Accepted Manuscript Title: Stem Cells and the Reproductive System: Historical Perspective and Future Directions Author: Cindy M.P. Duke Hugh S. Taylor PII: DOI: Reference:
S0378-5122(13)00282-X http://dx.doi.org/doi:10.1016/j.maturitas.2013.08.012 MAT 6036
To appear in:
Maturitas
Received date: Accepted date:
28-8-2013 29-8-2013
Please cite this article as: Duke CMP, Taylor HS, Stem Cells and the Reproductive System: Historical Perspective and Future Directions, Maturitas (2013), http://dx.doi.org/10.1016/j.maturitas.2013.08.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Stem Cells and the Reproductive System: Historical Perspective and Future Directions
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Cindy M.P. Duke and Hugh S. Taylor Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven Connecticut, USA
Corresponding Author: Hugh S. Taylor M.D
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Department of Obstetrics, Gynecology and Reproductive Sciences Yale University School of Medicine 333 Cedar Street, FMB 329K New Haven Connecticut, 06520 USA Email: [email protected]
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Stem Cells and the Reproductive System: Historical Perspective and Future Directions
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Cindy M.P. Duke and Hugh S. Taylor Department of Obstetrics, Gynecology and Reproductive Sciences, Yale School of Medicine, New Haven Connecticut, USA
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Abstract
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Recent findings in stem cell biology have presented new perspectives and opportunities for the treatment of reproductive disease. In a departure from the long held dogma of embryologically fixed numbers of oocytes, current literature suggests that human ovaries contain stem cells which form new oocytes even in adulthood and that these stem cells can be cultured in vitro to develop into mature oocytes. These findings have provided new hope and broader options for fertility preservation. Evidence of endometrial regeneration by bone marrow stem cells in endometrial tissue of women who received bone marrow transplant highlight potential for the novel treatments of uterine disorders and supports new theories for the etiology of endometriosis ectopic transdifferentiation of stem cells. Further, endometrial derived stem cells have been demonstrated to be useful in the treatment of several chronic and often debilitating diseases, including Parkinson’s Disease and Diabetes. Other cells that may present future therapeutic benefits for a myriad of disease states include placental and fetal cells which enter maternal circulation during pregnancy and can later promote parenchymal regeneration in maternal tissue. These findings highlight novel functions of the uterus and ovaries. They demonstrate that the uterus is a dynamic organ permeable to fetal stem cells capable of transdifferentiation as well as a renewable source of multipotent stem cells. While we still have much to understand about stem cells, their potential applications in reproductive biology and medicine are countless.
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Introduction
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Adult stem cells
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Stem cells have the capacity to renew themselves and differentiate into many different cell types that make up an organism[1] and can be described based on their characteristics and origin (Totipotent, pluripotent, mulitpotent or unipotent). Of these subtypes, much work has focused on pluripotent and mulitpotent cells known as Embryonic and Adult stem cells. Totipotent stem cells have the potential to differentiate into all the cells and tissues that make up an embryo and support the development of the fetus (for example, the zygote, or fertilized egg). Pluripotent stem cells have the potential to give rise to cells derived from all three germ layers (endoderm, ectoderm and mesoderm); embryonic stem (ES) cells are an example. Multipotent stem cells are capable of producing a limited range of differentiated cell lineages appropriate to their location; examples include somatic or adult stem cells. Unipotent stem cells are capable of differentiating along one lineage; these include epidermal stem cells and spermatogonial stem cells of the testis. ES cells are derived from the inner cell mass of the blastocysts from a developing embryo and were first isolated from the mouse model in 1981. They can be maintained in tissue culture under conditions where they can be propagated indefinitely as pluripotent ES cells [2,3]. ES cells are pluripotent stem cells. Research involving ES cells has opened up the possibility of using them as the foundation for "designer" tissue and organ engineering. However, some ethical considerations regarding the instrumental use of embryos for the isolation of stem cells are sharply debated and consequently curtailed much progress in research in this area. One alternative is to explore the use of non-embryonic stem cells as a source. In this paper, we will review non embryonic stem cell derivatives and their potential roles in the reproductive system.
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The main difference between blastocyst-derived (embryonic) pluripotent stem cells and multipotent stem cells from adult animals is in the number of types of differentiated cells that can be produced. Originally, adult stem cells were thought to have a limited potential for production of differentiated derivatives. However, studies on adult stem cell plasticity have questioned that view. For example, it has been demonstrated that neural stem cells can produce a variety of blood cell types including myeloid and lymphoid cells as well as early hematopoietic cells which are normally derived from the dermis; additionally, clones of individual cells can proliferate and differentiate in culture to produce neurons, glia, smooth muscle cells and adipocytes [4,5]. This is important because bone marrow is a mesodermal derived tissue consisting of various hematopoietic cellular components supported by a microenvironment composed of stromal cells embedded in a complex extracellular matrix. It contains hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs); the latter of which are particularly developmentally flexible and able to contribute usefully to several recipient organs. For example, bone marrow stem cells can generate cells bearing neuronal antigens and bone-marrow-derived or extra-hepatic stem cells can differentiate into hepatocytes [6-8]. These studies were amongst the first to show that some adult stem cells are not lineage restricted and thus, changed our view of Adult stem cells. Adult stem cells, in addition to their usual progeny in their organ of residence, are able to differentiate into other cell types in a new location; including cells not normally present in their organ of origin.
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The concept of plasticity of stem cells has presented the exciting possibility of repairing an individual’s failing organ by transplanting/locally delivering bone marrow cells. A number of studies have shown that bone marrow cells can in turn generate de novo myocardium in the heart, or when engrafted into the kidney, can differentiate into renal parenchymal cells, or new hepatocytes if engrafted to the liver [8-12]. These studies show that bone marrow derived stem cells may be involved and thereby can be engineered for use in the regeneration of damaged tissue. This concept then begs the question, “how can adult stem cells make lineage determination?” To this end, several theories have been proposed to explain the fate of stem cells and led to the notion of an Adult stem cell Niche.
