Heart failure is the leading cause of death and disability in the western world. According to the American Heart Association, nearly 5 million people are in heart failure and about 550,000 new cases are diagnosed each year in the United States alone. The definitive treatment is heart transplantation; yet, donor organ shortage and the need for immunosuppression limit clinical impact and outcomes. As a result, each year 25-50,000 patients in the US die while waiting for a donor heart. Autologous tissue-engineered hearts stemming from a patient's own cells could potentially overcome all these hurdles by providing viable tissue grafts needing no immunosuppressive therapy. In preliminary studies, we developed a novel technique to isolate acellular whole heart extracellular matrix (ECM) scaffolds from rat and porcine hearts by perfusion decellularization. These ECM scaffolds supported engraftment of fetal heart cells to form contractile tissue. Other groups have provided novel cardiac cell populations derived from adult human cells. We therefore propose to test the hypothesis that (1) the human heart's ECM can be isolated via perfusion decellularization from hearts that were found unsuitable for transplantation, and that (2) the resulting acellular scaffolds can be repopulated with adult stem cell derived cardiomyocytes to form viable tissue. Completion of the proposed research is expected to provide us with two basic milestones towards regeneration of functional human myocardium from patient derived cells: human ECM scaffolds and human cardiomyocytes. Further we will examine the ECM's role in supporting engraftment, differentiation and formation of functional tissue. Aside of the promise to regenerate viable human myocardium, the proposed experiments will provide a unique and novel in vitro system to evaluate the regenerative potential of different cell types, genes and small molecules in an isolated human heart model. (End of Abstract)