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The developing placenta, which in mice originates through the extraembryonic ectoderm (ExE), is essential for mammalian embryonic development. Yet unbiased characterization of the differentiation dynamics of the ExE and its interactions with the embryo proper remains incomplete. Here we develop a temporal single-cell model of mouse gastrulation that maps continuous and parallel differentiation in embryonic and extraembryonic lineages. This is matched with a three-way perturbation approach to target signalling from the embryo proper, the ExE alone, or both. We show that ExE specification involves early spatial and transcriptional bifurcation of uncommitted ectoplacental cone cells and chorion progenitors. Early BMP4 signalling from chorion progenitors is required for proper differentiation of uncommitted ectoplacental cone cells and later for their specification towards trophoblast giant cells. We also find biphasic regulation by BMP4 in the embryo. The early ExE-originating BMP4 signal is necessary for proper mesoendoderm bifurcation and for allantois and primordial germ cell specification. However, commencing at embryonic day 7.5, embryo-derived BMP4 restricts the primordial germ cell pool size by favouring differentiation of their extraembryonic mesoderm precursors towards an allantois fate. ExE and embryonic tissues are therefore entangled in time, space and signalling axes, highlighting the importance of their integrated understanding and modelling in vivo and in vitro. Temporally dynamic extraembryonic and embryonic BMP4 signalling shapes mouse embryo lineage choices.

The purpose of the present study was to process and assess the effect of hydrated amnion chorion membrane and dehydrated amnion chorion membrane on proliferation of periodontal ligament (PDL) fibroblast cells. The amnion chorion membrane (ACM) from placenta of 18 systemically healthy patients was obtained from the Department of Obstetrics and Gynaecology. They were processed as hydrated and dehydrated based on different processing methods. The Periodontal ligament cells were obtained from periodontal ligament of freshly extracted premolars of systemically healthy patients, due to orthodontic reasons. The PDL cells were further cultured in laboratory and were exposed to hydrated and dehydrated amnion chorion membrane. The MTT assay was performed to assess the proliferation of PDL fibroblast cells after 24 and 48 h. The hydrated and dehydrated amnion chorion membrane showed proliferation of PDL fibroblasts after 24 and 48 h. The proliferation of PDL fibroblasts in hydrated (p = 0.043) and dehydrated (p = 0.050) amnion chorion membrane was statistically significant at the end of 24 and 48 h respectively. On inter-group comparison dehydrated ACM showed significant proliferation of PDL fibroblasts after 24 (p=0.014) and 48 h (p=0.019). Within the limits of the present study, it can be concluded: both hydrated and dehydrated amnion chorion membrane showed proliferationof PDL fibroblast cells. However, dehydrated ACM showed significant proliferation of PDL fibroblasts.

Background Extracellular vesicles (EVs) are the foundation of modern regenerative medicine using a cell-free approach. While current research mainly explores EVs from biological fluids and cell culture supernatants, tissue-derived EVs hold great promise, but remain largely underexplored. Since healthy placental tissues such as the chorion are widely available after full-term delivery, ethically unobjectionable, and possess exceptional regenerative potential, we sought to compare the biological effects of EVs derived directly from chorion tissue with those from chorion-derived mesenchymal stromal cell EVs and plasma EVs. Method We compared the biological impact of EVs from various sources (chorion tissue CHO-Ti, MSCs from chorion CHO-MSC and platelet-poor plasma PPP) and isolated by various techniques on the gene expression of osteoarthritic chondrocytes. Additionally, we assessed the effect of enriched soluble proteins of CHO-MSC and CHO-Ti secretome vs. their EVs. EVs were characterized by particle number and size (NTA), protein content (BCA assay) and immunophenotype (flow cytometry). Changes in gene expression of chondrocytes were quantified by RT-qPCR. Results CHO-Ti-EVs and PPP-EVs showed particularly beneficial effect on the inflammatory process, with their biological impact surpassing that of CHO-MSC-EVs. Chondroprotective markers COL2A and ACAN were robustly upregulated by CHO-Ti-EVs and PPP-EVs but showed only modest or variable increases with CHO-MSC-EVs. COMP expression, however, was specifically enhanced by CHO-MSC-derived components. Furthermore, our results also indicate that the therapeutic properties of the CHO-Ti secretome are exclusively linked to EVs. Among CHO-MSC-EVs, purification combined with UC resulted in the highest purity, however EVs purified by SEC presented a more favourable surface marker profile and better biological effects. The observed variability suggests that different EV preparations harbour distinct subpopulations that influence regulatory pathways differently and highlight the importance of EV source and isolation methodology in determining biological activity. Conclusion CHO-Ti-EVs showed promising effects on cartilage regeneration and inflammation modulation, suggesting they may represent a viable alternative to plasma- and CHO-MSC-EVs. Moreover, the chorion represents a readily accessible and abundant source of perinatal tissue obtainable non-invasively after full-term delivery, further supporting the translational potential of CHO-Ti-EVs.

