J.J. Lysiak

Characteristic's of trophoblast cells migrating from first trimester chorionic villus explants and propagated in culture

We developed a method of propagating pure first trimester human trophoblast cells growing out of primary explants of mechanically derived chorionic villus fragments (Yagel et al, 1989; Graham et al, 1992). We have now extensively characterized these cells during their initial outgrowth and in long-term culture, employing a variety of markers and techniques as outlined below. By double label immunofluorescence using epithelial (cytokeratin) and mesenchymal (vimentin) cell markers, we identified the chorionic villus migrant cell populations as pure trophoblast (39 per cent of outgrowths) or a mixture of trophoblast and fibroblast (61 per cent). Further phenotyping of the pure trophoblast outgrowths by double label immunostaining using anti-cytokeratin antibody and a panel of other primary antisera revealed that these cells exhibit a variety of markers characteristic of extravillous invasive trophoblast cells in situ: insulin-like growth factor (IGF)-II, NDOG-5, proliferating cell nuclear antigen (PCNA), human leucocyte antigen framework antigen (W6/32) and a distinct set of integrins including alpha 1, alpha 3, alpha 5, alpha v and beta 1 subunits and alpha v beta 3/beta 5 vitonectin receptor. They were negative for alpha 6 and beta 4 integrin subunits. Immunogold electron microscopy of explants grown on type IV collagen gel revealed the production of conventional and oncofetal types of fibronectin by mononucleate trophoblast cells and human placental lactogen by multinucleate cells. Immunolabelling, flow cytometry and immunoprecipitation revealed that this phenotypic profile was retained with complete fidelity in the long-term culture; thus, trophoblasts migrating out of first trimester chorionic villus explants and their propagated progeny belong to the invasive extravillous trophoblast of the placenta.

Localization of transforming growth factor β and its natural inhibitor decorin in the human placenta and decidua throughout gestation

Transforming growth factor beta (TGF beta) produced at the human fetomaternal interface has been shown to play a crucial role in controlling trophoblast invasion of the uterus. Decorin, a naturally occurring chondroitin-dermatan sulphate proteoglycan which binds TGF beta can inhibit its activity. In this study, immunohistochemical techniques were used to determine the locations of TGF beta and decorin within the human placenta and decidua throughout normal gestation. In addition, sites of TGF beta 1 mRNA synthesis were identified in early and late placenta by in situ hybridization. Results revealed the presence of immunoreactive TGF beta in the cytoplasm of villous syncytiotrophoblast and extravillous trophoblast cells throughout gestation. TGF beta immunostaining was absent from villous cytotrophoblast at all gestational ages examined. The extracellular matrix (ECM) of the villous core at all stages of gestation and cells of the cytotrophoblastic shell of the term placenta were immunoreactive for TGF beta. Within decidual tissue, TGF beta was primarily localized in the ECM during the first trimester and only a small proportion of decidual cells exhibited intracellular labelling. At later gestational ages the majority of decidual cells showed intracellular labelling accompanied by a decrease in ECM staining. This switch may reflect increased TGF beta synthesis by the decidual cells, decreased release, or altered TGF beta binding to one or more ECM proteins. In situ hybridization indicated that TGF beta 1 mRNA was primarily localized to the syncytiotrophoblast cell layer with low intensity signals present in extravillous trophoblast cells, in trophoblast cell columns, and in large decidual cells. At term, TGF beta 1 mRNA was located in both the syncytiotrophoblast and villous mesenchymal cells. Decorin was immunolocalized to the ECM of the mesenchymal core of the chorionic villi throughout gestation and no immunoreactivity was observed in either villous or extravillous trophoblast. In the first trimester decidua, decorin was localized to the ECM whereas decidual cells, decidual leucocytes, and the uterine epithelium were negative. At later gestational ages, the ECM as well as a few decidual cells displayed weak immunoreactivity. A strong co-localization of TGF beta and decorin in the ECM of first trimester decidual tissue suggests that decorin may aid TGF beta storage or limit its activity in the ECM.

Activation of maternal killer cells in the pregnant uterus with chronic indomethacin therapy, IL-2 therapy, or a combination therapy is associated with embryonic demise.

We have previously shown that NK lineage cells migrate to the murine decidua of pregnancy; but with advancing gestation, they are progressively inactivated in situ by prostaglandins of the E series (PGE2) secreted by decidual cells and decidual macrophages. We have also shown that the same mechanism inactivates all killer lineage cells in the human decidua, and that this inactivation is at least in part due to a down-regulation of IL-2 receptors and an inhibition of IL-2 production in situ. We examined whether chronic indomethacin therapy (to block prostaglandin synthesis), or a systemic administration of a high dose of IL-2, or a combination of both agents administered to pregnant mice could activate killer cells in situ and interfere with the progress of pregnancy; and if so, whether there was a causal relationship between the two events. Pregnant CD1 mice (Day 5 of gestation) were subjected to chronic indomethacin therapy (14 micrograms/ml in drinking water up to Day 15, or 50 micrograms twice daily sc or ip up to Day 10), high dose IL-2 therapy (25,000 Cetus U of human recombinant IL-2, ip every 8 or 12 hr for 3-5 days), or a combination of the two. These treatments led to pregnancy loss in 89-100% of mice, in contrast to 1% loss in control, vehicle-treated mice. Uterine mononuclear cells isolated from the embryo resorption sites exhibited high killer activity against YAC-1 lymphoma as well as murine trophoblast targets, with NK-like phenotype (Asialo GM-1+, Thy-1-) after indomethacin therapy and LAK-like phenotype (AGM-1+, Thy-1+) after IL-2 or indomethacin + IL-2 therapy. That AGM-1+ killer cells resulted in the pregnancy loss was suggested by the findings that in two of three separate experiments, iv injections of AGM-1 ab into pregnant indomethacin + IL-2-treated mice nearly completely prevented the fetoplacental demise (reducing it to 7.7% from 100%). These results reveal that PGE2-mediated inactivation of killer lineage cells in the decidua in situ is conducive to the survival of the conceptus.

