Tetsuo Maruyama

Stem cell-like properties of the endometrial side population: Implication in endometrial regeneration

Background The human endometrium undergoes cyclical regeneration throughout a womans reproductive life. Ectopic implantation of endometrial cells through retrograde menstruation gives rise to endometriotic lesions which affect approximately 10% of reproductive-aged women. The high regenerative capacity of the human endometrium at eutopic and ectopic sites suggests the existence of stem/progenitor cells and a unique angiogenic system. The objective of this study was to isolate and characterize putative endometrial stem/progenitor cells and to address how they might be involved in the physiology of endometrium. Methodology/Principal Findings We found that approximately 2% of the total cells obtained from human endometrium displayed a side population (SP) phenotype, as determined by flow cytometric analysis of Hoechst-stained cells. The endometrial SP (ESP) cells exhibited preferential expression of several endothelial cell markers compared to endometrial main population (EMP) cells. A medium specific for endothelial cell culture enabled ESP cells to proliferate and differentiate into various types of endometrial cells, including glandular epithelial, stromal and endothelial cells in vitro, whereas in the same medium, EMP cells differentiated only into stromal cells. Furthermore, ESP cells, but not EMP cells, reconstituted organized endometrial tissue with well-delineated glandular structures when transplanted under the kidney capsule of severely immunodeficient mice. Notably, ESP cells generated endothelial cells that migrated into the mouse kidney parenchyma and formed mature blood vessels. This potential for in vivo angiogenesis and endometrial cell regeneration was more prominent in the ESP fraction than in the EMP fraction, as the latter mainly gave rise to stromal cells in vivo. Conclusions/Significance These results indicate that putative endometrial stem cells are highly enriched in the ESP cells. These unique characteristics suggest that ESP cells might drive physiological endometrial regeneration and be involved in the pathogenesis of endometriosis.

Side population in human uterine myometrium displays phenotypic and functional characteristics of myometrial stem cells

Over the course of pregnancy, the human uterus undergoes a 500- to 1,000-fold increase in volume and a 24-fold increase in weight. The uterine smooth muscle layer or myometrium is remodeled, and both cell hypertrophy and hyperplasia are evident. The origin of the new smooth muscle cells, however, is unclear. They may arise from existing smooth muscle cells, or they may be the product of stem cell differentiation. This study describes a subset of myometrial cells isolated from nonpregnant human myometrium that represents the myometrial stem cell population. This was characterized as side population of myometrial cells (myoSP) by a distinct Hoechst dye efflux pattern. In contrast to the main population of myometrial cells (myoMP), myoSP resided in quiescence, underexpressed or lacked myometrial cell markers, and could proliferate and eventually differentiate into mature myometrial cells in vitro only under low oxygen concentration. Although myoMP displayed mature myometrial phenotypes before and after in vitro cultivation, only myoSP, not myoMP, generated functional human myometrial tissues efficiently when transplanted into the uteri of severely immunodeficient mice. Finally, myoSP were multipotent and made to differentiate into osteocytes and adipocytes in vitro under the appropriate differentiation-inducing conditions. Thus, myoSP exhibited phenotypic and functional characteristics of myometrial stem cells. Study of myoSP will improve the understanding of myometrial physiology and the pathogenesis of myometrium-derived diseases such as leiomyoma. myoSP may also represent a novel source of biological material that could be used in the reconstruction of not only the human uterus but also other organs as well.

