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progenitor cells: Implications for uterine physiology and pathology
Placenta 34, Supplement A, Trophoblast Research, Vol. 27 (2013) S68eS72
Contents lists available at SciVerse ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Review: Human uterine stem/progenitor cells: Implications for uterine physiology and pathology T. Maruyama*, K. Miyazaki, H. Masuda, M. Ono, H. Uchida, Y. Yoshimura Department of Obstetrics and Gynecology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 17 December 2012
The human uterus is composed of the endometrial lining and the myometrium. The endometrium, in particular the functionalis layer, regenerates and regresses with each menstrual cycle under hormonal control. A mouse xenograft model has been developed in which the functional changes of the endometrium are reproduced. The myometrium possesses similar plasticity, critical to permit the changes connected with uterine expansion and involution associated with pregnancy. Regeneration and remodeling in the uterus are likely achieved through endometrial and myometrial stem cell systems. Putative stem/progenitor cells in humans and rodents recently have been identified, isolated and characterized. Their roles in endometrial physiology and pathophysiology are presently under study. These stem/progenitor cells ultimately may provide a novel means by which to produce tissues and organs in vitro and in vivo. Ó 2013 Published by IFPA and Elsevier Ltd.
Keywords: Stem cells Endometrium Myometrium Endometriosis Leiomyoma Hypoxia
1. Introduction
2. Endometrial stem cells
Adult stem cells or tissue-specific stem cells are undifferentiated cells which are retained following the completion of embryonic development [1,2]. They are capable of both self-renewal and asymmetric cell division, which results in some cells undergoing terminal differentiation. Somatic stem cells are critical for the maintenance of organ structure and function in that they are required for the replacement of apoptotic cells within organs and for tissue regeneration following damage [1,2]. Somatic stem cells have been identified, through unique cell surface markers, in a wide range of tissues and organs. In the case of the uterus, candidate populations of somatic stem cells have been isolated from the endometrium and myometrium with the “side population (SP) technique” [3,4], which identifies cells based on their ability to efflux Hoechst dye [5e7]. This article reviews current studies on endometrial and myometrial stem/progenitor cells particularly focusing on side population cells isolated from human endometrium and myometrium, discusses their possible roles in endometrial and myometrial physiology and addresses the role of these cells in the pathogenesis of disorders including endometriosis and leiomyomas.
The human endometrium undergoes cyclical breakdown and regeneration under the influence of estrogen and progesterone. Retrograde shedding and ectopic implantation of menstrual endometrial cells and tissue fragments outside of the uterus forms the basis of the implantation theory of endometriosis [8e10]. Exploitation of this theory has enabled in vivo modeling of the human endometrium. Explanted single cell suspensions of human endometrial cells are sufficient to establish functional endometrial tissue in immunodeficient NOD/SCID/gcnull (NOG) mouse xenograft models [11]. In this model, the endometrial tissue, which is established beneath the kidney capsule, recapitulates the morphological and functional changes of the menstrual cycle in response to treatment with estrogen and progesterone [11]. This observation illustrates the remarkable regenerative capacity of endometrial cells and alludes to the presence of endometrial stem cells and a unique system of angiogenesis. Indeed, it has been postulated that the endometrium contains such a pool of multipotent stem cells within the deep basalis layer from which endometrial cell component arises [12,13]. Recently, many groups including ours, through a variety of methods, have identified, isolated, and/or characterized putative endometrial stem/progenitor cells capable of pluripotent differentiation [4,10]. Endometrial side population (endoSP) cells are candidate stem cells that exhibit the potential for differentiation into glandular, stromal, endothelial and smooth muscle cells in vitro and
* Corresponding author. Tel./fax: þ81 3 5363 3578. E-mail address: [email protected] (T. Maruyama). 0143-4004/$ e see front matter Ó 2013 Published by IFPA and Elsevier Ltd. http://dx.doi.org/10.1016/j.placenta.2012.12.010
T. Maruyama et al. / Placenta 34, Supplement A, Trophoblast Research, Vol. 27 (2013) S68eS72
in vivo. They are isolated based on the expression and function of a universal stem cell marker, the ATP-binding cassette sub-family G member 2 (ABCG2) [14]. These ABCG2þ cells largely correspond to endSP cells. They reside in the vascular wall of endometrial small vessels of both the functional and basal layers, and have endothelial progenitor cell (EPC)-like properties [14]. It is plausible that the putative endometrial stem/progenitor cells within the side population may initially trigger neovascularization followed by propagation and differentiation into various cell components of the human endometrium [14]. As endSP cells are also found in the functional layer of the human endometrium [14], this layer may also contribute to endometrial renewal [3]. Stem cell characteristics differ based on the method used to isolate them, and are frequently not reproducible across laboratories. Endometrial side population cells reported in the literature differ with respect to the expression pattern of surface markers, clonal efficiency, preference for culture conditions, and localization in the eutopic normal endometrium [14e17]. Thus, it is unclear how many types of stem cells exist in the human endometrium and what role each plays. To achieve a consensus definition of the endometrial stem cell, our group has recently established a novel in vivo endometrial stem cell assay in which multipotential differentiation can be identified through cell tracking [18]. In this assay, endSP and nonendSP cells (namely endometrial main population cells, endMP 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 then subjected to excision of the kidneys. We found that all of the grafts reconstituted endometriumlike tissues under the kidney capsule. 