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

作　　者：彭历芝 位庆帅 马瑗锾 许金海 蒋斌[2] 曾园山[1,2] 曾湘[1,2] 丁英[1,2] PENG Li-zhi;WEI Qing-shuai;MA Yuan-huan;XU Jin-hai;JIANG Bin;ZENG Yuan-shan;ZENG Xiang;DING Ying(Department of Histoembryology and Cell Biology,Zhongshan School of Medicine,Sun Yat-sen University,Guanzhou 510080,China;Guangdong Province Key Labor atory of Brain Function and Disease,Zhongshan School of Medicine,Sun Yat-sen University,Guangzhou 510080,China)

机构地区：[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

出　　处：《中山大学学报（医学科学版）》2023年第1期18-25,共8页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(81891003,81891002)。

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。【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

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 

分 类 号：R318[医药卫生—生物医学工程]