Public Abstract:
One of the most potentially powerful aspects of regenerative medicine is stem cell therapy. In this therapy, healthy tissues derived from stem cells will be implanted into patients with damaged tissue in order to restore function. However, there is currently a risk of immune rejection. Induced pluripotent stem cells (iPS) have the potential to revolutionize regenerative medicine. By reprogramming a patient's own cells into pluripotent stem cells, stem cell therapies can be performed with little to no risk of rejection. However, this nuclear reprogramming process is not well understood at a mechanistic level. Also, all procedures developed to date use cancer-related genes. This has raised fears that current iPS cells could potentially have a high cancer risk if used therapeutically. In this proposed research, we intend to study the reprogramming process and determine if and when deleterious mutations are occurring. We will use the obtained knowledge to develop safer methods of generating iPS cells.
We will perform large-scale screening of mutations that have potential deleterious effects in iPS cells reprogrammed with a number of different methods. For the mutations found, we will also use a highly sensitive assay to quantify the frequencies of these mutations in the intermediate stages of the reprogramming. These information will help us to understand how do the mutations occur and propagate. Finally, we seek to develop a method for deriving genetically safe iPS cells by manipulating the activities of the mutated genes during the reprogramming process.
This proposed project will not only help us to gain additional mechanistic insights on nuclear reprogramming, but also allow great progress towards functional stem cell therapy, as safe IPS cells will be available for therapeutic use.
Statement of Benefit to California:
Over the past decade, California has made great progress in stem cell research thanks to Proposition 71. Research into stem cell properties and applications has made the promise of regenerative cell therapies almost a reality. Induced pluripotent stem (iPS) cells offer the promise of treatments or cures for diseases that affect millions of people without a risk of immune rejection, including Alzheimer's disease, heart disease, organ failure, and spinal cord injury. However, before these cells can be used therapeutically in the clinic, a better understanding of the mechanisms of generating iPS and the safety of the resultant cells must be gained. Our work will greatly improve understanding of the reprogramming process with respect to genomic mutations and integrity by determining the relative safety of a variety of available iPS reprogramming techniques. We will work towards creating iPS reprogramming methods that have been proven to be non-tumorigenic. Through this research, we hope to clear one of the biggest hurdles stopping iPS cells: the risk of cancer. Due to the huge potential of stem cell therapies in regenerative medicine, this work has applications to a large number of diseases and genetic disorders that affect Californians, from infants to senior citizens.
Review Summary:
EXECUTIVE SUMMARY
The goal of the proposed research is to investigate a little-studied aspect of cellular reprogramming: the possible production of deleterious genomic mutations that may occur during the generation of induced pluripotent stem cells (iPSC). The applicant will use a targeted genomic sequencing method to characterize iPS-specific mutations that occur during nuclear reprogramming. Further experiments will characterize the frequency and timing of mutations and assess whether mutations are selected for during the reprogramming process. Additional studies will investigate whether modulating the expression of mutated genes will improve reprogramming efficiency and reduce mutational frequency, thereby enhancing the safety of cellular reprogramming.
Reviewers believed that this proposal addresses a novel aspect of cellular reprogramming and investigates the important problem of genetic integrity of reprogrammed cells. They also recognized some substantial preliminary data, which supports the existence of such mutations in iPSC. However, reviewers were unconvinced about the significance of the problem as no functional or deleterious effects of the lesions have been demonstrated and, importantly, the ability to generate a viable animal from mouse iPSC indicates substantial genomic integrity of the cells.
The project was judged to be generally feasible with logical and achievable aims. It employs a sophisticated, state-of-the-art sequencing strategy previously developed and used by the PI in several large-scale projects. However, reviewers were concerned that the proposed approach would not actually allow determination of the mutation rate and frequency, rare mutations would not likely be detected by the proposed method, and alternative plans were not well developed. Furthermore, reviewers were unconvinced that the hypothesis concerning selection of mutations during reprogramming would be adequately and appropriately tested by the proposed experiments.
Reviewers found the PI to be highly qualified to carry out the proposed research. He/she has expertise in the relevant experimental approaches and a good publication record. The research team appears capable and qualified, and a named collaborator brings appropriate expertise in iPSC technology.
In summary, this is a novel proposal to investigate genomic instability resulting from cellular reprogramming. Strengths of the proposal include the potential importance of the problem, substantial preliminary data, and the expertise of the PI. Weaknesses include major concerns about the functional significance of the study and important aspects of the project’s feasibility.
