Basic Biology III
Embryonic stem cells are pluripotent (i.e. they can be differentiated into any adult cell type). Recent, major breakthroughs have been made into making adult cells (e.g. fibroblasts from skin) pluripotent (called induced pluripotency). The most successful method in humans involves introducing genes that confer pluripotency. Other methods are the use of proteins or drug-like compounds. However, all of these methods have disadvantages and are not yet safe for human use. Physical stimuli that alter cell function are called biophysical stimuli. These include mechanical manipulation, fluid flow, temperature changes, change in pressure of oxygen, and electromagnetic pulses among others. While it is known that biophysical stimuli can differentiate stem cells into a variety of cell types almost nothing is known about the reverse: i.e whether biophysical stimuli can induce pluripotency or a more stem-like behavior in adult cells. If we can induce pluripotency through biophysical means, several bottlenecks in tissue regeneration, gene therapy and drug discovery can be resolved. Our laboratory already has preliminary results showing electromagnetic pulses, controlled heating, and low oxygen can affect and reverse cell differentiation. In this proposal we aim to establish the effect of select biophysical stimuli on manipulating cell differentiation states towards a more stem cell-like nature, to identify potential non-genetic mechanisms by which these stimuli may act, and to determine whether these stimuli can enhance existing methods that are used to induce pluripotency. This will be achieved through 3 aims. Aim 1 is to establish the effects of mechanical stimuli, fluid flow and perfusion, electromagnetic fields, hypoxia and controlled heat shock on markers of differentiation in differentiated and undifferentiated embryonic stem cells and adult cells. Aim 2 is to determine how the biophysical stimulation affects the non-genetic regulation of cell differentiation. Aim 3 is to enhance existing methods of reprogramming of cells to become more stem-like by combining biophysical stimuli with genetic factors. Specific biophysical stimuli will be selected to substitute for one or more genetic factors as well as to enhance overall reprogramming efficiency. Reprogramming of cells through biophysical stimuli will lead to a clinically safer methods of generating stem cell-like cells from adult cells. Reprogramming can be achieved remotely or in situ if non-contact biophysical stimuli such as electromagnetic pulses are effective. Targeting reprogramming of specific cell types or reprogramming of partially differentiated cells may be possible and even desirable since many clinical applications do not require fully pluripotent cells. Our approach may also enhance the efficacy of existing methods of reprogramming or in parallel and in combination could result in more clinically acceptable treatments.
Statement of Benefit to California:
California has been at the forefront of biomedical research for more than 40 years and is internationally recognized as the biotechnology center of the world. The recent debate over the moral and the ethical issues of stem cell research has hampered the progress of scientific discoveries in this field, especially in the US. CIRM is a unique institute that fosters ethical stem cell research in California. CIRM has also serves as an exemplary model for similar programs in other states and countries. This grant proposal falls under the mission statement of the CIRM for funding innovative research. The proposal will utilize novel techniques for reprogramming of pluripotency in somatic cells. The proposed methods are likely to be increase the clinical safety and enhance the efficiency of current techniques. This proposal will also expand the field in a new direction and integrate multidisciplinary methods. If successful, this will further validate the significance of the CIRM program and will help maintain California's leading position at the cutting edge of biomedical research. Reducing the medical and economic burden of large numbers of patients who will benefit from this technology is of great significance.
Project Synopsis: In this proposal, the Principal Investigator (PI) will explore the relationship between biophysical stimuli, epigenetic regulation, and maintenance of stem cell pluripotency. This project is based on data indicating that biophysical stimuli, including mechanical stress, oxygen tension, temperature, and electromagnetic pulses, can significantly affect differentiation to a variety of cell and tissue types. In Aim 1, the PI proposes to investigate how markers of pluripotency and differentiation are affected by biophysical stimuli. In the second aim, the PI proposes to examine how biophysical stimuli influence epigenetic pathways that regulate pluripotency. In Aim 3, the PI proposes to enhance the process of cellular reprogramming to pluripotency by combining biophysical stimuli with transcription factor transduction. Significance and Innovation: - Establishing a clinically relevant cell source and improving efficiency of reprogramming to pluripotency are important goals; however, some reviewers felt that the development of a new reprogramming method based on the use of biophysical forces would not constitute a major unresolved need of the field. - The reviewers expressed mixed opinions about the study╒s potential impact; some believed the proposal might advance the production of induced pluripotent stem cells (iPSCs), while others were not convinced that the project would have a significant impact on the field. - Some reviewers believed the examination of biophysical forces on pluripotency efficiency was a novel investigation, but others judged the proposal as not particularly innovative and too phenomenological. - The proposed studies were mechanistically focused. Feasibility and Experimental Design: - This proposal lacked adequate organization and the quality of the experimental design was problematic. - The proposal reports a series of observations without appropriate quantification. More details and thorough preliminary data, as well as appropriate statistical analysis, are needed to draw proper conclusions regarding whether or not biophysical stimuli affect the regulation of pluripotency. - While the proposed experiments followed a logical progression, the entire project would be unsuccessful if the biophysical data on pluripotency induction (in Aim 1) were not supportive. - Some reviewers felt that the specific aims were not designed logically and that alternative approaches were not adequately explored, whereas others believed the specific aims and alternative approaches were appropriate. - The research environment was judged as appropriate for the proposed study. Principal Investigator (PI) and Research Team: - The PI has an extensive track record in the cartilage biology field. - Though the quality of the preliminary data indicated this team is capable of culturing human pluripotent stem cells, there was concern that the core research team may lack adequate expertise in pluripotent stem cell biology, particularly in assessing functional outcomes of pluripotency. Responsiveness to the RFA: - The proposal was responsive to the goals and criteria of the RFA.
- A motion was made to move this application into Tier 1, Recommended for Funding. Reviewers provided a brief synopsis of the application and then discussed whether the uniqueness of this approach to reprogramming merited funding despite the scientific concerns of the panel. The panel noted that reviewers were concerned about the quality of the preliminary data but acknowledged that the methylome analysis was well done. Some reviewers thought this a novel approach to reprogramming that may be effective while others were unconvinced that a new technology for reprogramming was needed for advancing the field. The motion failed.