Arrangement into layers and mechanobiology of multi-cell co-culture models of the uterine wall.

STUDY QUESTION: Can a co-culture of three cell types mimic the in vivo layers of the uterine wall? SUMMARY ANSWER: Three protocols tested for co-culture of endometrial epithelial cells (EEC), endometrial stromal cells (ESC), and myometrial smooth muscle cells (MSMC) led to formation of the distinct layers that are characteristic of the structure of the uterine wall in vivo. WHAT IS KNOWN ALREADY: We previously showed that a layer-by-layer co-culture of EEC and MSMC responded to peristaltic wall shear stresses (WSS) by increasing the polymerization of F-actin in both layers. Other studies showed that WSS induced significant cellular alterations in epithelial and endothelial cells. STUDY DESIGN, SIZE, DURATION: Human EEC and ESC cell lines and primary MSMC were co-cultured on a collagen-coated synthetic membrane in custom-designed wells. The co-culture model, created by seeding a mixture of all cells at once, was exposed to steady WSS of 0.5 dyne/cm2 for 10 and 30 min. PARTICIPANTS/MATERIALS, SETTING, METHODS: The co-culture of the three different cells was seeded either layer-by-layer or as a mixture of all cells at once. Validation of the models was by specific immunofluorescence staining and confocal microscopy. Alterations of the cytoskeletal F-actin in response to WSS were analyzed from the 2-dimensional confocal images through the Z-stacks following a previously published algorithm. MAIN RESULTS AND THE ROLE OF CHANCE: We generated three multi-cell in vitro models of the uterine wall with distinct layers of EEC, ESC, and MSMC that mimic the in vivo morphology. Exposure of the mixed seeding model to WSS induced increased polymerization of F-actin in all the three layers relative to the unexposed controls. Moreover, the increased polymerization of F-actin was higher (P-value < 0.05) when the length of exposure was increased from 10 to 30 min. Furthermore, the inner layers of ESC and MSMC, which are not in direct contact with the applied shearing fluid, also increased their F-actin polymerization. LARGE SCALE DATA: N/A. LIMITATIONS, RESONS FOR CAUTION: The mixed seeding co-culture model was exposed to steady WSS of one magnitude, whereas the uterus is a dynamic organ with intra-uterine peristaltic fluid motions that vary in vivo with different time-dependent magnitude. Further in vitro studies may explore the response to peristaltic WSS or other physical and/or hormonal perturbations that may mimic the spectrum of pathophysiological aspects. WIDER IMPLICATIONS OF THE FINDINGS: Numerous in vitro models were developed in order to mimic the human endometrium and endometrium-myometrium interface (EMI) region. The present co-culture models seem to be the first constructed from EEC, ESC, and MSMC on a collagen-coated synthetic membrane. These multi-cell in vitro models better represent the complex in vivo anatomy of the EMI region. The mixed seeding multi-cell in vitro model may easily be implemented in controlled studies of uterine function in reproduction and the pathogenesis of diseases. STUDY FINDING/COMPETING INTEREST(S): This study was supported in part by Tel Aviv University funds. All authors declare no conflict of interest.

A Method for Screening a Kinase Inhibitor Drug Library on ASCs.

Breast cancer is an ongoing issue due to its high mortality rates. Obesity enhances the problems associated with breast cancer, meaning there must be a biological connection between them. This crosstalk may be the adipose-derived stem cell. If we can interrupt the communication between adipose-derived stromal/stem cells (ASCs) and breast cancer, we may be able to prevent cancer propagation. Specific kinase inhibition may allow us to downregulate signals, preventing ASC-mediated cancer growth. This chapter provides a critical method for screening a kinase inhibitor drug library for hits on ASCs.

Mesenchymal stem cell secretome alters gene expression and upregulates motility of human endometrial stromal cells.

