Endometrial stem/progenitor cells in menstrual blood and peritoneal fluid of women with and without endometriosis.

RESEARCH QUESTION: Are endometrial stem/progenitor cells shed into uterine menstrual blood (UMB) and the peritoneal cavity in women with and without endometriosis during menstruation? DESIGN: Women with (n = 32) and without endometriosis (n = 29) at laparoscopy (total 61), carried out during the menstrual (n = 41) and non-menstrual phase (n = 20) were recruited. The UMB, peritoneal fluid and peripheral blood were analysed by clonogenicity assay and flow cytometry to quantify the concentrations of endometrial clonogenic cells, SUSD2+ mesenchymal stem cells (eMSC) and N-cadherin+ epithelial progenitor cells (eEPC). RESULTS: Clonogenic endometrial cells, eMSC and eEPC were found in most UMB samples at similar concentrations in women with and without endometriosis. In contrast, 62.5% of women with endometriosis and 75.0% without (controls) had clonogenic cells in peritoneal fluid samples during menses. The eMSC were present in the peritoneal fluid of 76.9% of women with endometriosis and 44.4% without, and eEPC were found in the peritoneal fluid of 60.0% of women with and 25.0% without endometriosis during menses. Median clonogenic, eMSC and eEPC concentrations in peritoneal fluid were not significantly different between groups. More clonogenic cells persisted beyond the menstrual phase in the peritoneal fluid of women with endometriosis (menstrual 119/ml [0-1360/ml] versus non-menstrual 8.5/ml [0-387/ml]; P = 0.277) compared with controls (menstrual 76.5/ml [1-1378/ml] versus non-menstrual 0/ml [0-14/ml]; P = 0.0362). No clonogenic endometrial cells were found in peripheral blood. CONCLUSIONS: Clonogenic endometrial cells, SUSD2+ eMSC and N-cadherin+ eEPC are present in UMB and the peritoneal fluid of women with and without endometriosis. Further study of the function of these cells may shed light on the cellular origins of endometriosis.

Endometrial stem/progenitor cells: the first 10 years.

BACKGROUND: The existence of stem/progenitor cells in the endometrium was postulated many years ago, but the first functional evidence was only published in 2004. The identification of rare epithelial and stromal populations of clonogenic cells in human endometrium has opened an active area of research on endometrial stem/progenitor cells in the subsequent 10 years. METHODS: The published literature was searched using the PubMed database with the search terms 'endometrial stem cells and menstrual blood stem cells' until December 2014. RESULTS: Endometrial epithelial stem/progenitor cells have been identified as clonogenic cells in human and as label-retaining or CD44(+) cells in mouse endometrium, but their characterization has been modest. In contrast, endometrial mesenchymal stem/stromal cells (MSCs) have been well characterized and show similar properties to bone marrow MSCs. Specific markers for their enrichment have been identified, CD146(+)PDGFRß(+) (platelet-derived growth factor receptor beta) and SUSD2(+) (sushi domain containing-2), which detected their perivascular location and likely pericyte identity in endometrial basalis and functionalis vessels. Transcriptomics and secretomics of SUSD2(+) cells confirm their perivascular phenotype. Stromal fibroblasts cultured from endometrial tissue or menstrual blood also have some MSC characteristics and demonstrate broad multilineage differentiation potential for mesodermal, endodermal and ectodermal lineages, indicating their plasticity. Side population (SP) cells are a mixed population, although predominantly vascular cells, which exhibit adult stem cell properties, including tissue reconstitution. There is some evidence that bone marrow cells contribute a small population of endometrial epithelial and stromal cells. The discovery of specific markers for endometrial stem/progenitor cells has enabled the examination of their role in endometrial proliferative disorders, including endometriosis, adenomyosis and Asherman's syndrome. Endometrial MSCs (eMSCs) and menstrual blood stromal fibroblasts are an attractive source of MSCs for regenerative medicine because of their relative ease of acquisition with minimal morbidity. Their homologous and non-homologous use as autologous and allogeneic cells for therapeutic purposes is currently being assessed in preclinical animal models of pelvic organ prolapse and phase I/II clinical trials for cardiac failure. eMSCs and stromal fibroblasts also exhibit non-stem cell-associated immunomodulatory and anti-inflammatory properties, further emphasizing their desirable properties for cell-based therapies. CONCLUSIONS: Much has been learnt about endometrial stem/progenitor cells in the 10 years since their discovery, although several unresolved issues remain. These include rationalizing the terminology and diagnostic characteristics used for distinguishing perivascular stem/progenitor cells from stromal fibroblasts, which also have considerable differentiation potential. The hierarchical relationship between clonogenic epithelial progenitor cells, endometrial and decidual SP cells, CD146(+)PDGFR-ß(+) and SUSD2(+) cells and menstrual blood stromal fibroblasts still needs to be resolved. Developing more genetic animal models for investigating the role of endometrial stem/progenitor cells in endometrial disorders is required, as well as elucidating which bone marrow cells contribute to endometrial tissue. Deep sequencing and epigenetic profiling of enriched populations of endometrial stem/progenitor cells and their differentiated progeny at the population and single-cell level will shed new light on the regulation and function of endometrial stem/progenitor cells.

