Endometrial stem/progenitor cells

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1 bs_bs_banner doi: /jog J. Obstet. Gynaecol. Res. Vol. 40, No. 9: , September 2014 Endometrial stem/progenitor cells Tetsuo Maruyama Department of Obstetrics and Gynecology, School of Medicine, Keio University, Tokyo, Japan Abstract Human endometrium regenerates and regresses with each menstrual cycle under hormonal control throughout a woman s reproductive life. The cyclical regeneration and remodeling potentials allude to the existence of stem/progenitor cells in the endometrium. There is increasing evidence that human endometrium contains small numbers of stem-like cells capable of self-renewal, multiple differentiation and tissue reconstitution. Although the precise identity of endometrial stem/progenitor cells remains elusive, these cells are thought to play pivotal role(s) in the physiological remodeling and regeneration of the human endometrium and also in the pathogenesis of endometrium-associated diseases, such as endometriosis. Key words: endometrial regeneration, endometriosis, side population, stem/progenitor cells. Introduction Tissue (-specific) stem cells, also known as adult stem cells or somatic stem cells, are undifferentiated cells that are retained throughout the body after the completion of embryonic development. 1,2 They are able to selfrenew indefinitely and multiply by asymmetric cell division, eventually giving rise to all the cell types of the organ/tissue from which they originate through terminal differentiation. Tissue stem cells play critical role(s) in the maintenance of organ structure and function in that they are required for the replacement of dying cells within organs and for tissue regeneration following physiological and pathological injuries. 1,2 The human endometrium undergoes cyclic repetitive changes, including proliferation, differentiation, tissue breakdown, and shedding (menstruation) more than 400 times throughout a woman s reproductive life. 3 Based on these features, one or more endometrium-specific tissue stem cell systems have long been believed to be critical for the regeneration and remodeling properties of the endometrium. 4 6 This article reviews current studies on endometrial stem/progenitor cells, particularly focusing on side population cells isolated from human endometrium, and discusses their possible roles in endometrial physiology and also in the pathogenesis of endometriosis. Regeneration of Human Endometrium Current paradigm and confronting implications in human endometrial regeneration Human endometrial tissue is structurally and functionally divided into two major compartments. The upper two-thirds (functional layer) are shed during menstruation in response to progesterone withdrawal and regenerate in the subsequent cycle. 7 The lower basal layer comprises the bases of the endometrial glands surrounded by dense stroma. It remains after menstruation and is believed to provide cells for generating a new functional layer each month. 7 The restructuring of the functional layer is critical for the development of a tissue ready for implantation or for menstruation. 3 Post-menstrual regeneration of the human endometrium is dependent upon estrogen. In the basal layer, transforming growth factor-α, epidermal growth factor and platelet-derived growth factor (PDGF) provide mitogenic stimuli to epithelial cells. 8 On day 2 of menstruation, epithelial cells begin to grow at the stumps of the glands. After 4 days, most of the surface is covered Reprint request to: Dr Tetsuo Maruyama, Department of Obstetrics and Gynecology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo , Japan. tetsuo@a5.keio.jp 2014 The Author 2015

2 T. Maruyama by a layer of epithelium that initiated growth at the edges of cone-shaped glands. By the end of the 6th day, the process of epithelialization is virtually complete. 9 The regrowth of endometrial vessels is critical in animals possessing spiral arterioles. A diverse assemblage of signaling molecules and receptors (including fibroblast growth factors, angiogenin, angiopoietins, the vascular endothelial growth factor family and the ephrins and their cognate receptors) modulate angiogenesis and the remodeling of the vessel network. 10 Recent histological studies, however, have reappraised the role of the glandular epithelium generated from the basal layer in resurfacing the menstrual endometrium Endometrial surface epithelial regeneration was demonstrated to occur as a consequence of cellular differentiation from stromal cells, but not direct extension from the residual basal epithelial glands. 11,12 Other studies suggested that new surface epithelium might engulf remnants of the shedding functional layer during menstruation. 