Localization of amphiregulin in the human placenta and decidua throughout gestation: Role in trophoblast growth

Amphiregulin (AR) is a growth regulatory glycoprotein with significant amino acid homology to members of the epidermal growth factor (EGF) family. Its effects are mediated via the EGF receptor tyrosine kinase or through specific nuclear targeting sequences. In this study, the localization of immunoreactive AR was examined in paraformaldehyde-glutaraldehyde fixed, paraffin-embedded human placentae and decidua obtained at various stages from 11 weeks to term pregnancy using the avidin-biotin complex/peroxidase method. In addition, the effects of AR on trophoblast proliferation were evaluated from 3HTdR uptake by first trimester human trophoblast cells. Results revealed immunolocalization of AR to the nucleus as well as the cytoplasm of the syncytiotrophoblast cell layer of chorionic villi until approximately week 18 of gestation after which no immunostaining was detected. Villous and extravillous cytotrophoblast cells as well as decidual tissue were negative for AR at all gestational ages examined. In the presence of exogenous AR, there was a dose-dependent increase in proliferation of the trophoblast at AR concentrations ranging from 1-100 ng/ml. These results suggest that AR may be an important paracrine or juxtacrine growth stimulatory molecule for cytotrophoblast cells in situ early in gestation.

Role of transforming growth factor-α (TGFα) and epidermal growth factor (EGF) on proliferation and invasion by first trimester human trophoblast

Summary By employing pure cultures of first trimester human trophoblast cells we have examined the effects of EGF and TGFα on trophoblast cell proliferation and invasiveness. Both exogenous EGF and TGFα were able to stimulate trophoblast proliferation ( 3 H-TdR incorporation). Anti-TGFα neutralizing Ab had no effect on proliferation indicating the lack of significant endogenous TGFα production in these cultures. Anti-EGF-receptor blocking Ab significantly decreased trophoblast proliferation indicating the presence of endogenous EGF or another EGF-receptor ligand, capable of binding to the EGF-receptor, and enhancing trophoblast growth. EGF and TGFα did not alter trophoblast invasiveness in a Matrigel invasion assay although an increase in the transcription of invasion regulating molecules, type IV collagenases and their inhibitors TIMP1 and TIMP2 was noted. Both growth factors may be required for normal placental growth in situ . These studies reveal that the two important biological processes, e.g., trophoblast proliferation and invasion required for placental development are not necessarily linked.

Role of Locally Produced Growth Factors in Human Placental Growth and Invasion with Special Reference to Transforming Growth Factors

Anatomically, the human fetomaternal interface consists of the placenta, a fetally derived organ, and the decidua, a maternally derived tissue. Physical as well as molecular interactions at this interface hold the secrets to two important biological riddles: (i) What protects the placenta, a fetally derived organ and thus genetically disparate from the mother, from destruction by the mother’s immune system? and (ii) What protects the uterus from overinvasion by the placenta, which is a highly invasive tumorlike structure? The present chapter focuses largely on our studies related to the second riddle.

Regulation of NM23 gene expression in the normal and malignant trophoblast by growth factors

Summary nm23-H1 is a nucleoside diphosphate kinase with a functional role in the microtubular assembly and disassembly during cell proliferation and in G-protein dependent signal transduction. Gene expression for this kinase has been reported to be inversely correlated with metastatic ability of tumor cells. Normal trophoblast cells are highly invasive but non-metastatic. Numerous growth factors, e.g., TGF-α, EGF, TGF-β, and CSF-1, present at the fetomaternal interface, influence trophoblast functions such as proliferation, differentiation, and invasion. In this study, we investigated the influence of the above growth factors on nm23-H1 mRNA expression by first trimester normal trophoblast and two choriocarcinoma cell lines, JAr and JEG-3. The expression was found to be abundant in the normal trophoblast as well as choriocarcinomas, indicating that this expression was not suppressed in malignant trophoblast cells. We found that growth factors (e.g., TGFα, EGF, and CSF-1) which stirnulate normal trophoblast proliferation also stimulated nm23-H1 expression in the normal trophoblast indicating that the expression may be coupled with cell proliferation. A stimulatory effect noted with a neutralizing anti-TGF-β antibody can also be explained on the basis of stimulation of trophoblast proliferation due to removal of the anti-proliferative action of endogenous TGF-β. The expression of nm23H-1 in choriocarcinoma cells was unaltered by the growth factors except for some stimulation of JEG-3 cells noted with TGF-β. Since anti-TGF-β Ab also stimulated the expression in JEG-3 cells, we propose that TGF-β may have a dual influence in this case, one related to signal transduction, another to its anti-proliferation.