Isolation and culture of epithelial progenitors and mesenchymal stem cells from human endometrium.

Human endometrium is a highly regenerative tissue undergoing more than 400 cycles of growth, differentiation, and shedding during a woman's reproductive years. Endometrial regeneration is likely mediated by adult stem/progenitor cells. This study investigated key stem cell properties of individual clonogenic epithelial and stromal cells obtained from human endometrium. Single-cell suspensions of endometrial epithelial or stromal cells were obtained from hysterectomy tissues from 15 women experiencing normal menstrual cycles, and were cultured at clonal density (10 cells/cm(2)) or limiting dilution. The adult stem cell properties-self-renewal, high proliferative potential, and differentiation of single epithelial and stromal cells-were assessed by harvesting individual colonies and undertaking serial clonal culture, serial passaging, and culture in differentiation-induction media, respectively. Lineage differentiation markers were examined by RT-PCR, immunocytochemistry, and flow cytometry. Rare single human endometrial EpCAM(+) epithelial cells and EpCAM(-) stromal cells demonstrated self-renewal by serially cloning >3 times and underwent >30 population doublings over 4 mo in culture. Clonally derived epithelial cells differentiated into cytokeratin(+) gland-like structures in three dimensional culture. Single stromal cells were multipotent, as their progeny differentiated into smooth muscle cells, adipocytes, chondrocytes, and osteoblasts. Stromal clones expressed mesenchymal stem cell (MSC) markers ITGB1 (CD29), CD44, NT5E (CD73), THY1 (CD90), ENG (CD105), PDGFRB (CD140B), MCAM (CD146) but not endothelial or hemopoietic markers PECAM1 (CD31), CD34, PTPRC (CD45). Adult human endometrium contains rare epithelial progenitors and MSCs, likely responsible for its immense regenerative capacity, which may also have critical roles in the development of endometriosis and endometrial cancer. Human endometrium may provide a readily available source of MSCs for cell-based therapies.

Hormone and growth factor signaling in endometrial renewal: role of stem/progenitor cells.

The human endometrium is a dynamic remodeling tissue undergoing more than 400 cycles of regeneration, differentiation and shedding during a woman's reproductive years. The co-ordinated and sequential actions of estrogen and progesterone direct these major remodeling events preparing a receptive endometrium for blastocyst implantation on a monthly basis. Adult stem/progenitor cells are likely responsible for endometrial regeneration. Functional approaches have been used to identify candidate endometrial stem/progenitor cells, as there are no specific stem cell markers. Rare populations of human endometrial epithelial and stromal colony-forming cells/units (CFU) and side population (SP) cells have been identified. Several growth factors are required for CFU activity: epidermal growth factor (EGF), transforming growth factor alpha (TGFalpha) and platelet-derived growth factor BB (PDGF-BB) for both epithelial and stromal CFU, and basic fibroblast growth factor (bFGF) for stromal, but not epithelial CFU. A sub-population of human endometrial stromal cells with mesenchymal stem cell properties of CFU activity and multilineage (fat, muscle, cartilage and bone) differentiation have been isolated by their co-expression of CD146 and PDGF-receptor beta. Candidate epithelial and stromal stem/progenitor cells have been identified in mouse endometrium as rare label retaining cells (LRCs) in the luminal epithelium and as perivascular cells at the endometrial-myometrial junction, respectively. While epithelial and most stromal LRC do not express estrogen receptor alpha (Esr1), they rapidly proliferate on estrogen stimulation, most likely mediated by neighbouring Esr1-expressing niche cells. It is likely that these newly identified endometrial stem/progenitor cells may play key roles in the development of gynecological diseases associated with abnormal endometrial proliferation such as endometriosis and endometrial cancer.