In brief: Endometrial stromal cell motility is fundamental to regeneration and repair of this tissue and crucial for successful reproduction. This paper shows a role for the mesenchymal stem cell (MSC) secretome in enhancing endometrial stromal cell motility. Abstract: Cyclic regeneration and repair of the endometrium are crucial for successful reproduction. Mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSC) and umbilical cord (UC-MSC) facilitate tissue repair via their secretome, which contains growth factors and cytokines that promote wound healing. Despite the implication of MSCs in endometrial regeneration and repair, mechanisms remain unclear. This study tested the hypothesis that the BM-MSC and UC-MSC secretomes upregulate human endometrial stromal cell (HESC) proliferation, migration, and invasion and activate pathways to increase HESC motility. BM-MSCs were purchased from ATCC and cultured from the BM aspirate of three healthy female donors. UC-MSCs were cultured from umbilical cords of two healthy male term infants. Using indirect co-culture of MSCs and hTERT-immortalized HESCs via a transwell system, we demonstrated that co-culture of HESCs with BM-MSCs or UC-MSCs from all donors significantly increased HESC migration and invasion, whereas effects on HESC proliferation varied among BM-MSC and UC-MSC donors. Analysis of gene expression by mRNA sequencing and RT-qPCR showed that expression of CCL2 and HGF was upregulated in HESCs that had been cocultured with BM-MSCs or UC-MSCs. Validation studies revealed that exposure to recombinant CCL2 for 48 h significantly increased HESC migration and invasion. Increased HESC motility by the BM-MSC and UC-MSC secretome appears to be mediated in part by upregulated HESC CCL2 expression. Our data support the potential for leveraging MSC secretome as a novel cell-free therapy to treat disorders of endometrial regeneration.

Distinct Adipogenic and Fibrogenic Differentiation Capacities of Mesenchymal Stromal Cells from Pancreas and White Adipose Tissue.

Pancreatic steatosis associates with ß-cell failure and may participate in the development of type-2-diabetes. Our previous studies have shown that diabetes-susceptible mice accumulate more adipocytes in the pancreas than diabetes-resistant mice. In addition, we have demonstrated that the co-culture of pancreatic islets and adipocytes affect insulin secretion. The aim of this current study was to elucidate if and to what extent pancreas-resident mesenchymal stromal cells (MSCs) with adipogenic progenitor potential differ from the corresponding stromal-type cells of the inguinal white adipose tissue (iWAT). miRNA (miRNome) and mRNA expression (transcriptome) analyses of MSCs isolated by flow cytometry of both tissues revealed 121 differentially expressed miRNAs and 1227 differentially expressed genes (DEGs). Target prediction analysis estimated 510 DEGs to be regulated by 58 differentially expressed miRNAs. Pathway analyses of DEGs and miRNA target genes showed unique transcriptional and miRNA signatures in pancreas (pMSCs) and iWAT MSCs (iwatMSCs), for instance fibrogenic and adipogenic differentiation, respectively. Accordingly, iwatMSCs revealed a higher adipogenic lineage commitment, whereas pMSCs showed an elevated fibrogenesis. As a low degree of adipogenesis was also observed in pMSCs of diabetes-susceptible mice, we conclude that the development of pancreatic steatosis has to be induced by other factors not related to cell-autonomous transcriptomic changes and miRNA-based signals.

Role of Serine Proteases at the Tumor-Stroma Interface.

During tumor development, invasion and metastasis, the intimate interaction between tumor and stroma shapes the tumor microenvironment and dictates the fate of tumor cells. Stromal cells can also influence anti-tumor immunity and response to immunotherapy. Understanding the molecular mechanisms that govern this complex and dynamic interplay, thus is important for cancer diagnosis and therapy. Proteolytic enzymes that are expressed and secreted by both cancer and stromal cells play important roles in modulating tumor-stromal interaction. Among, several serine proteases such as fibroblast activation protein, urokinase-type plasminogen activator, kallikrein-related peptidases, and granzymes have attracted great attention owing to their elevated expression and dysregulated activity in the tumor microenvironment. This review highlights the role of serine proteases that are mainly derived from stromal cells in tumor progression and associated theranostic applications.

