Tetrabromobisphenol A perturbs cell fate decisions via BMP signaling in the early embryonic development of zebrafish.

Tetrabromobisphenol A (TBBPA) readily accumulates in the egg yolk of aquatic oviparous animals and is transferred to their embryos. Early embryogenesis is vital for organ formation and subsequent development. The developmental toxicity of TBBPA in aquatic animals has been extensively reported. However, few studies have assessed the toxic effects of TBBPA in the early embryonic development. In this work, we found that TBBPA perturbed cell fate decisions along the dorsal-ventral (DV) axis during gastrulation, further disrupting early organogenesis in the entire embryo. TBBPA exposure increased the number of embryonic cells that acquired a ventral cell fate, which formed epidermis, blood and heart tissues. In return, the number of embryonic cells that acquired a dorsal cell fate was greatly decreased, causing the TBBPA-exposed embryos to develop a small brain and small eyes. We revealed that TBBPA elevated the activity gradient of bone morphogenetic protein (BMP) signaling which is responsible for cell fate specification along the DV axis, with up-regulation of BMP ligands (bmp4, bmp7a) and target genes (szl) and promotion signal transduction through phosphorylation of Smad1/5. As the function of BMP signaling in embryogenesis is highly conserved among many vertebrates, these findings highlight the ecological and health risks of TBBPA.

Differentially Expressed Genes and Enriched Signaling Pathways in the Adipose Tissue of Obese People.

As the prevalence of obesity increases, so does the occurrence of obesity-related complications, such as cardiovascular and cerebrovascular diseases, diabetes, and some cancers. Increased adipose tissue is the main cause of harm in obesity. To better understand obesity and its related complications, we analyzed the mRNA expression profiles of adipose tissues from 126 patients with obesity and 275 non-obese controls. Using an integrated bioinformatics method, we explored the functions of 113 differentially expressed genes (DEGs) between them. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses revealed that upregulated DEGs were enriched in immune cell chemotaxis, complement-related cascade activation, and various inflammatory signaling pathways, while downregulated DEGs enriched in nutrient metabolism. The CIBERSORT algorithm indicated that an increase in macrophages may be the main cause of adipose tissue inflammation, while decreased γδ T cells reduce sympathetic action, leading to dysregulation of adipocyte thermogenesis. A protein-protein interaction network was constructed using the STRING database, and the top 10 hub genes were identified using the cytoHubba plug-in in Cytoscape. All were confirmed to be obesity-related using a separate dataset. In addition, we identified chemicals related to these hub genes that may contribute to obesity. In conclusion, we have successfully identified several hub genes in the development of obesity, which provide insights into the possible mechanisms controlling obesity and its related complications, as well as potential biomarkers and therapeutic targets for further research.

In situ High-Throughput Single-Cell Analysis Reveals the Crosstalk between Nanoparticle-Induced Cell Responses.

Nanomaterials are widely used in a variety of industrial, biological, and medical applications. Therefore, high concerns about their possible impact on human and environmental health have been raised. Here, we describe a high-throughput single-cell imaging method to reveal the crosstalk among quantum dot (QDot)-induced ROS generation, apoptosis, and changes in nucleus size in macrophages. In triple marker combinations, we assessed the correlations of three QDot-induced cellular responses via divided subsets based on single-cell analysis. In contrast to the results obtained from the cell population, we demonstrated that the change in nucleus size was positively correlated with ROS generation. We found that QDot exposure induced ROS generation, which led to cell apoptosis, followed by a change in nucleus size. In general, these observations on crosstalk of cellular responses provide detailed insights into the heterogeneity of nanoparticle exposure.

Tetrabromobisphenol A induces THR ß-mediated inflammation and uterine injury in mice at environmentally relevant exposure concentrations.

