Rachel W.S. Chan

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 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.

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. Abbreviation LRC: label-retaining cell.

Identification of Cells with Colony-Forming Activity, Self-Renewal Capacity, and Multipotency in Ovarian Endometriosis

Endometriosis, the growth of endometrial tissue outside the uterine cavity, is a common gynecological disorder affecting 10% to 15% of women in their reproductive years. Retrograde menstrual shedding containing endometrial stem/progenitor cells has been postulated to be involved in its pathogenesis. In this study, we identified putative endometriotic stem/progenitor cells by their colony-forming potential, self-renewal capacity, and multipotency. Purified epithelial and stromal cells isolated from ovarian endometriotic cysts formed large and small colony-forming units (CFUs) in clonogenic assay. The colony-forming activity of epithelial and stromal cells was found to differ greatly between autologous endometrium and ovarian endometrioma samples. The large CFUs could propagate more than the small CFUs. The endometriotic epithelial small CFUs expressed epithelial markers (epithelial cell adhesion molecule, cytokeratin, and α6 integrin); only occasional large CFUs expressed α6 integrin. Aside from the expression of fibroblast markers, stromal CFUs also expressed three somatic stem cell markers: sal-like 4, CD133, and Musashi-1. Endometriotic stromal cells derived from large CFUs could differentiate into four mesenchymal lineages when cultured in the respective inducing-media, as determined by histochemical staining and RT-PCR of lineage specific markers. These findings demonstrate that ovarian endometrioma contains a subset of cells displaying somatic stem cell properties.

Role of Label Retaining Cells in Estrogen-Induced Endometrial Regeneration

Candidate stem/progenitor cells have been identified in mouse endometrium as label-retaining cells (LRCs). The role of endometrial stem/progenitor cells in initiating estrogen-stimulated endometrial growth in prepubertal and cycling mice was investigated following a single 17β-estradiol (E2) injection in bromodeoxyuridine (BrdU)-labeled and -chased (LRC), ovariectomised mice. Proliferating (BrdU+/Ki-67+) and mitotic (BrdU+/PH3+) epithelial LRCs were first detected in prepubertal mice 8 hours following E2 treatment, initiating the proliferative response. In contrast, all epithelial LRCs and 16% of epithelial cells in cycling mice proliferated within 2 hours. In cycling mice, 12% of stromal LRCs initiated a proliferative response 8 hours after E2. Proliferating epithelial LRCs and most stromal LRCs (85%) lacked estrogen receptor-α (ESR1). These findings suggest that endometrial epithelial LRCs function as stem/progenitor cells by receiving proliferative signals from neighboring ESR1+ niche cells to initiate the growth of the epithelium during development, while mature epithelial cells may undergo self-replication in cycling endometrium.

Glycodelin-A modulates cytokine production of peripheral blood natural killer cells

Glycodelin-A increased the secretion of interleukin-6, interleukin-13, and granulocyte-macrophage colony-stimulating factor from natural killer cells in the peripheral blood but does not affect their viability, cell death, and cytotoxicity. These data suggest that glycodelin-A contributes to the cytokine shift in early pregnancy.

Up-regulation of endocrine gland-derived vascular endothelial growth factor but not vascular endothelial growth factor in human ectopic endometriotic tissue

OBJECTIVE To study the expression of vascular endothelial growth factor (VEGF), endocrine gland-derived VEGF (EG-VEGF/PK1), and its receptors (PKR1 and PKR2) in eutopic and ectopic endometrial tissues. DESIGN A case-control study. SETTING University reproduction unit. PATIENT(S) Infertile women undergoing diagnostic laparoscopy for tubal patency. INTERVENTION(S) Endometrial and endometriotic tissue sampling from women with and without endometriosis. MAIN OUTCOME MEASURE(S) Quantitative polymerase chain reaction (PCR) analysis of genes in eutopic and ectopic endometrial tissues. The EG-VEGF protein was studied by immunohistochemistry. RESULT(S) In normal endometrium, EG-VEGF messenger RNA (mRNA) expression was 50-fold higher in the secretory than in the proliferative phase, but that of PKR1 was 6-fold higher in the latter than in the former. The PKR2 transcript was detected in the proliferative but not the secretory endometrium. In patients with endometriosis, eutopic endometrial PKR2 transcript level was 4-fold higher in the proliferative than in the secretory phase. No differences in EG-VEGF or PKR1 were found in proliferative versus secretory endometrium in these patients. There were no significant differences in the expression of EG-VEGF in eutopic endometrium of normal women and in those with endometriosis. In the paired laser-captured microdissected eutopic endometrial and ectopic endometriotic samples, a significantly higher EG-VEGF, but not VEGF, transcript level was detected in the ectopic when compared with eutopic samples; whereas the expressions of PKR1 and PKR2 were barely detectable. The H-scoring confirmed that the stroma of endometriotic samples had a significantly higher EG-VEGF protein expression than that in the paired eutopic endometrium. CONCLUSION(S) High levels of EG-VEGF expression may play an important role in angiogenesis in endometriotic tissues.