14 In support of this, gene profiling of laser capture microdissected epithelium and stroma of menstrual endometrium revealed the biosynthesis of extracellular matrix together with high expression of adhesion proteins in the fragments from the degraded functionalis, suggesting the potential of the lysed stroma for ectopic and eutopic implantation. 15 Endometrium can also regenerate after parturition, operative removal and in postmenopausal women taking estrogen replacement therapy. 6 Although mice do not menstruate, endometrial regeneration takes place after parturition in mice as it does in humans. Cell fate mapping studies using genetically engineered mice demonstrated that the stromal mesenchymal-toepithelial transition (MET) at least in part contributes to endometrial epithelial tissue regeneration following parturition. 16,17 These findings are consistent with previous findings from humans showing that small stromal cells differentiate into or generate epithelial cells during re-epithelialization after menstruation. 11,12 Experimental model for endometrial regeneration and angiogenesis Differences between humans and rodents complicate the studies of the regenerative and angiogenic processes in the endometrium. Thus, data obtained in rodents might not apply to human physiology. Using in vitro approaches to modeling physiologic processes might not accurately reflect the normal processes of tissue shedding and regeneration. To circumvent these limitations, we developed a novel mouse model for human endometrial regeneration and regression that depends upon the use of null severely immunodeficient NOD/SCID/γ c (NOG) mice. 18 We transplanted beneath the kidney capsule of NOG mice a limited number of human singledispersed endometrial cells (SDEC) that included epithelial, stromal, endothelial and immune cells. We observed regeneration of functional endometrial tissue when treatment with estrogen was included. 18 The artificial endometrium mimics normal endometrium, displaying hormone-dependent processes. They included estrogen-dependent cellular proliferation and progesterone-induced differentiation. Moreover, we observed that tissue breakdown occurred following withdrawal of progesterone, processes analogous to menstruation. In this endometrial model, the mouse kidney parenchyma was invaded by human blood vessels. 18 In fact, the vasculature from the human tissue formed chimeric vessels with the host endothelium, resulting in a functional circulatory system. 18 This observation illustrates the remarkable regenerative capacity of endometrial cells and further suggests the existence of endometrial stem cells and a unique system of angiogenesis. 18 Bioluminescent imaging (BLI) can be used to track tumor, hematopoietic and neural cells. 19,20 We used the technique to analyze endometrial model tissues formed by genetically engineered SDEC. 18 We transplanted SDEC beneath the kidney capsule (on the dorsal side) of ovariectomized NOG mice. The SDEC had been infected with a lentivirus carrying a variant luciferase reporter gene. Using in vivo BLI, we successfully assessed the estrogen-dependent growth, progesterone withdrawal-induced breakdown, and antiestrogen-mediated growth arrest of the tissue derived from lentiviral-engineered cells in a non-invasive and real-time manner. 18 Thus, by combining transplantation of SDEC with lentivirus-mediated cell engineering, we created an animal model suitable for the study of endometrial physiology/ pathophysiology. The model may also be appropriate for analysis of gene expression in endometrial disorders, drug testing and various types of neoplastic disease. Candidate Human Endometrial Stem Cells It has long been believed that endometrial regeneration is mediated by endometrial stem/progenitor cells. 4,5, The Author

3 Endometrial stem/progenitor cells The evidence supporting this hypothesis is based on clinical observations, studies of cell proliferation and analyses of gland monoclonality. 6,22 Loss of the endometrial functionalis layer at menstruation and its subsequent regeneration from the endometrial basalis indicates that cell proliferation in the two layers is significantly different. 23 Furthermore, the results suggest that endometrial stem cells are probably present in the basalis. 4,5,21 Several laboratories, including ours, have used a variety of methods to identify, isolate and/or characterized putative endometrial stem/progenitor cells capable of pluripotent differentiation. 24,25 These studies are summarized below. Clonogenic cells Clonogenicity at extremely low seeding densities is a time-honored method of conducting in vitro characterization of stem/progenitor cells. 