Oestrogen synthetase (aromatase) and hormone secretion in primary cultures of human placental trophoblast cells. Effects of cyclic AMP addition at the start of culture in attached and unattached cell populations

Term placental trophoblast cells, released by trypsin digestion of placental villi, purified on a Percoll gradient and grown in serum-containing medium, differentiate within 24 to 48 h in culture from mononucleated cytotrophoblast-like cells at the start of culture to highly multinucleated giant (syncytiotrophoblast-like) cells that are more active in hormonogenesis. To determine the changes in hormone biosynthesis and secretion that occur early in the trophoblast differentiation process in vitro, freshly isolated placental cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 20 per cent FBS, in the presence and absence of cAMP for up to 48 h. Cell attachment and growth, oestrogen synthetase (aromatase) activity in attached and unattached cells, and secretion of human chorionic gonadotropin (hCG) and progesterone were studied. The aromatase specific activity, low in freshly isolated cells, increased fourfold in attached cells by 3 h, and achieved a 10- to 15-fold increase by 40 to 48 h. In attached cells grown with cAMP, aromatase activity was further stimulated by about fourfold, relative to the control. The aromatase activity of the unattached cells removed from the culture dishes at various times up to 48 h showed a biphasic response: the activity decreased by 18 h and then increased back to the fresh cell levels. The effect of cAMP on aromatase in these unattached cells was manifested by a two-fold stimulation of activity by 18 h, relative to control unattached cells. Secretion of hCG from both attached and unattached cells remained at a low level (less than 200 ng/mg protein) in control cells; in the presence of cAMP, hCG secretion was stimulated by tenfold after 40 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibody reaction patterns in first trimester placenta: implications for trophoblast isolation and purity screening

The aim of this immunohistochemical and cytochemical study was to select specific antibodies to establish an efficient purification protocol for first trimester trophoblast and for subsequent purity screening of isolated trophoblast cells. The reactivity of antibodies to various cytokeratin filaments, glycoprotein CD9, fibroblast specific antigen (FSA), common leukocyte antigen CD45RB and macrophage antigens CD163, CD68 and CD14 were studied on cryosections of placental tissue. Among the cytokeratins tested, cytokeratin 7 was the only keratin filament type, which was not expressed in placental mesenchymal cells, but in all trophoblast subpopulations. Since anti-CD9, in addition to mesenchymal cells, also strongly labels extravillous cytotrophoblast cells, whereas the antibody to FSA only reacts with mesenchymal cells, anti-FSA is suitable as a depletion antibody for mesenchymal cells. Among the macrophage markers anti-CD163 was the most specific for Hofbauer cells. CD45RB was expressed on maternal and fetal leukocytes as well as on Hofbauer cells. Isolated first trimester placental cell preparations that have been collected from a density gradient contained up to 45 per cent non-trophoblast cells. Immunocytochemistry using antibodies to CK7, FSA, vimentin, CD45RB and CD163 demonstrated that subsequent immunodepletion with antibodies to CD45RB and FSA increased the purity of the trophoblast preparation to greater than 98 per cent.According to this study trophoblasts from first trimester placentae should be identified by cytokeratin antibodies specific for the isoform 7. Purification of isolated trophoblasts by density gradient alone does not result in a sufficient degree of purity.     

Placental protein 13 and decidual zones of necrosis: an immunologic diversion that may be linked to preeclampsia

We evaluated the role of placental protein 13 (PP13; galectin 13) in the process of trophoblast invasion and decidual necrosis. Immunohistochemical analysis for PP13, immune cells, human placental lactogen, cytokeratin, and apoptosis markers was performed on 20 elective pregnancy termination specimens between 6 and 15 weeks of gestation. Placental protein 13 was localized to syncytiotrophoblasts in the chorionic villi and to occasional multinucleated luminal trophoblasts within converted decidual spiral arterioles. Cytotrophoblasts, anchoring trophoblasts, and invasive trophoblasts did not stain for PP13. Extracellular PP13 aggregates were found around decidual veins associated with T-cell-, neutrophil- and macrophage-containing decidual zones of necrosis (ZONEs). We hypothesize that PP13 is secreted into the intervillus space, drains through the decidua basalis veins, and forms perivenous PP13 aggregates which attract and activate maternal immune cells. Thus, syncytiotrophoblast-derived PP13 may create a ZONE that facilitates trophoblast invasion and conversion of the maternal spiral arterioles.     

