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Author contributions: D.M.J.: conception and design, collection and/or assembly of data, data analysis and interpretation, and manuscript writing; D.C. and A.M.S.: conception and design, collection and/or assembly of data, and data analysis and interpretation; D.Y.P. and A.G.: conception and design, and manuscript writing; A.J. and M.P.: data analysis and interpretation; O.N.W.: manuscript writing; and S.M.: collection and/or assembly of data, conception and design, data analysis and interpretation, manuscript writing, and final approval of manuscript.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS January 22, 2013.
Synchronous with massive shifts in reproductive hormones, the uterus and its lining the endometrium expand to accommodate a growing fetus during pregnancy. In the absence of an embryo the endometrium, composed of epithelium and stroma, undergoes numerous hormonally regulated cycles of breakdown and regeneration. The hormonally mediated regenerative capacity of the endometrium suggests that signals that govern the growth of endometrial progenitors must be regulated by estrogen and progesterone. Here, we report an antigenic profile for isolation of mouse endometrial epithelial progenitors. These cells are EpCAM+CD44+ITGA6hiThy1−PECAM1−PTPRC−Ter119−, comprise a minor subpopulation of total endometrial epithelia and possess a gene expression profile that is unique and different from other cells of the endometrium. The epithelial progenitors of the endometrium could regenerate in vivo, undergo multilineage differentiation and proliferate. We show that the number of endometrial epithelial progenitors is regulated by reproductive hormones. Coadministration of estrogen and progesterone dramatically expanded the endometrial epithelial progenitor cell pool. This effect was not observed when estrogen or progesterone was administered alone. Despite the remarkable sensitivity to hormonal signals, endometrial epithelial progenitors do not express estrogen or progesterone receptors. Therefore, their hormonal regulation must be mediated through paracrine signals resulting from binding of steroid hormones to the progenitor cell niche. Discovery of signaling defects in endometrial epithelial progenitors or their niche can lead to development of better therapies in diseases of the endometrium. STEM CELLS2013;31:808–822
Hormonally regulated diseases of the endometrium are common. Endometriosis, a benign chronic condition characterized by endometrial growth outside the uterus, affects up to 10% of reproductive age women causing pain, infertility, and tumors . Endometrial carcinoma, the leading gynecologic cancer in developed countries , results from overgrowth of endometrial epithelium. Studies dating back to the 1970s hypothesized the existence of uterine epithelia with stem activity that could regenerate the endometrial epithelial lining in response to estrogen and progesterone . These cells were hypothesized to be a precursor for endometrial cancer when exposed to imbalances of the steroid hormones .
The evidence for existence of adult endometrial epithelial stem cells has been indirect and debated [4–6]. Subsets of short-lived label retaining cells (LRCs) were identified in the mouse endometrium suggesting that endometrial cells have varying proliferative capacity [7, 8]. Only 0.2% of human endometrial epithelia formed colonies in a two-dimensional assay suggesting that not all endometrial epithelia have equal regenerative potential . Subpopulations of Hoechst dye-excluding human endometrial cells, composed of mixtures of stroma and epithelium, formed endometrial glands in vivo  and colonies in vitro . These data support the presence of an adult progenitor cell pool in the endometrium but do not provide direct evidence for their existence. A possible extrauterine endometrial precursor has been proposed based on identification of bone marrow-derived cells in the endometrium of transplantation recipients [12, 13]. While cells from the bone marrow may contribute to the endometrial cell pool, direct evidence demonstrating stem-like activity in the epithelium derived from these cells is lacking.
There are similarities and differences between the mouse and human endometrium. In both species during reproductive years, the stroma and epithelia undergo cell loss and regrowth in a cyclic manner in response to steroid hormones [7, 14]. Ovulation induces a rise in progesterone, and in the absence of implantation, progesterone levels decline resulting in endometrial turnover. Humans undergo endometrial shedding while in mice apoptosis and reabsorption of the endometrium occurs [15, 16]. To support cyclic endometrial regeneration, a residual pool of progenitors must be maintained in both species.
Using an in vivo transplantation and regeneration assay established by our group, here we provide evidence for the existence of an adult mouse endometrial epithelial progenitor population. The total number of these epithelial progenitors fluctuated in response to two reproductive hormones: estrogen and progesterone. While sensitive to hormonal signals, endometrial epithelial progenitors did not express estrogen receptor alpha (ERα) or the progesterone receptor isoforms (PRA and PRB) suggesting that estrogen and progesterone regulate the total number of these cells via paracrine signals.
MATERIALS AND METHODS
Wild type (WT) C57BL/6, β-actin green fluorescent protein (C57BL/6-Tg[ACTbEGFP]1Osb), β-actin DsRed [C57BL/6-Tg(ACTB-DsRed.MST)1Nagy/J], and CB17Scid/Scid mice were from Jackson Laboratory (Bar Harbor, ME, www.jax.org). Mice were maintained in accordance with UCLA, Division of Laboratory Animal Medicine guidelines. All animal experiments were approved by the UCLA Animal Research Committee.