6,26 Clonogenicity has been demonstrated for many adult stem cell types. 6,26 Chan et al. were the first to identify clonogenic human endometrial epithelial and stromal cells in vitro. 8 Human endometrial clonogenic cells have multilineage capacity, that is, they can differentiate in vitro into mesenchymal lineages, including adipocytes, smooth muscle cells, chondrocytes and osteoblasts. 27 These results suggest that rare endometrial clonogenic cells have stem-cell-like properties, including self-renewal and multipotency. These properties likely explain the cyclical and regenerative capacity of human endometrium. Side population Side population (SP) cells have a characteristic ability to pump specific molecules into the extracellular medium via the ATP-binding cassette transporter G2 (ABCG2). 28,29 For example, after treatment with the DNA-binding dye Hoechst 33342, the concentration of the dye in SP cells is lower than in non-sp cells. Dual wavelength flow cytometric analysis shows that SP cells constitute a low proportion of tissue. SP cells are present in a range of adult tissues, suggesting that this phenotype might be common to adult stem cells. 29 SP cells are found in endometrial cell populations Endometrial SP (ESP) cells exhibit preferential expression of several endothelial cell markers compared to endometrial non-sp (endometrial main population [EMP]) cells. 32 In vitro, ESP cells can proliferate and differentiate into glandular epithelial, stromal and endothelial endometrial cells. In the same medium, EMP cells can only differentiate into stromal cells. 32 When transplanted under the kidney capsule of NOG mice, ESP cells (but not EMP cells) generate endometrial tissue with well-delineated glandular structures. 32 Importantly, ESP cells generate endothelial cells that migrate into the mouse kidney parenchyma, forming mature blood vessels. 32 The capacity for in vivo angiogenesis and endometrial cell regeneration was greater in ESP than EMP cells. 32 Endometrial ABCG2 + cells (similar to ESP cells) are located in the vascular wall of endometrial small vessels in the functional and basal layers. They have properties of endothelial progenitor-like cells. 32 It has been hypothesized that human ESP cells might trigger neovascularization and differentiation into various cell components of the endometrium. 32 Given the fact that ESP cells are found in the functional layer, 32 this layer might contribute to renewal of the endometrium. 13 The nature of stem cells appears to depend on the method used to isolate them. Furthermore, in vitro experiments, such as clonogenic and differentiation assays, do not necessarily provide an appropriate microenvironment (stem cell niche) required for maintenance of endometrial stem cell properties. Thus, the characteristics of stem cells often vary between laboratories. 24,25 For example, in several reports, ESP cells have differed in their surface marker expression, clonal efficiency, preference for culture conditions and localization in the eutopic normal endometrium. 30,32 34 Thus, it is not clear whether there are multiple types of stem cells in the human endometrium and, if so, whether they have distinct roles. To achieve a consensus definition of endometrial stem cells, we utilized our model of endometrial regeneration described above in combination with the use of cell tracking. 35 ESP and EMP cells were infected with lentivirus carrying tandem Tomato (TdTom), a red fluorescent protein, together with red-emitting firefly luciferase (Figs 1,2). They were mixed with unlabeled whole endometrial cells possibly capable of providing stem cell niche and transplanted under the kidney capsule of ovariectomized NOG mice (Fig. 2). These mice were treated with estradiol and progesterone for 8 weeks, after which the kidneys were examined (Fig. 2). All of the grafts reconstituted endometriumlike tissues under the kidney capsules (Fig. 2a). Greater numbers of TdTom-positive cells were found in glandular, stromal, and endothelial tissues of the generated endometrium when ESP cells were transplanted compared to EMP cells (Fig. 2d). These data support the in vivo multiple differentiation potentials of ESP. Thus, we have postulated that ESP cells are likely candidate stem 2014 The Author 2017