Human amniotic fluid transferrin stimulates progesterone production by human trophoblast cells in vitro

OBJECTIVE: During pregnancy transferrin plays a key role as an iron transport protein to serve the increased fetal demands of iron. Transferrin is also present in relatively high concentrations in amniotic fluid [6], showing a different glycosylation compared with serum transferrin. The biological function of human amniotic fluid transferrin (hAFT) is still unknown. In addition trophoblast cells also synthesise transferrin. Transferrin synthesised by the trophoblast shows a special glycosylation. We found identical carbohydrate structure of hAFT and trophoblast transferrin. We investigated the influence of hAFT on the progesterone-, cortisol- and hCG-release of trophoblasts in culture compared with the influence of human holo- and apo-serum transferrin on the release of these hormones. MATERIAL AND METHODS: Cytotrophoblast cells were prepared from human term placentae by standard trypsin-DNAse dispersion of villous tissue followed by a percoll gradient centrifugation step. When placed in culture, the trophoblasts were incubated with varying concentrations (50-300 micrograms/ml) of human amniotic fluid- and serum-transferrin. Unstimulated cells of each placenta used as controls. Culture supernatants were assayed for progesterone, hCG and cortisol by enzyme-immunometric methods. RESULTS: Our results show, that the release of progesterone increased in hAFT-treated cell cultures compared to untreated cell cultures. Holo- and apo-serumtransferrin did not show any effect on the progesterone release by trophoblast cells in vitro. Neither hAFT nor holo- and apo-serum transferrin had any effect on the cortisol- and hCG-release in vitro. CONCLUSIONS: Progesterone is a marker for differentiation of trophoblasts in syncytiotrophoblasts. Only hAFT stimulates the progesterone production. We suggest, that hAFT can modulate the endocrine function of trophoblast cells in culture by regulating progesterone production.     

Effects of various growth factors on the growth of trophoblast cells in long-term culture]

Trophoblasts taken from placental tissue of the 1st trimester and molar tissue, and GCH-1 (gestational choriocarcinoma cell line) cells were cultured in collagen coated dishes. The medium used was a mixture of DME and Ham's F-12 (1:1), containing EGF, PDGF, insulin, GM-CSF, IL-1, -2, -3, PGE1 and PGE2 in various concentrations. 3H-TdR uptake of the cultured cells was measured as a marker of cell growth. The growth of normal trophoblasts was enhanced by PDGF or insulin, and remarkably by EGF + PDGF + insulin + GM-CSF. The growth of molar trophoblasts was accelerated by EGF or insulin. Under these culture conditions, normal trophoblasts have been successfully cultured and maintained for 12 months and molar trophoblasts for 9 months to date. The cultured cells were identified with trophoblasts by immunohistochemical staining with CAM 5.2 monoclonal antibody. No effect of growth factors was observed in GCH-1 cells.     

Deep trophoblast invasion and spiral artery remodelling in the placental bed of the lowland gorilla

In contrast to baboon or rhesus macaque, trophoblast invasion in the human placental bed occurs by the interstitial as well as the endovascular route and reaches as deep as the inner myometrium. We here describe two rare specimens of gorilla placenta. In the light of recent findings in the chimpanzee, we postulated the occurrence of deep invasion in gorilla pregnancy. Tissues were processed for histology (PAS, orcein), lectin staining (Ulex europaeus agglutinin 1) and immunohistochemistry (cytokeratin 7/17, α-actin). A specimen of young but undetermined gestational age included deep placental bed tissue, showing interstitial and spiral artery invasion of the inner myometrium as well as the decidua. The cell density and depth of trophoblast invasion was equivalent to a human placental bed of 10-14 weeks. Intraluminal trophoblasts were not seen in any of the invaded vessels, allowing no definite conclusions about the origin of the intramural trophoblast and the time-course of spiral artery invasion. A different late second trimester placenta specimen showed scattered extravillous trophoblast in the basal plate and underlying decidua, as well as a remodelled spiral artery containing intramural trophoblast. Absence of inner myometrial tissue precluded assessment of invasion depth in this later specimen. Despite the limited material we can conclude that key aspects of trophoblast invasion are shared by the three hominid species: gorilla, chimpanzee and human.     

