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TISSUE-SPECIFIC STEM CELLS

Identification of Candidate Murine Esophageal Stem Cells Using a Combination of Cell Kinetic Studies and Cell Surface Markers

^aSurgical Oncology Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia;
^bUniversity of Melbourne Department of Surgery, St. Vincent's Hospital, Fitzroy, Victoria, Australia;
^cEpithelial Stem Cell Biology Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia

Key Words. Esophagus • Stem cells • Transferrin receptor • ₆ Integrin • Keratinocyte • Epithelial cell

Correspondence: Wayne A. Phillips, Ph.D., Centre for Cancer Genomics and Predictive Medicine, Peter MacCallum Cancer Centre, Locked Bag 1, A'Beckett Street, Melbourne, VIC 8006, Australia. Telephone: +61-3-9656 1842; Fax: +61-3-9656 1411; e-mail: wayne.phillips{at}petermac.org

Received July 12, 2006; accepted for publication October 3, 2006.
First published online in STEM CELLS EXPRESS   October 12, 2006.

	ABSTRACT

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The identification and characterization of esophageal stem cells are critical to our understanding of the biology of the esophageal epithelium in health and disease. However, the proliferative compartment within the mouse esophageal epithelium remains poorly characterized. Here, we report that the basal cells of the mouse esophagus can be separated into three phenotypically and functionally distinct subpopulations based on the expression of ₆ integrin and transferrin receptor (CD71). Cells that express high levels of ₆ integrin and low levels of CD71, termed ₆^briCD71^dim, are a minor subpopulation of small and undifferentiated cells that are enriched for label-retaining cells and thus represent a putative esophageal stem cell population. Conversely, cells expressing high levels of both ₆ integrin and CD71 (₆^briCD71^bri), the majority of basal esophageal cells, are enriched for actively cycling cells and therefore represent a transit-amplifying population. Kinetic analyses revealed that a third cell population, which is ₆ integrin-dim and CD71-bright (₆^dim), is destined to leave the basal layer and differentiate.

	INTRODUCTION

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In recent years, much attention has been devoted to resolving different classes of proliferative cells, particularly stem cells in a variety of mammalian tissues. Continuously renewing tissues like the hemopoietic system, the intestinal epithelium, and stratified epithelia are organized in a hierarchical fashion with the undifferentiated, quiescent, long-lived, multipotent, and self-renewing stem cells at one end of the hierarchy and fully mature, terminally differentiated cells at the other [1, 2]. Stem cells are important not only because of their potent ability to regenerate their own tissue but because their extensive capacity for proliferation implicates them in wound healing and tumorigenesis [3].

It is well-accepted that tissue renewal in the hemopoietic system and the skin is achieved through the combined proliferative activity of a minor population of stem cells and a large pool of actively dividing, short-lived, transit-amplifying cells and that these populations can be discerned by cell kinetic analyses and/or cell surface markers [4–6]. However, little work has been done on the esophageal epithelium despite the clinical importance of this tissue.

The epithelia of the mouse and rat esophagus can be divided into two compartments, the basal layer composed of densely packed columnar cells and the superficial spinous and granular cell layers composed of large polyhedral and flattened keratinizing cells, respectively. Tritiated thymidine-labeling studies revealed that all cells in the basal layer, and only cells in the basal layer, are capable of undergoing cell division [7]. Furthermore, cell division in the basal layer, and cell migration from it (i.e., differentiation), appears to be stochastic. Therefore, it was deduced that "the basal cells are the stem cells of the esophageal epithelia" [7]. This contention remains to be proven experimentally, although it seems unlikely that all basal cells represent stem cells. Indeed, in virtually all other stratified epithelia, it has been possible to demonstrate that stem cells are relatively rare cells within the basal layer. For example, we have previously shown that basal cells of the mouse and human epidermis can be divided into different populations on the basis of ₆ integrin (₆) and transferrin receptor (CD71) expression. Using these markers, we clearly identified three populations in the interfollicular epidermis: ₆^bri CD71^dim cells, which are rare, undifferentiated, quiescent, long-lived cells with high proliferative potential in vitro, representing putative stem cells; ₆^bri CD71^bri cells, which are a rapidly cycling population containing the majority of basal cells and representing transit-amplifying cells; and ₆^dim cells, which are the early differentiating cells [4, 8]. Given the structural similarity between interfollicular epidermis and esophagus, we have investigated the usefulness of these markers in fractionating murine basal esophageal cells into primitive stem cells and their more committed progeny. We initially sought to distinguish esophageal cells using in vivo cell turnover studies and then endeavored to confirm these differences using in vitro, and tissue-reconstitution, assays.