Endometrial stem cells.

PURPOSE OF REVIEW: The human endometrium is a dynamic tissue, which undergoes cycles of growth and regression with each menstrual cycle. Endometrial regeneration also follows parturition and extensive resection and occurs in postmenopausal women taking estrogen replacement therapy. It is likely that adult stem/progenitor cells are responsible for this remarkable regenerative capacity. This review discusses the first published evidence for the existence of endometrial stem/progenitor cells in human and mouse endometrium. RECENT FINDINGS: Functional approaches have been used to identify candidate endometrial epithelial and stromal stem/progenitor cells, due to lack of known specific endometrial stem cell markers. Rare clonogenic cells and side population cells have been identified in human endometrial cell populations. In mouse endometrium, rare label-retaining cells have also been identified. The ability of transplanted human endometrial cells to grow endometrial tissue in animal hosts also suggests the presence of stem/progenitor cells. SUMMARY: These initial studies providing the first functional evidence for epithelial and stromal stem/progenitor cells in human and mouse endometrium lay the groundwork for further studies to characterize their stem cell properties. They also provide the impetus to discover specific markers that will enable their prospective isolation and allow their location in normal and pathological endometrium to be determined.

Clonogenicity of human endometrial epithelial and stromal cells.

The human endometrium regenerates from the lower basalis layer, a germinal compartment that persists after menstruation to give rise to the new upper functionalis layer. Because adult stem cells are present in tissues that undergo regeneration, we hypothesized that human endometrium contains small populations of epithelial and stromal stem cells responsible for cyclical regeneration of endometrial glands and stroma and that these cells would exhibit clonogenicity, a stem-cell property. The aims of this study were to determine 1) the clonogenic activity of human endometrial epithelial and stromal cells, 2) which growth factors support this clonogenic activity, and 3) determine the cellular phenotypes of the clones. Endometrial tissue was obtained from women undergoing hysterectomy. Purified single- cell suspensions of epithelial and stromal cells were cultured at cloning density (300-500/cm(2)) in serum medium or in serum- free medium supplemented with one of eight growth factors. Small numbers of epithelial (0.22%) and stromal cells (1.25%) initiated colonies in serum-containing medium. The majority of colonies were small, containing large, loosely arranged cells, and 37% of epithelial and 1 in 60 of stromal colonies were classified as large, comprising small, densely packed cells. In serum-free medium, transforming growth factor-alpha (TGF alpha), epidermal growth factor (EGF), platelet-derived growth factor-BB (PDGF-BB) strongly supported clonogenicity of epithelial cells, while leukemia-inhibitory factor (LIF), hepatocyte growth factor (HGF), stem-cell factor (SCF), insulin-like growth factor-I (IGF- I) were weakly supportive, and basic fibroblast growth factor (bFGF) was without effect. TGF alpha, EGF, PDGF-BB, and bFGF supported stromal cell clonogenicity, while HGF, SCF, LIF, and IGF- I were without effect. Small epithelial colonies expressed three epithelial markers but not stromal markers; however, large epithelial colonies showed little reactivity for all markers except alpha(6)-integrin. All stromal colonies contained fibroblasts, expressing stromal markers, and in some colonies, myofibroblasts were also identified. This analysis of human endometrium has demonstrated the presence of rare clonogenic epithelial and stromal cells with high proliferative potential, providing the first evidence for the existence of putative endometrial epithelial and stromal stem cells.