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Keywords:

  • Mouse endometrium;
  • Endometrial stem cells;
  • Stem cell niche;
  • Label-retaining cells;
  • Epithelial stem cell;
  • Stromal stem cell

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Human and mouse endometrium (lining of the uterus) undergo cycles of growth and regression as part of each reproductive cycle. A well-known method to identify somatic stem/progenitor cells and their location in the stem cell niche is the label-retaining cell (LRC) approach. We hypothesized that mouse endometrium contains small populations of both epithelial and stromal somatic stem/progenitor cells that may be detected by the LRC technique. The overall objective of this study was to identify and quantify LRCs in mouse endometrium, to determine their location, and to identify their niche in this highly regenerative tissue. Endometrium was labeled for 3 days with bromodeoxyuridine (BrdU) in postnatal day 3 (P3) mice prior to gland development and prepubertal (P19) mice after glands had formed, followed by chase periods of up to 12 weeks. After an 8-week chase, 3% of epithelial nuclei immunostained with BrdU antibody and were considered epithelial LRCs. These were primarily located in the luminal epithelium. Epithelial LRCs did not express estrogen receptor-α (ER-α). Stromal LRCs (6%) were found adjacent to luminal epithelium, at the endometrial-myometrial junction, and near blood vessels after a 12-week chase. Stromal LRCs were stem cell antigen-1, CD45, and some (16%) expressed ER-α, indicating their capacity to respond to estrogen and transmit paracrine signals to epithelial cells for endometrial epithelium regeneration. Both epithelial LRCs and some stromal LRCs, mainly located at the endometrial-myometrial junction, were recruited into the cell cycle after estrogen-stimulated endometrial regeneration, indicating a functional response to proliferative signals. This study has demonstrated for the first time the presence of both epithelial and stromal LRCs in mouse endometrium, suggesting that these stem-like cells may be responsible for endometrial regeneration.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Somatic stem cells are rare cells, residing in specific niches in many tissues and organs [1]. The stem cell niche provides a specialized microenvironment that regulates fate determination of somatic stem cells. Somatic stem cells are defined by their functional properties, in particular their ability to self-renew and undergo uni- or multilineage differentiation, producing both identical daughter cells and one or more mature functional progeny [2]. A reliable way to identify somatic stem cells and their location in the stem cell niche is to take advantage of their slow-cycling, quiescent nature using the label-retaining cell (LRC) approach [36]. LRCs are cells that retain a DNA synthesis label after a prolonged chase period. Rapidly dividing transit amplifying cells dilute their nuclear label with each cell division during the chase period, but somatic stem cells retain the label after a long chase due to infrequent cycling. Stem cells are usually recruited into cell cycle when there is increased demand for cell replacement associated with cellular turnover or tissue injury. Proliferating cells observed in tissue are the more differentiated, transit-amplifying population. LRCs have been identified as somatic stem cells in the epidermis by demonstrating their clonogenic properties [7]. LRCs have also been detected in several other tissues and organs, including breast [8], skin [9], kidney [10], and the prostate [11]. Using this approach, the precise location of rare stem/progenitor cells can be established, and their phenotype and that of the neighboring cells that form the stem cell niche can also be identified.

The human endometrium or mucosal lining of the uterus is known for its remarkable regenerative capacity during the reproductive years of a woman's life [12]. The endometrium regenerates from the lower basal layer, which persists after each menstruation and gives rise to the new upper functional layer. This cyclical regeneration, differentiation, and regression is regulated by sex steroid hormones [13]. The endometrium comprises a luminal epithelium from which extend the tubular glands that traverse through the functional and basal layer to the endometrial-myometrial junction. Endometrial glands have a secretory role to nourish the implanting blastocyst, and they are supported by a stroma, consisting of stromal fibroblasts, vascular cells, and leukocytes [14, 15]. During menstruation, re-epithelialization of the luminal surface epithelium occurs from the exposed mouths of remnant basal glands within 24–48 hours, and glandular and stromal tissue begins to grow only when the endometrial wound is completely re-epithelialized. Since the endometrium is one of the few tissues in the adult human that undergoes cyclical shedding and rapid regrowth, the presence of stem cells has been speculated for many years [14]. However, only recently has it been demonstrated that human endometrium contains small populations of both epithelial and stromal cells with stem cell-like function [16, 17]. At present, no specific endometrial stem cell surface markers have been identified, and therefore nothing is known about the location or regulation of human endometrial stem cells [18].

Although the LRC approach cannot be used for studies in humans, it offers a valuable alternative for locating somatic stem cells in laboratory animals. We have used the LRC technique to identify somatic stem/progenitor cells in mouse endometrium. Although the overall organization of the mouse endometrium is similar to human, in that it contains both glands and supporting stroma, it differs in that there is no basal layer, and mice do not menstruate. However, mouse endometrium does undergo cycles of cellular proliferation and apoptosis during its 4-day estrus reproductive cycle and can be induced to undergo substantial regeneration on administration of estrogen following ovariectomy [19, 20]. The mouse uterus develops prenatally from the Müllerian ducts, two simple tubes comprising urogenital mesenchyme lined with coelomic epithelium. Further development occurs after birth, when the surface epithelium invaginates into the underlying mesenchyme on postnatal day 5 to commence formation of the glands [21, 22]. A similar process occurs in humans during fetal life. This early development of endometrial glands is independent of sex steroid hormones. Around day 21, mouse endometrial epithelium expresses estrogen receptor-α (ER-α) and can respond to estrogen. Female mice reach sexual maturity and commence estrous cycles from day 35.