	MATERIALS AND METHODS

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Isolation of Primary Murine Esophageal Keratinocytes
To obtain esophageal keratinocytes, female C57/B6 mice, 7–10 weeks of age, were culled at 6:00 p.m. (unless otherwise indicated), and the esophagi were removed and incubated in dispase II (6 mg/ml; Roche Diagnostics Australia Pty. Ltd., Castle Hill, Australia, http://www.rochediagnostics.com.au) in phosphate-buffered saline (PBS) with 12 µg/ml penicillin, 160 µg/ml gentamicin, and 0.6 µg/ml fluconazole for 6–16 hours at 4°C. The mucosa was then removed with sterile forceps and incubated in 5 ml of prewarmed (37°C) trypsin (2.5 mg/ml in PBS; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) for 4 minutes with agitation on a magnetic stirrer with a sterile stir bar. The trypsin was then quenched with 200 µg/ml soybean trypsin inhibitor (Sigma-Aldrich), and the suspension was passed through a 40-µm cell strainer (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Cells were centrifuged and resuspended in keratinocyte basal medium (KBM) (Cambrex Bio Science Walkersville, Inc., Walkersville, MD, http://www.cambrex.com) with 20 mg/ml bovine serum albumin (BSA) (Sigma-Aldrich) in PBS. A small proportion of the cells were found to be CD45-positive (0.34% ± 0.05%, mean ± SEM; n = 7) by fluorescence-activated cell sorting (FACS) analysis, and these were gated out in all subsequent experiments. The remaining cells stained positive for keratin-14 [8], confirming their epithelial origin.

Analysis of Cell Surface Markers
Freshly isolated primary murine esophageal keratinocytes (1 x 10⁷ cells per milliliter) were stained with ₆-fluorescein isothiocyanate (FITC) (GoH3, 1:100; Becton, Dickinson and Company) and CD71-phycoerythrin (CD71-PE) (C2; 2 µg/ml; Becton, Dickinson and Company) for 30 minutes on ice, washed once, and resuspended at 1 x 10⁷ cells per milliliter in KBM containing 20 mg/ml BSA. Cells were either analyzed on a Becton Dickinson LSRII flow cytometer or sorted using a Becton Dickinson FACS^Diva (see supplemental online data for FACS setup). Nonviable cells were excluded using 1 µg/ml viability dye, either 7-amino-actinomycin D or fluorogold (both from Invitrogen Corporation, Carlsbad, CA, http://www.invitrogen.com), and CD45-PE-CY7 (30-F11, 0.4 µg/ml; Becton, Dickinson and Company) was used to exclude CD45-positive cells. Isotype-matched negative controls and single-color controls were included with each experiment.

Bromodeoxyuridine Labeling and Cell Cycle Analysis
C57/B6 mice (7–9 weeks old) were pulse-labeled by a single injection of 100 µg/kg bromodeoxyuridine (BrdU) at 6:00 a.m. At the indicated times after labeling, mice were sacrificed and the esophageal keratinocytes were isolated, sorted based on ₆, CD71, and CD45 expression, and fixed in 70% (vol/vol) ethanol. The cells were then washed with PBS and incubated in 10 mg/ml BSA for 1–2 hours. DNA hydrolysis was performed by incubating the cells in 2 M HCl at room temperature for 30 minutes followed by neutralization of the acid with 0.1 M sodium tetraborate (Sigma-Aldrich). The cells were then washed once in PBS and stained with anti-BrdU (B44; 1:100, 25°C, 30 minutes; Becton, Dickinson and Company) and detected with goat anti-mouse immunoglobulin G (IgG)-Alexa Fluor 647 (1 µg/ml; Invitrogen Corporation). DNA was stained using 2 µg/ml 4',6-diamidino-2-phenylindole (Sigma-Aldrich) so as to simultaneously determine cell cycle status and BrdU incorporation using an LSRII analyzer. Esophageal cells from animals that had not been injected with BrdU were used as controls.