Preparation of Dissociated Uteri
Mouse uteri were dissected, cut into fragments, washed in 5 mM EDTA, and incubated in 1% trypsin for 45 minutes at 4°C. Trypsinization was stopped with Dulbecco's modified Eagle's medium (DMEM)/10% fetal bovine serum (FBS). Luminal epithelia was separated from underlying stroma with a fine forceps and saved. Remaining uterine tissue was minced. Both uterine fractions (luminal epithelium and minced uterine fragments) were digested with 0.8 mg/ml collagenase in DMEM/10% FBS with 5 μg/ml insulin and 0.5 mg/ml DNase for 1.5 hours at 37°C. Digested fractions were passed through 22-gauge syringes, filtered through 40-μm cell strainers, and recombined.
Human endometrial specimens from hysterectomies performed for benign indications were obtained through UCLA institutional review board approved protocols. The endometrial lining was isolated from specimens by scraping the endometrium and some of the underlying myometrium. Isolated tissue was minced and digested with 0.8 mg/ml collagenase in DMEM/10% FBS with 5 μg/ml insulin and 0.5 mg/ml DNase for 1.5 hours at 37°C. Digested tissue was washed with 1× phosphate buffered saline (PBS), then incubated 5 minutes in prewarmed 0.05% trypsin-EDTA and passed five times through a 20-gauge needle. After trypsin was neutralized with DMEM/10% FBS, cells were passed through a 40-μm cell strainer yielding single cells.
Endometrial regeneration was performed as described . Fluorescence-activated cell sorting (FACS) isolated epithelia and cultured neonatal stroma were mixed and resuspended in collagen (354236, BDBiosciences; San Jose, CA, www.bdbiosciences.com) and dispensed into grafts. Endometrial grafts were implanted under the kidney capsule of oophorectomized CB17Scid/Scid mice and regenerated for 8 weeks with an estrogen pellet (0.72-mg β-estradiol per pellet). A progesterone pulse (2.5 mg per day) was administered for the final 8 days of regeneration.
For serial transplantation, regenerated primary endometrial grafts were minced and digested into single cells. Dissociated total and DsRed single cells from the primary grafts were counted, combined with WT neonatal stroma, and reimplanted subrenally as a secondary graft. Similarly, cells harvested from secondary grafts were used to generate tertiary grafts.
For limiting dilution experiments, enumerated sorted DsRed epithelia were placed in 10-fold serial dilutions in the regeneration assay. Regeneration, defined as the formation of pankeratin or DsRed positive glandular structures, was scored for each graft and analyzed using extreme limiting dilution (http://bioinf.wehi.edu.au/software/elda/) .
Frozen or formalin-fixed and paraffin-embedded tissue was stained with antibodies (supporting information Table 1). To quantify expression of antigens, 5-10 high-power fields of view were scored per sample and averaged.
Endometrial cells were suspended in DMEM/5% FBS and stained with antibody (supporting information Table 2). For cell cycle analysis, cells were incubated with 20 μM Hoecsht 33342 at 37°C for 30 minutes, then washed and stained with cell surface antibodies. Intracellular FACS Ki67 staining was performed using the FITC-Ki67 kit from BDBiosciences (BD 556026).
Hormone Ablation and Supplementation in Experimental Mice
Hormone deprivation was achieved by surgical removal of ovaries followed by a 3-4-week rest period. For hormone supplementation, hormone pellets (90-day time-release, 0.72-mg β-estradiol per pellet; 60-day time-release, 100-mg progesterone per pellet; Innovative Research of America, Sarasota, FL, www.innovrsrch.com) were implanted subcutaneously for 2-3 weeks. Serum plasma hormone levels were measured using estradiol and progesterone EIA kits (Cayman Chemical, Ann Arbor, MI, www.caymanchem.com).
Quantitative Polymerase Chain Reaction
Quantitative polymerase chain reaction (PCR) was performed as described previously . Primers are outlined in supporting information Table 3.
Isolated cell fractions were lysed and equal amounts of protein were fractionated on SDS-PAGE gels and transferred to nitrocellulose membrane. Blocked blots were incubated overnight at 4°C in primary antibody and 1 hour at room temperature in secondary antibody (supporting information Table 4).
RNA Seq Analysis
Uterine tissue from three separate groups of mice was FACS sorted to yield biologic triplicates for endometrial epithelial progenitor cell (EEPC), non-EEPC, and stromal cell fractions. RNA was isolated using the Qiagen (Valencia, CA, www.qiagen.com) RNAmicro kit, and RNA integrity was confirmed on an Agilent (Santa Clara, CA, www.agilent.com) 2100 Bioanalyzer. Index-tagged cDNA libraries were generated from 250 ng RNA samples using the TruSeq RNA Sample Preparation kit (Illumina, San Diego, CA, www.illumina.com). Index-tagged libraries were pooled and sequenced at three per lane on an Illumina HiSeq 2000 to generate single-end 50-bp reads. RNA-seq reads were mapped against the mouse genome using TopHat allowing only unique reads with up to two mismatches , using the Ensembl annotation as a guide. Gene expression values were calculated with HTSeq, and adjusted p-values for differential expression between groups were calculated with DESeq. Gene expression was compared using RPKM values .
For hierarchal clustering, the gene expression set was first filtered. Genes with RPKM values < 1 were eliminated from the analysis set, and gene expression differences of greater than twofold between EEPC and non-EEPC with adjusted p-values < .05 were selected. Clustering was performed using Cluster3.0 (http://bonsai.hgc.jp/∼mdehoon/software/cluster/software.htm). Genes were centered and normalized, and average linkage clustering was performed. Resulting heat maps were visualized using JavaTreeview (http://jtreeview.sourceforge.net/). Gene clusters were analyzed using the DAVID Bioinformatics resource [20, 21]. Plots of RNA seq reads were examined using the UCSC genome browser (http://genome.ucsc.edu) .