We hypothesized that mouse endometrium contains small populations of both epithelial and stromal somatic stem-like cells that can be detected by the LRC technique. The overall objective of this study was to identify and quantify LRCs in mouse endometrium, to determine their location, and to identify their niche in this highly regenerative tissue. An important technical consideration of the LRC technique is to initially label the majority of cells with a DNA synthesis label in the tissue of interest. Two approaches were used for labeling during postnatal life, prior to development of endometrial glands, and in prepubertal mice after endometrial development. Specific aims were as follows: 1) to compare postnatal and prepubertal labeling of endometrium with bromodeoxyuridine (BrdU) for the detection of LRC; 2) to locate epithelial and stromal LRCs in mouse endometrium; 3) to identify LRC phenotypes using double immunolabeling techniques; and 4) to demonstrate the functionality of endometrial LRCs.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Animals and Housing Conditions

All mouse husbandry and experimental procedures were conducted in compliance with protocols established by the Monash Medical Centre Animal Ethics Committee A. Mice had access to food and water ad libitum and were housed under controlled environmental conditions at 20°C with a 12-hour dark/light cycle.

BrdU Labeling Mice

Two protocols were established to determine the optimal age for labeling the majority of mouse endometrial cells with BrdU. For postnatal labeling, 3-day-old (P3) C57BL/6J mice were subcutaneously injected with BrdU (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) twice daily, at 9 a.m. and 4 p.m. for 3 consecutive days (50 μg/g of body weight; total dose, 450 μg per mouse) as described [23]. Mice were then allowed to grow without further labeling. Four hours after the last injection, three female mice were sacrificed to determine the initial BrdU labeling at day 0. Chase periods for postnatal BrdU-labeled mice were 1, 2, 3, 4, 8, and 12 weeks (Fig. 1A). Tissues were harvested and analyzed for BrdU-labeled nuclei by immunohistochemistry (IHC) and immunofluorescence (IF).

For prepubertal labeling, P19 C57/CBA female mice were subcutaneously implanted with mini osmotic pumps (model 1007D; Alzet Corp, Cupertino, CA, http://www.alzet.com) containing 3.5 mg/ml BrdU in 0.9% saline (42 μg/g; total dose, 383 μg per mouse). After 3 days, the pumps were surgically removed; 4 hours later, two animals were sacrificed and endometrium was harvested to establish maximum labeling at day 0 of chase. Chase periods for the remaining animals were for 1, 4, and 10 weeks (Fig. 1B).

Some P3 BrdU-labeled mice were ovariectomized after 4-week (n = 3) and 8-week (n = 3) chase periods, and 1 week later, mice received a subcutaneous injection of estrogen (100 ng per 20 g of mouse) or vehicle (peanut oil). Uterine tissues were harvested 0 and 8 hours after the estrogen injection.

Tissue Preparation

Mice were euthanized by cervical dislocation, and the uterine horns were harvested. The right horn was fixed with either 10% formalin or 4% paraformaldehyde for 2 hours and processed into paraffin blocks by standard techniques. The left horn was frozen in Tissue Tek optimal cutting temperature (OCT) compound (Sakura, Finetechnical Co., Tokyo, http://www.sakuraus.com) on dry ice and stored at −80°C until required.

Antibodies

Supplemental online Table 1 details the source, clone, and concentration of primary and secondary antibodies used for IHC and IF protocols. Isotype-matched negative control IgGs at the same concentration as the primary antibodies were included in every staining run. Positive controls for stem cell antigen-1 (Sca-1) were performed on mouse spleen and pregnant mouse endometrium at day 5 (E5).

BrdU Immunohistochemistry Protocol

Single-BrdU IHC was performed as previously described [24]. Paraffin sections (5 μm) were used for all protocols. Antigen retrieval for BrdU IHC was performed by microwaving in 0.1 M citrate buffer, pH 6.0, for 20 minutes and cooling to room temperature (RT) for 30 minutes. Slides were incubated with 0.1 M HCl for 45 minutes and with 0.3% hydrogen peroxide (H2O2) (Orion Laboratories Pty. Ltd., Welshpool, WA, Australia, http://www.orion-group.net) for 10 minutes to quench endogenous peroxidase, followed by protein blocking agent (Immunon Thermo Shandon, Pittsburgh, PA, http://www.thermo.com) for 10 minutes. Sections were then incubated with sheep anti-BrdU primary antibody diluted in 0.1% phosphate-buffered saline (PBS)/bovine serum albumin for 1 hour, followed by appropriate biotinylated donkey anti-sheep secondary antibody for 1 hour. Slides were then covered with peroxidase-conjugated streptavidin (DakoCytomation) for 15 minutes and visualized with diaminobenzidine (Sigma-Al-drich) chromogen after 5 minutes. Slides were lightly counterstained with Mayer's hematoxylin (Amber Scientific, Belmont, WA, Australia, http://members.ozemail.com.au/∼ambersc) for 30 seconds, washed with distilled H2O, and coverslipped. Washing steps using PBS were conducted between each step, and all incubations were conducted at RT unless otherwise specified. Sections were examined under a Zeiss microscope (Axioskop; Carl Zeiss, Oberkochen, Germany, http://www.zeiss.com), and images were captured using a digital video camera (Fujix; Fuji, Tokyo, http://www.fujix-net.co.jp).

