Characterization of the Limbal Epithelial Stem Cell Niche: Novel Imaging Techniq - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:53 |只看该作者 |倒序浏览 |打印

作者：Alex J. Shortta,b,c, Genevieve A. Seckera,b, Peter M. Munrob, Peng T. Khawb,c, Stephen J. Tufta,c, Julie T. Danielsa,b作者单位：aCells for Sight Transplantation and Research Programme,bOcular Repair and Regeneration Biology Unit, UCL Institute of Ophthalmology, London, United Kingdom;cMoorfields Eye Hospital NHS Foundation Trust, London, United Kingdom ; A% k1 O) m% t$ l% m( E7 `9 {& O
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      【摘要】
   It is anticipated that stem cell (SC) therapy will enable the regeneration of diseased tissues and organs. Understanding SC niches is an essential step toward realizing this goal. By virtue of its optical transparency and physical separation of SC and transient amplifying cell compartments, the human cornea provides a unique opportunity to visualize and observe a population of adult stem cells, limbal epithelial stem cells (LESCs), in their niche environment. To date, the characteristics of the LESC niche have remained unclear. State-of-the-art imaging techniques were used to construct a three-dimensional (3D) view of the entire human corneal limbus and identify the structural characteristics of the LESC niche. Two distinct candidate LESC niche structures were identified. Cells within these structures express high levels of the putative limbal stem cell markers p63 and ABCG2; however, current methods cannot identify for certain which exact cells within this cell population are truly LESCs. These structures could be located and observed in vivo in normal human subjects, but not in patients with clinically diagnosed corneal LESC deficiency. The distribution of these structures around the corneal circumference is not uniform. Biopsies targeted to limbal regions rich in LESC niche structures yielded significantly higher numbers of LESCs in culture. Our findings demonstrate how adult stem cell niches can be identified and observed in vivo in humans and provide new biological insight into the importance of LESC niche structures in maintaining normal LESC function. Finally, the concept of targeted biopsy of adult SC niches improves stem cell yield and may prove to be essential for the successful development of novel adult stem cell therapies.

Disclosure of potential conflicts of interest is found at the end of this article. 3 r* e0 R; Y) o' H5 f
      【关键词】 Stem cell Cornea Limbus cornea Somatic stem cell biology Human Stem cell transplantation Three-dimensional imaging8 N9 f+ y+ y0 c3 n5 O. Y- ~" i8 F
   INTRODUCTION
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Recent advances in stem cell research have raised the prospect of a new era of regenerative medicine in which stem cells will enable the regeneration of diseased tissue and organs. Currently, there are substantial ethical, regulatory, and practical obstacles to the use of embryonic stem cells for therapeutic purposes. The alternative of using somatic or "adult" stem cells has major advantages in terms of immediate clinical application, but to realize their potential, substantial progress must be made in understanding the biology of these cells.

One key area of research is the identification and investigation of adult stem cell niches. Adult stem cells exhibit intrinsic properties that influence their behavior, but they also depend on specialized environmental niches to maintain them in an undifferentiated state and regulate their functions. Understanding these stem cell-niche interactions is essential if the goal of developing new adult stem cell therapies is to be fully realized. Some characteristics of adult stem cell niches in the gut, skin, and bone marrow have already been identified (reviewed in ), but progress in this area is hampered by the fact that, to date, it has not been possible to identify and observe adult stem cells within their niches in vivo in humans. Indeed, most studies in this area employ animal models and/or histopathological analysis of tissue specimens.
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In skin, the continuous desquamation of superficial cells and replenishment of basal cells is made possible by interfollicular epidermal keratinocyte stem cells . This pioneering adult stem cell therapy has placed ophthalmology at the forefront of stem cell research and therapeutic delivery.

