Basic Biology III
The understanding of the biological basis of stem cell differentiation into specialized cell types and 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, states is a promising avenue to cure fatal muscular diseases. Furthermore, the facile generation of large culture of muscle stem cells from skeletal muscles of patients affected by fatal neuromuscular diseases is important for in vitro disease modeling, behind the obvious direct therapeutic potential. 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:
This proposal is providing the basic knowledge that is necessary for the generation of muscle stem cells from embryonic or adult somatic cells, for translational purpose (cell mediated regeneration of diseased muscles) or in vitro modeling of neuromuscular diseases using patient-derived cells. As such, this study is an important step toward the applications of fundamental principles in stem cell biology to regenerative medicine in neuromuscular diseases. 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.
Project Synopsis: In this application, the Principal Investigator (PI) proposes to characterize the molecular network that controls the bidirectional transition between pluripotency and commitment to the skeletal muscle lineage. In the first aim, the PI will elucidate the mechanism by which a key myogenic regulatory protein extinguishes pluripotency in human embryonic stem cells (hESC) as they differentiate into muscle cells. The second aim focuses on an exploration of functional antagonism between regulatory factors governing pluripotency and differentiation. In the third aim, the PI will attempt to identify epigenetic signatures that predict generation of muscle stem cells suitable for therapeutic applications or disease modeling. Significance & Innovation: - The proposal is based on an intriguing but highly speculative hypothesis that the pluripotent and myogenic states are mutually antagonistic. This premise seems unlikely, as the regulatory programs governing those states are separated by considerable developmental distance. - The scientific rationale is based on insights gained from ectopic expression of transcription and chromatin remodeling factors. The significance of data that will emerge from such a contrived system is not clear. - The proposal is innovative within the context of myogenesis, but an opportunity has been missed to potentially look for more widely relevant control mechanisms that could be used to identify bidirectional controls in other differentiation pathways. - If successful, the proposed research could lead to improved methods for generating muscle cells from hESC or other cell sources. Feasibility & Experimental Design: - While the preliminary data support the validity of the applicant’s observations and the technical capabilities of the team, they do not convincingly support the assertion of functional antagonism apart from ectopic expression of powerful transcription factors. - Aim 3 is highly risky. There are no preliminary data to support the premise, and there are no experiments proposed to determine the functionality of the epigenetic signatures identified in muscle stem cells. - There is no discussion of potential difficulties or alternative strategies. - Some reviewers found the proposal difficult to read and noted excessively small figures. Principal Investigator (PI) and Research Team: - The PI has a strong track record in myogenesis and muscle stem cell biology and is well placed to coordinate this project. An effort level of 35% has been committed. - Two strong collaborators have been recruited to provide additional stem cell expertise and facilities, but their particular roles are not well defined. An additional collaborator provides important large-scale genomics expertise. - The research facilities are excellent and costs appear reasonable. Responsiveness to RFA: - The proposal utilizes various types of human stem cells and directly explores molecular mechanisms governing cell fate and identity. The application adequately and appropriately addresses the RFA.