Tetrabromobisphenol A (TBBPA) is a widely used flame retardant, but the adverse outcomes induced by TBBPA has not been fully elucidated. In this study, TBBPA was detected in 54.9% of 102 female Chinese volunteers with an average serum concentration of 0.34 ng/mL. To investigate whether TBBPA induces adverse outcomes at environmentally relevant exposure doses, the mice were exposed to TBBPA for 14 and 28 days. The internal doses of TBBPA in mice serum were nearly the internal doses in volunteers. TBBPA significantly increased the secretion of some pro-inflammatory cytokines and suppressed immune responses in mice under such serum concentrations after 14- and 28-days exposure. Interestingly, uterine edema was observed in TBBPA-treated mice. In primary uterine cells model, the results showed TBBPA exposure suppressed THRß expression, leading to the activation of the inflammatory PI3K/NF-κB signaling pathway. Our findings indicated that the uterus is the susceptible target organ of TBBPA and TBBPA exposure might increase risk of uterine cancer through deregulating inflammation pathways.

Tetrabromobisphenol A Perturbs Erythropoiesis and Impairs Blood Circulation in Zebrafish Embryos.

Tetrabromobisphenol A (TBBPA), a ubiquitous environmental pollutant, has been implicated in developmental toxicity of aquatic animals. However, the impact of TBBPA on development and the related mechanism have not been fully elucidated. In this study, using a live imaging technique and transgenic labeling of zebrafish embryos, we described the toxic effects of TBBPA on hematopoietic development in zebrafish. We demonstrated that TBBPA induced erythroid precursor expansion in the intermediate cell mass (ICM), which perturbed the onset of blood circulation at 24-26 hours postfertilization (hpf). Consequently, excessive blood cells accumulated in the posterior blood island (PBI) and vascular cells formed defective caudal veins (CVs), preventing blood cell flow to the heart at 32-34 hpf. We found that the one-cell to 50% epiboly stage was the most sensitive period to TBBPA exposure during hematopoietic development. Furthermore, our results demonstrated that PBI malformation induced by TBBPA resulted from effects on erythroid precursor cells, which might involve THR signaling in complex ways. These findings will improve the understanding of TBBPA-induced developmental toxicity in teleost.

A pilot study of mothers and infants reveals fetal sex differences in the placental transfer efficiency of heavy metals.

It has been demonstrated that heavy metals cross the placental barrier and exert potentially harmful fetal effects. Although previous studies showed sex differences in response to similar intrauterine environments, little is known about fetal sex-related differences in placental transfer and accumulation of heavy metals. This study aimed to reveal the sex-specific risk of fetal exposure to heavy metals in pregnant women. We detected the exposure levels of eight heavy metals in 64 paired mother-infant maternal blood, cord blood and placental tissue samples. We found that the placental transfer efficiency (PTE) of titanium (Ti) and silver (Ag) was significantly higher in the group with male fetuses than that with female fetuses. The group with male fetuses had a larger placental:maternal blood ratio of Ag levels than the group with female fetuses, indicating fetal sex-related differences in placental transfer and accumulation of Ag. Prospective research should focus on the sex differences of adverse health effects induced by heavy metals and other pollutants.

Ultralong AgNWs-induced toxicity in A549 cells and the important roles of ROS and autophagy.

Safety concerns have been raised with regard to silver nanowires (AgNWs) because of their extensive applications. Recently, ultralong AgNWs have shown physical properties superior to those of short AgNWs. However, little is known about their toxicity and potential risks. In this study, we demonstrated a series of ultralong AgNWs-induced biological effects in human lung cancer epithelial cells (A549). Ultralong AgNWs treatments induced ROS generation, mitochondria-mediated apoptosis, and self-protective autophagy at nonlethal concentrations. In contrast to some previous reports, apoptosis was found not to correlate with the reduction of intracellular ROS. Measuring the processing of ROS generation, apoptosis and autophagy, we demonstrated that ROS not only enhance mitochondrial damage, but also raise protective autophagic flux in ultralong AgNW-treated cells. Moreover, ultralong AgNWs were found to be internalized into the cytoplasm of the epithelial cells. This study not only investigates ultralong AgNWs-induced cytotoxicity but also pinpoints ROS as a key signal in mechanisms of their toxicity.

