Basic Biology II
Humans and other mammals are unable to regenerate significant portions of muscles of the body or heart lost to trauma or ischemic injury, for example from motor vehicle accidents, surgery or heart attacks. Despite the fact that we know there are stem cells in our muscles, these do not suffice to replace the large amounts of tissue that can be lost in many types of injuries. Even more dramatically, in the heart, there is no significant ability to regenerate damaged tissue. Whereas humans may be said to have poor regenerative capacity, some animals have an extraordinary ability to replace damaged or lost body parts such as a limb, a jaw or a large part of the heart. Such regenerative vertebrates include urodele amphibians (salamanders and newts) or fish such as zebrafish. These animals excel at regeneration by utilizing mechanism that is distinct from classical stem cell based regeneration as we currently understand it in mammals. Regeneration in newts and zebrafish is based at least in part on dedifferentiation and proliferation of lineage-committed cells to regenerate skeletal muscle and the heart. Dedifferentiation is thought to provide large numbers of progenitor cells that proliferate at the site of an injury or an amputation and contribute to the regenerating structure. By contrast, resident stem cells that exist in humans and other mammals represent a small proportion of the total cells in a given tissue and it is easy to imagine how much proliferation would be required for these scarce cells to rebuild a complete organ such as the heart or a structure such as a limb. But what if we could recruit all of the cells at the site of an injury as our regenerative counterparts do? The overall goal of this proposal is to mimic the "regeneration by dedifferentiation" paradigm in human muscle cells and to enhance regeneration in vivo. While the basal growth control mechanisms in mammals such as ourselves are similar to those in regenerative organisms, we have added levels of complexity that, it is our hypothesis, contribute to suppressing regeneration. The goal of this proposal is to transiently target growth regulators that are present in mammals but conspicuously absent in regenerative organisms to induce differentiated cells such as cardiomyocytes to become progenitors that can expand at the site of an injury and then replace damaged tissue. This approach, if successful has the distinct advantage of mimicking a process that already exists in nature and as such may be readily transitioned to medical approaches in humans.
Statement of Benefit to California:
Humans are unable to regenerate significant portions of muscles of the body or heart lost to trauma or ischemic injury, for example from motor vehicle accidents and surgery or from heart attacks. Despite the fact that we know there are stem cells in our muscles, these do not suffice to replace the large amounts of tissue that can be lost from in many types of injuries. Even more dramatically, in the heart, there is no significant ability to regenerate damaged tissue. In California, as elsewhere, debilitating injuries and heart disease take a staggering toll on patients, their families and on health care spending. The research described in this proposal has significant potential to benefit Californians. First, and foremost, the experiments are aimed at improving our ability to regenerate skeletal muscle and heart. If this research is successful, Californians will directly benefit from the translation to therapy. The people of California will be the first benefactors of new solutions generated by this CIRM funded research.
EXECUTIVE SUMMARY The goal of this proposal is to investigate methods for inducing de-differentiation of mature skeletal and cardiac muscle cells that then could contribute to repair of damaged muscle. Based on evolutionary considerations, comparing mammals to vertebrates that readily regenerate several organs, the applicant hypothesizes that inhibiting two tumor suppressor genes will promote muscle de-differentiation. In Specific Aim 1, the applicant proposes to test this hypothesis using RNA interference in primary human skeletal muscle cells, in murine cardiomyocytes, and possibly in cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs). In Aim 2, the applicant will test the hypothesis in mice by local viral delivery of shRNA constructs and in a genetic model. Finally, in Aim 3, the applicant proposes to test additional inhibitors and vary the timing of gene suppression in order to optimize de-differentiation in human muscle cells. Reviewers agreed that the development of strategies to induce regeneration of skeletal and cardiac muscle tissue is an important goal in stem cell biology and for regenerative medicine. However, they raised questions about the scientific rationale for several key aspects of the proposal. One major concern was that the genes being targeted are tumor suppressors that regulate pathways implicated in muscle tumors. It is possible that inhibiting these genes may lead to a dramatic increase in tumorigenesis. In addition, reviewers questioned the applicant’s decision to study both skeletal and cardiac muscle. They noted that there are significant differences in the differentiation of these two tissues that may not allow results from one system to easily translate to the other. In addition, skeletal muscle exhibits a significantly greater capacity for regeneration in response to injury and harbors a well-characterized resident stem cell population. Reviewers found the proposal to be innovative but highly speculative. They did not find the “evolutionary rationale” described by the applicant to be well supported by data. They also expressed some doubts that suppression of a single molecular pathway could promote sufficient de-differentiation to enable significant tissue repair. Considering these limitations, reviewers found the proposal to be very high-risk. Reviewers described several weaknesses in the research plan. In general, they found it to be overambitious and lacking sufficient detail. Reviewers noted that Aim 1 describes a very complex and time consuming set of experiments involving many muscle cell lines and many treatment and analysis parameters. Taken together with the need for optimization, this is an enormous amount of work. Given the ambitious nature of this Aim, reviewers expressed concern that no preliminary data on cultured human cells or on cardiac cells were included, and that some of the needed shRNA and reporter constructs are not yet in hand. Furthermore, they were surprised by the applicant’s hesitation regarding the availability of hESC- or hiPSC-derived cardiomyocytes, as such cells should be obtainable. Reviewers were concerned that Aim 2 might also be overambitious. It is unclear whether the applicant has generated the mouse model to be used in this Aim. If not, a significant amount of time will be required to characterize this genetically complex model. Reviewers also worried about the efficiency of the proposed viral shRNA delivery method and the timing of shRNA expression. Finally, reviewers found Aim 3 to be severely underdeveloped relative to the other Aims. They would have appreciated greater experimental detail for this Aim, including clarification if, and if so, how human cardiac cell samples would be obtained. The reviewers described the applicant as a junior investigator with a modest publication record. They were concerned that the research team does not have sufficient expertise to carry out the proposed studies. They noted that several senior researchers are named as collaborators in the application but letters of support are not provided. Overall, while reviewers appreciated that this proposal addresses important goals in regenerative medicine, they raised a number of concerns about the scientific rationale and research plan that caused them to doubt its feasibility.