Neural differentiation of mouse embryonic stem cells as a tool to assess developmental neurotoxicity in vitro

► Method for fast and efficient differentiation of mouse stem cells into neural cells. ► Neural cells express structural proteins associated with postsynaptic sites and presynaptic vesicles, as well as neurotransmitters. ► Agonists of glutamate and dopamine receptors resulted in a marked increase in...

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Published in:	Neurotoxicology (Park Forest South) 2012-10, Vol.33 (5), p.1135-1146
Main Authors:	Visan, Anke, Hayess, Katrin, Sittner, Dana, Pohl, Elena E., Riebeling, Christian, Slawik, Birgitta, Gulich, Konrad, Oelgeschläger, Michael, Luch, Andreas, Seiler, Andrea E.M.
Format:	Article
Language:	eng
Subjects:	2',3'-Cyclic-Nucleotide Phosphodiesterases - metabolism alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid - pharmacology Animals Astrocytes - drug effects Astrocytes - metabolism Biological and medical sciences Calcium - metabolism Cell Differentiation - drug effects Cell Differentiation - physiology Cell Line Cell Proliferation Cell Survival Chlorpyrifos - toxicity Developmental neurotoxicity Disks Large Homolog 4 Protein Dopamine - pharmacology Embryonic stem cells Embryonic Stem Cells - drug effects Embryonic Stem Cells - physiology Flow Cytometry Glial Fibrillary Acidic Protein - metabolism Guanylate Kinases - metabolism In vitro developmental neurotoxicity testing Medical sciences Membrane Proteins - metabolism Methylmercury Compounds - toxicity Mice Microtubule-Associated Proteins - metabolism N-Methylaspartate - pharmacology Neurons - drug effects Neurons - metabolism Oligodendroglia - drug effects Oligodendroglia - metabolism Organometallic Compounds - toxicity Sodium Glutamate - toxicity Time Factors Toxicology Tubulin - metabolism Tyrosine 3-Monooxygenase - metabolism
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Summary:	► Method for fast and efficient differentiation of mouse stem cells into neural cells. ► Neural cells express structural proteins associated with postsynaptic sites and presynaptic vesicles, as well as neurotransmitters. ► Agonists of glutamate and dopamine receptors resulted in a marked increase in intracellular Ca2+ levels. ► In vitro system to detect adverse effects of neurodevelopmental toxicants. Mouse embryonic stem cells (mESCs) represent an attractive cellular system for in vitro studies in developmental biology as well as toxicology because of their potential to differentiate into all fetal cell lineages. The present study aims to establish an in vitro system for developmental neurotoxicity testing employing mESCs. We developed a robust and reproducible protocol for fast and efficient differentiation of the mESC line D3 into neural cells, optimized with regard to chemical testing. Morphological examination and immunocytochemical staining confirmed the presence of different neural cell types, including neural progenitors, neurons, astrocytes, oligodendrocytes, and radial glial cells. Neurons derived from D3 cells expressed the synaptic proteins PSD95 and synaptophysin, and the neurotransmitters serotonin and γ-aminobutyric acid. Calcium ion imaging revealed the presence of functionally active glutamate and dopamine receptors. In addition, flow cytometry analysis of the neuron-specific marker protein MAP2 on day 12 after induction of differentiation demonstrated a concentration dependent effect of the neurodevelopmental toxicants methylmercury chloride, chlorpyrifos, and lead acetate on neuronal differentiation. The current study shows that D3 mESCs differentiate efficiently into neural cells involving a neurosphere-like state and that this system is suitable to detect adverse effects of neurodevelopmental toxicants. Therefore, we propose that the protocol for differentiation of mESCs into neural cells described here could constitute one component of an in vitro testing strategy for developmental neurotoxicity.
ISSN:	0161-813X 1872-9711