Progesterone inactivation in decidual stromal cells: A mechanism for inflammation-induced parturition.

The process of human parturition involves inflammation at the interface where fetal chorion trophoblast cells interact with maternal decidual stromal (DS) cells and maternal immune cells in the decidua (endometrium of pregnancy). This study tested the hypothesis that inflammation at the chorion-decidua interface (CDI) induces labor by negating the capacity for progesterone (P4) to block labor and that this is mediated by inactivation of P4 in DS cells by aldo-keto reductase family 1 member C1 (AKR1C1). In human, Rhesus macaque, and mouse CDI, AKR1C1 expression increased in association with term and preterm labor. In a human DS cell line and in explant cultures of term human fetal membranes containing the CDI, the prolabor inflammatory cytokine, interleukin-1ß (IL-1ß), and media conditioned by LPS-stimulated macrophages increased AKR1C1 expression and coordinately reduced nuclear P4 levels and P4 responsiveness. Loss of P4 responsiveness was overcome by inhibition of AKR1C1 activity, inhibition of AKR1C1 expression, and bypassing AKR1C1 activity with a P4 analog that is not metabolized by AKR1C1. Increased P4 activity in response to AKR1C1 inhibition was prevented by the P4 receptor antagonist RU486. Pharmacologic inhibition of AKR1C1 activity prevented parturition in a mouse model of inflammation-induced preterm parturition. The data suggest that inflammatory stimuli at the CDI drive labor by inducing AKR1C1-mediated P4 inactivation in DS cells and that inhibiting and/or bypassing of AKR1C1-mediated P4 inactivation is a plausible therapeutic strategy to mitigate the risk of inflammation-associated preterm birth.

PL-hMSC and CH-hMSC derived soluble factors inhibit proliferation but improve hGBM cell migration by activating TGF-ß and inhibiting Wnt signaling.

Glioblastoma multiforme (GBM) is one of the most common and aggressive brain tumors. GBM resists most chemotherapeutic agents, resulting in a high mortality rate in patients. Human mesenchymal stem cells (hMSCs), which are parts of the cancer stroma, have been shown to be involved in the development and progression of GBM. However, different sources of hMSCs might affect GBM cells differently. In the present study, we established hMSCs from placenta (PL-hMSC) and chorion (CH-hMSC) to study the effects of their released soluble factors on the proliferation, migration, invasion, gene expression, and survival of human GBM cells, U251. We found that the soluble factors derived from CH-hMSCs and PL-hMSCs suppressed the proliferation of U251 cells in a dose-dependent manner. In contrast, soluble factors derived from both hMSC sources increased U251 migration without affecting their invasive property. The soluble factors derived from these hMSCs decreased the expression levels of CyclinD1, E2Fs and MYC genes that promote GBM cell proliferation but increased the expression level of TWIST gene, which promotes EMT and GBM cell migration. The functional study suggests that both hMSCs might exert their effects, at least in part, by activating TGF-ß and suppressing Wnt/ß-catenin signaling in U251 cells. Our study provides a better understanding of the interaction between GBM cells and gestational tissue-derived hMSCs. This knowledge might be used to develop safer and more effective stem cell therapy that improves the survival and quality of life of patients with GBM by manipulating the interaction between hMSCs and GBM cells.

Membrane inflammasome activation by choriodecidual Ureaplasma parvum infection without intra-amniotic infection in a Non-Human Primate model†.

