Neural Stem Cells and Neuroimaging Group, Department of Neurobiology, Hellenic Pasteur Institute, Athens, Greece.
Methods Mol Biol. 2020;2155:41-61. doi: 10.1007/978-1-0716-0655-1_4.
Direct neuronal reprogramming, rewiring the epigenetic and transcriptional network of a differentiated cell type to neuron, apart from being a very promising approach for the treatment of brain injury and neurodegeneration, also offers a prime opportunity to investigate the molecular underpinnings of neuronal cell fate determination, as the precise molecular mechanisms that establish neuronal fate and diversity at the transcriptional and epigenetic level are incompletely understood. Recent studies from a number of groups, including ours, have shown that astrocytes can be directly reprogrammed into functional neurons in vitro and in vivo following ectopic overexpression of combinations of transcription factors, neurogenic proteins, miRNAs, and small chemical molecules.In this chapter we describe the protocols for in vitro converting primary cortical astrocytes of mouse and human origin to induced neurons, through forced expression of two neurogenic molecules, either each one alone or in combination: the master regulatory bHLH proneural transcription factor NEUROGENIN-2 (NEUROG2) and the neurogenic protein CEND1. Forced expression of each one of the two neurogenic proteins in primary astrocytes via retroviral gene transfer results in their direct conversion to subtype-specific induced neurons, while simultaneous coexpression of both molecules drives them predominantly toward acquisition of a neural precursor cell (NPC) state. Although mouse and human astrocytes exhibit differences in their reprogramming rate and particular characteristics, they can both get efficiently in vitro transdifferentiated to NPCs and induced neurons upon NEUROG2 or/and CEND1 forced expression using the reprogramming protocols described in the chapter, presenting valuable cellular platforms for mechanistic studies and in vivo applications to restore neurodegeneration.
直接神经元重编程，将分化细胞类型的表观遗传和转录网络重编为神经元，除了是治疗脑损伤和神经退行性变的极有前途的方法之外，还为研究神经元细胞命运决定的分子基础提供了绝佳机会，因为建立神经元命运和转录水平上的多样性的确切分子机制尚未完全理解。最近包括我们在内的多个研究小组的研究表明，在外源性过表达转录因子、神经发生蛋白、miRNA 和小化学分子的组合后，星形胶质细胞可以在体外和体内直接重编程为功能性神经元。在本章中，我们描述了通过强制表达两种神经发生分子（单独或组合）将原代皮质星形胶质细胞（源自小鼠和人类）体外转化为诱导神经元的协议：主要调节性碱性螺旋-环-螺旋（bHLH）神经前体细胞转录因子神经生成素-2（NEUROG2）和神经发生蛋白 CEND1。通过逆转录病毒基因转移在原代星形胶质细胞中强制表达这两种神经发生蛋白中的每一种，都会导致其直接转化为特定亚型的诱导神经元，而同时共表达这两种分子则主要促使它们获得神经前体细胞（NPC）状态。尽管小鼠和人类星形胶质细胞在其重编程率和特定特征方面存在差异，但它们都可以使用本章中描述的重编程方案，通过强制表达 NEUROG2 或/和 CEND1 有效地在体外向 NPC 和诱导神经元转化，为机制研究和体内应用提供了有价值的细胞平台，以恢复神经退行性变。