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 endSP cells than those with TdTom-labeled endMP cells, indicating the in vivo multiple differentiation potentials of endSP. We postulate, therefore, that endSP cells are genuine endometrial stem/progenitor cells [18]. SP-based cell isolation is, however, limited in terms of extreme expensiveness of keeping and using ultraviolet (UV) laser-equipped flow cytometry, cell damage caused by UV exposure, and toxicity of Hoechst dye [7]. Thus, to overcome these obstacles and to facilitate possible future clinical use, it is urgently necessary to identify surface markers to permit selection of the endometrial stem/progenitor cell population. Indeed, Gargett’s group has identified endometrial mesenchymal stem cell-specific surface markers such as CD146, CD140b/ PDGFR-b, and W5C5 [19,20]. We have investigated the expression of CD146, CD140b, and W5C5 in endSP cells [18]. We found that there was no difference in the percentages of W5C5-positive cells, purportedly putative human endometrial mesenchymal stem-like cells [20], between endSP and endMP fractions. In contrast, CD140bþCD146þ cells, reportedly putative human endometrial mesenchymal stem-like cells [19], were significantly more abundant in the endSP fraction than in endMP cells [18] substantiating the stem/progenitor cell property of endSP cells. Endometriosis is endometrium or endometrium-like tissues located outside the uterine cavity. It is frequently associated with a variety of symptoms including dysmenorrhea and dyspareunia [8,9]. Theoretical explanations of endometriosis include retrograde menstruation, lymphatic and vascular metastasis, iatrogenic direct implantation, coelomic metaplasia, embryonic rest, and mesenchymal cell differentiation (induction) [10]. Each theory, however, does not in itself account for all types of endometriotic lesions, implying multiple mechanisms [10]. In light of the role of
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endometrial stem cells in eutopic endometrial regeneration and differentiation [3,4], a novel mechanism for the origin of endometriotic lesions is that they arise from translocated endometrial stem/progenitor cells [3,4,10,21,22]. Gargett, Sasson and Taylor originally proposed a possible role for endometrial stem cells in the pathogenesis of endometriosis [21,22]. The identification of endSP cells by our group and others [14,17] further strengthens this concept of a “stem cell theory”. Additionally, these observations suggest that the endSP also contains the endometriosis-initiating (EMI) cell as they possess the following properties. First, they are present within the functional layer which is translocated during retrograde menstruation (implantation theory). Second, they possess the capacity for attachment, migration and angiogenesis essential for survival at an ectopic site. Third, they possess the multiple differentiation potential to reconstitute the endometrium at an ectopic site. We have shown that endSP cells have all of these characteristics [14]. In addition to lending further credence to the stem cell theory of endometriosis, our endSP cell observations also provide support to the retrograde menstruation theory [10]. 3. Myometrial stem/progenitor cells The human uterus expands its volume up to 1000-fold and its weight more than 20-fold over the course of pregnancy. In humans and rodents, this growth is the result of both hyperplasia and hypertrophy [23]. The uterus involutes postpartum as a result of extensive myometrial cell apoptosis followed by regeneration in order to maintain organ integrity [24]. These processes may occur over 20 times throughout a woman’s reproductive life. This regeneration is poorly understood; it is unknown if new smooth muscle cells arise from differentiated cells or from tissue-specific stem cells. To investigate myometrial stem/progenitor cells, we isolated SP cells from non-pregnant human myometria from patients undergoing hysterectomy [25]. The tissue was mechanically and enzymatically digested to produce cell suspensions that were stained with Hoechst dye and subjected to flow cytometric sorting to isolate the minority fraction of myometrial SP cells (myoSP). The expression level of ABCG2 mRNA was significantly higher in the myoSP than in the main population of myometrial cells (myoMP) [25]. MyoSP also had very low expression levels of estrogen-receptora and progesterone receptor, as well as smooth muscle cell-specific markers such as calponin and smoothelin. These observations are consistent with an undifferentiated state [25]. About 98% of myoSP (but only 20% of myoMP) were in the G0 phase of the cell cycle [25], a trait characteristic of hematopoietic and other tissue-specific stem cells. They were capable of multipotent differentiation when subject to various stimuli, and regenerated myometrium-like tissue when transplanted into both the non-pregnant and pregnant uterus in an immunodeficient mouse xenograft model. These findings were not observed in myoMP. Additionally, transcripts of octamer-binding transcription factor 4 (OCT4)/POU5F1, an embryonic stem cell marker, are more abundant in myoSP than in myoMP [26]. Interestingly, myoSP cells preferentially expand in vitro under 2% oxygen tension in comparison to a 20% O2 environment [25]. These stem cells are well suited for growth in a hypoxic environment, as hypoxia is known to stimulate somatic stem cell growth [27]. This observation supports the hypothesis that myoSP cells are indeed somatic stem cells and likely contribute to uterine enlargement. Mechanical stretching of the uterine wall does result in hypoxia [28]. Therefore, it is plausible that pregnancy-induced mechanical stretching leading to hypoxia may promote the proliferation of myoSP and further uterine enlargement. It is well known that hypoxia regulates the proliferation, differentiation and function of trophoblast and placenta [29]. Hypoxia
