Comparison of differentiation potential of ovarian surface epithelial stem cells into Oocyte-like cells between human- and mice-derived cells.

Malignant transformation of the ovarian surface epithelium (OSE) accounts for most ovarian carcinoma. Detection of preneoplastic changes in the OSE leading to overt malignancy is important in prevention and management of ovarian cancer. We identified OSE proteins with altered expression derived from women with a family history (FH) of ovarian and/or breast cancer and mutations in the BRCA1 tumor suppressor gene. Proteins from SV-40-transformed FH-OSE cell lines and control OSE lines derived from women without such histories (non-family history) were separated by two-dimensional PAGE. Gels were analyzed, a protein data base was created, and proteins were characterized according to their molecular weight, isoelectric point, and relative abundance. Mass spectrometry was performed on tryptic protein digests, and data bases were searched for known proteins with the same theoretical tryptic peptide masses. Several proteins showed altered expression in the FH-OSE cells. Beta-tubulin and to a lesser extent ubiquitin carboxyl-terminal hydrolase and glyoxalase 1 appeared to be up-regulated. In contrast, proteins suppressed in FH lines include the 27-kDa heat shock protein, translationally controlled tumor protein, and several proteins associated with actin modification such as actin prepeptide, F-actin capping protein alpha subunit, and cofilin. Sequencing of several cofilin gel spots revealed phosphorylation of serine 3, a post-translational modification associated with decreased actin binding and cytoskeletal reorganization. Two-dimensional Western blots probed with cofilin antibody showed multiple protein spots with isoelectric points of 6-9 pH units. Blots of one-dimensional gels showed a significant reduction in cofilin expression in three FH lines when compared with three non-family history lines (p < or = 0.05). Identification of these and other OSE proteins may be useful in detecting changes suggestive of increased risk of developing preneoplastic disease and defining the possible role(s) of the BRCA1 gene in regulation of OSE cell function.

Ovarian cancers present as three different types, epithelial, germ cell, and stromal, with epithelial cancers consisting ~90% of cases. As a result, it is believed that the majority of cancers that develop in the ovary arise from the ovarian surface epithelium. Previous studies have shown that exposure of mice to exogenous estradiol (E2) causes preneoplastic changes in ovarian surface epithelial (OSE) cell morphology and accelerates ovarian tumor onset. Clinically, estrogenic compounds are used for treatments such as hormone replacement therapy for relief of menopausal symptoms or as a form of contraceptive such as birth control pills. However, women using estrogen-only hormone replacement are at higher risk of ovarian cancer. Thus, the aim of this project is to determine the mechanisms by which estrogen has detrimental effects that promote an increased risk of ovarian cancer, specifically by elucidating the actions of E2 on the OSE. The pathways controlling the premalignant transformation are unknown, but exogenous E2 promote stratification and apparent loss of cellular polarity in the OSE layer. We hypothesized that this change in phenotype may be due to decreases in disabled-2 (Dab2), as Dab2 is required for epithelial cells to maintain polarity and a genome-wide screen has identified estrogen response elements in regions proximal to the transcriptional start site of Dab2 in mice and humans. Dab2 is an adaptor protein that is found in a variety of tissues and is highly expressed in breast and ovarian tissue but its expression is lost in the majority of ovarian carcinomas. To determine if Dab2 mediates the actions of E2, we examined the effects of E2 exposure on Dab2 expression (by qPCR) in cultures of normal mouse OSE (mOSE), SV40 T antigen-immortalized mOSE, and in a mouse ovarian cancer cell line (MASE). None of the cell lines showed a change in Dab2 expression in response to E2 over a range of doses (0-1000 nM) and treatment periods (0-72 hours). To determine the effects of E2 exposure on Dab2 expression in vivo, SCID mice were intraperitoneally injected with MASE cells and inserted subcutaneously with a 6o-day timed release 0.25 mg E2 pellet. Tumors showed a significant 8-fold decrease in Dab2 mRNA expression in response to E2 treatment. Tumors from the tgCAG-LS-TAg transgenic model of ovarian cancer also showed a trend for E2 suppression of Dab2 expression. Immunohistochemical detection of Dab2 shows that it is present in normal human and mouse OSE, but is lost in areas of stratified mOSE that occur at higher incidence after 60 days of exogenous E2 treatment. These results demonstrate that E2 exposure decreases Dab2 expression in both OSE and ovarian cancers in vivo, but not in vitro. The lack of response in vitro suggests that growing OSE cells on plastic is not a good model to study changes in mOSE polarity, a result that is consistent with previous studies using mammary epithelial cells. Current experiments are investigating alternative culture systems to develop a better model for the study of OSE cell polarity and epithelial cell architecture.