The cornea possesses two unique characteristics that make it ideally suited as a model system for studying adult stem cells in humans. Firstly, it is optically transparent, and, hence, noninvasive imaging of LESCs in humans is possible. Secondly, LESCs are found at the corneal limbus (the peripheral extent of the cornea) and are anatomically segregated from their transient amplifying cell progeny, which migrate centrally to cover the paracentral and central cornea . However, the total number and distribution of LESCs within the niche is unknown. Furthermore the presence of a specialized adult stem cell niche within the human corneal limbus has not been conclusively demonstrated to date. The aims of this study were to use state-of-the-art imaging techniques to identify the structural characteristics and regional distribution of the LESC niche and to determine whether this niche can be located and observed in vivo in humans, thus allowing limbal biopsies to be targeted to potentially LESC-rich limbal regions.
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METHODS

Tissue for In Vitro Imaging, Immunofluorescence, and Cell Culture Studies
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Cadaveric human corneas with research consent were obtained from the Moorfields Eye Hospital Lions Eye Bank (U.K.), North Carolina Eye Bank (U.S.), and San Diego Eye Bank (U.S.). These were utilized for in vitro confocal microscopy, cell culture, and immunofluorescence studies. Corneal orientation was determined before dissection of the globe using the position of the extraocular muscles. Scanning electron microscopy (SEM) studies were performed using corneoscleral rims discarded after penetrating keratoplasty. All experiments on human tissue were approved by our institutional research ethics committee.

In Vitro Confocal Microscopy and Three-Dimensional Reconstruction of Limbal Structure% d& h, F- ]' U; g
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Ten cadaveric human corneas were obtained from eight donors (age range, 18¨C77 years; mean, 56.9 years; median, 66 years). Corneas were oriented, dissected into quarters, fixed (within 72 hours of death), and stained with propidium iodide (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) to label nuclei and fluorescein isothiocyanate (FITC)-phalloidin (Sigma-Aldrich) to label the actin cytoskeleton. Segments were then whole-mounted on glass slides and coverslipped. Z-stack image series were collected through the entire corneal limbal epithelium and superficial limbal stroma using a Zeiss LSM510 confocal microscope (Carl Zeiss, Jena, Germany, http://www.zeiss.com). Image series were converted into AVI movie format using Windows Movie Maker (Microsoft, Redmond, WA, http://www.microsoft.com) and also reconstructed in three-dimensional (3D) imaging using Volocity 3.0 software (Improvision, Coventry, U.K., http://www.inprovision.com) for evaluation. To examine regional differences in the structure of the limbus, a continuous array of overlapping images collected at the level of the basal epithelial layer were combined to create a 360¡ã montage of the corneal limbus (Adobe Photoshop 7.0, Adobe, San Jose, CA, http://www.adobe.com).0 {1 s2 n7 H4 X7 P5 e, V, t/ D

Whole Mount Immunofluorescence! _5 Q' U0 O0 [1 ?9 y

Two human corneas from different donors were fixed with 4% paraformaldehyde for 1 hour at room temperature then divided into quarters, washed, and blocked using 20% goat serum in phosphate-buffered saline (PBS) at room temperature for 2 hours. They were then incubated with primary antibodies to CD31/platelet endothelial cell adhesion molecule-1 (PECAM-1; Abcam, Cambridge, U.K., http://www.abcam.com) at 4¡ãC overnight. After washing, specimens were incubated with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-mouse IgG 1:200 (Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com) and 50 µg/ml FITC-phalloidin (Sigma-Aldrich) for 2 hours at room temperature. The specimens were washed and mounted in Mowiol 4-88 (Sigma-Aldrich), coverslipped, and examined using a Zeiss LSM 510 confocal microscope.
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Scanning Electron Microscopy of Corneal Limbal Stroma/ [9 ]2 P1 B2 y( W* t* C

