构建具有突触传递潜能的iPSC源性抑制性神经网络组织

彭历芝; 位庆帅; 马瑗锾; 许金海; 蒋斌; 曾园山; 曾湘; 丁英

doi:10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).20221220.001

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构建具有突触传递潜能的iPSC源性抑制性神经网络组织

基础研究 | 更新时间：2024-02-19

- 构建具有突触传递潜能的iPSC源性抑制性神经网络组织
- Construction of iPSC-derived Inhibitory Neural Network Tissue with Synaptic Transmission Potentials
- 中山大学学报（医学科学版） 2023年44卷第1期页码：18-25
- 作者机构：
  
  1.中山大学中山医学院组织胚胎学与细胞生物学系，广东广州510080
  2.中山大学中山医学院广东省脑功能与脑疾病研究重点实验室，广东广州510080
- 作者简介：
  
  彭历芝，硕士生，研究方向：iPSC源性抑制性神经网络构建及其治疗脊髓损伤的研究，E-mail：penglizhi1997@163.com
- 基金信息：
  
  国家自然科学基金(81891003;81891002)
- DOI：10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).20221220.001
  中图分类号： R318
- 纸质出版日期：2023-01-20，
  
  收稿日期：2022-10-26，
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彭历芝,位庆帅,马瑗锾等.构建具有突触传递潜能的iPSC源性抑制性神经网络组织[J].中山大学学报（医学科学版）,2023,44(01):18-25.

PENG Li-zhi,WEI Qing-shuai,MA Yuan-huan,et al.Construction of iPSC-derived Inhibitory Neural Network Tissue with Synaptic Transmission Potentials[J].Journal of Sun Yat-sen University(Medical Sciences),2023,44(01):18-25.
彭历芝,位庆帅,马瑗锾等.构建具有突触传递潜能的iPSC源性抑制性神经网络组织[J].中山大学学报（医学科学版）,2023,44(01):18-25. DOI： 10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).20221220.001.

PENG Li-zhi,WEI Qing-shuai,MA Yuan-huan,et al.Construction of iPSC-derived Inhibitory Neural Network Tissue with Synaptic Transmission Potentials[J].Journal of Sun Yat-sen University(Medical Sciences),2023,44(01):18-25. DOI： 10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).20221220.001.

摘要

目的

利用人诱导多能干细胞（hiPSCs）定向分化为γ-氨基丁酸能（GABA）神经前体细胞后种植到脱细胞视神经（DON）材料内，以构建出具有突触形成潜能的hiPSC源性抑制性神经网络组织。为研究与治疗修复中枢神经系统损伤提供一种新的组织工程产品。

方法

采用分步定向诱导和组织工程构建技术相结合的方法。将hiPSCs体外定向诱导为GABA能神经前体细胞（hNPCs）后，种植于DON材料上三维培养，在特定神经元诱导环境下，将其进一步分化为GABA能神经元。应用透射电镜和全细胞膜片钳技术分别检测hiPSCs分化的神经元之间能否形成类突触结构，以及这些神经元是否具有自发性抑制性突触后电流。从结构与功能角度证实这些hiPSCs分化的神经元可形成具有突触传递潜能的抑制性神经网络组织。

结果

在体外将hiPSCs成功诱导出GABA能表型的抑制性神经元，且其培养28 d仍保持良好活力。透射电镜下，观察到三维材料上hiPSC源性神经细胞突起之间形成许多的细胞连接，其中一些为类突触样结构，表现为：一侧细胞突起的细胞膜稍增厚并且其内侧面胞质含有少量囊泡，形成类突触前成分的结构；其对侧为另一个细胞突起的细胞膜局部增厚，形成类突触后膜的结构。全细胞膜片钳检测可记录到已分化的hiPSC源性神经细胞具有产生动作电位和自发性抑制性突触后电流的能力。

结论

本研究结果表明，在体外诱导hiPSCs定向分化为GABA能神经前体细胞后种植到DON材料内三维培养，可成功构建成具有突触传递潜能的hiPSC源性抑制性神经网络组织，这将为后期研究与治疗中枢神经系统损伤提供一种新型干细胞组织工程来源的神经组织。

Abstract

Objective

Directed differentiation of human induced pluripotent stem cells （hiPSCs） into spinal cord γ-aminobutyric acid （GABA）-ergic progenitor cells were implanted into an decellularized optical nerve （DON） bioscaffold to construct a hiPSC-derived inhibitory neural network tissue with synaptic activities. This study aimed to provide a novel stem cell-based tissue engineering product for the study and the repair of central nervous system injury.

Methods

The combination of stepwise directional induction and tissue engineering technology was applied in this study. After hiPSCs were directionally induced into human neural progenitor cells （hNPCs） in vitro， they were seeded into a DON for three-dimensional culture， allowing further differentiation into inhibitory GABAergic neurons under the specific neuronal induction environment. Transmission electron microscopy and whole cell patch clamp technique were used to detect whether the hiPSCs differentiated neurons could form synapse-like structures and whether these neurons had spontaneous inhibitory postsynaptic currents， respectively， in order to validate that the hiPSC-derived neurons would form neural networks with synaptic transmission potentials from a structural and functional perspective.

Results

The inhibitory neurons of GABAergic phenotype were successfully induced from hiPSCs in vitro， and maintained good viability after 28 days of culture. With the transmission electron microscopy， it was observed that many cell junctions were formed between hiPSC-derived neural cells in the three-dimensional materials， some of which presented a synapse- like structure， manifested as the slight thickness of cell membrane and a small number of vesicles within one side of the cell junctions， the typical structure of a presynatic component， and focal thickness of the membrane of the other side of the cell junctions， a typical structure of a postsynaptic component. According to whole-cell patch-clamp recording， the hiPSC-derived neurons had the capability to generate action potentials and spontaneous inhibitory postsynaptic currents were recorded in this biotissue.

