During reproductive life, the human endometrium undergoes around 480 cycles of growth, breakdown and regeneration should pregnancy not be achieved. This outstanding regenerative capacity is the basis for women's cycling and its dysfunction may be involved in the etiology of pathological disorders. Therefore, the human endometrial tissue must rely on a remarkable endometrial somatic stem cells (SSC) population. Here we explore the hypothesis that human endometrial side population (SP) cells correspond to somatic stem cells. We isolated, identified and characterized the SP corresponding to the stromal and epithelial compartments using endometrial SP genes signature, immunophenotyping and characteristic telomerase pattern. We analyzed the clonogenic activity of SP cells under hypoxic conditions and the differentiation capacity in vitro to adipogenic and osteogenic lineages. Finally, we demonstrated the functional capability of endometrial SP to develop human endometrium after subcutaneous injection in NOD-SCID mice. Briefly, SP cells of human endometrium from epithelial and stromal compartments display genotypic, phenotypic and functional features of SSC.

Endometrial regeneration is mediated, at least in part, by the existence of a specialized somatic stem cell (SSC) population recently identified by several groups using the side population (SP) technique. We previously demonstrated that endometrial SP displays genotypic, phenotypic and the functional capability to develop human endometrium after subcutaneous injection in NOD-SCID mice. We have now established seven human endometrial SP (hESP) cell lines (ICE 1–7): four from the epithelial and three from the stromal fraction, respectively. SP cell lines were generated under hypoxic conditions based on their cloning efficiency ability, cultured for 12–15 passages (20 weeks) and cryopreserved. Cell lines displayed normal 46XX karyotype, intermediate telomerase activity pattern and expressed mRNAs encoding proteins that are considered characteristic of undifferentiated cells (Oct-4, GDF3, DNMT3B, Nanog, GABR3) and those of mesodermal origin (WT1, Cardiac Actin, Enolase, Globin, REN). Phenotype analysis corroborated their epithelial (CD9+) or stromal (vimentin+) cell origin and mesenchymal (CD90+, CD73+ and CD45−) attributes. Markers considered characteristic of ectoderm or endoderm were not detected. Cells did not express either estrogen receptor alpha (ERα) or progesterone receptor (PR). The hESP cell lines were able to differentiate in vitro into adipocytes and osteocytes, which confirmed their mesenchymal origin. Finally, we demonstrated their ability to generate human endometrium when transplanted beneath the renal capsule of NOD-SCID mice. These findings confirm that SP cells exhibit key features of human endometrial SSC and open up new possibilities for the understanding of gynecological disorders such as endometriosis or Asherman syndrome. Our cell lines can be a valuable model to investigate new targets for endometrium proliferation in endometriosis.

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Uterine leiomyomas, also known as uterine fibroids, are the most common pelvic tumors, occurring in nearly 70% of all reproductive-aged women and are the leading indication for hysterectomy worldwide. The development of uterine leiomyomas involve a complex and heterogeneous constellation of hormones, growth factors, stem cells, genetic, and epigenetic abnormalities. An increasing body of evidence emphasizes the important contribution of epigenetics in the pathogenesis of leiomyomas. Genome-wide methylation analysis demonstrates that a subset of estrogen receptor (ER) response genes exhibit abnormal hypermethylation levels that are inversely correlated with their RNA expression. Several tumor suppressor genes, including Kruppel-like factor 11 (KLF11), deleted in lung and esophageal cancer 1 (DLEC1), keratin 19 (KRT19), and death-associated protein kinase 1 (DAPK1) also display higher hypermethylation levels in leiomyomas when compared to adjacent normal tissues. The important role of active DNA demethylation was recently identified with regard to the ten-eleven translocation protein 1 and teneleven translocation protein 3-mediated elevated levels of 5-hydroxymethylcytosine in leiomyoma. In addition, both histone deacetylase and histone methyltransferase are reported to be involved in the biology of leiomyomas. A number of deregulated microRNAs have been identified in leiomyomas, leading to an altered expression of their targets. More recently, the existence of side population (SP) cells with characteristics of tumor-initiating cells have been characterized in leiomyomas. These SP cells exhibit a tumorigenic capacity in immunodeficient mice when exposed to 17b-estradiol and progesterone, giving rise to fibroid-like tissue in vivo. These new findings will likely enhance our understanding of the crucial role epigenetics plays in the pathogenesis of uterine leiomyomas as well as point the way to novel therapeutic options.