Long noncoding RNA ADAMTS9-AS1 represses ferroptosis of endometrial stromal cells by regulating the miR-6516-5p/GPX4 axis in endometriosis.

Endometriosis (EMs) is one of the most frequent diseases of reproductive-age women and is characterized by the growth of endometrial tissues beyond the uterus. The enhanced proliferative and migratory potential of endometrial stromal cells (ESCs) plays an important role in the progression of EMs. Mounting studies have demonstrated that long noncoding RNAs (lncRNAs) exert an important role in regulating the development and progression of EMs. Given the aberrant expression of lncRNA ADAMTS9-AS1 in ectopic endometrium (ecEM), we investigated the biological effect of ADAMTS9-AS1 on ESC proliferation and migration and explored the underlying mechanism. The current data showed that ADAMTS9-AS1 expression was significantly upregulated in ecEM compared with eutopic endometrium (euEM) in patients with EMs and in a murine model of EMs. Functionally, ADAMTS9-AS1 knockdown in ectopic ESCs (EESCs) decreased cell viability and migration, whereas ADAMTS9-AS1 overexpression in normal ESCs (NESCs) enhanced cell viability and migration. More importantly, the effect of ADAMTS9-AS1 inhibition on decreasing ESC viability was significantly blocked by ferrostatin-1 (Fer-1, a ferroptosis inhibitor), and ADAMTS9-AS1 overexpression repressed erastin (a ferroptosis activator)-induced cell death. Furthermore, the regulatory role of ADAMTS9-AS1 in ferroptosis was defined and evidenced by increased reactive oxygen species (ROS) levels and malonyl dialdehyde (MDA) content and decreased expression of glutathione peroxidase 4 (GPX4) after ADAMTS9-AS1 inhibition. Mechanistically, ADAMTS9-AS1 functioned as a competing endogenous RNA (ceRNA) by sponging miR-6516-5p to derepress the expression of GPX4, the critical repressor of ferroptosis. Taken together, these results demonstrate that upregulated ADAMTS9-AS1 accelerates ESC proliferation and migration by regulating miR-6516-5p/GPX4-dependent ferroptosis and may be a potential target for the treatment of EMs.

GnRH antagonist weakens endometrial stromal cells growth ability by decreasing c-kit receptor expression.

BACKGROUND: Several surveys have reported that patients treated with gonadotropin-releasing hormone antagonist (GnRH-ant) protocol showed a significantly lower rate of implantation and clinical pregnancy compared to GnRH agonist (GnRH-a) protocol during in vitro fertilization-fresh embryo transfer. Subsequent studies imputed this poor outcome to the negative effects of GnRH-ant on endometrial receptive. However, the mechanisms were not fully understood. METHODS: The clinical data of 2815 patients undergoing fresh embryo transfer in our center were analyzed. Human endometrial stromal cells (ESCs) from healthy women undergoing elective pregnancy termination of a normal pregnancy at 8-10 weeks gestation were treated with GnRH-analogs or imatinib (c-kit receptor inhibitor). CCK8 and Flow cytometry were used to investigated the growth ability of ESCs. Immunofluorescence staining and western blot was used to detected the target proteins. RESULTS: The clinical data showed that the endometrial thickness on HCG Day were significantly lower in GnRH-ant group. Although no difference of embryo quality in these two groups, GnRH-ant group showed remarkably decreased rate of HCG positive, embryo implantation and pregnancy. Moreover, GnRH-ant significantly reduced the proliferation and induced the apoptosis of ESCs. Furthermore, the expression and activation of c-kit receptor, which played pivotal roles during embryo implantation, were observably decreased by GnRH-ant. Inhibiting the activation of c-kit by imatinib remarkably suppressed the proliferation and promoted the apoptosis of ESCs. Additionally, the phosphorylation of AKT and expression of Cyclin D1, which were closely related with cellular growth, were distinctly lessened after treating with imatinib. CONCLUSIONS: In summary, our study showed that GnRH-ant weakened the activization of c-kit receptor by decreasing its expression, causing the impaired growth ability of ESCs. Our findings provided a new insight into the effects of GnRH-ant on endometrium.

