The understanding of the biological basis of adult somatic cell reprogramming to a pluripotent state is opening new horizons in regenerative medicine and is a key step toward “personalized” stem cell-based therapies to treat diseases for which there is currently no cure.
Genetic neuromuscular disorders include a large number of diseases (e.g. muscular dystrophies) that invariably lead children to the wheelchair and eventually death. Likewise, the age-related decline in muscle mass and function has detrimental effect on the overall performance and life span in elderly populations. Stem cell-mediated regeneration of muscles in these patients is the most awaited therapeutic approach, but effective applications are limited by technical and ethical issues. Thus, the potential to reprogram diseased muscles back to early embryonic, stem cell-like state is a promising avenue to cure fatal muscular diseases. Quite paradoxically, no evidence exists that skeletal muscle cells could be reprogrammed into pluripotent stem cells with the existing technologies, suggesting that skeletal muscle cells are resistant to induced pluripotency.
Our preliminary evidence indeed indicates that skeletal muscles are refractory to induced pluripotency and points to an “epigenetic restriction” of pluripotency that is established during the commitment toward a terminal differentiated phenotype. This proposal will shed light on the mechanism underpinning the antagonism between the cellular factors that induce pluripotency and those factors and events that restrict pluripotency, promote differentiation into skeletal muscles and maintain the terminally differentiated phenotype typical of skeletal muscles.
This study will fill an important gap existing in our current knowledge on the basic mechanism that governs the transition between the pluripotency typical of stem cells and the cellular specialization of differentiated cells, and viceversa. The knowledge generated by our proposal will create the molecular rationale for the generation of muscle stem cells from hESCs and from skeletal muscles. As such, it will have a strong impact in the regenerative medicine for neuromuscular diseases, as it will provide the molecular insight to devise optimal strategies for stem-cell mediated regeneration in the treatment of muscular disorders.
Statement of Benefit to California:
The increased life span in the population of developing countries and states, such as California, poses a number of new issues related to the health control and social assistance in elderly population. For instance, the age-associated muscle atrophy (sarcopenia), and the muscle catabolism occurring as a consequence of chronic diseases (i.e. cancer cachexia, AIDS or chronic infections, terminal stages of cardiovascular diseases) or prolonged pharmacological treatments (i.e. chemotherapy) lead to a reduced performance, increased morbidity, and request for medical and social assistance for an increasing percentage of the Californian population.
Thus, the identification of pharmacological strategies toward regenerating aged or diseased skeletal muscles is a critical task for the development of future health strategies in California.
Furthermore, the identification of stem cell-mediated strategies in regenerative medicine will fuel hopes for the treatment of genetic neuromuscular diseases, such as muscular dystrophies, and will reduce the emotional, social and economic impact that patients confined to wheel chair have on public opinion and health.
More in general, the discovery of pharmacological applications for stem cell employment in neuromuscular diseases will help to establish a leadership in regenerative medicine, will inspire new technologies and will give the impetus to new initiatives attracting financial resources and a new generation of stem cell scientists in California.
The generation of stem cell scientists is particularly important to create and propagate in the future a productive environment fueling the research in regenerative medicine. This proposal will also be instrumental to train and commit to the stem cell research new MD and PhD scientists that will provide a valuable resource to propel the advances in regenerative medicine in California.
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.