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Adult stem cell niche
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The notion that tissue stem cells reside within a specific anatomical location termed niche arose from studies of transplanted hematopoietic progenitors which suggested that a subset of tissue cells and extracellular substrates can indefinitely house one or more stem cells and control them in an undifferentiated state in vivo [13,14]. In this system, secreted factors by other cell types, cell-cell interactions mediated by integral membrane proteins, Integrins and extracellular matrix are believed to influence the survival and overall development of these undifferentiated cells into committed cells. Control of lineage determination is believed to be governed by intrinsic and extrinsic factors. Intrinsic factors include: the proteins responsible for setting up asymmetric cell divisions, nuclear factors (e.g TGF ß superfamily, ß1 integrin receptor) controlling gene expression and chromosomal modifications in stem and nonstem daughters as well as clocks that may set the number of rounds of division within the transit amplifying population. Additionally, multiple positive and negative signals are received through cytokines and other receptors to further stabilize a committed pathway. Other extrinsic factors work through growth factors, stroma or external signals that control stem cell fate and make up the stem cell microenvironment, or niche. Adult stem cells occupy specific niches in tissues, where they respond to environmental cues provided by neighbor niche cells, such as epithelial niches and endodermal niches [15,16]. Some locations have more of these “inter niche” crosstalk. For example, near the base of the intestinal crypt in close association with niche myofibroblasts or in the hair bulge of the epidermis or in close association with Sertoli cells on the basement membrane of the seminiferous tubules.
Germline stem cells (GSCs) in the postnatal ovary in mammal GSCs are the self-renewing population of germ cells that serve as the source for gametogenesis. GSCs exist in diverse forms in many organisms, ranging from Drosophila melanogaster to mammals [17]. Spermatogonial stem cells are one kind of GSCs which maintain spermatogenesis throughout the entire reproductive life of a male and have been found in all metazoan species [17-19]. In Drosophila, GSCs, in females, maintain oocyte production in adult ovaries [20]. However, until recently, it was believed that ovaries of some vertebrates, especially those of mammals, did not contain self-renewing stem cells. In contrast to spermatogenesis, there was an apparent evolutionary disparity in female organisms. To date, the notion that new primary follicles can be formed in adult ovaries is a evolving topic with increasing evidence leaning away
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from the long held doctrine that female mammals are born with an embryologically predetermined finite population of non-growing primordial follicles and oocyte numbers decline throughout postnatal life, eventually leaving the ovaries devoid of germ cells [21-25]. In humans, this lifetime decline in oocyte number is accompanied by exhaustion of the follicle pool and menopause before the natural end of the female’s life [26]. Congruent to this dogma, it is believed that primordial follicles in the mammalian ovary are arrested in diplotene stage of meiosis I (oocytes), and are surrounded by a single, squamous layer of somatic cells [22]. However, in 2004, Johnson et al demonstrated the existence of proliferative GSCs that gave rise to oocytes and follicle production in the postnatal period of mammalian ovary [27]. In brief, to better define germ-cell dynamics in female mammals, they counted the numbers of healthy (non-atretic) and degenerating (atretic) follicles in ovaries of mice and showed that the numbers of non-atretic quiescent (primordial) and early growing (primary) prenatal follicles in single ovaries were higher than expected; also their rate of depletion in the immature ovary was less than anticipated. These results were consistent with past studies of follicle depletion in mice but unlike all previous studies of follicle depletion in other mammals [24]; juvenile and adult mouse ovaries continuing to possess mitotically active germ cells which continuously replenish the follicle pool. Morphological studies of the juvenile and adult mouse ovaries also revealed the presence of large ovoid cells resembling germ cells of fetal mouse ovaries, in the surface epithelial cell layer covering the ovary [27]. Immunohistochemical staining for mouse Vasa homologue (MVH), a gene expressed exclusively in germ cells of both vertebrate and invertebrate species, confirmed that these large ovoid cells were of a germline lineage [27]. Moreover, when small wedges of ovaries were then grafted from female mice with ubiquitous expression of green fluorescent protein (GFP) to wild-type ovaries, GFP-transgenic germ cells were observed to form follicles in wild-type ovaries several weeks after graft. These data established the existence of proliferative germ cells that sustain oocyte and follicle production in the postnatal mammalian ovary and demonstrated that oocytes are continuously formed in the adult ovary. This body of work not only represented a novel concept in ovarian biology of the mammalian female but also had significant clinical implications related to therapeutic expansion of the follicle reserve.
Origin of germ cells in adult ovary
The origin of oocytes (and primary follicles) in ovaries of adult mammalian females has been disputed for well over a century. In the 19th century, Weismann theorized that before embryonic cells were committed along specific pathways, a set of germ cells is set aside and are destined to give rise to the gametes. This theory was not questioned until many decades later when studies of mouse embryos, in which genetically marked cells were introduced at 4- and 8cell stage blastomere demonstrated that such cells can either become germ cells or somatic cells [31] suggesting that no specific germ cell commitment exists prior to implantation. During the 1990’s, it was demonstrated that egg and sperm cells (gametes) of the mouse differentiate from somatic lineage and cellular differentiation of grafted embryonic cells did not depend on the source of the grafts but rather where they have been placed [32,33]. Later studies by other groups using the mouse model confirmed that germ cells can be derived from mouse somatic cell lines in vitro and found that functional (embryo forming) mouse oocytes and sperm can be derived from mouse stem cells in culture [34-36].
