Kajihara Ryutaro, Numakawa Tadahiro, Era Takumi
Department of Cell Modulation, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan.
Department of Biomedical Laboratory Sciences, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.
Bio Protoc. 2021 Feb 5;11(3):e3914. doi: 10.21769/BioProtoc.3914.
Human induced pluripotent stem cells (iPSCs) and their progeny displaying tissue-specific characteristics have paved the way for regenerative medicine and research in various fields such as the elucidation of the pathological mechanism of diseases and the discovery of drug candidates. iPSC-derived neurons are particularly valuable as it is difficult to analyze neural cells obtained from the central nervous system in humans. For neuronal induction with iPSCs, one of the commonly used approaches is the isolation and expansion of neural rosettes, following the formation of embryonic bodies (EBs). However, this process is laborious, inefficient, and requires further purification of the cells. To overcome these limitations, we have developed an efficient neural induction method that allows for the generation of neural stem/progenitor cells (NSCs/NPCs) from iPSCs within 7 days and of functional mature neurons. Our method yields a PAX6-positive homogeneous cell population, a cortical NSCs/NPCs, and the resultant NSCs/NPCs can be cryopreserved, expanded, and differentiated into functional mature neurons. Moreover, our protocol will be less expensive than other methods since the protocol requires fewer neural supplements during neural induction. This article also presents the FM1-43 imaging assay, which is useful for the presynaptic assessment of the iPSCs-derived human neurons. This protocol provides a quick and simplified way to generate NSCs/NPCs and neurons, enabling researchers to establish cellular models to study brain disease pathology.
人诱导多能干细胞（iPSC）及其具有组织特异性特征的后代为再生医学以及疾病病理机制阐释和候选药物发现等各个领域的研究铺平了道路。iPSC来源的神经元尤为珍贵，因为从人类中枢神经系统获取的神经细胞难以进行分析。对于用iPSC诱导神经元，常用方法之一是在形成胚状体（EB）后分离并扩增神经玫瑰花结。然而，这个过程费力、低效，并且需要对细胞进行进一步纯化。为克服这些局限性，我们开发了一种高效的神经诱导方法，该方法能在7天内从iPSC生成神经干细胞/祖细胞（NSC/NPC）以及功能性成熟神经元。我们的方法产生一个PAX6阳性的同质细胞群体，即皮质NSC/NPC，并且由此产生的NSC/NPC可以冷冻保存、扩增，并分化为功能性成熟神经元。此外，我们的方案成本会低于其他方法，因为该方案在神经诱导过程中所需的神经补充物更少。本文还介绍了FM1-43成像检测法，该方法对于iPSC来源的人类神经元的突触前评估很有用。此方案提供了一种快速且简化的生成NSC/NPC和神经元的方法，使研究人员能够建立细胞模型来研究脑部疾病病理学。