In Vitro Cell Growth Assays
To culture mouse and human endometrial cells in three-dimensional assays, FACS isolated single cell epithelial preparations were suspended in equal volumes of PrEGM media (Lonza, Allendale, NJ, www.lonza.com) and Matrigel (BD Biosciences). Suspensions were plated around the rim of 12-well tissue culture plate, allowed to solidify, and then overlaid with warm PrEGM. Hollow spheres were counted after 11 days. To test for self-renewal, spheres were released from matrigel by incubation in 1 mg/ml dispase in PrEGM at 37°C. Liberated spheres were pelleted by centrifugation, washed in PBS, incubated for 5 minutes in 0.05% trypsin-EDTA, and passed through a 22-gauge syringe five times to yield single cells. Cells were counted by hemocytometer and replated. For CD44 blocking experiments, cells were incubated with anti-CD44 or IgG2b isotype at 10 μg/ml throughout the duration of in vitro culture. For adherence assays, sterilized glass coverslips were coated with fibronectin or collagen. Enumerated cells were plated on coated coverslips in PrEGM. After 24 hours, unbound cells were counted and used to calculate percentage of bound cells by subtraction from original number of input cells.
Results are expressed as mean ± SD. To determine significance, comparisons were performed using a two-tailed t test for groups of two or one-way analysis of variance with Tukey honestly significant difference test for three or more groups.
Subpopulations of Mouse Endometrial Epithelia Have Long-Term Self-Renewing In Vivo Regenerative Activity
The uterus is composed of three predominant cellular compartments: endometrial epithelium, endometrial stroma (together called the endometrium), and the myometrium. To identify endometrial epithelial progenitors, we established in vivo assays for growth of total endometrial epithelia. Traditionally the endometrial epithelium is broken down into two compartments, glandular and luminal, based on their geographic localization in the endometrium. This geographic subdivision may have functional implications as the formation of endometrial glands by luminal branching is required for establishment of an endometrium receptive to implantation (reviewed in [23, 24]). Based on immunohistochemistry, all endometrial epithelia, glandular and luminal, expressed epithelial cell adhesion marker (EpCAM) (Fig. 1A a,b&e and supporting information Fig. S1A a&b). Endometrial epithelia expressed integrin α6 (ITGA6) and integrin β1 (ITGB1) at their basal surface and were bound to a laminin-marked basement membrane (supporting information Fig. S1A). Endometrial stroma was marked with Thy1 (Fig. 1Ac&f). EpCAM was used to isolate all epithelia from whole mouse uteri dissociated in to single cells (supporting information Fig. S1B). Hematopoietic and endothelial cells were excluded using the lineage depletion markers protein tyrosine phosphatase receptor C (PTPRC, lymphoid), platelet/endothelial cell adhesion marker 1 (PECAM1, endothelial), and Ter119 (red blood cells) . Thy1 was used to exclude endometrial stroma. Since a small fraction of EpCAM+Thy1− cells were Lin positive (supporting information Fig. S1C), the EpCAM+Thy1−Lin− fraction was used to stringently isolate endometrial epithelia (Fig. 1A). The EpCAM+Thy1−Lin− cells were confirmed to be epithelial based on expression of E-cadherin and a lack of expression of stromal (desmin) or myometrial (smooth muscle actin-SMA Fig. 1Ad) markers in a quantitative-PCR assay (Fig. 1A). The EpCAM−Thy1+Lin− and EpCAM−Thy1−Lin− fractions were identified as endometrial stromal and myometrial cells based on expression of desmin and SMA, respectively (Fig. 1A).
To assay for cells capable of forming functional endometrial epithelial structures, an in vivo regeneration model from dissociated uterine epithelia and stroma was used (Fig. 1B). This assay is adapted from a model previously reported by our group where luminal epithelia from an adult DsRed mouse in combination with neonatal WT stroma regenerated into endometrial tissue in the subrenal space of an immunodeficient mouse . Here, to assay for growth of all endometrial epithelia (glandular and luminal), FACS isolated endometrial epithelia from dissociated uterine cellular preparations were regenerated in vivo in a similar assay (Fig. 1B). This approach ensured that all endometrial epithelia (glandular and luminal) would be included in this in vivo regeneration assay. As reported previously , endometrial epithelia regenerated in this assay were marked with red fluorescent protein demonstrating that regenerated epithelia derive from the DsRed adult donor tissue (Fig. 1B).
The antigenic profile EpCAM+Thy1−Lin− was used for isolation of endometrial epithelia from group housed mice. The mouse reproductive estrous cycle lasts 4-5 days  and depending on the stage of the estrous cycle there may be variations in the growth capacity of the endometrial epithelia. Based on daily examination of vaginal cytology , mice housed in groups of five were in various stages of the estrous cycle (supporting information Fig. S1D). Therefore, endometrial epithelia harvested from 5 to 10 unstaged mice were pooled for most experiments. This approach ensured that a representative sample of endometrial epithelia from various stages of the estrous cycle would be used in our analyses.