Studies of Mesenchymal Cells from 1st Trimester Human Placenta: Expression of Cytokeratin Outside the Trophoblast Lineage

A fibroblast cell strain that expressed cytokeratins 8 and 18 was isolated from explanted first trimester placenta. Its properties were consistent with an origin in the villous fibroblast-myofibroblast lineage, including expression of vimentin, smooth muscle alpha-actin and fibroblast surface protein. The cells grew rapidly in vitro, and exhibited tightly aligned bipolar morphology at confluence, absence of multi-nucleated cells, lack of secreted chorionic gonadotrophin, and absence of HLA G, placental alkaline phosphatase and pregnancy-specific beta-1 glycoprotein (SP1). On these criteria it was concluded they were not trophoblasts. On the basis of their morphology, cytokeratin expression and absence of CD34 and endoglin it was concluded they were not endothelial cells. They lacked desmin and smooth muscle myosin and so were not vascular smooth muscle cells. Further mesenchymal cell isolates were studied to determine the generality of these findings. Phenotypic heterogeneity was a consistent characteristic, but cytokeratin-positive cells were always present both in first trimester and term strains. Desmin was absent from almost all the cells isolated using the protocols employed, despite its occurrence in a significant subpopulation of cells in the villous stroma. Cytokeratins 8 and 18 can be observed in the stromal compartment of both first trimester and term placental villi. Cytokeratin has hitherto been regarded as a highly reliable marker for cells of the trophoblast lineage in vitro. These observations suggest that care should be taken in characterization of placental cell isolates; trophoblasts should be identified by the presence of cytokeratin 7 in preference to cytokeratin 8/18. The functional significance of cytokeratin expression in placental mesenchymal cells remains to be established.     

A fibrin matrix modulates the proliferation, hormone secretion and morphologic differentiation of cultured human placental trophoblast.

Term placental trophoblast epithelialize fibrin deposits attached to villi in vitro and trophoblast cultured on a fibrin matrix form an epithelial bilayer typical of the trophoblast layer on term villi. We compared the morphology of cells grown on fibrin with cells grown on substrates of type IV collagen, laminin, type I collagen, or Matrigel. We also used autoradiography, hormone assays, electron microscopy, and immunofluorescence to determine what functional activities were influenced by trophoblast-fibrin interactions. Cultured cellular trophoblast from term placentae differentiated to form syncytial trophoblast and to secrete estrogen, progesterone, and hCG in the presence or absence of matrices. Trophoblast proliferation was lower in cells grown on matrices and was inversely related to cell height after 24 h in culture. Cells grown on fibrin remained the tallest and had the lowest labelling index. Cells grown for 72 h on fibrin had the most dilated rough endoplasmic reticulum but the lowest media hormone levels. Only cells grown on a fibrin matrix formed a basal lamina-like structure at the trophoblast-substrate interface, and only a fibrin matrix facilitated trophoblast to form an epithelial bilayer in culture. However, this histology was not accompanied by a change in the amount of syncytial trophoblast formed by the cells grown on fibrin. The results suggest that a fibrin matrix uniquely modulates the trophoblast phenotype, away from the secretion of placental specific products like hCG in favour of a repair-oriented phenotype that forms basement membrane and a trophoblast bilayer.     

Establishment of mouse embryo culture system on collagen gel layer

In order to study the morphological features of mouse embryos in the early developmental stage, we first established an in vitro culture system applying a collagen gel layer, and then observed the morphology of the embryos cultured in this system. Embryos after hatching were attached to the collagen gel layer and grew to the egg cylinder stage. By means of morphological analysis of embryos cultured for 3 or 4 days, some interesting characteristics, such as processes and villi of the mural trophoblast, lacunae formation in the mural trophoblast, steroid synthesis of the mural and polar trophoblasts and desmosome or intermediate junctions between the mural and the polar trophoblasts, were revealed. Moreover, no morphological difference was observed between cells derived from the inner cell mass. From those findings, it has been concluded that the established culture system is useful in observing the morphology of early embryonal development and also in maintaining the viability of the embryo.     

Fas ligand expression by maternal decidual cells is negatively correlated with the abundance of leukocytes present at the maternal-fetal interface