Corneoscleral rims were decellularized by incubation in 100 mM EDTA for 2 hours at 37¡ãC followed by gentle mechanical abrasion of the epithelium. Specimens were fixed, processed for scanning electron microscopy, coated with gold palladium, and imaged in a JEOL 6100 SEM (Jeol Ltd., Tokyo, http://www.jeol.com) operating at 15 kV. This protocol effectively removed virtually all epithelial cells. The integrity of the underlying basal lamina and stroma were assessed by scanning and transmission electron microscopy and were found to be unaffected by the decellularization protocol. Eighteen rims from 15 donors (age range, 3¨C70 years; mean, 46.5 years; median, 52 years) were examined. A continuous array of overlapping images collected at x25 magnification were combined to create a 360¡ã montage of the corneal limbus (Adobe Photoshop 7.0)." j* u' d( @6 [
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Culture of Limbal Epithelial Holoclone (Stem Cell) Colonies from Corneal Limbal Biopsies% Y$ V( ?1 B+ ^& f% U$ `  |6 k
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Clock hours were marked out on normal corneas using gentian violet. Confocal imaging of the limbus was then performed in an aseptic manner using the Heidelberg Retinal Tomograph (HRT) II and Rostock corneal module (Heidelberg Gmbh, Heidelberg, Germany, http://www.heidelberg.com). Areas that contained large numbers of proposed niche structures¡ªlimbal crypts (LCs) and focal stromal projections (FSPs)¡ªand areas in which these were absent were identified. The locations of these were noted using the clock hours. The features used to designate and area as containing LCs and FSPs were the presence of their distinct morphological characteristics (as demonstrated in Figs. 1, 2, and 6) and the presence of multiple structures in close proximity. This information was then used to target punch biopsies, 3 mm in diameter, to regions that were either rich in, or devoid of, these proposed niche structures. In total, 30 biopsies (16 from niche rich regions and 14 from non-niche regions) were taken from five donor corneas (age range, 27¨C79 years; mean, 63.2; median, 66 years). Biopsies were incubated with 2.4 units/ml dispase II (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com) at 37¡ãC for 60 minutes followed by gentle scraping to separate the epithelium from the limbal stroma. A single-cell suspension was obtained by incubation with 0.5 g of trypsin and 0.2 g of EDTA-4Na/L (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) at 37¡ãC for 3¨C8 minutes. Feeder layers of growth-arrested 3T3-J2 cells were prepared by incubation with 4 µg/ml mitomycin-C for 2 hours and seeded at 4 x 104 cells per cm2. All epithelial cells isolated from each biopsy were seeded onto separate feeder layers and cultured in a humidified atmosphere and 5% CO2 in Dulbecco's modified Eagle's medium and Ham's F12 basal medium (2:1 mixture) containing glutamine (4 mM), fetal calf serum (10%), hydrocortisone (0.4 µg/ml), cholera toxin (0.1 nM), insulin (5 µg/ml), penicillin/streptomycin (50 units/ml each), adenine (0.18 mM), triiodothyronine (2 nM), transferrin (5 µg/ml), and epidermal growth factor (10 ng/ml). After 10¨C12 days, cultures were fixed with 100% methanol at ¨C20¡ãC for 1 hour, rehydrated, and stained with 2% Rhodamine B. Colonies were examined under a dissecting microscope. Holoclone colonies formed by clonal expansion of single limbal epithelial cells were identified and counted as previously described . Results are expressed as the number of holoclone colonies per square millimeter of tissue biopsy.

