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Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay
BARRIER, M., S. C. JEFFAY, H. P. NICHOLS, K. J. CHANDLER, M. Hoopes, K. Slentz-Kesler, AND E. S. HUNTER. Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay. REPRODUCTIVE TOXICOLOGY. Elsevier Science Ltd, New York, NY, 31(4):383-391, (2011).
This assay was developed to build on existing mouse embryonic stem cell models and create an improved higher-throughput model system that evaluates chemical-induced effects on both stem cell viability and differentiation and facilitates analysis of associated toxicity pathways.
The Embryonic Stem Cell Test (EST) is an assay which evaluates xenobiotic-induced effects using three endpoints: mouse embryonic stem cell (mESC) differentiation, mESC viability, and 3T3-cell viability. Our research goal was to develop an improved high-throughput assay by establishing a model system that evaluates chemical-induced effects on both stem cell viability and differentiation using a stem cell culture technique. This model system would have quantitative markers of differentiation and cell number, and would improve efficiency of chemical assessments as compared to the EST. To this end, we developed an adherent cell differentiation and cytotoxicity (ACDC) assay. Pluripotent Jl mESCs were plated in multi-well plates as a single cell suspension and cultured in differentiation medium for 24 hours. Following this attachment period, mESCs were cultured with control, vehicle or chemical containing media. At the end ofthe 9-day culture, cell number and cardiomyocyte differentiation are determined in each well. Cardiomyocyte differentiation is quantified using In-Cell Western analysis for a,B myosin heavy chain (MHC) protein. Time course evaluations of cell number and MHC expression were used to characterize the system and determine an appropriate day to evaluate the effects of chemical exposure. TaqMan® Mouse Stem Cell Pluripotency Arrays were used to characterize the expression of 96 genes including biomarkers of pluripotency maintenance, sternness and differentiation on mESC samples across 12 days of differentiation culture. When J1 mESCs were maintained in pluripotent culture conditions for a total of 27 passages (67 days), they continued to retain a normal 33 karyotype as well as the potential to differentiate into cardiomyocytes. Acetic acid, 5-fluorouracil and bromochloroacetic acid were evaluated with both the EST and ACDC assay systems. Both systems were able to distinguish the relative potency of these compounds, while the ACDC assay did so with a technique more amenable to higher-throughput processing. In summary, the ACDC assay is a technique that can be used to evaluate the effects of chemical exposure on mouse embryonic stem cell differentiation and cell number.