Conclusions

The results of this study indicated that hiPSCs can be induced to differentiate into GABAergic progenitor cells in vitro and can successfully construct iPSC-derived inhibitory neural network tissue with synaptic transmission after implanted into a DON for three-dimensional culture. This study would provide a novel neural network tissue for future research and treatment of central nervous system injury by stem cell tissue engineering technology.

关键词

人诱导多能干细胞γ-氨基丁酸能神经元神经网络组织

Keywords

human induced pluripotent stem cells （hiPSCs）γ- Aminobutyric acid neuronsneural network tissue

references

Takahashi K， Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors［J］. Cell， 2006， 126（4）： 663-676.

Petit I， Kesner NS， Karry R， et al. Induced pluripotent stem cells from hair follicles as a cellular model for neurodevelopmental disorders［J］. Stem Cell Res， 2012， 8（1）： 134-140.

Yamanaka S. Pluripotent stem cell-based cell therapy- promise and challenges［J］. Cell Stem Cell， 2020， 27（4）： 523-531.

Zeng X， Qiu XC， Ma YH， et al. Integration of donor mesenchymal stem cell-derived neuron-like cells into host neural network after rat spinal cord transection［J］. Biomaterials， 2015， 53： 184-201.

Sun J-H， Li G， Wu T-T， et al. Decellularization optimizes the inhibitory microenvironment of the optic nerve to support neurite growth［J］. Biomaterials， 2020， 258： 120289.

Zhu H， Sun T， Wang Y， et al. Directed differentiation of porcine induced pluripotent stem cells into forebrain GABAergic neuron progenitors［J］. J South Med Univ， 2021， 41（6）： 820-827.

Gong C， Zheng X， Guo F， et al. Human spinal GABA neurons alleviate spasticity and improve locomotion in rats with spinal cord injury［J］. Cell Rep， 2021， 34（12）： 108889.

Treiman DM. GABAergic mechanisms in epilepsy［J］. Epilepsia， 2001， 42 Suppl 3： 8-12.

Basbaum AI， Braz JM. Cell transplants to treat the "disease" of neuropathic pain and itch［J］. Pain， 2016， 157（2）： S42-S47.

Hwang I， Hahm SC， Choi KA， et al. Intrathecal transplantation of embryonic stem cell-derived spinal GABAergic neural precursor cells attenuates neuropathic pain in a spinal cord injury rat model［J］. Cell Transplant， 2016， 25（3）： 593-607.

Braz JM， Juarez-Salinas D， Ross SE， et al. Transplant restoration of spinal cord inhibitory controls ameliorates neuropathic itch［J］. J Clin Invest， 2014， 124（8）： 3612-3616.

Jacobson LH， Vlachou S， Slattery DA， et al. The Gamma-Aminobutyric Acid B Receptor in Depression and Reward［J］. Biol Psychiatry， 2018， 83（11）： 963-976.

陈舒婷，任力，闵苏. 氨基丁酸能神经元在丙泊酚缓解抑郁模型大鼠电休克后脑损伤中的作用［J］. 中国神经精神疾病杂志， 2020， 46（3）： 159-165.

Chen ST， Ren L， Min S. The role of GABAergic neurons in propofol-mediated alleviation of electroconvulsive shock-induced learning and memory impairment in rats with depression after electroshock［J］. Chin J Nerv Ment Dis， 2020， 46（3）： 159-165.

Sun ZQ， Sun L， Tu LX. GABA（B） receptor-mediated PI3K/Akt signaling pathway alleviates oxidative stress and neuronal cell injury in a rat model of Alzheimer's disease［J］. J Alzheimers Dis， 2020， 76（4）： 1513-1526.

钟斯然，邝琦，覃宁，等. ATRNL1在阿尔茨海默病中的基因功能分析及ceRNA网络预测［J］. 重庆医科大学学报， 2022， 47（7）： 871-876.

Zhong SR，Kuang Q，Qin N，et al. Gene function analysis of ATRNL1 and ceRNA network construction in Alzheimer’s disease［J］. J Chongqing Med Univ， 2022， 47（7）： 871-876.

Tyagi RK， Bisht R， Pant J， et al. Possible role of GABA-B receptor modulation in MPTP induced Parkinson's disease in rats［J］. Exp Toxicol Pathol， 2015， 67（2）： 211-217.

Maccioni P， Colombo G. Potential of GABA（B） receptor positive allosteric modulators in the treatment of alcohol use disorder［J］. CNS Drugs， 2019， 33（2）： 107-123.

Kalinichev M， Girard F， Haddouk H， et al. The drug candidate， ADX71441， is a novel， potent and selective positive allosteric modulator of the GABA（B） receptor with a potential for treatment of anxiety， pain and spasticity［J］. Neuropharmacology， 2017， 114： 34-47.

Glausier JR， Lewis DA. GABA and schizophrenia： Where we stand and where we need to go［J］. Schizophr Res， 2017， 181： 2-3.

Lehmann A， Jensen JM， Boeckxstaens GE. GABA（B） receptor agonism as a novel therapeutic modality in the treatment of gastroesophageal reflux disease［M］//Blackburn TP. GABAb Receptor Pharmacology： A Tribute to Norman Bowery. Elsevier Academic Press Inc， 525 B Street， Suite 1900， San Diego， Ca 92101-4495 USA. 2010： 287-313.

Li G， Zhang B， Sun JH， et al. An NT-3-releasing bioscaffold supports the formation of TrkC-modified neural stem cell-derived neural network tissue with efficacy in repairing spinal cord injury［J］. Bioact Mater， 2021， 6（11）： 3766-3781.

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