No abstract

Precise cell fate decisions during differentiation of uterine tissues from the embryonic Müllerian duct are critical for normal fertility. Wnt-7a, a member of the Wnt family of secreted signaling molecules that can signal through a canonical beta-catenin pathway, is necessary for the correct differentiation of both anterior/posterior and radial axes of the uterus. In order to investigate the role of beta-catenin directly in mouse uterine development, we have generated mice that are deficient in beta-catenin expression in the embryonic Müllerian duct. We have found that conditional deletion of beta-catenin in the Müllerian duct mesenchyme before postnatal differentiation of the uterine layers results in a phenotype that is distinct from the phenotype observed by deletion of Wnt-7a. Shortly after birth, the uteri of the conditional mutants appear smaller and less organized. The uteri of adult conditional beta-catenin mutants are grossly deficient in smooth muscle of the myometrium, which has been replaced by adipose, a phenotype resembling human lipoleiomyoma. We also show that the adipocytes in the uteri of mice conditionally deleted for beta-catenin are derived from Müllerian inhibiting substance type II receptor-expressing cells suggesting that they share a common origin with the uterine smooth muscle cells. These results describe the first molecular evidence linking disruption of beta-catenin expression in mesenchymal cells with a switch from myogenesis to adipogenesis in vivo.

Ovulation induces cyclic rupture and regenerative repair of the ovarian coelomic epithelium. This process of repeated disruption and repair accompanied by complex remodeling typifies a somatic stem/progenitor cell-mediated process. Using BrdU incorporation and doxycycline inducible histone2B-green fluorescent protein pulse–chase techniques, we identify a label-retaining cell population in the coelomic epithelium of the adult mouse ovary as candidate somatic stem/progenitor cells. The identified population exhibits quiescence with asymmetric label retention, functional response to estrous cycling in vivo by proliferation, enhanced growth characteristics by in vitro colony formation, and cytoprotective mechanisms by enrichment for the side population. Together, these characteristics identify the label-retaining cell population as a candidate for the putative somatic stem/progenitor cells of the coelomic epithelium of the mouse ovary.

Cancer stem cells are proposed to be tumor-initiating cells capable of tumorigenesis, recurrence, metastasis, and drug resistance, and, like somatic stem cells, are thought to be capable of unlimited selfrenewal and, when stimulated, proliferation and differentiation. Here we select cells by expression of a panel of markers to enrich for a population with stem cell-like characteristics. A panel of eight was initially selected from 95 human cell surface antigens as each was shared among human ovarian primary cancers, ovarian cancer cell lines, and normal fimbria. A total of 150 combinations of markers were reduced to a panel of three-CD44, CD24, and Epcam-which selected, in three ovarian cancer cell lines, those cells which best formed colonies. Cells expressing CD44, CD24, and Epcam exhibited stem cell characteristics of shorter tumor-free intervals in vivo after limiting dilution, and enhanced migration in invasion assays in vitro. Also, doxorubicin, cisplatin, and paclitaxel increased this enriched population which, conversely, was significantly inhibited by Mülle-rian inhibiting substance (MIS) or the MIS mimetic SP600125. These findings demonstrate that flow cytometry can be used to detect a population which shows differential drug sensitivity, and imply that treatment of patients can be individualized to target both stem/progenitor cell enriched and nonenriched subpopulations. The findings also suggest that this population, amenable to isolation by flow cytometry, can be used to screen for novel treatment paradigms, including biologic agents such as MIS, which will improve outcomes for patients with ovarian cancer.anthrapyrazolone | chemotherapy resistance | stem/progenitor cell-enriched populations A s evidence is accumulating to indicate that cancer could be a stem cell disease (1-4), it is becoming increasingly important to be able to identify cancer stem/progenitor cells and to develop treatment modalities that specifically target the stem cell enriched population, coupled with treatments effective against the larger population not enriched for stem cells. The concept of cancer stem cells has opened new areas of research in carcinogenesis, but has the more immediate translational potential of uncovering new treatment targets.We previously identified somatic label-retaining cells with stem cell features in ovarian surface epithelium (5), and with others (6, 7), postulate that somatic stem cells or their immediate progenitors can revert to cancer stem cells (8). It is also possible that the stem cells may remain the same, but that signals which control the stem/progenitor cell activity may change. Several recent studies have demonstrated that cancer stem cells may confer chemotherapeutic resistant ovarian tumor growth and metastasis (2, 9).Ovarian cancer is diagnosed in approximately 25,000 new cases per year in the United States and is associated with a 50% mortality rate (10, 11); more than 90% of cases are epithelial in origin (12, 13). Epithelial ovarian cancers fall into four main subtypes: mucinous,...