Impact of growth hormone-releasing hormone antagonist on decidual stromal cell growth and apoptosis in vitro†.

Endometrial stromal cells remodeling is critical during human pregnancy. Growth hormone-releasing hormone and its functional receptor have been shown to be expressed in gynecological cancer cells and eutopic endometrial stromal cells. Recent studies have demonstrated the potential clinical uses of antagonists of growth hormone-releasing hormone as effective antitumor agents because of its directly antagonistic effect on the locally produced growth hormone-releasing hormone in gynecological tumors. However, the impact of growth hormone-releasing hormone antagonists on normal endometrial stromal cell growth remained to be elucidated. The aim of this study was to investigate the effect of a growth hormone-releasing hormone antagonist (JMR-132) on cell proliferation and apoptosis of human decidual stromal cells and the underlying molecular mechanisms. Our results showed that growth hormone-releasing hormone and the splice variant 1 of growth hormone-releasing hormone receptor are expressed in human decidual stromal cells isolated from the decidual tissues of early pregnant women receiving surgical abortion. In addition, treatment of stroma cells with JMR-132 induced cell apoptosis with increasing cleaved caspase-3 and caspase-9 activities and decrease cell viability in a time- and dose-dependent manner. Using a dual inhibition approach (pharmacological inhibitors and siRNA-mediated knockdown), we showed that JMR-132-induced activation of apoptotic signals are mediated by the activation of ERK1/2 and JNK signaling pathways and the subsequent upregulation of GADD45alpha. Taken together, JMR-132 suppresses cell survival of decidual stromal cells by inducing apoptosis through the activation of ERK1/2- and JNK-mediated upregulation of GADD45alpha in human endometrial stromal cells. Our findings provide new insights into the potential impact of growth hormone-releasing hormone antagonist on the decidual programming in humans.

Understanding and improving cellular immunotherapies against cancer: From cell-manufacturing to tumor-immune models.

The tumor microenvironment (TME) is shaped by dynamic metabolic and immune interactions between precancerous and cancerous tumor cells and stromal cells like epithelial cells, fibroblasts, endothelial cells, and hematopoietically-derived immune cells. The metabolic states of the TME, including the hypoxic and acidic niches, influence the immunosuppressive phenotypes of the stromal and immune cells, which confers resistance to both host-mediated tumor killing and therapeutics. Numerous in vitro TME platforms for studying immunotherapies, including cell therapies, are being developed. However, we do not yet understand which immune and stromal components are most critical and how much model complexity is needed to answer specific questions. In addition, scalable sourcing and quality-control of appropriate TME cells for reproducibly manufacturing these platforms remain challenging. In this regard, lessons from the manufacturing of immunomodulatory cell therapies could provide helpful guidance. Although immune cell therapies have shown unprecedented results in hematological cancers and hold promise in solid tumors, their manufacture poses significant scale, cost, and quality control challenges. This review first provides an overview of the in vivo TME, discussing the most influential cell populations in the tumor-immune landscape. Next, we summarize current approaches for cell therapies against cancers and the relevant manufacturing platforms. We then evaluate current immune-tumor models of the TME and immunotherapies, highlighting the complexity, architecture, function, and cell sources. Finally, we present the technical and fundamental knowledge gaps in both cell manufacturing systems and immune-TME models that must be addressed to elucidate the interactions between endogenous tumor immunity and exogenous engineered immunity.

PGE2 and Thrombin Induce Myofibroblast Transdifferentiation via Activin A and CTGF in Endometrial Stromal Cells.