Figure 1. Structure of limbal crypts (LCs). (A): En face optical section collected from a corneal limbal wholemount at a superficial, suprabasal level using confocal microscopy. The tissue was stained with fluorescein isothiocyanate (FITC)-phalloidin (green) to label the actin cytoskeleton. (B): Corresponding optical section collected at a deeper level demonstrating the presence of LCs (arrows). (C, D): Scanning electron microscopy of decellularized corneal limbus clearly demonstrates the manner in which the limbal stroma is specialized to enclose the LCs. (E): En face optical section collected from a corneal limbal wholemount stained with FITC-phalloidin (green) and anti-CD31 (platelet endothelial cell adhesion molecule-1) antibody (red) to identify blood vessels. This demonstrated the presence of a complex vascular plexus that is intimately associated with the LCs (arrows). (F): En face optical section and (G) tangential tissue section through LCs, illustrating the distribution of limbal epithelial stem cells and the proposed niche. The red dots indicate the distribution of cells that have a small diameter, high nuclear to cytoplasmic ratio, and express high levels of p63 and ABCG2, all of which are characteristics of limbal epithelial stem cells. Arrows indicate the locations of a LC. Abbreviation: CO, location of the peripheral cornea. Scale bars = 50 µm.
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Figure 2. Structure of focal stromal projections (FSPs). (A): En face optical section collected from a corneal limbal wholemount stained with fluorescein isothiocyanate (FITC)-phalloidin (green) to label the actin cytoskeleton. FSPs (arrows) comprise a focal protrusion of the limbal stroma into the overlying limbal epithelium. (B): Optical section collected from a corneal limbal wholemount stained with FITC-phalloidin (green) and anti CD31 (platelet endothelial cell adhesion molecule-1; red). Arrows indicate the presence of an FSP. (C): Scanning electron microscopy (SEM) image of decellularized corneal limbus demonstrating the presence and location of FSPs (arrows) at the corneal end of limbal crypts (arrowheads). This spatial relationship between the structures was consistently observed. (D): High-power SEM of FSPs. (E): En face optical section and (F) tangential tissue section through FSPs (arrows), illustrating the distribution of limbal epithelial stem cells and the proposed niche. The red dots indicate the distribution of cells that have a small diameter, high nuclear to cytoplasmic ratio and express p63 and ABCG2, all of which are characteristics of limbal epithelial stem cells. Abbreviation: CO, location of the peripheral cornea. Scale bars = 50 µm.
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Immunofluorescence Studies of Histological Tissue Sections
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Fresh human corneas (n = 4) were fixed within 36 hours of death using 3.2% paraformaldehyde at 4¡ãC for 1 hour and then frozen at ¨C80¡ãC until analyzed. Corneas were dissected into four quadrants, and serial sections were cut tangential to the corneal circumference, starting in the cornea. The start of the corneal limbus was defined as the termination of Bowman's membrane. Ten-micron-thick frozen sections were cut from all four quadrants, collected onto polylysine-coated microscope slides (Fisher Scientific International, Hampton, NH, http://www.fisherscientific.com), allowed to air dry for 60 seconds, immersed in methanol at ¨C20¡ãC for 20 minutes, and washed and blocked using 20% goat serum in PBS at room temperature for 90 minutes. Sections were then incubated with primary antibodies to ABCG2 (Chemicon, Temecula, CA, http://www.chemicon.com), p63 clone 4A4 (Abcam) or p63 (Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com) at 4¡ãC overnight. After washing, sections were incubated with secondary antibodies (FITC-conjugated goat anti-mouse IgG or TRITC-conjugated goat anti-rabbit IgG, 1:200; Jackson Immunoresearch) and 1 µg/ml 4'-6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) for 2 hours at room temperature. Slides were washed and mounted in Mowiol 4-88 (Sigma-Aldrich) and coverslipped. Sections were examined and images collected using a Zeiss LSM 510 confocal microscope.

In Vivo Confocal Imaging of the Human LESC Niche Structures( w7 v, B- M1 @; T* ~8 Y. U9 P* x

In vivo confocal microscopy was performed on the corneas of normal subjects to determine whether LCs and FSPs could be identified in vivo. Ethics committee approval and informed consent were obtained for all in vivo human studies. The corneal limbus was examined in vivo using the HRT II with the Rostock corneal module . This scanning laser confocal microscope allows the collection of en face optical sections at varying depths within the cornea and corneal limbus. Initially, the corneal limbus of 10 normal volunteers (six male, four female; age range, 22¨C59 years; mean, 44.7 years; median, 47 years) were examined. Subsequently, we examined the corneal limbus of eight patients with LESC deficiency (six male, two female; age range, 32¨C80 years; mean, 47.5 years; median, 42 years) due to aniridia (n = 4) or chemical burns (n = 4). A minimum of 10 volume scans were collected from each of the superior, inferior, nasal, and temporal corneal limbal quadrants. Each volume scan comprised a series of images starting at the limbal epithelial surface and continuing to a depth of 180 µm, with images collected at 1.5-µm intervals.' Z& F3 `6 z+ d. [0 q# u
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Quantification of Findings
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Extent of Proposed Niche Structures. The 360¡ã corneal limbal montages constructed from confocal and SEM images were imported into Adobe Photoshop 7.0. The extent of corneal limbus containing putative niche structures was quantified in degrees of arc using the geometric center of the montage as the center point of the arc.4 J  K5 D, n5 K* B1 T* X, O
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Nuclear to Cytoplasmic Ratio and Cell Size Measurements. Five representative images of each of the proposed niche structures identified (LCs and FSPs) were randomly selected from each of the 10 donors. The nuclear to cytoplasmic ratio (NCR) and cell size of 250 of the cells lining the bases and edges of LCs (as demonstrated in Fig. 1F and 1G) were measured using ImageJ (NIH, Rockville, MD, http://www.nih.gov) and Zeiss LSM 510 software. Similarly, the NCR and cell size of 250 basal cells immediately attached to the edges of focal stromal projections (as demonstrated in Fig. 2E and 2F) were measured. The NCR and cell sizes of these two groups of cells were compared first with 250 suprabasal cells that were located immediately adjacent to them within LCs and FSPs, and second with basal limbal cells that did not reside within LCs and FSPs.2 W7 N& M3 q/ J) w
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Statistical Analysis- `& N& P) D' s& }0 P5 s9 g2 `* L0 z0 {