Intrauterine infection is a significant cause of neonatal morbidity and mortality. Ureaplasma parvum is a microorganism commonly isolated from cases of preterm birth and preterm premature rupture of membranes (pPROM). However, the mechanisms of early stage ascending reproductive tract infection remain poorly understood. To examine inflammation in fetal (chorioamnionic) membranes we utilized a non-human primate (NHP) model of choriodecidual U. parvum infection. Eight chronically catheterized pregnant rhesus macaques underwent maternal-fetal catheterization surgery at ~105-112 days gestation and choriodecidual inoculation with U. parvum (105 CFU/mL, n =4) or sterile media (controls; n = 4) starting at 115-119 days, repeated at 5-day intervals until C-section at 136-140 days (term=167 days). The average inoculation to delivery interval was 21 days, and Ureaplasma infection of the amniotic fluid (AF) was undetectable in all animals. Choriodecidual Ureaplasma infection resulted in increased fetal membrane expression of MMP-9 and PTGS2, but did not result in preterm labor or increased concentrations of AF pro-inflammatory cytokines. However, membrane expression of inflammasome sensors, NLRP3, NLRC4, AIM2, and NOD2, and adaptor ASC (PYCARD) gene expression were significantly increased. Gene expression of IL-1ß, IL-18, IL-18R1  , CASPASE-1, and pro-CASPASE-1 protein increased with Ureaplasma infection. Downstream inflammatory genes MYD88 and NFκB (Nuclear factor kappa-light-chain-enhancer of activated B cells) were also significantly upregulated. These results demonstrate that choriodecidual Ureaplasma infection, can cause activation of inflammasome complexes and pathways associated with pPROM and preterm labor prior to microbes being detectable in the AF.

Mesothelial fusion mediates chorioallantoic membrane formation.

In amniotic vertebrates (birds, reptiles and mammals), an extraembryonic structure called the chorioallantoic membrane (CAM) functions as respiratory organ for embryonic development. The CAM is derived from fusion between two pre-existing membranes, the allantois, a hindgut diverticulum and a reservoir for metabolic waste, and the chorion which marks the embryo's external boundary. Modified CAM in eutherian mammals, including humans, gives rise to chorioallantoic placenta. Despite its importance, little is known about cellular and molecular mechanisms mediating CAM formation and maturation. In this work, using the avian model, we focused on the early phase of CAM morphogenesis when the allantois and chorion meet and initiate fusion. We report here that chicken chorioallantoic fusion takes place when the allantois reaches the size of 2.5-3.0 mm in diameter and in about 6 hours between E3.75 and E4. Electron microscopy and immunofluorescence analyses suggested that before fusion, in both the allantois and chorion, an epithelial-shaped mesothelial layer is present, which dissolves after fusion, presumably by undergoing epithelial-mesenchymal transition. The fusion process per se, however, is independent of allantoic growth, circulation, or its connection to the developing mesonephros. Mesoderm cells derived from the allantois and chorion can intermingle post-fusion, and chorionic ectoderm cells exhibit a specialized sub-apical intercellular interface, possibly to facilitate infiltration of allantois-derived vascular progenitors into the chorionic ectoderm territory for optimal oxygen transport. Finally, we investigated chorioallantoic fusion-like process in primates, with limited numbers of archived human and fresh macaque samples. We summarize the similarities and differences of CAM formation among different amniote groups and propose that mesothelial epithelial-mesenchymal transition mediates chorioallantoic fusion in most amniotic vertebrates. Further study is needed to clarify tissue morphogenesis leading to chorioallantoic fusion in primates. Elucidating molecular mechanisms regulating mesothelial integrity and epithelial-mesenchymal transition will also help understand mesothelial diseases in the adult, including mesothelioma, ovarian cancer and fibrosis. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.

Chorion-derived perinatal mesenchymal stem cells improve cardiac function and vascular regeneration: Preferential treatment for ischemic heart disease.

