Towards the differentiation of mouse embryonic stem cells into functional neurons for developmental neurotoxicity testing in vitro
2009
Visan,A. | Hayess,K. | Slawik,B. | Sittner,D. | Pohl,E.E. | Spielmann,H. | Luch,A. | Seiler,A.
allemand. Mouse embryonic stem (ES) cells are derived from the inner cell mass of growing blastocysts. These cells are considered pluripotent and capable of differentiating into a variety of endodermal, mesodermal and ectodermal cell types including neurons.This unique feature makes ES cells a favorable tool for studying developmental processes and the pathways affected upon exposure to toxic compounds. In the present study, we have developed a new and efficient protocol for differentiating murine ES cells into neural cells. This method is based on the formation of neuronal spheres and takes only about 12 to 14 days to produce mature neurons. The differentiation process has been well characterized my means of immunofluorescence staining of selected marker proteinsspecific for neuronal precursor cells as well as mature neurons and glial cells. To fully investigate the potential of our method we also determined transmitter phenotypes and performed Ca2+ imaging to confirm that functional neurotransmitter receptors are present. Currently our efforts are focused on ES cell differentiation into neurons on the surface of microelectrode arrays.This approach will be used for extracellular recording of electrical activity patterns in neural networks that have been formed. In comparison to previous protocols our method significantly shortens the differentiation time needed for the development ofmature neurons from ES cells. At the same time, the number of maturing cells is sufficiently high to be applicable in developmental neurotoxicity testing.
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