Length and diameter-dependent phagocytosis and cytotoxicity of long silver nanowires in macrophages.

Long silver nanowires (AgNWs, >5 µm) have shown promising applications in next generation biomaterials. However, the toxicity of long AgNWs is not well characterized in terms of their size. In this study, five AgNWs types, including SAgNW30 (length: 5-10 µm; diameter: 30 nm), MAgNW30 (length: 20-30 µm; diameter: 30 nm), LAgNW30 (length: ∼100 µm; diameter: 30 nm), LAgNW50 (length: ∼100 µm; diameter: 50 nm), and LAgNW100 (length: ∼100 µm; diameter: 100 nm), were used to investigate the size-dependent phagocytosis and cytotoxicity in macrophage. It showed that SAgNW30, MAgNW30, LAgNW30 can be fully phagocytosed by macrophages, but LAgNW50 and LAgNW100 frustrated the phagocytosis. It demonstrated that LAgNW30 can be internalized into macrophage in a curly manner. The size-dependent cytotoxicity was observed in cell viability, apoptosis, mitochondrial damage, phenotypic transition, and inflammatory response in AgNWs-treated macrophage. The AgNWs-induced cytotoxicity was depended on their length and diameter, increased gradually in the order of SAgNW30 > MAgNW30 > LAgNW30 > LAgNW50 > LAgNW100. The findings presented here will assist in the evaluation of the size-dependent cytotoxicity mediated by long AgNWs.

Generation of Functional Hepatocytes from Human Adipose-Derived MYC+ KLF4+ GMNN+ Stem Cells Analyzed by Single-Cell RNA-Seq Profiling.

Cell transplantation holds considerable promise for end-stage liver diseases but identifying a suitable, transplantable cell type has been problematic. Here, we describe a novel type of mesenchymal stem cells (MSCs) from human adipose tissue. These cells are different from previously reported MSCs, they are in the euchromatin state with epigenetic multipotency, and express pluripotent markers MYC, KLF4, and GMNN. Most of the genes associated with germ layer specification are modified by H3K4me3 or co-modified by H3K4me3 and H3K27me3. We named this new type of MSCs as adult multipotent adipose-derived stem cells (M-ADSCs). Using a four-step nonviral system, M-ADSCs can be efficiently Induced into hepatocyte like cells with expression of hepatocyte markers, drug metabolizing enzymes and transporters, and the other basic functional properties including albumin (ALB) secretion, glycogen storage, detoxification, low-density lipoprotein intake, and lipids accumulation. In vivo both M-ADSCs-derived hepatoblasts and hepatocytes could form vascularized liver-like tissue, secrete ALB and express metabolic enzymes. Single-cell RNA-seq was used to investigate the important stages in this conversion. M-ADSCs could be converted to a functionally multipotent state during the preinduction stage without undergoing reprogramming process. Our findings provide important insights into mechanisms underlying cell development and conversion. Stem Cells Translational Medicine 2018;7:792-805.

Non-cytotoxic nanomolar concentrations of bisphenol A induce human mesenchymal stem cell adipogenesis and osteogenesis.

Bisphenol A (BPA) is a typical endocrine disrupting chemical with extensive applications, and has been correlated with various hazardous health effects, including obesity and other metabolic-related diseases. Human mesenchymal stem cells (hMSCs), due to their abilities to differentiate into adipocytes and osteoblasts, can be a good in vitro model to assess chemical-dependent toxicity on adipogenesis or osteogenesis. Here, we employed hMSCs as an evaluation system to assess BPA-related effects on cell viability, oxidative stress induction, self-renewal, and differentiation. Our results revealed that low concentrations (1 and 10 nM) of BPA did not impair cell proliferation nor self-renewal capacity, but stimulated adipogenesis and osteogenesis. Our findings support the concern of BPA contributing to the epidemic of obesity, and also reveal its underlying toxicity on osteogenesis.