Endometriosis is characterized by inflammation and fibrotic changes. Our previous study using a mouse model showed that proinflammatory factors present in peritoneal hemorrhage exacerbated inflammation in endometriosis-like grafts, at least in part through the activation of prostaglandin (PG) E2 receptor and protease-activated receptor (PAR). In addition, menstruation-related factors, PGE2 and thrombin (P/T), a PAR1 agonist induced epithelial-mesenchymal transition (EMT) of endometrial cells under hypoxia. However, the molecular mechanisms by which P/T induce development of endometriosis have not been fully characterized. To investigate the effects of P/T, RNA extracted from endometrial stromal cells (ESCs) treated with P/T were subjected to RNA sequence analysis, and identified activin A, FOS, and GATA2 as upregulated genes. Activin A increased the expression of connective tissue growth factor (CTGF) and mesenchymal marker genes in ESCs. CTGF induced the expression of fibrosis marker type I collagen, fibronectin, and α-smooth muscle actin (αSMA), indicating fibroblast to myofibroblast transdifferentiation (FMT) of ESCs. In addition, activin A, FOS, GATA2, CTGF, and αSMA were localized in endometriosis lesions. Taken together, our data show that P/T induces changes resembling EMT and FMT in ectopic ESCs derived from retrograde menstruation, and that these are associated with fibrotic changes in the lesions. Pharmacological means that block P/T-induced activin A and CTGF signaling may be strategies to inhibit fibrosis in endometriotic lesions.

Development of an exosomal gene signature to detect residual disease in dogs with osteosarcoma using a novel xenograft platform and machine learning.

Osteosarcoma has a guarded prognosis. A major hurdle in developing more effective osteosarcoma therapies is the lack of disease-specific biomarkers to predict risk, prognosis, or therapeutic response. Exosomes are secreted extracellular microvesicles emerging as powerful diagnostic tools. However, their clinical application is precluded by challenges in identifying disease-associated cargo from the vastly larger background of normal exosome cargo. We developed a method using canine osteosarcoma in mouse xenografts to distinguish tumor-derived from host-response exosomal messenger RNAs (mRNAs). The model allows for the identification of canine osteosarcoma-specific gene signatures by RNA sequencing and a species-differentiating bioinformatics pipeline. An osteosarcoma-associated signature consisting of five gene transcripts (SKA2, NEU1, PAF1, PSMG2, and NOB1) was validated in dogs with spontaneous osteosarcoma by real-time quantitative reverse transcription PCR (qRT-PCR), while a machine learning model assigned dogs into healthy or disease groups. Serum/plasma exosomes were isolated from 53 dogs in distinct clinical groups ("healthy", "osteosarcoma", "other bone tumor", or "non-neoplastic disease"). Pre-treatment samples from osteosarcoma cases were used as the training set, and a validation set from post-treatment samples was used for testing, classifying as "osteosarcoma detected" or "osteosarcoma-NOT detected". Dogs in a validation set whose post-treatment samples were classified as "osteosarcoma-NOT detected" had longer remissions, up to 15 months after treatment. In conclusion, we identified a gene signature predictive of molecular remissions with potential applications in the early detection and minimal residual disease settings. These results provide proof of concept for our discovery platform and its utilization in future studies to inform cancer risk, diagnosis, prognosis, and therapeutic response.

Cell-to-cell contact-mediated regulation of tumor behavior in the tumor microenvironment.

Tumor growth and progression are complex processes mediated by mutual interactions between cancer cells and their surrounding stroma that include diverse cell types and acellular components, which form the tumor microenvironment. In this environment, direct intercellular communications play important roles in the regulation of the biological behaviors of tumors. However, the underlying molecular mechanisms are insufficiently defined. We used an in vitro coculture system to identify genes that were specifically expressed at higher levels in cancer cells associated with stromal cells. Major examples included epithelial membrane protein 1 (EMP1) and stomatin, which positively and negatively regulate tumor progression, respectively. EMP1 promotes tumor cell migration and metastasis via activation of the small GTPase Rac1, while stomatin strongly suppresses cell proliferation and induces apoptosis of cancer cells via inhibition of Akt signaling. Here we highlight important aspects of EMP1, stomatin, and their family members in cancer biology. Furthermore, we consider the molecules that participate in intercellular communications and signaling transduction between cancer cells and stromal cells, which may affect the phenotypes of cancer cells in the tumor microenvironment.