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promotes the proliferation of putative endometrial stem/progenitor cells like myoSP [17]. Therefore, oxygen tension, through its effects on the trophoblast, placenta and uterus, may be a critical determinant of a successful pregnancy. Leiomyomas are the most common benign tumor of the female reproductive tract and are the most common reason for hysterectomy [30]. Clinical symptoms are present in about half of patients and include menstrual bleeding, pelvic pain, urinary frequency, and infertility [30]. Despite their high prevalence, the exact pathogenesis of leiomyomas is poorly understood. Both benign and malignant tumors contain a small fraction of tumor stem cells [31]. Through asymmetric division, these cells
generate tumor-initiating daughter cells and non-tumorigenic cells that make up most of the tumor. The clonality of leiomyomas [32] argues strongly for their development from a single dysregulated cell [33,34]. Although the underlying pathogenesis of myomas is not well understood, hypoxia is thought to play a role [35e37]. Tissue hypoxia is frequently a feature of leiomyomas [38]. Low levels of oxygen (1e5% O2) upregulate secreted frizzled-related protein 1, a modulator of Wnt signaling. Frizzled-related protein 1 is antiapoptotic in leiomyoma cells but not in myometrial cells [37]. During menstruation, the myometrium becomes hypoxic due to contractions and vasoconstriction, which likely preferentially
Fig. 1. Possible roles of uterine stem/progenitor cells in uterine physiology and pathology. A. Possible roles of endometrial stem/progenitor cells in the pathogenesis of various types of endometriosis. B. Possible roles of myometrial stem/progenitor cells in pregnancy-induced uterine remodeling and pathogenesis of leiomyoma.
T. Maruyama et al. / Placenta 34, Supplement A, Trophoblast Research, Vol. 27 (2013) S68eS72
promotes the division of myoSP cells. This may provide a mechanism by which myoSP cells give rise to leiomyomas [25]. We suggest that menstruation-induced hypoxic conditions could promote the proliferation of an individual cell (a myoSP cell). This cell could then acquire cytogenetic abnormalities including MED12 mutations [39] that ultimately result in the development of a leiomyoma. This hypothesis is consistent with the inverse relationship between the risk of myomas and suppression of the hypoxic insult of menstruation [40], for example, by pregnancy. Recently, it has been reported that SP cells are also present in leiomyoma [41e43], and that leiomyoma SP cells have the capacity to generate leiomyomas [42]. Thus, we propose that myoSP cells are in fact candidates for myoma-initiating cells. It is plausible that uterine hypoxia in pregnancy may partially explain the growth of leiomyomas that frequently occurs in pregnancy. Further studies are needed to elucidate whether or not the leiomyoma SP represent a population distinct from myoSP cells. 4. Concluding remarks Increasing evidence indicates that putative stem/progenitor cells are present in the human uterus and may contribute to both physiologic and pathologic processes. The possible physiological and pathological roles are illustrated in Fig. 1. The understanding of human stem cells is still limited. For instance, endSP and myoSP are different in terms of expression patterns of surface markers and the ability of tissue reconstitution [14,18,25]; however, it remains to be elucidated exactly how different and similar they are. Although the SP phenotype is an important indicator to permit selection of these stem cells, identification of the specific surface markers characterizing these cells remains critically important and therefore is emerging [19,20]. This is a prerequisite for understanding the mechanisms underlying normal uterine physiology and pathological processes including endometriosis and leiomyomas. Finally, given their mesenchymal stem cell-like properties, uterine stem/ progenitor cells may represent a novel source of biological material useful for the reconstruction of the human uterus and other solid organs [44]. Conflicts of interest The authors declare no conflicts of interest. Acknowledgments We thank members of our research group, Hideyuki Okano and Yumi Matsuzaki, for their generous assistance and collaboration with this project. We 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. and Y.Y.); Grantin-aid from Keio University Sakaguchi-Memorial Medical Science Fund (to T.M.); Grant-in-aid from the Japan Medical Association (to T.M.); and Grant-in-aid from the Uehara Memorial Foundation (to T.M.). References [1] Weissman IL. Stem cells: units of development, units of regeneration, and units in evolution. Cell 2000;100(1):157e68. [2] Li L, Clevers H. Coexistence of quiescent and active adult stem cells in mammals. Science (New York, NY) 2010;327(5965):542e5. [3] Maruyama T, Masuda H, Ono M, Kajitani T, Yoshimura Y. Human uterine stem/ progenitor cells: their possible role in uterine physiology and pathology. Reproduction 2010;140(1):11e22. [4] Gargett CE, Masuda H. Adult stem cells in the endometrium. Mol Hum Reprod 2010;16(11):818e34.
