Epigenetic reprogramming of a somatic cell into pluripotent stem cells has raised enormous interest in the scientific community because of the multitude of applications in basic biology and clinical research. Particularly important are the impacts on regenerative medicine, as reprogramming would allow the generation of patient-derived (and thus individualized) stem cells that could be used to differentiate into functional donor cells for transplantation therapies. For many years epigenetic reprogramming was only achievable in animals through somatic cell nuclear transfer (SCNT) into enucleated oocytes, a technically and logistically challenging and very inefficient procedure. To this day, epigenetic reprogramming through SCNT has not been successful in human despite great efforts. In August 2006, Shinya Yamanaka has reported for the first time that retroviral overexpression of 4 transcription factors is sufficient to induce pluripotent stem (iPS) cells from mouse fibroblasts. Similarly to the mouse cells, defined factor-induced reprogramming was then shown to work also in human cells. This paved the way for potential clinical application as for the first time patient-specific stem cells were created.
However, in both mouse and human systems the efficiency of reprogramming is extremely low. This indicates that scientists have to rely on uncontrolled events leading to reprogramming. Before we will be able to use these iPS cells in a clinical setting we will therefore have to have a much better knowledge of the reprogramming process to be able to improve the efficiency. Once we know exactly what is going on in these reprogramming cells, have absolute control over the process and can reach efficiencies close to 100%, the generation of human iPS cells from somatic cells will very likely be a safe procedure.
This research proposal addresses some fundamental questions why the reprogramming process is so inefficient. In this study we propose to optimize the expression of the reprogramming factors, to identify the best donor cell population for reprogramming, and finally to attempt to provide a list of new factors that can improve the reprogramming efficiency.
Statement of Benefit to California:
The benefits of this research to California are two fold: 1) it stimulates the economy by directly creating 5 jobs or salary support for California citizens and supporting California business because of regular purchase of consumables and research equipment. 2) At the same time the money is spent to support a research project that aims to improve the development of a novel type of pluripotent stem cells that could be used to treat a variety of diseases such as neurodegenerative diseases, diabetes, spinal chord injury, and genetic skin and muscle diseases.
The impact of such a novel stem cell therapy on the California health system would be enormous. The number of patients suffering from neurodegenerative diseases alone are stunning: Currently 1-2% of the population older than 65 years is diagnosed with Parkinson's disease a devastating disease affecting cognition and body movements and 20% of people older than 75 years suffer from Alzheimer's disease a progredient neurodegenerative disease leading to loss of higher cognitive functions.
Any progress towards relieving the major symptoms of patients suffering from these neurodegenerative diseases will be of immense benefit to the State of California and its citizens.
In addition, all the tools and reagents that we develop will be made widely available to Californian researchers and we have selected a California-based company for potential commercialization. We hope that California-based physicians will be at the forefront of developing this promising avenue of research. We expect that the money expended on this research will benefit the Californian research community and the tools and reagents we develop will help accelerate the research of our colleagues in both California and worldwide.
The goal of the research described by this proposal is to better characterize the process by which cells are reprogrammed to generate induced pluripotent stem cells (iPSCs) and to identify conditions and additional factors that increase reprogramming efficiency. In a first specific aim, the applicant will determine the optimal expression levels of the four established reprogramming factors. The second aim will be to analyze the influence of epigenetic state on reprogramming efficiency. In the third aim, novel factors that improve reprogramming efficiency will be identified using unbiased genetic screening approaches and analysis of candidate pathway and chromatin remodeling components.
This proposal addresses a significant problem, important for the use of iPSC in the development of cell therapies. However reviewer enthusiasm was weakened by their estimation that the proposed study would provide only incremental progress in elucidating mechanisms underlying cellular reprogramming. Additionally, reviewers felt that the proposal lacked particular novelty, since numerous other labs are currently pursuing the same questions. There was also concern that the project was overly ambitious in its plan to study both mouse and human cells.
Reviewers expressed significant reservations about the proposal’s feasibility and experimental design. Although the significant amount of preliminary data was recognized as a key strength, reviewers felt the proposal was very open ended, extremely ambitious and unlikely to be completed within the time proposed, and highly unlikely to be accomplished using the resources requested. Reviewers were concerned with the lack of compelling rationale for many of the proposed approaches and aspects of the experimental design. For example, the heavy emphasis on number of retroviral integration sites and the choice of cells for reprogramming studies were inadequately justified. Furthermore, it was unclear to reviewers how the PI would use knowledge of the optimal ratio of each of the ectopically expressed transcription factors to generate more efficient reprogramming and whether this information would provide new insights into the mechanism of reprogramming. A further concern was the lack of adequate experimental detail in the description of the third specific aim.
The applicant is a newly independent scientist who is extremely well trained and who demonstrated excellent productivity as a postdoctoral researcher. The research environment for the proposed study is exceptional. The PI has also secured the collaboration of eminent scientists with his/her research team.
In summary, this proposal seeks to contribute to a better understanding of the reprogramming mechanisms and to identify novel factors that increase reprogramming efficiency. The project addresses a critical issue; however, the proposal suffers deficiencies in the experimental design and feasibility that severely diminished reviewers’ enthusiasm.