Global gene expression profiling of preimplantation embryos

Preimplantation development is marked by four major events: the transition of maternal transcripts to zygotic transcripts, compaction, the first lineage differentiation into inner cell mass and trophectoderm, and implantation. The scarcity of the materials of preimplantation embryos, both in size (diameter < 100 μm) and in quantity (only a few to tens of oocytes from each ovulation), has hampered molecular analysis of preimplantation embryos. Recent progress in RNA amplification methods and microarray platforms, including genes unique to preimplantation embryos, allow us to apply global gene expression profiling to the study of preimplantation embryos. Our gene expression profiling during preimplantation development revealed the distinctive patterns of maternal RNA degradation and embryonic gene activation, including two major transient waves of de novo transcription. The first wave corresponds to zygotic genome activation (ZGA). The second wave, mid-preimplantation gene activation (MGA), contributes dramatic morphological changes during late preimplantation development. Further expression profiling of embryos treated with inhibitors of transcription or translation revealed that the translation of maternal RNA is required for the initiation of ZGA, suggesting a cascade of gene activation from maternal RNA/protein sets to ZGA gene sets and thence to MGA gene sets. To date, several reports of microarray experiments using mouse and human preimplantation embryos have been published. The identification of a large number of genes and multiple signaling pathways involved at each developmental stage by such global gene expression profiling accelerates understanding of molecular mechanisms underlining totipotency/pluripotency and programs of early mammalian development.

Human uterine stem/progenitor cells: their possible role in uterine physiology and pathology

The human uterus mainly consists of the endometrium and the outer smooth muscle layer termed the myometrium. The uterus harbours the exceptional and remarkable regenerative ability responsible for cyclical regeneration and remodelling throughout the reproductive life. The uterus must swiftly and cooperatively enlarge to hold the growing foetus during pregnancy. Furthermore, the endometrium, in particular the functionalis layer, must also regenerate, differentiate and regress with each menstrual cycle under hormonal control. Endometrial regeneration from the basal layer is thought to contribute to replacement of the functionalis layer followed by its slough off during menses and parturition. These morphological and functional features of human endometrium can be reproduced in murine models in which severely immunodeficient mice are xenotransplanted with dispersed human endometrial cells under the kidney capsule. The uterine myometrium possesses the similar plasticity of the endometrium. This is demonstrated by multiple cycles of pregnancy-induced enlargement and regression after parturition. It is likely that regeneration and remodelling in the female reproductive tract are achieved presumably through endometrial and myometrial stem cell systems. Recent evidence now supports the existence of these stem cell systems in humans. Here, we will review our current understanding of uterine stem/progenitor cells. We also propose a novel hypothetical model in which stem cell activities explain the physiological remodelling and regeneration of the human uterus and the pathogenesis of gynaecological diseases such as endometriosis.

Paracrine activation of WNT/β-catenin pathway in uterine leiomyoma stem cells promotes tumor growth

Significance Stem cells and the ovarian steroids estrogen and progesterone are essential for leiomyoma tissue growth. The underlying mechanisms are unknown, particularly because leiomyoma stem cells are deficient in estrogen and progesterone receptors. Expression of these receptors is much higher in surrounding mature myometrial or leiomyoma smooth muscle cells. Here, we demonstrate that wingless-type (WNT) acts as a paracrine signal from estrogen/progesterone receptor-rich mature cells to activate the canonical β-catenin pathway in leiomyoma stem cells. Our findings suggest a paracrine role for the canonical WNT pathway in the growth of leiomyoma tumor. Uterine leiomyomas are extremely common estrogen and progesterone-dependent tumors of the myometrium and cause irregular uterine bleeding, severe anemia, and recurrent pregnancy loss in 15–30% of reproductive-age women. Each leiomyoma is thought to arise from a single mutated myometrial smooth muscle stem cell. Leiomyoma side-population (LMSP) cells comprising 1% of all tumor cells and displaying tumor-initiating stem cell characteristics are essential for estrogen- and progesterone-dependent in vivo growth of tumors, although they have remarkably lower estrogen/progesterone receptor levels than mature myometrial or leiomyoma cells. However, how estrogen/progesterone regulates the growth of LMSP cells via mature neighboring cells is unknown. Here, we demonstrate a critical paracrine role of the wingless-type (WNT)/β-catenin pathway in estrogen/progesterone-dependent tumorigenesis, involving LMSP and differentiated myometrial or leiomyoma cells. Estrogen/progesterone treatment of mature myometrial cells induced expression of WNT11 and WNT16, which remained constitutively elevated in leiomyoma tissues. In LMSP cells cocultured with mature myometrial cells, estrogen-progesterone selectively induced nuclear translocation of β-catenin and induced transcriptional activity of its heterodimeric partner T-cell factor and their target gene AXIN2, leading to the proliferation of LMSP cells. This effect could be blocked by a WNT antagonist. Ectopic expression of inhibitor of β-catenin and T-cell factor 4 in LMSP cells, but not in mature leiomyoma cells, blocked the estrogen/progesterone-dependent growth of human tumors in vivo. We uncovered a paracrine role of the WNT/β-catenin pathway that enables mature myometrial or leiomyoma cells to send mitogenic signals to neighboring tissue stem cells in response to estrogen and progesterone, leading to the growth of uterine leiomyomas.

