3 J4 @$ D6 Y* O. z$ N

Although most of the cells in each region analyzed were BrdU negative, we were able to identify 1889 limbal epithelial cells as label-retaining. All 1,889 LRCs were scored for both BrdU and integrin expression: 762 for 9-integrin, 715 for ß1-integrin, and 412 for ß4-integrin. To determine if there was an association between the retention of BrdU within the LRCs and the expression of integrins in these cells, mean integrin scores were determined for each BrdU score level (0.5, 1, 2, and 3), and Pearson correlation coefficients were calculated and tested. All bivariate associations were checked with scatter plots for distributional assumptions. Data showed significant positive correlation for both ß1- and ß4-integrin expression with BrdU label retention (r = 0.26, p
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The amount of BrdU that each cell retained varied; of all the cells evaluated, there was a nearly equal distribution among the four possible BrdU scores assigned: 501 scored 0.5 (27%), 528 scored 1 (28%), 369 scored 2 (20%), and 491 scored 3 (26%). Because cells with higher BrdU scores had proliferated fewer times after incorporating label, they were considered to be the most stem cell-like; cells with scores of 0.5, although still retaining label, were closer to losing label than those with higher BrdU scores. Although it was possible that a slow-cycling stem cell might have acquired less label during the initial labeling period, it was unlikely that such cells made up a large percentage of the total LRCs counted in this study given the significant positive correlation between the BrdU score and the ß1- and ß4-integrin scores.
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The image shown in Figure 3 suggests that LRCs were often present in close proximity to 9-integrin¨Cpositive cells. To assess how likely it was for 9-integrin¨Cpositive cells to be near LRCs, we identified and analyzed nine cells with BrdU scores of 3 that were negative for 9-integrin. This was straightforward because 75% of the cells that scored 3 for BrdU expressed no 9-integrin (Fig. 5A). For all nine of the LRCs assessed, we observed at least one9-integrin¨Cpositive cell within 10¨C20 µm of the LRC nucleus; for seven of nine LRCs, there was an 9-integrin¨Cpositive cell within 12.5 µm of the LRC nucleus. Since six of the nine LRCs assessed were from the superior or inferior region, where 9-integrin¨Cpositive cells are rare, these data imply a close relationship between 9-integrin¨Cpositive cells and the LRCs at the limbus. In our previous studies, we showed that 9-integrin¨Cpositive cells were present at the anatomical niche at the limbus, but we could not prove that the LRCs were found at the same site. Although we now show that the 9-integrin¨Cpositive cells were not CESCs, our results show that a close relationship exists between the LRCs and the 9-integrin¨Cpositive cells.2 ?4 ?$ L0 M, a7 U1 V

To approximate the percentage of cells that were label retaining, first, the total number of cells in each region was estimated directly from images such as those in Figure 4A and 4F; this number was found to be approximately 484 cells per region. Because each region was 167 x 167 µm, this estimate yielded an average cross-sectional cell size of 7.8 µm2. To calculate the total number of cells in all the random regions sampled, 484 was multiplied by 108 (12 regions per eye x 3 integrins x 3 eyes per integrin, or 108 regions) to yield 52,272 cells. Using the total number of estimated BrdU-positive cells from all regions (1,889; Fig. 5A¨C5C), the percentage of LRCs was calculated to be 3.6%. If only the cells with BrdU scores of 3 were considered to be the most stem cell¨Clike (Fig. 5A¨C5C), 0.94% of the cells were LRCs. Since we assume that not all of the CESCs were labeled initially with BrdU, this estimate must be considered lower than the value in vivo. Therefore, our data suggest that the percent CESCs at the limbus ranges from 0.94 to 3.6%, which is in accordance with the literature stating that stem cells make up a small percentage of the total cells in the limbus .6 C) o. L* N, X+ x; z  B2 M

