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Evaluation of a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay (Keystone Sym)
Citation:
BARRIER, M., S. C. JEFFAY, H. P. NICHOLS, K. J. CHANDLER, M. Hoopes, AND E. S. HUNTER. Evaluation of a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay (Keystone Sym). Presented at Keystone Symposium on Stem Cell Differentiation and Dedifferentiation, Keystone, CO, February 15 - 20, 2010.
Impact/Purpose:
The goal of this research is to establish an in vitro technique using embryonic stem cells that can be used to evaluate the effects of chemical exposure on differentiation and cell proliferation/death. This system would improve throughput and have quanititative markers of early differentiation and cell number.
Description:
Our goal is to establish an in vitro model system to evaluate chemical effects using a single stem cell culture technique that would improve throughput and provide quantitative markers of differentiation and cell number. To this end, we have used an adherent cell differentiation and cytotoxicity (ACDC) technique to evaluate the effects of xenobiotics. Pluripotent J1 mouse embryonic stem cells were plated as a single cell suspension in 96-well plates at 1000 cells/well and cultured in differentiation medium for 24 hours. Cells were then retreated with control or chemical containing media and cultured an additional 8 days. Each well was then assessed for cell number (DRAQ5/Sapphire 700 staining) and cardiomyocyte differentiation (quantitative In-Cell Western analysis for myosin heavy chain protein normalized by cell number). Cells were collected from a duplicate plate for quantitative real-time PCR analysis of gene expression patterns in a panel of markers for stem cells and multiple differentiation endpoints. A comparison of the ACDC technique to the established Embryonic Stem cell Test showed similar responses to a panel of test chemicals with known embryotoxic potential. The ACDC assay is a technique that can be used to evaluate the effects of chemical exposure on differentiation and cell proliferation/death in an embryonic stem cell approach. [This abstract does not necessarily reflect EPA policy.]