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Evaluation of a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay (SOT)
BARRIER, M., S. C. JEFFAY, H. P. NICHOLS, M. L. HOOPES, AND E. S. HUNTER. Evaluation of a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay (SOT). Presented at Society of Toxicology 49th Annual meeting, Salt Lake City, UT, March 07 - 11, 2010.
Our research goal is to establish a model system that would evaluate chemical effects using a single stem cell culture technique to improve throughput and have a quantitative marker of differentiation and cell number.
The Embryonic Stem Cell Test (EST) has been used to evaluate the effects of xenobiotics using three endpoints, stem cell differentiation, stem cell viability and 3T3-cell viability. Our research goal is to establish amodel system that would evaluate chemical effects using a single stem cell culture technique to improve throughput and have a quantitative marker of differentiation and cell number. To this end, we have used an adherent culture differentiation-cytotoxicity (ACDC) technique to evaluate the effects of xenobiotics. J1-mouse pluripotent embryonic stem cells (l X 10""3 cells/well) were plated in 96-well plates as a single cell suspension and cultured in differentiation medium for 24 hours. After the 24-hour attachment period, medium was replaced with control, vehicle or chemical containing media to complete a 9-day culture. At the end of culture, cell number (DRAQ5/Sapphire 700 staining) and cardiomyocyte differentiation (quantitative in-cell Western analysis for myosin heavy chain divided by the number of cells in the well) were assessed in each well. The effects of acetic acid (no effects at concentrations < 10mM) and 5-flurouraci1 (similar effects on both endpoints; ACDC Cell number reduction (EC50) 0.85 uM, ACDC differentiation (ED50) -0.49 uM) were evaluated in both assay systems. Bromochloroacetic acid produced effect on differentiation (ED50 = 135IlM) at a lower concentration than required to produced effects on cell number (EC50 = 3341lM) in the ACDC assay. In the EST, BCA produced effects on beating hearts at a higher concentration (Ebeating heart50 = 5111lM) than required to produced cytotoxicity (EC50 = 106IlM). Differences in the method ofcardiomyocyte analysis(presence ofbeating heartcells/plate-ESTcomparedtoanassessment ofcardiomyocytes/cellACDC), stem cell line (D3-EST compared to J1-ACDC), and method of differentiation (hanging drop/EB -EST compared to adherent culture-ACDC) may be important in comparing assay results. The ACDC assay is a technique that can be used to evaluate the effects of chemical exposure on differentiation and cell proliferation/death in embryonic stem cell approach. [This work is approved by EPA but does not necessarily reflect official Agency policy].