Characterisation of neurons derived from a cortical human neural stem cell line CTX0E16.
Characterisation of neurons derived from a cortical human neural stem cell line CTX0E16.
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DOI：
10.1186/s13287-015-0136-8
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发表时间：
2015-08-22
影响因子：
7.5
通讯作者：
Srivastava DP
中科院分区：
文献类型：
--
作者：
Anderson GW;Deans PJ;Taylor RD;Raval P;Chen D;Lowder H;Murkerji S;Andreae LC;Williams BP;Srivastava DP
条件永生化人类神经祖细胞 (hNPC) 是研究健康和疾病生理机制的天然神经细胞的强大来源。然而，为了认识这些细胞的效用，确定它们是否保留其起源组织的特征并在分化时产生适当的神经细胞类型至关重要。为此，我们对条件永生化、皮质衍生的人类 NPC 系 CTX0E16 进行了表征，研究了分化神经元的分子和细胞表型，以确定它们是否具有皮质谷氨酸能神经元的特征。通过评估几种神经命运标记物的表达并检查发育中的神经元形态来表征分化的 CTX0E16 细胞。通过 q- 和 RT-PCR 评估神经递质受体、信号蛋白和相关蛋白的表达，并通过 Ca2+ 成像、电生理学和响应神经递质配体应用的 ERK 信号评估进行补充。最后，评估分化神经元形成假定突触和响应活动依赖性刺激的能力。 CTX0E16 hNPC 的分化主要导致表达皮质和谷氨酸能（兴奋性）命运标记的神经元的产生，并具有典型的极化神经元形态。基因表达分析证实分化后皮质蛋白、谷氨酸能蛋白和信号蛋白的表达上调。 CTX0E16 神经元在应用外源神经递质后表现出 Ca2+ 和 ERK1/2 反应，并在 6 周后表现出自发的 Ca2+ 瞬变和与未成熟神经元一致的电生理特性。分化的 CTX0E16 神经元还表达一系列突触前和突触后蛋白质，这些蛋白质沿着远端树突共定位，此外，响应神经元活动的调节而表现出结构可塑性。总而言之，这些发现表明 CTX0E16 hNPC 系是皮质神经元的强大来源，其显示出与谷氨酸能表型一致的功能特性。因此，CTX0E16 神经元可用于研究皮质细胞功能，此外，由于这些神经元表达一系列疾病相关基因，因此它们代表了研究健康和疾病中天然人类细胞的神经发育机制的理想平台。本文的在线版本 (doi:10.1186/s13287-015-0136-8) 包含补充材料，可供授权用户使用。
Conditionally immortalised human neural progenitor cells (hNPCs) represent a robust source of native neural cells to investigate physiological mechanisms in both health and disease. However, in order to recognise the utility of such cells, it is critical to determine whether they retain characteristics of their tissue of origin and generate appropriate neural cell types upon differentiation. To this end, we have characterised the conditionally immortalised, cortically-derived, human NPC line, CTX0E16, investigating the molecular and cellular phenotype of differentiated neurons to determine whether they possess characteristics of cortical glutamatergic neurons. Differentiated CTX0E16 cells were characterised by assessing expression of several neural fates markers, and examination of developing neuronal morphology. Expression of neurotransmitter receptors, signalling proteins and related proteins were assessed by q- and RT-PCR and complemented by Ca2+ imaging, electrophysiology and assessment of ERK signalling in response to neurotransmitter ligand application. Finally, differentiated neurons were assessed for their ability to form putative synapses and to respond to activity-dependent stimulation. Differentiation of CTX0E16 hNPCs predominately resulted in the generation of neurons expressing markers of cortical and glutamatergic (excitatory) fate, and with a typical polarized neuronal morphology. Gene expression analysis confirmed an upregulation in the expression of cortical, glutamatergic and signalling proteins following differentiation. CTX0E16 neurons demonstrated Ca2+ and ERK1/2 responses following exogenous neurotransmitter application, and after 6 weeks displayed spontaneous Ca2+ transients and electrophysiological properties consistent with that of immature neurons. Differentiated CTX0E16 neurons also expressed a range of pre- and post-synaptic proteins that co-localized along distal dendrites, and moreover, displayed structural plasticity in response to modulation of neuronal activity. Taken together, these findings demonstrate that the CTX0E16 hNPC line is a robust source of cortical neurons, which display functional properties consistent with a glutamatergic phenotype. Thus CTX0E16 neurons can be used to study cortical cell function, and furthermore, as these neurons express a range of disease-associated genes, they represent an ideal platform with which to investigate neurodevelopmental mechanisms in native human cells in health and disease. The online version of this article (doi:10.1186/s13287-015-0136-8) contains supplementary material, which is available to authorized users.