Regeneration and adult stem cells in the human female reproductive tract.

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 through a combination of cell hypertrophy and hyperplasia. 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. The human uterus, which is composed mainly of myometrial cells, exhibits an expansion in size over the course of pregnancy. Both myometrial hyperplasia (an increase in cell number) and hypertrophy (an increase in cell size) contribute to the dramatic growth of the pregnant uterus ( Ramsey 1994 ; Shynlova et al 2006 ). In humans, most growth results from stretch-induced myometrial hypertrophy. Uterine growth during the first weeks of pregnancy, however, is accomplished by myometrial hyperplasia with a smaller contribution from hypertrophy ( Ramsey 1994 ). Similarly in rats, myometrial hyperplasia is high during early gestation and decreases dramatically later; and myometrial hypertrophy is low at the beginning of pregnancy but increases as gestation progresses ( Shynlova et al 2006 ). These changes repeat with each successive pregnancy. The presence of stem cells in other areas of the body that undergo continual renewal such as the bone marrow, gut, and skeletal muscle suggests that the changes in the uterus may not be attributable to the hypertrophy and hyperplasia of existing myometrial cells alone ( Körbling and Estrov 2003 ). The human subendometrial myometrium originates from the Müllerian duct, whereas the outer myometrium has a non-Müllerian origin ( Gargett 2007 ); however, both are derived from the mesenchyme ( Konishi et al 1984 ). Mesenchymal stem cells (MSCs) are defined as self-renewable, multipotent progenitor cells with the capacity to differentiate into several distinct mesenchymal lineages ( He et al 2007 ). Recently, a SP population of cells has also been identified in the myometrium. However, there exist some phenotypic and functional differences between the myometrial SP (myoSP) and MSCs. First, although myoSP express several MSC markers including CD90, CD73, CD105, and STRO-1, the majority of myoSP are CD34-positive and CD44-negative, whereas MSCs are negative for CD34 and positive for CD44 ( Deans and Moseley 2000 ). Second, unlike bone marrow- or peripheral blood-derived MSCs ( Wakitani et al 1995 ; Gang et al 2004 ), myoSP do not generate skeletal muscle cells when transplanted into intact or chemically injured skeletal muscles of NOG mice ( Ono et al 2007 ). Further studies are required to elucidate why myoSP behaves differently from MSCs in response to the microenvironment and/or its niche in various tissues and organs. Interestingly, although myoSP never proliferates efficiently in vitro in a normoxic (20% O 2 ) environment, it proliferates efficiently in vitro under 2% oxygen tension. Leiomyomas are the most common gynecological tumors in women of reproductive age, and are associated with a variety of symptoms including abnormal uterine bleeding, pelvic pain, urinary frequency, impaired fertility, and spontaneous abortion. They are clonal in origin ( Walker and Stewart 2005 ) and their development is thought to be induced and promoted by hypoxia ( Fukuhara et al 2002 ; Pavlovich and Schmidt 2004 ). Low oxygen tension (1%–5% O 2 ) dramatically upregulates secreted frizzled-related protein 1, a modulator of Wnt signaling. Secreted frizzled-related protein 1 exerts antiapoptotic effects in leiomyoma cells but not myometrial cells ( Fukuhara et al 2002 ). Myometrial contraction and vasoconstriction that occur during menstruation render the myometrium hypoxic. It is possible that repeated menstruation-induced hypoxia may select a single cell such as a myoSP to proliferate and acquire cytogenetic abnormalities that would ultimately result in the development of a leiomyoma. Leiomyomas occasionally contain adipogenic components and are referred to as lipoleiomyomas ( Wang et al 2006 ). Very rarely, they also become ossified, which is consistent with the potential of myoSP to differentiate not only into myocytes but also into adipocytes and osteocytes.

Arango and colleagues (2005) reported that Müllerian duct mesenchyme-specific disruption of β-catenin resulted in a progressive turnover of the uterine myometrium to adipose tissue. This supports the possibility that putative myometrial stem cells, with the potential for differentiation into adipocytes in the absence of β-catenin, may exist in the myometrium. These cells may give rise to lipoleiomyomas. Indeed, the same group has recently isolated a myometrial SP from the mouse uterus and provided evidence that this SP contains putative myometrial stem/progenitor cells derived from the Müllerian duct mesenchyme ( Szotek et al 2007 ). This finding supports our results on the enrichment of stem cells in myoSP in humans ( Ono et al 2007 ). The embryonic Müllerian ducts, or paramesonephric ducts, are derived from the coelomic epithelium in the bilateral urogenital ridges, during differentiation of the bipotential gonad ( Teixeira et al 2001 ). In female embryos, the absence of Müllerian inhibiting substance (MIS) allows the Müllerian ducts to persist and to differentiate into the internal female reproductive tract structures ( Teixeira et al 2001 ). After birth, the mesenchyme of the primitive uterine tube differentiates into two layers of the adult uterus: the endometrial stroma and the myometrial muscle layers ( Kurita et al 2001 ). By postnatal day 15, the myometrium is well developed and the endometrial glands are visibly coiled, comparable to those observed in the adult uterus. The adult uterus undergoes repeated cycles of cellular proliferation and degeneration in response to hormonal signals during a normal mammalian reproductive life span. These changes involve signaling through β-catenin. β-catenin has two roles: as an intracellular transcriptional cofactor of the canonical Wnt signaling cascade and as a structural adaptor protein linking cadherins to the actin cytoskeleton in cell-cell adhesion ( Nelson and Nusse 2004 ). The progressive smooth muscle atrophy and resultant adipogenesis in the conditional β-catenin-deleted myometrium suggests that this cell fate switch is likely due to the dysregulation of a previously uncharacterized, intrinsic, myometrial maintenance and repair mechanism. This mechanism presumably involves perturbed stem or progenitor cell activity. Cell lineage and early differentiation markers in the mouse myometrium are largely uncharacterized. Although much effort has focused on the events surrounding the endometrium during the estrous cycle and at pregnancy, little is known about the normal mechanisms of repair and remodeling in the myometrium. Stromal LRCs are present near the endometrial epithelium, in proximity to the myometrial-stromal junction, and in a perivascular distribution ( Chan and Gargett 2006 ). Myometrial LRCs are present at the periphery of the longitudinal muscle bundles, a location similar to where Pax7-expressing satellite or stem cells in skeletal muscle are found. In vitro and in vivo evidence suggests that either of the two known biological functions of β-catenin, Wnt signaling and cell-cell junctions, may be involved in the muscle-to-fat cell fate switch ( Ross et al 2000 ; Kennell and MacDougald 2005 ) Disruption of the Wnt canonical signaling pathway induces adipogenesis and causes the transdifferentiation of myoblasts into adipocytes. The mechanism of this likely involves the disruption of β-catenin-mediated inhibition of adipogenic transcription factors ( Ross et al 2000 ). Another possibility is that β-catenin cell-cell junctions play a role in the maintenance of the myometrial stem cell niche ( Szotek et al 2007 ). Disruption of this niche may result in a cell fate switch from smooth muscle myogenesis to adipogenesis.

