Stem Cell Toxicology

National Institute Of Environmental Health Sciences

Investigators

Linked publications & trials

Abstract

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. Research efforts focus on diseases and conditions associated with exposure to NTP-relevant chemicals and their effects at various life stages. Studies use 2D and 3D formats of human pluripotent stem cells (PSCs), adult SC and cancer SCs (CSCs), and other mature cell lines. 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 human 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), imaging, and proteomics platforms to examine chemical-induced alterations in gene expression, morphology, and protein expression, respectively, 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 artificial intelligence (AI) (e.g. hierarchical clustering and supervised classification analysis by machine learning; deep learning) and high-content imaging techniques, we have shown that this screening platform has a highly impressive success rate for identifying developmental toxicants. We have recently collected NGS data from EB models exposed to 30 teratogens, and from neural organoids exposed to arsenic; these data are being analyzed. The 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, renal, colonic, and hepato lineages. Efforts on creating neural, cardiac, and colon organoids have been successful. We have begun testing various chemicals during the development of these organoids and are prepping multiple manuscripts from the data. Together, these studies using pluripotent SCs will help determine effects of chemicals on embryonic development, germ-layer differentiation, and/or teratogenicity, lineage specification, and/or organogenesis. Two postdoctoral fellows in the lab were each recently awarded an NIH FARE award for their exciting work with these projects. With members of the Molecular Genomics Core (DIR) we have recently initiated a project to explore circulating cell-free biomolecules (i.e. DNA, RNA, miRNAs, proteins) that are either free-floating or packaged within exosomes. These studies will help to (1) identify potential circulating biomarkers or genetic signatures of temporal differentiation of pluripotent stem cells (PSCs) to specific cells types/lineages, (2) determine chemically-induced alterations in these biomarkers, and (3) determine molecular signatures. Using our PSC models and directed differentiation protocols we will identify chemical hazard and study the embryonic/developmental onset of neural and cardiac diseases, as well as cancer. These studies will enable DNTP to screen for circulating nucleic acids and biomolecules to help identify genetic signatures or biomarkers of disease progression, in a non-invasive and rapid manner, thereby allowing for monitoring disease initiation and progression caused by NTP-relevant chemicals. Inorganic carcinogens i.e. arsenic (As) and cadmium (Cd) 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 have also begun testing these inorganics on our hESC models to investigate their toxics effects during early development. We are working with the Predictive Toxicology 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. Novel findings implicate the activation of endogenous retroviruses in As-transformed cells that may have incorporated KRAS. These studies are nearing completion. To a lesser extent than previous years, we use our in vitro cancer models to discover and define the mechanisms involved in SC targeting and transformation by inorganics and NTP-relevant carcinogens. 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. With collaborators at Harvard and in Japan we examined 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. This study is complete, and a manuscript is in preparation.