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Cells in many tissues, such as bone, muscle, and placenta, fuse into syncytia to acquire new functions and transcriptional programs. While it is known that fused cells are specialized, it is unclear whether cell-fusion itself contributes to programmatic-changes that generate the new cellular state. Here, we address this by employing a fusogen-mediated, cell-fusion system to create syncytia from undifferentiated cells. RNA-Seq analysis reveals VSV-G-induced cell fusion precedes transcriptional changes. To gain mechanistic insights, we measure the plasma membrane surface area after cell-fusion and observe it diminishes through increases in endocytosis. Consequently, glucose transporters internalize, and cytoplasmic glucose and ATP transiently decrease. This reduced energetic state activates AMPK, which inhibits YAP1, causing transcriptional-reprogramming and cell-cycle arrest. Impairing either endocytosis or AMPK activity prevents YAP1 inhibition and cell-cycle arrest after fusion. Together, these data demonstrate plasma membrane diminishment upon cell-fusion causes transient nutrient stress that may promote transcriptional-reprogramming independent from extrinsic cues., Cells in many tissues fuse into syncytia acquiring new functions. By investigating whether physical remodelling promotes differentiation, here, the authors show that plasma membrane diminution post-fusion causes transient nutrient stress that inhibits YAP1 activity and may reduce proliferation-promoting transcription.

We sought to examine placentas enriched for trophoblast inclusions (TIs) in order to characterize, quantify, and examine the interrelations between subtypes of TIs to better understand their underlying biology. We examined a cohort of 600 placentas from deliveries between 20(6) and 43(0) weeks of gestation. Forty-five percent of the placentas had at least one TI in the two slides examined. Four percent of the placentas had 10 or more TIs and two placentas had more than 70 TIs. Four distinct TI types were observed: inclusionoids (early forming inclusions), inclusions, calcified inclusions, and calcified bodies. We suggest this reflects a developmental trajectory of TI maturation, the timing of which might be useful when comparing TI expression to clinical outcomes.

Objective Trophoblast inclusions—cross sections of abnormal trophoblast bilayer infoldings—have previously been associated with aneuploidy, placenta accreta, and prematurity. This study was conducted to establish the relationship between trophoblast inclusions and a range of placental, pregnancy, and birth outcomes in a patient population with high smoking and alcohol exposure. Specifically, we sought to evaluate the association between the presence of trophoblast inclusions and 1) three primary birth outcomes: full-term birth, preterm birth, and stillbirth; 2) gestational age at delivery; and 3) specific placental pathologies. Methods Two slides containing chorionic villi were evaluated from 589 placentas that were collected from Stellenbosch University in Cape Town, South Africa as part of the prospective, multicenter cohort Safe Passage Study of the Prenatal Alcohol and SIDS and Stillbirth Network. The subsample included 307 full-term live births, 212 preterm live births, and 70 stillbirths. Results We found that the odds of identifying at least one trophoblast inclusion across two slides of chorionic villi was significantly higher for placentas from preterm compared to term liveborn deliveries (OR = 1.74; 95% CI: 1.22, 2.49, p = 0.002), with an even greater odds ratio for placentas from stillborn compared to term liveborn deliveries (OR = 4.95; 95% CI: 2.78, 8.80, p < 0.001). Gestational age at delivery was inversely associated with trophoblast inclusion frequency. Trophoblast inclusions were significantly associated with small for gestational age birthweight, induction of labor, villous edema, placental infarction, and inflammation of the chorionic plate. Conclusions The novel associations that we report warrant further investigation in order to understand the complex network of biological mechanisms through which the factors that lead to trophoblast inclusions may influence or reflect the trajectory and health of a pregnancy. Ultimately, this line of research may provide critical insights that could inform both clinical and research applications.

