The cell movements underlying the morphogenesis of the embryonic endoderm, the tissue that will give rise to the respiratory and digestive tracts, are complex and not well understood. Using live imaging combined with genetic labeling, we investigated the cell behaviors and fate of the visceral endoderm during gut endoderm formation in the mouse gastrula. Contrary to the prevailing view, our data reveal no mass displacement of visceral endoderm to extraembryonic regions concomitant with the emergence of epiblast-derived definitive endoderm. Instead, we observed dispersal of the visceral endoderm epithelium and extensive mixing between cells of visceral endoderm and epiblast origin. Visceral endoderm cells remained associated with the epiblast and were incorporated into the early gut tube. Our findings suggest that the segregation of extraembryonic and embryonic tissues within the mammalian embryo is not as strict as believed and that a lineage previously defined as exclusively extraembryonic contributes cells to the embryo.
Expression and function of the follicle-stimulating hormone receptor (FSHR) in females were long thought to be limited to the ovary. Here, however, we identify extragonadal FSHR in both the human female reproductive tract and the placenta, and test its physiological relevance in mice. We show that in nonpregnant women FSHR is present on: endothelial cells of blood vessels in the endometrium, myometrium, and cervix; endometrial glands of the proliferative and secretory endometrium; cervical glands and the cervical stroma; and (at low levels) stromal cells and muscle fibers of the myometrium. In pregnant women, placental FSHR was detected as early as 8-10 wk of gestation and continued through term. It was expressed on: endothelial cells in fetal portions of the placenta and the umbilical cord; epithelial cells of the amnion; decidualized cells surrounding the maternal arteries in the maternal decidua; and the stromal cells and muscle fibers of the myometrium, with particularly strong expression at term. These findings suggest that FSHR expression is upregulated during decidualization and upregulated in myometrium as a function of pregnancy. The presence of FSHR in the placental vasculature suggests a role in placental angiogenesis. Analysis of genetically modified mice in which Fshr is lacking in fetal portions of the placenta revealed adverse effects on fetoplacental development. Our data further demonstrate FSHB and CGA mRNAs in placenta and uterus, consistent with potential local sources of FSH. Collectively, our data suggest heretofore unappreciated roles of extragonadal FSHR in female reproductive physiology.
Extraembryonic endoderm (XEN) cells are stem cell lines derived from primitive endoderm cells of inner cell mass in blastocysts. These cells have self-renewal properties and differentiate into visceral endoderm (VE) and parietal endoderm (PE) of the yolk sac. Recently, it has been reported that XEN cells can contribute to fetal embryonic endoderm, and their unique potency has been evaluated. In this study, we have described the induction and characterization of new canine stem cell lines that closely resemble to XEN cells. These cells, which we designated canine induced XEN (ciXEN)-like cells, were induced from canine embryonic fibroblasts by introducing four transgenes. ciXEN-like cells expressed XEN markers, which could be maintained over 50 passages in N2B27 medium supplemented with inhibitors of mitogen-activated protein kinase p38 and transforming growth factor-beta 1. Our ciXEN-like cells were maintained without transgene expression and exhibited upregulated expression of VE and PE markers in feeder-free conditions. The cells differentiated from ciXEN-like cells using a coculture system showed multiple nuclei and expressed albumin protein, similar to characteristics of hepatocytes. Furthermore, these cells expressed the adult hepatocyte marker, CYP3A4 . Interestingly, these cells also formed a net structure expressing the bile epithelium capillary marker, multidrug resistance-associated protein 2. Thus, we have demonstrated the induction of a new canine stem cell line, ciXEN-like cells, which could form an embryonic endodermal cell layer. Our ciXEN-like cells may be a helpful tool to study the canine embryo development and represent a promising cell source for proceeding human and canine regenerative medicine.