1 × 105 EpCAM+Thy1−Lin− or 1 × 105 EpCAM−Thy1±Lin− cells from DsRed uteri were combined with 2.5 × 105 WT neonatal stroma and placed in the regeneration assay. EpCAM+Thy1−Lin− cells contained almost all the in vivo regenerative activity demonstrated by formation of multiple endometrial epithelial structures (35 ± 2 EpCAM+Thy1−Lin− vs. 2 ± 2 EpCAM−Thy1±Lin−, p = .015) (Fig. 1C a vs. d and supporting information Fig. S1E). These findings confirm that the EpCAM+Thy1−Lin− antigenic profile marks epithelial cells capable of regenerating endometrial epithelial structures. We hypothesized that a subpopulation of endometrial epithelia would have stem-like activity with a capacity to self-renew and proliferate in vivo. To test this hypothesis, regenerated endometrial epithelial cells were serially passaged in vivo as secondary and tertiary grafts (Fig. 1D). Isolated color-marked endometrial epithelia from DsRed transgenic mice were used to regenerate primary grafts. One in 3,570 endometrial epithelia were capable of forming a gland in vivo and this number is comparable to previously reported mammary repopulating units of 1/1,400-2,000  to 1/4,900 . Regenerated tissue was dissociated into single cells and retransplanted with fresh WT stroma as secondary and then tertiary grafts. Importantly, new epithelia were not supplemented in these grafts to assess the serial retransplantability of existing regenerated epithelia. Based on an approximate estimation, 1 in 3,500 endometrial epithelia was capable of serial retransplantation in vivo (Fig. 1D). The fact that endometrial epithelia were capable of in vivo serial transplantation, establishes the existence of a long-term self-renewing endometrial epithelial population.
Hormonal Withdrawal Enriches for a Population of Endometrial Epithelia with Increased Regenerative Activity
To isolate and identify endometrial epithelial progenitors, we set out to define a population of endometrial epithelia enriched for regenerative activity. With the cessation of ovarian hormones, endometrial atrophy ensues in human and mouse; yet hormone supplementation causes remarkable regeneration of the remaining endometrial epithelia [28, 29]. These observations suggest that subsets of adult endometrial epithelia have stem-like activity and can survive without ovarian hormones. We hypothesized that hormonal deprivation would lead to loss of differentiated endometrial epithelia causing a relative increase in the percentage of endometrial epithelial progenitors. Reproductive age mice were hormonally deprived by surgical removal of their ovaries. The growth of endometrial epithelia from hormonally deprived and reproductive hormonally intact mice was compared. Hormonal deprivation dramatically decreased uterine size, accompanied by a 10-fold reduction in total number of endometrial epithelia (Fig. 2A). The surviving endometrial epithelia were enriched for a population of cells capable of in vivo regenerative activity demonstrated by increased growth potential of hormonally deprived compared to equal numbers of hormonally intact epithelia (1 in 69 hormonally deprived vs. 1 in 862 reproductive endometrial epithelia, p < .0001) (Fig. 2B and supporting information Fig. S2). These findings suggest that subsets of endometrial epithelial progenitors are resilient to hormonal deprivation. Results also demonstrate that hormonally deprived endometrial epithelia were enriched for progenitors capable of in vivo regeneration.
Signaling through the Wnt pathway is critical for the uterine development [30, 31]. For example, deletion of Wnt7a resulted in developmental abnormalities in the endometrial epithelium and infertility . The expression of β-catenin, a downstream target of Wnt (reviewed in ) was examined in intact and hormonally deprived uteri. Concentrated expression of membranous β-catenin was detected in the endometrial epithelial glands and luminal crypts of hormonally intact mice (Fig. 3A a-d). A similar distribution of increased but also nuclear β-catenin was detected in hormonally deprived mice (Fig. 3A e-h). The mRNA expression level of Wnt/β-catenin target genes [33–40] was compared in endometrial epithelia of hormonally deprived versus hormonally intact uteri (Fig. 3B). Higher expression of several Wnt/β-catenin target genes was observed in hormonally deprived endometrial epithelia (Fig. 3B) previously shown to be enriched for increased in vivo regenerative activity (Fig. 2B). These findings suggest the Wnt pathway may be activated in endometrial epithelial progenitors.
Basally Located CD44-Marked Cells are the Endometrial Epithelial Progenitors
The cell surface marker CD44 was one of the differentially expressed Wnt regulated genes found through q-PCR analysis, with a 12-fold increase in CD44 transcript levels detected in hormonally deprived epithelia (Fig. 3B). Over-expression of CD44 has been reported in human endometrial carcinoma  and more recently, this cell surface marker has been used to isolate a cancer initiating population in endometrial cancer cell lines . To test if CD44 was a candidate cell surface marker of normal mouse endometrial epithelial progenitors, its expression was examined in hormonally deprived and intact mouse uterine sections (Fig. 4A). In both hormonal conditions, CD44 positive cells were distributed as single cells in the luminal epithelium, in some luminal epithelial crypts and in some but not all glands (Fig. 4A). These cells were in close contact with the basement membrane marked with ITGA6 (Fig. 4A b&e). Similar to the human endometrium , CD44 was detected not only in the endometrial epithelia but also in endometrial stroma (EpCAM−CD44+ cells, Fig. 4A); however, these stromal cells were not included in our regenerations as they were EpCAM negative. We quantified the number of CD44 positive cells in hormonally intact and deprived endometrial epithelia by immunohistochemistry and FACS analysis (Fig. 4B, 4C). By both methods, hormonal deprivation led to an approximate twofold increase in CD44 positive endometrial epithelia (Fig. 4B, 4C).