The normal ovarian surface epithelium (OSE) is a primitive epithelium made up by a single layer of mesothelial-type epithelial cells. When these cells get trapped in the ovarian stroma, expression of epithelial specific markers, such as E-cadherin, are induced. Most epithelial cells are also characterized by the ability to form tight junctions (TJ). Incomplete TJ have earlier been demonstrated in the OSE by electron microscopy studies. We have investigated expression and localization of the TJ proteins ZO-1, occludin, and claudin-1 in tissue biopsies from normal human ovaries and OSE in culture. The dynamics of TJ formation were studied in human OSE cultured on porous filters in culture inserts by measuring trans epithelial resistance (TER) including Ca(2+) switch experiments. Confluent OSE cells were also analyzed by electron microscopy. The results show that normal human OSE has expression of all three TJ proteins investigated. These proteins, ZO-1, occludin, and claudin-1, were localized to OSE cell borders both in ovarian biopsies and in cultured OSE. There was no difference in this regard between fertile and postmenopausal women. Cells in culture were polarized and presented junctional complexes seen by electron microscopy. In the Ca(2+) switch experiments, removing free Ca(2+) transiently, TER decreased significantly (P < 0.05) in the Ca(2+)-free group compared with nontreated OSE. TER was fully restored after 24 h. N-cadherin but not E-cadherin was expressed in the OSE and localized to the cell borders. We conclude that normal human OSE express and form functional TJ both in vivo and vitro. This report also describes a method to study the influence of ovarian-derived mediators on TJ in cultured OSE.

The majority of ovarian cancers (>90%) are believed to derive from the ovarian surface epithelium (OSE); a single layer covering the entire surface of the ovary. At ovulation, the OSE cell layer undergoes an inflammatory response, involving cell death and growth, in order to overcome ovarian surface rupture. Abnormalities during these processes are believed to contribute to the development of tumours. Using primary cultures of OSE cells, we have compared anti-inflammatory and proliferative responses directly between human and ovine OSE cells to further establish the use of ovine OSE cells as a suitable model system for the study of human OSE cells. In order to compare effects of inflammatory stimulation, expression and activity of 11betahydroxysteroid dehydrogenase (11betaHSD) type 1 was measured in OSE cells in response to interleukin (IL)-1alpha. As previously identified in human OSE cells, treatment of ovine OSE cells with IL-1alpha stimulated a concomitant increase of 11betaHSD type 1 mRNA (31-fold; P <0.05) and oxoreductase activity, indicating an increased production of anti-inflammatory cortisol. To compare the growth of human and ovine OSE cells, OSE cell number was measured in response to treatment with gonadotropins or growth factors. In the presence of FSH, LH or human chorionic gonadotropin (hCG), ovine and human OSE cell growth was similarly stimulated >1.2-fold (P <0.05). In the presence of connective tissue growth factor (CTGF) and more significantly insulin growth factor I (IGF-I), human and ovine OSE cell growth was also similarly stimulated >1.2-fold (P <0.05) and >1.5-fold (P <0.01), respectively. The induction of both human and ovine OSE cell growth by IGF-I or hCG was further shown to be dependent on activation of the MAP kinase/extracellular-signal-regulated kinase (ERK) pathway. Stimulation of ovine OSE cell growth by hepatocyte growth factor (HGF) was similarly shown to be ERK-dependent; however, for human OSE cells, HGF only mildly stimulated ERK phosphorylation and failed to stimulate OSE cell growth. The demonstration that human and ovine OSE cells share similarities at the level of cell signalling, gene expression and cellular growth supports the use of ovine OSE cells as a suitable model for the study of human OSE cells.

