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Development of an invitro technique to use mouse embryonic stem cell in evaluating effects of xenobiotics
Citation:
BARRIER, M., S. C. JEFFAY, H. P. NICHOLS, K. SLENTZ-KESLER, AND E. S. HUNTER. Development of an invitro technique to use mouse embryonic stem cell in evaluating effects of xenobiotics. Presented at NC Society of Toxicology, RTP, NC, February 26, 2009.
Impact/Purpose:
To be presented at the NC SOT Meeting
Description:
Our goal has been to develop a high-throughput, in vitro technique for evaluating the effects of xenobiotics using mouse embryonic stem cells (mESCs). We began with the Embryonic Stem Cell Test (EST), which is used to predict the embryotoxic potential of a test compound by combining the EC50 data from cytotoxicity assays in undifferentiated mESCs and differentiated mouse cells with the ED50 data from a differentiation assay in mESCs. The EST differentiation assay involves the formation of embryoid bodies (EBs), which are multicellular aggregates formed in a suspension culture of mESCs. These EBs must be seeded, transferred to suspension culture, and plated for growth, then the characteristics of the cell outgrowths evaluated for inhibition of differentiation after 10 days of treatment with the test compound. The EST analysis of differentiation involves a qualitative process of visually monitoring the presence of beating cardiomyocytes in each EB, which can be time consuming and subjective. To better quantify the effects of xenobiotics, quantitative analyses of gene and protein markers of stem and differentiated cells have been used. We added an “in-cell western” analysis for a quantitative analysis of a cardiac-specific marker of differentiation, Myosin Heavy Chain (MHC), in the EBs. In addition to a differentiation assay, the EST also requires a cytotoxicity assay being performed in a separate plate with adherent cells. We took the information we learned from the EST and created a higher-throughput combined adherent cell differentiation/cytotoxicity assay (ACDC) using mESCs. We placed mESCs in a 96-well plate and treated them with a test chemical at various concentrations. We then utilized an in-cell western analysis to determine the effects on differentiation and cytotoxicity in the same plate by using an MHC antibody as a cardiac-specific marker of differentiation and a DNA stain set (DRAQ5 and Sapphire 700) as an indicator of cell number. A comparative analysis of both the EST and ACDC assays with a set of test chemicals (Acetic Acid, 5-Fluorouracil, Bromochloroacetic Acid) found comparable effects of these xenobiotics on differentiation and cytotoxicity in these techniques. This abstract does not necessarily reflect U.S. EPA policy.