The visceral endoderm (VE) is an epithelial tissue in the early postimplantation mouse embryo that encapsulates the pluripotent epiblast distally and the extraembryonic ectoderm proximally. In addition to facilitating nutrient exchange before the establishment of a circulation, the VE is critical for patterning the epiblast. Since VE is derived from the primitive endoderm (PrE) of the blastocyst, and PrE-derived eXtraembryonic ENdoderm (XEN) cells can be propagated , XEN cells should provide an important tool for identifying factors that direct VE differentiation. In this study, we demonstrated that BMP4 signaling induces the formation of a polarized epithelium in XEN cells. This morphological transition was reversible, and was associated with the acquisition of a molecular signature comparable to extraembryonic (ex) VE. Resembling exVE which will form the endoderm of the visceral yolk sac, BMP4-treated XEN cells regulated hematopoiesis by stimulating the expansion of primitive erythroid progenitors. We also observed that LIF exerted an antagonistic effect on BMP4-induced XEN cell differentiation, thereby impacting the extrinsic conditions used for the isolation and maintenance of XEN cells in an undifferentiated state. Taken together, our data suggest that XEN cells can be differentiated towards an exVE identity upon BMP4 stimulation and therefore represent a valuable tool for investigating PrE lineage differentiation. ► BMP signaling promotes extraembryonic endoderm stem (XEN) cell epithelialization. ► BMP4-treated XEN cells acquire an extraembryonic visceral endoderm identity. ► Effect of BMP4 is reversible and is antagonized by LIF signaling. ► Fluctuation of expression reveals a dynamic equilibrium upon BMP4 stimulation. ► XEN cells promote primitive erythroid progenitor (EryP) expansion.
Mesenchymal stem cells (MSCs) have a broad differentiation potential. We aimed to determine if MSCs are present in fetal membranes and placental tissue and to assess their potential to differentiate into neurogenic and mesodermal lineages. MSCs isolated from first and third trimester chorion and amnion and first trimester chorionic villi and characterized morphologically and by flourescence-activated cell sorting analysis. Their ability to mature under different culture conditions into various cells of mesodermal and neuroectodermal cell lines was assessed by immuno- and cytochemical staining. Independent of gestational age, cells isolated from fetal membranes and placenta showed typical MSC phenotype (positive for CD166, CD105, CD90, CD73, CD49e, CD44, CD29, CD13, MHC I; negative for CD14, CD34, CD45, MHC II) and were able to differentiate into mesodermal cells expressing cell markers/cytologic staining consistent with mature chondroblasts, osteoblasts, adipocytes, or myocytes and into neuronal cells presenting markers of various stages of maturation. The differentiation pattern was mainly dependent on cell type. Mesenchymal cells from chorion, amnion, and villous stroma can be differentiated into neurogenic, chondrogenic, osteogenic, adipogenic, and myogenic lineage. Placental tissue obtained during prenatal chorionic villous sampling or at delivery might be an ideal source for autologous stem cell graft for peripartum neuroregeneration and other clinical issues.
Abstract The ability of stem cells to differentiate into multiple cell lineages has ushered in exciting possibilities for stem cell based therapies that would be used to regenerate and repair damaged tissues and organs. Stem cells isolated from the embryo, fetus, adult and also the umbilical cord and placenta are being widely tested. Recent studies show that human fetal membranes also harbour cells with stem cell like properties. The amnion and chorion contain stromal cells that display characteristics and differentiation potential similar to that of adult, bone marrow derived mesenchymal stem cells. Amniotic epithelial cells share some of the features of pluripotent embryonic stem cells and multipotent mesenchymal stem cells and differentiate into multiple cell lineages in vitro . Amniotic epithelial cells also produce numerous substances that could augment tissue regeneration and repair. This review will focus on the stem cell like properties of stromal and epithelial cells derived from human fetal membranes and their potential use in stem cell based therapies.