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However, as previously mentioned, the paradigm that all primary follicles in adult mammalian (especially primates) females were formed during fetal life is still supported by a sizable number of scientists, primarily because of the lack of direct evidence on formation of new primary follicles in adult mammalian ovaries [37]. They argue that Weissmann’s theory may fit for invertebrates (C. Elegans and Drosophila) and some lower vertebrates (Zebra fish and frogs) but not mice and possibly mammals in general. In Human studies, Bukovsky et al. reported that germ cells can originate from surface epithelium (SE) cells which cover the tunica albuginea (TA) [28]. In these studies, ovaries from twelve adult women (average age of 32 years) were investigated by single, double, and triple color immunohistochemistry. The results show that cytokeratin (CK) positive mesenchymal cells in ovarian TA differentiate into SE cells by a mesenchymal-epithelial transition. Segments of SE directly associated with ovarian cortex form solid epithelial cords, which fragment into small epithelial nests descending into the lower ovarian cortex and associate with zona pellucida(ZP) positive oocytes strongly favoring the concept that the pool of primary follicles in adult human ovaries does not represent a static but rather a dynamic population of differentiating and regressing structures. An essential mission of such follicular turnover might be elimination of spontaneous or environmentally induced genetic alterations of oocytes in resting primary follicles. This same group also reported that new primary follicles are formed by assembly of oocytes within nests of primitive granulosa cells in the ovarian cortex. In this study, cells were scraped from the surface of ovaries and cultured for 5 to 6 days, in the presence or absence of estrogenic stimuli. The OSE cells cultured in medium differentiated into small granulosa cells as well as epithelial, neural and mesenchymal type cells. OSE cells cultured in medium containing Phenol red, which is estrogenic, differentiated directly into large cells with an oocyte phenotype and exhibited germinal vesicle breakdown, expulsion of the polar body, and surface expression of zona pellucida proteins, i.e. characteristics of secondary oocytes. These results show that granulosa cells and oocytes may develop directly from cultured OSE cells derived from adult human ovaries; these studies also confirmed in vivo observations that adult human ovaries contain mesenchymal cells which are bipotent progenitors, able to become both primitive granulosa and germ cells. Recent work by the Tilly group has also shown that GSCs from human female ovaries can be isolated, xenografted and propagated to oocytes in vivo [38, 39]. These data suggest the possibility of developing new strategies for the egg preservation and treatment of female infertility and importantly, Stem cell transfer from the fetus
The presence of fetal cells in maternal circulation was first described more than a century ago and has been repeatedly confirmed by many investigators [40, 43]. Fetal cells enter the maternal circulation during all pregnancies and may persist in maternal blood and tissue for decades, thus creating a state of physiologic microchimerism in the parous woman [41]. Circulating materno-fetal cells are detected as CD34 and CD38 positive cells and represent progenitor cells derived from the fetus [41,42]. The observations of feto-maternal traffic and maternal organ chimera raised the question of what could be the role of fetal stem cells in the mother’s body? Microchimeric cells of fetal origin have been identified in the peripheral blood of patients with autoimmune diseases such as systemic sclerosis (SSc) [44]. However, whether these cells are integrally involved in the pathogenesis of SSc, or if fetal microchimeric cells are just an associated marker of inflammation is unclear. Increased numbers of microchimeric fetal
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Stem Cell within the Uterus
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cells have also been identified in some pregnancy specific diseases: pre-term labor, preeclampsia and aneuploidy [45]. Even in these instances, there is speculation that the increased number of fetal microchimeric cells in the maternal circulation is more a reflection of the abnormalities within the structure of the placenta rather than a direct result of the disease process. In 2001, a team led by Bianchi discovered that male cells, presumably from their sons, were seen in thyroid sections in women [46]. Essentially, male cells were seen individually or in clusters in all (including inflammatory) thyroid disease biopsies that were examined. In one patient with a progressively enlarging goiter, fully differentiated male thyroid follicles were noted to be closely attached to and indistinguishable from the rest of the thyroid. Later work by this group, reported that XY+ microchimeric cells in maternal tissue, acquired most likely through pregnancy, expressed leukocyte, hepatocyte and epithelial markers [47] suggesting that pregnancy may result in the physiologic acquisition of a fetal cell population with the capacity for multi-lineage differentiation. This study also showed that hepatocytes of fetal stem cell origin were identified in liver tissue of one woman with liver injury and in another woman following hepatic transplantation. Similar findings have been noted in non-human studies suggesting that in a state where the tissue injury is chronic, fetal cell microchimerism may be established more frequently or easily, and also suggests that microchimeric cells are involved in tissue repair. Although the functional contribution of these fetal-derived cells remains to be determined, the possibility that newly implanted or persistent fetal stem cells may promote tissue regeneration in maternal disease states in novel and exciting.
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The mammalian uterine endometrium is one of the most dynamic human tissue types and sites. It consists of a glandular epithelium and stroma that are completely renewed in each monthly menstrual cycle. Endometrial stem cells are thought to reside in the basalis layer and serve as a source of cells that differentiate to form the endometrium. Cyclic endometrial renewal depends on a small pool of tissue-specific multipotential stem cells [48]. Under systemic hormonal changes, stem cells migrate and give rise to a group of progenitor cells that become committed to specific types of differentiated cells, e.g. epithelial, stromal and vascular. These endogenous stem cells allow the rapid regeneration of the endometrium necessary to support pregnancy [49]. Disorders of these endometrial stem cells may contribute to common proliferative disorders such as endometriosis and cancer. Work from our group has demonstrated, in a murine model, the ability to differentiate endometrial stromal stem cells (ESSC) into morphologically and functionally glucose responsive insulin secreting cells that are indistinguishable from pancreatic beta cells. This provides new possible therapeutic strategies and applications for treatment of diabetes (e.g use in augmentative therapies to achieve better glycemic control if not euglycemia) [50]. Phase I study designs are in progress to further assess the role of this treatment in humans. Other novel work being undertaken by our group include the differentiation of human derived endometrial stem cells into dopaminergic neurons capable of restoring dopamine levels in a murine Parkinson’s disease model; further primate studies evaluating clinical responses to this therapy are currently underway [51]. The uterus is a particularly rich source of stem cells and one that is easily
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accessible. Uterine stem cells are present after menopause and may be banked from hysterectomy specimens or menstrual blood for use in men or women without a uterus. Another potential source of endometrial cells is the bone marrow. Our group has provided novel evidence of endometrial regeneration in bone marrow transplant recipients who received marrow from a single-HLA antigen mismatched bone marrow transplant for leukemia. Donor-derived endometrial epithelial cells and stromal cells were detected in endometrial samples of bone marrow recipients by RT-PCR and immunohistochemistry. Cyclic mobilization of bone marrow derived stem cells may be a normal physiologic process [52]. These findings have potential advantages for the treatment of many uterine disorders including Asherman’s syndrome. It also defines a new theory for the etiology of endometriosis - ectopic transdifferentiation of stem cells. We have shown that bone marrow derived stem cells can contribute to endometriosis, perhaps explaining the occurrence of disease outside of the peritoneal cavity. Placenta and stem cells
