EMBRYONIC STEM CELL DERIVED CARDIAC MYOCYTES: DEVELOPMENTAL STUDIES

胚胎干细胞衍生的心肌细胞：发育研究

• 批准号: 6431481
• 负责人: Kenneth R Boheler
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6431481
• 关键词: calcium channel; calcium indicator; cell differentiation; confocal scanning microscopy; embryo /fetus tissue /cell culture; embryonic stem cell; gene targeting; heart cell; heart contraction; laboratory mouse; receptor coupling; sarcolemma; sarcoplasmic reticulum

SUMMARY OF WORK This research area involves a model of in vitro differentiation of cardiomyocytes originating from embryonic stem cells (R1). The research is aimed at generating cardiac-lineage specific cells to understand the role of proteins important for excitation-contraction-relaxation coupling in ventricular myocytes. We have been able to successfully differentiate pluripotent ES cells into embryoid bodies containing contracting cardiac-like cells. Immunofluorescence analyses of the differentiating ES-derived cardiac cells using monoclonal and polyclonal antibodies have been successfully performed for sarcomeric actins, troponin T; specific work on identifying the earliest stages of ryanodine receptor, SR CaATPase, phospholamban and dihydropyridine receptor expression have also been successful. We have established several techniques to measure the mRNA contents of these proteins, all of which are being used to develop a molecular model for the development of EC coupling and relaxation in early and late differentiating ES cells. Using puromycin resistance cassettes with cardiac-restrited promoters, we have generated a number of ES cell lines that, when differentiated, can be used, at least partially, to select for ventricular-specific cell lineages. We have also developed a number of ES cell clones with a targeted the mouse ryanodine receptor 2 (RyR2) gene which have been utilized with Cre Recombinase to introdude of loxP flanking cDNA constructs. Using these cells, we have co-transfected cDNA constructs containing puromycin resistance cassettes flanked by loxP sites with an expression vector for Cre Recombinase. After cotransfection, positive selection techniques have been used to identify clones where the targeted RyR2 alleles have been replaced by puromycin resistance cassettes. This demonstrates that floxed RyR2 exon can be successfully deleted and that a puromycin resistance cassette can be inserted into the same locus at high efficiency. New constructs are now being prepared to form chimeras between the endogenous mouse gene and a human cDNA construct. This system will ultimately allow us to rapidly create chimeric mutants for RyR2 so that structure-function characteristics of this protein can be studied on isolated cells. A number of the clonal cell lines have now been partially characterized. This work is being performed in collaboration with the Excitation Contraction Coupling Unit of the Laboratory of Cardiovascular Science who have performed a number of preliminary studies using the techniques of voltage clamping and confocal microscopy to analyze calcium spark formation, calcium transients and function of calcium handling proteins.

工作摘要该研究领域涉及源自胚胎干细胞（R1）的心肌细胞的体外分化模型。该研究旨在生成心脏谱系特异性细胞，以了解对心室肌细胞兴奋-收缩-舒张耦合重要的蛋白质的作用。我们已经能够成功地将多能胚胎干细胞分化成含有收缩的心脏样细胞的胚状体。使用单克隆和多克隆抗体对分化的 ES 来源的心肌细胞进行了免疫荧光分析，已成功对肌节肌动蛋白、肌钙蛋白 T 进行了免疫荧光分析。鉴定兰尼碱受体、SR CaATP酶、受磷蛋白和二氢吡啶受体表达的最早阶段的具体工作也取得了成功。我们已经建立了多种技术来测量这些蛋白质的 mRNA 含量，所有这些技术都用于开发早期和晚期分化 ES 细胞中 EC 偶联和松弛发展的分子模型。使用具有心脏受限启动子的嘌呤霉素抗性盒，我们产生了许多ES细胞系，这些细胞系在分化时可用于（至少部分地）选择心室特异性细胞谱系。我们还开发了许多带有靶向小鼠兰尼碱受体 2 (RyR2) 基因的 ES 细胞克隆，这些克隆已与 Cre 重组酶一起使用以引入 loxP 侧翼 cDNA 构建体。使用这些细胞，我们共转染了含有侧翼为 loxP 位点的嘌呤霉素抗性盒的 cDNA 构建体以及 Cre 重组酶的表达载体。共转染后，使用正选技术来鉴定其中目标 RyR2 等位基因已被嘌呤霉素抗性盒取代的克隆。这表明可以成功删除 floxed RyR2 外显子，并且可以将嘌呤霉素抗性盒高效插入同一基因座。目前正在制备新的构建体，以形成内源性小鼠基因和人类 cDNA 构建体之间的嵌合体。该系统最终将使我们能够快速创建 RyR2 的嵌合突变体，以便可以在分离的细胞上研究该蛋白质的结构功能特征。 许多克隆细胞系现已得到部分表征。这项工作是与心血管科学实验室的激发收缩耦合单元合作进行的，该单元使用电压钳和共聚焦显微镜技术进行了许多初步研究，以分析钙火花形成、钙瞬变和钙处理蛋白的功能。