BACKGROUND: Stem cell therapy has emerged as a novel treatment for heart failure after myocardial infarction (Ml). Bone marrow-derived mesenchymal stem cells (BM-MSCs) are commonly considered because of their accessibility and usability. However, their therapeutic potential remains controversial. In our previous in vitro study, chorion-derived mesenchymal stem cells (C-MSCs) and umbilical cord-derived mesenchymal stem cells (UC-MSCs) demonstrated an ability to differentiate into cardiomyocytes and neural cells, respectively. Thus, we examined whether C-MSCs had a better differentiation potential in an MI animal model. METHODS: MI was induced by ligation of the left anterior descending artery, and DiI-labeled MSCs were injected into the border of the infarcted myocardium. The left ventricular ejection fraction (LVEF) and fractional shortening (FS) were measured using echocardiograms. Masson's Trichrome staining was performed to evaluate the viable myocardium. Alpha-sarcomeric actin (α-SA), cardiac troponin-T (cTnT), and isolectin were immunolabeled to evaluate differentiation and capillary formation. RESULTS: After 8 weeks, the LVEF and FS significantly increased to a greater extent in the C-MSC-injected group with maintenance of viable myocardium, as compared to in the control, UC-MSC-, and BM-MSC-injected groups (p < 0.05). Compared to UC-MSCs and BM-MSCs, C-MSCs significantly increased the capillary density (p < 0.05) and demonstrated higher expressions of cTnT and α-SA. CONCLUSIONS: In conclusion, compared to UC-MSCs and BM-MSCs, C-MSCs showed a better therapeutic efficacy in a rat MI model.

Study of mesenchymal stem cells derived from lung-resident, bone marrow and chorion for treatment of LPS-induced acute lung injury.

BACKGROUND: Mesenchymal stem cells (MSCs) have been shown to improve acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). However, the optimal source of MSCs for cell-based therapy remains unknown. To determine which kind of MSCs are more effective, we compared the effects of rat lung resident MSC (LRMSC), human chorion-derived MSC (HMSC-C) and human bone marrow derived MSC (HMSC-BM) in LPS-induced ALI in mice. METHODS: LPS (Pseudomonas aeruginosa) was used to induce ALI model. All three kinds of MSCs were administered via tail vein 4 h after LPS instillation. The mice were sacrificed 48 h after LPS instillation. H&E staining of lung section, wet-to-dry weight ratio of lung tissue, ratio of regulatory T cells (Tregs) and Th17 cells, and total protein concentration, leukocytes counting and cytokines in bronchoalveolar lavage fluid (BALF) were evaluated. RESULTS: The data showed that compared with LRMSC and HMSC-BM, HMSC-C more significantly attenuated lung injury, upregulated the Tregs/Th17 cells ratio, and inhibited release of inflammatory cytokines (IL-1ß, IL-6 and TNF-α) and recruitment of neutrophils and macrophages into alveolus. CONCLUSIONS: Although all three kinds of LRMSC, HMSC-C and HMSC-BM are protective against LPS-induced lung injury, HMSC-C was more effective than LRMSC and HMSC-BM to treat LPS-induced lung injury.

Comprehensive analysis of human chorionic membrane extracts regulating mesenchymal stem cells during osteogenesis.

OBJECTIVE: Human chorionic membrane extracts (CMEs) from placenta are known to be a natural biomaterial for bone regeneration, with their excellent osteogenic efficacy on osteoblasts. However, little is known about the regulatory mechanism involved. METHODS AND RESULTS: We have shown the in vitro and in vivo bone-forming ability of CME using human osteoblasts and bone defect animal models, suggesting that CME greatly enhances osteogenesis by providing an osteoconductive environment for the osteogenesis of osteoblasts. Proteomic analysis revealed that CME contained several osteogenesis-related stimulators such as osteopontin, osteomodulin, Thy-1, netrin 4, retinol-binding protein and DJ-1. Additionally, 23 growth factors/growth factor-related proteins were found in CME, which may trigger mitogen-activated protein kinase (MAPK) signalling as a specific cellular signalling pathway for osteogenic differentiation. Microarray analysis showed four interaction networks (chemokine, Wnt signalling, angiogenesis and ossification), indicating the possibility that CME can promote osteogenic differentiation through a non-canonical Wnt-mediated CXCL signalling-dependent pathway. CONCLUSIONS: The results of this study showed the function and mechanism of action of CME during the osteogenesis of osteoblasts and highlighted a novel strategy for the use of CME as a biocompatible therapeutic material for bone regeneration.

Small Extracellular Vesicles Derived from Human Chorionic MSCs as Modern Perspective towards Cell-Free Therapy.