Glucocorticoid receptor-mediated regulation of MMP9 gene expression in human ovarian surface epithelial cells

Ovarian surface epithelial (OSE) cells express multiple nuclear hormone receptor genes, including those encoding thyroid hormone and estrogen receptors (TR and ER, respectively). Ovarian cancer is hormone-dependent, and epidemiological evidence links hyperthyroidism, inflammation of the ovarian surface, and increased risk of ovarian cancer. The objective of this study was to assess T3 action on human OSE cells in vitro, asking 1) is there evidence for (pre)receptor control, 2) is T3 inflammatory, and 3) does T3 affect ER expression? Immunohistochemical analysis of fixed human ovaries and in vitro analysis of human OSE primary cell cultures were performed. Twelve women aged 29-50 yr (median, 41 yr) undergoing elective gynecological surgery for nonmalignant conditions were studied. Messenger RNA transcripts for TRalpha1, TRalpha2, TRbeta1, and T3 activating deiodinase 2 and inactivating deiodinase 3 were present in primary OSE cell cultures by RT-PCR. TRalpha and TRbeta proteins were also localized to intact OSE by immunohistochemistry. Treatment of OSE cell cultures for 24 h with T3 caused dose-dependent mRNA expression of inflammation-associated genes: cyclooxygenase-2, matrix metalloproteinase-9, and 11betahydroxysteroid dehydrogenase type 1, determined by quantitative RT-PCR. Finally, treatment with T3 dose dependently stimulated ERalpha mRNA expression without affecting ERbeta1 or ERbeta2. The ovarian surface is a potential T3 target. T3 exerts direct inflammatory effects on OSE cell function in vitro. OSE cell responses to T3 include increased expression of ERalpha mRNA, which encodes the ER isoform most strongly associated with ovarian cancer. This could help explain suggested epidemiological links between hyperthyroidism and ovarian cancer.

The ovarian surface epithelium (OSE) represents a minute fraction of the cell mass of the ovary but gives rise to over 80% of human ovarian carcinomas. No experimental models for the study of human OSE exist. To characterize OSE cells in culture, explants of ovarian surface from normal ovary of premenopausal women were grown on plastic, glass, and collagen gel in 25% fetal bovine serum/Waymouth's medium 752/1. About 25% of explants produced epithelial outgrowths. Morphologically, these outgrowths resembled OSE in vivo and endothelial and mesothelial cells in culture, but they differed from cultured ovarian stromal, granulosa, and luteal cells. Only OSE among ovarian cell types were intensely keratin positive by immunofluorescence. Keratin also distinguished OSE cells from the keratin-negative endothelial cells. Most but not all OSE colonies tested showed 17 beta-hydroxysteroid dehydrogenase (HSD) activity, which was absent in peritoneal mesothelial cells. Colonies from most patients were limited to a few millimetres and became stationary within a few weeks. Changes that accompanied cessation of growth included senescence, increased keratin content, or the formation of multicellular papillary aggregates. With time, OSE cells tended to assume a fibroblast-like morphology but remained keratin positive and continued to resemble OSE by scanning electron microscopy (SEM). Subcultured OSE cells persisted in a stationary keratin-positive form for many weeks. Throughout this study, all pavementlike epithelial outgrowths that were contiguous with an explant stained for keratin; thus, such colonies can be assumed to be OSE. Conversely, fibroblast-shaped cells may represent OSE as indicated by keratin content and SEM appearance. The methods presented here permit culture of normal human OSE under conditions in which the cells exhibit morphologic plasticity, variable 17 beta-HSD activity, and presence of keratin.

