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.

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.

Hypoxia alters early gestation human cytotrophoblast differentiation/invasion in vitro and models the placental defects that occur in preeclampsia.

During normal human pregnancy a subpopulation of fetal cytotrophoblast stem cells differentiate and invade the uterus and its arterioles. In the pregnancy disease preeclampsia, cytotrophoblast differentiation is abnormal and invasion is shallow. Thus, the placenta is relatively hypoxic. We investigated whether lowering oxygen tension affects cytotrophoblast differentiation and invasion. Previously we showed that when early gestation cytotrophoblast stem cells are cultured under standard conditions (20% O2) they differentiate/invade, replicating many aspects of the in vivo process. Specifically, the cells proliferate at a low rate and rapidly invade extracellular matrix (ECM) substrates, a phenomenon that requires switching their repertoire of integrin cell-ECM receptors, which are stage-specific antigens that mark specific transitions in the differentiation process. In this study we found that lowering oxygen tension to 2% did not change many of the cells' basic processes. However, there was a marked increase in their incorporation of [3H]thymidine and 5-bromo-2'-deoxyuridine (BrdU). Moreover, they failed to invade ECM substrates, due at least in part to their inability to completely switch their integrin repertoire. These changes mimic many of the alterations in cytotrophoblast differentiation/invasion that occur in preeclampsia, suggesting that oxygen tension plays an important role in regulating these processes in vivo.

Human cytotrophoblasts adopt a vascular phenotype as they differentiate. A strategy for successful endovascular invasion?

Establishment of the human placenta requires that fetal cytotrophoblast stem cells in anchoring chorionic villi become invasive. These cytotrophoblasts aggregate into cell columns and invade both the uterine interstitium and vasculature, anchoring the fetus to the mother and establishing blood flow to the placenta. Cytotrophoblasts colonizing spiral arterioles replace maternal endothelium as far as the first third of the myometrium. We show here that differentiating cytotrophoblasts transform their adhesion receptor phenotype so as to resemble the endothelial cells they replace. Cytotrophoblasts in cell columns show reduced E-cadherin staining and express VE-(endothelial) cadherin, platelet-endothelial adhesion molecule-1, vascular endothelial adhesion molecule-1, and alpha-4-integrins. Cytotrophoblasts in the uterine interstitium and maternal vasculature continue to express these receptors, and, like endothelial cells during angiogenesis, also stain for alphaVbeta3. In functional studies, alphaVbeta3 and VE-cadherin enhance, while E-cadherin restrains, cytotrophoblast invasiveness. Cytotrophoblasts expressing alpha4 integrins bound immobilized VCAM-1 in vitro, suggesting that this receptor-pair could mediate cytotrophoblast-endothelium or cytotrophoblast-cytotrophoblast interactions in vivo, during endovascular invasion. In the pregnancy disorder preeclampsia, in which endovascular invasion remains superficial, cytotrophoblasts fail to express most of these endothelial markers (Zhou et al., 1997. J. Clin. Invest. 99:2152-2164.), suggesting that this adhesion phenotype switch is required for successful endovascular invasion and normal placentation.

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.

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 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.

Coincubation--an experimental approach to the study of decidual-trophoblast interaction.

Coincubation of trophoblast and decidual tissue explants was used for the study of placental-endometrial interaction in early pregnancy. To this end two types of experiments were performed: coincubation with (type A) and without (type B) direct tissue contact. The rate of incorporation of [14C]leucine into cytosol proteins in both tissues was employed for the estimation of total protein synthesis. Prolactin production in vitro was used as a specific marker of the decidual and hCG of the trophoblast cell function. The results show that the type A coincubation experiments produced a strong inhibition of cytosol protein synthesis in both tissues. PRL production by the decidual tissue and hCG production by the trophoblast tissue was reduced. In the type B coincubation experiments protein synthesis and prolactin production by the decidual tissue remained within the control range. In the trophoblast tissue explants protein synthesis and hCG production were depressed. The degree of inhibition was, however, lower than that in type-A experiments. Based on these results it was concluded that the in vitro model of coincubation of trophoblast and decidual tissue explants is suitable for the study of the role of tissue interactions in vitro.

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.

Villous culture of first trimester human placenta--model to study extravillous trophoblast (EVT) differentiation.

During implantation and subsequent placentation the human extravillous trophoblast (EVT) cells invade the endometrium and maternal vasculature within the uterus. The origin of the EVT and signals triggering its differentiation, migration and invasion are poorly understood. First and second trimester human chorionic villi explants were used as a source of EVT and a variety of substrates which resemble extracellular matrix (ECM) in vivo have been tested to induce EVT differentiation and migration. The obtained results demonstrate that villous explants from both 5-7 and 8-10 weeks of gestation give rise to EVT cells in vitro if maintained on the surface of Matrigel or decidual extract supplemented collagen gel. Fetal calf serum (FCS) supplemented media was essential for EVT differentiation and villous trophoblast viability. Immunostaining of both EVT cells and cells from the cytotrophoblastic column with monoclonal antibody Ki67 (cell proliferation marker) indicate that EVT cells differentiate in vitro by proliferation from the tip of anchoring villi. These mononucleated, round-shaped, migrating cells are HLA-A,B,C class I antigen (W6/32) antibody and low molecular weight cytokeratin positive, and do not immunostain with PAI-1 (plasminogen activator inhibitor) and HPL antibodies. Differentiation of EVT was restricted to first trimester villous tissue; explants from second trimester placentae did not give rise to EVT. Tissue viability as monitored by glucose utilization, lactate, progesterone and hCG production rates correlated with EVT differentiation. The production rates for hCG demonstrated significant variation among individual placentae and was maintained constant for 10 days consistently only in explants cultured on decidual extract supplemented collagen matrix. The described villous tissue culture system may be, therefore, a unique in vitro model to study proliferation and differentiation of EVT from cytotrophoblastic columns, the regulation of EVT proliferation and differentiation, the role of ECM in the induction of the migration and the interaction of extravillous and villous trophoblast at the level of the cytotrophoblastic column.

