hVGAT-mCherry: A novel molecular tool for analysis of GABAergic neurons derived from human pluripotent stem cells.
hVGAT-mCherry: A novel molecular tool for analysis of GABAergic neurons derived from human pluripotent stem cells.
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DOI：
10.1016/j.mcn.2015.08.007
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发表时间：
2015-09
期刊：
影响因子：
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通讯作者：
Dykxhoorn DM
中科院分区：
文献类型：
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作者：
DeRosa BA;Belle KC;Thomas BJ;Cukier HN;Pericak-Vance MA;Vance JM;Dykxhoorn DM
gaba能突触传递在发育过程中神经元回路的组装中起着关键作用，并负责维持大脑成熟至成年期间兴奋和抑制信号之间的平衡。重要的是，gaba能神经元功能和信号的缺陷与许多神经系统疾病有关，包括自闭症谱系障碍、精神分裂症和癫痫。利用基于患者特异性诱导多能干细胞（iPSC）的神经系统疾病模型，现在有可能研究受影响的人类神经元中gaba能功能缺陷的疾病机制。为此，能够从人类多能干细胞中标记和纯化有活力的gaba能神经元的工具将具有很大的价值。为了解决在iPSC细胞系体外分化中鉴定和分离有活力GABA能神经元的工具需求，研究人员开发了一种细胞类型特异性启动子驱动的荧光报告构建，该构建利用人囊泡GABA转运蛋白（hVGAT）启动子驱动mCherry在表达vgat的神经元中特异性表达。用hVGAT启动子- mcherry慢病毒报告基因转导ipsc衍生的前脑神经元构建特异性标记的gaba能神经元。免疫细胞化学分析显示，hVGAT-mCherry表达细胞与内源性VGAT、GABA和GAD67的GABA能神经元标记物显著共标记。来自VGAT启动子的mCherry在几种皮层中间神经元亚型中的表达水平相似。此外，开发了一种有效且可重复的方案，以促进荧光活化细胞分选（FACS）介导的高产量活vgat阳性细胞的纯化。这些研究表明，hVGAT-mCherry报告结构是研究从人多能干细胞体外分化的gaba能神经元的有效工具。这种方法可以提供一种从疾病特异性hiPSCs中获得大量活的gaba能神经元的方法，可用于功能分析或小分子文库的高通量筛选。
GABAergic synaptic transmission is known to play a critical role in the assembly of neuronal circuits during development and is responsible for maintaining the balance between excitatory and inhibitory signaling in the brain during maturation into adulthood. Importantly, defects in GABAergic neuronal function and signaling have been linked to a number of neurological diseases, including autism spectrum disorders, schizophrenia, and epilepsy. With patient-specific induced pluripotent stem cell (iPSC)-based models of neurological disease, it is now possible to investigate the disease mechanisms that underlie deficits in GABAergic function in affected human neurons. To that end, tools that enable the labeling and purification of viable GABAergic neurons from human pluripotent stem cells would be of great value. To address the need for tools that facilitate the identification and isolation of viable GABAergic neurons from the in vitro differentiation of iPSC lines, a cell type-specific promoter-driven fluorescent reporter construct was developed that utilizes the human vesicular GABA transporter (hVGAT) promoter to drive the expression of mCherry specifically in VGAT-expressing neurons. The transduction of iPSC-derived forebrain neuronal cultures with the hVGAT promoter-mCherry lentiviral reporter construct specifically labeled GABAergic neurons. Immunocytochemical analysis of hVGAT-mCherry expression cells showed significant co-labelling with the GABAergic neuronal markers for endogenous VGAT, GABA, and GAD67. Expression of mCherry from the VGAT promoter showed expression in several cortical interneuron subtypes to similar levels. In addition, an effective and reproducible protocol was developed to facilitate the fluorescent activated cell sorting (FACS)-mediated purification of high yields of viable VGAT-positive cells. These studies demonstrate the utility of the hVGAT-mCherry reporter construct as an effective tool for studying GABAergic neurons differentiated in vitro from human pluripotent stem cells. This approach could provide a means of obtaining large quantities of viable GABAergic neurons derived from disease-specific hiPSCs that could be used for functional assays or high-throughput screening of small molecule libraries.