Uterine fibroids are the most common gynecologic tumors with a significant medical and financial burden. Several genetic, hormonal, and biological factors have been shown to contribute to the development and growth of fibroid tumors. Of these factors, estrogen is particularly critical since fibroids are considered estrogen dependent because no prepubertal cases have been described in the literature and tumors tend to regress after menopause. Understanding the role of estrogen in fibroids is not only important for understanding the pathobiology of fibroids but also for the development of successful therapeutics. In this review, we discuss the types and structure of estrogen receptors (nuclear and membrane bound, including α and β receptors and G protein-coupled estrogen receptor 1 GPER1). Estrogen-signaling pathways in fibroids include genomic (direct and indirect) and nongenomic including Ras-Raf-MEK (MAPK/Erk Kinase)-mitogen-activated protein kinase (MAPK) and phosphatidylinositide 3-kinase (PI3K)-phosphatidylinositol-3,4,5-trisphosphate (PIP3)-Akt (Protein kinase B)-mammalian target of rapamycin (mTOR) pathways; shortly Ras-Raf-MEK-MAPK and PI3K-PIP3-Akt-mTOR pathways. Several aberrations in estrogen receptors and signaling pathways are implicated in fibroid pathobiology. Current therapeutic and research agents targeting ERs/signaling include gonadotropin-releasing hormone (GnRH) agonists, GnRH antagonists, aromatase inhibitors, selective ER modulators, gene therapy, and others. Future research can identify potential targets for the development of novel treatments. In particular, epigenomics of estrogen activity and individualized (precision) medicine appear to be attractive areas for future research.

Objective To identify and characterize myometrial/fibroid stem cells by specific stem cell markers in human myometrium, and to better understand the stem cell contribution in the development of uterine fibroids. Design Prospective experimental human and animal study. Setting University research laboratory. Patients Women undergoing hysterectomy for treatment of symptomatic uterine fibroids. Animals Female NOD/SCID/IL-2Rγnull mice. Interventions Identification and isolation of stem cells from human fibroids (F) and adjacent myometrium (MyoF) tissues using Stro-1/CD44 specific surface markers. Main Outcome Measures Flow cytometry, semi- quantitative polymerase chain reaction, clonogenicity assays, cell culture, molecular analysis, immunocyto- histochemistry, in vitro differentiation, and xenotransplantation assays. Results Using Stro-1/CD44 surface markers, we were able to isolate stem cells from MyoF and F tissues. The undifferentiated status of isolated cells was confirmed by the expression of ABCG2 transporter, as well as additional stem cell markers OCT4, NANOG and GDB3, and the low expression of steroid receptors ERα and PR-A/PR-B. Mesodermal cell origin was established by the presence of typical mesenchymal markers (CD90, CD105, and CD73) and absence of hematopoietic stem cell markers (CD34, CD45), and confirmed by the ability of these cells to differentiate in vitro into adipocytes, osteocytes and chondrocytes. Finally, their functional capability to form fibroid-like lesions was established in xenotransplantation mouse model. The injected cells labeled with superparamagnetic iron oxide (SPIO) were tracked by both magnetic resonance imaging (MRI) and fluorescence imaging, thus demonstrating the regenerative potential of putative fibroid stem cells in vivo. Conclusion We have demonstrated that Stro-1/CD44 can be used as specific surface markers to enrich a subpopulation of myometrial/fibroids cells, exhibiting key features of stem/progenitor cells. These findings offer a useful tool to better understanding the initiation of uterine fibroids, and may lead the establishment of effective therapeutic options.

Despite the high prevalence and major negative impact of uterine fibroids (UFs) on women's health, their pathogenesis remains largely unknown. While tumor-initiating cells have been previously isolated from UFs, the cell of origin for these tumors in normal myometrium has not been identified. We isolated cells with Stro1/CD44 surface markers from normal myometrium expressing stem cell markers Oct-4/c-kit/nanog that exhibited the properties of myometrial stem/progenitor-like cells (MSCs). Using a murine model for UFs, we showed that the cervix was a hypoxic "niche" and primary site (96%) for fibroid development in these animals. The pool size of these MSCs also responded to environmental cues, contracting with age and expanding in response to developmental environmental exposures that promote fibroid development. Translating these findings to women, the number of MSCs in unaffected human myometrium correlated with risk for developing UFs. Caucasian (CC) women with fibroids had increased numbers of MSCs relative to CC women without fibroids, and African-American (AA) women at highest risk for these tumors had the highest number of MSCs: AA-with fibroids > CC-with fibroids > AA-without fibroids > CC-without fibroids. These data identify Stro1 /CD44 MSCs as MSC/progenitor cell for UFs, and a target for ethnic and environmental factors that increase UF risk. Stem Cells 2017;35:666-678.

More than a decade after their discovery, the knowledge of uterine stem cells and their niches is crystalising into novel therapeutic approaches aiming to treat with cells those conditions that cannot be cured with drugs, particularly the currently incurable uterine pathologies. Additional work and improvements are needed, but the basis has been formed for this therapeutic application of uterine cells.