More than 95% of ovarian cancers originate from the epithelial cells on the surface of the ovary, which are termed ovarian surface epithelium (OSE). These OSE cells are modified peritoneal mesothelial cells separated from underlying ovarian surface stromal tissue by a basal lamina of dense collagenous connective tissue. Mesenchymal-epithelial cell interactions between stromal cells and OSE cells are postulated to be important for normal OSE biology and for the onset of ovarian cancer. Hepatocyte growth factor (HGF) is a mesenchymal-derived growth factor that mediates mesenchymal-epithelial cell interactions in a number of different tissues. The current study was an investigation of the expression and actions of HGF in normal OSE and ovarian cancer. Human epithelial cells from borderline and stage III ovarian cancer cases were found to express HGF protein in the epithelial cell component by immunocytochemistry analysis. The stromal cell component of human ovarian tumors contained little or no HGF immunostaining. Normal bovine ovaries have a similar physiology and endocrinology to human ovaries and are used as a model system to investigate normal OSE functions. HGF protein was detected in the OSE from both normal human and bovine ovaries. Adjacent ovarian stromal tissue contained light but positive HGF immunostaining. RNA was collected from normal bovine ovarian stromal cells to examine HGF gene expression. HGF transcripts were detected in cultured OSE and stromal cells by Northern blot analysis. Using a quantitative reverse transcription-polymerase chain reaction procedure, HGF gene expression was found to be high in freshly isolated OSE but low in freshly isolated stroma. Levels of HGF gene expression after culture of stroma increased. Observations indicate that normal OSE express high levels of HGF in vivo and in vitro. Expression of HGF by normal epithelial cells versus stromal cells was unexpected and suggests that HGF may be important in an autocrine regulation of OSE. HGF actions on normal OSE cells and ovarian cancer cells were investigated. HGF was found to stimulate the growth of normal OSE cells in a manner similar to such growth stimulated by epidermal growth factor. Two ovarian cancer cell lines, SKOV3 and OCC1, were also stimulated to grow in response to HGF. This observation suggests that HGF may be involved in sustaining growth of ovarian tumors. These results are the first to demonstrate the production and action of HGF in normal OSE cells and ovarian cancer cells. This appears to be an example of HGF production by an epithelial cell, such that a mesenchymal-epithelial mixed phenotype is present. The autocrine stimulation of OSE growth by the local production and action of HGF provides insight into how the OSE may develop abnormal growth characteristics involved in the onset and progression of ovarian cancer.

Negative Effect of Platelet Rich Plasma on the Differentiation of Synovium-Derived Mesenchymal Stem Cells

Evaluating differentiation potential of the human menstrual blood-derived stem cells from infertile women into oocyte-like cells

The present study was undertaken to detect, characterize, and study differentiation potential of stem cells in adult rabbit, sheep, monkey, and menopausal human ovarian surface epithelium (OSE). Two distinct populations of putative stem cells (PSCs) of variable size were detected in scraped OSE, one being smaller and other similar in size to the surrounding red blood cells in the scraped OSE. The smaller 1-3 μm very small embryonic-like PSCs were pluripotent in nature with nuclear Oct-4 and cell surface SSEA-4, whereas the bigger 4-7 μm cells with cytoplasmic localization of Oct-4 and minimal expression of SSEA-4 were possibly the tissue committed progenitor stem cells. Pluripotent gene transcripts of Oct-4, Oct-4A, Nanog, Sox-2, TERT, and Stat-3 in human and sheep OSE were detected by reverse transcriptase-polymerase chain reaction. The PSCs underwent spontaneous differentiation into oocyte-like structures, parthenote-like structures, embryoid body-like structures, cells with neuronal-like phenotype, and embryonic stem cell-like colonies, whereas the epithelial cells transformed into mesenchymal phenotype by epithelial-mesenchymal transition in 3 weeks of OSE culture. Germ cell markers like c-Kit, DAZL, GDF-9, VASA, and ZP4 were immuno-localized in oocyte-like structures. In conclusion, as opposed to the existing view of OSE being a bipotent source of oocytes and granulosa cells, mammalian ovaries harbor distinct very small embryonic-like PSCs and tissue committed progenitor stem cells population that have the potential to develop into oocyte-like structures in vitro, whereas mesenchymal fibroblasts appear to form supporting granulosa-like somatic cells. Research at the single-cell level, including complete gene expression profiling, is required to further confirm whether postnatal oogenesis is a conserved phenomenon in adult mammals.

