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. ? ? ?
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