Integrin α4-positive human trophoblast progenitors: functional characterization and transcriptional regulation.

STUDY QUESTION: What are the functional characteristics and transcriptional regulators of human trophoblast progenitor cells (TBPCs)? SUMMARY ANSWER: TBPC lines established from the human smooth chorion by cell sorting for integrin α4 expressed markers of stemness and trophoblast (TB) stage-specific antigens, invaded Matrigel substrates and contributed to the cytotrophoblasts (CTBs) layer of smooth chorion explants with high-mobility group protein HMGI-C (HMGA2) and transcription factor GATA-4 (GATA4) controlling their progenitor state and TB identity. WHAT IS KNOWN ALREADY: Previously, we reported the derivation of TBPC lines by trypsinization of colonies that formed in cultures of chorionic mesenchyme cells that were treated with an activin nodal inhibitor. Microarray analyses showed that, among integrins, α4 was most highly expressed, and identified HMGA2 and GATA4 as potential transcriptional regulators. STUDY DESIGN, SIZE, DURATION: The aim of this study was to streamline TBPC derivation across gestation. High-cell surface expression of integrin α4 enabled the use of a fluorescence-activated cell sorter (FACS) approach for TBPC isolation from the human smooth chorion (n = 6 lines). To confirm their TBPC identity, we profiled their expression of stemness and TB markers, and growth factor receptors. At a functional level, we assayed their invasive capacity (n = 3) and tropism for the CTB layer of the smooth chorion (n = 3). At a molecular level, we studied the roles of HMGA2 and GATA4. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Cells were enzymatically disassociated from the human smooth chorion across gestation. FACS was used to isolate the integrin α4-positive population. In total, we established six TBPC lines, two per trimester. Their identity was determined by immunolocalization of a suite of antigens. Function was assessed via Matrigel invasion and co-culture with explants of the human smooth chorion. An siRNA approach was used to down-regulate HMGA2 and GATA4 expression and the results were confirmed by immunoblotting and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses. The endpoints analyzed included proliferation, as determined by 5-bromo-2'-deoxyuridine (BrDU) incorporation, and the expression of stage-specific antigens and hormones, as determined by qRT-PCR and immunostaining approaches. MAIN RESULTS AND THE ROLE OF CHANCE: As with the original cell lines, the progenitors expressed a combination of human embryonic stem cell and TB markers. Upon differentiation, they primarily formed CTBs, which were capable of Matrigel invasion. Co-culture of the cells with smooth chorion explants enabled their migration through the mesenchyme after which they intercalated within the chorionic CTB layer. Down-regulation of HMGA2 showed that this DNA-binding protein governed their self-renewal. Both HMGA2 and GATA4 had pleitropic effects on the cells' progenitor state and TB identity. LIMITATIONS, REASONS FOR CAUTION: This study supported our hypothesis that TBPCs from the chorionic mesenchyme can contribute to the subpopulation of CTBs that reside in the smooth chorion. In the absence of in vivo data, which is difficult to obtain in humans, the results have the limitations common to all in vitro studies. WIDER IMPLICATIONS OF THE FINDINGS: The accepted view is that progenitors reside among the villous CTB subpopulation. Here, we show that TBPCs also reside in the mesenchymal layer of the smooth chorion throughout gestation. We theorize that they can contribute to the CTB layer in this region. This phenomenon may be particularly important in pathological situations when CTBs of the smooth chorion might provide a functional reserve for CTBs of the placenta proper. STUDY FUNDING/COMPETING INTERESTS: Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under award P50HD055764. O.G., N.L., K.O., A.P., T.G.-G., M.K., A.B., M.G. have nothing to disclose. S.J.F. received licensing fees and royalties from SeraCare Life Sciences for trisomic TBPC lines that were derived according to the methods described in this manuscript. TRIAL REGISTRATION NUMBER: N/A.

The role of chorionic cytotrophoblasts in the smooth chorion fusion with parietal decidua.