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[5] Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 1996;183(4):1797e806. [6] Challen GA, Little MH. A side order of stem cells: the SP phenotype. Stem Cells 2006;24(1):3e12. [7] Golebiewska A, Brons NH, Bjerkvig R, Niclou SP. Critical appraisal of the side population assay in stem cell and cancer stem cell research. Cell Stem Cell 2011;8(2):136e47. [8] Giudice LC, Kao LC. Endometriosis. Lancet 2004;364(9447):1789e99. [9] Bulun SE. Endometriosis. N Engl J Med 2009;360(3):268e79. [10] Maruyama T, Yoshimura Y. Stem cell theory for the pathogenesis of endometriosis. Front Biosci 2012;E4:2754e63. [11] Masuda H, Maruyama T, Hiratsu E, Yamane J, Iwanami A, Nagashima T, et al. Noninvasive and real-time assessment of reconstructed functional human immunodeficient mice. Proc Natl Acad Sci U S endometrium in NOD/SCID/gnull c A 2007;104(6):1925e30. [12] Padykula HA. Regeneration in the primate uterus: the role of stem cells. Ann N Y Acad Sci 1991;622:47e56. [13] Gargett CE, Chan RW, Schwab KE. Hormone and growth factor signaling in endometrial renewal: role of stem/progenitor cells. Mol Cell Endocrinol 2008; 288(1e2):22e9. [14] Masuda H, Matsuzaki Y, Hiratsu E, Ono M, Nagashima T, Kajitani T, et al. Stem cell-like properties of the endometrial side population: implication in endometrial regeneration. PLoS ONE 2010;5(4):e10387. [15] Kato K, Yoshimoto M, Kato K, Adachi S, Yamayoshi A, Arima T, et al. Characterization of side-population cells in human normal endometrium. Hum Reprod 2007;22(5):1214e23. [16] Tsuji S, Yoshimoto M, Takahashi K, Noda Y, Nakahata T, Heike T. Side population cells contribute to the genesis of human endometrium. Fertil Steril 2008;90(4 Suppl.):1528e37. [17] Cervello I, Gil-Sanchis C, Mas A, Delgado-Rosas F, Martinez-Conejero JA, Galan A, et al. Human endometrial side population cells exhibit genotypic, phenotypic and functional features of somatic stem cells. PLoS One 2010;5(6):e10964. [18] Miyazaki K, Maruyama T, Masuda H, Yamasaki A, Uchida S, Oda H, et al. Stem cell-like differentiation potentials of endometrial side population cells as revealed by a newly developed in vivo endometrial stem cell assay. PLoS One 2012;7(12):e50749. [19] Schwab KE, Gargett CE. Co-expression of two perivascular cell markers isolates mesenchymal stem-like cells from human endometrium. Hum Reprod 2007;22(11):2903e11. [20] Masuda H, Anwar SS, Buhring HJ, Rao JR, Gargett CE. A novel marker of human endometrial mesenchymal stem-like cells. Cell Transplant 2012;21:2201e14. [21] Sasson IE, Taylor HS. Stem cells and the pathogenesis of endometriosis. Ann N Y Acad Sci 2008;1127:106e15. [22] Gargett CE. Uterine stem cells: what is the evidence? Hum Reprod Update 2007;13(1):87e101. [23] Ramsey EM. In: Chard T, Grudzinskas JG, editors. Anatomy of the human uterus. Cambridge: Cambridge University Press; 1994. p. 18e40. [24] Shynlova O, Oldenhof A, Dorogin A, Xu Q, Mu J, Nashman N, et al. Myometrial apoptosis: activation of the caspase cascade in the pregnant rat myometrium at midgestation. Biol Reprod 2006;74(5):839e49. [25] Ono M, Maruyama T, Masuda H, Kajitani T, Nagashima T, Arase T, et al. Side population in human uterine myometrium displays phenotypic and functional characteristics of myometrial stem cells. Proc Natl Acad Sci U S A 2007; 104(47):18700e5. [26] Ono M, Kajitani T, Uchida H, Arase T, Oda H, Nishikawa-Uchida S, et al. OCT4 expression in human uterine myometrial stem/progenitor cells. Hum Reprod 2010;25(8):2059e67. [27] Mohyeldin A, Garzon-Muvdi T, Quinones-Hinojosa A. Oxygen in stem cell biology: a critical component of the stem cell niche. Cell Stem Cell 2010;7(2): 150e61. [28] Shynlova O, Dorogin A, Lye SJ. Stretch-induced uterine myocyte differentiation during rat pregnancy: involvement of caspase activation. Biol Reprod 2010;82(6):1248e55. [29] Red-Horse K, Zhou Y, Genbacev O, Prakobphol A, Foulk R, McMaster M, et al. Trophoblast differentiation during embryo implantation and formation