4 T. Maruyama Figure 1 Experimental protocol for preparation of endometrial cells and in vivo endometrial stem cell assay (adopted from Miyazaki et al. 35 ). (a) Summary of procedures for the preparation of epithelium-enriched and stroma-enriched fractions from cycling human endometrium and isolation of endometrial side population (ESP) and endometrial main population (EMP) cells from the mixture of both fractions (single-dispersed endometrial cells [SDEC]). The two panels on the right illustrate representative flow cytometric distributions of ESP and EMP in SDEC stained with Hoechst in the (upper) absence and (lower) presence of 50-μM reserpine. (b) Summary of procedures for in vivo endometrial stem cell assay. E2 + P4, treatment with estradiol in combination with progesterone. NOG, NOD/SCID/γ c null ; TdTom, tandem Tomato. cells with the capacity to differentiate into glandular, stromal, endothelial and smooth muscle cells. 35 Mesenchymal stem/stromal cells SP-based cell isolation is relatively costly and laborintensive. 36 To facilitate the possible clinical use of endometrial precursors, it is necessary to identify surface markers to permit direct selection of endometrial stem/progenitor cell populations. Garget s group found that endometrial mesenchymal stem cells expressed CD146, CD140b/PDGFR-β, and W5C5. 27,37 In our hands, there was no difference between ESP and EMP fractions in the percentages of W5C5-positive cells. 37 In contrast, CD140b + CD146 + cells, candidate human endometrial mesenchymal stem-like cells, 27 were more abundant in the ESP The Author

5 Endometrial stem/progenitor cells (a) (b) (c) (d) Figure 2 Macroscopic and microscopic findings of human endometrium-like tissues reconstituted in the in vivo stem cell assay (adopted from Miyazaki et al. 35 ). (a,b) Representative macroscopic images of the transplanted site (arrowheads) of NOD/SCID/γ c null mice 8 weeks after xenotransplantation of tandem Tomato (TdTom)-endometrial side population (ESP) cells. (c) Hematoxylin eosin staining was performed on the transplanted lesion. Scale bar, 100 μm. (d) Representative immunofluorescent staining of the endometrial constructs derived from (upper panels) TdTom-ESP and (lower panels) TdTom-endometrial main population (EMP) using antibodies against vimentin (Vm), and TdTom followed by Hoechst (HO) staining. Note that localization of TdTom-expressing cells was focal (dotted line) in the endometrial constructs derived from TdTom-ESP, whereas much fewer TdTom-expressing cells were sporadically distributed in TdTom-EMPderived constructs. Scale bars, 100 μm. fraction than in EMP cells 35 substantiating the stem/ progenitor cell properties of ESP cells. Bone-marrow-derived cells Bone marrow might be a source of human endometrial epithelial and stromal stem/progenitor cells. 38,39 Clinical studies have demonstrated that chimerism (ranging from 0.2% to 52%) exists in the endometrial glands and stroma following transplants of single-antigen, human leukocyte antigen-mismatched bone marrow. 38 Thus, bone marrow stem cells might well have a role in endometrial regeneration, at least in terms of cellular turnover and inflammatory stimuli. 38 Nevertheless, the contribution of bone marrow stem cells to endometrial regeneration is likely smaller than that of stem/ progenitor cells residing in the endometrium itself. 7,40 Importantly, in women who underwent bone marrow transplantation from a male donor, XY + endometrial cells were identified in the non-sp, but not in the SP progenitor fraction. 41 Most glands are exclusively of donor or recipient origin, though some chimerism is present within individual glands. Thus, not all are monoclonal, findings that are consistent with the methylation pattern of individual glands. 42 Possible Roles of Endometrial Stem/ Progenitor Cells in Endometrial Regeneration Endometrial stem/progenitor cells are likely located in the basalis. 6,13 However, it is entirely possible that they 2014 The Author 2019

6 T. Maruyama are also present in the functionalis. Indeed, human ABCG2 + cells can be found in the endothelium of both the functional and basal layers of the endometrium. 30,32 Moreover, human CD146 + and PDGF-Rb + mesenchymal stem-like cells are located perivascularly in the functionalis and basalis layers of the endometrium. 27 Furthermore, studies have shown that endometrial surface epithelial regeneration is achieved through cellular differentiation from stromal cells and not proliferation from basal epithelial glands. 11 ESP cells have been shown to generate endometrial epithelial and stromal cells in vitro and in vivo. 33,34 It is possible that ESP cells (alone and/or tissues) might be trapped within the uterine cavity after menstruation. Such cells might contribute to endometrial regeneration. It is also possible that endometrial stem/progenitor daughter cells undergo mesenchymal-to-epithelial transition (MET) and EMT and thereby contribute to endometrial regeneration during the human menstrual cycle. 16,17 Possible Roles of Endometrial Stem/ Progenitor Cells in the Pathogenesis of Endometriosis The presence of endometrium-like tissues outside the uterus is termed endometriosis. It can be accompanied by a variety of symptoms, including dysmenorrhea and dyspareunia. 