Pregnant women appear to be at increased risk for severe outcomes associated with COVID-19, but the pathophysiology underlying this increased morbidity and its potential impact on the developing fetus is not well understood. In this study of pregnant women with and without COVID-19, we assessed viral and immune dynamics at the placenta during maternal SARS-CoV-2 infection. Amongst uninfected women, ACE2 was detected by immunohistochemistry in syncytiotrophoblast cells of the normal placenta during early pregnancy but was rarely seen in healthy placentas at full term. Term placentas from women infected with SARS-CoV-2, however, displayed a significant increase in ACE2 levels. Using immortalized cell lines and primary isolated placental cells, we determined the vulnerability of various placental cell types to direct infection by SARS-CoV-2in vitro. Yet, despite the susceptibility of placental cells to SARS-CoV-2 infection, viral RNA was detected in the placentas of only a subset (∼13%) of women in this cohort. Through single cell transcriptomic analyses, we found that the maternal-fetal interface of SARS-CoV-2-infected women exhibited markers associated with pregnancy complications, such as preeclampsia, and robust immune responses, including increased activation of placental NK and T cells and increased expression of interferon-related genes. Overall, this study suggests that SARS-CoV-2 is associated with immune activation at the maternal-fetal interface even in the absence of detectable local viral invasion. While this likely represents a protective mechanism shielding the placenta from infection, inflammatory changes in the placenta may also contribute to poor pregnancy outcomes and thus warrant further investigation.

Serotonin [5-hydroxytryptamine (5-HT)] is essential to intrauterine development, but its source is debated. We used immunocytochemistry to gauge 5-HT, its biosynthetic enzyme tryptophan hydroxylase 1 (TPH1); an importer (serotonin transporter, 5-HTT/SERT/SLC6A); other transporters [P-glycoprotein 1 (P-gp/ABCB1), OCT3/SLC22A3, and gap junction connexin-43]; and the 5-HT degradative enzyme monoamine oxidase A (MAOA) in sections of placentas. In humans, 5-HT was faintly stained only in first-trimester trophoblasts, whereas TPH1 was not seen at any stage. SERT was expressed in syncytiotrophoblasts and, more strongly, in cytotrophoblasts. MAOA was prominent in syncytiotrophoblasts, OCT3 and gap junctions were stained in cytotrophoblasts, and P-gp was present at the apical surfaces of both epithelia. 5-HT added to cultured placental explants accumulated in the trophoblast epithelium and reached the villus core vessels. Trophoblast uptake was blocked by the SERT inhibitor escitalopram. Inhibition of gap junctions with heptanol prevented the accumulation of 5-HT in cytotrophoblasts, whereas blocking OCT3 with decynium-22 and P-gp with mitotane led to its accumulation in cytotrophoblasts. Reducing 5-HT destruction by inhibiting MAOA with clorgyline increased the accumulation of 5-HT throughout the villus. In the mouse fetus, intravascular platelets stained prominently for 5-HT at day 13.5, whereas the placenta and yolk sac endoderm were both negative. TPH1 was not detected, but SERT was prominent in these mouse tissues. We conclude that serotonin is conveyed from the maternal blood stream through syncytiotrophoblasts, cytotrophoblasts and the villus core to the fetus through a physiological pathway that involves at least SERT, gap junctions, P-gp, OCT3, and MAOA.

SummaryDeveloping cells divide and differentiate, and in many tissues, such as bone, muscle, and placenta, cells fuse acquiring specialized functions. While it is known that fused-cells are differentiated, it is unclear what mechanisms trigger the programmatic-change, and whether cell-fusion alone drives differentiation. To address this, we employed a fusogen-mediated cell-fusion system involving undifferentiated cells in tissue culture. RNA-seq analysis revealed cell-fusion initiates a dramatic transcriptional change towards differentiation. Dissecting the mechanisms causing this reprogramming, we observed that after cell-fusion plasma-membrane surface area decreases through increased endocytosis. Consequently, glucose-transporters are internalized, and cytoplasmic-glucose and ATP transiently decrease. This low-energetic state activates AMPK, which inhibits YAP1, causing cell-cycle arrest. Impairing either endocytosis or AMPK prevents YAP1 inhibition and cell-cycle arrest after fusion. Together these data suggest that cell-fusion-induced differentiation does not need to rely on extrinsic-cues; rather the plasma-membrane diminishment forced by the geometric-transformations of cell-fusion cause transient cell-starvation that induces differentiation.

