Caroline E. Gargett

Clonogenicity of Human Endometrial Epithelial and Stromal Cells

Abstract 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/cm2) 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-α (TGFα), 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. TGFα, 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 α6-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.

Isolation and Culture of Epithelial Progenitors and Mesenchymal Stem Cells from Human Endometrium

Abstract Human endometrium is a highly regenerative tissue undergoing more than 400 cycles of growth, differentiation, and shedding during a womans 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/cm2) 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.

Adult stem cells in the endometrium

Rare cells with adult stem cell activity were recently discovered in human endometrium. Endometrial stem/progenitor cell candidates include epithelial, mesenchymal and endothelial cells, and all may contribute to the rapid endometrial regeneration following menstruation, rather than a single candidate. Endometrial mesenchymal stem-like cells (eMSC) are prospectively isolated as CD146(+)PDGF-Rβ(+) cells and are found in both basalis and functionalis as perivascular cells. Epithelial progenitor cells are detected in colony forming unit assays but their identity awaits elucidation. They are postulated to reside in the basalis in gland bases. Endometrial stem/progenitor cells may be derived from endogenous stem cells, but emerging evidence suggests a bone marrow contribution. Endometrial endothelial progenitor cells are detected as side population cells, which express several endothelial cell markers and differentiate into endometrial glandular epithelial, stromal and endothelial cells. Investigating endometrial stem cell biology is crucial to understanding normal endometrial physiology and to determine their roles in endometrial proliferative diseases. The nature of endometriosis suggests that initiation of ectopic endometrial lesions involves endometrial stem/progenitor cells, a notion compatible with Sampsons retrograde menstruation theory and supported by the demonstration of eMSC in menstrual blood. Evidence of cancer stem cells (CSC) in endometrial cancer indicates that new avenues for developing therapeutic options targeting CSC may become available. We provide an overview of the accumulating evidence for endometrial stem/progenitor cells and their possible roles in endometrial proliferative disorders, and discuss the unresolved issues.

Identification of Label‐Retaining Cells in Mouse Endometrium

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.

The isoquinoline derivative KN-62 a potent antagonist of the P2Z-receptor of human lymphocytes.

Extracellular adenosine 5′‐triphosphate (ATP) is an agonist for a P2Z receptor on human lymphocytes which mediates opening of a cation‐selective ion channel, activation of phospholipase D and shedding of the adhesion molecule, L‐selectin, from the cell surface. The isoquinolinesulphonamides, KN‐62, (1‐[N, O‐bis(5‐isoquinolinesulphonyl)‐N‐methyl‐L‐tyrosyl]‐ 4‐phenylpiperazine), a selective antagonist of Ca2+/calmodulin‐dependent protein kinase II (CaMKII), and KN‐04, (N‐[1‐[N‐methyl‐p‐(5 isoquinoline sulphonyl)benzyl]‐2‐(4 phenylpiperazine)ethyl]‐5‐isoquinolinesulphonamide) an inactive analogue, were used to investigate the possible role of CaMKII in these diverse effects of extracellular ATP. KN‐62 potently antagonized ATP‐stimulated Ba2+ influx into fura‐2 loaded human lymphocytes with an IC50 of 12.7±1.5 nm (n=3) and complete inhibition of the flux at a concentration of 500 nm. Similarly, KN‐62 inhibited ATP‐stimulated ethidium+ uptake, measured by time resolved flow cytometry, with an IC50 of 13.1±2.6 nm (n=4) and complete inhibition of the flux at 500 nm. KN‐04 antagonized ATP‐stimulated Ba2+ influx with an IC50 of 17.3±2.7 nm (n=3). Similarly, KN‐04 inhibited ATP‐stimulated ethidium+ uptake with an IC50 of 37.2±8.9 nm (n=4). Both fluxes were completely inhibited at 500 nm KN‐04. ATP‐stimulated phospholipase D activity, measured in [3H]‐oleic acid‐labelled lymphocytes by the transphosphatidylation reaction, was antagonized by KN‐62 and KN‐04, with 50% inhibition at 5.9±1.2 and 9.7±2.8 nm (n=3), respectively. Both KN‐62 and KN‐04 inhibited ATP‐stimulated shedding of L‐selectin, measured by flow cytometric analysis of cell surface L‐selectin, with IC50 values of 31.5±4.5 and 78.7±10.8 nm (n=3), respectively. Neither of the isoquinolinesulphonamides (500 nm) inhibited phorbol ester‐ or ionomycin‐stimulated phospholipase D activity or phorbol ester‐induced shedding of L‐selectin. The inhibitory effect of KN‐62 or KN‐04 on P2Z‐mediated responses was slow in onset (5 min) and only partially reversed by washing the cells. Both KN‐62 and KN‐04 (at 500 nm) had no effect on uridine 5′‐triphosphate (UTP)‐stimulated Ca2+ transients in fura‐2 loaded human neutrophils, a response which is mediated by the P2Y2 receptor. Thus, KN‐62 and KN‐04 are potent antagonists of the P2Z receptor and at nanomolar concentrations inhibit all known responses mediated by the P2Z receptor of human lymphocytes. In contrast, KN‐62 and KN‐04 had no effect on responses mediated by the P2Y2 receptor of neutrophils. Moreover, since KN‐62 and KN‐04 are almost equipotent, the P2Z‐mediated responses do not involve CaMKII, but indicate that the isoquinolinesulphonamides are potent and direct inhibitors of the P2Z‐receptor.

