Endometrial stem cell-derived granulocyte-colony stimulating factor attenuates endometrial fibrosis via sonic hedgehog transcriptional activator Gli2.

Intrauterine adhesion (IUA) is characterized by endometrial fibrosis, which ultimately leads to menstrual abnormalities, infertility, and recurrent miscarriages. The Shh/Gli2 pathway plays a critical role in tissue fibrogenesis and regeneration; Gli2 activation induces profibrogenic effects in various tissues, such as the liver and kidney. However, the role of Gli2 in endometrial fibrosis remains unknown. The purpose of this study was to test the hypothesis that activated Gli2 promotes endometrial fibrosis. Endometrial samples from moderate and severe IUA patients exhibited significantly enhanced expression of Gli2 compared with normal endometrial samples and mild IUA samples. Transfection with overactive Gli2 plasmids induced higher fibrosis-related protein expression, while blocking Gli2 signaling with cyclopamine caused the opposite effect in endometriotic stromal cells (ESCs), including inducing cell-cycle arrest. Menstrual-derived stem cell conditioned medium (MenSCs-CM) reduced endometrial fibrosis by reducing Gli2 protein levels and causing cell-cycle arrest in ESCs through granulocyte-colony stimulating factor (G-CSF). The effect was weakened after neutralization with a G-CSF antibody. Gli2 overexpression reduced the effects of MenSC-CM and G-CSF on fibrosis and cell-cycle progression in vitro. The antifibrotic effect of G-CSF was also observed in murine model. These findings demonstrate that Gli2 signaling promotes endometrial fibrosis, and the inhibition of Gli2 through MenSCs-secreted G-CSF may be of therapeutic value for managing endometrial fibrosis.

Human endometrial stem cells confer enhanced myocardial salvage and regeneration by paracrine mechanisms.

Human endometrial stem cells (EnSCs) have the potential to be 'off the shelf' clinical reagents for the treatment of heart failure. Here, using an immunocompetent rat model of myocardial infarction (MI), we provide evidence that the functional benefits of EnSC transplantation are principally and possibly exclusively through a paracrine effect. Human EnSCs were delivered by intramyocardial injection into rats 30 min. after coronary ligation. EnSC therapy significantly preserved viable myocardium in the infarct zone and improved cardiac function at 28 days. Despite increased viable myocardium and vascular density, there was scant evidence of differentiation of EnSCs into any cardiovascular cell type. Cultured human EnSCs expressed a distinctive profile of cytokines that enhanced the survival, proliferation and function of endothelial cells in vitro. When injected into the peri-infarct zone, human EnSCs activated AKT, ERK1/2 and STAT3 and inhibited the p38 signalling pathway. EnSC therapy decreased apoptosis and promoted cell proliferation and c-kit+ cell recruitment in vivo. Myocardial protection and enhanced post-infarction regeneration by EnSCs is mediated primarily by paracrine effects conferred by secreted cytokines that activate survival pathways and recruit endogenous progenitor stem cells. Menstrual blood provides a potentially limitless source of biologically competent 'off the shelf' EnSCs for allogeneic myocardial regenerative medicine.

[CURATIVE EFFECT OF HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS BY INTRA-ARTICULAR INJECTION FOR DEGENERATIVE KNEE OSTEOARTHRITIS].

OBJECTIVE: To investigate the safety and efficacy of human umbilical cord mesenchymal stem cells (MSCs) by intra-articular injection for degenerative knee osteoarthritis. METHODS: Between January 2015 and January 2016, 36 patients with moderate or severe degenerative knee osteoarthritis were randomly devided into 2 groups (n=18). Intra-articular injection of 2.5-3.0 mL human umbilical cord MSCs suspension containing (2-3)×107 cells was performed once a month for 2 times as a course of treatment in the cell treatment group; sodium hyaluronate by intra-articular injection was used once a week for 5 times as a course of treatment in the control group. There was no significant difference in gender, age, body mass index, side, stage of osteoarthritis, course of disease, and preoperative Lysholm score of the knee joint, the Western Ontario and McMaster Universities osteoarthritis index (WOMAC), and SF-36 scale score between 2 groups (P>0.05). The clinical efficacy was evaluated by SF-36 scale score, Lysholm score, and WOMAC score. RESULTS: All patients of 2 groups received a course of treatment. The patients were followed up for 6 months. After injection, the incidences of pain and swelling in the cell treatment group were significantly higher than those in the control group (χ2=16.200, P=0.000; χ2=11.688, P=0.000), but no significant difference was found in the incidence of effusion (χ2=2.118, P=0.146). In the cell treatment group, Lysholm score at 1-6 months after treatment, WOMAC score and SF-36 scale score at 2-6 months after treatment were significantly better when compared with scores before treatment (P<0.05), and no recurrence of knee pain was observed during follow-up. In the control group, there was no significant difference in Lysholm score and SF-36 scale score between pre- and post-treatment (P>0.05); there were significant differences in WOMAC score between pre-treatment and at 1, 2, 3 months after treatment (P<0.05); at 3 months after treatment, 11 patients had joint pain symptoms again. No significant difference was found in the knee joint function score and SF-36 scale score at 1 and 2 months after treatment between 2 groups (P>0.05), but the scores of the cell treatment group were significantly better than those of the control group at 3 and 6 months (P<0.05). CONCLUSIONS: It can significantly improve the joint function and quality of life to use intra-articular injection of human umbilical cord MSCs for treating degenerative knee osteoarthritis. It takes effect after 1 month and the treatment effect can be sustained for 6 months.