Double BrdU and Specific Marker Immunohistochemistry

Several double IHC protocols were used in this study: CD31/BrdU [25], α-smooth muscle actin (α-SMA)/BrdU (established by J.E. Girling and F.L. Lederman, unpublished), Sca-1/BrdU, and ER-α/BrdU. For each protocol, the specific marker immunostaining procedure was conducted prior to the BrdU IHC protocol. Antigen retrieval for CD31 was with 0.1% pepsin at 37°C for 10 minutes, whereas Sca-1 and ER-α were with citrate buffer as described in BrdU IHC protocol. CD31, α-SMA, and ER-α primary antibodies were incubated for 1 hour at RT, whereas anti-Sca-1 antibody was left overnight at 4°C followed by its appropriate secondary antibody (details in supplemental online Table 1). 3-Amino-9-ethylcarbazole (AEC) chromogen (Zymed Laboratories, San Francisco, http://www.zymed.com) was used to detect ER-α, and Vector alkaline phosphatase substrate Kit III (Vector Laboratories Inc., Burlingame, CA, http://www.vectorlabs.com) was used for CD31. The chromogen used to detect BrdU in these double IHC protocols was first incubated with Dako LSAB alkaline phosphatase-conjugated streptavidin (DakoCytomation) for 15 minutes, followed by the Vector alkaline phosphatase substrate Kit III (Vector Laboratories Inc.) for 10 minutes, except for CD31/BrdU, which used AEC (Zymed Laboratories) as previously described [25].

Two-Color Immunofluorescence

Two-color immunofluorescent protocols were established on paraformaldehyde-fixed uterine sections obtained from BrdU-labeled mice. Sections were pretreated with HCl and blocking steps as described for BrdU IHC. For BrdU/CD45, both BrdU and CD45 primary antibodies were incubated concurrently for 1 hour, followed by concurrent incubations with the appropriate Alexa Fluor-conjugated secondary antibodies for 30 minutes in the dark (details and concentrations of primary, secondary antibodies and isotype control IgGs are given in supplemental online Table 1). Sections were then mounted with fluorescent mounting medium (DakoCytomation) and protected from light. For ER-α/BrdU, the same protocol was conducted except that the M.O.M. basic kit (Vector Laboratories Inc.) was used to prevent nonspecific binding of the ER-α monoclonal antibody to mouse tissue. After staining with BrdU, sections were incubated for 1 hour with M.O.M. mouse Ig blocking reagent, incubated with ER-α antibody in M.O.M. diluent for 30 minutes, and incubated with M.O.M. biotinylated anti-mouse IgG for 10 minutes followed by streptavidin Alexa Fluor 488 conjugate (Molecular Probes Inc., Carlsbad, CA, http://probes.invitrogen.com) for 30 minutes to detect ER-α. Nuclei were counterstained using Hoechst dye 33258 (4 μg/ml; Molecular Probes) for 1 minute. For Ki-67/BrdU, BrdU primary antibody was first incubated for 1 hour followed by Alexa Fluor 568-conjugated secondary antibody (supplemental online Table 1) for 30 minutes at RT in the dark. Slides were then incubated with Ki-67 primary antibody for 1 hour, followed by biotinylated secondary antibody (supplemental online Table 1) for 20 minutes and streptavidin Alexa Fluor 488 conjugate (Molecular Probes). Washing steps using PBS were conducted between each step, and all incubations were conducted at RT unless otherwise specified.

Dual immunofluorescence was detected using a Leica confocal microscope (Leica, Heerbrugg, Switzerland, http://www.leica.com). Images were captured with Leica confocal software version 2.5 and merged using Adobe Photoshop software version 8.0 (Adobe Systems Inc., San Jose, CA, http://www.adobe.com).

Enumeration of BrdU-Labeled Cells

Counting of BrdU-labeled cells was conducted in a blinded manner on three sections each of transverse and longitudinal sections from a single uterine horn from 2–6 mice per group for each time point. Images of the entire mouse uterus were captured by digital video camera (Fujix), and BrdU-labeled and -unlabeled nuclei in epithelial and stromal compartments were counted from the entire section of endometrium using Analytical Imaging Station software (version 3.0; AIS, St. Catharine's, ON, Canada, http://www.imagingresearch.com). At least 2,000 nuclei per uterine horn per mouse were counted at each time point. Only heavily immunostained BrdU nuclei were counted as labeled cells, and those that had a speckled appearance were considered to have undergone one or more cell divisions, with subsequent dilution of the label and were not counted as LRCs. The percentages of the BrdU-containing cells were determined by dividing the number of labeled nuclei by the total number of nuclei counted in each section. The results are depicted as means and standard errors.