Conditional deletion of ␤-catenin in the Mü llerian duct mesenchyme results in a degenerative uterus characterized by replacement of the myometrial smooth muscle with adipose tissue. We hypothesized that the mouse myometrium houses somatic smooth muscle progenitor cells that are hormonally responsive and necessary for remodeling and regeneration during estrous cycling and pregnancy. We surmise that the phenotype observed in ␤-catenin conditionally deleted mice is the result of dysregulation of these progenitor cells. The objective of this study was to identify the mouse myometrial smooth muscle progenitor cell and its niche, define the surface marker phenotype, and show a functional response of these cells to normal myometrial cycling. Uteri were labeled with 5-bromo-2-deoxyuridine

Recent data suggest that rare stem cell populations with the capacity to self renew and drive tumor formation are a feature of solid tumors. Several investigators have identified putative stem cells from solid tumors and cancer cell lines following isolation of a side population (SP) defined by dye exclusion. We investigated this parameter in our efforts to identify an endometrial cancer (EnCa) stem cell population. Multiple EnCa cell lines were assessed and verapamil sensitive SP and non-SP cells were isolated from two human EnCa cell lines. The functional significance of the SP and non-SP derived from AN3CA was evaluated in vitro and in vivo. SP cells proliferated at a significantly slower rate than the non-SP fraction, and a larger proportion of the SP cells were in the G 1 phase of the cell cycle as compared to the non-SP fraction. The SP fraction was more resistant to the chemotherapeutic agent paclitaxel. The SP comprised ~0.02% of the initial AN3CA cell population and this proportion of SP cells was maintained within the larger heterogeneous population following repeated passages of purified SP cells. These findings suggest that SP cells derived from the AN3CA cell line have the stem cell properties of low proliferative activity, chemoresistance and self-renewal. We also tested relative tumor formation activity of the SP and non-SP fractions. Only the SP fraction was tumorigenic. Additionally, we identified SP fractions in primary EnCa. Together these results are consistent with the hypothesis that EnCa contain a subpopulation of tumor initiating cells with stem like properties.

Examination of Müllerian inhibiting substance (MIS) signaling in the rat in vivo and in vitro revealed novel developmental stageand tissue-specific events that contributed to a window of MIS responsiveness in Müllerian duct regression. The MIS type II receptor (MISRII)-expressing cells are initially present in the coelomic epithelium of both male and female urogenital ridges, and then migrate into the mesenchyme surrounding the male Müllerian duct under the influence of MIS. Expression of the genes encoding MIS type I receptors, Alk2 and Alk3, is also spatiotemporally controlled; Alk2 expression appears earlier and increases predominantly in the coelomic epithelium, whereas Alk3 expression appears later and is restricted to the mesenchyme, suggesting sequential roles in Müllerian duct regression. MIS induces expression of Alk2, Alk3 and Smad8, but downregulates Smad5 in the urogenital ridge. Alk2-specific small interfering RNA (siRNA) blocks both the transition of MISRII expression from the coelomic epithelium to the mesenchyme and Müllerian duct regression in organ culture. Müllerian duct regression can also be inhibited or accelerated by siRNA targeting Smad8 and Smad5, respectively. Thus, the early action of MIS is to initiate an epithelial-tomesenchymal transition of MISRII-expressing cells and to specify the components of the receptor/SMAD signaling pathway by differentially regulating their expression. Development 133, 2359Development 133, -2369Development 133, (2006Development 133, ) doi:10.1242 Pediatric Surgical Research Laboratories, Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA. KEY WORDS: MIS, MIS type I/II receptor, SMAD, Epithelial-to-mesenchymal transition, RNA interference, Organ culture, Rat*Author for correspondence (e-mail: donahoe.patricia@mgh.harvard.edu) Accepted 30 March 2006DEVELOPMENT 2360 phosphorylated SMADs translocate into the nucleus complexed with SMAD4 and transcriptionally regulate specific sets of targeted genes (for reviews, see Massagué, 2000; Attisano and Wrana, 2002). MIS has been shown to activate SMAD1 (Gouédard et al., 2000;Clarke et al., 2001;Visser et al., 2001) and SMAD5 (Visser et al., 2001) in vitro, implying that R-SMADs 1, 5 and 8 may mediate Müllerian duct regression (Kobayashi and Behringer, 2003).The present study was undertaken to define when and where the MIS type I receptors are employed and to determine which SMADs transduce MIS signals in the urogenital ridge during Müllerian duct regression. We adapted RNA interference (RNAi) (Calegari et al., 2002;Sakai et al., 2003;Soutschek et al., 2004) to test functional activity of the components of the MIS signaling pathway in a urogenital ridge organ culture assay, which recapitulates the morphological events occurring in vivo during Müllerian duct regression . We show that ALK2-mediated MIS signaling induces migration of MISRII-expressing cells from the coelomic epithelium into the Müllerian duct mesenchyme, and thus is responsible for the sexual dimorp...

Uterine leiomyomas (also known as uterine fibroids) are the most common benign tumors of female reproductive tract and are the single most common indication for hysterectomies. Despite their high prevalence, the exact pathogenesis of these benign tumors is still unknown. One possible mechanism for leiomyoma formation is dysregulation of mesenchymal stem cell activity. Mesenchymal stem cells have been identified in both human and murine uteri and cancer stem cells have been identified in female reproductive malignancies. We compared stem/progenitor cell characteristics in both normal myometrium and the corresponding leiomyoma of patient's undergoing hysterectomies. We found that leiomyoma cells form fewer mesenchymal stem cell colonies and exhibit less Hoechst dye-excluding side population activity, which is a function associated with progenitor cells in other tissues, than cells isolated from normal myometrium. Whereas in normal myometrium we observed heterogeneous expression of CD90, a cell surface marker associated the with differentiation potential of uterine fibroblasts, in leiomyomas, we observed homogenous expression of CD90, suggesting leiomyoma cells are more terminally differentiated. Furthermore, we found that while leiomyoma cells could only produce CD90 expressing cells, both CD90+ and CD90− myometrial cells could reestablish their original heterogeneous CD90 profile when expanded in vitro. These results suggest that normal myometrium contains cells with stem/progenitor cell activities that are absent in leiomyomas.