Background Pregnant women are at increased risk for severe outcomes from coronavirus disease 2019 (COVID-19), but the pathophysiology underlying this increased morbidity and its potential effect on the developing fetus is not well understood. Methods We assessed placental histology, ACE2 expression, and viral and immune dynamics at the term placenta in pregnant women with and without respiratory severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Findings The majority (13 of 15) of placentas analyzed had no detectable viral RNA. ACE2 was detected by immunohistochemistry in syncytiotrophoblast cells of the normal placenta during early pregnancy but was rarely seen in healthy placentas at full term, suggesting that low ACE2 expression may protect the term placenta from viral infection. Using immortalized cell lines and primary isolated placental cells, we found that cytotrophoblasts, the trophoblast stem cells and precursors to syncytiotrophoblasts, rather than syncytiotrophoblasts or Hofbauer cells, are most vulnerable to SARS-CoV-2 infection in vitro. To better understand potential immune mechanisms shielding placental cells from infection in vivo, we performed bulk and single-cell transcriptomics analyses and found that the maternal-fetal interface of SARS-CoV-2-infected women exhibited robust immune responses, including increased activation of natural killer (NK) and T cells, increased expression of interferon-related genes, as well as markers associated with pregnancy complications such as preeclampsia. Conclusions SARS-CoV-2 infection in late pregnancy is associated with immune activation at the maternal-fetal interface even in the absence of detectable local viral invasion. Funding NIH (T32GM007205, F30HD093350, K23MH118999, R01AI157488, U01DA040588) and Fast Grant funding support from Emergent Ventures at the Mercatus Center., Graphical abstract, Context and significance Pregnant women with COVID-19 are at increased risk for severe illness and pregnancy complications compared with non-pregnant women. Researchers at Yale School of Medicine analyzed placentas from SARS-CoV-2-infected women at the time of delivery and found that, although placental cells are susceptible to infection in vitro, viral RNA is rarely detected in clinical samples. The Yale team observed local immune responses at the maternal-fetal interface, including upregulation of interferon pathways and activation of T and NK cells. Although placental immune activation during maternal SARS-CoV-2 infection likely represents a host defense mechanism of shielding the maternal-fetal interface from infection, these inflammatory changes may contribute to the increased risk for complications seen in COVID-19-affected pregnancies., COVID-19 is more severe in pregnant women and can lead to adverse fetal outcomes. Through histological and gene expression studies of placentas from infected women, Lu-Culligan et al. find that maternal SARS-CoV-2 infection during term pregnancy and delivery is associated with immune activation at the maternal-fetal interface even in the absence of detectable virus in the placenta.

e15602 Background: Serotonin (5-HT) is most often considered a neurotransmitter—but in reality its main function is to promote cell proliferation at the site of wound healing via platelet degranulation. We tested the hypothesis that blocking 5-HT production in cancer cells might lead to their death by exposure to a specific tryptophan hydroxylase (TPH) inhibitor: telotristat ethyl (TE). The advantage of this TPH inhibitor is that it does not cross the blood brain barrier and therefore can be used to treat peripheral cancers without danger of interfering with central nervous system 5-HT production. Methods: Three cancer cell lines were tested: liposarcoma (94T778), colon cancer (HT-29), and cholangiocarcinoma (TFK-1). The cells were grown with 0, 5, 15 and 30 µM TE, plated into four-chambered slides with an initial confluence of at least 30%, and cultured for 24-96 H. At the end of each 24 hour period slides were removed, washed, fixed, and stained with hematoxylin for density assessment. Experiments were performed in triplicate and repeated in at least three separate experiments. Cell confluence was determined under low power microscopic examination. Results: In the absence of TE the three cell lines increased their confluence from approximately 30% to 83±5.8% (94T778), 68±11% (HT-29), and 60±16% (TFK-1) over 96 H. When the 94T778 cells were exposed to increasing concentrations of TE their confluence decreased progressively. At a dose of 30 µM cell confluence at 24 H was 3±0%, at 48 H 0.3±0.6%, at 72 H 0±0%, and at 96 H 0±0%. For the HT-29 cells, cell confluence in the 30 µM TE groups were 12±8%, 3±3%, 7±5% and 2±1% at 24, 48, 72 and 96 H, respectively. For the TFK-1cells, cell confluence in the 30 µM TE groups were 7±3%, 20±14%, 17±14% and 24±16% at 24, 48, 72 and 96 H, respectively. Conclusions: Telotristat ethyl is a potent inhibitor of tumor cell line growth. However, the efficacy of this inhibition varies between cell lines. The liposarcoma cell line was most sensitive to TE, with the cholangiocarcinoma cell line being moderately impacted. The colon cancer cell line was intermediate between these two. The differences in response to TE may be related to the specific expression of TPH in any particular cell line, with cancers that are more dependent on 5-HT production being most impacted by TPH blockade. Just as ER PR positive breast cancers are sensitive to blockade of these receptors, TPH positive cancers may be successfully targeted by specific inhibition of the 5-HT biosynthetic pathway.

