Liu Yingfei, Wang Jinzhao, Südhof Thomas C, Wernig Marius
Institute for Stem Cell Biology and Regenerative Medicine, Departments of Pathology and Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, USA.
Shaanxi Provincial People's Hospital, Xi'an, China.
Nat Protoc. 2023 Oct;18(10):2954-2974. doi: 10.1038/s41596-023-00863-2. Epub 2023 Aug 18.
The production of induced neuronal (iN) cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells by the forced expression of proneural transcription factors is rapid, efficient and reproducible. The ability to generate large numbers of human neurons in such a robust manner enables large-scale studies of human neural differentiation and neuropsychiatric diseases. Surprisingly, similar transcription factor-based approaches for converting mouse ESCs into iN cells have been challenging, primarily because of low cell survival. Here, we provide a detailed approach for the efficient and reproducible generation of functional iN cells from mouse ESC cultures by the genetically induced expression of neurogenin-2. The resulting iN cells display mature pre- and postsynaptic specializations and form synaptic networks. Our method provides the basis for studying neuronal development and enables the direct comparison of cellular phenotypes in mouse and human neurons generated in an equivalent way. The procedure requires 14 d and can be carried out by users with expertise in stem cell culture.
通过强制表达神经前体转录因子，从人类胚胎干细胞（ESCs）和诱导多能干细胞中生产诱导神经元（iN）细胞的过程快速、高效且可重复。以这种稳健方式生成大量人类神经元的能力使得能够对人类神经分化和神经精神疾病进行大规模研究。令人惊讶的是，基于类似转录因子的方法将小鼠胚胎干细胞转化为诱导神经元细胞一直具有挑战性，主要原因是细胞存活率低。在这里，我们提供了一种详细方法，通过基因诱导神经生成素-2的表达，从小鼠胚胎干细胞培养物中高效且可重复地生成功能性诱导神经元细胞。所产生的诱导神经元细胞表现出成熟的突触前和突触后特化，并形成突触网络。我们的方法为研究神经元发育提供了基础，并能够直接比较以相同方式生成的小鼠和人类神经元的细胞表型。该过程需要14天，具有干细胞培养专业知识的用户即可进行操作。