For label-retention studies, groups of mice were injected intraperintoneally with BrdU (100 µg/kg) daily for 7 days, and labeled cells were visualized in tissue sections using a BrdU In Situ Detection Kit (Becton, Dickinson and Company) and a Tyramide Signal Amplification Kit (PerkinElmer Life and Analytical Sciences, Boston, http://www.perkinelmer.com). At various times (1–200 hours) after labeling, individual mice were sacrificed, esophageal keratinocytes were isolated, and the loss of BrdU from sorted cell populations was assessed by flow cytometric analysis using anti-BrdU. Overton histogram subtraction was used to establish the percentage of BrdU-positive cells in experimental versus unlabeled control animals.

Limiting Dilution Assay
Mouse esophageal epithelial cells were sorted directly into 96-well plates coated with 20 µg/ml collagen IV (Sigma-Aldrich) using single-cell mode and doublet discrimination on the FACS^Diva. They were plated at 3, 10, and 30 cells per well in replicates of 24 or 48 (except for the CD71⁺⁺⁺ population, which was plated at 30, 100, and 300 cells per well). The plates were centrifuged at 200g for 5 minutes to encourage cell adherence. Cells were then cultured in KBM supplemented with 5 µg/ml insulin (Sigma-Aldrich), 0.5 µg/ml hydrocortisone (Sigma-Aldrich), 70 µg/ml bovine pituitary extract (Hammond Cell Tech, Windsor, CA, https://secure2.wahju.com/hammond), 10 ng/ml epidermal growth factor (Sigma-Aldrich), 2 mg/ml BSA, 100 ng/ml cholera toxin (Sigma-Aldrich), 5 µg/ml transferrin (Sigma-Aldrich), 20 pM triiodothyronine (Sigma-Aldrich), 2.4 µg/ml penicillin, 32 µg/ml gentamicin, and 0.6 µg/ml fluconazole for 7 days at 37°C, 5% CO₂. The plates were then washed, fixed with 5% (vol/vol) formalin, and stained with toluidine blue. Wells were scored as positive when there was at least one colony with at least 32 cells. According to Poisson distribution, at a limiting dilution of one colony, 37% of the wells are expected to be negative. Extrapolation of the 37% negative point on the y-axis intersects the x-axis at the number of cells required to produce a colony [9].

In Vivo Tissue Reconstitution
The ability of the cell populations to regenerate a stratified epithelium was assessed using an in vivo transplant model as described previously [10]. Briefly, 1 x 10⁵ freshly sorted cells from each population were resuspended in 30 µl of KBM with supplements and inoculated into devitalized rat tracheas, which were then implanted under the dorsal skin of SCID (severe combined immunodeficient) mice. The trachea were retrieved after 3–5 weeks, processed as previously described [10], and stained with hematoxylin and eosin.

Immunofluorescence Staining
Cryostat sections of mouse esophagus were fixed in acetone for 10 minutes at –20°C, blocked, and stained with rat anti-mouse CD71-FITC (clone R17 217.1.4; Caltag Laboratories, South San Francisco, CA, http://www.caltag.com), ₆-FITC (both at 1:100), or mouse anti-K4 (1:1,000). Sections were counterstained with propidium iodide.

	RESULTS

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Phenotyping of Mouse Esophageal Keratinocytes
The ₆-CD71 phenotype of primary mouse esophageal keratinocytes is shown in Figure 1. It contains four discernable populations: the ₆^briCD71^bri population, which constitutes the majority of isolated cells (72.3% ± 2.1%, mean ± SEM; n = 7), and three smaller populations, ₆^briCD71^dim (7.8% ± 1.2%), ₆^dimCD71^bri (referred to hereafter as ₆^dim; 8.6% ± 0.9%), and CD71 very bright (CD71⁺⁺⁺; 11.2% ± 2.6%).

View larger version (58K): [in this window] [in a new window]   Figure 1. Phenotype of mouse esophageal keratinocytes. Primary mouse esophageal epithelial cells were double-labeled with anti-CD71 (phycoerythrin) and anti-6 integrin (fluorescein isothiocyanate) and analyzed by flow cytometry. The dot-plot indicating the identified subpopulations is representative of at least seven independent experiments. Abbreviations: +/71–, 6briCD71dim cells; +/71+, 6briCD71bri cells; –/71+, 6dim cells; 71+++, CD71+++ cells.