Coating with spermine-pullulan polymer enhances adenoviral transduction of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are adult stem cells with multilineage potential, which makes them attractive tools for regenerative medicine applications. Efficient gene transfer into MSCs is essential not only for basic research in developmental biology but also for therapeutic applications involving gene-modification in regenerative medicine. Adenovirus vectors (Advs) can efficiently and transiently introduce an exogenous gene into many cell types via their primary receptors, the coxsackievirus and adenovirus receptors, but not into MSCs, which are deficient in coxsackievirus and adenovirus receptors expression. To overcome this problem, we developed an Adv coated with a spermine-pullulan (SP) cationic polymer and investigated its physicochemical properties and internalization mechanisms. We demonstrated that the SP coating could enhance adenoviral transduction of MSCs without detectable cytotoxicity or effects on differentiation. Our results argue in favor of the potentiality of the SP-coated Adv as a prototype vector for efficient and safe transduction of MSCs.

Role of IL-17 Pathways in Immune Privilege: A RNA Deep Sequencing Analysis of the Mice Testis Exposure to Fluoride.

We sequenced RNA transcripts from the testicles of healthy male mice, divided into a control group with distilled water and two experimental groups with 50 and 100 mg/l NaF in drinking water for 56 days. Bowtie/Tophat were used to align 50-bp paired-end reads into transcripts, Cufflinks to measure the relative abundance of each transcript and IPA to analyze RNA-Sequencing data. In the 100 mg/l NaF-treated group, four pathways related to IL-17, TGF-ß and other cellular growth factor pathways were overexpressed. The mRNA expression of IL-17RA, IL-17RC, MAP2K1, MAP2K2, MAP2K3 and MAPKAPK2, monitored by qRT-PCR, increased remarkably in the 100 mg/L NaF group and coincided with the result of RNA-Sequencing. Fluoride exposure could disrupt spermatogenesis and testicles in male mice by influencing many signaling pathways and genes, which work on the immune signal transduction and cellular metabolism. The high expression of the IL-17 signal pathway was a response to the invasion of the testicular immune system due to extracellular fluoride. The PI3-kinase/AKT, MAPKs and the cytokines in TGF-ß family were contributed to control the IL-17 pathway activation and maintain the immune privilege and spermatogenesis. All the findings provided new ideas for further molecular researches of fluorosis on the reproduction and immune response mechanism.

Optimization of Reference Genes for Normalization of Reverse Transcription Quantitative Real-Time Polymerase Chain Reaction Results in Senescence Study of Mesenchymal Stem Cells.

Recently, it has been suggested that cellular senescence is associated with stem cell exhaustion, which reduces the regenerative potential of tissues and contributes to aging and age-related diseases. Mesenchymal stem cells (MSCs) attract a large amount of attention in stem cell research and regeneration medicine because they possess multiple advantages and senescent MSCs could be one of the most useful stem cell models in aging studies. It is important to quantitatively evaluate senescence markers to both identify and study the mechanisms involved in MSC senescence. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is currently the most widely used tool to quantify the mRNA levels of markers. However, no report has demonstrated the optimal reference genes that should be used to normalize RT-qPCR in senescence studies of MSCs. In this study, we compared 16 commonly used reference genes (GAPDH, ACTB, RPL13A, TBP, B2M, GUSB, RPLPO, YWHAZ, RPS18, EEF1A1, ATP5F1, HPRT1, PGK1, TFRC, UBC, and PPIA) in proliferating or replicative-senescent human adipose-derived MSCs (hAD-MSCs) that were isolated from seven healthy donors aged 29-59 years old. Three algorithms (geNorm, NormFinder, and BestKeeper) were used to determine the most optimal reference gene. The results showed that PPIA exhibited the most stable expression during senescence, while the widely used ACTB exhibited the lowest stability. We also confirmed that different reference genes lead to different evaluations of senescence markers. Our work ensures that results obtained from senescence studies of hAD-MSCs will be appropriately evaluated in both basic research and clinical trials.

