Cytogenomics of six human trophoblastic cell lines.

Trophoblastic cell lines are established models used to examine human placenta physiology and disease. We performed concurrent cytogenetic analyses of six established and well-studied trophoblastic cell lines including JAR, BeWo, JEG-3, AC-1M59, HTR8/SVneo, and ACH-3P. All cell lines showed near triploid or tetraploid karyotypes with unique inter- and intra-clonal aberrations, which result possibly from long-term culture or defects in the placenta or its malignant choriocarcinoma origin. Variable aneuploidy in 'standard' cell lines is under-appreciated and may not reflect the in vivo situation. It has the potential to negatively impact our understanding of normal cell function and cause disagreement between studies.

Are uterine natural killer and plasma cells in infertility patients associated with endometriosis, repeated implantation failure, or recurrent pregnancy loss?

PURPOSE: Infertility is a debilitating situation that millions of women around the world suffer from, but the causal relationship between infertility and endometriosis is still unclear. We hypothesize that the immune cell populations of uterine natural killer cells (uNK) and plasma cells (PC) which define chronic endometritis could differ in patients with or without endometriosis and therefore be the link to endometriosis-associated infertility. METHODS: Our retrospective study includes 173 patients that underwent an endometrial scratching in the secretory phase of the menstrual cycle and subsequently immunohistochemical examination for uNK cells and PC. Sixty-seven patients were diagnosed with endometriosis, 106 served as the control cohort. RESULTS: The risk for an elevated number of uNK cells in women with endometriosis is not increased as compared to the control group. Our findings suggest that patients with endometriosis are 1.3 times more likely to have chronic endometritis (CE) as compared to those without and that the treatment with doxycycline might increase pregnancy rates. Endometriosis and an increased number of uNK cells seem to be unrelated. CONCLUSIONS: In contrast to the lately published connection between endometriosis, infertility and increased uNK cells, we could not find any evidence that patients with endometriosis are more prone to elevated uterine uNK cells. Counting of PC in endometrial biopsies might be a new approach in the search of biomarkers for the nonsurgical diagnosis of endometriosis since our findings suggest a connection.

PIM kinases 1, 2 and 3 in intracellular LIF signaling, proliferation and apoptosis in trophoblastic cells.

Proviral insertion in murine (PIM) lymphoma proteins are mainly regulated by the Janus Kinase/Signal Transducer Activator of Transcription (JAK/STAT) signaling pathway, which can be activated by members of the Interleukin-6 (IL-6) family, including Leukemia Inhibitory Factor (LIF). Aim of the study was to compare PIM1, PIM2 and PIM3 expression and potential cellular functions in human first and third trimester trophoblast cells, the immortalized first trimester extravillous trophoblast cell line HTR8/SVneo and the choriocarcinoma cell line JEG-3. Expression was analyzed by qPCR and immunochemical staining. Functions were evaluated by PIM inhibition followed by analysis of kinetics of cell viability as assessed by MTS assay, proliferation by BrdU assay, and apoptosis by Western blotting for BAD, BCL-XL, (cleaved) PARP, CASP3 and c-MYC. Apoptosis and necrosis were tested by flow cytometry (annexin V/propidium iodide staining). All analyzed PIM kinases are expressed in primary trophoblast cells and both cell lines and are regulated upon stimulation with LIF. Inhibition of PIM kinases significantly reduces viability and proliferation and induces apoptosis. Simultaneously, phosphorylation of c-MYC was reduced. These results demonstrate the involvement of PIM kinases in LIF-induced regulation in different trophoblastic cell lines which may indicate similar functions in primary cells.

Uterine natural killer cells in patients with idiopathic recurrent miscarriage.

PROBLEM: Uterine natural killer (uNK) cells are major players during implantation and early pregnancy. The aim of our study was to analyze uNK cell concentration in the endometrium of idiopathic recurrent miscarriage (iRM) patients and fertile controls. METHOD OF STUDY: Out of n=130 couples with ≥3 consecutive, clinical RM screened according to a standardized diagnostic protocol, n=58 patients with iRM were identified. Endometrial biopsies were investigated in patients and n=17 fertile women (controls) via immunohistochemistry. RESULTS: Compared to controls, the concentration of uNK cells was significantly higher in iRM patients (257±212 vs. 148±73 uNK cells/mm², P=.04). IRM patients showed a higher prevalence of >300 uNK cells/mm² than controls (34.5% vs. 5.9%, P=.02). In 88% of controls and 62% of iRM patients, uNK cells were detected within the range of 40-300/mm². CONCLUSION: Idiopathic recurrent miscarriage patients showed higher uNK cell levels than controls supporting the possible impact of uNK cells in the pathophysiology of miscarriage. Our cutoff levels might help to select RM patients which may benefit from immunomodulatory treatment.