BACKGROUND/PURPOSE: Human placenta and chorion are rapidly growing transient embryonic organs built from diverse cell populations that are of either, ectodermal [placenta and chorion specific trophoblast (TB) cells], or mesodermal origin [villous core and chorionic mesenchyme]. The development of placenta and chorion is synchronized from the earliest phase of implantation. Little is known about the formative stages of the human chorion, in particular the steps between the formation of a smooth chorion and its fusion with the parietal decidua. METHODS: We examined the available histological material using immunohistochemistry, and further analyzed in vitro the characteristics of the recently established and reported human self-renewing trophoblast progenitor cells (TBPC) derived from chorionic mesoderm. RESULTS: Here, we provided evidence that the mechanism by which smooth chorion fuses with parietal decidua is the invasion of smooth chorionic cytotrophoblasts (schCTBs) into the uterine wall opposite to the implantation side. This process, which partially replicates some of the mechanisms of the blastocyst implantation, leads to the formation of a new zone of contacts between fetal and maternal cells. CONCLUSION: We propose the schCTBs invasion of the parietal decidua as a mechanism of 'fusion' of the membranes, and that schCTBs in vivo contribute to the pool of the invasive schCTB.

Human placental explants in culture: approaches and assessments.

Placental explant cultures in vitro are useful for studying tissue functions including cellular uptake, production and release of secretory components, cell interactions, proliferation, growth and differentiation, gene delivery, pharmacology, toxicology, and disease processes. A variety of culture conditions are required to mimic in utero environments at different times of gestation including differing oxygen partial pressures, extracellular matrices and culture medium. Optimization of explant methods is examined for first and third trimester human placental tissue and the biological processes under investigation.

The adverse effects of maternal smoking on the human placenta: a review.

Studies of placental pathologies associated with maternal cigarette smoking have led to many interesting observations. For example, maternal smoking impairs human placental development by changing the balance between cytotrophoblast (CTB) proliferation and differentiation. It is likely that chronic exposure to tobacco constituents in early pregnancy can affect placental development directly or indirectly by reducing blood flow, which creates a pathologically hypoxic environment. To understand this process at a molecular level, tissue samples from non-smoking and smoking mothers were studied to determine whether active and/or passive cigarette smoke exposure affects CTB expression of molecules that govern cellular responses to oxygen tension: the von Hippel-Lindau tumor suppressor protein (pVHL), hypoxia-inducible transcription factors (HIFs) and vascular endothelial growth factor-A (VEGF). The results show that maternal smoking dysregulates CTB expression of all three types of molecules. In addition, cell columns and proliferating cells were reduced while there was a corresponding increase in cell islands. All three phenomena were most obvious in the placentas of heavy smokers. Interestingly, a subset of the aforementioned effects can be detected in samples obtained from women who were passively exposed to cigarette smoke during pregnancy. These observations suggest that tobacco constituents exert direct effects on CTB proliferation and differentiation and help to explain the mechanisms by which smoking negatively effects human pregnancy outcome.

Trophoblast origin of hCG isoforms: cytotrophoblasts are the primary source of choriocarcinoma-like hCG.