Melatonin as a local regulator of human placental function

Abstract:  Melatonin plays a critical role in a variety of mammalian reproductive processes not only acting on the central nervous system but also behaving as a peripheral physiologic regulator. To address the relevance of melatonin to the maintenance of pregnancy at the feto‐maternal interface, we investigated the expression of two types of membrane melatonin receptors, MT1 and MT2, as well as arylalkylamine N‐acetyltransferase (AA‐NAT) and hydroxyindole‐O‐methyltransferase (HIOMT), the two enzymes required for the conversion of serotonin to melatonin, in the human placenta and the effect of melatonin on the release of human chorionic gonadotropin (hCG) from cultured human trophoblast cells. RT‐PCR analysis and DNA sequencing revealed that transcripts of MT1, MT2, AA‐NAT, and HIOMT were present in the first‐trimester human placenta. We also found that melatonin significantly potentiated hCG secretion at optimal concentrations. These results suggest that melatonin may regulate human placental function in a paracrine/autocrine manner, providing evidence for a novel role in human reproduction.

Studies Using an in Vitro Model Show Evidence of Involvement of Epithelial-Mesenchymal Transition of Human Endometrial Epithelial Cells in Human Embryo Implantation

Human embryo implantation is a critical multistep process consisting of embryo apposition/adhesion, followed by penetration and invasion. Through embryo penetration, the endometrial epithelial cell barrier is disrupted and remodeled by an unknown mechanism. We have previously developed an in vitro model for human embryo implantation employing the human choriocarcinoma cell line JAR and the human endometrial adenocarcinoma cell line Ishikawa. Using this model we have shown that stimulation with ovarian steroid hormones (17β-estradiol and progesterone, E2P4) and suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, enhances the attachment and adhesion of JAR spheroids to Ishikawa. In the present study we showed that the attachment and adhesion of JAR spheroids and treatment with E2P4 or SAHA individually induce the epithelial-mesenchymal transition (EMT) in Ishikawa cells. This was evident by up-regulation of N-cadherin and vimentin, a mesenchymal cell marker, and concomitant down-regulation of E-cadherin in Ishikawa cells. Stimulation with E2P4 or SAHA accelerated Ishikawa cell motility, increased JAR spheroid outgrowth, and enhanced the unique redistribution of N-cadherin, which was most prominent in proximity to the adhered spheroids. Moreover, an N-cadherin functional blocking antibody attenuated all events but not JAR spheroid adhesion. These results collectively provide evidence suggesting that E2P4- and implanting embryo-induced EMT of endometrial epithelial cells may play a pivotal role in the subsequent processes of human embryo implantation with functional control of N-cadherin.

The UDP-Glucose Receptor P2RY14 Triggers Innate Mucosal Immunity in the Female Reproductive Tract by Inducing IL-8

Innate mucosal immune responses, including recognition of pathogen-associated molecular patterns through Toll-like receptors, play an important role in preventing infection in the female reproductive tract (FRT). Damaged cells release nucleotides, including ATP and uridine 5′-diphosphoglucose (UDP-glucose), during inflammation and mechanical stress. We show in this report that P2RY14, a membrane receptor for UDP-glucose, is exclusively expressed in the epithelium, but not the stroma, of the FRT in humans and mice. P2RY14 and several proinflammatory cytokines, such as IL-8, are up-regulated in the endometria of patients with pelvic inflammatory disease. UDP-glucose stimulated IL-8 production via P2RY14 in human endometrial epithelial cells but not stromal cells. Furthermore, UDP-glucose enhanced neutrophil chemotaxis in the presence of a human endometrial epithelial cell line in an IL-8-dependent manner. Administration of UDP-glucose into the mouse uterus induced expression of macrophage inflammatory protein-2 and keratinocyte-derived cytokine, two murine chemokines that are functional homologues of IL-8, and augmented endometrial neutrophil recruitment. Reduced expression of P2RY14 by small interfering RNA gene silencing attenuated LPS- or UDP-glucose-induced leukocytosis in the mouse uterus. These results suggest that UDP-glucose and its receptor P2RY14 are key front line players able to trigger innate mucosal immune responses in the FRT bypassing the recognition of pathogen-associated molecular patterns. Our findings would significantly impact the strategic design of therapies to modulate mucosal immunity by targeting P2RY14.

