Department of Physiological Genomics, Institute of Physiology, Ludwig-Maximilians University Munich, Munich, Germany.
Nat Protoc. 2011 Feb;6(2):214-28. doi: 10.1038/nprot.2010.188. Epub 2011 Feb 3.
Instructing glial cells to generate neurons may prove to be a strategy to replace neurons that have degenerated. Here, we describe a robust protocol for the efficient in vitro conversion of postnatal astroglia from the mouse cerebral cortex into functional, synapse-forming neurons. This protocol involves two steps: (i) expansion of astroglial cells (7 d) and (ii) astroglia-to-neuron conversion induced by persistent and strong retroviral expression of Neurog2 (encoding neurogenin-2) or Mash1 (also referred to as achaete-scute complex homolog 1 or Ascl1) and/or distal-less homeobox 2 (Dlx2) for generation of glutamatergic or GABAergic neurons, respectively (7-21 d for different degrees of maturity). Our protocol of astroglia-to-neuron conversion by a single neurogenic transcription factor provides a stringent experimental system to study the specification of a selective neuronal subtype, thus offering an alternative to the use of embryonic or neural stem cells. Moreover, it can be a useful model for studies of lineage conversion from non-neuronal cells, with potential for brain regenerative medicine.
指导神经胶质细胞生成神经元可能被证明是一种替代已经退化的神经元的策略。在这里，我们描述了一种从出生后小鼠大脑皮层中体外高效转化成功能性、形成突触神经元的星形胶质细胞的稳健方案。该方案包括两个步骤：（i）星形胶质细胞的扩增（7 天）和（ii）通过持续和强烈的逆转录病毒表达 Neurog2（编码神经生成素-2）或 Mash1（也称为achaete-scute 复合物同源物 1 或 Ascl1）和/或远侧同源盒 2（Dlx2）诱导的星形胶质细胞到神经元的转化，分别产生谷氨酸能或 GABA 能神经元（不同成熟程度的 7-21 天）。我们的星形胶质细胞到神经元的转化方案由单个神经发生转录因子提供了一个严格的实验系统，用于研究选择性神经元亚型的特化，因此提供了替代胚胎或神经干细胞的方法。此外，它可能是用于研究非神经元细胞谱系转化的有用模型，具有脑再生医学的潜力。
