Endoderm Induction of Cardiac Myocytes from ES Cells

产品说明：多能胚胎干（ES）细胞再生心脏组织的潜力引起了人们对其治疗应用的极大兴趣。ES细胞的临床应用需要满足几个问题，包括如何从ES细胞诱导心肌细胞。为此，调节胚胎心肌细胞分化的机制提供了线索。利用基于本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）调节的培养基诱导几乎所有来自小鼠ES细胞的胚状体（EB）的心脏发生，并且单个EB在心肌细胞中富集>80%。拟议的工作将通过四个具体目标的执行来扩展这些发现。首先，正在确定由来源于胚胎组织的细胞系调节的培养基是否可以模拟胚胎外植体的心源性作用，所述细胞系可以以足以进行广泛的生物化学和功能分析的体积获得。还将确定由胚胎外植体或由它们衍生的细胞系调节的培养基是否可以类似地诱导人ES细胞（系#WA01，WiCell，麦迪逊WI）中的心脏发生。其次，为了鉴定化学上确定的心源性“鸡尾酒”，将通过多种细胞和生物化学方法表征由心源性细胞系调节的培养基。在第三个目标中，将精确确定由条件培养基诱导的ES衍生的心肌细胞（下文称为ES-肌细胞）的数量和表型身份，目的是为移植研究提供最佳的群组。最后，移植的ES-肌细胞在结构上和功能上修复梗死心肌的能力将根据分化状态（预分化对多能性）、最佳供体细胞数量、增殖潜力和存活率进行评价。这项工作将有助于阐明如何利用ES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.