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EVALUATION OF HUMAN NEURAL PROGENITOR CELLS FOR DEVELOPMENTAL NEUROTOXICITY SCREENING: TIME COURSE OF EFFECTS ON CELL PROLIFERATION AND VIABILITY.
Citation:
BREIER, J. M. AND T. J. SHAFER. EVALUATION OF HUMAN NEURAL PROGENITOR CELLS FOR DEVELOPMENTAL NEUROTOXICITY SCREENING: TIME COURSE OF EFFECTS ON CELL PROLIFERATION AND VIABILITY. Presented at Society of Toxicology, Seattle, WA, March 16 - 20, 2008.
Impact/Purpose:
The goal of the present study was to determine the optimal time for proliferation and cytotoxicity assays.
Description:
Current testing methods for developmental neurotoxicity (DNT) make evaluation of the effects of large numbers of chemicals impractical and prohibitively expensive. As such, we are evaluating human neural progenitor cells (NPCs) as a screen for DNT. ReNcell CX (ReN CX) cells are an immortalized NPC line that differentiates into neurons and glia. The goal of the present study was to determine the optimal time for proliferation and cytotoxicity assays. Cell proliferation (BrdU incorporation) and viability (propidium iodide staining) were assessed in cells treated with a selected set of compounds (1 nM – 100 μM) for 4, 24, or 48 hrs, and each endpoint was quantified using an automated, high content imaging approach. Removal of EGF and FGF-2 from the expansion media, which initiates differentiation, decreased BrdU incorporation 12% (4 hrs), 22% (24 hrs), and 41% (48 hrs) without affecting viability. The anti-proliferatives aphidicolin and hydroxyurea inhibited BrdU incorporation most potently at 4 hrs (IC50aph = 0.21 μM; IC50HU = 23.81 μM) compared to incubation times of 24 or 48 hrs; decreases in viability were also seen at 24 (100 μM) and 48 hrs (1-100 μM). The developmental neurotoxicants methylmercury, trans-retinoic acid, and cadmium potently inhibited only proliferation at 4 hrs, but both proliferation and viability at 24 and 48 hrs, indicating that a 4-hr treatment may be most appropriate to study anti-proliferative effects in the absence of corresponding decreases in viability. Amoxicillin, acetaminophen, diphenhydramine saccharin, sorbitol and glyphosphate (≤ 30 μM) were without effect on proliferation or viability at all treatment times. These data demonstrate the utility of ReN CX cells for screening for proliferation and viability using automated, high-content methods.