Postsynaptic structure formation of human iPS cell-derived neurons takes longer than presynaptic formation during neural differentiation in vitro.
Postsynaptic structure formation of human iPS cell-derived neurons takes longer than presynaptic formation during neural differentiation in vitro.
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DOI：
10.1186/s13041-021-00851-1
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发表时间：
2021-10-11
期刊：
影响因子：
3.6
通讯作者：
Kanemura Y
中科院分区：
文献类型：
--
作者：
Togo K;Fukusumi H;Shofuda T;Ohnishi H;Yamazaki H;Hayashi MK;Kawasaki N;Takei N;Nakazawa T;Saito Y;Baba K;Hashimoto H;Sekino Y;Shirao T;Mochizuki H;Kanemura Y
利用人类诱导多能干细胞（hiPSC-神经元）分化的神经元生成成熟突触结构，有望应用于人类细胞突触的生理学研究以及导致突触功能异常的疾病的病理学研究。尽管有报道称，随着神经元的成熟，突触本身从未成熟状态转变为成熟状态，但很少有报道清楚地表明人类干细胞衍生的神经元何时以及如何转变为成熟的突触结构。本研究旨在阐明 hiPSC 神经元的突触形成过程。我们繁殖了 hiPSC 衍生的神经祖细胞 (hiPSC-NPC)，通过双重 SMAD 抑制将腹侧后脑的局部标记表达为神经球，然后在体外将其分化为 hiPSC 神经元。经过 49 天的体外分化后，hiPSC 神经元在转录物和蛋白质水平上显着表达突触前和突触后标记。然而，突触后标记物的表达低于正常人或正常大鼠脑组织，免疫染色分析显示其相对适度且低于突触前标记物，并且其在突触结构中的定位不足。使用微电极阵列的神经生理学分析还表明，分化 49 天时 hiPSC 神经元没有产生突触活动。通过免疫染色对亚型标记物进行分析表明，大多数 hiPSC 神经元表达囊泡谷氨酸转运蛋白 2 (VGLUT2)。胆碱能神经元存活所需的 NGF 的存在与否对其细胞分级没有影响。这些结果表明，在 hiPSC 神经元的突触发生过程中，突触前结构的形成并不是突触后结构形成的唯一要求，并且突触后标记的 mRNA 表达与其成熟结构的形成不相关。从技术上讲，我们还在多中心环境中证实了我们的神经元分化方法具有一定程度的稳健性和重现性，这将有助于未来的研究。对于干细胞衍生的神经元来说，具有成熟突触后结构的突触形成仍然是一个有趣的问题，并且本方法可用于从 hiPSC-NPC 获得早期且质量稳定的神经元培养物。在线版本包含可在 10.1186/s13041-021-00851-1 获取的补充材料。
The generation of mature synaptic structures using neurons differentiated from human-induced pluripotent stem cells (hiPSC-neurons) is expected to be applied to physiological studies of synapses in human cells and to pathological studies of diseases that cause abnormal synaptic function. Although it has been reported that synapses themselves change from an immature to a mature state as neurons mature, there are few reports that clearly show when and how human stem cell-derived neurons change to mature synaptic structures. This study was designed to elucidate the synapse formation process of hiPSC-neurons. We propagated hiPSC-derived neural progenitor cells (hiPSC-NPCs) that expressed localized markers of the ventral hindbrain as neurospheres by dual SMAD inhibition and then differentiated them into hiPSC-neurons in vitro. After 49 days of in vitro differentiation, hiPSC-neurons significantly expressed pre- and postsynaptic markers at both the transcript and protein levels. However, the expression of postsynaptic markers was lower than in normal human or normal rat brain tissues, and immunostaining analysis showed that it was relatively modest and was lower than that of presynaptic markers and that its localization in synaptic structures was insufficient. Neurophysiological analysis using a microelectrode array also revealed that no synaptic activity was generated on hiPSC-neurons at 49 days of differentiation. Analysis of subtype markers by immunostaining revealed that most hiPSC-neurons expressed vesicular glutamate transporter 2 (VGLUT2). The presence or absence of NGF, which is required for the survival of cholinergic neurons, had no effect on their cell fractionation. These results suggest that during the synaptogenesis of hiPSC-neurons, the formation of presynaptic structures is not the only requirement for the formation of postsynaptic structures and that the mRNA expression of postsynaptic markers does not correlate with the formation of their mature structures. Technically, we also confirmed a certain level of robustness and reproducibility of our neuronal differentiation method in a multicenter setting, which will be helpful for future research. Synapse formation with mature postsynaptic structures will remain an interesting issue for stem cell-derived neurons, and the present method can be used to obtain early and stable quality neuronal cultures from hiPSC-NPCs. The online version contains supplementary material available at 10.1186/s13041-021-00851-1.
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影响因子：
2.7
作者：
Fukusumi, Hayato;Handa, Yukako;Kanemura, Yonehiro
通讯作者：
Kanemura, Yonehiro
DOI：
10.1016/0169-328x(93)90154-h
发表时间：
1993-07-01
期刊：
MOLECULAR BRAIN RESEARCH
影响因子：
--
作者：
KOJIMA, N;SHIRAO, T;OBATA, K
通讯作者：
OBATA, K
影响因子：
5.3
作者：
Rocco ML;Soligo M;Manni L;Aloe L
通讯作者：
Aloe L
影响因子：
56.9
作者：
Bellocchio, EE;Reimer, RJ;Edwards, RH
通讯作者：
Edwards, RH
影响因子：
4.2
作者：
Kanemura, Y;Mori, H;Miyake, J
通讯作者：
Miyake, J