Biella G, Di Febo F, Goffredo D, Moiana A, Taglietti V, Conti L, Cattaneo E, Toselli M
Department of Cellular and Molecular Physiological and Pharmacological Sciences, University of Pavia, Via Forlanini 6, I-27100 Pavia, Italy.
Neuroscience. 2007 Oct 12;149(1):38-52. doi: 10.1016/j.neuroscience.2007.07.021. Epub 2007 Jul 20.
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.
最近发现了一群源自小鼠胚胎干细胞（ES）的神经干细胞（命名为NS细胞），它们表现出类似于放射状胶质细胞群体的特征，并且可以在单层中均匀扩增，同时在多次传代后仍保持稳定且具有高度神经源性。这一新群体提供了一个独特的体外系统，用于研究干细胞失去多能性并终末分化时发生的生理事件。在此，我们分析了在体外长期扩增的分化NS细胞兴奋性特性出现的时间、质量和数量。为此，我们研究了电压依赖性Na(+)电流的生物物理特性，作为分化NS细胞向产生完全功能性神经元的神经元成熟阶段的电生理读数，因为神经元电压门控Na(+)通道的表达是神经元分化的一个重要标志，并且对神经系统中的信号传递至关重要。使用膜片钳技术的全细胞和单通道细胞贴附变体，我们发现NS细胞中的Na(+)电流在体外神经元分化过程中表现出显著的电生理变化，主要包括Na(+)电流密度的增加以及稳态激活和失活曲线分别向更负和更正电位的偏移。Na(+)通道系统的变化与分化NS细胞产生动作电位的能力密切相关，因此可以用作经历功能性神经元成熟的ES衍生NS细胞的合适电生理标记。
