Quantitative assessment of neurite outgrowth in human embryonic stem cell-derived hN2 cells using automated high-content image analysis.
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 hN2 human embryonic stem cell (hESC)-derived neural cells and uses automated high-content image analysis to measure neurite outgrowth in vitro. At 24h post-plating hN2 cells express a number of protein markers indicative of a neuronal phenotype, including: nestin, beta(III)-tubulin, microtubule-associated protein 2 (MAP2) and phosphorylated neurofilaments. Neurite outgrowth in hN2 cells proceeded rapidly, with a majority of cells extending one to three neurites by 48h in culture. In addition, concentration-dependent decreases in neurite outgrowth and ATP-content were observed following treatment of hN2 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 of hN2 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 chemical effects on neurite outgrowth in a non-transformed human-derived cells and provide an alternative to the use of primary rodent neural cultures or immortalized clonal cell lines.
Harrill
Joshua A
JA
Systems Biology Branch, Integrated Systems Toxicology Division, National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency, Research Triangle Park, NC 27711, United States.
Freudenrich
Theresa M
TM
Machacek
Dave W
DW
Stice
Steven L
SL
Mundy
William R
WR
D000255:Adenosine Triphosphate / Q000378:metabolism; D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000704:Analysis of Variance; D020126:Brefeldin A / Q000494:pharmacology; D002070:Butadienes / Q000494:pharmacology; D002461:Cell Line, Transformed; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007091:Image Processing, Computer-Assisted; D018021:Lithium Chloride / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D016501:Neurites / Q000187:drug effects / Q000473:pathology*; D009474:Neurons / Q000473:pathology*; D009570:Nitriles / Q000494:pharmacology; D013234:Stem Cells / Q000187:drug effects / Q000473:pathology*; D014638:Vanadates / Q000494:pharmacology
D000276:Adjuvants, Immunologic; D002070:Butadienes; D004791:Enzyme Inhibitors; D009419:Nerve Tissue Proteins; D009570:Nitriles; C113580:U 0126; D020126:Brefeldin A; D014638:Vanadates; D000255:Adenosine Triphosphate; D018021:Lithium Chloride