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Quantitative assessment of neurite outgrowth in human embryonic stem cell derived hN2 cells using automated high-content image analysis
Citation:
HARRILL, J., T. M. FREUDENRICH, D. Machacek, S. Stice, AND W. R. MUNDY. Quantitative assessment of neurite outgrowth in human embryonic stem cell derived hN2 cells using automated high-content image analysis. NEUROTOXICOLOGY. Intox Press, Inc, Little Rock, AR, 31(3):277-290, (2010).
Impact/Purpose:
This manuscript describes the application of human embryonic neuronal progenitor cell line, hN2 and automated dendrite measurement for as a potential system for characterizing developmental neurotoxic compounds. These cells are of particular interest as they may overcome many of the concerns (e.g. species, availability, and phenotype) faced by other models.
Description:
Throughout development neurons undergo a number of morphological changes including neurite outgrowth from the cell body. Exposure to neurotoxic chemicals that interfere with this process may result in permanent deficits in nervous system function. Traditionally, rodent primary neural cultures and immortalized human and non-human clonal cell lines have been used to investigate the molecular mechanisms controlling neurite outgrowth and examine chemical effects on this process. The present study characterizes the molecular phenotype of the hN2™ human embryonic stem cell (hESC)-derived neural cells and uses automated high-content image analysis to measure neurite outgrowth in vitro. At 24 h post-seeding hN2™ cells express a number of protein markers indicative of a neuronal phenotype, including: nestin, Pm-tubulin, microtubule-associated protein 2 (MAP2) and phosphorylated neurofilaments. Neurite outgrowth in hN2™ cells proceeded rapidly, with a majority ofcells extending one to three neurites of~40-60 urn in length by 48 h in culture. In addition dose-dependent decreases in neurite outgrowth and cell viability were observed following treatment ofhN2™ cells with either bisindolylmaleimide I, U0126, lithium chloride, sodium orthovanadate and brefeldin A, all of which have previously been shown to inhibit neurite outgrowth in primary rodent neural cultures. Overall, the molecular phenotype, rate of neurite outgrowth and sensitivity ofhN2™ cells to neurite outgrowth inhibitors were comparable to other in vitro models previously characterized in the literature. hN2™ cells provide a model in which to investigate chemicals effects on neurite outgrowth in the context of human biology and provide an alternative to the use ofprimary rodent neural cultures or immortalized clonal cell lines.