Zika virus (ZIKV) infection during pregnancy is associated with adverse fetal outcomes, including microcephaly, growth restriction, and fetal demise. Type I interferons (IFNs) are essential for host resistance against ZIKV, and IFN-α/β receptor (IFNAR)-deficient mice are highly susceptible to ZIKV infection. Severe fetal growth restriction with placental damage and fetal resorption is observed after ZIKV infection of type I IFN receptor knockout (Ifnar1-/-) dams mated with wild-type sires, resulting in fetuses with functional type I IFN signaling. The role of type I IFNs in limiting or mediating ZIKV disease within this congenital infection model remains unknown. In this study, we challenged Ifnar1-/- dams mated with Ifnar1+/- sires with ZIKV. This breeding scheme enabled us to examine pregnant dams that carry a mixture of fetuses that express (Ifnar1+/-) or do not express IFNAR (Ifnar1-/-) within the same uterus. Virus replicated to a higher titer in the placenta of Ifnar1-/- than within the Ifnar1+/- concepti. Yet, rather unexpectedly, we found that only Ifnar1+/- fetuses were resorbed after ZIKV infection during early pregnancy, whereas their Ifnar1-/- littermates continue to develop. Analyses of the fetus and placenta revealed that, after ZIKV infection, IFNAR signaling in the conceptus inhibits development of the placental labyrinth, resulting in abnormal architecture of the maternal-fetal barrier. Exposure of midgestation human chorionic villous explants to type I IFN, but not type III IFNs, altered placental morphology and induced cytoskeletal rearrangements within the villous core. Our results implicate type I IFNs as a possible mediator of pregnancy complications, including spontaneous abortions and growth restriction, in the context of congenital viral infections.

Maternal physiologic stress during gestation has been reported to be associated with negative developmental outcomes, including intra-uterine growth restriction and reduced birth weight, which can impact postnatal development, behavior and health. The human fetus is partially protected from elevated cortisol exposure by placental 11 β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), which oxidizes bioactive cortisol into bio-inactive cortisone. Importantly, despite the critical protective role hypothesized for 11β-HSD2, the onset of its placental expression has yet to be clearly established. To this aim, we present immunocytochemical analysis of placentas collected 3–6 weeks post-conception. 11β-HSD2 was present as early as 3 weeks post-conception in syncytiotrophoblasts, where most maternal–fetal exchange occurs, and in columnar epithelial cells encircling uterine endometrial glands, which provide early histiopathic nutrition to the embryo. 11β-HSD2 expression in these critical maternal–fetal exchange areas is consistent with its hypothesized protective role. Future studies should investigate the mechanisms that may modulate embryonic glucocorticoid exposure earlier, immediately post-conception.

We evaluated the role of placental protein 13 (PP13; galectin 13) in the process of trophoblast invasion and decidual necrosis. Immunohistochemical analysis for PP13, immune cells, human placental lactogen, cytokeratin, and apoptosis markers was performed on 20 elective pregnancy termination specimens between 6 and 15 weeks of gestation. Placental protein 13 was localized to syncytiotrophoblasts in the chorionic villi and to occasional multinucleated luminal trophoblasts within converted decidual spiral arterioles. Cytotrophoblasts, anchoring trophoblasts, and invasive trophoblasts did not stain for PP13. Extracellular PP13 aggregates were found around decidual veins associated with T-cell-, neutrophil- and macrophage-containing decidual zones of necrosis (ZONEs). We hypothesize that PP13 is secreted into the intervillus space, drains through the decidua basalis veins, and forms perivenous PP13 aggregates which attract and activate maternal immune cells. Thus, syncytiotrophoblast-derived PP13 may create a ZONE that facilitates trophoblast invasion and conversion of the maternal spiral arterioles.