Priorities for Endometriosis Research: Recommendations From an International Consensus Workshop

Endometriosis is an estrogen-dependent disorder where endometrial tissue forms lesions outside the uterus. Endometriosis affects an estimated 10% of women in the reproductive-age group, rising to 30% to 50% in patients with infertility and/or pain, with significant impact on their physical, mental, and social well-being. There is no known cure, and most current medical treatments are not suitable long term due to their side-effect profiles. Endometriosis has an estimated annual cost in the United States of

Identification of surface markers for prospective isolation of human endometrial stromal colony-forming cells

18.8 to

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

22 billion (2002 figures). Although endometriosis was first described more than 100 years ago, current knowledge of its pathogenesis, spontaneous evolution, and the pathophysiology of the related infertility and pelvic pain, remain unclear. A consensus workshop was convened following the 10th World Congress on Endometriosis to establish recommendations for priorities in endometriosis research. One major issue identified as impacting on the capacity to undertake endometriosis research is the need for multidisciplinary expertise. A total of 25 recommendations for research have been developed, grouped under 5 subheadings: (1) diagnosis, (2) classification and prognosis, (3) treatment and outcome, (4) epidemiology, and (5) pathophysiology. Endometriosis research is underfunded relative to other diseases with high health care burdens. This may be due to the practical difficulties of developing competitive research proposals on a complex and poorly understood disease, which affects only women. By producing this consensus international research priorities statement it is the hope of the workshop participants that researchers will be encouraged to develop new interdisciplinary research proposals that will attract increased funding support for work on endometriosis.

A novel marker of human endometrial mesenchymal stem-like cells

BACKGROUND Human endometrium is a highly regenerative tissue. We hypothesized that the source of endometrial stromal and vascular regeneration is a resident stromal stem/progenitor cell population. Putative human endometrial stromal stem/progenitor cells have been identified using clonal assays, a retrospective functional stem cell assay. Therefore, the aim of this study was to screen potential stem cell markers for the prospective isolation of human endometrial stromal stem/progenitor cells and to determine their capacity to identify colony-forming stromal cells. METHODS Single-cell suspensions of human endometrial stromal cells were sorted using fluorescence-activated cell sorting into positive and negative populations based on STRO-1, CD133, CD90 or CD146 expression for clonal assays. All markers were immunolocalized in human endometrium. RESULTS Small populations (2-9%) of human endometrial stromal cells expressed each of the markers. Only CD146(+) cells were enriched for colony-forming cells, and CD90(hi) cells showed a trend for greater enrichment compared with CD90(lo) cells. STRO-1 and CD146 were localized to perivascular cells of the endometrium. CD90 was strongly expressed by functionalis stroma and perivascular cells, but only weakly expressed in the basalis stroma. CD133 was expressed by epithelial cells of the endometrium, rather than by stroma or perivascular cells. CONCLUSIONS This study identified CD146 as a marker of colony-forming human endometrial stromal cells supporting the concept that human endometrium contains a population of candidate stromal stem/progenitor cells.

Endometrial stem cells

The human endometrium is a dynamic remodeling tissue undergoing more than 400 cycles of regeneration, differentiation and shedding during a womans 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.