Involvement of STAT1 in proliferation and invasiveness of trophoblastic cells.

Trophoblast proliferation and invasion are controlled by cytokines and growth factors present at the implantation site. Members of the Interleukin-6 (IL-6) family of cytokines trigger their effects through activation of intracellular cascades including the Janus Kinase/Signal Transducer and Activator of Transcription (JAK-STAT) pathway. Functions of several STAT molecules in trophoblast cells have been described, but the role of STAT1 remained unclear. Here, potential functions of STAT1 and its activation by Oncostatin M (OSM) have been investigated in an in vitro model. STAT1 expression and phosphorylation were analyzed in human term placenta tissue by immunohistochemistry. HTR-8/SVneo cells (immortalized human extravillous trophoblast cells) were stimulated with OSM, IL-6, IL-11, Leukemia Inhibitory Factor (LIF) and Granulocyte Macrophage Colony-Stimulating Factor. Expression and phosphorylation of STAT1 were analyzed by Western blotting and immunocytochemistry. Fludarabine and STAT1 siRNA were employed for STAT1 depletion. STAT1 transcriptional activity was evaluated by DNA-binding capacity assay. Cell viability and invasion were assessed by MTS and Matrigel assays, respectively. STAT1 was expressed in villous and extravillous trophoblast cells. Low phosphorylation was detectable exclusively in extravillous trophoblast cells. Only OSM and LIF induced phosphorylation of STAT1 in the in vitro model. Challenge with OSM increased cell invasion but not proliferation. Inhibition of STAT1 by fludarabine treatment or STAT1 siRNA transfection reduced cell viability and invasiveness in presence and absence of OSM. These results indicate the potential involvement of STAT1 in the regulation of trophoblast behavior. Furthermore, STAT 1 functions are more efficiently inhibited by blocking its expression than its phosphorylation.

Unique trophoblast stem cell- and pluripotency marker staining patterns depending on gestational age and placenta-associated pregnancy complications.

Preeclampsia (PE) and intrauterine growth retardation (IUGR) are rare but severe pregnancy complications that are associated with placental insufficiency often resulting in premature birth. The clinical pathologies are related to gross placental pathologies and trophoblastic deficiencies that might derive from inflammatory processes and oxidative stress injury. The mesenchymal core of placental villi has been identified as a possible niche for trophoblast progenitor cells that are called upon to replenish the injured syncytiotrophoblast layer. These progenitor cells are known to express trophoblast stem cell (CDX2) and pluripotency (SOX2, NANOG and OCT4A) markers, however only little data is available characterizing the expression of these transcription factors beyond the blastocyst stage. We aimed to describe the expression of these factors in healthy 1st and 3rd trimester placentae as well as PE, IUGR and combined PE+IUGR placentae. We analyzed 8 respective samples derived from 1st trimester (elective abortions), and 3rd trimester (healthy controls, PE, IUGR and combined PE+IUGR). We accomplished immunoperoxidase staining to detect the stem cell markers: CDX2 (trophectoderm), SOX2, NANOG and OCT4A (embryonal). Immunoreative scoring was used for objective analyses of staining patterns. All markers display clearly elevated signals in 1st trimester villous samples as compared to healthy 3rd trimester counterparts. Especially CDX2 and NANOG were specific to the cytotrophoblast layer and the mesenchymal core. Specific and differential expression patterns were visible in the villous/extravillous compartment of each placenta-associated pregnancy complication (PE: pan elevated expression; IUGR elevated SOX2 in basal plate; combined PE+IUGR pan loss of expression). Reduction of stem cell transcription factor expression in term placentae indicates temporal regulation, and probably a specific function which is yet to be elucidated. The differential expression patterns within placentae complicated with placenta-associated pregnancy complications indicate that PE, IUGR and combined PE+IUGR are separate entities. It is unclear whether the alterations are the cause or the effect of the clinical pathology.

Intranuclear crosstalk between extracellular regulated kinase1/2 and signal transducer and activator of transcription 3 regulates JEG-3 choriocarcinoma cell invasion and proliferation.