Subsequent pregnancy outcomes in recurrent miscarriage patients with a paternal or maternal carrier of a structural chromosome rearrangement

AbstractInformation concerning the prognosis of subsequent pregnancies in patients with reciprocal translocations is limited. This study was performed to determine the percentage success rate with first pregnancies after ascertainment of a carrier status. A total of 2,382 couples with a history of two or more consecutive miscarriages were studied in multicenters. The prevalence of an abnormal chromosome in either partner was examined, and subsequent success rates were compared between cases with and without an abnormal karyotype in either partner. A total of 129 couples (5.4%) had an abnormal karyotype in one partner excluding inversion 9 in 44 men and in 85 women. Thus, 2,253 couples had a normal karyotype in both partner. Eighty-five (3.6%) had translocations, 13 being Robertsonian translocations. Twenty-nine of the 46 cases (63.0%) who became pregnant with reciprocal translocations in either partner experienced a live birth with natural conception. In contrast, 950 of 1,207 cases (78.7%) with normal chromosomes had successful live births, the difference being significant (P = 0.019). No infant with an unbalanced translocation was found in 29 cases of successful pregnancy following recurrent miscarriage. Pregnancy prognosis was worsened with either maternal or paternal reciprocal translocations. Explanation of the success rate with natural conception should be provided before the subsequent pregnancy after ascertainment of carrier status.

Stem Cell-Like Differentiation Potentials of Endometrial Side Population Cells as Revealed by a Newly Developed In Vivo Endometrial Stem Cell Assay

Background Endometrial stem/progenitor cells contribute to the cyclical regeneration of human endometrium throughout a womans reproductive life. Although the candidate cell populations have been extensively studied, no consensus exists regarding which endometrial population represents the stem/progenitor cell fraction in terms of in vivo stem cell activity. We have previously reported that human endometrial side population cells (ESP), but not endometrial main population cells (EMP), exhibit stem cell-like properties, including in vivo reconstitution of endometrium-like tissues when xenotransplanted into immunodeficient mice. The reconstitution efficiency, however, was low presumably because ESP cells alone could not provide a sufficient microenvironment (niche) to support their stem cell activity. The objective of this study was to establish a novel in vivo endometrial stem cell assay employing cell tracking and tissue reconstitution systems and to examine the stem cell properties of ESP through use of this assay. Methodology/Principal Findings ESP and EMP cells isolated from whole endometrial cells were infected with lentivirus to express tandem Tomato (TdTom), a red fluorescent protein. They were mixed with unlabeled whole endometrial cells and then transplanted under the kidney capsule of ovariectomized immunodeficient mice. These mice were treated with estradiol and progesterone for eight weeks and nephrectomized. All of the grafts reconstituted endometrium-like tissues under the kidney capsules. Immunofluorescence revealed that TdTom-positive cells were significantly more abundant in the glandular, stromal, and endothelial cells of the reconstituted endometrium in mice transplanted with TdTom-labeled ESP cells than those with TdTom-labeled EMP cells. Conclusions/Significance We have established a novel in vivo endometrial stem cell assay in which multi-potential differentiation can be identified through cell tracking during in vivo endometrial tissue reconstitution. Using this assay, we demonstrated that ESP cells differentiated into multiple endometrial lineages in the niche provided by whole endometrial cells, indicating that ESP cells are genuine endometrial stem/progenitor cells.