We have previously demonstrated that a hyperglycosylated isoform of chorionic gonadotropin (hCG) (B152 hCG) is detected in the blood and urine in early pregnancy and is subsequently rapidly replaced by the hCG isoform (B109 hCG) characteristic of later pregnancy. In the current study we have extended our work on the origin of these isoforms. We have used a combination of in situ and in vitro approaches. Localization studies in placental tissues showed that monoclonal antibody B109 stained very specifically syncytiotrophoblast (STBs) from first and second trimester tissues. At term, STBs exhibited no B109 staining at all. Immunostaining with B152 antibody, that recognize the hyperglycosylated isoform of hCG, revealed only punctate staining of STBs in most villi of first trimester tissue. Both antibodies B109 and B152 failed to stain cytotrophoblasts (CTBs). To assess the functional relevance of these observations we analyzed conditioned media from purified CTBs using two immunometric assays, one of which (B152-B207*) has primary specificity for the hyperglycosylated, choriocarcinoma-like hCG and the other (B109-B108*) having primary specificity for the later pregnancy hCG isoform. Regardless of gestational age, isolated CTBs secreted predominantly B152 hCG isoform in contrast to placental villi (predominantly STBs), which released primarily the B109 hCG isoform. Isolated CTBs, however, failed to immunostain with both B109 and B152 antibodies. To resolve this contradiction, we cultured CTBs in the presence of brefeldin A, a drug known to block secretion by inhibiting protein translocation from the endoplasmic reticulum to the Golgi vesicles. Brefeldin A treated CTBs stained strongly with B109 and did not stain or stained weakly with B152 antibody. We assume that treatment with brefeldin A impaired glycosylation of beta subunit and consequently inhibited the production of hyperglycosylated form of hCG recognized by B152. In summary, our in vitro experiments indicate that both isoforms of hCG are produced by villus CTBs and that the dominant isoform is the one recognized by antibody B152. STBs produce primarily the less glycosylated B109 hCG isoform. This data suggests that at the beginning of pregnancy villus CTBs are the major source of the B152 hCG isoform. This finding is supported by our clinical data that show that the dominant hCG isoform in the blood and urine of pregnant women in the first 6 weeks of pregnancy is recognized by B152 (). The inversion of the B152/B109 ratio observed after 6-7 weeks of pregnancy can be explained by the reduction of number of villus CTBs and/or by maturation of STBs.

To proliferate or to divide - to be or not to be.

Placental growth depends on cytotrophoblast (CTB) proliferation. A portion of CTBs, which retain an undifferentiated phenotype throughout pregnancy, provide a reservoir of stem cells. The remaining CTBs give rise to the two major trophoblast subpopulations, syncytiotrophoblast (STB) and invasive CTBs. Some of the molecular mechanisms that govern human CTB differentiation and invasion are well understood. These include an upstream suites of transcription factors such as Mash-2, Hand-1 and Gem1, and a downstream set of effectors such as adhesion molecules, proteinases, and the trophoblast major histocompatibility antigen HLA-G. In comparison, much less is known about how CTB proliferation is coordinated with differentiation. We immunolocalized markers that are specifically expressed during all of the key transitions and phases of the cell cycle in tissue sections of the maternal-fetal interface. We mapped cell cycle progression in both populations with the goal of understanding the mechanisms that maintain a pool of CTB stem cells (villus CTBs) and govern CTB exit from the cell cycle during differentiation along the invasive pathway. The results showed that as CTBs differentiate/invade or differentiate/fuse, they down-regulate the expression of molecules that are associated with mitosis and up-regulate the expression of a number of inhibitors that engineer permanent withdrawal from the cell cycle. Multinucleate STB coexpressed an unusual repertoire of markers that are usually segregated to distinct portions of the cell cycle. One of the regulatory factors involved in the regulation of CTB proliferation during early pregnancy is oxygen. During much of the first trimester of pregnancy there is little endovascular invasion, so maternal blood flow to the placenta is at minimum. This creates hypoxic environment in which placental development occurs. During this period, placental mass increases much more rapidly than that of the embryo proper. Both in vivo and in vitro data suggest that oxygen tension might regulate cytotrophoblast proliferation and differentiation along the invasive pathway. The fact that hypoxia stimulates CTBs, but not other cells, to undergo mitosis could help account for the discrepancy in size between the embryo and the placenta, which continues well into the second trimester of pregnancy. As cytotrophoblast invade the uterine blood vessels, they encounter a steep, positive oxygen tension gradient and exit gradually from the cell cycle. Interesting gestation-related changes were also observed by the second trimester, many fewer CTB stem cells and cells in cell column expressed mitotic markers. There was a reciprocal increase in the number of column CTBs that expressed inhibitors. Together, these data suggest that CTB proliferation, like differentiation, is part of a developmental program that is timed to precede development of the embryo/fetus.