More BrdU Is Present in the Superior and Inferior Quadrants
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BrdU-positive cells were also evaluated for how they were distributed across quadrants in nine individual eyes (Fig. 6A). Data suggest that, overall, LRCs were more common in the superior and inferior quadrants than in the nasal and temporal quadrants. To evaluate whether these differences were significant, the distribution of BrdU was calculated by quadrant in each eye relative to the total BrdU-positive cells for that eye, and data were analyzed for significance using 2 tests. As presented in Figure 6B, the inferior and superior quadrants of the mouse eye had significantly more LRCs than the nasal and temporal quadrants (p values . [, V/ @5 u' V" ~2 D
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Figure 6. Percentage of BrdU retention is higher in the superior and inferior quadrants than in the nasal and temporal quadrants. (A): The total numbers of BrdU-positive cells (summing all cells with scores of 0.5¨C3) analyzed by eye and by quadrant, independent of integrin or BrdU score. Although there is variability among the nine eyes studied in detail, the trend is that more BrdU-positive cells are present in the superior and inferior quadrants and fewer are present in the nasal and temporal quadrants. The percentages represent the distribution of BrdU-positive cells across quadrants for each eye. (B): There were significant differences (p 1 l& f1 F% D( t2 }9 [
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DISCUSSION
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Corneal Epithelial LRCs as Putative CESCs) y4 [  b7 D# T7 K
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Until fate mapping of individual corneal epithelial stem-like cells and functional reconstitution of an adult mouse cornea can be performed, there will remain controversy regarding the identification of CESCs. Corneal epithelium, like the epidermis, has an important barrier function but, in addition, the corneal epithelium plays roles in maintaining the immune-privileged status of the cornea, its avascular nature, clarity, and ability to heal wounds with minimal scarring. Full functional reconstitution of the ocular surface remains a significant hurdle. In mouse skin, slow-cycling epithelial cells were capable of reconstituting the epidermis, hair follicles, and sebaceous glands of the mouse, and authors concluded that the slow-cycling cells were epidermal stem cells . Retention of BrdU label is a tool we use in this report to probe the integrin expression status of a subpopulation of cells we propose are the CESCs. We recognize that not every slow-cycling cell is a stem cell nor has every stem cell in the cornea been labeled with BrdU. Direct proof that the slow-cycling LRCs we study in the mouse are actually the CESCs will have to wait until there are better methods for both fate mapping and reconstitution of the mouse ocular surface.
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Progress in characterizing the properties of the adult stem cells in the mouse cornea has been slower than that in skin as a result of difficulties in establishing mouse cornea epithelial cells in culture. Methods for the culture of mouse keratinocytes were originally developed over 25 years ago , we were only able to get cells to grow as spheroids (data not shown). Without reliable methods for the culture of mouse corneal epithelium, exploiting mouse genetics to study the CESCs is difficult.
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CESCs Were Not Uniformly Distributed Around the Limbus# w$ M: y8 m- o2 L4 x

Corneal epithelial cells that retained BrdU label for prolonged chase times were located at the adult mouse limbus; no BrdU-positive cells were found in the epithelial cells of the central cornea. Numerous mesenchymal LRCs can be observed in the corneal stroma, some closely associated with the epithelial LRCs. These mesenchymal cells retain abundant BrdU, suggesting that they divide less frequently after acquiring label than do the corneal epithelial basal cells. Looking at the distribution of epithelial LRCs as a function of quadrant, we observed that there were significant differences among quadrants. The superior and inferior quadrants had more LRCs than did the temporal and nasal quadrants, and there were no significant differences between inferior and superior or nasal and temporal quadrants. This observation could have resulted from several different causes. The presence of eyelids could protect stem cells in the inferior and superior quadrants but leave stem cells in the nasal and temporal quadrants more exposed to damage from UV light, which can induce apoptosis . Mice may be born with more stem cells at the superior and inferior quadrants and fewer at the nasal and temporal quadrants. Another possibility is that there is a slower rate of cell proliferation and turnover in adults at the superior and inferior regions than at the nasal and temporal regions; more cells retain label overall when they proliferate less frequently.5 ^3 B. ]8 @3 z- c2 j1 d
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Integrins as Potential Survival and Retention Factors for Epithelial Stem Cells

Jones and colleagues .
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Whereas individual LRCs are enriched in ß1- and ß4-integrin relative to other basal cells at the limbus, basal cells from the central cornea express as much or more of these integrins. If this is so, then using integrin expression or function alone as a means for enriching for stem-like cells from the cornea could prove difficult. Li and colleagues , has a stem-like population of cells that exhibits the property of rapid adhesion. It will be interesting to determine if these cultured human corneal cells express high levels of several integrins or just those that mediate attachment to type IV collagen.( b* O6 n! N9 w7 m. }
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If expression of ß1- and ß4-integrin mediates retention and survival of epidermal stem cells and corneal LRCs at their niche, what roles are played by 9-integrin in maintaining the LRCs at the limbus? The data presented here suggest that, as the LRCs divide, lose label, and become committed to terminal differentiation, they acquire more 9-integrin and less ß1- and ß4-integrin. They also show that LRCs remain in close proximity to 9-integrin-positive cells. We know that, following large wounds, a partial stem cell deficiency develops that is accompanied by a loss of 9-integrin at sites where conjunctival goblet cells have invaded .

Studies have shown that when cells are induced to express 9-integrin, migration is enhanced . Taken together, data from several different types of experimental systems provide strong support for a role for 9-integrin in enhancing epithelial cell migration. Since CESCs remain quiescent and nonmigratory, their expression of 9-integrin should be low. However, the progeny of the CESCs must migrate away from the niche, and we propose that the expression of 9-integrin on cells derived from the CESCs facilitates their departure from the niche.& u: p( `* N; C# Y. U2 j

