Trophoblast differentiation, invasion and hormone secretion in a three-dimensional in vitro implantation model with rhesus monkey embryos.

BACKGROUND: The initiation of primate embryo invasion into the endometrium and the formation of the placenta from trophoblasts, fetal mesenchyme, and vascular components are essential for the establishment of a successful pregnancy. The mechanisms which direct morphogenesis of the chorionic villi, and the interactions between trophectoderm-derived trophoblasts and the fetal mesenchyme to direct these processes during placentation are not well understood due to a dearth of systems to examine and manipulate real-time primate implantation. Here we describe an in vitro three-dimensional (3-D) model to study implantation which utilized IVF-generated rhesus monkey embryos cultured in a Matrigel explant system. METHODS: Blastocyst stage embryos were embedded in a 3-D microenvironment of a Matrigel carrier and co-cultured with a feeder layer of cells generating conditioned medium. Throughout the course of embryo co-culture embryo growth and secretions were monitored. Embedded embryos were then sectioned and stained for markers of trophoblast function and differentiation. RESULTS: Signs of implantation were observed including enlargement of the embryo mass, and invasion and proliferation of trophoblast outgrowths. Expression of chorionic gonadotropin defined by immunohistochemical staining, and secretion of chorionic gonadotropin and progesterone coincident with the appearance of trophoblast outgrowths, supported the conclusion that a trophoblast cell lineage formed from implanted embryos. Positive staining for selected markers including Ki67, MHC class I, NeuN, CD31, vonWillebrand Factor and Vimentin, suggest growth and differentiation of the embryo following embedding. CONCLUSIONS: This 3-D in vitro system will facilitate further study of primate embryo biology, with potential to provide a platform for study of genes related to implantation defects and trophoblast differentiation.

Xanthohumol decreases Notch1 expression and cell growth by cell cycle arrest and induction of apoptosis in epithelial ovarian cancer cell lines.

OBJECTIVE: Notch1 signaling is active in ovarian cancer and is a promising pathway for new therapies in ovarian cancer. We have previously detected high Notch1 expression in ovarian tumors. Xanthohumol has been shown to inhibit cancer cell growth and invasion, including Kaposi's sarcoma, which also highly expresses Notch1. We hypothesized that the Notch1 signaling pathway is targeted by xanthohumol leading to decreased ovarian cancer cell growth. METHODS: SKOV3 and OVCAR3 cells were utilized. MTT growth assays were conducted following treatment with xanthohumol. Quantitative RT-PCR and Western blot analyses were conducted to assess Notch1 down-regulation. Luciferase reporter assays were performed to assess functional down-regulation of Notch1. Cell cycle analysis was performed by flow cytometry. RESULTS: Significant growth inhibition and down-regulation of Notch1 transcription and protein expression were found following xanthohumol treatment. In addition, xanthohumol increased Hes6 transcription and decreased Hes1 transcription, known downstream targets of Notch 1. These observations were associated with cell cycle inhibition as demonstrated by an increase in p21 expression and S and G2/M cell cycle arrest confirmed by an increase in phosphorylated cdc2. Furthermore, an increase in the apoptotic markers, cleaved caspase-3 and cleaved PARP were observed. CONCLUSION: Xanthohumol was a potent inhibitor of ovarian cancer cell growth, and our results suggest that xanthohumol may be influencing the Notch1 pathway. These findings suggest that xanthohumol could be useful as a therapeutic agent in ovarian cancer.

Expression of indoleamine 2,3-dioxygenase in the rhesus monkey and common marmoset.