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During development, blood begins to form not only in the embryo itself, but also in the placenta [53,54]. Experiments in mice revealed that the placenta harbors a large supply of hematopoietic (blood-forming) stem cells. These cells, which appear very early in development, are able to generate more blood stem cells and can give rise to a complete hematopoietic system when transplanted into an adult. Unlike other sites where blood stem cells are found during embryonic development, such as the liver, the stem cells in the placenta can increase in number without giving rise to mature, specialized cells. Over the last 30 years, colonization has been a long-accepted theory which proposes that the yolk sac was the sole source of hematopoiesis in the mammalian embryo. According to this theory, one yolk sac-derived hematopoietic stem cell (HSC) colonized fetal liver to initiate definitive hematopoiesis and subsequently colonize bone marrow at the neonatal stages to support adult hematopoiesis [55]. However, in the 1990s, accumulating evidence pointed to another site of hematopoiesis in the aorta-gonad-mesonephros (AGM) of mouse embryos [56]. Later studies also indicated that the placenta contains numerous clonogenic hematopoietic progenitors; suggesting that the placenta may function as a hematopoietic organ during development. Other studies have simultaneously reported that pluripotent HSCs can be detected in the placental labyrinth region in MDI gestation of the murine embryo [53,54]. The onset of HSC activity in the placenta coincides with that in the AGM region and the yolk sac. However, the expansion of HSC pool size in placenta is greater than 15-fold than in AGM. The expansion of the HSC pool in the placenta occurs prior to and during the initial expansion of HSCs in the fetal liver. The size of the placental HSC pool diminished while the HSC pool in the fetal liver continued to expand. These data suggest that placenta is another site contributing to the establishment of the mammalian definitive hematopoietic system. A common feature of these three hematopoietic sites, yolk sac, AGM, and placenta, is that the induction and generation of hematopoietic cells coincides with that of endothelial cells [57]. Indeed, coexpression of endothelial cell markers CD31/CD34 and hematopoietic stem cell markers Kit/CD34 (and Sca-1, partially) is found in the hematopoietic cells in the yolk sac [54,58,59]. HSCs derived from different sites have different functional capacities. These differences in developmental potency may result from differences in their inherited intrinsic programs or may be due to different microenvironmental signals. Unlike the adult HSC niche, which imposes a restriction signal for
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HSC proliferation, the placenta niche promotes rapid expansion but without inducing myeloerythroid lineage differentiation, and therefore strongly suggests that the placental labyrinth provides crucial niche signals to induce fast expansion of HSCs. Is there a Relationship between Ovarian Cancer and Stem Cells?
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As early as 2001, it was hypothesized that all stem cells may be regulated by similar selfrenewal signaling pathways. Moreover, one leading hypothesis is that tumors may originate from the transformation of normal stem cells; ergo cancer cells may include cancer stem cells---rare cells with indefinite potential for self-renewal that drive tumorigenesis [60]. Stem cells have the ability to renew themselves and cancer can be thought of as a disease of dysregulated selfrenewal. Therefore, it is highly probable that there are common mechanisms such - as telomerase activity - for achieving these processes. Telomeres are maintained by the novel ribonucleoprotein enzyme telomerase. Telomerase appears to be reactivated in almost all human cancers but is repressed in most somatic human cells; leading to telomere loss during replicative aging in vivo and in vitro. Telomerase activity prevents the replication-dependent loss of telomere repeats and prevents cellular senescence in highly proliferative germline cells in early embryonal tissues and in many cancers. Significant levels of telomerase have been found in CD34+CD38 + human hematopoietic progenitor cells [61]. Many pathways that are classically associated with cancer may also regulate and preserve normal stem cell development; these include signaling pathways (Wnt, Notch, Sonoc hedgehog (Shh)), the HOX family of regulatory genes and the anti-apoptotic oncogene bcl-2 [62-67]. Ovarian cancer is an extremely aggressive disease and the cellular mechanisms underlying the increasing aggressiveness associated with ovarian cancer progression are poorly understood. In 2005, a study reported that lineage infidelity of epithelial ovarian cancers (EOCs) is controlled by HOXA9, HOXA10 and HOXA11 genes that specify regional identity in the reproductive tract [68]. Several HOX genes, including HOXA9 and HOXA10, are expressed in primitive hematopoietic cells, implying a role in early hematopoietic differentiation [69]. It is believed that HOXA9, HOXA10 and HOXA11 are not expressed in normal ovarian surface epithelium (OSE) but rather by a group of OSE in adult ovary is a source of germ cells. Therefore it is possible that HOX genes expressed in OSE cells play a role in cell differentiation and may even be a cancer stem cell. Another report showed stem cell transformation can be the underlying cause of ovarian cancer [70]. One study demonstrated direct evidence that the aggressiveness of human ovarian cancer may be a result of transformation and dysfunction of stem cells in the ovary [70]. They showed that a single tumorigenic clone was isolated among a mixed population of cells derived from the ascites of a patient with advanced ovarian cancer and during the course of the study, another clone underwent spontaneous transformation in culture, providing a model of disease progression. Both the transformed clones possessed stem cell-like characteristics and differentiated to grow in an anchorage-independent manner in vitro (as spheroids) although further maturation and tissue-specific differentiation was arrested. Significantly, tumors established from these clones in animal models are histologically similar to those in the human disease and even on serial transplantation, continue to establish tumors; thereby confirming their identity as tumor stem cells. These findings suggest that stem cell transformation can be the underlying cause of ovarian cancer and continuing stochastic events of
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stem and progenitor cell transformation define the increasing aggression that is characteristically associated with the disease. Conclusion
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Our understanding of stem cells and their specific existence and role in the reproductive system has expanded significantly in the past 20 years and our studies of stem cell biology has already led to significant advances in therapeutic options including oocyte cryopreservation. However, many key questions remain. Radiotherapy, high-dose chemotherapy and bone-marrow transplantation have resulted in an increased number of long-term cancer survivors. Since the ovaries and germ cells are very sensitive to cytotoxic treatment, especially to alkylating agents and radiation, ovarian failure and infertility are common side-effects [71]. For young female cancer survivors, as a result of many advances in stem cell research, there are now several potential treatments available to avoid premature sterility. The methods for preserving fertility include oocyte cryopreservation, embryo cryopreservation and cryopreservation of ovarian cortical tissue. Our expanding knowledge on the broad niches and diverse applications for stem cells within the different organs of the female reproductive system present many exciting targets for their use not only in advanced reproductive techniques for the prevention and treatment of infertility but also for other chronic disease states like Diabetes and Parkinson’s Disease. Additionally, the discovery and refined ability to retrieve and differentiate endometrial stem cells presents exciting possibilities for using a renewable and relatively easily accessible niche (in female patients at least) for targeting these therapeutic strategies.