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.

Extracellular vesicles from maternal uterine cells exposed to risk factors cause fetal inflammatory response.

BACKGROUND: Fetal cell-derived exosomes (extracellular vesicles, 40-160 nm) are communication channels that can signal parturition by inducing inflammatory changes in maternal decidua and myometrium. Little is known about maternal cell-derived exosomes and their functional roles on the fetal side. This study isolated and characterized exosomes from decidual and myometrial cells grown under normal and inflammatory/oxidative stress conditions and determined their impact on fetal membrane cells. METHODS: Decidual and myometrial cells were grown under standard culture conditions (control) or exposed for 48 h to cigarette smoke extract or tumor necrosis factor-α, as proxies for oxidative stress and inflammation, respectively. Exosomes were isolated from media (differential ultra-centrifugation followed by size exclusion chromatography), quantified (nano particle tracking analysis), and characterized in terms of their size and morphology (cryo-electron microscopy), markers (dot blot), and cargo contents (proteomics followed by bioinformatics analysis). Maternal exosomes (109/mL) were used to treat amnion epithelial cells and chorion trophoblast cells for 24 h. The exosome uptake by fetal cells (confocal microscopy) and the cytokine response (enzyme-linked immunosorbent assays for IL-6, IL-10, and TNF-α) was determined. RESULTS: Exosomes from both decidual and myometrial cells were round and expressed tetraspanins and endosomal sorting complexes required for transport (ESCRT) protein markers. The size and quantity was not different between control and treated cell exosomes. Proteomic analysis identified several common proteins in exosomes, as well as unique proteins based on cell type and treatment. Compared to control exosomes, pro-inflammatory cytokine release was higher in both amnion epithelial cell and chorion trophoblast cell media when the cells had been exposed to exosomes from decidual or myometrial cells treated with either cigarette smoke extract or tumor necrosis factor-α. In chorion trophoblast cells, anti-inflammatory IL-10 was increased by exosomes from both decidual and myometrial cells. CONCLUSION: Various pathophysiological conditions cause maternal exosomes to carry inflammatory mediators that can result in cell type dependent fetal inflammatory response. Video Abstract.

Effects of hatching enzymes on egg envelope digestion in the male-brooding seahorse.

In the present study, we aimed to evaluate the effects of hatching enzymes on the egg envelope digestion during the hatching period in the male brooding seahorse. The complementary DNAs encoding two hatching-enzyme genes, high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE), were cloned and functionally characterized from the lined seahorse (Hippocampus erectus). The genomic-synteny analysis confirmed that teleosts shared LCE gene synteny. In contrast, the genomic location of HCE was found to be conserved with pipefish, but not other teleosts, suggesting that translocation into a novel genomic location occurred. Whole-mount in situ hybridization showed that HCE and LCE mRNAs were expressed in hatching gland cells. To determine the digestion mechanisms of HCE and LCE in hatching, recombinant HCE and LCE were generated and their enzyme activities were examined using fertilized egg envelopes and synthetic peptides. Seahorse HCE and LCE independently digested and softened the egg envelopes of the lined seahorse. Although the egg envelope was digested more following HCE and LCE co-treatment, envelope solubilization was not observed. Indeed, both HCE and LCE showed similar substrate specificities toward four different synthetic peptides designed from the cleavage sites of egg envelope proteins. HCE and LCE proteins from other euteleostean fishes showed different specificities, and the egg envelope was solubilized by the cooperative action of HCE and LCE. These results suggest that the function of LCE was degenerated in the lined seahorse. Our results imply a digestion mechanism for evolutionary adaptation in ovoviviparous fish with male pregnancy.

Chorion and amnion/chorion membranes in oral and periodontal surgery: A systematic review.