When analyzing differences in cell sizes and nuclear to cytoplasmic ratios, and differences in the number of holoclone colonies cultured from different biopsy regions, a two-tailed t test was used to compare groups. Where means are reported, they are followed by the SE.$ o4 K2 k) h$ D3 {& \1 u4 e
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In Vitro Characterization of Limbal Crypts and Focal Stromal Projections

Confocal microscopy and SEM of cadaveric human corneas revealed the presence of two distinct structures that warranted further investigation as a potential niche for LESCs.0 i- J' l5 P# s8 ~, y

Limbal Crypts. Distinct invaginations of epithelial cells extended from the peripheral cornea into the corneal limbal stroma (Fig. 1B; supplemental online data). We termed these structures limbal crypts. They are similar to the rete pegs of the epidermis in that they were downward projections of the limbal epithelium into the limbal stroma, but in other respects, they have similarities to the epithelial crypts of the gut in that they are extremely well circumscribed, are shorter in length than rete pegs, and are polarized so far as they opened onto the corneal rather than the conjunctival surface. The stroma that surrounds LCs is highly cellular and contains a distinct vascular supply that is closely associated with the crypts (Fig. 1E; supplemental online data). SEM images of decellularized corneal limbus in a region rich in LCs demonstrated the manner in which the limbal stroma is specialized to enclose the LCs (Fig. 1C, 1D). The previously described limbal palisades of Vogt  enclose the LCs laterally and are continuous posteriorly, with a less prominent ridge of limbal stroma that encircles the posterior aspects of the palisades. Limbal stroma borders the inferior aspect of the LCs.' X: e# j* h- E6 d, I0 ^
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Focal Stromal Projections. These are fingerlike projections of stroma containing a central blood vessel (Fig. 2B), that extend upward into the corneal limbal epithelium and are surrounded by small, tightly packed basal cells (Fig. 2A; supplemental online data). The structure of FSPs was further demonstrated on SEM (Fig. 2B, 2C) and 3D reconstruction of the confocal optical sections (supplemental online data). FSPs are located at the corneal end of, but are distinct from, the LCs (Fig. 3).
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Figure 3. Three-hundred-sixty-degree limbal montages. A continuous array of overlapping images collected using confocal microscopy (A, C) and scanning electron microscopy (B, D) were combined to create 360¡ã montages of the corneal limbus (L). These demonstrated that not all regions of the corneal L contain limbal crypts (LCs) (identified by arrows in C, D) and focal stromal projections (FSPs) (identified by arrowheads in C, D). These were located only in the superior and inferior limbal regions, extending to a variable degree nasally and temporally, but were never observed on the horizontal meridian nasally and temporally; that is, the areas of corneal L most exposed to sunlight. Note that FSPs are located at the corneal end of, but are distinct from, the LCs. In (A, C), the tissue was stained with fluorescein isothiocyanate-phalloidin (green) to label the actin cytoskeleton and propidium iodide (red to label nuclei). Abbreviations: FSP, focal stromal projection; L, limbus; LC, limbal crypt; PC, peripheral cornea; S, sclera. Scale bar = 2 mm.3 h/ i  q' _! K! m" `
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Nuclear to Cytoplasmic Ratio and Size of Cells Within LCs and FSPs
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The cells lining the edges and bases of LCs (highlighted in Fig. 1F, 1G) are significantly smaller (mean diameter, 9.48 ¡À 0.12 µm; range 5.24¨C19.48 µm) than suprabasal cells that were located immediately adjacent to them within LCs (mean diameter, 16.80 ¡À 0.21 µm; range, 10.27¨C29.87 µm; p
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The cells that are directly adherent to FSPs (highlighted in Fig. 2E, 2F) are significantly smaller (mean diameter, 9.25 ¡À 0.10 µm; range, 6.21¨C15.22 µm) and have a higher nuclear to cytoplasmic ratio (mean, 0.76 ¡À 0.01; range, 0.50¨C0.98) than suprabasal cells that were located immediately adjacent to them (mean diameter, 18.09 ¡À 0.23 µm; range, 10.1¨C29.76 µm; p / S4 y  Z: j$ |& p: N
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There was no significant difference in cells size or NCR between the cells lining the edges or bases of LCs (Fig. 1F, 1G) and FSPs (Fig. 2E, 2F). These values suggest that these cells may represent LESCs.$ T0 ^6 ?% }1 k+ }8 F# C" w
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Regional Variation in Distribution of LCs and FSPs

