Construction of hiPSC-derived Excitatory Neural Network-like Tissue

SU Qi-song; LI Ge; XU Jin-hai; JIANG Bin; WEI Qing-shuai; ZENG Xiang; ZENG Yuan-shan; ZHU Ping

doi:10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).20230529.001

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Construction of hiPSC-derived Excitatory Neural Network-like Tissue

Basic Research | 更新时间：2024-02-19

- Construction of hiPSC-derived Excitatory Neural Network-like Tissue
- Journal of Sun Yat-sen University(Medical Sciences) Vol. 44, Issue 4, Pages: 625-633(2023)
- 作者机构：
  
  1.华南理工大学生物科学与工程学院，广东广州510006
  2.中山大学中山医学院组织学与胚胎学教研室，广东广州510080
  3.广东省心血管病研究所，南方医科大学附属广东省人民医院//广东省医学科学院，广东广州 510100
  4.医学研究部，南方医科大学附属广东省人民医院//广东省医学科学院，广东广州 510100
  5.广东省心脏病发病机制与精准防治重点实验室//广州市心脏病发病机制与防治重点实验室，广东广州 510100
  6.广东省脑功能与脑疾病重点实验室，广东广州 510080
- 作者简介：
  
  ZENG Yuan-shan， E-mail： zengysh@mail.sysu.edu.cn
  ZHU Ping， E-mail： tanganqier@163.com
- 基金信息：
- DOI：10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).20230529.001
  CLC： R318
- Published：20 July 2023，
  
  Received：08 February 2023，
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苏琪淞,李戈,许金海等.兴奋性hiPSC源性类神经网络组织的构建[J].中山大学学报（医学科学版）,2023,44(04):625-633.

SU Qi-song,LI Ge,XU Jin-hai,et al.Construction of hiPSC-derived Excitatory Neural Network-like Tissue[J].Journal of Sun Yat-sen University(Medical Sciences),2023,44(04):625-633.
苏琪淞,李戈,许金海等.兴奋性hiPSC源性类神经网络组织的构建[J].中山大学学报（医学科学版）,2023,44(04):625-633. DOI： 10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).20230529.001.

SU Qi-song,LI Ge,XU Jin-hai,et al.Construction of hiPSC-derived Excitatory Neural Network-like Tissue[J].Journal of Sun Yat-sen University(Medical Sciences),2023,44(04):625-633. DOI： 10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).20230529.001.

摘要

目的

将人诱导多能干细胞源性神经前体细胞（hiPSC-NPCs）种植于去细胞视神经（ DON）上，在体外构建一种具有突触传递功能的类神经网络组织，为神经组织损伤的修复提供一种有效途径。

方法

通过定向诱导和组织工程技术，联合应用hiPSCs和3D DON支架，构建一种新的类神经网络组织。定向诱导人诱导多能干细胞（hiPSCs）向人神经前体细胞（ hNPCs）及神经元方向分化，使用免疫荧光染色鉴定细胞分化效率。制备3D DON支架，借助扫描电镜、Tunnel染色，鉴定支架形貌及细胞相容性。将诱导的hiPSC-NPCs种植于DON支架中，借助免疫荧光染色、扫描电镜、透射电镜和膜片钳对构建的类神经网络组织进行形态学观察和功能学鉴定。

结果

①免疫细胞化学染色结果提示，诱导的hiPSC-NPCs在体外大部分向神经元方向分化，成功构建了一种以神经元为主的神经网络。②扫描电镜和免疫组织化学结果提示，在体外成功构建了一种以兴奋性神经元为主的类神经网络组织。③免疫组织化学染色、透射电镜和膜片钳结果提示，构建的类神经网络组织能够传递兴奋性突触信息。

结论

利用天然来源的具有均匀孔道分布的DON生物支架材料和hiPSC-NPCs的有机结合，构建了一种以兴奋性神经元为主的具有突触传递功能的类神经网络组织，这种神经网络可作为脊髓损伤（spinal cord injury， SCI）等神经组织损伤后组织替换疗法的有利工具，具有广阔的临床应用前景。

Abstract

Objective

To construct a neural network-like tissue with the potential of synaptic formation in vitro by seeding human induced pluripotent stem cell-derived neural precursor cells （hiPSC-NPCs） on decellularized optic nerve （DON）， so as to provide a promising approach for repair of nerve tissue injury.

Methods

Through directional induction and tissue engineering technology， human induced pluripotent stem cells （hiPSCs） and 3D DON scaffolds were combined to construct neural network-like tissues. Then the hiPSCs were directionally induced into human neural precursor cells （hNPCs） and neurons. Immunofluorescence staining was used to identify cell differentiation efficiency. 3D DON scaffolds were prepared. Morphology and cytocompatibility of scaffolds were identified by scanning electron microscopy and Tunnel staining. Induced hiPSC-NPCs were seeded on DON scaffolds. Immunofluorescence staining， scanning electron microscopy， transmission electron microscopy and patch clamp were used to observe the morphology and functional identification of constructed neural network tissues.

Results

①The results of immunofluorescence staining suggested that most of hiPSC-NPCs differentiated into neurons in vitro. We had successfully constructed a neural network dominated by neurons. ② The results of scanning electron microscopy and immunohistochemistry suggested that a neural network-like tissue with predominating excitatory neurons in vitro was successfully constructed. ③The results of immunohistochemical staining， transmission electron microscopy and patch clamp indicated that the neural network-like tissue had synaptic transmission function.

Conclusion

A neural network-like tissue mainly composed of excitatory neurons has been constructed by the combination of natural uniform-channel DON scaffold and hiPSC-NPCs， which has the function of synaptic transmission. This neural network plays a significant role in stem cell derived replacement therapy， and offers a promising prospect for repair of spinal cord injury （SCI） and other neural tissue injuries.

关键词

人诱导多能干细胞源性神经前体细胞去细胞视神经突触兴奋性神经元类神经网络组织

Keywords

hiPSC-NPCsDONsynapseexcitatory neuronsneural network-like tissue

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Related Author

SU Qi-song

LI Ge

XU Jin-hai

JIANG Bin

WEI Qing-shuai

ZENG Xiang

ZENG Yuan-shan

ZHU Ping

Related Institution

School of Biology and Biological Engineering， South China University of Technology

Department of Histology and Embryology， Zhongshan School of Medicine， Sun Yat-sen University

Guangdong Cardiovascular Institute， Guangdong Provincial People's Hospital//Guangdong Academy of Medical Sciences， Southern Medical University

Medical Research Institute， Guangdong Provincial People's Hospital //Guangdong Academy of Medical Sciences， Southern Medical University

Guangdong Provincial Key Laboratory of Pathogenesis， Targeted Prevention and Treatment of Heart Disease//Guangzhou Key Laboratory of Cardiac Pathogenesis and Prevention

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