小鼠诱导性多能干细胞的神经细胞分化与突触连接的建立及其功能分析

doi:10.16098/j.issn.0529-1356.2015.01.002

解剖学报 ›› 2015, Vol. 46 ›› Issue (1): 6-12.doi: 10.16098/j.issn.0529-1356.2015.01.002

小鼠诱导性多能干细胞的神经细胞分化与突触连接的建立及其功能分析

范文娟¹ 陈旭东¹ 袁磊¹ 饶淑梅¹ 王福青^1* 邓锦波^2*

1.漯河医学高等专科学校分子医学实验室，河南漯河 462002;2.河南大学神经生物学研究所，河南开封 475400

收稿日期:2014-04-30 修回日期:2014-07-15 出版日期:2015-02-06 发布日期:2015-02-06
通讯作者: 王福青,邓锦波 E-mail:fwj81@163.com
基金资助:
诱导多能干细胞促小脑平行纤维损伤后再生及突触重建的实验性研究;诱导多能干细胞脑内移植向功能性神经细胞分化的实验性研究;诱导多能干细胞移植对宫内缺氧新生鼠脑损伤后功能恢复的实验研究

Neural differentiation and synapse formation in mouse induced pluripotent stem cells

FAN Wen-juan¹ CHEN Xu-dong¹YUAN Lei¹RAO Shu-mei¹ WANG Fu-qing ^1* DENG Jin-bo^2*

1. Laboratory of Molecular Medicine, Luohe Medical College, He’nan Luohe 462002, China;2. Institute of Neurobiology, He’nan University, He’nan Kaifeng 475004, China

Received:2014-04-30 Revised:2014-07-15 Online:2015-02-06 Published:2015-02-06
Contact: WANG Fu-qing,DENG Jin-bo E-mail:fwj81@163.com

摘要/Abstract

摘要：

目的探讨小鼠诱导性多能干细胞（iPSCs）在体外某些因素的诱导下能否分化成功能性的神经细胞，以及神经细胞之间突触的发生与建立。
方法首先将iPSCs悬浮培养形成拟胚体，利用维甲酸(RA)将其诱导分化成神经前体细胞，最后撤去RA贴壁培养，利用免疫荧光染色技术观察小鼠iPSCs在体外分化成神经细胞以及神经细胞之间突触发生的形态特征，利用FM1-43染色技术分析突触末端的功能活性。结果小鼠iPSCs能够在RA的诱导下向神经细胞分化，这些神经细胞可以被成熟神经元与胶质细胞标记物标记，新分化的神经元还可以观察到树突棘及突触连接的形成，在去极化刺激下突触活动增强，表现为轴突末端大量FM1-43阳性内吞颗粒。结论由小鼠iPSCs分化而来的神经细胞在体外形成突触连接的过程，提示其可以进一步分化为功能性的神经元和神经胶质细胞。

关键词: 诱导性多能干细胞, 神经细胞分化, 突触形成, 免疫荧光, 小鼠

Abstract:

Objective To observe whether mouse induced pluripotent stem cells (iPSCs) efficiently can differentiate to functional neurons and formation synapse in vitro. Methods Mouse iPSCs were pre-differentiated into neural stem cells by using retinoic acid (RA) after embryoid body (EB) formation. After RA removed, immunofluorescence staining was used to study the synaptogenesis between neurons，and FM1-43 staining was used to show synaptic terminal with functional activity. Results Neural precursors matured faster, differentiated to functional neurons that stained positively for mature neuronal and glial markers under adherent culture, and iPSCs-derived neurons formed dendritic spines and synaptic connections by morphological analyses. Under depolarization, the activity of synapsis was enhanced and a large number of FM1-43 endocytosis particles were in axon terminal. Conclusion Our results reveal that the processes involved in the formation of synapses in mouse iPSCs differentiate into functional neurons and glia, which may have important implications for neurodevelopmental studies, safety pharmacological studies, and autologous cell transplantation.

Key words: Induced pluripotent stem cell, Neural differentiation, Synapse formation, Immunofluorescence, Mouse

范文娟陈旭东袁磊饶淑梅王福青* 邓锦波*. 小鼠诱导性多能干细胞的神经细胞分化与突触连接的建立及其功能分析[J]. 解剖学报, 2015, 46(1): 6-12.

FAN Wen-juan CHEN Xu-dong YUAN Lei RAO Shu-mei WANG Fu-qing* DENG Jin-bo*. Neural differentiation and synapse formation in mouse induced pluripotent stem cells[J]. AAS, 2015, 46(1): 6-12.

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小鼠诱导性多能干细胞的神经细胞分化与突触连接的建立及其功能分析

Neural differentiation and synapse formation in mouse induced pluripotent stem cells

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