In vitro differentiation and ultrastructure of human extravillous trophoblast (EVT) cells.

Tissue explants of anchoring villi from the first trimester placentae cultured on extracellular matrix (Matrigel) give rise to EVT cells in vitro. This study was designed to address two issues important for further application of the described in vitro model: first, were the observed EVT cells derived by cell proliferation in vitro and second, what is the degree of homology between the in vivo and the in vitro differentiated EVT cells. The cultures (tissue and matrix) were prepared for light and electron microscopic (EM) examinations. Semi-thin sections from Spurr epoxy resin-embedded tissue were used to 'pop-off' the selected area for EM examination. Cell proliferation in vitro was assessed immunohistochemically using proliferative cell nuclear antigen (PCNA) antibodies. Since positive hPL immunostaining has been consistently demonstrated in the invasive subpopulation of EVT cells from placental bed in situ, hPL staining was used as a marker of EVT cell differentiation in vitro. It has been demonstrated that PCNA antibodies immunostained nuclei of cytotrophoblast cells from cell column at the base of the anchoring villi, indicating that these cells expressed proliferative activity in vitro. Cytotrophoblast proliferation resulted in the formation of the flattened zone of cell outgrowths at the tip of anchoring villi. Cells from the distal layer of the cell column detached gradually and migrated into the surrounding matrix. These cells appeared as individual, round-shaped EVT cells with smooth surface cell membrane. Their cytoplasm was rich in glycogen and contained large lipid droplets and flattened cisternae of the RER. Positive PCNA immunostaining, along with the presence of mitotic figures, indicated that EVT cells in vitro retained the ability for cell proliferation. As a result of cell proliferation and migration, the number of EVT cells increased during the culture period of 4 days. EVT cell glycogen content and lipid stores decreased progressively as they migrated into the matrix. Individual EVT cells, as well as EVT cell clusters, became surrounded by the clear zone of digested matrix. Some cells started to express strong positive staining with hPL antibodies as soon as they had migrated outside the villous explant. By day 4 of culture, a small percentage of EVT cells (about 5-10%) ceased to migrate, firmly attached to the substratum and appeared as irregular shaped cells with filopodia-like projections. Their cytoplasm contained dilated cisternae of RER, a small number of glycogen granules and bundles of actin-like filaments located in the cytoplasm inside the plasma membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.

Effect of morphine on hCG release by first trimester human trophoblast in vitro.

Opiate synthesis by human placental cells and the presence of kappa-type opiate binding sites in the syncytiotrophoblast brush border membrane may indicate the possible role of morphine-like substances in the autocrine regulation of trophoblast cell metabolism. This study was undertaken to examine the in vitro effect of morphine on hCG (human chorionic gonadotrophin) and hPL (human placental lactogen) release by 1st and 3rd trimester placental tissue explants. The results have shown that morphine (100 nM) significantly stimulated hCG secretion by 6-8 weeks old trophoblast and was without effect on hPL. Hormone secretion by term placental tissue explants was unaffected by morphine treatment. Based on these results we assume that opiates may have a role in the local (autocrine and/or paracrine) regulation of hCG secretion in early gestation.

Human trophoblast cultures: models for implantation and peri-implantation toxicology.

Implantation is the process that leads from blastocyst attachment to its embedding in the uterine wall. It is widely believed that failure of implantation is a common cause of pregnancy loss. Toxic agents can interfere directly with the process of implantation and therefore may account for unexplained implantation failures. Our knowledge of human implantation remains limited, mainly due to the lack of adequate experimental models. Studies of mechanisms underlying implantation in humans are by nature and for ethical reasons restricted to in vitro models. The aim of this review is to provide a critical evaluation of various in vitro models of implantation in humans, as well as essential background knowledge required for application of these models to the assessment of peri-implantation toxicity. Particular attention has been devoted to cell-cell and cell-matrix interactions as possible endpoints in the screening of toxic agents.

Maternal smoking inhibits early human cytotrophoblast differentiation.

Differentiation of the specialized epithelial cells of the placenta, termed cytotrophoblasts, is a particularly important aspect of placental development during the first trimester of pregnancy. During this process cytotrophoblast stem cells either fuse to form the syncytium or aggregate to form cell columns that adhere to, then invade the uterus. We found that chorionic villi from early gestation placentas of mothers who smoke showed a marked reduction in cell columns, a defect that could not be corrected by placing them in culture. We used two different in vitro models to determine if nicotine plays a role in the etiology of this defect. Exposing early gestation chorionic villi from nonsmoking women to nicotine inhibited subsequent cell column formation in vitro. Nicotine also inhibited normal first trimester cytotrophoblast invasion, apparently by reducing the ability of treated cells to synthesize and activate the 92 kDa type IV collagenase, an important mediator of invasion in vitro. These results suggest that maternal cigarette smoking inhibits the trophoblast differentiation pathway that leads to column formation and uterine invasion. This effect, which is due at least in part to the effects of nicotine, may contribute to the growth retardation observed in fetuses of mothers who smoke during pregnancy.

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.

Healing effect of human trophoblast on indolent wounds.

This study reports on the results of a trial in which the cytosol extract of trophoblast obtained from legal abortions was used for the treatment of indolent leg ulcers and severe chronic cystitis caused by irradiation therapy.