Preterm delivery is the leading cause of perinatal mortality and morbidity. Current tocolytics target myometrial contractions, a late step in the labor cascade. Identifying earlier events in parturition may lead to more effective therapeutic strategies. We hypothesized that inflammatory events in decidua (the maternal-fetal interface), characterized by leucocyte infiltration, are an early event during term and preterm labor (PTL). Leucocyte abundance in decidua of human pregnancies was quantified following term labor and PTL (idiopathic and infection associated), in conjunction with investigation of temporal inflammatory events in rat uterus during the peri-labor period and in PTL induced by mifepristone. In human decidua, macrophage numbers were 4-fold higher in term labor (P < 0.01) and 2.5-fold higher in non-infection-associated PTL (P < 0.05) than in term nonlaboring samples. Neutrophil abundance was unchanged with labor but elevated in PTL with infection (5- to 53-fold increase; P < 0.01). T and NK cells were more abundant in idiopathic PTL than TL (P < 0.05). In rat, decidual macrophage infiltration increased 4.5-fold 12 h prior to labor and remained elevated during labor and early postpartum (P < 0.01). Decidual infiltration preceded that of the myometrium and was 4-fold higher (P < 0.01). In rat PTL, decidual macrophage numbers were also elevated (P < 0.01) and exceeded those of the myometrium (P < 0.05). These studies show for the first time that leucocytes infiltrate decidua during labor at term and preterm, supporting a role for leucocyte-derived inflammatory mediators in decidual activation. In the rat, this occurred prior to labor, suggesting it is an early event during parturition and thus a potential target for intervention.
Conceptus (embryo and associated extraembryonic membranes) implantation and development require a reciprocal biochemical and physical interactions between the extraembryonic membranes and the endometrium. However, the enzymatic antioxidative pathways controlling reactive oxygen species production at the endometrial–extraembryonic membrane interface early in pregnancy are not known. We aimed therefore to determine the content of malondialdehyde, as biomarkers of lipid peroxidation, and the activities of the major antioxidant enzymes, copper–zinc containing and manganese containing superoxide dismutases, catalase and glutathione peroxidase, in sheep extraembryonic membranes, caruncular and intercaruncular endometrium zones sampled at specific stages of pregnancy corresponding to the conceptus implantation (day 16) and the early post-implantation period (day 21). Malondialdehyde content in caruncular, intercaruncular and extraembryonic tissues was not different between stages of the pregnancy. Extraembryonic membranes demonstrated increased manganese containing superoxide dismutase and glutathione peroxidase activities, whereas catalase activity in these tissues decreased from day 16 to day 21. Caruncular tissues demonstrated increased manganese containing superoxide dismutase activity from day 16 to day 21. Intercaruncular tissues demonstrated increased copper–zinc containing superoxide dismutase, manganese containing superoxide dismutase and catalase activities from day 16 to day 21. The ovine extraembryonic membranes exhibit dynamic changes in enzymatic antioxidative pathways different from those of endometrial tissues during the transition from implantation to post-implantation period. This biochemical data provides novel insights into the developmental changes in antioxidative pathways of extraembryonic membranes and endometrium during early conceptus development.
During the peri-implantation and early placentation periods in pigs, conceptuses (embryo and its extra-embryonic membranes) undergo dramatic morphological changes and differentiation that require the exchange of nutrients (histotroph) and gasses across the trophectoderm and a true epitheliochorial placenta. Of these nutrients, arginine (Arg), leucine (Leu), and glutamine (Gln) are essential components of histotroph; however, little is known about changes in their total amounts in the uterine lumen of cyclic and pregnant gilts and their effects on cell signaling cascades. Therefore, we determined quantities of Arg, Leu, and Gln in uterine luminal fluids and found that total recoverable amounts of these amino acids increased in pregnant but not cyclic gilts between Days 12 and 15 after onset of estrus. We hypothesized that Arg, Leu, and Gln have differential effects on hypertrophy, hyperplasia, and differentiated functions of trophectoderm cells that are critical to conceptus development. Primary porcine trophectoderm (pTr) cells treated with either Arg, Leu, or Gln had increased abundance of phosphorylated RPS6K, RPS6, and EIF4EBP1 compared to basal levels, and this effect was maintained for up to 120 min. When pTr cells were treated with Arg, Leu, and Gln, low levels of pRPS6K and pEIF4EBP1 were detected in the cytosol, but the abundance of nuclear pRPS6K increased. Immunofluorescence analyses revealed abundant amounts of pRPS6 protein in the cytoplasm of pTr cells treated with Arg, Leu, and Gln. These amino acids also increased proliferation of pTr cells. Furthermore, when Arg, Leu, and Gln were combined with siRNAs for either MTOR, RPTOR, or RICTOR, effects of those amino acids on proliferation of pTr cells were significantly inhibited. Collectively, these results indicate that Arg, Leu, and Gln act coordinately to stimulate proliferation of pTr cells through activation of the MTOR-RPS6K-RPS6-EIF4EBP1 signal transduction pathway.