We hypothesized that CD44 positive cells in contact with the basement membrane contained the endometrial epithelial progenitors. We examined the in vivo growth of EpCAM+Lin−CD44+ITGA6hi endometrial epithelia compared to EpCAM+Lin−CD44−ITGA6hi/EpCAM+Lin−CD44±ITGA6lo epithelia (Fig. 5A and supporting information Fig. S3A). To exclude endometrial stroma, Thy1 was added to the lineage depletion markers (supporting information Fig. S3A). Enrichment for CD44 was verified in the EpCAM+Lin−CD44+ITGA6hi cellular pool (supporting information Fig. S3B). To estimate the number of endometrial epithelia capable of forming endometrial epithelial structures in vivo, 10-fold serial dilutions of epithelia were placed in the regeneration assay (Fig. 5A). The EpCAM+Lin−CD44+ITGA6hi population was clearly enriched for cells capable of in vivo growth compared to the depleted fraction (Fig. 5A). One in 109 EpCAM+Lin−CD44+ITGA6hi cells formed an endometrial epithelial structure in vivo compared to 1 in 3,124 EpCAM+Lin−CD44−ITGA6hi/EpCAM+Lin−CD44±ITGA6lo epithelia (Fig. 5B). Previously we found that 1 in 862 hormonally intact endometrial epithelia regenerated endometrial epithelial structures in vivo (Fig. 2B). Collectively, these results suggest that selection with EpCAM+Lin−CD44+ITGA6hi antigenic profile resulted in an approximate eightfold enrichment in endometrial epithelia with in vivo regenerative activity.
Analysis of grafts regenerated from EpCAM+Lin−CD44+ITGA6hi cells revealed that majority of regenerated epithelia were EpCAM and ITGA6 positive but CD44 negative (Fig. 5C). Only a small fraction of regenerated cells maintained the EpCAM/CD44 double positive progenitor signature (0.8 ± 0.1%) (Fig. 5C a-c). Similarly, these grafts contained a small subpopulation of ITGA6/CD44 dual positive cells (Fig. 5C d-f). Pankeratin marked all regenerated endometrial epithelia similar to the native endometrium (supporting information Fig. S3C). These results demonstrate that EpCAM+Lin−CD44+ITGA6hi cells are capable of self-renewal, proliferation, and multilineage differentiation in vivo. The eightfold increase in the in vivo regenerative activity of the EPCAM+Lin−CD44+ITGA6hi cells coupled with their ability to undergo multilineage differentiation suggests that this population is enriched for murine EEPCs. The remaining fraction of endometrial epithelia (EpCAM+Lin−CD44−ITGA6hi/EpCAM+Lin−CD44±ITGA6lo) had an approximately fourfold decrease in their in vivo regenerative potential compared to all endometrial epithelia and this cellular fraction will be called non-EEPC.
To assess if the Wnt signaling pathway was activated in endometrial progenitors the expression of β-catenin protein, a well known target of Wnt signaling  was measured by Western blot (Fig. 5D). A relatively greater expression of total β-catenin was detected in the EECP cells compared to the non-EEPC and endometrial stroma (Fig. 5D). A β-catenin degradation product was detected only in the non-EEPC fraction . These findings suggest that the Wnt/β-catenin pathway is primarily activated in the endometrial epithelial progenitors.
The Transcriptome of Uterine Epithelial Progenitors is Unique in the Endometrium
Thus far, we have demonstrated that the EEPC are the cellular population in the endometrium capable of regenerating endometrial epithelial structures. To test if this biologic behavior is accompanied with a unique transcriptional profile, RNA-sequencing was performed to compare the transcriptomes of EEPC, non-EEPC and endometrial stroma. Among 36,872 genes examined, 11,140 transcripts were expressed in the epithelial cell fractions (RPKM > 1.0). Of these transcripts, 332 were differentially expressed in EEPC versus non-EEPC. Differentially regulated genes were defined as transcripts that varied by greater than twofold and were significantly different among sample replicates (adjusted p-value < .05).
Hierarchal clustering of the differentially regulated gene set revealed four distinct groups of genes (Fig. 6Aa and supporting information Table S5). Cluster A contained 135 genes more highly expressed in non-EEPC compared to EEPC and stroma. This cluster was enriched for genes encoding secreted proteins, and genes involved in immune response and host defense (Fig. 6Ab). The detection of secreted protein genes may reflect the differentiated nature of non-EEPC as a secretory epithelium. Expression of immune defense related genes in non-EEPC may be a protective mechanism to prevent uterine infections given that the uterus is regularly exposed to foreign fluids [45, 46].
