Advances in human stem cell technology have ushered in a new era of regenerative medicine. Researchers have recently reported the ability to generate induced pluripotent stem cells (iPS) from human somatic tissue. Patient- and disease-specific iPS cell lines will be valuable tools for the research and medical communities. For example, disease-specific iPS lines could help researchers understand the molecular pathogenesis of a specific disease and screen potential drug candidates. Optimized methods for developing patient-specific iPS lines would allow scientists and clinicians to transplant tissues with minimal risk of complications associated with immune rejection.
However, many technical hurdles need to be addressed before the potential of stem cell technology can be fully realized. Improved technologies for producing normal and disease-specific iPS cells in a rapid, efficient, cost-effective manner are necessary. Our proposal aims to solve many of the technical challenges associated with patient- and disease-specific iPS production. Specifically, we are proposing to optimize and scale-up proof-of-concept studies for application in disease-specific iPS cell production. To this end, we are proposing the following specific aims:
(1) Collection of disease-specific tissues from patient samples. Disease-specific tissues will be collected from patients. These studies will be undertaken using cardiovascular disease as a pilot disease model.
(2) Development of disease-specific iPS cells. We have developed protocols for iPS cell production from human neonatal skin-derived cells. Results from these proof-of-concept studies have been published. We propose to scale-up and optimize these existing iPS protocols for the generation of disease-specific iPS cells.
(3) Characterization of disease-specific iPS cells. We are proposing to study the heterogeneity of human disease-specific iPS cells, using molecular and cellular techniques. Following this characterization, we will attempt to identify surrogate markers that can be used to eliminate embryonic stem cell-like, non-pluripotent cells from iPS cells. These surrogate markers will be used to prospectively identify and isolate pluripotent iPS cells, and will contribute to the overall efficiency of the iPS methodology.
The iPS lines that are generated as part of this project will be available for use by cardiovascular disease researchers. More importantly, these optimized methods will enable additional disease-specific iPS lines to be generated in an efficient and cost-effective manner. The methods and tools developed as part of this project will be broadly applicable to disease etiology studies and will ultimately accelerate the pace of medical research.
Statement of Benefit to California:
The State of California, like the entire United States, faces significant challenges to its health care system. Health care costs are rising dramatically within California and the United States. Health spending in California reached $169 billion in 2004, or 11 percent of the state’s economy (California Healthcare Foundation, 2006). These health care costs will continue to rise as California’s population ages and requires treatment for chronic diseases, such as cancer, cardiovascular disease, diabetes, Alzheimer’s disease and others. Clearly, there is a need for improved technologies and tools that can be used to address this pressing state and national concern.
Advances in human stem cell technology have ushered in a new era of regenerative medicine. In theory, stem cell technology could ultimately be used to treat chronic disorders, regenerate patient-specific tissues, screen new drug candidates, and shed light on the molecular pathogenesis of disease. Researchers have recently reported the ability to induce pluripotent stem cells (iPS) from human somatic tissue. The continued development of patient- and disease-specific iPS cells will be a valuable tool for studying disease etiology and translating stem cell technology from the research lab to the medical clinic. However, there are many technical hurdles that need to be addressed before the potential of iPS technology can be fully realized.
Improved technologies for producing normal and disease-specific iPS cells in a rapid, efficient, cost-effective manner are necessary. Our proposal aims to solve many of the technical challenges associated with patient- and disease-specific iPS production. As part of this research, we will optimize the methods and techniques necessary to generate patient- and disease-specific iPS cells in an industrialized fashion. If successful, this work will benefit the State of California in four specific areas. First, iPS lines that are created under this grant will be made available to CIRM researchers. Second, the methods that are optimized as part of this grant will increase the efficiency and cost-effectiveness of iPS cell production. These methods will address a critical need in the field of stem cell research. Third, this work will enable novel research and therapies to be developed for diseases, which will ultimately improve the health of millions of Californians. Finally, the proposed project will stimulate the state economy and contribute to California’s standing as a leader in the biotechnology industry.
This application focuses on the development of disease-specific stem cell lines for investigating pathological mechanisms and for eventually translating basic stem cell research to the clinic. In the first aim, the Principal Investigator (PI) proposes to obtain tissues from patients suffering from various cardiovascular diseases. Induced pluripotent stem cells (iPSCs) will be generated from these tissues, and the efficiency of this process will be assessed as a function of culture condition and cell source. Finally, iPS cells will be characterized by a variety of criteria including gene expression profiling, long-term expansion capacity, karyotype stability, and genomic/epigenomic analyses.
The reviewers agreed that the proposed technology was aimed at an important roadblock in stem cell science, the need for efficient iPSC methodologies for producing stem cell disease models. Reviewers’ enthusiasm was diminished by the recent publication of a paper describing generation of iPS cells for several diseases (Park et al., Cell, 2008), but they agreed that there is still a great need for more efficient, broadly applicable technologies for developing disease-specific iPS cell lines. Importantly, however, reviewers commented that the application failed to provide a clear plan for implementation of the technology in this proposal. In addition, although the PI repeatedly uses the term “scale up” throughout the text, there doesn’t appear to be any intent to scale-up production of the cells in terms of numbers or volume. Finally, it was unclear why the applicant chose to focus on cardiovascular diseases and which cardiovascular diseases would be chosen, which diminished the reviewers’ ability to assess the project’s impact and feasibility.
Reviewers appreciated the straightforward, logical approach presented in this application, and felt that the characterization experiments were rigorous and robust. However, several weaknesses in the research plan diminished their enthusiasm. The PI did not provide sufficient details of the potential difficulties that might be encountered and what alternative approaches might be utilized. In addition, reviewers commented that the application did not adequately discuss controls – it was unclear what benchmarks the PI would use when assessing efficiency of reprogramming and other characteristics of the iPS cells to be generated. In addition to these omissions, the reviewers were uncertain that the described iPSC protocols would be translatable to other cell types, and it was unclear how non-pluripotent cells would be removed from induced cell lines. Finally, the PI did not adequately justify the assumption that naive stem cell sources would be more efficient for iPSC generation than those that are currently in use. The application did not take into account the possibility adult stem cells could be less efficient at reprogramming than previously used somatic, differentiated cell types, such as fibroblasts.
The reviewers noted that the research team members are in the early stages of their careers and do not have a long track record by which to judge their capabilities. The PI has direct, hands-on experience with iPSC generation from tissues, and the reviewers felt that this unique qualification represents a significant asset. However, there was some uncertainty as to who would be performing the actual experiments in addition to the PI. One reviewer felt that the team would greatly benefit from the inclusion of a pathologist.
The overall budget was considered to be appropriate, although the individual components were somewhat controversial. While the personnel costs were considered modest, certain supply requests were thought to be excessive. One reviewer indicated that the travel costs for the second year exceeded the maximum stipulated amount.
In summary, while acknowledging the need for improved iPSC technologies, the reviewers were not convinced that this effort would contribute significantly to the field. The proposal was straightforward but lacking in details, and both the proposed use and the general applicability of the described methods were questioned.