Myocardial Infarct leads to loss of cardiomyocytes and often results in heart failure. Cardiomyocyte grafting can generate new myocardium in injured hearts. For humans however, it is desirable to derive cardiomyocytes from stem cells. My project involves the use of the murine R1 ES cell line to study cardiomyocyte differentiation. Cells are cultured in hanging drops as aggregates (embryoid bodies, EB). Aggregates are grown in suspension culture for 5 days and then plated onto gelatin-coated 12 well plates. RT-PCR analysis showed that the early stem cell marker Oct 4 was markedly down regulated compared to undifferentiated ES cells. Brachyury T, an early mesoderm marker was highly expressed in 2 day old EBs. At later timepoints Brachyury T expression was reduced. The first beating cells were observed between 3 and 5 days after plating the EBs. With the onset of contraction in the differentiating ES cells the cradiac transcription factors Nkx2.5, GATA4, Mef2a, b, c, and d, dHAND, and the contractile proteins Myosin Light Chain 2v, cardiac alpha actin, and cardiac troponin I were up regulated. One objective is to repair injured tissue by grafting ES cell-derived cardiomyocytes into the heart. This requires the isolation of cardiomyocytes from hetereogeneous EB. In order to select differentiated cardiomyocytes, ES cells were stably transfected with a construct conatining the Enhanced Green Fluorescent Protein (EGFP) under the transcriptional control of the cardiac alpha-actin promoter. Fluorescent cells were detected in 10 to 12 day old EBs, coincident with the appearance of the first beating cells. Current studies focus on the dissociation of EB and FACS analysis of EGFP-labeled early stage cardiomyocytes.