Preterm premature rupture of membranes is a leading contributor to maternal and neonatal morbidity and death. Epidemiologic and experimental studies have demonstrated that thrombin causes fetal membrane weakening and subsequently preterm premature rupture of membranes. Although blood is suspected to be the likely source of thrombin in fetal membranes and amniotic fluid of patients with preterm premature rupture of membranes, this has not been proved. is emerging as a pathogen involved in prematurity, which includes preterm premature rupture of membranes; however, until now, prothrombin production that has been induced directly by bacteria in fetal membranes has not been described. This study was designed to investigate whether exposure can induce prothrombin production in fetal membranes cells. Primary fetal membrane cells (amnion epithelial, chorion trophoblast, and decidua stromal) or full-thickness fetal membrane tissue explants from elective, term, uncomplicated cesarean deliveries were harvested. Cells or tissue explants were infected with live (1×10 , 1×10 or 1×10 colony-forming units per milliliter) or lipopolysaccharide ( J5, L-5014; Sigma Chemical Company, St. Louis, MO; 100 ng/mL or 1000 ng/mL) for 24 hours. Tissue explants were fixed for immunohistochemistry staining of thrombin/prothrombin. Fetal membrane cells were fixed for confocal immunofluorescent staining of the biomarkers of fetal membrane cell types and thrombin/prothrombin. Protein and messenger RNA were harvested from the cells and tissue explants for Western blot or quantitative reverse transcription polymerase chain reaction to quantify thrombin/prothrombin protein or messenger RNA production, respectively. Data are presented as mean values ± standard errors of mean. Data were analyzed using 1-way analysis of variance with post hoc Dunnett’s test. Prothrombin production and localization were confirmed by Western blot and immunostainings in all primary fetal membrane cells and tissue explants. Immunofluorescence observations revealed a perinuclear localization of prothrombin in amnion epithelial cells. Localization of prothrombin in chorion and decidua cells was perinuclear and cytoplasmic. Prothrombin messenger RNA and protein expression in fetal membranes were increased significantly by , but not lipopolysaccharide, treatments in a dose-dependent manner. Specifically, at a dose of 1×10 colony-forming units/mL significantly increased both prothrombin messenger RNA (fold changes in amnion: 4.1±1.9; chorion: 5.7±4.2; decidua: 10.0±5.4; fetal membrane: 9.2±3.0) and protein expression (fold changes in amnion: 138.0±44.0; chorion: 139.6±15.1; decidua: 56.9±29.1; fetal membrane: 133.1±40.0) compared with untreated control subjects. at a dose of 1×10 colony-forming units/mL significantly up-regulated prothrombin protein expression in chorion cells (fold change: 54.9±5.3) and prothrombin messenger RNA expression in decidua cells (fold change: 4.4±1.9). Our results demonstrate that prothrombin can be produced directly by fetal membrane amnion, chorion, and decidua cells. Further, prothrombin production can be stimulated by exposure in fetal membranes. These findings represent a potential novel underlying mechanism of –induced rupture of fetal membranes.