The aim of this study was to perform a systematic review on the clinical applications where chorion membrane (CM) and amnion/chorion membrane (ACM) were used for oral tissue regeneration procedures. Selection of articles was carried out by two evaluators in Pubmed and Scopus databases, and Outcomes (PICO) method was used to select the relevant articles. Clinical studies reporting the use of CM or ACM for oral soft and hard tissue regeneration were included. The research involved 21 studies conducted on 375 human patients. Seven clinical applications of CM and ACM in oral and periodontal surgery were identified: gingival recession treatment, intrabony and furcation defect treatment, alveolar ridge preservation, keratinized gum width augmentation around dental implants, maxillary sinus membrane repair, and large bone defect reconstruction. CM and ACM were compared to negative controls (conventional surgeries without membrane) or to the following materials: collagen membranes, dense polytetrafluoroethylene membranes, platelet-rich fibrin membranes, amnion membranes, and to a bone substitute. Several studies support the use of CM and ACM as an efficient alternative to current techniques for periodontal and oral soft tissue regeneration procedures. However, further studies are necessary to increase the level of evidence and especially to demonstrate their role for bone regeneration.

Expression profile of proinflammatory mediators in the placenta of mares during physiological detachment and retention of fetal membranes.

Physiological parturition is characterized by sterile, inflammatory-like processes. During parturition, the placenta expresses various proinflammatory mediators, such as chemokines and IL-17. Nevertheless, inflammatory processes present in the parturient mare are poorly characterized. The aim of this study was to investigate the expression of selected chemokines and IL-17 in the allantochorion and the endometrium of mares that retained fetal membranes (RFM) and expelled them physiologically. We hypothesized that the expression of these mediators may be altered in the placenta of mares with RFM and result in RFM occurrence. Differences in mRNA expression in the placenta of investigated groups of mares were detected for CCL2, CCL3, CCL4, CCL8, CXCL1, CXCL8, CXCL10, CX3CL1 and IL-17. There were no differences in mRNA expression of CCL5 and CXCL6. Gene ontology network analysis showed enrichment in genes related to leukocyte migration, cell chemotaxis and response to chemokine in tissues of RFM mares. Analysis of association network suggested denotations between CXCL6, CXCL8, CXCL1, CCL5, CCL4, CX3CL1 and CXCL10. Moreover, possible inhibition of CXCL10 by IL-17A and prostaglandin peroxide synthase 2 (PTGS2) by CXCL1 was detected. Our results suggest that, based on differences in chemokines and IL-17 expression, recruited subsets of leukocytes might differ between the analyzed groups of mares, which in turn may impair the separation of fetal membranes in the group of RFM mares. In addition, the results of the expression analysis suggest that macrophages might be one of the most abundant cells infiltrating the equine placenta during the expulsion of fetal membranes. Furthermore, we suspect that the synthesis of PTGS2 might be inhibited in mares with RFM.

ABCA1 plays an anti-inflammatory role by affecting TLR4 at the feto-maternal interface.

AIMS: This study aimed to evaluate the function and pathway of ATP-binding cassette transporter member A1 (ABCA1)-induced anti-inflammatory response in cells at the feto-maternal interface. MAIN METHODS: The primary amniotic mesenchymal cells (AMCs), chorion cells and decidual cells were isolated from placental membranes of women with uncomplicated pregnancies at full-term (not in labor) using enzymatic digestion. Flow cytometry was used to measure the purity of isolated cells. Immunofluorescence assay was performed to detect the location of ABCA1 and toll-like receptor 4 (TLR4). Reverse transcription PCR and western blotting analyses were used to examine ABCA1, TLR4 and inflammatory factor expression in primary cells. ELISA was used to detect cytokine secretions from the primary cells. KEY FINDINGS: ABCA1 and TLR4 were mainly located in the cell nucleus and cytoplasm of feto-maternal interface cells. ABCA1 expression remained the highest in chorion cells, medium in decidual cells, and weakest in AMCs. Upregulated expression of ABCA1 decreased expression of TLR4 and the levels of pro-inflammatory factors, but increased cytoprotective factors in all cell types. In contrast, downregulated expression of ABCA1 increased the expression of TLR4 and pro-inflammatory factors, but decreased the levels of cytoprotective factors. Downregulated ABCA1 expression followed by decreased TLR4 expression using a small interference RNA (siRNA) induced reduction of interleukin (IL)-1ß and tumor necrosis factor-α (TNF-α) in all cell types. SIGNIFICANCE: ABCA1 at feto-maternal interface acts as an anti-inflammatory role by reducing the expression of TLR4 in uncomplicated pregnancies. ABCA1 might be a potential therapeutic target for preventing gestational diseases.