of the maternalefetal interface. J Clin Invest 2004;114(6):744e54. [30] Okolo S. Incidence, aetiology and epidemiology of uterine fibroids. Best Pract Res Clin Obstet Gynaecol 2008;22(4):571e88. [31] Sugihara E, Saya H. Complexity of cancer stem cells. Int J Cancer, in press. [32] Walker CL, Stewart EA. Uterine fibroids: the elephant in the room. Science (New York, NY) 2005;308(5728):1589e92. [33] Townsend DE, Sparkes RS, Baluda MC, McClelland G. Unicellular histogenesis of uterine leiomyomas as determined by electrophoresis by glucose-6phosphate dehydrogenase. Am J Obstet Gynecol 1970;107(8):1168e73. [34] Lobel MK, Somasundaram P, Morton CC. The genetic heterogeneity of uterine leiomyomata. Obstet Gynecol Clin North Am 2006;33(1):13e39. [35] Fujii S, Konishi I, Horiuchi A, Orii A, Nikaido T. In: Brosens IA, Lunenfeld B, Donnez J, editors. Mesenchymal cell differentiation: speculation about the histogenesis of uterine leiomyomas. New York: The Parthenon Publishing Group; 1999. p. 3e15. [36] Pavlovich CP, Schmidt LS. Searching for the hereditary causes of renal-cell carcinoma. Nat Rev Cancer 2004;4(5):381e93. [37] Fukuhara K, Kariya M, Kita M, Shime H, Kanamori T, Kosaka C, et al. Secreted frizzled related protein 1 is overexpressed in uterine leiomyomas, associated
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with a high estrogenic environment and unrelated to proliferative activity. J Clin Endocrinol Metab 2002;87(4):1729e36. [38] Mayer A, Hoeckel M, von Wallbrunn A, Horn LC, Wree A, Vaupel P. HIFmediated hypoxic response is missing in severely hypoxic uterine leiomyomas. Adv Exp Med Biol 2010;662:399e405. [39] Makinen N, Mehine M, Tolvanen J, Kaasinen E, Li Y, Lehtonen HJ, et al. MED12, the mediator complex subunit 12 gene, is mutated at high frequency in uterine leiomyomas. Science (New York, NY) 2011;334(6053): 252e5. [40] Flake GP, Andersen J, Dixon D. Etiology and pathogenesis of uterine leiomyomas: a review. Environ Health Perspect 2003;111(8):1037e54.
[41] Chang HL, Senaratne TN, Zhang L, Szotek PP, Stewart E, Dombkowski D, et al. Uterine leiomyomas exhibit fewer stem/progenitor cell characteristics when compared with corresponding normal myometrium. Reprod Sci 2010;17(2): 158e67. [42] Ono M, Qiang W, Serna VA, Yin P, Coon JSt, Navarro A, et al. Role of stem cells in human uterine leiomyoma growth. PLoS One 2012;7(5):e36935. [43] Mas A, Cervello I, Gil-Sanchis C, Faus A, Ferro J, Pellicer A, et al. Identification of novel genes implicated in leiomyoma formation by wide genomic analysis of side population cells. Reprod Sci 2012;19:226A. [44] Gargett CE, Ye L. Endometrial reconstruction from stem cells. Fertil Steril 2012;98(1):11e20.
Contents lists available at SciVerse ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Review: Human uterine stem/progenitor cells: Implications for uterine physiology and pathology T. Maruyama*, K. Miyazaki, H. Masuda, M. Ono, H. Uchida, Y. Yoshimura Department of Obstetrics and Gynecology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 17 December 2012
The human uterus is composed of the endometrial lining and the myometrium. The endometrium, in particular the functionalis layer, regenerates and regresses with each menstrual cycle under hormonal control. A mouse xenograft model has been developed in which the functional changes of the endometrium are reproduced. The myometrium possesses similar plasticity, critical to permit the changes connected with uterine expansion and involution associated with pregnancy. Regeneration and remodeling in the uterus are likely achieved through endometrial and myometrial stem cell systems. Putative stem/progenitor cells in humans and rodents recently have been identified, isolated and characterized. Their roles in endometrial physiology and pathophysiology are presently under study. These stem/progenitor cells ultimately may provide a novel means by which to produce tissues and organs in vitro and in vivo. Ó 2013 Published by IFPA and Elsevier Ltd.