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S0378-5122(13)00282-X http://dx.doi.org/doi:10.1016/j.maturitas.2013.08.012 MAT 6036
To appear in:
Maturitas
Received date: Accepted date:
28-8-2013 29-8-2013
Please cite this article as: Duke CMP, Taylor HS, Stem Cells and the Reproductive System: Historical Perspective and Future Directions, Maturitas (2013), http://dx.doi.org/10.1016/j.maturitas.2013.08.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Stem Cells and the Reproductive System: Historical Perspective and Future Directions
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Cindy M.P. Duke and Hugh S. Taylor Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven Connecticut, USA
Corresponding Author: Hugh S. Taylor M.D
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Department of Obstetrics, Gynecology and Reproductive Sciences Yale University School of Medicine 333 Cedar Street, FMB 329K New Haven Connecticut, 06520 USA Email: [email protected]
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Stem Cells and the Reproductive System: Historical Perspective and Future Directions
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Cindy M.P. Duke and Hugh S. Taylor Department of Obstetrics, Gynecology and Reproductive Sciences, Yale School of Medicine, New Haven Connecticut, USA
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Abstract
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Recent findings in stem cell biology have presented new perspectives and opportunities for the treatment of reproductive disease. In a departure from the long held dogma of embryologically fixed numbers of oocytes, current literature suggests that human ovaries contain stem cells which form new oocytes even in adulthood and that these stem cells can be cultured in vitro to develop into mature oocytes. These findings have provided new hope and broader options for fertility preservation. Evidence of endometrial regeneration by bone marrow stem cells in endometrial tissue of women who received bone marrow transplant highlight potential for the novel treatments of uterine disorders and supports new theories for the etiology of endometriosis ectopic transdifferentiation of stem cells. Further, endometrial derived stem cells have been demonstrated to be useful in the treatment of several chronic and often debilitating diseases, including Parkinson’s Disease and Diabetes. Other cells that may present future therapeutic benefits for a myriad of disease states include placental and fetal cells which enter maternal circulation during pregnancy and can later promote parenchymal regeneration in maternal tissue. These findings highlight novel functions of the uterus and ovaries. They demonstrate that the uterus is a dynamic organ permeable to fetal stem cells capable of transdifferentiation as well as a renewable source of multipotent stem cells. While we still have much to understand about stem cells, their potential applications in reproductive biology and medicine are countless.
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Introduction
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Adult stem cells
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Stem cells have the capacity to renew themselves and differentiate into many different cell types that make up an organism[1] and can be described based on their characteristics and origin (Totipotent, pluripotent, mulitpotent or unipotent). Of these subtypes, much work has focused on pluripotent and mulitpotent cells known as Embryonic and Adult stem cells. Totipotent stem cells have the potential to differentiate into all the cells and tissues that make up an embryo and support the development of the fetus (for example, the zygote, or fertilized egg). Pluripotent stem cells have the potential to give rise to cells derived from all three germ layers (endoderm, ectoderm and mesoderm); embryonic stem (ES) cells are an example. Multipotent stem cells are capable of producing a limited range of differentiated cell lineages appropriate to their location; examples include somatic or adult stem cells. Unipotent stem cells are capable of differentiating along one lineage; these include epidermal stem cells and spermatogonial stem cells of the testis. ES cells are derived from the inner cell mass of the blastocysts from a developing embryo and were first isolated from the mouse model in 1981. They can be maintained in tissue culture under conditions where they can be propagated indefinitely as pluripotent ES cells [2,3]. ES cells are pluripotent stem cells. Research involving ES cells has opened up the possibility of using them as the foundation for "designer" tissue and organ engineering. However, some ethical considerations regarding the instrumental use of embryos for the isolation of stem cells are sharply debated and consequently curtailed much progress in research in this area. One alternative is to explore the use of non-embryonic stem cells as a source. In this paper, we will review non embryonic stem cell derivatives and their potential roles in the reproductive system.
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The main difference between blastocyst-derived (embryonic) pluripotent stem cells and multipotent stem cells from adult animals is in the number of types of differentiated cells that can be produced. Originally, adult stem cells were thought to have a limited potential for production of differentiated derivatives. However, studies on adult stem cell plasticity have questioned that view. For example, it has been demonstrated that neural stem cells can produce a variety of blood cell types including myeloid and lymphoid cells as well as early hematopoietic cells which are normally derived from the dermis; additionally, clones of individual cells can proliferate and differentiate in culture to produce neurons, glia, smooth muscle cells and adipocytes [4,5]. This is important because bone marrow is a mesodermal derived tissue consisting of various hematopoietic cellular components supported by a microenvironment composed of stromal cells embedded in a complex extracellular matrix. It contains hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs); the latter of which are particularly developmentally flexible and able to contribute usefully to several recipient organs. For example, bone marrow stem cells can generate cells bearing neuronal antigens and bone-marrow-derived or extra-hepatic stem cells can differentiate into hepatocytes [6-8]. These studies were amongst the first to show that some adult stem cells are not lineage restricted and thus, changed our view of Adult stem cells. Adult stem cells, in addition to their usual progeny in their organ of residence, are able to differentiate into other cell types in a new location; including cells not normally present in their organ of origin.
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The concept of plasticity of stem cells has presented the exciting possibility of repairing an individual’s failing organ by transplanting/locally delivering bone marrow cells. A number of studies have shown that bone marrow cells can in turn generate de novo myocardium in the heart, or when engrafted into the kidney, can differentiate into renal parenchymal cells, or new hepatocytes if engrafted to the liver [8-12]. These studies show that bone marrow derived stem cells may be involved and thereby can be engineered for use in the regeneration of damaged tissue. This concept then begs the question, “how can adult stem cells make lineage determination?” To this end, several theories have been proposed to explain the fate of stem cells and led to the notion of an Adult stem cell Niche.