Structural and Functional Equivalency Between Lyopreserved and Cryopreserved Chorions with Viable Cells.

Objective: Clinical studies have demonstrated that the use of cryopreserved amnion or trophoblast (TR)-free chorion, containing viable cells, in the treatment of chronic wounds results in high rate of wound closure. Recently, a new lyopreservation method has been developed for preservation of amnion that also retains the endogenous viable cells. The objective of this study was to use this method for lyopreservation of TR-free chorionic membrane (viable lyopreserved chorionic membrane [VLCM]) and compare it with the viable cryopreserved chorionic membrane (VCCM). A second objective was to investigate the immunogenicity of chorion, an important question that has not been fully addressed. Approach: Chorion immunogenicity was tested in vitro in a mixed lymphocyte reaction and lipopolysaccharide (LPS) challenge assay, and in vivo in a mouse subcutaneous pocket implantation model. VLCM tissue structure was assessed histologically, growth factor content by multiplex assay, and cell viability by LIVE/DEAD cell fluorescent staining. Inhibition of tumor necrosis factor α secretion by LPS-activated THP-1 cells and endothelial cell tubule formation assays were performed to evaluate the anti-inflammatory and proangiogenic properties, respectively. An in vivo rabbit abdominal adhesion model was used to evaluate the antifibrotic properties. Results: Chorionic membrane without trophoblast (CM) was shown to be nonimmunogenic. Tissue architecture, growth factors, and cell viability of fresh CM were maintained in VLCM and VCCM. In vitro studies showed that anti-inflammatory and angiogenic properties were retained in VLCM. Furthermore, VLCM prevents formation of postsurgical adhesions in a rabbit abdominal surgical adhesion model. Innovation: Characterization of structural and functional properties of VLCM is reported for the first time. Conclusion: Similar to VCCM, VLCM retains native components of fresh CM, including collagen-rich extracellular matrix, growth factors, and viable cells. In vitro and in vivo models demonstrate that VLCM is anti-inflammatory, proangiogenic and antifibrotic. Results of this study support the structural and functional equivalency between VLCM and VCCM.

New insights in equine steroidogenesis: an in-depth look at steroid signaling in the placenta.

Steroid production varies widely among species, with these differences becoming more pronounced during pregnancy. As a result, each species has its own distinct pattern of steroids, steroidogenic enzymes, receptors, and transporters to support its individual physiological requirements. Although the circulating steroid profile is well characterized during equine pregnancy, there is much yet to be explored regarding the factors that support steroidogenesis and steroid signaling. To obtain a holistic view of steroid-related transcripts, we sequenced chorioallantois (45 days, 4 months, 6 months, 10 months, 11 months, and post-partum) and endometrium (4 months, 6 months, 10 months, 11 months, and diestrus) throughout gestation, then looked in-depth at transcripts related to steroid synthesis, conjugation, transportation, and signaling. Key findings include: 1) differential expression of HSD17B isoforms among tissues (HSD17B1 high in the chorioallantois, while HSD17B2 is the dominant form in the endometrium) 2) a novel isoform with homology to SULT1A1 is the predominant sulfotransferase transcript in the chorioallantois; and 3) nuclear estrogen (ESR1, ESR2) and progesterone (PGR) expression is minimal to nonexistant in the chorioallantois and pregnant endometrium. Additionally, several hypotheses have been formed, including the possibility that the 45-day chorioallantois is able to synthesize steroids de novo from acetate and that horses utilize glucuronidation to clear estrogens from the endometrium during estrous, but not during pregnancy. In summary, these findings represent an in-depth look at equine steroid-related transcripts through gestation, providing novel hypotheses and future directions for equine endocrine research.