CESCs Give Rise to 9-Integrin-Positive Early Transient Amplifying Cells (9 eTACs) and eTACS That Are Rapidly Cycling and Function to Maintain the Limbal Niche
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To account for the data presented in this paper and that presented previously in our studies of integrin expression during corneal wound healing, we propose the model presented in Figure 7. We hypothesize that the slow-cycling LRCs we have evaluated in this study are the CESCs. Under homeostatic conditions, quiescent CESCs are enriched in the ß1-integrin family and 6ß4-integrin. The two major ß1-integrin family integrins on the skin and ocular surface are 2ß1-integrin and 3ß1-integrin. 2ß1-integrin knockout mice have no skin or ocular phenotype, and wound healing proceeds normally , we suggest that the ß1-integrin family member upregulated on CESCs is 3ß1-integrin and that the immediate progeny of asymmetric division of the CESCs are CESCs and 9 eTACs.( t0 F- X: T* y0 O7 T
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Figure 7. A model showing the relationships among the CESCs, 9 eTACs, eTACs, and TACs on the mouse ocular surface. The CESCs express high levels of 6ß4- and 3ß1-integrin. Data from 5-bromo-2-deoxyuridin studies show that as these slow-cycling cells divide, they progressively lose 6ß4-integrin and 3ß1-integrin and gain expression of 9ß1-integrin. Both the 9 eTACs and eTACs that make up most of the cells present at the limbal niche have lower levels of 6ß4-integrin and 3ß1-integrin on their surface than the TACs that make up most of the cells in the basal layer of the central cornea. The CESCs, 9 eTACs, and eTACs overlie a basement membrane with a composition of extracellular matrix proteins that is distinct from the that of the basement membrane underlying the epithelial cells at the central cornea and conjunctiva. Abbreviations: CESC, corneal epithelial stem cell; 9 eTAC, 9-integrin¨Cpositive early transient amplifying cell; eTAC, early transient amplifying cell; TAC, transient amplifying cell.6 w+ n( O$ ], w1 J& E

The 9 eTACs express lower amounts of 3ß1- and 6ß4-integrin; when 9 eTACs divide, they give rise to eTACs that do not express 9-integrin and retain lower levels of 3ß1 and 6ß4. It is not clear from our data whether CESCs could give rise directly to eTACS, bypassing the stage in which the cells are 9-integrin positive. eTACs do not retain label because they are rapidly cycling. They make up the majority of the limbal basal cells and account for the overall lower expression level of epithelial integrins at the limbus. eTACS give rise to TACs and, eventually, all the cells of the corneal epithelium. Whereas, in the basal cell layer, TACs express high levels of 3ß1- and 6ß4-integrin, this expression decreases as cells leave the basal cell layer and terminally differentiate.( l1 I8 \9 O4 H9 Z) l5 S9 v

The CESCs, 9 eTACs, and eTACs overlie a basement membrane zone (BMZ) unique in that it contains tenascin-C .7 f1 Y2 E/ Q1 O% c- N% x
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Assuming that our data extend to the human cornea, 9 eTACs are likely enriched not only in 9-integrin, but also in -enolase, p63, and ABCG2. The basal cell layer of skin also expresses 9-integrin . However, when mouse epidermal keratinocytes and human corneal epithelial cells are placed in culture, 9-integrin expression is rapidly downregulated (data not shown). Because both human skin and corneal epithelial cells grow well in culture and, in the mouse, epidermal keratinocytes can reconstitute mouse skin, we know that 9-integrin expression is not permanently downregulated in cultured keratinocytes. Taken together, these observations suggest that to maintain 9-integrin expression in keratinocytes, growth or survival factors present in the basement membrane or mesenchyme are required.+ l1 ~+ p" [# w( i! ~% |

The slow-cycling CESCs cells make up a very small percentage of the total cells in the basal cell layer at the limbus (0.94%¨C3.6%) and, as a result, it has been impossible to study the molecular properties of this cell population in situ within its niche at the limbus without a molecular marker. By developing a method for the simultaneous assessment of BrdU and integrins within whole mounts of the adult mouse cornea using confocal imaging and 3D reconstruction, we were able to use the slow-cycling nature of the CESCs as a means for their identification within their niche at the limbus. After identifying the putative CESCs based on retention of label, we assessed their expression of 9-, ß1-, and ß4-integrin and found that the cells that retained the most label had the highest levels of ß1- and ß4-integrin and the lowest levels of 9-integrin. We also found that the putative CESCs were more abundant in the inferior and superior regions of the cornea than in the nasal and temporal regions. Although proof that the LRCs analyzed are in fact the CESCs will require development of better mouse corneal epithelial cell culture methods to permit ocular surface reconstruction of the mouse cornea, these results implicate cell adhesion to specific niche components as important factors in maintaining the CESCs.: {# l) i) A$ v- n* X) V7 }- T" y
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ACKNOWLEDGMENTS. e- y+ |/ }2 h/ k1 f9 B- X
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We thank Dr. Robyn Rufner, the director of CMIA at The George Washington University, and Dr. Anastas Popratiloff for help with the confocal microscopy and image analysis. In addition, we thank Dr. Stuart Yuspa for helpful comments on the manuscript. This work was sponsored by NIH/National Eye Institute grants RO1-EY13559-03 to M.A.S.4 E  U; n  Z$ _6 u/ C- y& H$ Q
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DISCLOSURES
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The authors indicate no potential conflicts of interest.' O/ l2 n# m0 z% g6 l6 g3 k
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