Assessment of Bisphenol A (BPA) neurotoxicity in vitro with mouse embryonic stem cells.

The adverse effects of environmental pollution on our well-being have been intensively studied with many in vitro and in vivo systems. In our group, we focus on stem cell toxicology due to the multitude of embryonic stem cell (ESC) properties which can be exerted in toxicity assays. In fact, ESCs can differentiate in culture to mimic embryonic development in vivo, or specifically to virtually any kind of somatic cells. Here, we used the toxicant Bisphenol A (BPA), a chemical known as a hazard to infants and children, and showed that our stem cell toxicology system was able to efficiently recapitulate most of the toxic effects of BPA previously detected by in vitro system or animal tests. More precisely, we demonstrated that BPA affected the proper specification of germ layers during our in vitro mimicking of the embryonic development, as well as the establishment of neural ectoderm and neural progenitor cells.

Vitamin E attenuates silver nanoparticle-induced effects on body weight and neurotoxicity in rats.

Silver nanoparticles (AgNPs) are one of the most commonly used nanomaterials; however, it remains unclear whether AgNPs induce neurotoxicity. Here, we investigated the potential neurological effects of AgNPs and the neuroprotective effect of vitamin E (VE). We found that intranasal instillation of AgNPs in neonatal Sprague-Dawley rats caused significant body weight loss. Moreover, histological examinations revealed activation of neuroglial cells with concomitant destruction of the granular layer of the cerebellum. Furthermore, western blot analyses showed an increase in the levels of the glial fibrillary acidic protein (GFAP), a marker of astrocyte activation. These observations suggest that AgNPs have significant neurotoxic effects on the rat cerebellum. Strikingly, oral administration of VE counterbalanced the toxic effects triggered by AgNPs. Taken together, our findings suggest that nasal administration of AgNPs may produce neurotoxicity in rats, and that VE supplementation attenuates these effects.

Polyethyleneimine-coating enhances adenoviral transduction of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are non-hematopoietic cells with multi-lineage potential, which makes them attractive targets for regenerative medicine applications. Efficient gene transfer into MSCs is essential for basic research in developmental biology and for therapeutic applications involving gene-modification in regenerative medicine. Adenovirus vectors (Advs) can efficiently and transiently introduce an exogenous gene into many cell types via their primary receptors, the coxsackievirus and adenovirus receptors (CARs), but not into MSCs, which lack CAR expression. To overcome this problem, an Adv coated with cationic polymer polyethyleneimine (PEI) was developed. In this study, we demonstrated that PEI coating with an optimal ratio can enhance adenoviral transduction of MSCs without cytotoxicity. We also investigated the physicochemical properties and internalization mechanisms of the PEI-coated Adv. These results could help to evaluate the potentiality of the PEI-coated Adv as a prototype vector for efficient and safe transduction into MSCs.

Macrophage mannose receptor-specific gene delivery vehicle for macrophage engineering.

Macrophages are the most plastic cells in the hematopoietic system and they exhibit great functional diversity. They have been extensively applied in anti-inflammatory, anti-fibrotic and anti-cancer therapies. However, the application of macrophages is limited by the efficiency of their engineering. The macrophage mannose receptor (MMR, CD206), a C-type lectin receptor, is ubiquitously expressed on macrophages and has a high affinity for mannose oligosaccharides. In the present study, we developed a novel non-viral vehicle with specific affinity for MMR. Mannan was cationized with spermine at a grafted ratio of ∼12% to deliver DNA and was characterized as a stable system for delivery. This spermine-mannan (SM)-based delivery system was evaluated as a biocompatible vehicle with superior transfection efficiency on murine macrophages, up to 28.5-fold higher than spermine-pullulan, 11.5-fold higher than polyethylenimine and 3.0-fold higher than Lipofectamine™ 2000. We confirmed that the SM-based delivery system for macrophages transfection was MMR-specific and we described the intracellular transport of the delivery system. To our knowledge, this is the first study using SM to demonstrate a mannose receptor-specific gene delivery system, thereby highlighting the potential of a novel specific non-viral delivery vehicle for macrophage engineering.