Keywords: Stem cells Endometrium Myometrium Endometriosis Leiomyoma Hypoxia
1. Introduction
2. Endometrial stem cells
Adult stem cells or tissue-specific stem cells are undifferentiated cells which are retained following the completion of embryonic development [1,2]. They are capable of both self-renewal and asymmetric cell division, which results in some cells undergoing terminal differentiation. Somatic stem cells are critical for the maintenance of organ structure and function in that they are required for the replacement of apoptotic cells within organs and for tissue regeneration following damage [1,2]. Somatic stem cells have been identified, through unique cell surface markers, in a wide range of tissues and organs. In the case of the uterus, candidate populations of somatic stem cells have been isolated from the endometrium and myometrium with the “side population (SP) technique” [3,4], which identifies cells based on their ability to efflux Hoechst dye [5e7]. This article reviews current studies on endometrial and myometrial stem/progenitor cells particularly focusing on side population cells isolated from human endometrium and myometrium, discusses their possible roles in endometrial and myometrial physiology and addresses the role of these cells in the pathogenesis of disorders including endometriosis and leiomyomas.
The human endometrium undergoes cyclical breakdown and regeneration under the influence of estrogen and progesterone. Retrograde shedding and ectopic implantation of menstrual endometrial cells and tissue fragments outside of the uterus forms the basis of the implantation theory of endometriosis [8e10]. Exploitation of this theory has enabled in vivo modeling of the human endometrium. Explanted single cell suspensions of human endometrial cells are sufficient to establish functional endometrial tissue in immunodeficient NOD/SCID/gcnull (NOG) mouse xenograft models [11]. In this model, the endometrial tissue, which is established beneath the kidney capsule, recapitulates the morphological and functional changes of the menstrual cycle in response to treatment with estrogen and progesterone [11]. This observation illustrates the remarkable regenerative capacity of endometrial cells and alludes to the presence of endometrial stem cells and a unique system of angiogenesis. Indeed, it has been postulated that the endometrium contains such a pool of multipotent stem cells within the deep basalis layer from which endometrial cell component arises [12,13]. Recently, many groups including ours, through a variety of methods, have identified, isolated, and/or characterized putative endometrial stem/progenitor cells capable of pluripotent differentiation [4,10]. Endometrial side population (endoSP) cells are candidate stem cells that exhibit the potential for differentiation into glandular, stromal, endothelial and smooth muscle cells in vitro and
* Corresponding author. Tel./fax: þ81 3 5363 3578. E-mail address: [email protected] (T. Maruyama). 0143-4004/$ e see front matter Ó 2013 Published by IFPA and Elsevier Ltd. http://dx.doi.org/10.1016/j.placenta.2012.12.010
T. Maruyama et al. / Placenta 34, Supplement A, Trophoblast Research, Vol. 27 (2013) S68eS72
in vivo. They are isolated based on the expression and function of a universal stem cell marker, the ATP-binding cassette sub-family G member 2 (ABCG2) [14]. These ABCG2þ cells largely correspond to endSP cells. They reside in the vascular wall of endometrial small vessels of both the functional and basal layers, and have endothelial progenitor cell (EPC)-like properties [14]. It is plausible that the putative endometrial stem/progenitor cells within the side population may initially trigger neovascularization followed by propagation and differentiation into various cell components of the human endometrium [14]. As endSP cells are also found in the functional layer of the human endometrium [14], this layer may also contribute to endometrial renewal [3]. Stem cell characteristics differ based on the method used to isolate them, and are frequently not reproducible across laboratories. Endometrial side population cells reported in the literature differ with respect to the expression pattern of surface markers, clonal efficiency, preference for culture conditions, and localization in the eutopic normal endometrium [14e17]. Thus, it is unclear how many types of stem cells exist in the human endometrium and what role each plays. To achieve a consensus definition of the endometrial stem cell, our group has recently established a novel in vivo endometrial stem cell assay in which multipotential differentiation can be identified through cell tracking [18]. In this assay, endSP and nonendSP cells (namely endometrial main population cells, endMP 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 then subjected to excision of the kidneys. We found that all of the grafts reconstituted endometriumlike tissues under the kidney capsule. 