Enumeration of ER-α/BrdU Coexpressing Cells

Slides double-immunostained with ER-α/BrdU were viewed under a light microbscope (Axioskop), and the number of BrdU+ cells coexpressing ER-α were counted using Analytical Imaging Station software (version 3.0; AIS). The percentage of colocalizing cells was calculated from three sections each of postnatal labeled mice at 8 and 12 weeks of chase for epithelial and stromal LRCs, respectively.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Mouse Endometrium Contains Label-Retaining Epithelial and Stromal Cells

At postnatal day 3, the mouse endometrium comprises a simple uterine tube lined with a luminal epithelium (Fig. 1C), which begins to invaginate into the underlying mesenchyme at days 5–6 to form longitudinal epithelial folds [26]. These invaginations are precursors to uterine glands, which begin to form as focal outgrowths of the luminal epithelium. By day 19, the luminal contour is more complex in shape, and incompletely developed glands are observed (Fig. 1D).

An important consideration in establishing the LRC technique in endometrial tissue was to determine the most appropriate age and stage of endometrial development for adequate labeling of the majority of cells, including the candidate stem cells. Two approaches were investigated: 1) postnatal day 3 (P3; Fig. 1A), where labeling occurs prior to glandular development, and 2) prepubertal day 19 (P19; Fig. 1B), when endometrial glands are well developed, based on LRC studies in other mouse tissues [4, 7, 11, 23, 27]. Administration of BrdU had no effect on subsequent growth or body weight. Mice receiving BrdU injections showed normal growth, weighing 8.5 ± 0.4 g (n = 5) compared with vehicle-injected controls, 9.1 ± 0.9 g (n = 5), at 3 weeks of age. On day 0, after labeling of P3 mice was completed, the majority of epithelial (72.3% ± 1.5%, n = 3) and stromal (64.4% ± 2.3%, n = 3) cells stained for BrdU (Fig. 2A) when glandular development commences. Nuclear staining was apparent in the stroma, glandular epithelium, and luminal epithelium (Fig. 3A, 3B). The percentage of epithelial LRCs in P3-labeled endometrium declined progressively from approx 72%–2% over the course of 8 weeks, reaching 0% at 12 weeks of chase in normal cycling mice, and from 64%–6% for stromal cells over a 12-week chase of postnatal-labeled mice. Figure 2A shows the decline in the percentage of BrdU-labeled epithelial and stromal cells over the entire 12-week chase period. During this time, labeled cells proliferated as the endometrium grew and glands developed. After 4 weeks of chase, proliferation of endometrial cells became cyclic as mice reached puberty. By this stage, BrdU staining was diffuse and showed diminished intensity in many epithelial and stromal cells (data not shown). Four weeks after initial BrdU labeling, there was a rapid decline in epithelial LRCs compared with the initial labeling (day 0), and by 8 weeks only occasional epithelial LRCs were observed. These were mainly located in the luminal epithelium (Fig. 3C), but there were some in the glandular epithelium (data not shown). After a 12-week chase, no epithelial LRCs were detected, despite counting at least 2,000 nuclei in six sections (Fig. 2A). Occasional stromal LRCs were present after a 12-week chase (Fig. 3D), and 31.3% ± 2.6% (n = 3) were observed near blood vessels (Fig. 4A), whereas 40.2% ± 0.8% (n = 3) were near the endometrial-myometrial junction (Fig. 3E). Some appeared underneath the luminal epithelium (Fig. 3D). Vehicle-treated mice displayed no BrdU staining at any stages of the labeling or chase (Fig. 3F).

Mini osmotic pumps were used for BrdU labeling of P19 mice for 3 days, since these mice were large enough to accommodate the surgery and the devices. At this age, endometrial cells continue to proliferate during the final stages of prepubertal growth, as well as during natural cycles regulated by estrogen. The percentage of LRCs observed in the endometrium of prepubertal labeled mice showed a similar pattern to that observed in P3-labeled mice over the 12 weeks (Fig. 2B), although it was less at each time point examined. There was a trend to lesser initial labeling for epithelial (55.0%, n = 2) and stromal (41.3%, n = 2) cells at 0 weeks (Fig. 3H, 3I) of prepubertal mice compared with P3 mice. Labeled epithelial cells showed a linear rapid decline during the first 4 weeks of the chase (Fig. 2B). At 4 weeks of chase, only an occasional BrdU-labeled epithelial nucleus was observed, and these were located in the luminal epithelium (0.2% ± 0.2%, n = 6; Figs. 2B, 3J). A rapid decline was observed for labeled stromal cells in the first week, followed by a gradual decline for longer chase times. By 10 weeks, epithelial LRCs were not detected, but 8.8% ± 1.6% (n = 4) of stromal LRCs were observed (Fig. 3K). As for P3-labeled mice, stromal LRCs were located near blood vessels, underneath the luminal epithelium, and at the endometrial-myometrial junction.

To our knowledge, these data demonstrate for the first time that BrdU labeling was successful in mouse endometrium in both postnatal and prepubertal labeled mice and that epithelial and stromal LRCs are present. Since a higher percentage of initial labeling occurred with postnatal mice, subsequent studies to further characterize the phenotype of endometrial LRCs used P3-labeled mice.