Generation of highly purified neural stem cells from human adipose-derived mesenchymal stem cells by Sox1 activation.

Neural stem cells (NSCs) are ideal candidates in stem cell-based therapy for neurodegenerative diseases. However, it is unfeasible to get enough quantity of NSCs for clinical application. Generation of NSCs from human adipose-derived mesenchymal stem cells (hAD-MSCs) will provide a solution to this problem. Currently, the differentiation of hAD-MSCs into highly purified NSCs with biological functions is rarely reported. In our study, we established a three-step NSC-inducing protocol, in which hAD-MSCs were induced to generate NSCs with high purity after sequentially cultured in the pre-inducing medium (Step1), the N2B27 medium (Step2), and the N2B27 medium supplement with basic fibroblast growth factor and epidermal growth factor (Step3). These hAD-MSC-derived NSCs (adNSCs) can form neurospheres and highly express Sox1, Pax6, Nestin, and Vimentin; the proportion was 96.1% ± 1.3%, 96.8% ± 1.7%, 96.2% ± 1.3%, and 97.2% ± 2.5%, respectively, as detected by flow cytometry. These adNSCs can further differentiate into astrocytes, oligodendrocytes, and functional neurons, which were able to generate tetrodotoxin-sensitive sodium current. Additionally, we found that the neural differentiation of hAD-MSCs were significantly suppressed by Sox1 interference, and what's more, Step1 was a key step for the following induction, probably because it was associated with the initiation and nuclear translocation of Sox1, an important transcriptional factor for neural development. Finally, we observed that bone morphogenetic protein signal was inhibited, and Wnt/ß-catenin signal was activated during inducing process, and both signals were related with Sox1 expression. In conclusion, we successfully established a three-step inducing protocol to derive NSCs from hAD-MSCs with high purity by Sox1 activation. These findings might enable to acquire enough autologous transplantable NSCs for the therapy of neurodegenerative diseases in clinic.

Co-transfection gene delivery of dendritic cells induced effective lymph node targeting and anti-tumor vaccination.

PURPOSE: Successful genetically engineered Dendritic Cell (DC) can enhance DC's antigen presentation and lymph node migration. The present study aims to genetically engineer a DC using an efficient non-viral gene delivery vector to induce a highly efficient antigen presentation and lymph node targeting in vivo. METHODS: Spermine-dextran (SD), a cationic polysaccharide vector, was used to prepare a gene delivery system for DC engineering. Transfection efficiency, nuclear trafficking, and safety of the SD/DNA complex were evaluated. A vaccine prepared by engineering DC with SD/gp100, a plasmid encoding melanoma-associated antigen, was injected subcutaneously into mice to evaluate the tumor suppression. The migration of the engineered DCs was also evaluated in vitro and in vivo. RESULTS: SD/DNA complex has a better transfection behavior in vitro than commercially purchased reagents. The DC vaccine co-transfected with plasmid coding CCR7, a chemokine receptor essential for DC migration, and plasmid coding gp100 displayed superior tumor suppression than that with plasmid coding gp100 alone. Migration assay demonstrated that DC transfected with SD/CCR7 can promote DC migration capacity. CONCLUSIONS: The study is the first to report the application of nonviral vector SD to co-transfect DC with gp100 and CCR7-coding plasmid to induce both the capacity of antigen presentation and lymph node targeting.