Patch-clamp recordings and calcium imaging followed by single-cell PCR reveal the developmental profile of 13 genes in iPSC-derived human neurons.
Patch-clamp recordings and calcium imaging followed by single-cell PCR reveal the developmental profile of 13 genes in iPSC-derived human neurons.
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DOI：
10.1016/j.scr.2013.09.014
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发表时间：
2014-01
影响因子：
1.2
通讯作者：
Antic, Srdjan D.
中科院分区：
文献类型：
--
作者：
Belinsky, Glenn S.;Rich, Matthew T.;Sirois, Carissa L.;Short, Shaina M.;Pedrosa, Erika;Lachman, Herbert M.;Antic, Srdjan D.
分子遗传学研究通常在均质化的生物样品上进行，导致非神经元细胞的污染。为了改善神经元的表达谱，我们将斑片记录与单细胞PCR相结合。两个iPSC系（健康受试者和22q11.2缺失）分化成神经元。从第13天至第88天，对229个人细胞进行贴片电极记录，然后对13个基因进行捕获和单细胞PCR：ACTB、HPRT、vGLUT 1、βTUBIII、COMT、DISC 1、GAD 1、PAX 6、DTNBP 1、ERBB 4、FOXP 1、FOXP 2和GIRK 2。来源于两种iPSC系的神经元表达βTUBIII，激发动作电位，并在vGLUT 1、GAD 1和GIRK 2出现前约2周经历自发去极化（UP状态）。多位点钙成像显示这些UP状态在hESC-H9衍生的神经元之间不同步。FOXP 1、FOXP 2和vGLUT 1的表达在培养50天后丧失，与其他持续表达的基因相反。当基因表达与电生理学相结合时，两个基因子集是明显的;那些与自发去极化无关的基因（包括vGLUT 1、GIRK 2、FOXP 2和DISC 1）和那些与自发去极化相关的基因（GAD 1和ERBB 4）。结果表明，在神经元发育的最早阶段，在细胞对细胞的基础上，将联合收割机遗传分析与生理特征结合起来是有用的。
Molecular genetic studies are typically performed on homogenized biological samples, resulting in contamination from non-neuronal cells. To improve expression profiling of neurons we combined patch recordings with single-cell PCR. Two iPSC lines (healthy subject and 22q11.2 deletion), were differentiated into neurons. Patch electrode recordings were performed on 229 human cells from Day-13 to Day-88, followed by capture and single-cell PCR for 13 genes: ACTB, HPRT, vGLUT1, βTUBIII, COMT, DISC1, GAD1, PAX6, DTNBP1, ERBB4, FOXP1, FOXP2, and GIRK2. Neurons derived from both iPSC lines expressed βTUBIII, fired action potentials, and experienced spontaneous depolarizations (UP states) ~2 weeks before vGLUT1, GAD1 and GIRK2 appeared. Multisite calcium imaging revealed that these UP states were not synchronized among hESC-H9-derived neurons. The expression of FOXP1, FOXP2 and vGLUT1 was lost after 50 days in culture, in contrast to other continuously expressed genes. When gene expression was combined with electrophysiology, two subsets of genes were apparent; those irrelevant to spontaneous depolarizations (including vGLUT1, GIRK2, FOXP2 and DISC1) and those associated with spontaneous depolarizations (GAD1 and ERBB4). The results demonstrate that in the earliest stages of neuron development, it is useful to combine genetic analysis with physiological characterizations, on a cell-to-cell basis.
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影响因子：
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作者：
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