Invasiveness of trophoblast and choriocarcinoma cells is in part mediated via leukemia inhibitory factor- (LIF-) induced activation of signal transducer and activator of transcription 3 (STAT3). The regulation of STAT3 phosphorylation at its ser727 binding site, possible crosstalk with intracellular MAPK signaling, and their functional implications are the object of the present investigation. JEG-3 choriocarcinoma cells were cultured in presence/absence of LIF and the specific ERK1/2 inhibitor (U0126). Phosphorylation of signaling molecules (p-STAT3 (ser727 and tyr705) and p-ERK1/2 (thr 202/tyr 204)) was assessed per Western blot. Immunocytochemistry confirmed results, but also pinpointed the location of phosphorylated signaling molecules. STAT3 DNA-binding capacity was studied with a colorimetric ELISA-based assay. Cell viability and invasion capability were assessed by MTS and Matrigel assays. Our results demonstrate that LIF-induced phosphorylation of STAT3 (tyr705 and ser727) is significantly increased after blocking ERK1/2. STAT3 DNA-binding capacity and cell invasiveness are enhanced after LIF stimulation and ERK1/2 blockage. In contrast, proliferation is enhanced by LIF but reduced after ERK1/2 inhibition. The findings herein show that blocking ERK1/2 increases LIF-induced STAT3 phosphorylation and STAT3 DNA-binding capacity by an intranuclear crosstalk, which leads to enhanced invasiveness and reduced proliferation.

HTR8/SVneo cells display trophoblast progenitor cell-like characteristics indicative of self-renewal, repopulation activity, and expression of "stemness-" associated transcription factors.

INTRODUCTION: JEG3 is a choriocarcinoma--and HTR8/SVneo a transformed extravillous trophoblast--cell line often used to model the physiologically invasive extravillous trophoblast. Past studies suggest that these cell lines possess some stem or progenitor cell characteristics. Aim was to study whether these cells fulfill minimum criteria used to identify stem-like (progenitor) cells. In summary, we found that the expression profile of HTR8/SVneo (CDX2+, NOTCH1+, SOX2+, NANOG+, and OCT-) is distinct from JEG3 (CDX2+ and NOTCH1+) as seen only in human-serum blocked immunocytochemistry. This correlates with HTR8/SVneo's self-renewal capacities, as made visible via spheroid formation and multi-passagability in hanging drops protocols paralleling those used to maintain embryoid bodies. JEG3 displayed only low propensity to form and reform spheroids. HTR8/SVneo spheroids migrated to cover and seemingly repopulate human chorionic villi during confrontation cultures with placental explants in hanging drops. We conclude that HTR8/SVneo spheroid cells possess progenitor cell traits that are probably attained through corruption of "stemness-" associated transcription factor networks. Furthermore, trophoblastic cells are highly prone to unspecific binding, which is resistant to conventional blocking methods, but which can be alleviated through blockage with human serum.

Is galectin-1 a trigger for trophoblast cell fusion?: the MAP-kinase pathway and syncytium formation in trophoblast tumour cells BeWo.

Galectin-1 (gal-1), a member of the mammalian ß-galactoside-binding proteins, exerts biological effects by recognition of glycan ligands, including those involved in cell adhesion and growth regulation. In a previous study, we demonstrated that gal-1 induces cell differentiation processes on the membrane of choriocarcinoma cells BeWo, including the receptor tyrosine kinases, REarranged during transfection, janus kinase 2 and vascular endothelial growth factor receptor 3. Within this study, we examined which mitogen-activated protein kinases (MAPK) and serine/threonine kinases were phoshorylated by gal-1. Out of a number of 21 different MAPKs and other serine/threonine kinases, the stimulation of BeWo cells with gal-1 showed a significant alteration of signal intensity in extracellular-regulated kinases 1/2 (ERK1/2), Akt-3, Akt-pan and glycogen synthase kinase-α/ß (GSK-3α/ß). We demonstrated that gal-1 significantly inhibited ERK1/2, Akt-3/pan and GSK-3α/ß phosphorylation in BeWo cells and in addition induced Elk1 transcription factor activation. In contrast to gal-1 effects, MAPK inhibitor U0126 reduced syncytium formation of BeWo cells. The results of our data showed that phosphorylation of MAP kinases are involved in gal-1-induced signal transduction processes in BeWo cells. Additional results obtained with MAPK inhibitor U0126 close the gap between syncytium formation induced by gal-1 and MAPK activation in trophoblast cells. Furthermore, we demonstrated that gal-1 induces the activation of Elk1, a transcription factor that is activated by MAPK pathways.