Endoderm Induction of Cardiac Myocytes from ES Cells

描述：多能胚胎干细胞（ES）在心脏组织再生方面的潜力引起了人们对其治疗应用的极大兴趣。胚胎干细胞的临床应用需要满足几个问题，包括如何从胚胎干细胞诱导心肌细胞。为此，胚胎中调节心肌细胞分化的机制提供了线索。使用基于该实验室早期发现的方法，我们最近报道了由胚胎心前内胚层/中胚层（E/M-cm）调节的培养基在几乎所有来源于小鼠胚胎干细胞的胚状体（EBs）中诱导心脏发生，并且单个胚状体在心肌细胞中富集bbbb80 %。拟议的工作将通过四个具体目标的表现来扩展这些发现。首先，由胚胎组织衍生的细胞系调节的培养基是否能够模拟胚胎外植体的心源性作用，这种培养基可以获得足够的量用于广泛的生化和功能分析，目前正在确定。还将确定由胚胎外植体或由其衍生的细胞系调节的培养基是否能类似地诱导人类胚胎干细胞的心脏发生（细胞系#WA01， WiCell, Madison Wl）。其次，为了确定化学上定义的心源性“鸡尾酒”，由心源性细胞系调节的培养基将通过各种细胞和生化方法进行表征。在第三个目标中，将精确确定条件培养基诱导的es源性心肌细胞（以下简称ES-myocytes）的数量和表型特征，目的是为移植研究提供最佳队列。最后，移植es -肌细胞在结构和功能上修复梗死心肌的能力将根据分化状态（预分化与多能性）、最佳供体细胞数量、增殖潜力和存活率进行评估。这项工作将有助于阐明如何利用胚胎干细胞来修复因疾病或梗死而受损的心肌。

DESCRIPTION: The potential of pluripotent embryonic stem (ES) cells to regenerate cardiac tissue has caused extraordinary interest in their therapeutic application. Clinical utilization of ES cells requires satisfaction of several issues, including how cardiac myocytes can be induced from ES cells. Toward this end, mechanisms that regulate cardiac myocyte differentiation in the embryo have provided clues. Using an approach based on earlier findings from this laboratory, we recently reported that medium conditioned by embryonic precardiac endoderm/mesoderm (E/M-cm) induces cardiogenesis in virtually all embryoid bodies (EBs) derived from mouse ES cells, and that individual EBs are >80% enriched in cardiac myocytes. The proposed work will extend these findings via performance of four Specific Aims. First, whether medium conditioned by cell-lines derived from embryonic tissues, which can be obtained in volumes sufficient for extensive biochemical and functional analysis, can mimic the cardiogenic effect of embryonic explants is being determined. It will also be determined whether medium conditioned by embryonic explants or by cell-lines derived from them can similarly induce cardiogenesis in human ES cells (line #WA01, WiCell, Madison Wl). Second, in order to identify a chemically defined cardiogenic "cocktail", medium conditioned by cardiogenic cell-lines will be characterized via a variety of cellular and biochemical methods. In the third Aim, the number and phenotypic identity of ES-derived cardiac myocytes (hereafter ES-myocytes) that are induced by conditioned medium will be precisely determined, with the objective of providing optimal cohorts for transplantation studies. Lastly, the ability of transplanted ES-myocytes to structurally and functionally repair infarcted myocardium will be evaluated in terms of differentiation state (pre-differentiated v. pluripotent), optimal donor cell numbers, proliferative potential, and survival rate. This work will help elucidate how ES cells may be utilized to repair myocardium that is damaged by disease or infarction.