Label-Retaining Stromal Cells in Mouse Endometrium Awaken for Expansion and Repair After Parturition

Human and mouse endometrium undergo dramatic cellular reorganization during pregnancy and postpartum. Somatic stem cells maintain homeostasis of the tissue by providing a cell reservoir for regeneration. We hypothesized that endometrial cells with quiescent properties (stem/progenitor cells) were involved in the regeneration of the endometrial tissue. Given that stem cells divide infrequently, they can retain the DNA synthesis label [bromodeoxyuridine (BrdU)] after a prolonged chase period. In this study, prepubertal mice were pulsed with BrdU and after a 6-week chase a small population of label-retaining stromal cells (LRSC) was located primarily beneath the luminal epithelium, adjacent to blood vessels, and near the endometrial-myometrial junction. Marker analyses suggested that they were of mesenchymal origin expressing CD44(+), CD90(+), CD140b(+), CD146(+), and Sca-1(+). During pregnancy, nonproliferating LRSC predominately resided at the interimplantation/placental loci of the gestational endometrium. Immediately after parturition, a significant portion of the LRSC underwent proliferation (BrdU(+)/Ki-67(+)) and expressed total and active β-catenin. The β-catenin expression in the LRSC was transiently elevated at postpartum day (PPD) 1. The proliferation of LRSC resulted in a significant decline in the proportion of LRSC in the postpartum uterus. The LRSC returned to dormancy at PPD7, and the percentage of LRSC remained stable thereafter until 11 weeks. This study demonstrated that LRSC can respond efficiently to physiological stimuli upon initiation of uterine involution and return to its quiescent state after postpartum repair.

Co-culture with macrophages enhances the clonogenic and invasion activity of endometriotic stromal cells

To study the effect on endometrial and endometriotic cells after co‐culture with macrophages, using clonogenic, invasion and self‐renewal assays.

Spatial and temporal characterization of endometrial mesenchymal stem-like cells activity during the menstrual cycle

Abstract The human endometrium is a highly dynamic tissue with the ability to cyclically regenerate during the reproductive life. Endometrial mesenchymal stem‐like cells (eMSCs) located throughout the endometrium have shown to functionally contribute to endometrial regeneration. In this study we examine whether the menstrual cycle stage and the location in the endometrial bilayer (superficial and deep portions of the endometrium) has an effect on stem cell activities of eMSCs (CD140b+CD146+ cells). Here we show the percentage and clonogenic ability of eMSCs were constant in the various stages of the menstrual cycle (menstrual, proliferative and secretory). However, eMSCs from the menstrual endometrium underwent significantly more rounds of self‐renewal and enabled a greater total cell output than those from the secretory phase. Significantly more eMSCs were detected in the deeper portion of the endometrium compared to the superficial layer but their clonogenic and self‐renewal activities remained similar. Our findings suggest that eMSCs are activated in the menstrual phase for the cyclical regeneration of the endometrium. HighlightsThe percentages of endometrial mesenchymal‐like stem cells (eMSCs) were constant across the menstrual cycle.Menstruation eMSCs display superior self‐renewal and long‐term proliferative activities.More eMSCs reside in the deeper portion of the endometrium than the superficial layer.

84. Clonogenicity of human endometrial epithelial 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. We hypothesise that epithelial stem cells (ESCs) reside in the endometrial basalis from which new endometrial glands grow with each menstrual cycle. One property of stem cells is their clonogenic ability. The aims of this study were to determine 1) the clonogenic activity of human endometrial epithelial cells, 2) which growth factors support clonogenic activity and 3) compare clonogenic activity of various endometrial states. Endometrial tissue was obtained from 21 women undergoing hysterectomy from proliferative, secretory or inactive states. The entire endometrium was dissociated with collagenase to achieve single cell suspensions. Glandular epithelial cells were selected using Ber-EP4 Dynabeads and cultured at low seeding density (500 cell/cm) for 15 days in serum-containing medium (SM), or serum-free medium (SFM) containing either PDGF-BB, TGF-α, EGF, IGF-1, LIF, FGF-2, HGF or SCF on mouse fibroblast feeder layers. Colonies were stained and the cloning efficiencies were determined. Two types of colonies were observed: small loosely packed colonies (SC) containing large cells ( 4000 cells/colony). In SM, the clonogenicity was 0.20 ± 0.05% (n = 21), 0.08 ± 0.02% for LC and 0.12 ± 0.03% for SC. In SFM, TGF-α, EGF and PDGF strongly supported clonogenicity of epithelial cells in the proliferative phase 0.57 ± 0.20% (n = 7), 0.36 ± 0.14% (n = 8), 0.43 ± 0.14% (n = 7) respectively and in the secretory phase 0.47 ± 0.16% (n = 5), 0.60 ± 0.29% (n = 5), 0.53 ± 0.25% (n = 5). Clonogencity was generally lower in inactive endometrium for all growth factors. HGF, SCF and FGF-2 showed no support for clonogenic activity of epithelial cells from proliferative or inactive endometrium, while LIF and IGF-1 were weakly supportive. Variation between individual samples was high, possibly masking differences in clonogenicity between the endometrial states. This study provides preliminary evidence for the existence of ESCs in the human endometrium, identifies 5 growth factors supporting the clonogenic activity of ESCs and demonstrates some differences in clonogenic activity of epithelial cells from proliferative, secretory and inactive endometrium.