Premalignant alteration assessment in liver-like tissue derived from embryonic stem cells by aristolochic acid I exposure.

The in vitro predictive evaluation of chemical carcinogenicity based on hepatic premalignance has so far not been established. Here, we report a novel approach to investigate the premalignant events triggered by human carcinogen aristolochic acid I (AAI) in the liver-like tissue derived from mouse embryonic stem cells. By AAI exposure, the liver-like tissue exhibited the paracrine interleukin-6 phenotypic characteristics. Hepatocytes expressed STAT3/p-STAT3, c-Myc and Lin28B in parallel. Some of them displayed the dedifferentiation characteristics, such as full of α-fetoprotein granules, increase in size, and nucleocytoplasmic shuttle of Oct4. When these cells were injected into mice, the xenografts mostly displayed the uniform area of hepatic-like tissue with malignant nuclei. The hepatic malignant markers, α-fetoprotein, cytokeratin 7 and cytokeratin 19, were co-expressed in albumin-positive areas, respectively. In conclusion, we established an approach to predict the hepatic premalignance triggered by carcinogen AAI. This premalignant assay system might aid to evaluate the effects of potential carcinogens in liver, and probably to screen the protecting against hepatocarcinogenic efficacy of pharmaceuticals in vitro.

Hepatic Premalignant Alterations Triggered by Human Nephrotoxin Aristolochic Acid I in Canines.

Aristolochic acid I (AAI) existing in plant drugs from Aristolochia species is an environmental human carcinogen associated with urothelial cancer. Although gene association network analysis demonstrated gene expression profile changes in the liver of human TP53 knock-in mice after acute AAI exposure, to date, whether AAI causes hepatic tumorigenesis is still not confirmed. Here, we show that hepatic premalignant alterations appeared in canines after a 10-day AAI oral administration (3 mg/kg/day). We observed c-Myc oncoprotein and oncofetal RNA-binding protein Lin28B overexpressions accompanied by cancer progenitor-like cell formation in the liver by AAI exposure. Meanwhile, we found that forkhead box O1 (FOXO1) was robustly phosphorylated, thereby shuttling into the cytoplasm of hepatocytes. Furthermore, utilizing microarray and qRT-PCR analysis, we confirmed that microRNA expression significantly dysregulated in the liver treated with AAI. Among them, we particularly focused on the members in let-7 miRNAs and miR-23a clusters, the downstream of c-Myc and IL6 receptor (IL6R) signaling pathway linking the premalignant alteration. Strikingly, when IL6 was added in vitro, IL6R/NF-κB signaling activation contributed to the increase of FOXO1 phosphorylation by the let-7b inhibitor. Therefore, it highlights the new insight into the interplay of the network in hepatic tumorigenesis by AAI exposure, and also suggests that anti-premalignant therapy may be crucial for preventing AAI-induced hepatocarcinogenesis.

Metagenome-wide analysis of antibiotic resistance genes in a large cohort of human gut microbiota.

The human gut microbiota is a reservoir of antibiotic resistance genes, but little is known about their diversity and richness within the gut. Here we analyse the antibiotic resistance genes of gut microbiota from 162 individuals. We identify a total of 1,093 antibiotic resistance genes and find that Chinese individuals harbour the highest number and abundance of antibiotic resistance genes, followed by Danish and Spanish individuals. Single-nucleotide polymorphism-based analysis indicates that antibiotic resistance genes from the two European populations are more closely related while the Chinese ones are clustered separately. We also confirm high abundance of tetracycline resistance genes with this large cohort study. Our study provides a broad view of antibiotic resistance genes in the human gut microbiota.

Functional screening of antibiotic resistance genes from human gut microbiota reveals a novel gene fusion.

The human gut microbiota has a high density of bacteria that are considered a reservoir for antibiotic resistance genes (ARGs). In this study, one fosmid metagenomic library generated from the gut microbiota of four healthy humans was used to screen for ARGs against seven antibiotics. Eight new ARGs were obtained: one against amoxicillin, six against d-cycloserine, and one against kanamycin. The new amoxicillin resistance gene encodes a protein with 53% identity to a class D ß-lactamase from Riemerella anatipestifer RA-GD. The six new d-cycloserine resistance genes encode proteins with 73-81% identity to known d-alanine-d-alanine ligases. The new kanamycin resistance gene encodes a protein of 274 amino acids with an N-terminus (amino acids 1-189) that has 42% identity to the 6'-aminoglycoside acetyltransferase [AAC(6')] from Enterococcus hirae and a C-terminus (amino acids 190-274) with 35% identity to a hypothetical protein from Clostridiales sp. SSC/2. A functional study on the novel kanamycin resistance gene showed that only the N-terminus conferred kanamycin resistance. Our results showed that functional metagenomics is a useful tool for the identification of new ARGs.