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Investigation of a redox-sensitive predictive model of mouse embryonic stem cells differentiation using quantitative nuclease protection assays and glutathione redox status
Citation:
Chandler, K., J. Hanson, T. Knudsen, AND S. Hunter. Investigation of a redox-sensitive predictive model of mouse embryonic stem cells differentiation using quantitative nuclease protection assays and glutathione redox status. Presented at NIEHS Symposium on Unlocking thr Promise of Stem Cells, Research Triangle Park, NC, April 11, 2013.
Impact/Purpose:
These experiments document the importance of using multiple differentiation endpoints and support the linkage of our predictive model to altered differentiation in chemical profiling.
Description:
Investigation of a redox-sensitive predictive model of mouse embryonic stem cell differentiation via quantitative nuclease protection assays and glutathione redox status Chandler KJ,Hansen JM, Knudsen T,and Hunter ES 1. U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA. 2. Emory University School of Medicine, Atlanta, Georgia 30322. Mouse embryonic stem cells (mESCs) recapitulate developmental signals that occur in vivo and are amenable to high-throughput profiling of chemical-induced effects. In silica models of impaired mESC differentiation identified 19 ToxCast™ assays that distinguished chemical • effects on cardiomyocyte differentiation versus cytotoxicity (Wilcoxon rank sum, p=0.03). Taken together, these assays connect a redox-sensitive pathway(s) as a likely target of a chemical set that impaired mESC differentiation. To evaluate this predictive model, a custom quantitative nuclease protection assay (qNPA) array (HTG Molecular Diagnostics) of 41 redox-sensitive targets and differentiation markers was designed. The concentration-dependent effects of twelve ToxCast chemicals were evaluated using the qNPA array. The highest concentration produced AC20 cytotoxicity in mESCs and the remaining were half logarithmic decrements. Cells were harvested on days 4 and 9 of culture. The prediction model correctly identified that pyridaben would disrupt redox signaling in mESCs. Expression of the redox-responsive transcription factors Nrj2, Hifl a and Mtj2 increased upon exposure. Pyridaben showed significant concentration-dependent effects on markers for endoderm, ectoderm and mesoderm differentiation on day 4 of culture (decreased Fgf5, Otx2, and Fg/8 expression and increased Tbx3 expression) without producing a 50% decrease in cardiomyogenesis in the standard assay on day 9. Preliminary data evaluated the redox status (glutathione and glutathione disulfide) of mESCs at 3, 6, 9 and 24 hours following chemical exposure and indicated a unique pattern of redox status for predicted redox disrupting chemicals. These experiments document the importance of using multiple differentiation endpoints and support the linkage of our predictive model to altered differentiation in chemical profiling. This abstract does not reflect EPA policy.