SYNOPSIS: The average 5-year survival rate for individuals with congestive heart failure is < 50%. Attempts to compensate for the loss of cells or cellular function have been largely unsuccessful. The Specific Aims in this proposal are designed to isolate cardiomyocyte committed cells from hES cells and administer them into animal models of mycocardial infarction (MI). Cardiomyocyte progenitors will be marked using GFP expression driven by a cardiomyocyte-specific promoter. Optimal culture conditions for driving progenitor cells towards myocardial differentiation will be identified, and the ability to image these cells and track survival in murine models will be examined in Specific Aim 2. In Specific Aim 3, the safety and efficacy of the differentiated, tagged cardiomyocytes will be tested in porcine models of MI. The ultimate goal of these studies is to develop a rational clinical protocol for testing hESC-derived cardiomyocytes (hESC-CM) as therapeutics for congestive heart failure. IMPACT AND SIGNIFICANCE: This study addresses an important medical problem related to the isolation of cardiac progenitors from human ES cells, directing their differentiation, and then studying their localization and fate with advanced imaging techniques in well-defined small and large animal model systems. The significance and interest are therefore high. The investigators have identified three issues of interest in bringing hESCs to myocardial repair and, while they do not imply that these are the only important issues, they focus their attentions on them. Hence, they propose: 1) in vitro enrichment of hESC-CM; 2) in vivo small animal multi-modality imaging of hESC-CM survival, and 3) in vivo large animal delivery and multi-modality imaging of survival and functional improvement with hESC-CM. The significance of these steps is that they impact first on making sufficient numbers of appropriately differentiated cells available for the purpose of implantation, and second that they consider means for imaging the localization of cells and their functionality in situ. The successful performance of these studies should have a major impact on both our understanding of the differentiation of hESCs into myocytes and on our abilities to study the fate of the cells and of the organs in which they reside. The approach is interdisciplinary, and incorporates existing strengths on site at Stanford. QUALITY OF THE RESEARCH PLAN: There are both positive and negative aspects in the research plan. On the positive side, the investigators clearly enunciate the general tendency in the field to confuse cells being differentiated towards a functional cardiac lineage and the need to actually obtain a cell line that functions as a surrogate/replacement for lost myocardium. They have a clear understanding of the markers to be used to follow cell differentiation, and a strategy via the use of embryoid bodies. Based on preliminary data they anticipate 30-50% purification will be attained for murine and porcine studies, respectively (although it is not clear why this isn’t greater). The development of the chip array cell differentiation system in collaboration with Pat Brown to establish the appropriate conditions to optimize hESC differentiation is a major plus. Also the use of in vivo MRI and PET imaging should provide the information needed about cell localization in the murine model. Finally, the studies of functionality in the porcine heart are well-described and appear do-able. Overall, the plan includes experiments that focus on important steps on the way to myocardial repair and assessment of its efficacy. The data gathered and the technology being developed all should be of use. On the negative side, while the goals of the study are admirable, and the plan is solid, focused and logical, it is not terribly inspired in terms of the cellular isolation and other approaches. The proposal for cell isolation would gain from further depth, preliminary data, and a better consideration of alternative approaches. It is entirely possible that the methods proposed will not result in a sufficient number of cardiovascular progenitors or their progeny to allow subsequent study in vivo. Again, the preliminary data here could be stronger. In addition, important and very relevant subject areas are not carefully considered (e.g. amount of rejection, migration of cells elsewhere in the body). These areas are beyond the intent and the time frame allotted, but perhaps could be addressed in later (or parallel) work. The study as written is extremely ambitious, and it is not clear that the goals can be fully realized within the time span of the proposal. Finally, the experience of the investigators in the human ES cell arena is rather limited to date, although the existing expertise is available on site at Stanford. STRENGHTS: While this is a high risk project, the goals are meritorious and the quality of the preliminary data suggests that one may be justified in taking the risk. The major strengths are the experimental plan itself, and the preliminary data provided, which do a great deal to assuage concerns. Specifically the results studying mES cells transfected with the Nkx2.5:GFP construct, and the tracking of meschenymal cells in the porcine system are quite strong. The PI has made a reasonable choice of the markers for cell isolation, though they are identical to that of others. That this investigator is a highly respected cardiovascular researcher strengthens the feasibility of the proposed studies. They have assembled a good team of cardiologists, cardiac surgeons, and imagers, and the environment for these studies, particularly the in vivo imaging studies, is very solid. WEAKNESSES: The overriding weakness of this proposal is that it is not clear that the experimental approach will result in the isolation of sufficient quantities of the cardiac cell types of interest with preservation of their differentiation potential to allow the completion of Specific Aims II and III. To the extent that either in robustness or number the study falls short, this is a major weakness. Further, there is nothing very original in the work plan or its execution. The aims are quite ambitious and somewhat diffuse, and the investigator may have underestimated the difficulty of accomplishing the first specific aim. Another concern is that the protocols, while strongly “result-oriented” are not as strongly “mechanism-oriented.” In other words, while there may be useful outcomes, obtaining such outcomes may not be strongly associated with an understanding of why they have come out in this fashion. If results are positive, this may not be seen as a compelling flaw. If they are negative, it is difficult to understand how the investigators will use what they have learned to come up with alternative approaches. DISCUSSION: The techniques in this proposal are beautiful, but the proposal is results-oriented and not hypothesis-driven. Insofar as the robustness and number of isolated cells is critical, there are no guarantees they’ll get what they need for the animal studies, and the proposal lacks sufficient consideration of alternative plans for cell isolation. The team and environment are outstanding, and the preliminary data are strong; however, Reviewers feel that the applicants have underestimated the difficulty of achieving success in Aim 1. Should future funding opportunities arise, it would be best to focus a proposal the goals related to Specific Aim 1, and to attack this problem with multiple alternative and/or synergistic approaches and a new set of specific aims.