Plasma membrane-associated pY397FAK is a marker of cytotrophoblast invasion in vivo and in vitro.

During human pregnancy specialized placental cells of fetal origin, termed cytotrophoblasts, invade the uterus and its blood vessels. This tumor-like process anchors the conceptus to the mother and diverts the flow of uterine blood to the placenta. Previously, we showed that the expression of molecules with important functional roles, including a number of extracellular matrix integrin receptors, is precisely modulated during cytotrophoblast invasion in situ. Here we exploited this observation to study the role of the focal adhesion kinase (FAK), which transduces signals from the extracellular matrix and recruits additional signaling proteins to focal adhesions. Immunolocalization studies on tissue sections showed that FAK is expressed by cytotrophoblasts in all stages of differentiation. Because extracellular matrix-induced integrin clustering results in FAK (auto)phosphorylation on tyrosine 397 (Y397FAK), we also localized this form of the molecule. Immunolocalization experiments detected Y397FAK in a subset of cytotrophoblasts near the surface of the uterine wall. To assess the functional relevance of this observation, we used an adenovirus strategy to inhibit cytotrophoblast expression of FAK as the cells differentiated along the invasive pathway in vitro. Compared to control cells transduced with a wild-type virus, cytotrophoblasts that expressed antisense FAK exhibited a striking reduction in their ability to invade an extracellular matrix substrate. When cytotrophoblast differentiation was compromised (hypoxia in vitro, preeclampsia in vivo), Y397FAK levels associated with the plasma membrane were strikingly lower, although total FAK levels did not change. Together our results suggest that (auto)phosphorylation of Y397 on FAK is a critical component of the signaling pathway that mediates cytotrophoblast migration/invasion.

Human cytotrophoblast expression of the von Hippel-Lindau protein is downregulated during uterine invasion in situ and upregulated by hypoxia in vitro.

The von Hippel-Lindau tumor-suppressor protein (pVHL) regulates the stability of HIF1 alpha and HIF2 alpha and thus is pivotal in cellular responses to changes in oxygen tension. Paradoxically, human cytotrophoblasts proliferate under hypoxic conditions comparable to those measured in the early gestation placenta (2% O(2)), but differentiate into tumorlike invasive cells under well-oxygenated conditions such as those found in the uterus. We sought to explain this phenomenon in terms of pVHL expression. In situ, pVHL immunolocalized to villous cytotrophoblast stem cells, and expression was enhanced at sites of cell column initiation; in both of these relatively hypoxic locations, cytoplasmic staining for HIF2 alpha was also detected. As cytotrophoblasts attached to and invaded the uterus, which results in their increased exposure to oxygen, pVHL staining was abruptly downregulated concordant with localization of HIF2 alpha to the nucleus. In vitro, hypoxia (2% O(2)) upregulated cytotrophoblast pVHL expression together with HIF2 alpha, which localized to the cytoplasm; culture under well-oxygenated conditions greatly reduced levels of both molecules. These results, together with the placental defects previously observed in VHL(-/-) mice, suggest that pVHL is a component of the mechanism that transduces local differences in oxygen tension at the maternal-fetal interface to changes in the biological behavior of cytotrophoblasts. Furthermore, these data provide the first example of oxygen-dependent changes in pVHL abundance.

Conditioned medium from hypoxic cytotrophoblasts alters arterial function.

