This request for a Competing Supplement for Human Embryonic Stem Cell Research would complement and extend the Applicant's NIH-funded investigations of growth factor signaling pathways that drive heart muscle cell formation. Undertaking such a transition, from mouse models to human cells, is seen as instrumental towards the long-term goal of efficacious cardiac repair. Dr. Lior Gepstein, one of the founders of cardiac differentiation studies in human ES cells, serves as a Consultant to this proposal. In model organisms including flies, fish, frogs, avians, and mice, heart muscle formation is triggered universally thus far by secreted peptides of the bone morphogenetic protein (BMP) family. The primordial pathway for heart formation in flies involves a second positive-acting signal (wingless, the prototype for Wnts in vertebrates), but Wnts are said to be inhibitors of cardiogenesis in some species. Recent work by the applicant, in pluripotent mouse cells, suggests that Wnts could lie upstream in mammals from most if not all of the cardiogenic peptides reported to date (BMPs, fibroblast growth factor-8, and Cripto, founding member of the epidermal growth factor-Cripto FRL1 Cryptic family). For human embryonic stem cells, a potential source of human heart muscle cells with imminent importance for cardiac repair, the responsible cues and circuits remain conjectural. Defining steps that govern their recruitment to the cardiac fate is an urgent priority both intellectually, as a fundamental research question with inherent fascination, and pragmatically, as a possible step toward devising more efficient means to create human heart muscle for therapeutic goals. Building on the Applicant's funded work on growth factor signals that drive cardiac myogenesis in mouse pluripotent cells, we propose: (1) To investigate the cardiogenic potential of endogenous and exogenous BMP signaling proteins in human embryonic stem cells, in relation to downstream growth factors, transcription factors, and cardiac structural proteins. (2) To study, analogously, the cardiogenic potential of endogenous and exogenous Wnt signaling proteins in the cells, and their interplay with the BMP cascade. (3) To test the hypothesis that cardiogenic differentiation in human ES cells requires down-regulation of transcriptional repressors that are associated with "stemness." [unreadable] [unreadable]