The voltage-gated proton channel hHv1 is functionally expressed in human chorion-derived mesenchymal stem cells.

The voltage-gated proton channel Hv1 is widely expressed, among others, in immune and cancer cells, it provides an efficient cytosolic H+extrusion mechanism and regulates vital functions such as oxidative burst, migration and proliferation. Here we demonstrate the presence of human Hv1 (hHv1) in the placenta/chorion-derived mesenchymal stem cells (cMSCs) using RT-PCR. The voltage- and pH-dependent gating of the current is similar to that of hHv1 expressed in cell lines and that the current is blocked by 5-chloro-2-guanidinobenzimidazole (ClGBI) and activated by arachidonic acid (AA). Inhibition of hHv1 by ClGBI significantly decreases mineral matrix production of cMSCs induced by conditions mimicking physiological or pathological (inorganic phosphate, Pi) induction of osteogenesis. Wound healing assay and single cell motility analysis show that ClGBI significantly inhibits the migration of cMSCs. Thus, seminal functions of cMSCs are modulated by hHv1 which makes this channel as an attractive target for controlling advantages/disadvantages of MSCs therapy.

Granulocyte macrophage colony stimulating factor (GM-CSF), the critical intermediate of inflammation-induced fetal membrane weakening, primarily exerts its weakening effect on the choriodecidua rather than the amnion.

INTRODUCTION: We have previously demonstrated two associations of PPROM, (1) inflammation/infection (modeled by tumor necrosis factor (TNF)) and (2) decidual bleeding (modeled by thrombin), both decrease fetal membrane (FM) rupture strength in-vitro. Furthermore, Granulocyte-Macrophage-Colony-Stimulating-Factor (GM-CSF) induced by both TNF and thrombin is a critical intermediate, necessary and sufficient for weakening by either agent. The amnion is the strength component of FM and must weaken for FM to rupture. It is unclear whether GM-CSF weakens amnion (AM) directly, or initially targets choriodecidua (CD) which secondarily releases agents to act on amnion. METHODS: Full thickness FM fragments were treated with/without GM-CSF. Some were preincubated with alpha-lipoic acid (LA), a known inhibitor of FM weakening. The FM fragments were then strength-tested. Separately, FM fragments were initially separated to AM and CD. AM fragments were cultured with Medium ± GM-CSF and then strength-tested. In other experiments, CD fragments were cultured with Medium, GM-CSF, LA, or LA + GM-CSF. Conditioned medium from each group was then incubated with AM. AM was then strength-tested. Matrix Metalloproteinases (MMPs) and Tissue Inhibitors of Matrix Metalloproteinases (TIMPs) were analyzed by Mutiplex Elisa. RESULTS: GM-CSF weakened intact FM which was blocked by LA. GM-CSF did not weaken isolated AM. However, GM-CSF conditioned CD media weakened AM and this weakening was inhibited by LA. GM-CSF treatment of CD increased MMPs 2, 9, and 10, and decreased TIMPs 1-3. LA reversed these effects. CONCLUSIONS: GM-CSF does not weaken amnion directly; GM-CSF acts on CD to increase proteases and decrease anti-proteases which secondarily weaken the amnion.

Comparative proteome analysis of amniotic fluids and placentas from patients with idiopathic polyhydramnios.