OBJECTIVE: Our goal was to test the hypothesis that cytotrophoblasts, under low oxygen tension, release substances that affect vascular behavior. STUDY DESIGN: We studied the vascular response to the vasoconstrictors phenylephrine (receptor dependent) and potassium (receptor independent), the relaxation response to methacholine, and the vasomotor behavior of isolated resistance (mesenteric) arteries from early pregnant rats after incubation in conditioned medium from first-trimester cytotrophoblasts, maintained in standard or hypoxic (2%; 14 mm Hg) culture conditions. RESULTS: After incubation in medium from hypoxic cytotrophoblasts, arterial segments were more responsive to phenylephrine and to potassium-induced constriction but were less responsive to methacholine, and the vasomotor activity was increased compared with that found in vessels incubated in control medium. CONCLUSIONS: These changes in vascular behavior are similar to those reported in isolated arteries from women with preeclampsia. These studies provide evidence which suggests that the link between abnormal placentation and maternal vascular abnormality in preeclampsia is the elaboration of vasoactive factors by cytotrophoblasts in response to hypoxia.

Concordant in situ and in vitro data show that maternal cigarette smoking negatively regulates placental cytotrophoblast passage through the cell cycle.

Maternal cigarette smoking is associated with fetal growth restriction and other pregnancy complications. To investigate possible mechanisms involving the placenta, we studied the morphology of first trimester chorionic villi from mothers who smoked. In mothers who smoked > 20 cigarettes/day, floating villi showed focal defects including an absence of cytotrophoblast stem cells and an abnormal thinning of the syncytium. Anchoring villi displayed a striking increase in the number of cytotrophoblast columns that failed to reach the uterus or degenerated in the intervillous space. Many samples showed a significant reduction in the number of anchoring villi. Also, the number of Ki67-positive cytotrophoblasts was dramatically decreased, indicating that fewer cells were in S phase of the mitotic cycle. Together, these results suggested premature depletion of the cytotrophoblast stem cell population. To test this hypothesis, we exposed anchoring villi from nonsmokers to nicotine in vitro and analyzed the effects on cytotrophoblast passage through the cell cycle. Nicotine (0.23 to 6.0 microM) negatively affected the expression of a number of cell cycle regulators/markers and BrdU incorporation, without discernable effects on apoptosis. These results link abnormal placental development secondary to maternal cigarette smoking to a substantial decrease in the mitotic potential of cytotrophoblasts.

A repertoire of cell cycle regulators whose expression is coordinated with human cytotrophoblast differentiation.

Although placental development depends on careful coordination of trophoblast proliferation and differentiation, little is known about the mitotic regulators that are key to synchronizing these events. We immunolocalized a broad range of these regulators in tissue sections of the maternal-fetal interface (first trimester through term) that contained floating villi (which include cytotrophoblasts differentiating into syncytiotrophoblasts) and anchoring villi (which include cytotrophoblasts differentiating into invasive cells). Trophoblast populations at the maternal-fetal interface stained for 16 of the cell cycle regulators whose expression we studied. The staining patterns changed as a function of both differentiation and gestational age. Differentiation along the invasive pathway was associated with entrance into, then permanent withdrawal from, the cell cycle, as evidenced by the orchestrated expression of cyclins, their catalytic subunits, and inhibitors. Surprisingly, we found coexpression of molecules that regulate different portions of the cell cycle in the syncytium. These data, which constitute one of the few examples to date of in situ localization of an extensive repertoire of mitotic regulators, provide the basis for studies aimed at understanding factors that lead to abnormal placentation.

Human cytomegalovirus infection of placental cytotrophoblasts in vitro and in utero: implications for transmission and pathogenesis.

