EMBRYONIC STEM CELL DERIVED CARDIAC MYOCYTES: DEVELOPMENTAL STUDIES

工作概述本研究领域涉及胚胎干细胞(R1)来源的心肌细胞体外分化模型。这项研究的目的是生成心脏谱系特异性细胞，以了解在心室肌细胞中对兴奋-收缩-松弛偶联至关重要的蛋白质的作用。我们已经能够成功地将多能ES细胞分化为含有收缩的心脏样细胞的类胚体。已经成功地使用单抗和多克隆抗体对分化的ES来源的心肌细胞进行了肌节肌动蛋白和肌钙蛋白T的免疫荧光分析；在鉴定Ryanodine受体、SR CaATPase、磷蛋白和二氢吡啶受体表达的早期阶段的具体工作也取得了成功。我们已经建立了几种技术来测量这些蛋白质的mRNA含量，所有这些技术都被用来开发一个分子模型，用于早期和晚期分化的ES细胞中EC偶联和松弛的发展。使用带有心脏限制性启动子的嘌呤霉素抗性盒，我们已经产生了一些ES细胞系，当分化时，可以用于至少部分地选择心室特异性细胞系。我们还开发了一些以小鼠Ryanodine受体2(RyR2)基因为靶点的ES细胞克隆，这些克隆已经被Cre重组酶用来导入loxP侧翼的cDNA结构。利用这些细胞，我们用Cre重组酶的表达载体将含有嘌呤霉素抗性盒和loxP位点的cDNA共转染。共转染后，阳性选择技术已被用于鉴定目标RyR2等位基因已被嘌呤霉素耐药盒取代的克隆。这表明RyR2外显子可以成功缺失，并且可以高效地插入一个嘌呤霉素抗性盒。目前正在准备新的构建物，以在内源性小鼠基因和人类cdna构建物之间形成嵌合体。这个系统最终将允许我们快速创建RyR2的嵌合突变体，以便在分离的细胞上研究这种蛋白质的结构和功能特征。一些克隆细胞系现在已经被部分鉴定。这项工作是与心血管科学实验室的兴奋收缩耦合单元合作进行的，他们使用电压钳制和共聚焦显微镜技术进行了许多初步研究，以分析钙火花的形成、钙瞬变和钙处理蛋白的功能。

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.