Phenotype of Endometrial Label-Retaining Cells

The phenotype of endometrial epithelial and stromal LRCs and their surrounding niche cells was analyzed using dual immunolabeling to ascertain whether BrdU+ LRC expressed known markers of various cells types found in endometrium. Specifically, we examined for the expression of α-SMA (pericyte and smooth muscle marker), CD31 (endothelial marker), CD45 (leukocyte marker that detects all isoforms of CD45 exon A, B, and C), Sca-1 (stem cell marker), and ER-α on epithelial and stromal LRCs. For some phenotypic markers, individual BrdU+ cells were analyzed by confocal microscopy to establish true cola-beling with BrdU.

Given that endometrium is richly vascularized and some somatic organ-specific stem cells have been found in close association with blood vessels [2830], the relationship between BrdU-labeled stromal LRCs and endothelial cells was examined in postnatally labeled mice chased for 12 weeks. Double immunostaining with an antibody against BrdU and antibodies against CD31 or α-SMA showed that many stromal LRCs were in close proximity to endothelial cells, but CD31 and BrdU were not coexpressed in the same cells (Fig. 4A). Some stromal LRCs colocalized with α-SMA (Fig. 4B), and these are likely to be perivascular mural cells or pericytes.

BrdU+ LRCs did not coexpress with Sca-1, a known stem cell marker, suggesting that this marker is not expressed on mouse endometrial stem/progenitor cells (Fig. 4D). However, Sca-1 was abundantly expressed in positive control sections of pregnant mouse endometrium (E5), primarily in the decidual zone (Fig. 4C). Double immunostaining also confirmed that stromal LRCs were not leukocytes since they failed to express the leukocyte marker CD45 (Fig. 5A).

Since sex steroid hormones are regulators of endometrial growth and regression, we were interested to examine for the presence of the estrogen receptor ER-α in endometrial LRCs. In mouse endometrium, these receptors are expressed in both epithelial and stromal cells [31]. Epithelial LRCs did not coexpress ER-α. Figure 5B shows a typical epithelial LRC that did not express ER-α in 8-week-chased mouse endometrium, whereas all the neighboring luminal epithelium cells were ER-α+. In contrast, 15.7% ± 0.02% (n = 3) of stromal LRCs coexpressed ER-α in 12-week-chased mice, and these were mainly located in the three defined regions of the endometrium. They are near the luminal epithelium (Fig. 5C, 5D), around blood vessels (Fig. 4A, 4B), and near the endometrial-myometrial junction. The expression of ER-α in some stromal LRCs indicates that these cells are capable of responding to estrogen and that they may transmit paracrine signals to epithelial cells to stimulate regeneration of the endometrial epithelium [32, 33].

Endometrial Label-Retaining Cells Proliferate On Estrogen Stimulation

To determine whether endometrial LRCs were functional and could proliferate in response to estrogen, 4- and 8-week-chased BrdU-labeled mice were ovariectomized to regress the endometrium and then given an acute injection of estrogen. Double IF staining with BrdU to detect the LRCs and a proliferation marker, Ki-67, was then performed on endometrium harvested 8 hours after the estrogen injection. Epithelial LRCs located in the luminal epithelial (Fig. 4G) and some stromal LRCs located near the endometrial-myometrial junction (Fig. 4H) were found to coexpress Ki-67, indicating their ability to proliferate and function as precursor endometrial cells during endometrial regeneration. Although all epithelial LRCs were observed to proliferate in response to estrogen, only some stromal LRCs proliferated, and these were mainly located at the endometrial-myometrial junction, although not all in this location proliferated.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Using the LRC technique, we identified and located a small population of epithelial and stromal cells that exhibit a stem-like property (quiescence) in mouse endometrium. We found epithelial LRCs (3%) residing mainly in the luminal epithelium at 8 weeks of chase, although occasional LRCs were observed in glandular epithelium. Phenotypic markers showed that epithelial LRCs were Sca-1, CD45, and ER-α. At 12 weeks of chase, a small population of stromal LRCs (6%) was identified, and these were situated in three regions: adjacent to luminal epithelium, near the endometrial-myometrial junction, and near blood vessels. There were two stromal LRC phenotypes (the majority were Sca-1, CD45, ER-α, and one in six were Sca-1, CD45, ER-α), and both stromal LRC phenotypes reside in the three defined regions. Furthermore, some LRCs proliferated in response to estrogen-stimulated endometrial regeneration. For the first time, this study has demonstrated the presence of functional epithelial and stromal LRCs in mouse endometrium, characterizing their phenotype and partially identifying their respective niches.