Human cytomegalovirus (CMV) is the leading cause of prenatal viral infection. Affected infants may suffer intrauterine growth retardation and serious neurologic impairment. Analysis of spontaneously aborted conceptuses shows that CMV infects the placenta before the embryo or fetus. In the human hemochorial placenta, maternal blood directly contacts syncytiotrophoblasts that cover chorionic villi and cytotrophoblasts that invade uterine vessels, suggesting possible routes for CMV transmission. To test this hypothesis, we exposed first-trimester chorionic villi and isolated cytotrophoblasts to CMV in vitro. In chorionic villi, syncytiotrophoblasts did not become infected, although clusters of underlying cytotrophoblasts expressed viral proteins. In chorionic villi that were infected with CMV in utero, syncytiotrophoblasts were often spared, whereas cytotrophoblasts and other cells of the villous core expressed viral proteins. Isolated cytotrophoblasts were also permissive for CMV replication in vitro; significantly, infection subsequently impaired the cytotrophoblasts' ability to differentiate and invade. These results suggest two possible routes of CMV transmission to the fetus: (i) across syncytiotrophoblasts with subsequent infection of the underlying cytotrophoblasts and (ii) via invasive cytotrophoblasts within the uterine wall. Furthermore, the observation that CMV infection impairs critical aspects of cytotrophoblast function offers testable hypotheses for explaining the deleterious effects of this virus on pregnancy outcome.

Post-implantation differentiation and proliferation of cytotrophoblast cells: in vitro models--a review.

Cytotrophoblast cells, specialized placental cells, proliferate early in pregnancy and then differentiate into tumour-like cells that invade the uterus and its vasculature. We have established in vitro models of three-dimensional cultures for anchoring villi and cell islands on extracellular matrix in order to study regulation of cytotrophoblast cell differentiation and proliferation. It has been demonstrated that cytotrophoblast cells from cell islands and cell columns share the same characteristics and that their differentiation is triggered by interaction with the extracellular matrix. The fact that during much of the first trimester maternal blood flow to the placenta is at a minimum, suggests that oxygen tension might regulate cytotrophoblast proliferation and differentiation. Hypoxia, comparable to that encountered by early gestation cytotrophoblast cells in the intervillous space, stimulated the cells to enter the cell cycle and inhibited their differentiation along the invasive pathway. Thus, oxygen gradient and cell-matrix interactions at the maternal-fetal interface play an important role in the regulation of cytotrophoblast proliferation and differentiation.

Id-2 regulates critical aspects of human cytotrophoblast differentiation, invasion and migration.

During early human placental development, the conceptus attaches itself to the uterus through cytotrophoblast invasion. Invasive cytotrophoblast cells differentiate from precursor villous cytotrophoblasts, but the essential regulating factors in this process are unknown. Basic helix-loop-helix (bHLH) transcription factor dimers are essential regulators of mouse trophoblast development. We therefore examined the importance of this family of factors in the human placenta. In many cell lineages, bHLH factors are sequestered by members of the Id family, HLH proteins that lack the basic DNA binding domain (Inhibitor of DNA binding proteins (Id-1 to Id-4)). During differentiation of some tissues, Id expression declines, allowing bHLH factors to dimerize, bind DNA and trans-activate lineage-specific genes. To begin to study the role of bHLH transcription factors in human placental development, we first characterized Id expression in cytotrophoblast cells. The cells expressed Id-3 constitutively; Id-2 was downregulated, at the mRNA and protein levels, as the cells differentiated in culture and in situ, respectively. In cases when cytotrophoblast differentiation was compromised (in placentas from women with preeclampsia, or in cells grown under hypoxic conditions in culture), Id-2 expression was maintained. To assess the functional relevance of these correlations, we used an adenovirus vector to maintain Id-2 protein expression in cultured cytotrophoblasts. Compared to control (lacZ-expressing) cells, cytotrophoblasts transduced to constitutively express Id-2 retained characteristics of undifferentiated cells: (alpha)1 integrin expression was low and cyclin B expression was retained. Furthermore, invasion through Matrigel was partially inhibited and migration was strikingly enhanced in Id-2-expressing cells. These results suggest that Id-2 and the bHLH factors that it partners play important roles in human cytotrophoblast development.

Preeclampsia is associated with widespread apoptosis of placental cytotrophoblasts within the uterine wall.