The 60 genes in cluster B had greater expression in EEPC relative to non-EEPC and stroma. This cluster was enriched for transcripts involved in the cell cycle and cell division (Fig. 6Ab). Traditionally regenerative cells were considered to be quiescent, but recent studies have shown that in tissues with rapid cellular turnover regenerative cells are cycling [47, 48]. To determine whether EEPC were quiescent or cycling, two assays were performed: (a) cell cycle analysis and (b) measurement of proliferation using Ki67 expression. A higher percentage of cells were found in G2M/S in the EEPC compared to non-EEPC (supporting information Fig. S4A). Similarly, a greater percentage of cells were Ki67 positive in the EEPC pool compared to other endometrial epithelia (supporting information Fig. S4B). These findings suggest that EEPC are not quiescent but actively cycling. The high rate of proliferation in EEPC may support the rapid turnover of the endometrial lining during reproductive years. Higher mRNA levels of telomerase reverse transcriptase enzyme (TERT), the catalytic component of telomerase, have been reported in rapidly dividing stem cells such as those in the intestinal epithelium . Given that many EEPC were found to be cycling, the expression of TERT mRNA was measured by quantitative-PCR and compared in EEPC versus non-EEPC cells. TERT transcript levels were significantly elevated in EEPC compared to non-EEPC (supporting information Fig. S4C). Telomerase activity may help protect dividing EEPC from DNA attrition .
Cluster C contained 119 genes whose transcripts were highest in stroma and higher in EEPC compared to non-EEPC. Analysis of this cluster showed enrichment for transcripts involved in signal transduction, pattern binding, cell adhesion, and components of the extracellular matrix (Fig. 6Ab). These genes may be involved in localizing and maintaining the EEPC in its niche. Cluster D contained only 14 genes that were enriched in EEPC and stroma compared to non-EEPC. Similar to genes in cluster C, these transcripts were associated with cell surface proteins (Fig. 6Ab).
CD44 was expressed in all EEPC and some endometrial stromal cells (Fig. 4A). Multiple isoforms of CD44 exist based on splicing of its 10 variant exons (reviewed in ). The differential expression of CD44 splice variants in specific cells can regulate their adhesion and interaction with the microenvironment [52, 53]. We compared the expression of CD44 variants in EEPC, non-EEPC, and endometrial stroma by examining plots of the CD44 RNA sequencing reads. Clear differences between the abundance of CD44 variant exons were observed between the three cellular population and increased levels of CD44 variant transcripts were detected in EEPC (Fig. 6B). Functionally, EEPC had greater in vitro adherence capacity to fibronectin  and collagen [55, 56] compared to non-EEPC (supporting information Fig. S4D). This differential adherence capacity may be due to increased expression of CD44 and CD44 variants in the EEPC.
Collectively, we demonstrate that four cell surface markers (EpCAM, Thy1, CD44, and ITGA6) can divide the endometrium into three distinct cellular compartments: EEPC (epithelial progenitors), non-EEPC (differentiated epithelia), and stroma. These three compartments have different biologic properties in vivo coupled with distinct transcriptional profiles.
The Numbers of Endometrial Epithelial Progenitors are Regulated by Estrogen and Progesterone Through Paracrine Signals
The endometrium undergoes numerous cycles of breakdown and regeneration during reproductive years. This activity is tightly regulated with two steroid hormones: estrogen (E) and progesterone (P). Both hormones are necessary for embryonic implantation . To examine effects of E and P on the progenitors, the percentage of EEPC was measured in reproductive mice supplemented with placebo, E, P, or a combination of both hormones. Serum levels of E and P were measured in each experimental condition (supporting information Fig. S5A). The percentage of EEPC remained unchanged in placebo, E or P treated uteri (Fig. 7A and supporting information Fig. S5B). In contrast, when E and P were coadministered the percentage of EEPC doubled (Fig. 7A and supporting information Fig. S5B). Previously we observed a similar increase in the percentage of EEPC in hormonally deprived mice (Figs. 4B, 4C, 7A). To determine the biologic explanation for this unexpected result, a rise in the percentage of EEPC seen with both hormone deprivation and supplementation, we examined the total numbers of EEPC per uterus. To estimate the total number of EEPC per uterus, the percentage of EEPC was multiplied with total number of endometrial epithelia per uterus. Dual hormone supplementation resulted in a twofold rise in the number of EEPC while hormonal deprivation caused a twofold drop in EEPC compared to hormonally intact control (Fig. 7A). With hormonal deprivation the increased percentage of EEPC is explained by massive loss of the differentiated endometrial epithelia coupled with relative survival of many EEPC (Figs. 7A, 2A). In contrast, with dual hormone supplementation, the total number of endometrial epithelial remained unchanged while the number of progenitors doubled resulting in a net increase in the percentage of EEPC (Fig. 7A and supporting information Fig. S5B, S5C).
Given these results, we hypothesized that exposure to E and P would enhance the growth of endometrial epithelia. To test this hypothesis, the in vivo regenerative activity of E and P versus placebo-treated endometrial epithelia was compared (Fig. 7B and supporting information Fig. S5D). Estrogen and progesterone treatment enhanced the in vivo growth potential of endometrial epithelia sixfold (Fig. 7B and supporting information Fig. S5D). This hormone-induced growth augmentation could be due to (a) an increase in progenitors and/or (b) a heightened growth of existing progenitors. To test this hypothesis, the growth of EEPC was compared to total endometrial epithelia using an in vitro growth assay (supporting information Fig. S6 and Fig. 7C) adapted from previously established protocols [58, 59]. In this assay, isolated epithelia give rise to spheroid structures that express EpCAM similar to the native endometrial epithelium (supporting information Fig. S6A, S6B). The EEPC were the cells with sphere forming capacity in vitro (supporting information Fig. S6C). Blocking CD44 with a neutralizing antibody  decreased the sphere forming capacity of endometrial epithelia in this assay, suggesting a possible role for CD44 signaling in the proliferation of sphere forming cells (supporting information Fig. S6D). Cell growth in this assay was clonal (supporting information Fig. S6E, S6F). Cells within spheres were capable of serial passaging in vitro (supporting information Fig. S6G) suggesting that subsets of endometrial epithelia maintain clonal growth with time.
