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Evaluation of a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay (SOT)
Citation:
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.
Impact/Purpose:
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.
Description:
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].