The degrees of arc containing these proposed niche structures were quantified in ten 360¡ã confocal montages (Fig. 3A, 3C) and 18 SEM montages (Fig. 3B, 3D). The results are summarized in Table 1. These results demonstrate that LCs and FSPs are not uniformly distributed around the corneal circumference; rather, they are predominantly located in the superior and inferior corneal limbal quadrants and extend to a varying degree temporally and nasally. They were absent in the horizontal meridian of all donors.
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Table 1. Results of quantifying the degrees of arc containing limbal crypts and focal stromal projections4 h4 b! C$ s. E4 b$ e
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Culture of Limbal Epithelial Holoclone (Stem Cell) Colonies from Corneal Limbal Biopsies
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Limbal epithelial cells isolated from biopsies of LC-/FSP-rich regions gave rise to one or more holoclone colonies in 16 (100%) of 16 biopsies, whereas those from non-LC/-FSP regions gave rise to one or more holoclone colonies in only 3 (21%) of 14 biopsies. The number of holoclone colonies per square millimeter of biopsy was significantly greater (p " \' q) }+ w+ Y* D( w0 ^/ d1 }

Figure 4. Colony-forming ability of biopsies from LC-/FSP-rich regions versus biopsies from non-LC/-FSP regions. (A): Colony-forming efficiency (CFE) assay for a 3-mm diameter punch biopsy taken from an LC-/FSP-rich region. The plate demonstrates numerous macroscopically visible large colonies with smooth borders consistent with holoclone (stem cell) growth. In addition, several smaller irregular abortive meroclone (transient amplifying cell) colonies are present. (B): CFE assay for equivalent biopsy from a non-LC/-FSP region. Only abortive meroclone colonies are observed. (C): Bar chart demonstrating the mean number of holoclone colonies that were cultured from LC-/FSP-rich regions (16 biopsies) and from non-LC/-FSP regions (14 biopsies). Abbreviations: LC, limbal crypt; FSP, focal stromal projection. *, Statistically significant result (two tailed t test p 0 `/ J$ `  m5 m  Q$ Z* _+ O

Immunofluorescence Studies of Histological Tissue Sections' k6 G9 w% R5 Q# a' r
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Expression of the putative LESC markers ABCG2 and p63 were examined and compared between the central cornea, LC-/FSP-rich limbal regions and non-LC/-FSP limbal regions. ABCG2 and p63 were expressed only by limbal basal epithelial cells and not by the corneal epithelium. Levels of ABCG2 and p63 expression were markedly higher in LC-/FSP-rich regions than in non-LC/-FSP regions (Fig. 5). The highest levels of p63 and ABCG2 expression were observed in the cells highlighted in Figures 1F and 1G (LCs) and 2E and 2F (FSPs).