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 endSP cells than those with TdTom-labeled endMP cells, indicating the in vivo multiple differentiation potentials of endSP. We postulate, therefore, that endSP cells are genuine endometrial stem/progenitor cells [18]. SP-based cell isolation is, however, limited in terms of extreme expensiveness of keeping and using ultraviolet (UV) laser-equipped flow cytometry, cell damage caused by UV exposure, and toxicity of Hoechst dye [7]. Thus, to overcome these obstacles and to facilitate possible future clinical use, it is urgently necessary to identify surface markers to permit selection of the endometrial stem/progenitor cell population. Indeed, Gargett’s group has identified endometrial mesenchymal stem cell-specific surface markers such as CD146, CD140b/ PDGFR-b, and W5C5 [19,20]. We have investigated the expression of CD146, CD140b, and W5C5 in endSP cells [18]. We found that there was no difference in the percentages of W5C5-positive cells, purportedly putative human endometrial mesenchymal stem-like cells [20], between endSP and endMP fractions. In contrast, CD140bþCD146þ cells, reportedly putative human endometrial mesenchymal stem-like cells [19], were significantly more abundant in the endSP fraction than in endMP cells [18] substantiating the stem/progenitor cell property of endSP cells. Endometriosis is endometrium or endometrium-like tissues located outside the uterine cavity. It is frequently associated with a variety of symptoms including dysmenorrhea and dyspareunia [8,9]. Theoretical explanations of endometriosis include retrograde menstruation, lymphatic and vascular metastasis, iatrogenic direct implantation, coelomic metaplasia, embryonic rest, and mesenchymal cell differentiation (induction) [10]. Each theory, however, does not in itself account for all types of endometriotic lesions, implying multiple mechanisms [10]. In light of the role of
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endometrial stem cells in eutopic endometrial regeneration and differentiation [3,4], a novel mechanism for the origin of endometriotic lesions is that they arise from translocated endometrial stem/progenitor cells [3,4,10,21,22]. Gargett, Sasson and Taylor originally proposed a possible role for endometrial stem cells in the pathogenesis of endometriosis [21,22]. The identification of endSP cells by our group and others [14,17] further strengthens this concept of a “stem cell theory”. Additionally, these observations suggest that the endSP also contains the endometriosis-initiating (EMI) cell as they possess the following properties. First, they are present within the functional layer which is translocated during retrograde menstruation (implantation theory). Second, they possess the capacity for attachment, migration and angiogenesis essential for survival at an ectopic site. Third, they possess the multiple differentiation potential to reconstitute the endometrium at an ectopic site. We have shown that endSP cells have all of these characteristics [14]. In addition to lending further credence to the stem cell theory of endometriosis, our endSP cell observations also provide support to the retrograde menstruation theory [10]. 3. Myometrial stem/progenitor cells The human uterus expands its volume up to 1000-fold and its weight more than 20-fold over the course of pregnancy. In humans and rodents, this growth is the result of both hyperplasia and hypertrophy [23]. The uterus involutes postpartum as a result of extensive myometrial cell apoptosis followed by regeneration in order to maintain organ integrity [24]. These processes may occur over 20 times throughout a woman’s reproductive life. This regeneration is poorly understood; it is unknown if new smooth muscle cells arise from differentiated cells or from tissue-specific stem cells. To investigate myometrial stem/progenitor cells, we isolated SP cells from non-pregnant human myometria from patients undergoing hysterectomy [25]. The tissue was mechanically and enzymatically digested to produce cell suspensions that were stained with Hoechst dye and subjected to flow cytometric sorting to isolate the minority fraction of myometrial SP cells (myoSP). The expression level of ABCG2 mRNA was significantly higher in the myoSP than in the main population of myometrial cells (myoMP) [25]. MyoSP also had very low expression levels of estrogen-receptora and progesterone receptor, as well as smooth muscle cell-specific markers such as calponin and smoothelin. These observations are consistent with an undifferentiated state [25]. About 98% of myoSP (but only 20% of myoMP) were in the G0 phase of the cell cycle [25], a trait characteristic of hematopoietic and other tissue-specific stem cells. They were capable of multipotent differentiation when subject to various stimuli, and regenerated myometrium-like tissue when transplanted into both the non-pregnant and pregnant uterus in an immunodeficient mouse xenograft model. These findings were not observed in myoMP. Additionally, transcripts of octamer-binding transcription factor 4 (OCT4)/POU5F1, an embryonic stem cell marker, are more abundant in myoSP than in myoMP [26]. Interestingly, myoSP cells preferentially expand in vitro under 2% oxygen tension in comparison to a 20% O2 environment [25]. These stem cells are well suited for growth in a hypoxic environment, as hypoxia is known to stimulate somatic stem cell growth [27]. This observation supports the hypothesis that myoSP cells are indeed somatic stem cells and likely contribute to uterine enlargement. Mechanical stretching of the uterine wall does result in hypoxia [28]. Therefore, it is plausible that pregnancy-induced mechanical stretching leading to hypoxia may promote the proliferation of myoSP and further uterine enlargement. It is well known that hypoxia regulates the proliferation, differentiation and function of trophoblast and placenta [29]. Hypoxia
