Elsevier

Neuroscience

Volume 149, Issue 1, 12 October 2007, Pages 38-52
Neuroscience

Cellular neuroscience
Differentiating embryonic stem–derived neural stem cells show a maturation-dependent pattern of voltage-gated sodium current expression and graded action potentials

https://doi.org/10.1016/j.neuroscience.2007.07.021Get rights and content

Abstract

A population of mouse embryonic stem (ES)–derived neural stem cells (named NS cells) that exhibits traits reminiscent of radial glia-like cell population and that can be homogeneously expanded in monolayer while remaining stable and highly neurogenic over multiple passages has been recently discovered. This novel population has provided a unique in vitro system in which to investigate physiological events occurring as stem cells lose multipotency and terminally differentiate. Here we analysed the timing, quality and quantity of the appearance of the excitability properties of differentiating NS cells which have been long-term expanded in vitro. To this end, we studied the biophysical properties of voltage-dependent Na+ currents as an electrophysiological readout for neuronal maturation stages of differentiating NS cells toward the generation of fully functional neurons, since the expression of neuronal voltage-gated Na+ channels is an essential hallmark of neuronal differentiation and crucial for signal transmission in the nervous system. Using the whole cell and single-channel cell-attached variations of the patch-clamp technique we found that the Na+ currents in NS cells showed substantial electrophysiological changes during in vitro neuronal differentiation, consisting mainly in an increase of Na+ current density and in a shift of the steady-state activation and inactivation curves toward more negative and more positive potentials respectively. The changes in the Na+ channel system were closely related with the ability of differentiating NS cells to generate action potentials, and could therefore be exploited as an appropriate electrophysiological marker of ES-derived NS cells undergoing functional neuronal maturation.

Section snippets

ES-derived NS cell culture and differentiation

The ES-derived NS cells (LC1 cell line) were established from 46C ES cell line and normally grown as previously described (Conti et al., 2005). Briefly, established NS cells were routinely grown in Euromed-N medium (Euroclone, Celbio, Pero, Italy) supplemented with N2 mix (1%, GIBCO, Invitrogen, San Giuliano Milanese, Italy) and epidermal growth factor (EGF) and fibroblast growth factor (FGF-2) (10 ng/mL each; Peprotech, Tebu-Bio, D.B.A.Italia s.r.l., Segrate, Italy). They were passaged using

Differentiating ES-derived NS cells develop Na+ currents

In order to assess the in vitro differentiation of the NS cells into mature neurons, we first examined the time-dependent appearance of TTX-sensitive Na+ channels by means of immunocytochemical analyses. Analysis of the distribution of Na+ channel proteins in the membranes of NS cells under self-renewal conditions and after 2 (Fig. 1A), 4 (Fig. 1B) and 21 days (Fig. 1C) of exposure to neuronal differentiation indicated a gradual increase in the immunoreactive signal in the cultures. The Na+

Discussion

The intention of this study was to gain deeper understanding about the functional in vitro maturation of neurons derived from differentiating NS cells, a novel population of homogeneous neurogenic NS cells (Conti et al., 2005). We focused on the definition of specific electrophysiological properties, like voltage-activated Na+ currents and underlying regenerative electrical activity, that can be used as readouts of the maturation process. Overall, the results indicate that significant changes

Conclusion

In conclusion, the expression of neuron-like Na+ currents in in vitro differentiated NS cells, and the ability of a fraction of these cells to generate overshooting action potentials, can be considered as strong evidence supporting the belief that, when exposed to appropriate neuronal differentiating conditions, NS cells become electrophysiologically active neurons and acquire the typical properties of maturing neuronal cells. Thanks to the homogeneity and stability of NS cell system, we have

Acknowledgments

This work was supported by grants from the Italian Ministry for University and Research (MIUR) to V.T. (PRIN 2005 No. 2005051740_002) and L.C. (PRIN 2005 No. 2005051740_001), and by Eurostemcell (FP6, European Union) and FIRB (Fondo Incentivazione Ricerca di Base, MIUR) to E.C.

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