The abundance of leukocytes at the maternal-fetal interface could influence the fate and invasion of extravillous trophoblasts. However, the mechanism(s) involved in determining the number of leukocytes present at the maternal-fetal interface as well as the nature of the interactions between invading fetal trophoblasts, maternal leukocytes and decidual cells are not well understood. In the present studies, we examined Fas ligand (FasL)/Fas expression at the maternal-fetal interface in human placental tissues of early pregnancy by immunohistochemistry. The types of cells and their localization were also characterized by specific cell markers (cytokeratin, vimentin and CD45 for trophoblast, decidual cells and leukocytes, respectively). The cells undergoing apoptosis and specific apoptotic trophoblasts were detected by TUNEL assay and M30 cytoDEATH immunostaining, respectively. Using single or double immunostaining, we found that FasL expression in decidual cells was negatively correlated with the number of Fas-expressing leukocytes in the same region. Furthermore, the density of leukocytes had an inverse relationship with the number of interstitial trophoblasts present in the same area. We observed also that extravillous trophoblasts are viable despite expressing Fas and being in close proximity to decidual cell-derived FasL. These data support our hypothesis that maternal decidual cell-derived FasL may be involved in preventing the recruitment of Fas-bearing leukocytes at the maternal-fetal interface through apoptosis induction by Fas/FasL interaction, thereby promoting trophoblast invasion.     

新型体外滋养细胞胎盘屏障模型(PCB-HPT)的建立和鉴定

目的:建立具有极性和完整性的体外滋养细胞胎盘屏障模型。方法:以永生化的、通过系统鉴定的人早孕滋养细胞株构建胎盘屏障模型(PCB-HPT)。采用细胞生长周期评估细胞屏障构建时间;采用透射电镜评估滋养细胞屏障的极性;采用上皮细胞电阻测量评估滋养细胞屏障的完整性。结果:该滋养细胞胎盘屏障模型采用0.4μm孔径、0.47cm2NUNC插入式细胞培养皿培养,建议小室接种细胞总数为6×104cells,细胞屏障构建完整的培养时间为3d,屏障可维持36～48h。光镜下细胞培养至融合后具有典型的"铺路石"样外观;透射电镜观察显示,屏障具有极性,滋养细胞屏障可形成紧密连接并产生较高的跨内皮阻抗(TER)达600Ω;证实该屏障具有完整性。结论:建立了具有极性和完整性的体外滋养细胞胎盘屏障模型,为研究妊娠期间病原体和物质跨越胎盘屏障机制研究提供了体外研究模型。     

Progesterone and human placental lactogen inhibit leptin secretion on cultured trophoblast cells from human placentas at term.

The placenta is an important source of leptin production that contributes to the state of hyperleptinemia observed in pregnant women. Moreover, the synthesis of leptin and its receptors by syncytiotrophoblast cells suggests a potential paracrine or autocrine action of leptin in the placenta. In the present study we examined the effect of gestational hormones, human chorionic gonadotropin (hCG), human placental lactogen (hPL), progesterone and estradiol, on in vitro leptin release by human term trophoblast cells in culture. Placentas at term were obtained immediately after delivery from mothers with uncomplicated pregnancies. Leptin levels were measured by enzyme-linked immunosorbent assay in culture media of trophoblasts maintained in monolayer culture for 24, 48 and 72 h with different hormonal treatments or placebo. Treatment with hPL and progesterone led to a time- and dose-dependent decrease in leptin release that was statistically significant after 24 h, with a maximal effect after 72 h of incubation. In contrast, incubation with estradiol and hCG did not have exhibit any effect on leptin secretion at any of the doses and times assayed in this work. The results obtained in this study support that leptin can be considered a gestational hormone implied in the endocrine function of the placenta and that its secretion is at least partially regulated by steroid and peptidic reproductive hormones in trophoblast cells in vitro.     

Isolation and characterization of trophoblast from murine placenta

A discontinuous density gradient centrifugation method, devised to isolate enriched populations of trophoblast from murine definitive placentae, is described. It is concluded that the isolated adherent cells are trophoblast on the basis of the following characteristics: they are fetally derived, as determined by their donor glucose phosphate isomerase phenotype in embryo transfer experiments; epithelial cells, as shown by the presence of cytokeratin filaments and the absence of vimentin; negative for the stage-specific embryonic antigen-I (SSEA-I); and capable of progesterone secretion. Initially, they grew as individual polygonal cells, tending to form tight confluent monolayers with poorly defined intercellular boundaries. They were mono- or binucleate and increased their nuclear size with time. After two days, giant cells appeared to be formed from binucleated cells by nuclear fusion, and multinucleated cells appeared forming syncytia. Some of these cells also seemed to form giant cells. A low percentage (1 to 10 per cent) of contaminating cells, mainly macrophage-like cells, was observed. The isolated cells were a mixture of alkaline phosphatase- (AP-)positive and AP-negative cells, with some of the latter having phagocytic capacity. All were Fc receptor-negative. The possible identity of these cells in relation to trophoblast in the intact placenta is discussed. This method of isolating and characterizing trophoblast cells from the definitive mouse placenta will be a useful tool for studying the biology and immunology of trophoblast.     