INTRODUCTION: Idiopathic polyhydramnios (IPH) is an abnormal increase in amniotic fluid volume (AFV). This condition has unknown etiologies and is associated with various adverse pregnancy outcomes including maternal and fetal complication. This study aims to establish a comparative proteome profile for the human amniotic fluid (AF) of IPH and normal pregnancies and identify the responsible mediators and pathways that regulate AFV. METHODS: We first employed coupled isobaric tags for relative and absolute quantitation (iTRAQ) proteomics and bioinformatics analysis to examine the differentially expression proteins (DEPs) in the AF of IPH and normal pregnancies. Second, CUL5, HIP1, FSTL3, and LAMP2 proteins were selected for verification in amnion, chorion, and placental tissues by Western blot analysis. RESULTS: We identified 357 DEPs with 282 upregulated and 75 downregulated. Bioinformatics analysis revealed that cell, cellular process, and binding were the most enriched Gene Ontology terms. Amoebiasis, hematopoietic cell lineage, and NF-kappa B signaling pathway were the top significant pathways. In the verification procedure, FSTL3 protein had a highly significant expression in the amnion, chorion, and placentas of IPH and normal AFV groups (p < 0.05). DISCUSSION: Our results provide new insights into idiopathic polyhydramnios and offer fundamental points for future studies on AFV.

Spatiotemporal coordination of trophoblast and allantoic Rbpj signaling directs normal placental morphogenesis.

The placenta, responsible for the nutrient and gas exchange between the mother and fetus, is pivotal for successful pregnancy. It has been shown that Rbpj, the core transcriptional mediator of Notch signaling pathway, is required for normal placentation in mice. However, it remains largely unclear how Rbpj signaling in different placental compartments coordinates with other important regulators to ensure normal placental morphogenesis. In this study, we found that systemic deletion of Rbpj led to abnormal chorioallantoic morphogenesis and defective trophoblast differentiation in the ectoplacental cone (EPC). Employing mouse models with selective deletion of Rbpj in the allantois versus trophoblast, combining tetraploid aggregation assay, we demonstrated that allantois-expressed Rbpj is essential for chorioallantoic attachment and subsequent invagination of allantoic blood vessels into the chorionic ectoderm. Further studies uncovered that allantoic Rbpj regulates chorioallantoic fusion and morphogenesis via targeting Vcam1 in a Notch-dependent manner. Meanwhile, we also revealed that trophoblast-expressed Rbpj in EPC facilitates Mash2's transcriptional activity, promoting the specification of Tpbpα-positive trophoblasts, which differentiate into trophoblast subtypes responsible for interstitial and endovascular invasion at the later stage of placental development. Collectively, our study further shed light on the molecular network governing placental development and functions, highlighting the necessity of a spatiotemporal coordination of Rbpj signaling for normal placental morphogenesis.

Comparative Biological Effects of Human Amnion and Chorion Membrane Extracts on Human Adipose-Derived Stromal Cells.

Although therapies with human amnion/chorion are used to ameliorate acute and chronic wounds, it is unclear which component of the amnion/chorion tissue promotes wound healing. To characterize the comparative effects of amnion and chorion in wound healing, we used human adipose-derived stromal cells to assess cell viability, migration, and gel contraction after treatment with amnion membrane extract (AME) or chorion membrane extract (CME). We then correlated the possible effectors via AME and CME protein profiling, and compared them by enzyme-linked immunosorbent assay (ELISA), western blotting, and immunocytochemistry. Cell viability was significantly increased with 50 and 100 µg/mL AME treatment, but with CME treatment, a significant increase was only observed with 100 µg/mL. With CME treatment, cell migration was 2.22-fold greater than the control, and collagen gels showed 20% greater contraction. Compared to control, the expression levels of α-smooth muscle actin (SMA) and smooth muscle protein 22-alpha (SM22α) increased both with AME and CME treatments, whereas calponin expression decreased. Protein profiling revealed significantly higher tissue inhibitor of metalloproteinase-1 (TIMP-1), interleukin-8, exotoxin, and adiponectin levels in CME than in AME, and ELISA revealed 8-fold higher adiponectin levels in cells treated with CME than those treated with AME. Immunocytochemistry revealed that α-SMA, SM22α, and calponin were significantly higher in CME- than AME-treated cells; however, adiponectin treatment did not enhance α-SMA, SM22α, or calponin expression. In conclusion, amnion and chorion membrane extracts exerted differential effects on proliferation and contraction of human adipose-derived stromal cells. Amnion extract was superior at inducing cell proliferation and migration, whereas CME was superior at inducing cell contraction.