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EXECUTIVE SUMMARY The goal of the proposed research is to elucidate the molecular pathways that contribute to human embryonic stem cell (hESC) differentiation into skeletal muscles, and the converse: the generation of induced-pluripotent stem (iPS) cells from skeletal muscles. The principal investigator (PI) has proposed three specific aims that will focus on the molecular and epigenetic basis by which a defined molecular complex participates in these processes. Aim1 will employ gene expression profiling, chromatin analyses and proteomic technologies to elucidate the mechanism by which this complex extinguishes the pluripotent state in hESC. For Aim 2, the PI will employ similar strategies along with depletion studies to explore how this complex acts as a functional antagonist between factors controlling pluripotency and muscle specification. Finally, the PI will explore the cellular mechanisms that contribute to the resistance of terminally differentiated human skeletal myotubes to induced pluripotency (Aim 3). Reviewers commented that the facile generation of myogenic lineage cells from pluripotent cell sources would be very useful for disease modeling and potentially for future therapeutic approaches. Strategies to efficiently generate skeletal muscle lineages from hESC are currently lacking, and this project is aimed at uncovering new strategies for achieving this. However, the need to convert myoblasts to iPS cells, as also proposed here, is difficult to justify for translational research purposes, since fibroblasts and keratinocytes are easily obtained and can readily undergo induced pluripotency. Nonetheless, there is good reason to study this process for purposes of basic research, as general mechanisms and principles that erase somatic gene expression patterns may be obtained. The significance of the research lies in a comparative analysis of differentiation (hESCs to myoblasts) and reprogramming (induced pluripotency of myoblasts) to determine if common mechanisms are utilized in these opposite outcomes. If successful, the impact of this work would be a better understanding of the molecular mechanisms of differentiation and reprogramming. Reviewers felt this application is based on an interesting, but somewhat speculative premise. While they agreed that much of the rationale stems from well-supported observations, they were not convinced that a role for the relevant molecular complex in silencing pluripotency had been adequately established. As a result, they were uncertain whether the proposed approaches would lead to useful data being generated in Aim 1. The reviewers also identified a number of weaknesses in the experimental design. While the description of Aims 1 and 2 seemed mechanistically focused, the actual experiments proposed are highly descriptive and rely almost exclusively on profiling. No discussion of how targets would be analyzed or prioritized was included, and the rationale for proposing extensive profiling experiments was not strong. Reviewers were disappointed that time points were not discussed for the analysis of differentiation and iPSC efficiencies, particularly given the recent published findings suggesting that perhaps all cells in a given population could become reprogrammed, if given enough time. Furthermore, the proposed pluripotency assessment lacked rigor, as it was limited to marker analysis without functional follow up or teratoma assays. Finally, some reviewers were unsurprised by the observation that myotubes would be resistant to induced pluripotency, given their multinucleate and post-mitotic state, and therefore questioned the rationale for pursuing their reprogramming in Aim 3. They also worried that the results for this aim would be difficult to interpret, as the experimental approaches, although creative, are complex and no preliminary data were provided to support the feasibility of this approach. The PI is an Assistant Professor with a strong publication record in the myogenesis field and relevant expertise with the proposed approaches. His/her work has contributed significantly to the understanding of the molecular mechanisms by which myogenic specification occurs. The 35% commitment of the PI to this project is reasonable to achieve the proposed aims. The research location is excellent, with good access to core facilities and to highly talented colleagues; the expertise of the research team is adequate for the proposed research.