Indoleamine 2,3-dioxygenase (IDO) catalyzes the initial and rate-limiting step of tryptophan degradation along the kynurenine pathway, and is hypothesized to limit tryptophan availability at embryo implantation and prevent maternal T cell activation at the maternal-fetal interface. To determine if nonhuman primates are suitable models for investigating the role of IDO during pregnancy, we defined the expression of IDO in the rhesus monkey and common marmoset with particular attention to the female reproductive tract and placenta. IDO mRNA was detected by RT-PCR in the rhesus monkey term placenta, lung, small intestine, spleen, lymph node and nonpregnant uterus, and also in the common marmoset placenta. Immunohistochemical analysis of rhesus monkey tissues localized IDO to glandular epithelium of nonpregnant endometrium and first trimester decidua, vessel endothelium of nonpregnant myometrium, first trimester decidua and term decidua, and villous vessel endothelium and syncytiotrophoblast of term placenta. Western blot analysis confirmed IDO in rhesus monkey term placenta. In the common marmoset, IDO was detected in glandular epithelium of the nonpregnant uterus and in the decidua at day 60 and day 128 of gestation. IDO activity was higher in rhesus monkey and common marmoset decidua and placentas than in other tissues. Confirmation of IDO expression in rhesus monkey and common marmoset uterine and placental tissues supports the hypothesis that this enzyme regulates immune activation at the maternal-fetal interface and demonstrates that nonhuman primates may provide models with distinct similarities to human placentation to study the role of IDO in maternal-fetal immune dialogue.

Modulation of cytokine and chemokine secretions in rhesus monkey trophoblast co-culture with decidual but not peripheral blood monocyte-derived macrophages.

PROBLEM: Decidual macrophages are thought to promote pregnancy success, in part through interactions with invading trophoblast cells in hemochorial placentation. However, the factors that constitute this regulatory cross talk are not well understood. METHOD OF STUDY: Rhesus monkey decidual and peripheral blood-derived macrophages were co-cultured with primary Rhesus trophoblasts. Macrophage functions including cell-surface marker expression, antigen uptake and processing, in vitro migration, and cytokine and chemokine secretions were evaluated. RESULTS: While most macrophage functions were unchanged by trophoblast co-culture, changes in the secretion of selected cytokines and the migration of trophoblasts were noted when decidual (but generally, not peripheral blood monocyte-derived) macrophages were cultured with trophoblasts. In addition, basal secretion differed significantly between peripheral blood-derived and decidual macrophages for a broad spectrum of cytokines. When trophoblasts were pre-treated with an anti-Mamu-AG antibody, 25D3, there was no change in cytokine or chemokine secretion. CONCLUSION: Macrophage cytokine expression can be modulated by trophoblast co-culture, but it remains unclear how Mamu-AG is involved.

Suppression of Mamu-AG by RNA interference.

PROBLEM: The role of placental major histocompatibility complex (MHC) class I molecules in pregnancy is not well understood. Mamu-AG, the rhesus monkey homology of human leukocyte antigen (HLA)-G expressed in the human placenta, was targeted for degradation by RNA interference (RNAi), a powerful tool to aid in determining gene function, to determine the effect that this knockdown has on NK cell function. METHOD OF STUDY: A series of potential target short hairpin RNA (shRNA) sequences to suppress Mamu-AG expression was screened, which identified an optimal sequence to use in transfection experiments. Knockdown in two different Mamu-AG-expressing cell lines was measured by flow cytometry. Cytotoxicity assays were performed to correlate Mamu-AG expression with NK cell cytotoxicity. RESULTS: Decreased expression of Mamu-AG by short interfering RNA (siRNA) (70-80%) in cell types tested was associated with increased lysis of Mamu-AG target cells. CONCLUSION: Target sequences have been identified that knocked down Mamu-AG expression by RNAi and increased lysis by NK cells. This supports the concept that NK cell receptors recognize this placental non-classical MHC class I molecule.

Generation of macrophages from peripheral blood monocytes in the rhesus monkey.

Macrophages are found in tissues throughout the body and are important immune cells, however, these tissue macrophages are difficult to collect and study. Therefore, the ability to differentiate macrophages from peripheral blood precursors is an important research tool. Macrophage differentiation has been well studied in humans, but differentiation in the non-human primate is poorly characterized. Using human models is not always feasible for invasive experimental studies and, therefore, developing reliable protocols for the non-human primate model is important. We describe a method to differentiate macrophages in vitro in the rhesus monkey by culturing adherent peripheral blood mononuclear cells for five days in RPMI-1640 supplemented with 1% human serum, M-CSF, and IL-1beta. The resulting cells had a distinct macrophage phenotype, the ability to secrete cytokines in response to LPS, and antigen uptake and processing capabilities.