Differences in male (M) and female (F) neonates’ premature birth outcomes and placental trophoblast inflammation have been observed but are unknown to occur within the fetal membrane trophoblast layer (chorion trophoblasts [CTC]). This study examined whether sex-based differences in gene expression and inflammatory marker expression can be observed in CTCs under control or infectious inflammatory conditions modeling preterm birth. CTCs from six different patient-derived fetal membrane samples (3M/3F) were cultured and divided into experimental (Lipopolysaccharide [LPS]) and control groups for 6, 12, or 24 h. RNA from CTCs was subjected to RNA-seq, while cytokine multiplex or ELISA detected pro-/anti-inflammatory cytokines, progesterone, and soluble HLA-G in cell supernatants. CTC-M and CTC-F showed sex, time, and stimulant-dependent differential gene expression profiles. Cytokine analysis demonstrated a significantly lower IL-6 production in control CTC-M than in CTC-F. No sex-dependent responses were observed after LPS treatment regarding cytokines. CTC-M produced significantly lower progesterone than CTC-F. The theories of sexual dimorphism linked to placental inflammation may not extend to CTCs. This study supports that the chorion acts as a “great wall” protecting the fetus by being refractory to insults. Further examination into the weaknesses of the chorion barrier and sex-dependent responses of fetal membranes is needed.

The fetal membrane surrounds the fetus during pregnancy and is a thin tissue composed of two layers, the chorion and the amnion. While rupture of this membrane normally occurs at term, preterm rupture can result in increased risk of fetal mortality and morbidity, as well as danger of infection in the mother. Although structural changes have been observed in the membrane in such cases, the mechanical behaviour of the human fetal membrane in vivo remains poorly understood and is challenging to investigate experimentally. Therefore, the objective of this study was to develop simplified finite element models to investigate the mechanical behaviour and rupture of the fetal membrane, particularly its constituent layers, under various physiological conditions. It was found that modelling the chorion and amnion as a single layer predicts remarkably different behaviour compared with a more anatomically-accurate bilayer, significantly underestimating stress in the amnion and under-predicting the risk of membrane rupture. Additionally, reductions in chorion-amnion interface lubrication and chorion thickness (reported in cases of preterm rupture) both resulted in increased membrane stress. Interestingly, the inclusion of a weak zone in the fetal membrane that has been observed to develop overlying the cervix would likely cause it to fail at term, during labour. Finally, these findings support the theory that the amnion is the dominant structural component of the fetal membrane and is required to maintain its integrity. The results provide a novel insight into the mechanical effect of structural changes in the chorion and amnion, in cases of both normal and preterm rupture.

Cholangiocarcinoma (CCA) is an aggressive bile duct malignancy with poor survival rates due to late detection, rapid metastasis, and treatment resistance. Therefore, new therapeutic strategies are needed to improve treatment outcomes. Previous studies have reported that mesenchymal stem cells (MSCs) secrete several soluble factors that modulate intracellular signaling pathways that are critical for the regulation of cancer cell function. The present study aimed to investigate the effects and molecular mechanisms of conditioned media from chorion-derived human MSCs (CH-CM) and placenta-derived human MSCs (PL-CM) on the migration and invasion of human CCA cells. We found that both CH-CM and PL-CM suppress cell migration and invasion in three CCA cell lines (KKU100, KKU213A and KKU213B) by increasing E-cadherin expression and decreasing the expression of several factors involved in the epithelial–mesenchymal transition process, including ZEB1, ZEB2, N-cadherin, vimentin, and MMP-2. The effects of CH-CM and PL-CM were mediated, at least in part, through the suppression of the PI3K/AKT signaling pathway in CCA cells. Our findings suggest that soluble factors derived from CH-MSCs and PL-MSCs could be used in combination with other conventional treatments to diminish the invasiveness of CCA cells, thus improving the therapeutic outcome and increasing survival in CCA patients.

