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Effects of the EVCAM chemical validation library on differentiation using marker gene expression in lmouse embryonic stem cells
Jeffay, S., M. Barrier, H. Nichols, K. Chandler, A. V. Singh, T. Chandler, T. Knudson, AND E. S. HUNTER. Effects of the EVCAM chemical validation library on differentiation using marker gene expression in lmouse embryonic stem cells. Presented at Teratology Society Annual meeting, San Diego, CA, June 25 - 29, 2011.
The adherent cell differentiation and cytotoxicity (ACDC) assay was used to profile the effects ofthe ECVAM EST validation chemical library (19 compounds) on J1 mouse embryonic stem cells (mESC).
The adherent cell differentiation and cytotoxicity (ACDC) assay was used to profile the effects of the ECVAM EST validation chemical library (19 compounds) on J1 mouse embryonic stem cells (mESC). PCR-based TaqMan Low Density Arrays (TLDA) provided a high-content assessment of alteration in 21 pluripotency, "sternness", trophoblast and differentiation marker genes. Custom Mouse TLDAs were used to analyze the collected samples (3 concentrations per chemical and DMSO vehicle control). For chemicals that affected myosin heavy chain (MHC) protein levels, we chose the 3 lowest concentrations at which the first change in MHC was observed. For chemicals that did not affect MHC levels, samples concentrations were selected to cover the concentrations the first observed change in cell number or to cover the full concentration range tested. Highconcentrations(1OOuM-1OmM)altered patterns of gene expression even for all non/weak toxicants, except for D-camphor (1 and 50J.lM concentrations induced pluripotency, sternness and endodermal markers). For strong developmental toxicants, the effect on marker gene expression did not follow monotonic dose-response relationships. For example, hydroxyurea decreased expression of stem, endoderm and mesoderm markers at 1OJ.lM but increased expression of undifferentiated, stem, germ cell, endoderm and ectoderm marker genes at 100 J.lM, which was generally cytotoxic. Methotrexate decreased expression of endoderm, mesoderm and ectoderm markers at 1nM and but at 50nM increased pluripotency, undifferentiated, stem, germ cell and ectoderm markers. Retinoic acid increased endodermal and ectodermal markers, and decreased mesoderm markers, at 0.1 to 10 nM. These results show that gene expression markers in the J1 ACDC assay provide a sensitive method to characterize changes in marker genes and assess differentiation outcomes following xenobiotic exposure. This abstract does not represent EPA policy.