Figure 5. Results of immunofluorescence staining for the putative limbal epithelial stem cell markers ABCG2, p63 (clone 4A4), and p63 (n = 4). There was a notable difference in the expression of ABCG2 (green) and p63 (red) between areas of corneal limbus rich in LCs and FSPs and regions devoid of these structures. p63 and ABCG2 expression is markedly increased in cells lining the edges and bases of LCs and by cells adherent to FSPs (identified by arrowheads). There is markedly less p63 and ABCG2 expression in limbal basal cells that reside just outside these structures and in the nasal and temporal cornea, where these structures are completely absent (third column). Significantly more cells stained with greater intensity for these putative markers in areas containing these structures. Corneal sections were used as a negative control and sections were counterstained with DAPI (blue). Scale bar = 100 µm. Abbreviations: DAPI, 4'6-diamidino-2-phenylindole; FSP, focal stromal projection; LC, limbal crypt.) I/ a+ w3 I8 a( i$ q5 N( M7 l+ r
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In Vivo Confocal Imaging of Proposed LESC Niche Structures
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Of the 10 normal subjects examined, LCs were identified in all 10 subjects and FSPs in eight (Fig. 6). LCs and FSPs were identifiable only in the superior and inferior quadrants of the cornea. They were not detected nasally or temporally in the horizontal meridian. A video of a representative scan from the superior corneal limbus of a normal subject appears in the supplemental online data. In patients with LESC deficiency (n = 8), we were unable to identify LCs or FSPs in any patient, but instead we found a thinned epithelium, with prominent limbal vessels encroaching onto the corneal surface (Fig. 6).
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Figure 6. In vivo confocal microscopy of human limbus. In vivo confocal microscopy of corneal limbus of normal human volunteers (A, B) and patients with limbal epithelial stem cell deficiency (C, D). (A): In vivo confocal microscopic appearance of a limbal crypt (LC; arrowhead) and (B) collection of focal stromal projections (FSPs; arrows) in the superior corneal limbus of a normal subject. In both instances, the characteristic morphology of each can be clearly identified in vivo. (C, D): Representative images from the superior corneal limbus of two stem-cell-deficient patients. No LCs or FSPs can be seen. This was the case in all eight stem-cell-deficient patients examined. Scale bar = 50 µm.
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DISCUSSION
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We have employed state-of-the-art imaging techniques to characterize the structure and regional distribution of proposed novel LESC niche structures and have demonstrated how these can be located and observed in vivo in humans. We have also demonstrated that targeting of biopsies to these structures results in a significantly higher yield of LESCs in culture.7 W$ j1 |: [) d7 Z

The limbal palisades of Vogt have previously been proposed as the site of this niche . The advent of confocal microscopy has provided the opportunity to optically section the corneal limbus and create 3D reconstructions of the tissue. By applying these techniques together with SEM, we have for the first time performed a detailed 3D study of the entire human corneal limbus. This approach allowed us to identify previously unrecognized candidates for the LESC niche, LCs and FSPs, and has significantly advanced our understanding of the structure of this adult stem cell niche. Our findings show that the structures traditionally ascribed as the limbal niche (the limbal palisades of Vogt) are only one facet of the limbal niche architecture, forming the lateral walls of the stromal structures that encircle LCs. These results demonstrate how state-of-the-art 3D imaging can be used to search for and detect adult stem cells niches.
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It is widely accepted that LESCs are located in the basal layer of the corneal limbal epithelium. Although there is functional evidence that the rabbit corneal limbus serves as a niche . Thirdly, biopsies from LC-/FSP-rich regions give rise to significantly more limbal holoclone (stem cell) colonies in culture than do biopsies from non-LC/-FSP regions. Finally, we have demonstrated that the presence of these structures is associated with normal corneal epithelial function but that, in patients with LESC deficiency, because environmental insult (chemical injury) or genetic abnormalities (aniridia/pax6 haploinsufficiency) these structures are undetectable. These data provide a unique biological insight into the importance of this novel adult stem cell niche in maintaining normal LESC function.- |/ }* w. e" a
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The absence of a definitive marker for LESC has proved challenging to the study of the biology of and in particular to the localization of these adult stem cells  are not uniformly expressed by limbal basal cells but rather are expressed by clusters of cells. Our demonstration of regional variation in limbal structure may explain these apparently contradictory results. Furthermore, the search for a definitive LESC marker may have been hampered by the investigation of potential markers in inappropriate limbal regions.