Of the 3% of epithelial LRCs found at 8 weeks of chase, most were located in the luminal epithelium, with the occasional LRC in the glands. This surprising finding suggests that luminal epithelial rather than basal glandular epithelial stem/progenitor cells are responsible for the growth and regeneration of the mouse endometrial epithelium. Since no shedding occurs in mouse endometrium, it is likely that the luminal epithelium may be a possible niche for candidate epithelial endometrial stem/progenitor cells. However, this may differ for human endometrium, since the luminal/surface epithelium is shed during menstruation. Of the 6% of stromal LRCs observed at 12 weeks of chase, some were found in close association with luminal epithelium, and 40% were near the endometrial-myometrial junction. Even though mice do not have a defined basal endometrial layer, their location near the junction correlates with their postulated basal layer location in human endometrium [16]. One-third of stromal LRCs were also observed in a perivascular location. This is of particular interest, since they may be recruited from the bone marrow via the vasculature, as suggested by Taylor's study of HLA-mismatched bone marrow transplant recipients [34]. In Taylor's study, whole endometrial glands were of donor origin, supporting the concept of a precursor cell (stem cell) responsible for endometrial regeneration in human. These patients had graft-versus-host disease, a significant source of local damage known to recruit bone marrow stem cells into tissues. Whether bone marrow stem cells are the ultimate source of human endometrial stem/progenitor cells under normal physiological conditions is unknown. Our stromal LRCs were CD45, which suggests that under normal physiological conditions, the source of endometrial stem/progenitor cells in the mouse is not bone marrow. Alternatively, our stromal LRCs could be derived from plastic bone marrow stem cells that no longer express CD45. Similar to our discovery of LRCs in mouse endometrium, a recent abstract reported stromal LRCs in the mouse endometrium. Using a similar approach to our study, the Simón group detected 2.4% of stromal cells as LRCs after 16 weeks of chase, and they demonstrated that 0.6% of these cells colocalized with c-kit [35]. They failed to detect epithelial LRCs and claimed that a somatic stem cell population was present in murine endometrium. Whether a single source of stem/progenitor cells (stromal LRCs) is responsible for mouse endometrial regeneration remains to be determined. However, our data suggest that there are two separate stem-like cell populations in mouse endometrium with at least two distinct niches. Likewise, multiple somatic stem cell niches have been shown for the extensively studied hematopoietic stem cell [29].

Although epidermal LRC have been demonstrated to have clonogenic activity, it is well known that other long-lived, rarely cycling cells, such as leukocytes and endothelial cells, may be detected as LRCs, and these need to be excluded [7]. Our double immunostaining with the endothelial marker CD31 clearly demonstrates that stromal LRCs were not endothelial cells; however, it identified blood vessels as a component of the stromal LRC niche for at least one-third of stromal LRCs. This was further confirmed with α-smooth muscle actin double IHC, which indicated that a substantial proportion (30%) of stromal LRCs were perivascular and possibly pericytes or vascular smooth muscle cells. We demonstrated conclusively that endometrial stromal LRCs were not leukocytes of hematopoietic origin, as they were CD45. Neighboring cells in these niches may be crucially important in determining in vivo stem cell fates. Similar to epidermal stem cells and muscle satellite cells, we also discovered that a well-established stem cell marker, Sca-1, was not a marker of endometrial LRCs [6, 36]. The expression of Sca-1 in decidualized cells suggests that it may be a stromal differentiation marker rather than a stem cell marker in mouse endometrium.

Another key finding was the differential expression of ER-α in endometrial epithelial and stromal LRCs. We detected dual colocalization of ER-α in some stromal LRCs, but not in epithelial LRCs, although most mature luminal and glandular epithelial cells and many stromal cells express ER-α as expected [31]. Estrogen interaction with the 16% of stromal LRCs is likely to stimulate stromal proliferation, as well as proliferation of neighboring epithelial cells, via cell-cell interaction. It has been shown that estrogen binds to ER-α in endometrial stromal cells and triggers the production of paracrine factors, which then act on epithelial cells to stimulate mitogenesis [32]. Epithelial ER-α is neither necessary nor sufficient to mediate a mitogenic response of epithelial cells to estrogen [19, 32, 33]. The nature of the estrogen-induced stromal signal that induces epithelial proliferation is known to involve epidermal growth factor (EGF) [37, 38], and EGF supports clonal growth of putative human endometrial stem/progenitor cells [16, 17]. If human endometrial stromal stem-like cells express ER-α, they may mediate the proliferative response of atrophic postmenopausal endometrium to the exogenous estrogen component of hormone replacement therapy and may have a role in the development of endometrial hyperplasia and endometrial carcinoma. Clonogenic epithelial and stromal cells have been demonstrated in postmenopausal endometrium, but it is not known whether they express ER-α [17], and studies in breast have shown the expression of ER-α in breast cancer stem cells [39]. Investigation into the interaction between putative endometrial stromal stem/progenitor cells and mature epithelial cells will give insight into the etiologies of several female reproductive cancers, since estrogen is a permissive agent in the initiation and progression of these diseases [40].

Having identified LRCs in the mouse endometrium, it was important to determine whether these cells were functional and could respond to growth inducing signals. Using estrogen to induce the regeneration of regressed endometrium, we demonstrated coexpression of the proliferation marker Ki-67 with both epithelial and some stromal LRC within 8 hours. These findings suggest that estrogen recruits endometrial LRCs into cell cycle to initiate tissue regeneration. We found that 100% of epithelial LRC and 29% of stromal LRC located at the endometrial-myometrial junction proliferated in response to estrogen-stimulated endometrial regeneration. Similar responses were observed in small intestinal epithelial LRCs, where 90% entered the cell cycle, after irradiation-induced intestinal regeneration [41], and in mammary gland epithelial LRCs, where 83% proliferated in response to hormone-induced ductal growth [42]. Interestingly, stromal LRCs were observed in the stroma of mammary tissue, but none of these proliferated on hormonal stimulation [42]. Although the majority of endometrial stromal LRCs did not proliferate in response to estrogen, many of those located at the endometrial-myometrial junction did. It is more likely that this population represents the putative endometrial stromal stem/progenitor cell; however, more studies are required to provide definitive proof.

