Feldman D H, Thinschmidt J S, Peel A L, Papke R L, Reier P J
Department of Neuroscience, College of Medicine, University of Florida Brain Institute, Gainesville 32610, USA.
Exp Neurol. 1996 Aug;140(2):206-17. doi: 10.1006/exnr.1996.0130.
Multipotential progenitor cells grown from central nervous system (CNS) tissues in defined media supplemented with epidermal growth factor (EGF), when attached to a suitable substratum, differentiate to express neural and glial histochemical markers and morphologies. To assess the functional characteristics of such cells, expression of voltage-gated Na+ and K+ currents (INa, IK) was studied by whole-cell patch clamp methods in progenitors raised from postnatal rat forebrain. Undifferentiated cells were acutely dissociated from proliferative "spheres," and differentiated cells were studied 1-25 days after plating spheres onto polylysine/laminin-treated coverslips. INa and IK were detected together in 58%, INa alone in 11%, and IK alone in 19% of differentiated cells recorded with K(+)-containing pipettes. With internal Cs+ (to isolate INa), INa up to 45 pA/pF was observed in some cells within 1 day after plating. I Na ranged up to 150 pA/pF subsequently. Overall, 84% of cells expressed I Na, with an average of 38 pA/pF. INa had fast kinetics, as in neurons, but steadystate inactivation curves were strongly negative, resembling those of glial INa. Inward tail currents sensitive to [K+]out were observed upon repolarization after the 10-ms test pulse with internal Cs+, indicating the expression of K+ channels in 82% of cells. In contrast to the substantial currents observed in differentiating cells, little or no INa or Ik-tail currents were detected in recordings from cells acutely dissociated from spheres. Thus, in the presence of EGF, ionic currents develop early during differentiation induced by attachment to an appropriate substratum. Cells switched from EGF to basic fibroblast growth factor (bFGF) when plated onto coverslips showed greatly reduced proliferation and developed less neuron-like morphologies than cells plated in the presence of EGF. INa was observed in only 53% of bFGF-treated cells, with an average of 9 pA/pF. Thus, in contrast to reports that bFGF promotes neuronal differentiation in some CNS progenitor populations, our EGF-generated postnatal rat CNS progenitors do not develop neuronal characteristics when switched to medium containing bFGF. Thus, differentiated CNS progenitors can express a mix of neuronal and glial molecular, morphological, and electrophysiological properties that can be modified by culture conditions.
在添加表皮生长因子（EGF）的特定培养基中，从中枢神经系统（CNS）组织培养出的多能祖细胞，当附着于合适的基质上时，会分化并表达神经和胶质细胞的组织化学标志物及形态。为评估此类细胞的功能特性，采用全细胞膜片钳方法，对出生后大鼠前脑来源的祖细胞中电压门控性Na⁺和K⁺电流（INa、IK）的表达进行了研究。未分化细胞从增殖的“球体”中急性解离，将球体接种到经聚赖氨酸/层粘连蛋白处理的盖玻片上1 - 25天后，对分化细胞进行研究。用含K⁺的微电极记录时，在58%的分化细胞中同时检测到INa和IK，11%的细胞仅检测到INa，19%的细胞仅检测到IK。使用内部Cs⁺（以分离INa）时，接种后1天内，部分细胞中观察到高达45 pA/pF的INa。随后INa可达150 pA/pF。总体而言，84%的细胞表达INa，平均为38 pA/pF。INa具有快速动力学，如同神经元中的情况，但稳态失活曲线呈强负性，类似于胶质细胞的INa。用内部Cs⁺进行10 ms测试脉冲后复极化时，观察到对胞外[K⁺]敏感的内向尾电流，表明82%的细胞表达K⁺通道。与分化细胞中观察到的大量电流形成对比，从球体中急性解离的细胞记录中几乎未检测到INa或IK尾电流。因此，在EGF存在的情况下，附着于合适基质诱导分化的早期会形成离子电流。接种到盖玻片上后从EGF转换为碱性成纤维细胞生长因子（bFGF）的细胞，其增殖大幅降低，且与在EGF存在下接种的细胞相比，形成的神经元样形态较少。在仅53%的bFGF处理细胞中观察到INa，平均为9 pA/pF。因此，与某些报道称bFGF可促进一些CNS祖细胞群体的神经元分化相反，我们用EGF培养的出生后大鼠CNS祖细胞转换到含bFGF的培养基中时，不会形成神经元特征。因此，分化的CNS祖细胞可表达神经元和胶质细胞的分子、形态及电生理特性的混合特征，且这些特征可被培养条件所改变。