Extracellular vesicles of bovine small follicular fluid promote ovarian cortical stromal cell proliferation and steroidogenesis.

The aim of this study was to investigate the effects of extracellular vesicles (EVs) on the proliferation and steroid hormone synthesis of bovine ovarian cortical stromal cells in vitro. The release and uptake of EVs are the new mechanisms of cell-to-cell communication. Using reverse transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay, TUNEL and other experiments, we found that EVs in bovine follicular fluid can promote the proliferation and synthesis of androstenedione and progesterone in ovarian cortical stromal cells. Moreover, 100 µg/ml EVs caused the most significant effect. We conclude that EVs at 100 µg/ml can significantly promote the proliferation and synthesis of androstenedione and progesterone in ovarian cortical stromal cells. This research is of great significance for further elucidating the regulatory role of follicular fluid EVs in follicular development and atresia and for research on the interaction of ovarian stromal cells, granulosa cells and oocytes.

Interleukin-33 promotes invasiveness of human ovarian endometriotic stromal cells through the ST2/MAPK/MMP-9 pathway activated by 17ß-estradiol.

OBJECTIVE: Endometriosis is an estrogen-dependent, benign, and chronic gynecological disorder occurring in women of reproductive age. Although the pathogenesis of endometriosis is poorly understood, implantation theory indicates that viable endometrial cells shed from the endometrium into the pelvic peritoneum or ovaries, possibly through retrograde menstruation, and then reattach, invade, and damage other tissues. Interleukin (IL)-33, a new member of the IL-1 superfamily, is mainly upregulated by stromal cells following proinflammatory stimulation. Matrix metalloproteinases (MMPs) are involved in the degradation and reconstruction of the extracellular matrix. MMP-9 participates in the pathogenesis of endometriosis by promoting the invasion of endometriotic cells. This study investigated the effect of IL-33 on the cell invasion ability of and MMP-9 expression in human stromal cells derived from ovarian endometrioma (hOVEN-SCs). MATERIALS AND METHODS: We isolated hOVEN-SCs from human ovarian endometrioma. Gene expression was analyzed using the Illumina Human WG-6 v2 Expression BeadChips microarray platform and through reverse transcription-polymerase chain reaction. Cell migration and invasion were examined by performing the transwell chamber assay. RESULTS: We found that 17ß-estradiol could increase the expression of IL-33 and ST2 through the estrogen receptor pathway in hOVEN-SCs. Moreover, IL-33 upregulated MMP-9 expression in and enhanced the invasion ability of hOVEN-SCs through the ST2/MAPK signaling pathway. Our results showed that MMP-9 expression was essential for IL-33-induced cell invasion. CONCLUSION: Our main finding is that 17ß-estradiol could increase IL-33 expression through the estrogen receptor pathway and activate MMP-9 expression in and invasion ability of hOVEN-SCs through the IL-33/ST2/MAPK signaling pathway. The results of this study and further related studies may provide new strategies for the prevention and treatment of endometriosis.

Exosomes and their cargo are important regulators of cell function in endometriosis.

