Embryonic Stem Cell Derived Cardiac Myocytes: Developmen

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。该研究旨在产生心脏谱系特异性细胞，以了解对兴奋-收缩-舒张偶联和分化过程至关重要的蛋白质的作用，从而导致体外心肌细胞的形成。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状体。已成功地进行了肌节肌动蛋白，肌钙蛋白T的单克隆和多克隆抗体的分化ES衍生的心肌细胞的免疫荧光分析，具体的工作，确定最早阶段的兰尼碱受体，SR钙ATP酶，受磷蛋白和二氢吡啶受体的表达也已成功。我们已经建立了几种技术来测量这些蛋白质的mRNA含量，所有这些都被用来开发一个分子模型的EC耦合和松弛的早期和晚期分化的ES细胞的发展。使用嘌呤霉素抗性盒与心脏限制性启动子，我们已经产生了一些ES细胞系，分化时，可以使用，至少部分地，选择心室特异性细胞系。我们还开发了许多具有靶向小鼠ryanodine受体2（RyR 2）基因的ES细胞克隆，其已与Cre蛋白酶一起用于引入loxP侧翼cDNA构建体。使用这些细胞，我们共转染的cDNA构建体含有嘌呤霉素抗性盒侧翼loxP位点与表达载体的Cre蛋白酶。共转染后，阳性选择技术已被用于鉴定克隆，其中靶向RyR 2等位基因已被嘌呤霉素抗性盒取代。这证明了floxed RyR 2外显子可以成功缺失，并且嘌呤霉素抗性盒可以以高效率插入相同的基因座。新的构建体正在制备中，以形成小鼠内源基因和人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RyR 2的嵌合突变体，从而可以在分离的细胞上研究该蛋白质的结构-功能特征。一些克隆细胞系现已部分表征。RyR KO细胞的结果表明，ryanodine受体不是从ES细胞分化搏动心肌细胞所必需的，但它在调节心肌细胞的搏动速率中起着关键作用。 此外，我们已经使用基因组技术，如基因表达或微阵列的系列分析，以确定可能参与心肌细胞分化过程的转录本的数量。这些基因靶标是积极研究的主题。其目的是确定将促进体外心肌细胞发育的因素，或可用于修复体内受损心脏的因素。

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.