Our results demonstrate that putative LESCs are found lining the edges and bases of LCs. In addition, they were also located around the sides and tips of FSPs. Current methods cannot identify for certain which exact cells within this cell population are truly LESCs. The dual location and the difference in distribution of putative LESCs is interesting, but its significance is unclear. In the skin and the intestine, the stem cell compartment is located in the bases of crypts ." @, Y$ ]) z" J
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The presence of a rich vascular supply is a common feature of both LCs (Fig. 1E; supplemental online data) and FSPs (Fig. 2B). The presence of a dedicated vasculature network suggests that this is an essential component of the niche environment, supplying nutrients and possibly survival factors to LESCs.

In contrast to our findings, Dua et al. . These structures were not detected in any of the 38 corneas examined in the present study.

In vivo confocal microscopy has provided a significant advance in corneal imaging in human patients. We have used this technology to locate and observe an adult stem cell population in vivo for the first time. The ability to directly visualize LESC allowed us to study these cells in normal and in disease conditions. Although the identification of a definitive LESC-specific marker would be of immense value, there is no guarantee that this will be achieved. If, however, it were achieved, there would be extensive technical, safety, and regulatory obstacles to using an antibody to such a marker to identify stem cells in humans. In contrast, the parameters of structural niche characteristics, cell size, and nuclear to cytoplasmic ratio that we have employed are immediately translatable to clinical practice in the following ways. Firstly, documentation of the absence of LCs and FSPs can assist ophthalmologists in making the clinical diagnosis of LESC deficiency. Secondly, it enables the targeted biopsy of stem-cell-rich regions of corneal limbus for therapeutic stem cell cultures. At present, limbal biopsies are taken from areas in which limbal palisades can be identified clinically. However, in up to 20% of patients, limbal palisades cannot be identified clinically . Ex vivo expansion and therapeutic transplantation of LESCs is dependent on harvesting LESCs from the corneal limbus of living (autologous or allogeneic) or cadaveric (allogeneic) donors. Our findings demonstrate how this variability can be overcome and how the presence or absence of these proposed niche structures can be clearly and reliably documented. We are currently investigating whether such targeted biopsies result in an improved clinical outcome after transplantation of cultured LESCs in humans." e  i4 m; ~( X2 q+ l% ~+ N
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Transplantation of ex vivo cultured LESCs is one of the few adult human stem cell therapies to have been successfully and widely employed . Many of the issues and obstacles encountered during the development of this therapy are likely to be replicated during the development of future stem cell therapies for other tissues. The novel concept of applying high-resolution 3D imaging to locate adult stem cell niches may facilitate the identification and spatial localization of previously unrecognized niches in other tissues and organs. Our unique concept of targeted niche biopsy may prove to be essential for the successful development of novel adult stem cell therapies because harvesting an optimal population of cells will maximize culture efficiency and, possibly, clinical outcomes after transplantation.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST2 A6 x+ D8 H7 Y/ I( Q$ p; `
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The authors indicate no potential conflicts of interest.2 A8 v+ V. r6 H: |  R9 |

ACKNOWLEDGMENTS$ c% i5 H& D' M8 Y! c- }

This study was supported by a Clinical Research Training Fellowship (A.J.S.) by the UK Medical Research Council and from the Special Trustees of Moorfields Eye Hospital (A.J.S.) and the Eranda Foundation (G.A.S.). Funding for the Rostock adaptor and HRT II used to perform in vivo confocal microscopy was provided by Moorfields Eye Hospital Special Trustees. We thank Robin Howes for his assistance with performing the scanning electron microscopy studies.9 X) K) a2 n. b5 I* ]
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