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Adult stem cell niche
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The notion that tissue stem cells reside within a specific anatomical location termed niche arose from studies of transplanted hematopoietic progenitors which suggested that a subset of tissue cells and extracellular substrates can indefinitely house one or more stem cells and control them in an undifferentiated state in vivo [13,14]. In this system, secreted factors by other cell types, cell-cell interactions mediated by integral membrane proteins, Integrins and extracellular matrix are believed to influence the survival and overall development of these undifferentiated cells into committed cells. Control of lineage determination is believed to be governed by intrinsic and extrinsic factors. Intrinsic factors include: the proteins responsible for setting up asymmetric cell divisions, nuclear factors (e.g TGF ß superfamily, ß1 integrin receptor) controlling gene expression and chromosomal modifications in stem and nonstem daughters as well as clocks that may set the number of rounds of division within the transit amplifying population. Additionally, multiple positive and negative signals are received through cytokines and other receptors to further stabilize a committed pathway. Other extrinsic factors work through growth factors, stroma or external signals that control stem cell fate and make up the stem cell microenvironment, or niche. Adult stem cells occupy specific niches in tissues, where they respond to environmental cues provided by neighbor niche cells, such as epithelial niches and endodermal niches [15,16]. Some locations have more of these “inter niche” crosstalk. For example, near the base of the intestinal crypt in close association with niche myofibroblasts or in the hair bulge of the epidermis or in close association with Sertoli cells on the basement membrane of the seminiferous tubules.
Germline stem cells (GSCs) in the postnatal ovary in mammal GSCs are the self-renewing population of germ cells that serve as the source for gametogenesis. GSCs exist in diverse forms in many organisms, ranging from Drosophila melanogaster to mammals [17]. Spermatogonial stem cells are one kind of GSCs which maintain spermatogenesis throughout the entire reproductive life of a male and have been found in all metazoan species [17-19]. In Drosophila, GSCs, in females, maintain oocyte production in adult ovaries [20]. However, until recently, it was believed that ovaries of some vertebrates, especially those of mammals, did not contain self-renewing stem cells. In contrast to spermatogenesis, there was an apparent evolutionary disparity in female organisms. To date, the notion that new primary follicles can be formed in adult ovaries is a evolving topic with increasing evidence leaning away
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from the long held doctrine that female mammals are born with an embryologically predetermined finite population of non-growing primordial follicles and oocyte numbers decline throughout postnatal life, eventually leaving the ovaries devoid of germ cells [21-25]. In humans, this lifetime decline in oocyte number is accompanied by exhaustion of the follicle pool and menopause before the natural end of the female’s life [26]. Congruent to this dogma, it is believed that primordial follicles in the mammalian ovary are arrested in diplotene stage of meiosis I (oocytes), and are surrounded by a single, squamous layer of somatic cells [22]. However, in 2004, Johnson et al demonstrated the existence of proliferative GSCs that gave rise to oocytes and follicle production in the postnatal period of mammalian ovary [27]. In brief, to better define germ-cell dynamics in female mammals, they counted the numbers of healthy (non-atretic) and degenerating (atretic) follicles in ovaries of mice and showed that the numbers of non-atretic quiescent (primordial) and early growing (primary) prenatal follicles in single ovaries were higher than expected; also their rate of depletion in the immature ovary was less than anticipated. These results were consistent with past studies of follicle depletion in mice but unlike all previous studies of follicle depletion in other mammals [24]; juvenile and adult mouse ovaries continuing to possess mitotically active germ cells which continuously replenish the follicle pool. Morphological studies of the juvenile and adult mouse ovaries also revealed the presence of large ovoid cells resembling germ cells of fetal mouse ovaries, in the surface epithelial cell layer covering the ovary [27]. Immunohistochemical staining for mouse Vasa homologue (MVH), a gene expressed exclusively in germ cells of both vertebrate and invertebrate species, confirmed that these large ovoid cells were of a germline lineage [27]. Moreover, when small wedges of ovaries were then grafted from female mice with ubiquitous expression of green fluorescent protein (GFP) to wild-type ovaries, GFP-transgenic germ cells were observed to form follicles in wild-type ovaries several weeks after graft. These data established the existence of proliferative germ cells that sustain oocyte and follicle production in the postnatal mammalian ovary and demonstrated that oocytes are continuously formed in the adult ovary. This body of work not only represented a novel concept in ovarian biology of the mammalian female but also had significant clinical implications related to therapeutic expansion of the follicle reserve.
Origin of germ cells in adult ovary
The origin of oocytes (and primary follicles) in ovaries of adult mammalian females has been disputed for well over a century. In the 19th century, Weismann theorized that before embryonic cells were committed along specific pathways, a set of germ cells is set aside and are destined to give rise to the gametes. This theory was not questioned until many decades later when studies of mouse embryos, in which genetically marked cells were introduced at 4- and 8cell stage blastomere demonstrated that such cells can either become germ cells or somatic cells [31] suggesting that no specific germ cell commitment exists prior to implantation. During the 1990’s, it was demonstrated that egg and sperm cells (gametes) of the mouse differentiate from somatic lineage and cellular differentiation of grafted embryonic cells did not depend on the source of the grafts but rather where they have been placed [32,33]. Later studies by other groups using the mouse model confirmed that germ cells can be derived from mouse somatic cell lines in vitro and found that functional (embryo forming) mouse oocytes and sperm can be derived from mouse stem cells in culture [34-36].