Analysis of Cell Cycle
As has been demonstrated in previous studies [12], we detected a strong circadian rhythm to cell proliferation in the mouse esophagus, with 16.7% ± 1.2% (mean ± SEM) of cells cycling at 6:00 a.m. compared with only 1.8% ± 0.4% of cells cycling at 6:00 p.m. (n = 7; p < .001) (supplemental online Fig. 10A, 10B). Assessment of the cell cycle was therefore undertaken at 6:00 a.m., near the peak in the circadian rhythm. As shown in Figure 2A, the ₆^briCD71^bri population was clearly enriched for cycling cells (cells in S, G₂, or M phase), whereas the ₆^briCD71^dim, ₆^dim, and CD71⁺⁺⁺ populations had reduced levels of cycling cells. Consistent with this result, the ₆^briCD71^bri population had the greatest percentage of cells incorporating BrdU, thus confirming that this population has the greatest proportion of cycling cells (Fig. 2B).

View larger version (13K): [in this window] [in a new window]   Figure 2. Analysis of BrdU-labeled esophageal keratinocytes. Mice were pulse-labeled by a single intraperitoneal injection of BrdU (100 µg/kg) at 6:00 a.m., and esophageal keratinocytes were collected after 1 hour. Bivariate flow cytometric analysis was used to simultaneously assess (A) the percentage of cells in S + G2M phase of the cell cycle via DNA content and (B) the percentage of BrdU-positive cells in 6-CD71-defined subpopulations (mean ± SEM; n = 7). The percentages of BrdU-positive cells in each of the 6-CD71-defined subpopulations assessed at 1, 12, 24, and 36 hours after BrdU injection are shown in (C) (mean ± SEM; n = 3). Dotted line connected by indicates 6briCD71dim cells; dotted-and-dashed line connected by indicates 6briCD71bri cells; dashed line connected by indicates 6dimcells; unbroken line connected by indicates CD71+++ cells. Abbreviations: +/71–, 6briCD71dim cells; +/71+, 6briCD71bri cells; –/71+, 6dim cells; 71+++, CD71+++ cells; BrdU, bromodeoxyuridine; hrs, hours.

During the interval from 12 to 36 hours after the initial pulse, the percentage of labeled cells in the ₆^dim population decreased dramatically to 17.1% ± 5.2%; concomitantly, the proportion of BrdU-positive cells in the CD71⁺⁺⁺ population increased from less than 10% to more than 40%. Given that the absolute number of cells in both the ₆^dim and the CD71⁺⁺⁺ populations are approximately equivalent and that the level of label in the other two populations, ₆^briCD71^bri and ₆^briCD71^dim, remains essentially constant over this time period, we infer that the loss of labeled cells from the ₆^dim population is due to the migration of labeled cells into the CD71⁺⁺⁺ population.

Label-Retention Studies
Essentially complete labeling of esophageal keratinocytes was achieved by the injection of mice with BrdU daily for 7 days (supplemental online Fig. 11). The proportion of BrdU-positive cells in each population after chase periods ranging from 12 hours to 8 days was then used to establish the turnover time for each population. Using this technique, we were able to show that the ₆^briCD71^dim population retained a greater proportion of the BrdU compared with both the ₆^briCD71^bri and ₆^dim populations (Fig. 3A). Linear regression analysis demonstrates that the t_1/2 (time required for 50% of the cells to become BrdU-negative) is approximately 100 hours for the ₆^briCD71^dim population compared with approximately 75 and 70 hours for the ₆^briCD71^bri and ₆^dim populations, respectively (Fig. 3B, 3C).

View larger version (12K): [in this window] [in a new window]   Figure 3. Assessing cell turnover by label retention. Mice were labeled by daily injections of BrdU for 7 days. (A): The percentage of BrdU-positive cells in 6-CD71-defined subpopulations of esophageal keratinocytes freshly isolated after a 5-day chase period (mean ± SEM; n = 5). (B, C): Plots illustrating the kinetics of label retention in mouse esophageal cells after various chase times, as indicated, comparing the 6briCD71dim cell population (, dashed lines) with (B) 6briCD71bri and (C) 6dim cell populations (, dotted and dashed lines). Data shown are derived from at least three independent mice at each time point. p < .004 (B); p < .001 (C) (paired t test). Abbreviations: +/71–, 6briCD71dim cells; +/71+, 6briCD71bri cells; –/71+, 6dim cells; BrdU, bromodeoxyuridine; hrs, hours.

