通过发育中的耳蜗间隙连接特性产生具有自发Ca2+瞬态的功能性cx26 -间隙连接斑块形成细胞

Q2 Biochemistry, Genetics and Molecular Biology

Current Protocols in Stem Cell Biology Pub Date : 2019-10-28 DOI:10.1002/cpsc.100

Ichiro Fukunaga, Ayumi Fujimoto, Kaori Hatakeyama, Nagomi Kurebayashi, Katsuhisa Ikeda, Kazusaku Kamiya

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引用次数: 1

摘要

编码连接蛋白26 (CX26)的基因GJB2突变;也被称为间隙连接β 2)，是世界范围内遗传性耳聋最常见的原因。CX26在耳蜗非感觉细胞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(gap junction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靶点。然而，从胚胎干细胞/诱导多能干细胞(ES/iPS)中生成cx26gjc(如耳蜗支持细胞)至今尚未见报道。在这里，我们详细介绍了一种将iPS细胞分化为耳蜗中发现的功能性Cx26GJCs的新策略。描述了几种表征ips衍生的Cx26GJCs表型的方法，包括qRT-PCR，免疫组织学分析，形态学分析，刮片加载和染料转移试验以及钙成像。该体外模型可应用于内耳细胞疗法的建立和gjb2相关性听力损失的药物筛选。©2019 by John Wiley &基本方案:诱导小鼠干细胞生成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培养中GJB2和GJB6的高表达支持方案3:通过免疫染色表征不同分化阶段的细胞cx26 - gjp形成细胞诱导不同阶段细胞的超微结构分析支持方案5:干细胞衍生的cx26 - gjp形成细胞的功能分析

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Generation of Functional CX26–Gap-Junction-Plaque-Forming Cells with Spontaneous Ca2+ Transients via a Gap Junction Characteristic of Developing Cochlea

Mutation of the gene GJB2, 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 target GJB2-related hearing loss. © 2019 by John Wiley & Sons, Inc.

Basic Protocol: Induction of mouse stem cells to create CX26-GJP-forming cells

Support Protocol 1: Maintenance and passage of mouse induced pluripotent stem cells

Support Protocol 2: Screening for high GJB2 and GJB6 expression in SFEBq culture using quantitative real-time PCR

Support Protocol 3: Characterization of cells at different stages of differentiation by immunostaining

Support Protocol 4: Ultrastructural analyses of cells at different stages of CX26-GJP-forming cell induction

Support Protocol 5: Functional analyses of stem cell–derived CX26-GJP-forming cells

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来源期刊

Current Protocols in Stem Cell Biology Biochemistry, Genetics and Molecular Biology-Cell Biology

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期刊介绍： Published in affiliation with the International Society for Stem Cell Research (ISSCR), Current Protocols in Stem Cell Biology (CPSC) covers the most fundamental protocols and methods in the rapidly growing field of stem cell biology. Updated monthly, CPSC will constantly evolve with thelatest developments and breakthroughs in the field. Drawing on the expertise of leading researchers from around the world, Current Protocols in Stem Cell Biology includes methods and insights that will enhance the progress of global research.