There is now sufficient published evidence to conclude that rare populations of adult stem cells exist in the human myometrium and endometrium. The study of stem cells in the female reproductive tract, however, is still in its infancy. There is a pressing need to identify definitive markers for both myometrial and endometrial stem cells. A thorough characterization of uterine stem cells is a prerequisite for understanding the complex mechanisms underlying the morphogenesis and physiological regeneration of the female reproductive tract. Additionally, the techniques developed by our laboratory for culturing and differentiating uterine stem cells could be a starting point for using these cells for the regeneration of the uterus or other organs. Studying the underlying mechanisms involved in the uterine stem cell niche will improve the understanding of the pathophysiology of uterine cancer, hyperplasia, endometriosis, leiomyomas, and adenomyosis, and may change how these diseases are treated in the future.

The derivation of human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells has heralded a new era in stem cell research ( Shamblott et al 1998 ; Thomson et al 1998 ; Takahashi et al 2007 ). Candidate stem cells have now been identified throughout the reproductive system and include the germ stem cells in the testis ( Kanatsu-Shinohara et al 2004 ; Seandel et al 2007 ), as well as recently identified cell populations with adult stem cell activity in the human myometrium and endometrium ( Gargett 2007 ; Ono et al 2007 ). The discovery of those cells highlights the importance of the stem cell system in human reproduction. The study of the adult stem cells derived from the female reproductive tract, in particular, is a new avenue through which to investigate gynecological diseases including leiomyoma, endometriosis, and endometrial cancer. The human uterus is unique in that it possesses the tremendous regenerative capacity required for cyclical regeneration and remodeling throughout reproductive life. Not only must the uterus rapidly enlarge to accommodate the developing fetus, the endometrium must also regenerate with each menstrual cycle. Regeneration and remodeling in the female reproductive tract allude to the existence of myometrial and endometrial stem cell systems; however, the characteristics and function of these stem cells remain poorly understood. The concept that endometrial regeneration is mediated by endometrial stem/ progenitor cells was proposed many years ago ( Prianishnikov 1978 ; Padykula et al 1989 ). Since then, indirect evidence has accumulated from proliferation studies, clinical observations, and the demonstration of gland monoclonality ( Gargett 2007 ). Shedding of the endometrial functionalis layer at menstruation and its subsequent regeneration from the endometrial basalis suggests that the proliferation kinetics differ between the two layers ( Brenner et al 2003 ) and that putative endometrial stem cells reside in the basalis. Given that endometriotic lesions have a basalis phenotype ( Leyendecker et al 2002 ), it is likely that adult stem cells present in the basalis may implant ectopically through retrograde menstruation and may give rise to endometriotic lesions ( Sasson and Taylor 2008 ). Since the endometrium is comprised of glands, surface epithelium and supportive stroma, it is plausible that there may exist both epithelial and stromal stem/progenitor cells responsible for the regenerative capacity of the endometrium ( Chan et al 2004 ; Gargett 2007 ). Our recent xenograft study confirms the plasticity of endometrial cells. We have shown that a small number of singly dispersed human endometrial cells, transplanted beneath the kidney capsule of severely immunodeficient mice, regenerates functional endometrial tissue ( Masuda et al 2007 ). This artificially generated endometrium resembles the natural endometrium and contains human blood vessels which invade the mouse kidney parenchyma. Additionally, it mimics normal hormone dependent changes including proliferation, differentiation, and tissue breakdown (menstruation). We have also recently isolated a candidate population of adult stem cells from the human myometrium. Selection was performed using a side population method based on a distinct Hoechst dye efflux pattern ( Ono et al 2007 ). Characterization of these myometrial cells, along with the study of the mechanisms controlling their regeneration, will improve the understanding of the physiology and pathophysiology of the female reproductive tract.