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However, as previously mentioned, the paradigm that all primary follicles in adult mammalian (especially primates) females were formed during fetal life is still supported by a sizable number of scientists, primarily because of the lack of direct evidence on formation of new primary follicles in adult mammalian ovaries [37]. They argue that Weissmann’s theory may fit for invertebrates (C. Elegans and Drosophila) and some lower vertebrates (Zebra fish and frogs) but not mice and possibly mammals in general. In Human studies, Bukovsky et al. reported that germ cells can originate from surface epithelium (SE) cells which cover the tunica albuginea (TA) [28]. In these studies, ovaries from twelve adult women (average age of 32 years) were investigated by single, double, and triple color immunohistochemistry. The results show that cytokeratin (CK) positive mesenchymal cells in ovarian TA differentiate into SE cells by a mesenchymal-epithelial transition. Segments of SE directly associated with ovarian cortex form solid epithelial cords, which fragment into small epithelial nests descending into the lower ovarian cortex and associate with zona pellucida(ZP) positive oocytes strongly favoring the concept that the pool of primary follicles in adult human ovaries does not represent a static but rather a dynamic population of differentiating and regressing structures. An essential mission of such follicular turnover might be elimination of spontaneous or environmentally induced genetic alterations of oocytes in resting primary follicles. This same group also reported that new primary follicles are formed by assembly of oocytes within nests of primitive granulosa cells in the ovarian cortex. In this study, cells were scraped from the surface of ovaries and cultured for 5 to 6 days, in the presence or absence of estrogenic stimuli. The OSE cells cultured in medium differentiated into small granulosa cells as well as epithelial, neural and mesenchymal type cells. OSE cells cultured in medium containing Phenol red, which is estrogenic, differentiated directly into large cells with an oocyte phenotype and exhibited germinal vesicle breakdown, expulsion of the polar body, and surface expression of zona pellucida proteins, i.e. characteristics of secondary oocytes. These results show that granulosa cells and oocytes may develop directly from cultured OSE cells derived from adult human ovaries; these studies also confirmed in vivo observations that adult human ovaries contain mesenchymal cells which are bipotent progenitors, able to become both primitive granulosa and germ cells. Recent work by the Tilly group has also shown that GSCs from human female ovaries can be isolated, xenografted and propagated to oocytes in vivo [38, 39]. These data suggest the possibility of developing new strategies for the egg preservation and treatment of female infertility and importantly, Stem cell transfer from the fetus
The presence of fetal cells in maternal circulation was first described more than a century ago and has been repeatedly confirmed by many investigators [40, 43]. Fetal cells enter the maternal circulation during all pregnancies and may persist in maternal blood and tissue for decades, thus creating a state of physiologic microchimerism in the parous woman [41]. Circulating materno-fetal cells are detected as CD34 and CD38 positive cells and represent progenitor cells derived from the fetus [41,42]. The observations of feto-maternal traffic and maternal organ chimera raised the question of what could be the role of fetal stem cells in the mother’s body? Microchimeric cells of fetal origin have been identified in the peripheral blood of patients with autoimmune diseases such as systemic sclerosis (SSc) [44]. However, whether these cells are integrally involved in the pathogenesis of SSc, or if fetal microchimeric cells are just an associated marker of inflammation is unclear. Increased numbers of microchimeric fetal
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Stem Cell within the Uterus
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cells have also been identified in some pregnancy specific diseases: pre-term labor, preeclampsia and aneuploidy [45]. Even in these instances, there is speculation that the increased number of fetal microchimeric cells in the maternal circulation is more a reflection of the abnormalities within the structure of the placenta rather than a direct result of the disease process. In 2001, a team led by Bianchi discovered that male cells, presumably from their sons, were seen in thyroid sections in women [46]. Essentially, male cells were seen individually or in clusters in all (including inflammatory) thyroid disease biopsies that were examined. In one patient with a progressively enlarging goiter, fully differentiated male thyroid follicles were noted to be closely attached to and indistinguishable from the rest of the thyroid. Later work by this group, reported that XY+ microchimeric cells in maternal tissue, acquired most likely through pregnancy, expressed leukocyte, hepatocyte and epithelial markers [47] suggesting that pregnancy may result in the physiologic acquisition of a fetal cell population with the capacity for multi-lineage differentiation. This study also showed that hepatocytes of fetal stem cell origin were identified in liver tissue of one woman with liver injury and in another woman following hepatic transplantation. Similar findings have been noted in non-human studies suggesting that in a state where the tissue injury is chronic, fetal cell microchimerism may be established more frequently or easily, and also suggests that microchimeric cells are involved in tissue repair. Although the functional contribution of these fetal-derived cells remains to be determined, the possibility that newly implanted or persistent fetal stem cells may promote tissue regeneration in maternal disease states in novel and exciting.
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The mammalian uterine endometrium is one of the most dynamic human tissue types and sites. It consists of a glandular epithelium and stroma that are completely renewed in each monthly menstrual cycle. Endometrial stem cells are thought to reside in the basalis layer and serve as a source of cells that differentiate to form the endometrium. Cyclic endometrial renewal depends on a small pool of tissue-specific multipotential stem cells [48]. Under systemic hormonal changes, stem cells migrate and give rise to a group of progenitor cells that become committed to specific types of differentiated cells, e.g. epithelial, stromal and vascular. These endogenous stem cells allow the rapid regeneration of the endometrium necessary to support pregnancy [49]. Disorders of these endometrial stem cells may contribute to common proliferative disorders such as endometriosis and cancer. Work from our group has demonstrated, in a murine model, the ability to differentiate endometrial stromal stem cells (ESSC) into morphologically and functionally glucose responsive insulin secreting cells that are indistinguishable from pancreatic beta cells. This provides new possible therapeutic strategies and applications for treatment of diabetes (e.g use in augmentative therapies to achieve better glycemic control if not euglycemia) [50]. Phase I study designs are in progress to further assess the role of this treatment in humans. Other novel work being undertaken by our group include the differentiation of human derived endometrial stem cells into dopaminergic neurons capable of restoring dopamine levels in a murine Parkinson’s disease model; further primate studies evaluating clinical responses to this therapy are currently underway [51]. The uterus is a particularly rich source of stem cells and one that is easily
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accessible. Uterine stem cells are present after menopause and may be banked from hysterectomy specimens or menstrual blood for use in men or women without a uterus. Another potential source of endometrial cells is the bone marrow. Our group has provided novel evidence of endometrial regeneration in bone marrow transplant recipients who received marrow from a single-HLA antigen mismatched bone marrow transplant for leukemia. Donor-derived endometrial epithelial cells and stromal cells were detected in endometrial samples of bone marrow recipients by RT-PCR and immunohistochemistry. Cyclic mobilization of bone marrow derived stem cells may be a normal physiologic process [52]. These findings have potential advantages for the treatment of many uterine disorders including Asherman’s syndrome. It also defines a new theory for the etiology of endometriosis - ectopic transdifferentiation of stem cells. We have shown that bone marrow derived stem cells can contribute to endometriosis, perhaps explaining the occurrence of disease outside of the peritoneal cavity. Placenta and stem cells
