The mission of the Stem Cell Toxicology Group is to characterize responses to toxicants to elucidate mechanisms and identify the role of stem cells (SCs) in disease manifestation. The Group provides expertise in areas of SC biology and SC toxicology and works on National Toxicology Program (NTP) Laboratory mission-related projects that involve SCs. While SCs/cancer SCs (CSCs) and potential mechanisms of action in inorganic carcinogenesis has been the major focus, research efforts are extending beyond cancer to include those diseases and conditions associated with exposure to NTP-relevant chemicals as well as effects of these chemicals at various life stages. The potential of embryonic SCs (ESCs) and induced pluripotent SCs (iPSCs) in disease modeling, drug testing, drug development, and regenerative therapies have recently made SC biology one of the most active areas of research. We are using ESCs and iPSCs to examine effects of environmental toxicants on embryogenesis, developmental toxicology and reproductive toxicology. With the Epigenetics and Stem Cell Biology Lab (DIR) we have developed assays using both 3D and 2D models of human ESCs and iPSCs to screen environmental toxicants/chemicals to help discover and better predict developmental toxicants and teratogens. Ongoing studies are using high-throughput transcriptomics (HTT) platforms to examine chemical-induced gene expression alterations in hESCs. We have also developed a high-throughput screening platform using 3D embryoid bodies to screen 100+ NTP-relevant chemicals for teratogenic potential by examining effects on 37 hallmark genes involved in embryogenesis. Using hierarchical clustering and supervised classification analysis by machine learning we have shown that this screening platform appears to successfully identify developmental toxicants. Current focus of these studies and models is on developmental cardiotoxicity, neurotoxicity, and hepatotoxicity. We have also developed methods/protocols to derive organoids in the cardio, neuro, and hepato lineages. Preliminary efforts on creating neural organoids have been successful, and we are not awaiting results on the first chemical tests on neural organoid formation. Together, these studies using pluripotent SCs will help determine effects of chemicals on embryonic development, germ-layer differentiation, and/or teratogenicity, and/or organogenesis. Arsenic (As) and cadmium (Cd) are inorganic carcinogens that are major human health hazards and defining mechanisms is key to defining risk. We use mature (differentiated) and SC cell models of human target-relevant tissues of these carcinogens. Millions of people worldwide are exposed to unhealthy levels of these inorganics making elucidation of mechanisms critical. We have a few on-going projects/studies involving the role of SCs/CSCs in inorganic i.e. arsenic (As) and cadmium (Cd) carcinogenesis, although this area of research has recently become less of a focus of our group. We are working with the Biomolecular Screening Branch (NTP) using next-gen sequencing methods to further examine KRAS upregulation in As transformation. In these studies, the major driver of cell transformation is an increased level of KRAS. We performed a genome-wide evaluation of DNA methylation and gene expression, but observed genomic changes appear to be secondary to elevated KRAS. Data show that KRAS expression appears unaffected by any changes in proximal methylation clusters at this genomic locus. Preliminary findings implicate the activation of endogenous retroviruses in As-transformed cells that may have incorporated KRAS. Exosomes are extracellular vesicles that contain biomolecular cargo that can exert effects on recipient cells. Recently we isolated and characterized exosomes from As-transformed cells and found that exosomes released by As-transformed cells play a key role in regulating the tumor microenvironment. As altered both exosome quantity and cargo during malignant transformation. As-transformed cells secret exosomes carrying cancer-favoring biomolecules (KRAS, specific miRNAs) which appear to recruit and transform nearby SCs. Knockdown of many of these factors decreases the ability of the transformed cells to recruit the SCs into a CSC phenotype. These exosome methods are being expanded to studies within multiple NTPL groups to help identify disease biomarkers. We continue to use our in vitro cancer models to discover and define the mechanisms involved in SC targeting and transformation by inorganics. Unlike As, Cd initially selectively kills 90% of SCs during exposure to a non-toxic, but transforming, level for the heterogeneous parental lines. The remaining SCs rapidly re-emerge and undergo transformation. We are characterizing these putative Cd-CSCs, including defining the metabolic profiles during transformation of these cells as well as other (i.e. As) transformed SC/CSC models. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at a post-transcriptional level. We find inorganic carcinogens dysregulate miRNA expression, including changes that control RAS activation during malignant transformation suggesting miRNA-regulated RAS expression is a putative driver in transformation by some inorganics. These studies help determine roles of miRNAs and underlying epigenetic mechanisms involved in inorganic carcinogenesis and suggest possible miRNA biomarkers of transformation. Studies using miRNAs have been expanded to additional projects (i.e. ESC differentiation) to help identify stage- and/or disease-specific biomarkers. Polycyclic aromatic compounds (PACs) are environmental contaminants with extensive toxicities. With the Toxicology Branch (NTP), we characterized the toxicity of a broad range of PAHs and PAH mixtures. Initial studies (24 PACs, 5 cell lines) of structurally diverse PACs showed that, in general, activity levels of PACs corresponded across in vitro and in vivo testing platforms. These results offer promise for the development of an in vitro battery for predicting in vivo activity. We then screened 100 PAHs and PAH mixtures on 13 biologically diverse cell lines including non-tumorigenic and cancer cell lines. Crumb rubber (CR) is a major component of synthetic turfs. There is evidence of potential carcinogenic risk from playing on these turfs. CR is composed of ground tires and can contain volatile, semi-volatile, and non-volatile organic compounds, metals, and particulate matter. Major exposure pathways are dermal, inhalation and ingestion. With the Predictive Toxciology and Screening Group (NTPL) and the Toxicology Branch (NTP) we investigated the toxicity of several crumb rubber samples. We found these samples were cytotoxic to human lung and skin cells at relatively low doses and short exposure times. Similar results were found in an intestinal cell line. An NTP Research Report was published on these data. With collaborators at Harvard and in Japan we are examining a unique Japanese cohort that was acutely exposed to high levels of As during infancy in order evaluate the association between this developmental exposure and the resultant differential gene expression and signaling pathway alterations that have persisted into adulthood. The overall purpose was to help identify the genes and pathways most affected by this early-life exposure as a possible method to help identify potential early indicators of disease. As-poisoned subjects showed DNA methylation and signaling pathway alterations in blood cells that suggested adverse effects on immune regulation and function
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