Generation of Functional CX26?Gap‐Junction‐Plaque‐Forming Cells with Spontaneous Ca 2+ Transients via a Gap Junction Characteristic of Developing Cochlea
Generation of Functional CX26?Gap‐Junction‐Plaque‐Forming Cells with Spontaneous Ca 2+ Transients via a Gap Junction Characteristic of Developing Cochlea
复制标题
通过发育中耳蜗的间隙连接特性产生具有自发 Ca 2+ 瞬变的功能性 CX26？间隙连接斑块形成细胞
DOI：
10.1002/cpsc.100
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发表时间：
2019
影响因子：
--
通讯作者：
Kamiya Kazusaku
中科院分区：
文献类型：
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作者：
Fukunaga Ichiro;Fujimoto Ayumi;Hatakeyama Kaori;Kurebayashi Nagomi;Ikeda Katsuhisa;Kamiya Kazusaku
编码连接蛋白 26（CX26；也称为间隙连接 beta 2）的基因 GJB2 突变是全球遗传性耳聋的最常见原因。 CX26 在耳蜗非感觉细胞（例如耳蜗支持细胞）中表达，并在细胞-细胞边界形成间隙连接斑块 (GJP)。耳蜗CX26-GJP形成细胞（Cx26GJCs）被认为是治疗遗传性耳聋的重要治疗靶点。然而，迄今为止，尚未报道从胚胎干/诱导多能干 (ES/iPS) 细胞产生 Cx26GJC（例如耳蜗支持细胞）。在这里，我们详细介绍了一种将 iPS 细胞分化为功能性 Cx26GJC（例如耳蜗中的 Cx26GJC）的新策略。描述了表征 iPS 衍生的 Cx26GJC 表型的几种测定方法，包括 qRT-PCR、免疫组织学分析、形态学分析、刮擦加载和染料转移测定以及钙成像。这种体外模型可用于建立内耳细胞疗法和针对 GJB2 相关听力损失的药物筛选。 © 2019 John Wiley & Sons, Inc. 基本方案：诱导小鼠干细胞产生 CX26-GJP 形成细胞支持方案 1：小鼠诱导多能干细胞的维持和传代支持方案 2：使用定量实时 PCR 筛选 SFEBq 培养物中高 GJB2 和 GJB6 表达支持方案 3：不同分化阶段细胞的表征 通过免疫染色支持方案 4：CX26-GJP 形成细胞诱导不同阶段的细胞超微结构分析支持方案 5：干细胞来源的 CX26-GJP 形成细胞的功能分析
Mutation of the geneGJB2, encoding connexin 26 (CX26; also known as gap junction beta 2), is the most frequent cause of hereditary deafness worldwide. CX26 is expressed in cochlear nonsensory cells, such as cochlear supporting cells, and forms gap junction plaques (GJPs) at cell‐cell borders. Cochlear CX26‐GJP‐forming cells (Cx26GJCs) are thought to be an important therapeutic target for treatment of hereditary deafness. Nevertheless, the generation of Cx26GJCs—such as cochlear supporting cells—from embryonic stem/induced pluripotent stem (ES/iPS) cells has not been reported to date. Here, we detail a novel strategy for differentiating iPS cells into functional Cx26GJCs such as are found in cochlea. Several assays to characterize the phenotype of iPS‐derived Cx26GJCs are described, including qRT‐PCR, immunohistological analysis, morphological analysis, a scrape‐loading and dye transfer assay, and calcium imaging. This in vitro model has applications in the establishment of inner‐ear cell therapies and in drug screening to targetGJB2‐related hearing loss. © 2019 by John Wiley & Sons, Inc.Basic Protocol: Induction of mouse stem cells to create CX26‐GJP‐forming cellsSupport Protocol 1: Maintenance and passage of mouse induced pluripotent stem cellsSupport Protocol 2: Screening for highGJB2andGJB6expression in SFEBq culture using quantitative real‐time PCRSupport Protocol 3: Characterization of cells at different stages of differentiation by immunostainingSupport Protocol 4: Ultrastructural analyses of cells at different stages of CX26‐GJP‐forming cell inductionSupport Protocol 5: Functional analyses of stem cell–derived CX26‐GJP‐forming cells