View larger version (14K): [in this window] [in a new window]   Figure 4. Clonogenicity of 6 integrin-CD71-defined subpopulations of mouse esophageal cells. The indicated numbers of cells per well were plated, and after 7 days, wells were scored as positive if at least one colony with at least 32 cells was present. The number of cells required to produce a colony was calculated from the graph as the point at which 37% of the wells are negative (dotted lines). Limiting dilution graphs of (A) CD71+++ cells () compared with basal cells (all other populations) () and (B) 6briCD71dim () compared with 6briCD71bri () and 6dim () cells. Data shown are mean ± SEM; n = 3 for CD71+++ and n = 5 for all others. Abbreviations: +/71–, 6briCD71dim cells; +/71+, 6briCD71bri cells; –/71+, 6dim cells; 71+++, CD71+++ cells.

View larger version (83K): [in this window] [in a new window]   Figure 5. Esophageal tissue reconstitution in vivo. Transverse histological sections of rat trachea lumens lined with epithelium derived from 1 x 105 mouse esophageal cells and harvested at 5 weeks. (A): Total cells (magnification x2.5). (B): Total cells (magnification x10). (C): 6briCD71dim cells. (D): 6briCD71bri cells. (E): 6dim cells. (F): CD71+++ cells. Negative control tracheas not inoculated with cells do not form an epithelium (not shown). Scale bar = 100 µm. Abbreviations: c, trachea cartilage; e, epithelium; l, trachea lumen; s, submucosal layer.

View larger version (30K): [in this window] [in a new window]   Figure 6. In vivo localization of CD71 in the murine esophageal epithelium. Immunofluorescence images of a cross-section of mouse esophagus stained with anti-CD71-fluorescein isothiocyanate (green) and counterstained with propidium iodide (red) to identify nuclei. (A): Low-magnification image. (B): High-magnification image. Scale bar = 100 µm.

	DISCUSSION

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We have previously used the cell surface markers ₆ and CD71 to identify putative stem cells in the mouse epidermis [4, 8]. We now show that these markers can identify distinct cell populations in the mouse esophageal epithelium and have been able to elucidate a kinetic and functional hierarchy.

The CD71⁺⁺⁺ Population Is Enriched for Differentiated, Suprabasal Cells
Cell morphology, flow cytometry data (forward and side scatter), and staining for the differentiation marker keratin-4 are consistent with the conclusion that CD71⁺⁺⁺ cells are derived from the suprabasal compartment. This conclusion is supported by immunofluorescence studies demonstrating the highest CD71 staining in the suprabasal layers of the esophageal epithelium (Fig. 6). As would be expected of cells from the differentiated suprabasal compartment, the CD71⁺⁺⁺ cells exhibited the lowest clonogenicity in vitro and no tissue-reconstituting activity.

The ₆^dim Population of Esophageal Epithelial Cells Fulfils Criteria Expected of an EarlyDifferentiating Compartment
By definition, the early differentiating compartment is characterized by cells that have left the cycling pool but are not yet demonstrating all the characteristics of fully mature differentiated cells. Like the CD71⁺⁺⁺ cells, the ₆^dim cells appear to have a poor proliferative activity and a reduced capacity to reconstitute a normal esophageal epithelium in a tissue-reconstitution model (Fig. 7), suggesting that most have left the cell cycle and committed to differentiation. Indeed, although the ₆^dim cells express relatively low levels of the differentiation marker keratin-4, cell-tracking experiments suggest that these cells will eventually move into the CD71⁺⁺⁺ compartment. Thus, the ₆^dim population appears to be enriched for cells that, having just undergone a cell division (possibly their last), are now destined to leave the basement membrane and differentiate into suprabasal cells. This would be consistent with their cell surface phenotype, as downregulation of ₆ expression has been linked to detachment of keratinocytes from the basement membrane in other tissues [4]. Interestingly, the CD71⁺⁺⁺ population appears by flow cytometry to be ₆ integrin-bright; however, because this technique measures total cell-associated fluorescence, the apparent increase in total ₆ may not indicate an upregulation in the density of ₆ expression on the cell surface but rather may be a reflection of the increase in cell size.

