Like embryonic stem (ES) cells, induced pluripotent stem (iPS) cells can differentiate into every cell type in the body, providing enormous potential for regenerative medicine. Unlike ES cells, the derivation of iPS cells is more straightforward technically, and can be performed on human adult cells. This potentially obviates the need for donated eggs or embryos, and permits the ability to generate patient-specific stem cells for disease research, drug development, and new cell-based therapies - generating great excitement in the scientific community as well as with the public.
iPS cells hold great promise for regenerative medicine, but the cellular signaling that controls their derivation and function remains poorly understood. We are developing methods to measure protein phosphorylation (the most common mechanism of cellular signaling) in iPS cells, and we will use the key signaling events we identify to improve the speed and efficiency of iPS derivation, as well as the safety and utility of iPS cells for regenerative medicine. In addition to improved iPS cell protocols that will benefit basic science and clinical therapy, the methods we develop to measure protein phosphorylation in will make a valuable diagnostic test for iPS and iPS-derived cells to determine their safety and functionality before use in patient-specific regenerative therapy.
Our proposal will benefit California in three important ways:
First, we will advance the field of stem cell biology as demanded by the people of California when they voted for Proposition 71, the California Stem Cell Research and Cures Initiative, on November 2, 2004 to establish The California Institute for Regenerative Medicine (CIRM). The mission of CIRM is to support and advance stem cell research and regenerative medicine under the highest ethical and medical standards for the discovery and development of cures, therapies, diagnostics and research technologies to relieve human suffering from chronic disease and injury. Our proposal will add new, essential knowledge concerning the function and molecular mechanisms of induced pluripotent stem (iPS) cells. The recent discovery of iPS cells has opened new frontiers in patient-specific regenerative therapy, the study of embryonic development and cellular differentiation, and the ability to create disease-specific cell lines for drug testing and the study of disease mechanism. Our study of the kinase signaling networks that control their derivation and function will add new, essential knowledge to the field of stem cell biology that will be published in a timely manner and readily available.
Second, the improved methods, protocols, and techniques we identify to control iPS cell derivation and function will be of great utility for the translation of iPS cells to the clinic, which will provide health benefits to all Californians. Our studies will identify new methods for iPS cell derivation that improve their safety for regenerative therapy by reducing their oncogenic potential. We will also develop new methods to control their differentiation into specified lineages or cell types for use in regenerative medicine. Additionally, the high-dimensional flow cytometry techniques we develop to analyze stem cell biology will be a useful quality control test to use on human stem and stem-derived cells before their use in regenerative therapy.
Third, our research will benefit California’s robust biotechnology industry, not only by improving regenerative therapy, but also by identifying improved methods for the generation of disease-specific cell lines for drug testing and the study of disease mechanism. Strengthening the California biotechnology industry benefits all Californians, not only through improved drugs and therapies that benefit their health, but also by bringing more business to the state, increasing tax revenues, and providing much-needed employment opportunities.
The overall goal of this proposal is to elucidate the signaling networks underlying pluripotency and cell fate transitions using novel phospho-specific flow cytometry methods developed in the applicant’s laboratory. In order to map this “geography” of network states, a series of 4 specific aims has been proposed. First, the applicant will characterize differences in kinase signaling between pluripotent and differentiated cells using phospho-specific multi-parameter flow cytometry approaches to compare fibroblasts, embryonic stem cells (ES) and cells in which pluripotency has been induced (iPS). Second, kinase signaling events will be evaluated for each stage of the reprogramming process whereby somatic cells are de-differentiated towards a pluripotent state. For the third Aim, the applicant proposes to identify conditions that improve the speed, efficiency, and safety of iPS cell reprogramming. Finally, the kinase signaling networks that are required for differentiation of ES/iPS into hematopoietic stem cells (HSC) will be identified.
The reviewers were largely impressed with the potential of this effort to have a major impact on our understanding of stem cell biology. The applicant’s flow cytometry platform was considered a key innovation, as it should enable the analysis of phosphoprotein signaling events in ways that have not been possible with current methods. As kinase signaling networks have not yet been extensively mapped in the context of cell fate, the reviewers were extremely enthusiastic that this effort could lead to highly significant and meaningful findings. The inclusion of an aim to study hematopoietic lineage specification was also acknowledged for its potential to add novelty. While confident that a large amount of data would emerge from these descriptive studies, some reviewers were uncertain as to how this would directly translate into a new understanding of pluripotency and cell behavior. Others questioned the extent to which the emphasis on mouse cells would be useful for informing human biology. Despite these minor concerns relating to scope, the reviewers were generally confident that this effort would substantially advance the field.
In general, reviewers appreciated the logical and straightforward research plan that was supported by extensive and compelling preliminary data. Importantly, these studies demonstrated that the technology and reagents were both ready and available in the proposed setting for addressing the questions at hand. Despite these merits, some reviewers worried that the focus of the proposed research was overly broad and did not include adequate consideration of potential pitfalls and possible solutions. One reviewer described the plan as a fishing expedition that emphasized breadth over depth. Another reviewer offered a more specific criticism, wondering why the applicant did not mention or consider using knockout strains for many of the mouse fibroblast reprogramming studies that were proposed for Aim 3. While these caveats were noteworthy, the reviewers nonetheless remained confident in the overall feasibility of this effort.
The reviewers unanimously praised the qualifications of the principal investigator (PI) as an internationally recognized pioneer in the technologies to be exploited. The capabilities of the entire research team were judged to be outstanding. Most reviewers attributed their enthusiasm for this effort to the unique expertise and track record of these investigators.
In summary, the proposed effort represents an innovative, ambitious strategy for addressing important fundamental questions in stem cell biology. Despite some minor concerns relating to scope, the reviewers were confident in its overall feasibility based on compelling preliminary data and the unique qualifications of the research team.