Ovarian carcinomas are thought to arise in the ovarian surface epithelium (OSE). Although this tissue forms a simple epithelial covering on the ovarian surface, OSE cells exhibit some mesenchymal characteristics and contain little or no E-cadherin. However, E-cadherin is present in metaplastic OSE cells that resemble the more complex epithelia of the oviduct, endometrium and endocervix, and in primary epithelial ovarian carcinomas. To determine whether E-cadherin was a cause or consequence of OSE metaplasia, we expressed this cell-adhesion molecule in simian virus 40-immortalized OSE cells. In these cells the exogenous E-cadherin, all three catenins, and F-actin localized at sites of cell-cell contact, indicating the formation of functional adherens junctions. Unlike the parent OSE cell line, which had undergone a typical mesenchymal transformation in culture, E-cadherin-expressing cells contained cytokeratins and the tight-junction protein occludin. They also formed cobblestone monolayers in two-dimensional culture and simple epithelia in three-dimensional culture that produced CA125 and shed it into the culture medium. CA125 is a normal epithelial-differentiation product of the oviduct, endometrium, and endocervix, but not of normal OSE. It is also a tumor antigen that is produced by ovarian neoplasms and by metaplastic OSE. Thus, E-cadherin restored some normal characteristics of OSE, such as keratin, and it also induced epithelial-differentiation markers associated with weakly preneoplastic, metaplastic OSE and OSE-derived primary carcinomas. The results suggest an unexpected role for E-cadherin in ovarian neoplastic progression.

Initiation of meiosis is the most difficult aspect of inducing competent oocytes differentiation from human stem cells in vitro. Human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) were cultured with follicle fluid, cytokines and small molecule to induced oocyte-like cells (OLCs) formation through a three-step induction procedure. Expression of surface markers and differentiation potential of germ cells were analyzed in vitro by flow cytometry, gene expression, immunocytochemistry, western blotting and RNA Sequencing. To induce the differentiation of hiPSCs into OLCs, cells were firstly cultured with a primordial germ cell medium for 10 days. The cells exhibited similar morphological features to primordial germ cells (PGCs), high expressing of germ cell markers and primordial follicle development associated genes. The induced PGCs were then cultured with the primordial follicle-like cell medium for 5 days to form the induced follicle-like structures (iFLs), which retained both primordial oocytes-like cells and granulosa-like cells. In the third step, the detached iFLs were harvested and transferred to the OLC-medium for additional 10 days. The cultured cells developed cumulus-oocyte-complexes (COCs) structures and OLCs with different sizes (50–150 μm diameter) and a zona pellucida. The in vitro matured OLCs had polar bodies and were arrested at metaphase II (MII) stage. Some OLCs were self-activated and spontaneously developed into multiple-cell structures similar to preimplantation embryos, indicating that OLCs were parthenogenetically activated though in vitro fertilization potential of OLCs are yet to be proved. in vitro maturation of OLCs derived from hiPSCs provides a new means to study human germ cell formation and oogenesis.Graphical