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During development, blood begins to form not only in the embryo itself, but also in the placenta [53,54]. Experiments in mice revealed that the placenta harbors a large supply of hematopoietic (blood-forming) stem cells. These cells, which appear very early in development, are able to generate more blood stem cells and can give rise to a complete hematopoietic system when transplanted into an adult. Unlike other sites where blood stem cells are found during embryonic development, such as the liver, the stem cells in the placenta can increase in number without giving rise to mature, specialized cells. Over the last 30 years, colonization has been a long-accepted theory which proposes that the yolk sac was the sole source of hematopoiesis in the mammalian embryo. According to this theory, one yolk sac-derived hematopoietic stem cell (HSC) colonized fetal liver to initiate definitive hematopoiesis and subsequently colonize bone marrow at the neonatal stages to support adult hematopoiesis [55]. However, in the 1990s, accumulating evidence pointed to another site of hematopoiesis in the aorta-gonad-mesonephros (AGM) of mouse embryos [56]. Later studies also indicated that the placenta contains numerous clonogenic hematopoietic progenitors; suggesting that the placenta may function as a hematopoietic organ during development. Other studies have simultaneously reported that pluripotent HSCs can be detected in the placental labyrinth region in MDI gestation of the murine embryo [53,54]. The onset of HSC activity in the placenta coincides with that in the AGM region and the yolk sac. However, the expansion of HSC pool size in placenta is greater than 15-fold than in AGM. The expansion of the HSC pool in the placenta occurs prior to and during the initial expansion of HSCs in the fetal liver. The size of the placental HSC pool diminished while the HSC pool in the fetal liver continued to expand. These data suggest that placenta is another site contributing to the establishment of the mammalian definitive hematopoietic system. A common feature of these three hematopoietic sites, yolk sac, AGM, and placenta, is that the induction and generation of hematopoietic cells coincides with that of endothelial cells [57]. Indeed, coexpression of endothelial cell markers CD31/CD34 and hematopoietic stem cell markers Kit/CD34 (and Sca-1, partially) is found in the hematopoietic cells in the yolk sac [54,58,59]. HSCs derived from different sites have different functional capacities. These differences in developmental potency may result from differences in their inherited intrinsic programs or may be due to different microenvironmental signals. Unlike the adult HSC niche, which imposes a restriction signal for
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HSC proliferation, the placenta niche promotes rapid expansion but without inducing myeloerythroid lineage differentiation, and therefore strongly suggests that the placental labyrinth provides crucial niche signals to induce fast expansion of HSCs. Is there a Relationship between Ovarian Cancer and Stem Cells?
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As early as 2001, it was hypothesized that all stem cells may be regulated by similar selfrenewal signaling pathways. Moreover, one leading hypothesis is that tumors may originate from the transformation of normal stem cells; ergo cancer cells may include cancer stem cells---rare cells with indefinite potential for self-renewal that drive tumorigenesis [60]. Stem cells have the ability to renew themselves and cancer can be thought of as a disease of dysregulated selfrenewal. Therefore, it is highly probable that there are common mechanisms such - as telomerase activity - for achieving these processes. Telomeres are maintained by the novel ribonucleoprotein enzyme telomerase. Telomerase appears to be reactivated in almost all human cancers but is repressed in most somatic human cells; leading to telomere loss during replicative aging in vivo and in vitro. Telomerase activity prevents the replication-dependent loss of telomere repeats and prevents cellular senescence in highly proliferative germline cells in early embryonal tissues and in many cancers. Significant levels of telomerase have been found in CD34+CD38 + human hematopoietic progenitor cells [61]. Many pathways that are classically associated with cancer may also regulate and preserve normal stem cell development; these include signaling pathways (Wnt, Notch, Sonoc hedgehog (Shh)), the HOX family of regulatory genes and the anti-apoptotic oncogene bcl-2 [62-67]. Ovarian cancer is an extremely aggressive disease and the cellular mechanisms underlying the increasing aggressiveness associated with ovarian cancer progression are poorly understood. In 2005, a study reported that lineage infidelity of epithelial ovarian cancers (EOCs) is controlled by HOXA9, HOXA10 and HOXA11 genes that specify regional identity in the reproductive tract [68]. Several HOX genes, including HOXA9 and HOXA10, are expressed in primitive hematopoietic cells, implying a role in early hematopoietic differentiation [69]. It is believed that HOXA9, HOXA10 and HOXA11 are not expressed in normal ovarian surface epithelium (OSE) but rather by a group of OSE in adult ovary is a source of germ cells. Therefore it is possible that HOX genes expressed in OSE cells play a role in cell differentiation and may even be a cancer stem cell. Another report showed stem cell transformation can be the underlying cause of ovarian cancer [70]. One study demonstrated direct evidence that the aggressiveness of human ovarian cancer may be a result of transformation and dysfunction of stem cells in the ovary [70]. They showed that a single tumorigenic clone was isolated among a mixed population of cells derived from the ascites of a patient with advanced ovarian cancer and during the course of the study, another clone underwent spontaneous transformation in culture, providing a model of disease progression. Both the transformed clones possessed stem cell-like characteristics and differentiated to grow in an anchorage-independent manner in vitro (as spheroids) although further maturation and tissue-specific differentiation was arrested. Significantly, tumors established from these clones in animal models are histologically similar to those in the human disease and even on serial transplantation, continue to establish tumors; thereby confirming their identity as tumor stem cells. These findings suggest that stem cell transformation can be the underlying cause of ovarian cancer and continuing stochastic events of
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stem and progenitor cell transformation define the increasing aggression that is characteristically associated with the disease. Conclusion
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Our understanding of stem cells and their specific existence and role in the reproductive system has expanded significantly in the past 20 years and our studies of stem cell biology has already led to significant advances in therapeutic options including oocyte cryopreservation. However, many key questions remain. Radiotherapy, high-dose chemotherapy and bone-marrow transplantation have resulted in an increased number of long-term cancer survivors. Since the ovaries and germ cells are very sensitive to cytotoxic treatment, especially to alkylating agents and radiation, ovarian failure and infertility are common side-effects [71]. For young female cancer survivors, as a result of many advances in stem cell research, there are now several potential treatments available to avoid premature sterility. The methods for preserving fertility include oocyte cryopreservation, embryo cryopreservation and cryopreservation of ovarian cortical tissue. Our expanding knowledge on the broad niches and diverse applications for stem cells within the different organs of the female reproductive system present many exciting targets for their use not only in advanced reproductive techniques for the prevention and treatment of infertility but also for other chronic disease states like Diabetes and Parkinson’s Disease. Additionally, the discovery and refined ability to retrieve and differentiate endometrial stem cells presents exciting possibilities for using a renewable and relatively easily accessible niche (in female patients at least) for targeting these therapeutic strategies.
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