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cotransfection, positive selection techniques have been used to identify clones where the targeted RyR2 alleles have been replaced by puromycin resistance cassettes.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.

工作总结该研究领域涉及源自胚胎干细胞（R1）的心肌细胞体外分化模型。该研究旨在产生心脏差的特定细胞，以了解蛋白质在心室心肌细胞中对激发 - 收缩 - 脱毛偶联重要的作用。我们已经能够成功地将多能ES细胞分化为含有收缩心脏样细胞的胚胎体。使用单克隆和多克隆抗体对分化ES衍生的心脏细胞的免疫荧光分析已成功地针对肌动蛋白Troponin T进行了。鉴定瑞氨烷受体，SR CAATPase，Phospholamban和二氢吡啶受体表达的最早阶段的具体工作也已成功。我们已经建立了几种技术来测量这些蛋白质的mRNA含量，所有这些都用于开发一种分子模型，以开发EC偶联和放松的早期和晚期分化ES细胞。使用与心脏抑制启动子一起使用紫霉素抗性盒，我们产生了许多ES细胞系，当分化时，可以至少部分地使用中心特异性细胞谱系。我们还开发了许多ES细胞克隆，该克隆具有针对的小鼠ryanodine受体2（RYR2）基因，这些基因已与CRE重组酶一起使用，以介绍Loxp侧翼cDNA构建体。使用这些细胞，我们具有共转染的cDNA构建体，其中含有紫霉素耐药性盒，侧面是Loxp位点，其表达载体用于CRE重组酶。共转染后，已使用阳性选择技术来识别靶向的RYR2等位基因被嘌呤霉素耐药盒取代的克隆。这表明可以成功删除Floxed Ryr2外显子，并且可以在高效率下将紫霉素耐药性盒插入相同的基因座。现在，正在准备新的构建体，以在内源性小鼠基因和人cDNA构建体之间形成嵌合体。该系统最终将使我们能够快速为RYR2创建嵌合突变体，以便可以在分离的细胞上研究该蛋白质的结构功能特征。 现在已经部分表征了许多克隆细胞系。这项工作正在与心血管科学实验室的激发收缩耦合单元合作进行，他们使用电压夹紧和共聚焦显微镜进行了许多初步研究，以分析钙火花形成，钙瞬态和钙处理蛋白的功能。