Basic Biology II
$1 423 603
The possibility of generating patient-specific pluripotent stem cells has tremendous utility for treating patients with a wide variety of diseases. Recently, it has become possible to derive cells from patients and to subject them to a process known as cellular reprogramming to form induced pluripotent (iPS) cells specific to that patient. This technology, however, currently has some limitations. First, the reprogramming process is not very efficient. Second, the process required to reprogram cells entails introduction of genes that can promote cancer. Therefore, it is a critical goal to define the mechanisms that control the ability of cells to undergo reprogramming, to provide alternate strategies to generate iPS cells more efficiently and more safely. One approach recently demonstrated to enhance the efficiency of reprogramming is through inactivation of a gene known as p53 in the cells to be reprogrammed. Inactivation of p53 also allows iPS cells to be generated without the introduction of cancer-causing genes. Despite increasing efficiency of iPS cell formation, p53 inactivation also has some limitations, as it can cause chromosomal instability in the cell, contributing to cancer development itself. P53 affects a vast network of proteins in the cell, and therefore, if select components of that network could be targeted instead of p53 itself, it might provide a means to increase the efficiency of reprogramming without causing genetic instability and promoting cancer. Here, we propose to identify mediators of p53 function in limiting reprogramming to determine if it is possible to uncouple this function from its ability to maintain chromosomal stability. In this way, we could devise strategies to achieve an enhancement of reprogramming efficiency without the deleterious consequences of compromising chromosomal stability and promoting cancer. Inactivation of such key p53 reprogramming mediators may also allow generation of iPS cells without introducing cancer-causing genes, as does p53-deficiency. Thus, these experiments may lead to both improved efficiency and safety of reprogramming.
Statement of Benefit to California:
The use of stem cells to regenerate and restore tissues is an important strategy for treating a variety of diseases. However, a limitation of this strategy is the possibility of immune rejection of foreign cells by an individual’s body. Therefore, generation of stem cells specific to a given patient, which will not be rejected by that individual’s body, holds great promise for the treatment of a variety of human diseases. Recent technology called cellular reprogramming allows the conversion of cells derived from an individual into stem cells that can be used for therapeutic purposes. This technology still needs further development, in the sense that it is inefficient and not adequately safe. In this grant we explore the ways in which this process is controlled, with the aim of enhancing the efficiency and safety of this process, to facilitate its therapeutic applications. This technology should benefit Californians of a variety of ages, with a broad spectrum of diseases ranging from neurodegeneration to diabetes.
EXECUTIVE SUMMARY The goal of the proposed study is to understand how the tumor suppressor p53 inhibits cellular reprogramming to improve the derivation of induced pluripotent stem cells (iPSC). This protein is a key cellular regulator of cell cycle progression, senescence, genomic stability, and programmed cell death. As p53 inactivation has been previously shown to promote reprogramming, precise regulation of p53 might improve efficiency of iPSC derivation. The central focus of the planned research is on identifying p53 target genes specifically active in reprogramming and distinguishing these from targets involved in cellular senescence and tumor suppression. The first specific aim will be to use cells derived from mutant mouse strains to identify genes whose activation by p53 inhibits reprogramming. In a second aim, p53 targets important for iPSC reprogramming and cellular transformation will be assessed in human fibroblasts. Additionally, microRNAs regulated by p53 and involved in reprogramming will be identified. The proposed research is concerned with the important goal of improving the efficiency and safety of cellular reprogramming. However reviewers questioned the overall impact and significance of the study, since others have shown that inhibition of p53 is not required for reprogramming, and generation of iPSC has already been achieved without the need for the oncogenic reprogramming factors, cMyc and KLF4. Additionally, although the mutant mouse strains and fibroblast lines were recognized as unique and useful tools, the proposed approaches and methodology were not viewed as particularly innovative. In general, reviewers viewed the proposed study as technically feasible and following a logical research plan. However, a significant concern is that the entire project depends on results from the initial experiments and on a positive finding that p53 mutants will have differential capacities for restricting iPSC generation efficiency; preliminary data supporting this question would have strengthened the proposal. In addition, reviewers were concerned that p53 target genes may differ between mice and humans and that these and other significant differences between species could severely diminish value of the proposed study. The Principal Investigator is highly qualified, with significant expertise in the field and an impressive record of productivity. Reviewers viewed the PI as a major strength of the proposal. Additionally, the proposed research team, collaborators, and institutional environment were judged as excellent in quality and appropriate for the project. In summary, this proposal addresses the role of p53 in cellular reprogramming. Strengths of the proposal include the well-qualified PI and the previous generation of mouse strains with interesting lesions in p53. Weaknesses include serious reservations about the study’s significance and value in advancing the field and deficiencies with regards to important aspects of the project’s feasibility.