Embryonic Stem Cell Derived Cardiac Myocytes: Developmen

本研究领域涉及胚胎干细胞（R1）的心肌细胞体外分化模型。该研究旨在生成心脏谱系特异性细胞，以了解在体外导致心肌细胞形成的兴奋-收缩-松弛耦合和分化过程中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为含有收缩心脏样细胞的胚胎样体。利用单克隆抗体和多克隆抗体对es来源的分化心肌细胞进行了免疫荧光分析，成功地检测了肌动蛋白、肌钙蛋白T；在确定ryanodine受体、SR CaATPase、phospholamban和dihydropyridine受体的早期表达方面也取得了成功。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于开发早期和晚期分化ES细胞EC偶联和松弛发展的分子模型。使用具有心脏限制性启动子的嘌呤霉素耐药盒，我们已经产生了许多胚胎干细胞系，这些细胞系分化后，可以用于（至少部分地）选择心室特异性细胞系。我们还开发了一些针对小鼠RyR2基因的胚胎干细胞克隆，并利用Cre重组酶引入了loxP侧翼cDNA构建体。利用这些细胞，我们用Cre重组酶的表达载体共转染了含有嘌呤霉素耐药盒的cDNA构建体，其两侧是loxP位点。共转染后，使用阳性选择技术鉴定目标RyR2等位基因已被嘌呤霉素耐药磁带所取代的克隆。这表明可以成功地删除带螺纹的RyR2外显子，并且可以高效地将嘌呤霉素耐药盒插入同一位点。新的构建体正在准备在内源性小鼠基因和人类cDNA构建体之间形成嵌合体。该系统最终将使我们能够快速创建RyR2的嵌合突变体，从而可以在分离细胞上研究该蛋白的结构-功能特征。许多克隆细胞系目前已被部分鉴定。RyR KO细胞的研究结果表明，ryanodine受体在搏动心肌细胞与ES细胞的分化中不是必需的，但它在心肌细胞的搏动速率调节中起着关键作用。

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 and differentiation processes leading to the formation of cardiomyocytes in vitro. 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. The results with the RyR KO cells indicate that the ryanodine receptor is not necessary for the differentiation of beating cardiac myocytes from ES cells, but that it plays a critical role in the regulation of the beating rate of cardiac myocytes. Additionally, we have used genomic techniques like serial analysis of gene expression or microarrays to identify a number of transcripts that may be involved in the differentiation processes to cardiomyocytes. These gene targets are the subject of active investigation. The aim is to identify factors that will promote the development of cardiomyocytes in vitro, or which can be utilized to lead to repair of the damaged heart in vivo.