Human derived composite amnion-chorion membrane (ACM) has been used to facilitate wound healing due to reported anti-inflammatory properties and promotion of cell proliferation. This study aimed to assess the antimicrobial properties of the ACM using novel methods to visualize the antimicrobial efficacy of membranes in situ at different time points. Porcine Pericardium Collagen Membranes (PPCM) served as membrane controls. Circular pieces of the membranes were used in three different assays: insert, agar contact and glass-bottom well assays. Streptococcus gordonii were spotted onto the membranes and the plates were subsequently centrifuged to ensure direct bacterial contact with the membranes in the insert and agar contact assays, thus better mimicking bacterial adherence in the oral cavity. After incubation at 37 °C for 8, 24, and 48 hours, the membranes were dyed with the Live/Dead BacLight Bacterial Viability fluorescence stain and analyzed via confocal microscopy. The results demonstrated that the ACM completely inhibited bacterial growth at all time points, whereas the PPCM did not demonstrate any antimicrobial properties. Within the limits of this study, the ACM showed extremely high antimicrobial efficacy against oral streptococci. In addition, our methods may be useful in assessing antimicrobial properties for biomaterials with minimum diffusion ability, when traditional assessment methods are not applicable.

Mesenchymal stem cells (MSCs) are of great interest to scientists due to their application in cell therapy of many diseases, as well as regenerative medicine and tissue engineering. Recently, there has been growing evidence surrounding the research based on extracellular vesicles (EVs), especially small EVs (sEVs)/exosomes derived from MSCs. EVs/exosomes can be secreted by almost all cell types and various types of EVs show multiple functions. In addition, MSCs-derived exosomes have similar characteristics and biological activities to MSCs and their therapeutic applications are considered as a safe strategy in cell-free therapy. The aim of this study was the characterization of MSCs isolated from the chorion (CHo-MSCs) of human full-term placenta, as well as the isolation and analysis of small EVs obtained from these cells. Accordingly, in this study, the ability of small EVs’ uptake is indicated by synovial fibroblasts, osteoblasts and periosteum-derived MSCs. Improvement in the understanding of the structure, characteristics, mechanism of action and potential application of MSCs-derived small EVs can provide new insight into improved therapeutic strategies.

The impact of carbon nanotubes (CNTs) on the aquatic environment was investigated by examining the properties of raw CNTs under several environmental conditions and using developing zebrafish (Danio rerio) embryos. The agglomerate size for single‐walled CNTs (SWCNTs) was significantly larger at pH 11 or greater and was stable at temperatures from 4 to 40°C and salinities from 0 to 30 ppt. Exposure to SWCNTs induced a significant hatching delay in zebrafish embryos between 52 to 72 h postfertilization (hpf) at concentrations of greater than 120 mg/L, but 99% of the exposed embryos hatched by 75 hpf. Double‐walled CNTs also induced a hatching delay at concentrations of greater than 240 mg/L, but carbon black did not affect hatching at the concentrations tested. Molecular and cellular analysis showed that the embryonic development of the exposed embryos up to 96 hpf was not affected at SWCNT concentrations of up to 360 mg/L. Scanning‐electron microscopic inspection showed that the size of the pores on the embryo chorion was nanoscaled and that the size of SWCNT agglomerates was microscaled or larger, indicating that the chorion of zebrafish embryos was an effective protective barrier to SWCNT agglomerates. The hatching delay observed in this study likely was induced by the Co and Ni catalysts used in the production of SWCNTs that remained at trace concentrations after purification. This study suggests that materials associated with raw SWCNTs (perhaps metal contaminants) have the potential to affect aquatic life when released into the aquatic environment.

Morphology and physiology of fish embryos undergo dramatic changes during their development until the onset of feeding, supplied only by endogenous yolk reserves. For obtaining an insight how these restructuring processes are reflected by body mass related parameters, dry weights (dw), contents of the elements carbon and nitrogen and lipid and protein levels were quantified in different stages within the first four days of embryo development of the zebrafish (Danio rerio). The data show age dependent changes in tissue composition. Dry weights decreased significantly from 79μgdw/egg at 0hours post fertilization (hpf) to 61 μgdw/egg after 96 hpf. The amounts of total carbon fluctuated between 460 mg g-1 and 540 mg g-1 dw, nitrogen was at about 100 mg g-1 dw and total fatty acids were between 48-73 mg g-1 dw. In contrast to these parameters that remained relatively constant, the protein content, which was 240 mg g-1 at 0 hpf, showed an overall increase of about 40%. Comparisons of intact eggs and dechorionated embryos at stages prior to hatching (24, 30, 48 hpf) showed that the differences seen for dry weight and for carbon and nitrogen contents became smaller at more advanced stages, consistent with transition of material from the chorion to embryo tissue. Further, we determined the effect of 2,4-dinitrophenol at a subacutely toxic concentration (14 μM, LC10) as a model chemical challenge on the examined body mass related parameters. The compound caused significant decreases in phospholipid and glycolipid fatty acid contents along with a decrease in the phospholipid fatty acid unsaturation index. No major changes were observed for the other examined parameters. Lipidomic studies as performed here may thus be useful for determining subacute effects of lipophilic organic compounds on lipid metabolism and on cellular membranes of zebrafish embryos.