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RODENT AND HUMAN NEUROPROGENITOR CELLS FOR HIGH-CONTENT SCREENS OF CHEMICAL EFFECTS ON PROLIFERATION AND APOPTOSIS
Culbreth, M., T. Freudenrich, B. ROBINETTE, J. HARRILL, W. R. MUNDY, AND T. J. SHAFER. RODENT AND HUMAN NEUROPROGENITOR CELLS FOR HIGH-CONTENT SCREENS OF CHEMICAL EFFECTS ON PROLIFERATION AND APOPTOSIS. Presented at Society of Toxicology 49th Annual meeting, Salt Lake City, UT, March 07 - 11, 2010.
Effects of 4 chemicals on proliferation and apoptosis in mouse cortical neural stem cells (mCNS) and ReNcell CX Cells (ReN), an immortalized human cortical neuroprogenitor cell line, were examined using automated high content image analysis.
The objective of these experiments is to develop high-throughput screens for proliferation and apoptosis in order to compare rodent and human neuroprogenitor cell responses to potential developmental neurotoxicants. Effects of 4 chemicals on proliferation and apoptosis in mouse cortical neural stem cells (mCNS) and ReNcell CX Cells (ReN), an immortalized human cortical neuroprogenitor cell line, were examined using automated high content image analysis. Expression of neuroprogenitor cell markers Nestin and Sox2 were confirmed in proliferating cultures of both cell types. Cells were plated in 96-well plates and exposed (24 hrs) to 4 chemicals with known actions on proliferation and apoptosis; aphidicolin, staurosporine, etoposide, and actinomycin D. Both endpoints were immunohistochemically assessed with antibodies to BrdU in replicating DNA, activated p53 and activated Caspase 3. In both cell types, aphidicolin (1,3, 10pM) decreased BrdU intensity to 40-60% control and increased p53 and Caspase 3 intensity to < 200% control. Staurosporine (0.03, 0.1, 0.3 pM) decreased BrdU intensity to 35% control for mCNS and ReN, but increased p53 intensity 200-400% control in both cell types. Caspase 3 intensity decreased to 50% control in mCNS, but not ReN cells. Staurosporine also decreased the number of cells/well. In both cell types, etoposide (0.001,0.01,0.1 pM) decreased BrdU and Caspase 3 to -75% and -80% control, respectively but not p53 intensity. Actinomycin D, (0.001, 0.01, 0.1 pM) decreased BrdU intensity to 30% control in meNS but not ReN cells. By contrast, p53 and Caspase 3 increased to 400-800% and 400-1000% control, respectively in both cell types. These results demonstrate aphidicolin and actinomycin are positive controls for proliferation and apoptosis, and that this high-content assay for proliferation and apoptosis can be used in both human and mouse neuroprogenitor cells. (Supported by the SOT-Colgate-Palmolive Alternative Grant Award. This abstract does not reflect Agency Policy).