Coadministration of E and P doubled the growth of total endometrial epithelia but did not change the growth of EEPC (Fig. 7C). These results suggest that the enhanced growth potential induced by E and P cotreatment primarily results from increased numbers of EEPC but not qualitative changes in these progenitors. Pregnancy is a physiologic state accompanied with elevated levels of both E and P resulting in expansion and biochemical changes in the endometrial stroma called decidualization . The E and P induced expansion of EEPC could be a physiologic adaptation for a concomitant expansion of endometrial epithelia in a rapidly growing uterus during pregnancy.
To determine whether the mechanisms controlling the expansion of EEPC are cell autonomous or paracrine, the expression of ERα and PR was examined. Basally located CD44 positive cells did not express ERα or PR based on immunohistochemistry (Fig. 7D). These findings were confirmed by immunocytochemistry on isolated EEPC (supporting information Fig. S7). ERα and the progesterone receptor isoforms, PRA and PRB, were detected in non-EEPC and endometrial stroma but were absent in EEPC in Western blots (Fig. 7E). We did not detect a difference between mRNA levels of these hormone receptors in EEPC versus non-EEPC cells suggesting modulation of hormone receptors at a post-transcriptional level. These results suggest that hormonal signals promoting the proliferation of EEPC are paracrine and arise either from neighboring epithelia or uterine stroma.
Similar to the mouse endometrium, using the markers EpCAM and membrane metallo-endopeptidase (MME), we have fractionated human endometrium into an epithelial (EpCAM+MME−), stromal (EpCAM−MME+), and myometrial (EpCAM−MME−) compartments (supporting information Fig. S8A). Expression of CD44 has been reported in the secretory phase of the normal endometrium and human endometrial carcinoma [41, 61]. We detected basally located CD44 positive cells in both the proliferative and the secretory phase of the human endometrium (supporting information Fig. S8B). To assess if, basally located CD44 positive human endometrial epithelia were candidate progenitors, these cells were isolated from three normal reproductive age women undergoing hysterectomy for benign indication. Lineage depletion markers PTPRC (lymphoid), PECAM1 (endothelial), glycophorin A (GYPA, red blood cell), and MME (stroma) were used to exclude nonepithelial cells from human dissociated endometrial cellular preparations. The in vitro growth capacity of EpCAM+CD44+ITGA6hiLin− human endometrial epithelia was compared to EpCAM+CD44−ITGA6hiLin−/EpCAM+CD44+/−ITGA6loLin− and unfractionated endometrial epithelia (supporting information Fig. S8C). In all three clinical specimens, predominance of the in vitro sphere forming activity was found in the EpCAM+ CD44+ITGA6hiLin− fraction, suggesting that this cell surface signature enriches for cells progenitor activity (supporting information Fig. S8C). Similar to mouse endometrial epithelial progenitors, basally located human endometrial epithelia marked with CD44 were predominantly ERα and PR negative (supporting information Fig. S8D).
Over the last decade, investigators have attempted to identify adult endometrial progenitors with marginal success (reviewed in ). This is surprising given the remarkable strides made in defining the cellular hierarchy in other hormonally regulated tissues such as breast [62, 63] or the prostate epithelium [64–66]. Recent work has identified candidate cell surface markers for isolation of stromal stem-like population in the human endometrium. Human stromal cells marked with W5C5 (marking an unknown antigen) or the CD146 and PDGFRβ were found to have stem-like activity based on in vitro assays, differentiation potential, and limited in vivo growth [67, 68]. These cells did not differentiate into endometrial epithelia. Putative markers for isolation of human endometrial epithelial stem cells remain unknown.
Minimal progress has been made in identification of murine endometrial epithelial or stromal progenitors (reviewed in ). Epithelial or stromal LRCs in the endometrium or the fallopian tube have been considered as potential endometrial progenitors [7, 8, 69, 70] but functional in vivo stem activity of these cells has not been demonstrated. The challenges for analysis of LRCs as progenitors are twofold: (a) lack of in vivo growth assays that can examine regenerative activity of candidate progenitors [6, 69] and (b) absence of cell surface markers for live isolation of bromodeoxyuridine (BrdU)-marked LRCs. Therefore, a cell surface antigenic profile for isolation of murine endometrial epithelia or stromal cells has been unavailable.
Similar to the human endometrium [9, 71], we find that mouse endometrial epithelia express EpCAM. Subpopulations of EpCAM positive cells were capable of regenerative activity in an in vivo functional assay designed to test for clonal growth of endometrial epithelia. Some EpCAM positive cells could undergo serial transplantation, establishing proof for the existence of a long-term self-renewing endometrial epithelial progenitor population. To isolate the progenitor cells, we fractionated EpCAM positive endometrial cells based on expression of two other makers: CD44 and ITGA6. A minor subpopulation of the mouse endometrium, the EEPC marked with EpCAM+CD44+ITGA6hiThy1−PECAM1−PTPRC−Ter119− contained predominance of epithelia with in vivo growth capacity. This cellular pool must contain the endometrial epithelial stem cells demonstrated by the capacity of these cells to self-renew, differentiate and proliferate in vivo. One in 109 EEPC were capable of regenerating an endometrial gland in vivo. In the mammary system, where epithelial progenitors are also regulated with reproductive hormones [72, 73], 1 in 91  and 1 in 64  epithelial stem cells could regenerate a mammary gland in vivo as determined by limiting dilution analyses. Thus, our purification of EEPC is comparable to earlier studies in the mammary system. We plan to refine isolation of our epithelial progenitor population by mining the transcriptome of EEPC for identification of cell surface markers that could be used to further purify the stem population.