In establishing a protocol for detection of LRCs in mouse endometrium, we considered a number of practical issues. We chose to examine two key periods of endometrial growth to establish optimal labeling. Our results suggest that the window for optimal labeling occurs in postnatal mice, when the endometrium is hyperproliferative and the luminal epithelium of the postnatal uterus begins to extend into the mesenchyme to form the glands [21], rather than in prepubertal mice, when the endometrium becomes responsive to estrogen (day 21) [43]. We believe that postnatal labeling provides a better chance at labeling most of the quiescent stem-like cells in both glands and stroma when the endometrium is rapidly developing. Epithelial LRCs were detectable for shorter chase periods than stromal LRCs, indicating considerable epithelial stem-like cell turnover in endometrial glands as they proliferate in response to estrogen [43, 44]. The length of the chase period was another issue considered, since four cell divisions result in loss of label in transit amplifying cells to undetectable levels [45], whereas quiescent LRCs retain their label. Even though cycling mice do not menstruate, the endometrium undergoes constant turnover in response to sex steroids [4648]. Our experimental design ensured that both groups of mice reached adulthood and underwent several rounds of proliferation and regression before being examined for LRCs.

The LRC technique relies on the assumption that somatic stem cells are quiescent as they undergo infrequent cycling compared with the majority of tissue cells. Another explanation is that somatic stem cells retain their original template DNA strands and pass on newly synthesized strands to their progeny. These original DNA template strands persist through life protecting stem cells from acquiring mutations during DNA replication. In several elegant dual-pulse-labeling, dual-chase studies, it has been demonstrated that LRCs in the small intestine [41], mammary gland [42], and neural stem cells [49] selectively retain long-term-labeled DNA strands, passing newly synthesized DNA strands to their daughter cells destined for differentiation via asymmetric cell divisions. Although the current study did not distinguish between endometrial LRC quiescence and DNA template strand segregation, it is possible that epithelial LRCs and stromal LRCs located at the endometrial-myometrial junction would still retain the same level of BrdU intensity several days following estrogen-stimulated proliferation, when Ki-67 expression would no longer be detectable. Indeed, the percentage of epithelial and stromal LRCs from estrogen-stimulated mice was not significantly different 48 hours after hormonal treatment compared with pretreatment (unpublished observations). A longer second chase of 5–7 days is required to fully establish that endometrial epithelial and stromal LRCs segregate their DNA strands as do intestinal and mammary epithelial stem cells. Our study, however, did demonstrate that heavily stained epithelial and some stromal LRCs, capable of proliferation, could still be detected after prolonged single chase periods of 8 and 12 weeks, after postnatal-labeled endometrium had undergone gland morphogenesis and significant growth, as well as at least 4–5 and 10–12 estrus cycles, respectively. With each estrus cycle there is significant glandular and surface epithelial proliferation and apoptosis, and thus it is possible that epithelial LRCs had retained their original DNA template strands to maintain the intensity of BrdU immunostaining observed in the present study.

In conclusion, we have successfully adapted the well-established LRC method to identify candidate stem/progenitor cells in mouse endometrium. We have demonstrated the existence, location, and functionality of endometrial epithelial and stromal stem-like cells, providing the first data characterizing their respective niches. The ability to identify LRCs in mouse endometrium is a prerequisite for future studies to investigate whether they exhibit other stem cell properties. It will also assist in determining a role for stem cells in endometriosis, adenomyosis, and endometrial carcinoma, important gynecological diseases associated with abnormal endometrial cell proliferation. Further characterization of the putative endometrial stem cell niche may assist in the understanding of these common diseases and ultimately alter their treatment.

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Figure Figure 1.. Experimental protocols for the label-retaining cell technique in mouse endometrium. (A): Postnatal labeling. Mice were given a total of six bromodeoxyuridine (BrdU) injections over 3 days commencing at P3. (B): Prepubertal labeling. Mice were implanted with prefilled BrdU mini osmotic pumps on P19 for 3 days, as described in Material and Methods. Tissue was harvested after the chase periods as indicated ([DOWNWARDS ARROW]). (C): H&E staining of a transverse section of mouse uterus at day 3 showing the simple architecture of the postnatal uterus lined by a single layered luminal epithelium prior to glandular development. (D): Composite micrograph of a day 19 juvenile mouse uterus. Black arrows ([UPWARDS ARROW]) indicate the invaginations of the luminal epithelium into the mesenchyme to form glands. Scale bars = 50 μm. Abbreviations: ×6, times 6; CP, chase period; ge, glandular epithelium; le, luminal epithelium; m, mesenchyme; myo, myometrium; P, postnatal day; s, stroma; wks, weeks.