Endometriosis is a chronic oestrogen-dependent gynaecological disorder characterized by non-menstrual pelvic pain, infertility and the extrauterine growth of endometrial-like glands and stroma. It has been noted that the eutopic endometrium of women with endometriosis is functionally distinct from that of women without endometriosis. Moreover, ectopic endometrial implants are functionally different from the eutopic endometrium of women with endometriosis. However, the mechanisms directing these differences are ill-defined. It is proposed here that small membrane-bound extracellular vesicles called exosomes are important vehicles in the protection and transport of signalling molecules central to the dysregulation of endometrial function in women with endometriosis. Therefore, a critical review of the literature linking exosomes and their cargo to the pathobiology of endometriosis was conducted. Circulating peritoneal fluid and endometrial cell exosomes contained long non-coding RNA, miRNA and proteins involved in histone modification, angiogenesis and immune modulation that differed significantly in women with endometriosis compared with controls. Moreover, experimental evidence supports a role for exosomes and their cargo in angiogenesis, neurogenesis, immune modulation and endometrial stromal cell invasion. It is therefore suggested that exosomes play an important role in the pathophysiology of endometriosis.

Preimplantation factor modulates trophoblastic invasion throughout the decidualization of human endometrial stromal cells.

BACKGROUND: Successful human embryo implantation requires the differentiation of endometrial stromal cells (ESCs) into decidual cells during a process called decidualization. ESCs express specific markers of decidualization, including prolactin, insulin-like growth factor-binding protein-1 (IGFBP-1), and connexin-43. Decidual cells also control of trophoblast invasion by secreting various factors, such as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases. Preimplantation factor (PIF) is a recently identified, embryo-derived peptide with activities at the fetal-maternal interface. It creates a favorable pro-inflammatory environment in human endometrium and directly controls placental development by increasing the human trophoblastic cells' ability to invade the endometrium. We hypothesized that PIF's effects on the endometrium counteract its pro-invasive effects. METHODS: We tested sPIF effect on the expression of three decidualization markers by RT-qPCR and/or immunochemiluminescence assay. We examined sPIF effect on human ESC migration by performing an in vitro wound healing assay. We analyzed sPIF effect on endometrial control of human trophoblast invasion by performing a zymography and an invasion assay. RESULTS: Firstly, we found that a synthetic analog of PIF (sPIF) significantly upregulates the mRNA expression of IGFBP-1 and connexin-43, and prolactin secretion in ESCs - suggesting a pro-differentiation effect. Secondly, we showed that the HTR-8/SVneo trophoblastic cell line's invasive ability was low in the presence of conditioned media from ESCs cultured with sPIF. Thirdly, this PIF's anti-invasive action was associated with a specifically decrease in MMP-9 activity. CONCLUSION: Taken as a whole, our results suggest that PIF accentuates the decidualization process and the production of endometrial factors that limit trophoblast invasion. By controlling both trophoblast and endometrial cells, PIF therefore appears to be a pivotal player in the human embryo implantation process.

Upregulated Talin1 synergistically boosts ß-estradiol-induced proliferation and pro-angiogenesis of eutopic and ectopic endometrial stromal cells in adenomyosis.

Adenomyosis (ADS) is an estrogen-dependent gynecological disease with unspecified etiopathogenesis. Local hyperestrogenism may serve a key role in contributing to the origin of ADS. Talin1 is mostly identified to be overexpressed and involved in the progression of numerous human carcinomas through mediating cell proliferation, adhesion and motility. Whether Talin1 exerts an oncogenic role in the pathogenesis of ADS and puts an extra impact on the efficacy of estrogen, no relevant data are available yet. Here we demonstrated that the adenomyotic eutopic and ectopic endometrial stromal cells (ADS_Eu_ESC and ADS_Ec_ESC) treated with ß-estradiol (ß-E2) presented stronger proliferative and pro-angiogenetic capacities, accompanied by increased expression of PCNA, Ki67, VEGFB and ANGPTL4 proteins. Meanwhile, these promoting effects were partially abrogated by Fulvestrant (ICI 182780, an estrogen-receptor antagonist). Aberrantly upregulation of Talin1 mRNA and protein level was observed in ADS endometrial specimens and stromal cells. Through performing functional experiments in vitro, we further determined that merely overexpression of Talin1 (OV-Talin1) also enhanced ADS stromal cell proliferation and pro-angiogenesis, while the most pronounced facilitating effects were found in the co-intervention group of OV-Talin1 plus ß-E2 treatment. Results from the xenograft nude mice model showed that the hypodermic endometrial lesions from co-intervention group had the highest mean weight and volume, compared with that of individual OV-Talin1 or ß-E2 treatment. The expression levels of PCNA, Ki67, VEGFB and ANGPTL4 in the lesions were correspondingly elevated the most in the co-intervention group. Our findings unveiled that overexpressed Talin1 might cooperate withß-E2 in stimulating ADS endometrial stromal cell proliferation and neovascularization, synergistically promoting the growth and survival of ectopic lesions. These results may be beneficial to provide a new insight for clarifying the pathogenesis of ADS.

