Maja Weber

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/SVneos 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.

Extracellular vesicles in blood, milk and body fluids of the female and male urogenital tract and with special regard to reproduction

Abstract Extracellular vesicles (EVs) are released from almost all cells and tissues. They are able to transport substances (e.g. proteins, RNA or DNA) at higher concentrations than in their environment and may adhere in a receptor-controlled manner to specific cells or tissues in order to release their content into the respective target structure. Blood contains high concentrations of EVs mainly derived from platelets, and, at a smaller amount, from erythrocytes. The female and male reproductive tracts produce EVs which may be associated with fertility or infertility and are released into body fluids and mucosas of the urogenital organs. In this review, the currently relevant detection methods are presented and critically compared. During pregnancy, placenta-derived EVs are dynamically detectable in peripheral blood with changing profiles depending upon progress of pregnancy and different pregnancy-associated pathologies, such as preeclampsia. EVs offer novel non-invasive diagnostic tools which may reflect the situation of the placenta and the foetus. EVs in urine have the potential of reflecting urogenital diseases including cancers of the neighbouring organs. Several methods for detection, quantification and phenotyping of EVs have been established, which include electron microscopy, flow cytometry, ELISA-like methods, Western blotting and analyses based on Brownian motion. This review article summarises the current knowledge about EVs in blood and cord blood, in the different compartments of the male and female reproductive tracts, in trophoblast cells from normal and pre-eclamptic pregnancies, in placenta ex vivo perfusate, in the amniotic fluid, and in breast milk, as well as their potential effects on natural killer cells as possible targets.

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.

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 β-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/β (GSK-3α/β). We demonstrated that gal-1 significantly inhibited ERK1/2, Akt-3/pan and GSK-3α/β 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.

A New Enzyme-linked Sorbent Assay (ELSA) to Quantify Syncytiotrophoblast Extracellular Vesicles in Biological Fluids

The pregnancy‐associated disease preeclampsia is related to the release of syncytiotrophoblast extracellular vesicles (STBEV) by the placenta. To improve functional research on STBEV, reliable and specific methods are needed to quantify them. However, only a few quantification methods are available and accepted, though imperfect. For this purpose, we aimed to provide an enzyme‐linked sorbent assay (ELSA) to quantify STBEV in fluid samples based on their microvesicle characteristics and placental origin.

Stat3 and Socs3 expression patterns during murine placenta development.

Signal transducers and activators of transcription 3 (STAT3) has been identified as an important signal transducer in the invasive phenotype of the trophoblasts cells in in vitro studies. However, the in situ distribution and patterns of expression of this molecule in trophoblast cells during the development of the placenta are still under-elucidated. Mice uteri of gestational ages between 7 and 14 days of pregnancy (dop) were fixed in methacarn and processed with immunoperoxidase techniques for detection of STAT3 and its phosphorylation at serine (p-ser727) residues, as well as the suppressor of cytokine signaling 3 (SOCS3) expression. STAT3 was observed at 7 through 9 dop in both the antimesometrial and mesometrial deciduas, while continued immunoreactivity between 10 and 13 dop was seen only in the mesometrial decidua. In the placenta, STAT3 was detected in the cytotrophoblast cells of labyrinth and giant trophoblast cells between 10 and 14 dop. Immunoreactivity for STAT3 was also seen in trophoblast cells surrounding the maternal blood vessels. On days 10 and 11 of pregnancy, p-ser727 was detectable in the mesometrial decidua and in giant trophoblasts, while during 12-14 dop in the spongiotrophoblast region. In addition, SOCS3 was immunodetected in maternal and placental tissues, principally in the giant trophoblast cells during the whole period of the study. The present in situ study shows the distribution of STAT3, its serine activation and SOCS3 in different maternal and fetal compartments during murine placental development, thus further supporting the idea that they play a role during physiological placentation in mice.

The “killer cell story” in recurrent miscarriage: Association between activated peripheral lymphocytes and uterine natural killer cells

Peripheral and uterine NK cells (pNK, uNK) can be distinguished according to their receptor expression. Recent studies indicate an association of elevated pNK and uNK with recurrent miscarriage (RM). This study aimed to analyze pNK and uNK in patients with RM and healthy controls. Out of n=590 RM patients screened according to a standard diagnostic protocol, n=268 couples with ≥3 consecutive RM were identified. Subgroups consisted of n=151 primary RM (pRM), n=85 secondary RM (sRM), n=32 tertiary RM (tRM) and n=42 healthy controls. Finally, n=147 idiopathic RM (iRM) and n=121 non-iRM patients were identified. Peripheral blood levels of CD45+CD3-CD56+CD16+ NK cells were determined in non-pregnant patients and controls in the mid-luteal phase by FACS. In n=129 RM patients a uterine biopsy was taken to evaluate CD56+ NK cells by immunohistochemistry. PRM showed higher absolute pNK than sRM (median/μl (Q1;Q3): 234 (147;306) vs 176 (128;245), p=0.02). Further a trend towards higher pNK percentages in pRM was detected. UNK numbers did not differ between RM subgroups and did not correlate with pNK. However, the rate of highly elevated uNK was increased in iRM compared to non-iRM patients (p=0.04). Further, higher numbers of CD45+CD3-DR+ (p<0.01) and CD45+CD3+CD8+DR+ (p=0.04) peripheral lymphocytes were associated with higher uNK numbers. In conclusion, elevated pNK were present in pRM patients. Although pNK and uNK numbers did not correlate, the association between high CD45+CD3-DR+ and CD45+CD3+CD8+DR+ peripheral lymphocytes and uNK might indicate that activated NK, B and T cells provide cytokines for the differentiation of uNK.

Uterine natural killer cells in patients with idiopathic recurrent miscarriage

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.

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

ABSTRACT 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.

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.