Here, we show that a greater proportion of the EEPC are cycling and proliferating compared to the differentiated endometrial epithelia. The high rate of cellular division in these progenitors must be a biologic adaptation required to keep up with the demands of rapid turnover in the reproductive endometrium. In this respect, the endometrial progenitors may behave similar to intestinal epithelial and hematopoietic stem populations where a pulsed label such as BrdU is not retained [48, 74]. Many studies had attempted to use the label retention for identification of endometrial epithelial progenitors [7, 69, 70, 75]. These studies showed that LRCs could be detected up to 8 weeks in the endometrial epithelium  but failed to identify a long-term LRC in this compartment [7, 69, 70, 76]. Because long-term LRCs were not observed in the endometrial epithelium, it has been proposed that the progenitor/stem cell pool for these cells must reside elsewhere, either in the stroma/perivascular niche  or in the distal end of the fallopian tube  where LRCs can be detected. A major assumption in these studies is that endometrial epithelial progenitors are quiescent.
An alternative method for identification of stem cells is lineage tracing, but it is only feasible when relevant transgenic mice are available. There were two limitations with our combination of makers which made lineage tracing not possible: (a) there are no transgenic mice with promoters relevant to our markers and (b) because CD44 is also expressed in the endometrial stroma, lineage tracing or depletion of cells using the CD44 promoter would lead to difficulties interpreting the data. In previous studies, lineage tracing has been compared to in vivo regeneration as a method for identification of somatic stem cells. In a murine prostate model, the same epithelial stem cell population was capable of self-renewal and multilineage differentiation regardless of the method tested . While in vivo regeneration and lineage tracing did not yield identical results in terms of differentiation capacity of mouse mammary cells, the stem cell subset identified using limiting dilution transplantation was confirmed to be a stem cell population by lineage tracing . Taken together, in vivo regeneration coupled with serial transplantation is consistent with standards in the field for the identification of self-renewing epithelial progenitor and stem-like cells. We hope that analysis of the EEPC transcriptome will reveal promoters that can be used for lineage tracing in future studies.
Similar to the endometrial epithelial progenitors, mammary stem cells are ER/PR negative and expanded when E and P were coadministered [72, 73]. However, unlike mammary tissue where E and P increase the risk of breast cancer , P concomitant with E has protective effects on the endometrium . These differences may result from selective use of PR isoforms, PRA in the uterus and PRB in the mammary tissue, resulting in different transcriptional signals . The exquisite sensitivity of EEPCs to E and P suggests that the balance of these hormones may be critical for maintaining endometrial homeostasis. In fact, resistance of endometrial epithelium to progesterone has been implicated as a central mechanism in the pathophysiology of endometriosis, infertility and endometrial cancer . Mechanisms leading to progesterone resistance remain unknown. Estrogen induced proliferation of the endometrial epithelium occurs even with epithelial specific deletion of ERα, suggesting that the effects of estrogen on endometrial epithelium are paracrine and mediated through the stroma . During pregnancy, the antiproliferative effects of progesterone on the endometrial epithelium are proven to be paracrine . We find that murine EEPC and basally located CD44-marked human endometrial epithelia are ERα and PR negative, suggesting that signals regulating their growth may also be paracrine. Two plausible mechanisms for progesterone resistance may include (a) cell autonomous defects in EEPC that make them unable to respond to paracrine signals and/or (b) defects in stromal or surrounding non-EEPC rendering them incapable of PR mediated signaling and thus paracrine regulation of EEPC. Understanding hormone-mediated mechanisms that regulate growth of EEPC in their niche will likely have major implications in designing therapies for common yet poorly understood diseases of the endometrium.
We report the first description for dissociating the mouse uterus into three major cellular compartments: endometrial epithelium, endometrial stroma, and myometrium. We subdivide the endometrial epithelium into two functionally distinct cellular fractions: (a) the epithelial progenitor pool capable of self-renewal, multilineage differentiation, and propagation in vivo and (b) the differentiated endometrial epithelia. The progenitors comprise a small fraction of total endometrial epithelia and their numbers are tightly regulated with two female reproductive hormones estrogen and progesterone through paracrine signals. We suspect that defects in the normal crosstalk between these progenitors and their niche may play a critical role in endometriosis, endometrial cancer, and infertility. Findings in this article are currently being used as a stepping stone for discovery of these defects and therapeutic approaches that can correct them.
SM is supported by a VA CDA-2 Career Development Award, a gift from the Scholars in Translational Medicine Program, Mary Kay Foundation Award, PCF Young Investigators Award, STOP Cancer Award, the Broad Stem Cell Research Center, Research Award, the U54 CA 143,931 award and a Kimmel Translational Scholar Award. ASG is supported by the Joe and Ali Torre- Prostate Cancer Foundation Young Investigator Award. ONW is a Howard Hughes Medical Institute investigator.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
The authors indicate no potential conflicts of interest.