43,44 It has been suggested that endometriosis might be due to retrograde menstruation, iatrogenic direct implantation, lymphatic and vascular metastasis, embryonic rest, mesenchymal cell differentiation (induction) and coelomic metaplasia. 25 No theory, by itself, explains all types of endometriotic lesions. Thus, there might be multiple mechanisms. 25 The existence of endometrial stem cells suggests an alternative mechanism for endometriotic lesions, that is, they might originate from translocated endometrial stem/progenitor cells. 6,13,24,25,45 In support of this hypothesis, endometriotic lesions contain selfrenewing mesenchymal stem cells, express stem cell markers and show a monoclonal nature in individual glands. 25,46 Gargett, Sasson and Taylor suggested that endometrial stem cells participate in the pathogenesis of endometriosis. 6,45 Cells that initiate endometriosis would likely possess the following properties. First, they should be present within the functional layer, tissue that is translocated during retrograde menstruation (implantation theory). Second, they should be able to attach, migrate and initiate angiogenesis at an ectopic site. Finally, the cells should possess the capability to differentiate into multiple lineages in order to reconstitute the endometrium at an ectopic site. The identification of ESP cells by our group and others 30,32 34 supports the stem cell theory and suggests that ESP can initiate endometriosis. 32 The stem cell theory accounts for the limitations of the implantation theory and is consistent with the pathology of endometriosis. For instance, there is a high prevalence of peritoneal endometriosis. However, it is rather rare to observe the initial developmental steps of endometrial implantation, such as the attachment of endometrial tissue to the peritoneum Thus, if a small number of stem cells (and not endometrial tissue fragments) could initiate endometriotic lesions, detection of the event would be exceedingly difficult. Considering the low proportion of ESP cells, the chances are small that they could initiate an endometriotic lesion in a supportive microenvironment. This might explain the apparent discrepancy between the frequencies of endometriosis and retrograde menstruation. One might even speculate that ESP that were lost from the endometrial vascular walls during shedding might enter the circulation and migrate to ectopic sites, producing extrapelvic endometriotic lesions. Thus, our ESP cell observations support the stem cell theory of endometriosis and also strengthen the implantation theory. 25 Concluding Remarks Putative stem/progenitor cells are likely present in the human endometrium and probably play important physiologic and pathologic roles. A number of laboratories (including our own) have identified putative endometrial stem/progenitor cells through a variety of methods. 24,25 Nevertheless, it is not at all clear how many types of stem/progenitor cells are present in the human endometrium. For example, how do they vary in phenotype and function and is there a hierarchical relation between them? We suggest that the in vivo endometrial stem cell assay that we have developed 35 will be useful in determining the nature and roles of endometrial precursors. Supplementing the SP phenotype with specific surface markers remains critically important. 27,37 The existence of a defined endometrial stem cell immunophenotype will broaden our understanding of the mechanisms underlying normal and pathological endometrial physiology processes as well as endometriosis The Author

7 Endometrial stem/progenitor cells Acknowledgments I thank all members of my research group, Hideyuki Okano and Yumi Matsuzaki for their generous assistance and collaboration with this project. I acknowledge the secretarial assistance of Rika Shibata. This work was partly supported by Grants-in-aid from the Japan Society for the Promotion of Science (to T.M.); a Grant-in-aid from Keio University Sakaguchi- Memorial Medical Science Fund (to T.M.); a Grant-inaid from the Japan Medical Association (to T.M.); and a Grant-in-aid from the Uehara Memorial Foundation (to T.M.). Disclosure None declared. References 1. Weissman IL. Stem cells: Units of development, units of regeneration, and units in evolution. Cell 2000; 100: Li L, Clevers H. Coexistence of quiescent and active adult stem cells in mammals. Science 2010; 327: Maruyama T, Yoshimura Y. Molecular and cellular mechanisms for differentiation and regeneration of the uterine endometrium. Endocr J 2008; 55: Prianishnikov VA. 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