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Quantitative assessment of neurite outgrowth in human embryonic stem-cell derived neurons using automated high-content image analysis
Citation:
Harrill, J., T. Freudenrich, D. Machacek, S. Stice, AND W. R. MUNDY. Quantitative assessment of neurite outgrowth in human embryonic stem-cell derived neurons using automated high-content image analysis. Presented at Society of Toxicology 49th Annual Meeting, Salt Lake City, UT, March 07 - 11, 2010.
Impact/Purpose:
This study characterizes the molecular phenotype of human embryonic stem cell (hESC)-derived neural cells (hN2TM)and uses automated high-content image analysis to measure neurite outgrowth in vitro.
Description:
During development neurons undergo a number of morphological changes including neurite outgrowth from the cell body. Exposure to neurotoxicants that interfere with this process may cause in permanent deficits in nervous system function. While many studies have used rodent primary neural cultures, and human and non-human clonal cell lines to investigate mechanisms regulating neurite outgrowth and examine chemical effects on this process, few have used primary neurons of human origin. This study characterizes the molecular phenotype of human embryonic stem cell (hESC)-derived neural cells (hN2TM)and uses automated high-content image analysis to measure neurite outgrowth in vitro. At 24 h after plating hN2 ™ cells express a number of proteins indicative of a neuronal phenotype, including nestin, ~111-tubulin, microtubule-associated protein 2 and phosphorylated neurofilaments. Neurite outgrowth in hN2™ cells proceeded rapidly, with a majority of cells extending one to three neurites of 40-60 urn in length by 48 h. In addition dose-dependent decreases in neurite outgrowth and cell viability were observed following treatment of hN2 ™ cells with either bisindolylmaleimide 1, UO126, lithium chloride, sodium orthovanadate or brefeldin A, all of which have previously been shown to inhibit neurite outgrowth in rodent primary neural cultures. Overall, the molecular phenotype, rate of neurite outgrowth and sensitivity of hN2™ cells to neurite outgrowth inhibitors were comparable to other in vitro models. hN2™ cells provide a model for assessing chemicals effects on neurite outgrowth in the context of human biology and provide an alternative to the use ofprimary rodent neural cultures or clonal cell lines. This abstract does not necessarily reflect USEPA policy.