Human amniotic fluid glycoproteins expressing sialyl Lewis carbohydrate antigens stimulate progesterone production in human trophoblasts in vitro

BACKGROUND: Progesterone is thought to mediate immune modulator effects by regulating uterine responsiveness. The aim of the study was to clarify the effect of transferrin and glycodelin A (former name PP14) as sialyl Lewis X-expressing glycoproteins on the release of progesterone by trophoblast cells in vitro. METHODS: Cytotrophoblast cells were prepared from human term placentas by standard dispersion of villous tissue followed by a Percoll gradient centrifugation step. Trophoblasts were incubated with varying concentrations (50-300 microg/ml) of human amniotic fluid- and serum-transferrin as well as with glycodelin A. Culture supernatants were assayed for progesterone, human chorionic gonadotropin (hCG) and cortisol by enzyme immunometric methods. RESULTS: The release of progesterone is increased in amniotic fluid transferrin- and glycodelin A-treated trophoblast cell cultures compared to untreated trophoblast cells. There is no relation between transferrin and the hCG or cortisol production of trophoblast cells. CONCLUSION: The results suggest that sialyl Lewis carbohydrate antigen-expressing amniotic fluid glycoproteins modulate the endocrine function of trophoblasts in culture by upregulating progesterone production.     

The distribution of intermediate filament proteins, actin and desmoplakins in human placental tissue as revealed by polyclonal and monoclonal antibodies

The distribution of intermediate filament proteins (cytokeratin, vimentin, desmin), actin, and desmoplakins in various placental compartments was studied by immunofluorescence microscopy using polyclonal and monoclonal antibodies. Trophoblast cells (cytotrophoblast, syncytiotrophoblast, isolated trophoblast cells, trophoblastic giant cells) were strongly stained by all types of cytokeratin antibodies. Antibodies to desmoplakins revealed the presence of desmosomes at all membranes, except the basal membrane of cytotrophoblast cells, and at the basal as well as the lumen-oriented membrane of the syncytiotrophoblast. After disappearance of the cytotrophoblast cell layer the distribution of desmosomes in the syncytiotrophoblast was unaltered. Isolated trophoblast cells contained desmosomes around their entire circumference. Amnion epithelial cells were heterogeneous with respect to cytokeratin composition as revealed, for example, by polyclonal antibodies with a broad range of cytokeratin reactivity and by monoclonal antibodies to cytokeratin No. 18. With the latter, a heterogeneous staining of amnion epithelial cells was achieved. Desmosomes (spots reactive with desmoplakin antibodies) were present at the lateral membranes of the amnion epithelial cells. In addition, vimentin filaments were coexpressed in these cells. Large vessels of the chorionic plate and stem villi showed thick walls consisting of vimentin-, desmin- and actin-positive cells. They were surrounded by mantles rich in vimentin-, desmin- and actin-positive cells, resembling myofibroblasts. This indicates that these cells may play a role in villous contractility and modulation of the intervillous space with effect on both maternal and fetal placental circulation.     

N-glycans of human amniotic fluid transferrin stimulate progesterone production in human first trimester trophoblast cells in vitro

AIMS: During pregnancy, the placenta produces a variety of steroid hormones and proteins. Several of these substances have been shown to exert immunomodulatory effects. Progesterone is thought to mediate some of these effects by regulating uterine responsiveness. The aim of this study was to clarify the effect of amniotic fluid transferrin and its N-glycans on the release of progesterone by first trimester trophoblast cells in vitro. METHODS: Cytotrophoblast cells were prepared from human first trimester placentae by trypsin-DNAse dispersion of villous tissue followed by a percoll gradient centrifugation and depletion of CD45 positive cells by magnetic cell sorting. Trophoblasts were incubated with varying concentrations (50-300 microg/ml) of transferrin from human amniotic fluid and serum as well as with N-glycans obtained from amniotic fluid transferrin. Culture supernatants were assayed for progesterone by enzyme-immunometric methods. RESULTS: The release of progesterone increased in amniotic fluid transferrin- and N-glycan-treated trophoblast cell cultures compared to untreated trophoblast cells. There was no stimulating effect of serum transferrin on the progesterone production of trophoblast cells. CONCLUSIONS: The results suggest that amnion-transferrin and especially its N-glycans modulate the endocrine function of trophoblasts in culture by up regulating progesterone secretion.