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Figure Figure 2.. BrdU-labeled cells in postnatal and prepubertal mouse endometrium at various chase periods. Quantitation of BrdU-labeled cells in epithelial and stromal compartments at 0 (n = 3), 1 (n = 3), 2 (n = 6), 3 (n = 5), 4 (n = 4), 8 (n = 4), and 12 (n = 3) weeks of chase for postnatal mice (A) and 0 (n = 2), 1 (n = 6), 4 (n = 6), and 10 (n = 4) weeks of chase for prepubertal mice (B). BrdU-labeled cells are expressed as percentage of total epithelial or stromal cells and reported as mean ± SEM. Abbreviations: BrdU, bromodeoxyuridine; wks, weeks.

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Figure Figure 3.. Localization of label-retaining cells (LRCs) in postnatal and prepubertal-labeled mice. (A–G): Bromodeoxyuridine (BrdU) immunohistochemistry of postnatally labeled mouse uteri at 0 weeks of chase, showing the simple uterine structure of the postnatal uterus duct at day 6, with the majority of the epithelial and stromal cells labeled with BrdU (A); and 1 week of chase, showing the intensity of the staining at a higher magnification (B). Note the invagination of luminal epithelium into the stroma as endometrial glands develop. (C): Eight-week chase showing fewer BrdU-positive cells, and epithelial LRCs located in luminal epithelium (black open arrow). Note: Speckled BrdU-labeled cells in stromal and epithelial compartments are not considered LRCs, as they had undergone one or more cell divisions. (D): Twelve-week chase, where only stromal LRCs were observed (black arrows), some located beneath the luminal epithelium. (E): Stromal LRCs at 12 weeks of chase at higher magnification (black arrows) located near the endometrial-myometrial junction (dotted line). (F): Vehicle-treated mice at 12 weeks of chase. (G): Negative control for BrdU immunostaining. (H–K): BrdU immunohistochemistry of prepubertal-labeled mouse uteri at 0 weeks of chase after initial labeling (H); 1 week of chase, showing the progressive loss of BrdU-positive epithelial and stromal cells (I); 4 weeks of chase, showing epithelial LRC in the luminal epithelium (black open arrow) (J); and 10 weeks of chase with only stromal LRCs present (black arrows) (K). Dotted lines indicate endometrial-myometrial junction. Scale bars = 50 μm. Abbreviations: ge, glandular epithelium; le, luminal epithelium; m, mesenchyme; myo, myometrium; s, stroma; wks, weeks.

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Figure Figure 4.. Localization of cell lineage markers and putative stem cell marker on label-retaining cells (LRCs) in mouse endometrium. Postnatal BrdU-labeled mouse endometrium at 12 weeks of chase was double-immunostained for BrdU (brown) and CD31 (blue) to visualize endothelial cells (A); BrdU (blue) and α-smooth muscle actin (brown) to detect smooth muscle actin on pericytes and vascular smooth muscle cells of blood vessels (inset, negative control) (B); Sca-1 (brown) and BrdU (blue) (D). (E): Negative control for Sca-1 (IgG2a). (F): Coexpression of ER-α (brown) and BrdU (blue) in stromal LRCs of 12-week-chased mice was located near glands. Arrows show LRCs. (C): Positive control for Sca-1 (brown) and BrdU (blue) on day 5 pregnant mouse uterus. Endometrium from postnatal BrdU-labeled, 4-week-chased (G) and 8-week-chased (H) ovariectomized mice double-immunofluorescent-stained for BrdU (red) and Ki-67 (green) to visualize estrogen stimulated proliferation of epithelial LRCs (G) (red arrows) and stromal LRCs (H) (white arrows). Dotted line indicates endometrial-myometrial junction. Scale bars = 25 μm (A, B), 50 μm (C–F), and 40 μm (G, H). Abbreviations: BrdU, bromodeoxyuridine; bv, blood vessel; ge, glandular epithelium; le, luminal epithelium; myo, myometrium; s, stroma.

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Figure Figure 5.. Phenotype of BrdU-labeled cells using confocal microscopy. Postnatal BrdU-labeled mouse endometrium double-immunofluorescent-stained for BrdU (red) and CD45 (green) to visualize leukocytes after a 12-week chase (A); BrdU (red) and ER-α (green) showing lack of coexpression in a single epithelial LRC at 8 weeks (red arrows), but ER-α expression in mature epithelial cells (B); colocalization of BrdU (red) and ER-α (green) in some stromal LRCs at 12 weeks of chase (white arrows) (C); and at higher magnification (D). The x/z and y/z planes are shown on the far right and underneath the merged pictures, demonstrating true colocalization of the two markers. Scale bars = 16 μm (A, B, D) and 40 μm (C). Abbreviations: BrdU, bromodeoxyuridine; ER-α, estrogen receptor-α; ge, glandular epithelium; le, luminal epithelium; s, stroma.

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Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

We are grateful to Kjiana Schwab, Rachel Zillwood, Renea Taylor, Sarah Meachem, and the Animal House Staff at Monash Medical Centre for help and guidance in the establishment of the LRC method. We are also grateful to Stephen Firth from Monash University for technical assistance with the confocal microscopy. This work was supported by National Health and Medical Research Council of Australia grant 284344 (C.E.G.) and the Australian Stem Cell Centre (R.W.S.C.).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information
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SupplTable1.docx18KSupplementary Information Table

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