The hypertrophic cartilage induction influences the building-block capacity of human adipose stem/stromal cell spheroids for biofabrication.

As an alternative to the classical tissue engineering approach, bottom-up tissue engineering emerges using building blocks in bioassembly technologies. Spheroids can be used as building blocks to reach a highly complex ordered tissue by their fusion (bioassembly), representing the foundation of biofabrication. In this study, we analyzed the biomechanical properties and the fusion capacity of human adipose stem/stromal cell (ASC) we spheroids during an in vitro model of hypertrophic cartilage established by our research group. Hypertrophic induced-ASC spheroids showed a statistically significant higher Young's modulus at weeks 2 (P < .001) and 3 (P < .0005) compared with non-induced. After fusion, non-induced and induced-ASC spheroids increased the contact area and decreased their pairs' total length. At weeks 3 and 5, induced-ASC spheroids did not fuse completely, and the cells migrate preferentially in the fusion contact region. Alizarin red O staining showed the highest intensity of staining in the fused induced-ASC spheroids at week 5, together with intense staining for collagen type I and osteocalcin. Transmission electron microscopy and element content analysis (X-ray Energy Dispersive Spectroscopy) revealed in the fused quartet at week 3 a crystal-like structure. Hypertrophic induction interferes with the intrinsic capacity of spheroids to fuse. The measurements of contact between spheroids during the fusion process, together with the change in viscoelastic profile to the plastic, will impact the establishment of bioassembly protocols using hypertrophic induced-ASC spheroids as building blocks in biofabrication.

Microvesicles derived from human umbilical cord mesenchyme promote M2 macrophage polarization and ameliorate renal fibrosis following partial nephrectomy via hepatocyte growth factor.

The intraoperative ischemia in partial nephrectomy (PN) often leads to postoperative renal function impairment and fibrosis, which can be regulated by macrophage polarization. We have previously demonstrated that microvesicles derived from human Wharton's Jelly mesenchymal stromal cells (hWJMSC-MVs) attenuated renal ischemia-induced renal fibrosis and contained a substantial quantity of hepatocyte growth factor (HGF). Herein, we investigated whether MSC-MVs regulate macrophage polarization and ameliorate renal fibrosis following ischemia-PN via transferring HGF. A rat model of ischemia-PN was established by 45 min of left renal ischemia followed by removal of 1/3 upper left kidney. MSC-MVs were injected through the tail vein immediately after ischemia. Renal injury biomarkers were measured and histologic analysis was performed to analyze renal injury. A co-culture model of THP-1 macrophages and MSC-MVs was utilized. The expression of M1 markers and M2 markers were determined to evaluate macrophage polarization. MSC-MV administration significantly ameliorated renal inflammation, lesions, and fibrosis in ischemia-PN rats, and promoted M2 macrophage polarization both in rat remnant renal tissues and LPS-treated THP-1 cells. These effects of MSC-MVs were compromised when HGF expression was downregulated in MSC-MVs. Collectively, MSC-MVs promote M2 macrophage polarization and attenuate renal fibrosis following ischemia-PN via transferring HGF.