Preeclampsia is a serious pregnancy complication diagnosed by signs of widespread maternal endothelial dysfunction. In normal pregnancy, a subpopulation of placental cytotrophoblast stem cells executes an unusual differentiation program that leads to invasion of the uterus and its vasculature. This process attaches the conceptus to the uterine wall and starts the flow of maternal blood to the placenta. Preeclampsia is associated with abnormal cytotrophoblast differentiation, shallow invasion, and decreased blood flow to the placenta. To determine whether abnormal differentiation and/or hypoxia leads to cytotrophoblast apoptosis, we used the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) method to label DNA strand breaks in tissue sections of the placenta and the uterine wall to which it attaches. Control samples (n = 9) showed almost no apoptosis, but in samples from patients with preeclampsia, 15-50% of the cytotrophoblasts that invaded the uterine wall were labeled (8/9 samples). These same cells failed to stain for Bcl-2, a survival factor normally expressed by trophoblasts in both the placenta and the uterine wall. Our results show that preeclampsia is associated with widespread apoptosis of cytotrophoblasts that invade the uterus. The magnitude of programmed cell death in this population may account for the sudden onset of symptoms in some patients, as well as the associated coagulopathies.

Invasive cytotrophoblast apoptosis in pre-eclampsia.

Pre-eclampsia is a serious pregnancy complication diagnosed by signs of widespread maternal endothelial dysfunction. In normal pregnancy, a subpopulation of placental cytotrophoblast stem cells executes a differentiation programme that leads to invasion of the uterus and its vasculature. This process attaches the conceptus to the uterine wall and starts the flow of maternal blood to the placenta. In pre-eclampsia, cytotrophoblasts fail to differentiate along the invasive pathway. The functional consequences of this abnormality negatively affect interstitial and endovascular invasion, thereby compromising blood flow to the maternal-fetal interface. To determine whether abnormal differentiation and/or hypoxia leads to apoptosis of invasive cytotrophoblasts, we used the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) method to label DNA strand breaks in tissue sections of the placenta and the uterine wall to which it attaches. Control samples (n = 9) showed little or no apoptosis in any location, but in samples from patients with pre-eclampsia, 15-50% of the cytotrophoblast subpopulation that invaded the uterine wall was labelled (8/9 samples). These same cells failed to stain for Bcl-2, a survival factor normally expressed by trophoblasts in both the placenta and the uterine wall. Our results show that pre-eclampsia is associated with widespread apoptosis of cytotrophoblasts that invade the uterus. The magnitude of programmed cell death in this population may account for the sudden onset of symptoms in some patients, as well as the associated coagulopathies.

Oxygen regulates human cytotrophoblast differentiation and invasion: implications for endovascular invasion in normal pregnancy and in pre-eclampsia.

This review article focuses on the unique process by which the human placenta normally forms and how changes in this process can lead to serious pregnancy complications such as pre-eclampsia. One way to compare normal and pathologic pregnancies is to examine biopsy specimens of the placenta and placental bed for disease-associated morphological changes in cellular architecture. Our recent work has verified the decades-old observation that pre-eclampsia is associated with abnormally shallow placentation. We also discuss how these morphological observations prompted us to use a combination of in vitro modeling and in situ immunolocalization techniques to gain insights into the molecular bases of normal placentation and how these mechanisms go awry in pre-eclampsia.

Regulation of human placental development by oxygen tension.

Cytotrophoblasts, specialized placental cells, proliferate early in pregnancy and then differentiate into tumor-like cells that establish blood flow to the placenta by invading the uterus and its vasculature. In this study, cytotrophoblasts cultured under hypoxic conditions (2 percent oxygen), mimicking the environment near the uterine surface before 10 weeks of gestation, continued proliferating and differentiated poorly. When cultured in 20 percent oxygen, mimicking the environment near uterine arterioles, the cells stopped proliferating and differentiated normally. Thus, oxygen tension determines whether cytotrophoblasts proliferate or invade, thereby regulating placental growth and cellular architecture.