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promotes the proliferation of putative endometrial stem/progenitor cells like myoSP [17]. Therefore, oxygen tension, through its effects on the trophoblast, placenta and uterus, may be a critical determinant of a successful pregnancy. Leiomyomas are the most common benign tumor of the female reproductive tract and are the most common reason for hysterectomy [30]. Clinical symptoms are present in about half of patients and include menstrual bleeding, pelvic pain, urinary frequency, and infertility [30]. Despite their high prevalence, the exact pathogenesis of leiomyomas is poorly understood. Both benign and malignant tumors contain a small fraction of tumor stem cells [31]. Through asymmetric division, these cells
generate tumor-initiating daughter cells and non-tumorigenic cells that make up most of the tumor. The clonality of leiomyomas [32] argues strongly for their development from a single dysregulated cell [33,34]. Although the underlying pathogenesis of myomas is not well understood, hypoxia is thought to play a role [35e37]. Tissue hypoxia is frequently a feature of leiomyomas [38]. Low levels of oxygen (1e5% O2) upregulate secreted frizzled-related protein 1, a modulator of Wnt signaling. Frizzled-related protein 1 is antiapoptotic in leiomyoma cells but not in myometrial cells [37]. During menstruation, the myometrium becomes hypoxic due to contractions and vasoconstriction, which likely preferentially
Fig. 1. Possible roles of uterine stem/progenitor cells in uterine physiology and pathology. A. Possible roles of endometrial stem/progenitor cells in the pathogenesis of various types of endometriosis. B. Possible roles of myometrial stem/progenitor cells in pregnancy-induced uterine remodeling and pathogenesis of leiomyoma.
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promotes the division of myoSP cells. This may provide a mechanism by which myoSP cells give rise to leiomyomas [25]. We suggest that menstruation-induced hypoxic conditions could promote the proliferation of an individual cell (a myoSP cell). This cell could then acquire cytogenetic abnormalities including MED12 mutations [39] that ultimately result in the development of a leiomyoma. This hypothesis is consistent with the inverse relationship between the risk of myomas and suppression of the hypoxic insult of menstruation [40], for example, by pregnancy. Recently, it has been reported that SP cells are also present in leiomyoma [41e43], and that leiomyoma SP cells have the capacity to generate leiomyomas [42]. Thus, we propose that myoSP cells are in fact candidates for myoma-initiating cells. It is plausible that uterine hypoxia in pregnancy may partially explain the growth of leiomyomas that frequently occurs in pregnancy. Further studies are needed to elucidate whether or not the leiomyoma SP represent a population distinct from myoSP cells. 4. Concluding remarks Increasing evidence indicates that putative stem/progenitor cells are present in the human uterus and may contribute to both physiologic and pathologic processes. The possible physiological and pathological roles are illustrated in Fig. 1. The understanding of human stem cells is still limited. For instance, endSP and myoSP are different in terms of expression patterns of surface markers and the ability of tissue reconstitution [14,18,25]; however, it remains to be elucidated exactly how different and similar they are. Although the SP phenotype is an important indicator to permit selection of these stem cells, identification of the specific surface markers characterizing these cells remains critically important and therefore is emerging [19,20]. This is a prerequisite for understanding the mechanisms underlying normal uterine physiology and pathological processes including endometriosis and leiomyomas. Finally, given their mesenchymal stem cell-like properties, uterine stem/ progenitor cells may represent a novel source of biological material useful for the reconstruction of the human uterus and other solid organs [44]. Conflicts of interest The authors declare no conflicts of interest. Acknowledgments We thank members of our research group, Hideyuki Okano and Yumi Matsuzaki, for their generous assistance and collaboration with this project. We 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. and Y.Y.); Grantin-aid from Keio University Sakaguchi-Memorial Medical Science Fund (to T.M.); Grant-in-aid from the Japan Medical Association (to T.M.); and Grant-in-aid from the Uehara Memorial Foundation (to T.M.). References [1] Weissman IL. Stem cells: units of development, units of regeneration, and units in evolution. Cell 2000;100(1):157e68. [2] Li L, Clevers H. Coexistence of quiescent and active adult stem cells in mammals. Science (New York, NY) 2010;327(5965):542e5. [3] Maruyama T, Masuda H, Ono M, Kajitani T, Yoshimura Y. Human uterine stem/ progenitor cells: their possible role in uterine physiology and pathology. Reproduction 2010;140(1):11e22. [4] Gargett CE, Masuda H. Adult stem cells in the endometrium. Mol Hum Reprod 2010;16(11):818e34.
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