View larger version (24K): [in this window] [in a new window]   Figure 7. Proposed model of stem cell hierarchy in the esophageal epithelium of the mouse. Diagrammatic representation of the 6-CD71 phenotype illustrating the proposed parent-progeny/differentiation relationships between the 6-CD71-defined subpopulations. Arrows indicate movement of cells from the stem cell compartment, to the transit-amplifying, early differentiating, and finally the suprabasal differentiating compartments in the course of epithelial cell renewal and differentiation. Abbreviations: ED, early differentiating; SC, stem cell; SD, suprabasal differentiating; TA, transit-amplifying.

The ₆^briCD71^bri Population of Esophageal Epithelial Cells Fulfills Criteria Expected of a Transit-Amplifying Compartment

The ₆^briCD71^dim Population of Esophageal Epithelial Cells Fulfills Criteria Expected of a Stem Cell Compartment
The stem cell compartment is ultimately responsible for the renewal of tissues undergoing continuous turnover. The defining characteristics of stem cells are sustained self-renewal and tissue regeneration, although these criteria are often difficult to demonstrate experimentally.

Typically, epithelial stem cells tend to be small, quiescent, undifferentiated blast-like cells [8, 15]. Such characteristics most closely describe the ₆^briCD71^dim population. The ₆^briCD71^dim cells retain BrdU significantly longer after pulse-labeling than the other populations (Fig. 3), indicating a slower turnover rate. However, because the entire tissue is turning over very rapidly, even this putative stem cell population has a relatively high rate of cell turnover as compared with the hematopoietic system [16] or the epidermis [15]. The ₆^briCD71^dim cells efficiently regenerate a complete epithelium in the tissue-reconstitution assay (Fig. 5), demonstrating their potency. Furthermore, by serially passaging ₆^briCD71^dim cells through the tissue-reconstitution model, we not only were able to demonstrate the capacity for self-renewal but also increased long-term proliferative capacity compared with the other cell populations (data not shown).

Esophageal Stem Cell Hierarchy
Based on the data presented above, we propose a model of the esophageal stem cell hierarchy (outlined in Fig. 7) in which the ₆^briCD71^dim population represents the stem cell compartment. Cells then sequentially progress through the ₆^briCD71^bri (transit-amplifying) and ₆^dim (early differentiating) compartments, eventually emerging as mature, differentiated cells that constitute the suprabasal layers of the esophageal epithelium (CD71⁺⁺⁺).

Consistent with our model, CD71 expression in the basal layer of the mouse esophagus alternates between areas of low expression and regions of high expression, and the transition between the two appears to be gradual rather than abrupt (Fig. 6). This is consistent with the flow cytometric data, which suggest that there is a continuum of expression levels. We propose that the areas of lowest CD71 expression contain the putative stem cells and that these regions merge into regions of high expression, which contain progressively more committed transit-amplifying (₆^briCD71^bri) and early postmitotic (₆^dim and also CD71^bri) cells that ultimately transit into the suprabasal layers where CD71 expression is the highest.

Although our in vivo cell turnover studies were clearly able to differentiate between the ₆^briCD71^dim (stem) and ₆^briCD71^bri (transit-amplifying) cell populations, it has proven difficult to separate these populations using the tissue-reconstitution assay. Similarly, we have previously reported that extensive tissue-regenerative capacity measured over the course of 6–10 weeks can be elicited from keratinocyte stem, transit-amplifying, and early differentiating cells isolated from human foreskin [13]. Most likely, this can be attributed to the gradual loss of "stem-ness" as cells progress through the proliferative hierarchy prior to irreversible commitment to terminal differentiation. This notion was first proposed by Potten and Loeffler in the "diminishing stem-ness spiral model" for epithelia [17] and suggests that the basal cells of the esophagus are likely to be organized in a biological continuum whereby tissue-regenerating potency is lost gradually as cells progress from being stem cells to functional end cells. Furthermore, it is also important to recognize that liberation of cells from their normal tissue constraints, which is required to assay subsets of epithelial cells in vitro and in vivo, may result in revealing their potential ability for proliferation or tissue regeneration and may not necessarily reflect their actual properties in situ.

	DISCLOSURES

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The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

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We thank Ralph Rossi for invaluable help and advice with flow cytometry and cell sorting and Ivan Bertoncello for valuable discussions. D.C. is the recipient of a Surgeon-Scientist Fellowship from the Royal Australasian College of Surgeons and a postgraduate research scholarship from the National Health and Medical Research Council of Australia.

	REFERENCES

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