Most epithelial ovarian tumors appear to arise from the ovarian surface epithelium (OSE). Even though it has been suggested that estrogen may be associated with ovarian tumorigenesis, the exact role of estrogen in the regulation of apoptosis in neoplastic OSE cells remains uncertain. Immortalized OSE (IOSE) cell lines were generated from human normal OSE. These cell lines represent early neoplastic (IOSE-29), tumorigenic (IOSE-29EC), and late neoplastic (IOSE-29EC/T4 and IOSE-29EC/T5) transformation stages from human normal OSE. The present studies demonstrated that both mRNAs and proteins of estrogen receptor (ER) alpha and beta were expressed in IOSE cell lines. No difference was observed in normal OSE and IOSE-29 cells, whereas treatment with 17beta-estradiol (E(2); 10(-8)-10(-6) M) resulted in an increased thymidine incorporation and DNA content per culture in IOSE-29EC cells. This effect of E(2) was attenuated with tamoxifen treatment (10(-6) M), the estrogen antagonist, suggesting that the effect of E(2) is mediated through specific ERs. There was no stimulatory effect on thymidine incorporation before day 6, but after 6 days of E(2) treatment, thymidine incorporation was significantly increased. Because the ratio of thymidine incorporation to DNA content per culture did not change, this E(2) effect does not appear to indicate stimulation of proliferation but, rather, inhibition of apoptosis. In addition, treatment with tamoxifen (10(-6) M) induced apoptosis up to 3-fold in IOSE-29EC cells, whereas cotreatment with E(2) (10(-8)-10(-6) M) plus tamoxifen attenuated tamoxifen-induced apoptosis in a dose-dependent manner. Both proapoptotic bax and antiapoptotic bcl-2 at messenger RNA (mRNA) and protein levels were expressed in IOSE cell lines. Interestingly, treatments with E(2) resulted in a significant increase of bcl-2 mRNA and protein levels (2- and 1.7-fold, respectively), whereas no difference was observed in bax mRNA level. Thus, E(2) may enhance survival of IOSE-29EC by up-regulating bcl-2, and antiapoptotic bcl-2 may be a dominant regulator of apoptotic pathway in these cells. In conclusion, the present study indicates that early neoplastic (IOSE-29), tumorigenic (IOSE-29EC), and late neoplastic (IOSE-29EC/T4 and T5) OSE cells expressed both ERalpha and ERbeta at the mRNA and protein levels. In addition, E(2) prevented tamoxifen induced-apoptosis through ERs. The mechanism of E(2) action may be associated with up-regulation of bcl-2 gene at mRNA and protein levels. These results suggest that estrogen may play a role in ovarian tumorigenesis by preventing apoptosis in tumorigenic OSE cells.

Mutations in the BRCA1 and TP53 genes are early genetic events leading to (hereditary) ovarian carcinoma. The human ovarian surface epithelium (OSE) is considered the tissue of origin of at least a subset of these tumours. Therefore, OSE cell cultures derived from women harbouring BRCA1 germline mutations can be a potential model to study hereditary ovarian carcinogenesis. In fact, previous in vitro studies indicate phenotypical differences between OSE from women with and without such germline mutations. Therefore, we have assessed whether differences in the expression of BRCA1 and p53 proteins in cultured OSE cells could contribute to these observations. Thirty-two OSE cultures derived from women harbouring a BRCA1 mutation (Predisposed OSE [POSE]) and ten cultures from women without a cancer predisposition (Non predisposed OSE [NPOSE]) were grown under standard conditions. Immunocytochemistry was performed to assess the expression of the BRCA1- and p53 proteins. Ki67 immunocytochemical expression was assessed to determine possible differences in cell cycle status between the two groups. In addition, to study whether wild type p53 was expressed, induction of p53 by cis-platinum was assessed by Western blot. On the basis of Ki67 expression, three different groups were analyzed. In the group with all cultures that expressed Ki67 no significant difference was observed in BRCA1 (P = 0.19) and p53 expression (P = 0.09). In the group with moderate to high Ki67 expression no difference in BRCA1 expression (P = 0.50) was observed. However, p53 expression was significantly lower in the case group (P = 0.01). The same observation for p53 was made in the group with only high Ki67 expression (P = 0.02). Furthermore, the expression of both BRCA1 and p53 positively correlates with Ki67 expression. In POSE and NPOSE, p53 was induced by cis-platinum to a similar extent. Our study indicates differences in the expression of p53, but not in the expression of BRCA1 between POSE and NPOSE. In addition, our findings do suggest the absence of losses of the wild type BRCA1 and p53 genes in the studied OSE cultures. This indicates that losses in these genes cannot account for observed differences in phenotypical traits between POSE and NPOSE, but that differences in levels of p53 might contribute.