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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
范文娟 陈旭东 袁磊 饶淑梅 王福青* 邓锦波*
解剖学报 ›› 2015, Vol. 46 ›› Issue (1) : 6-12.
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
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。
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.
诱导性多能干细胞 / 神经细胞分化 / 突触形成 / 免疫荧光 / 小鼠
Induced pluripotent stem cell / Neural differentiation / Synapse formation / Immunofluorescence / Mouse
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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
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