Tools and Technologies I
Heart failure can result from either myocardial ischemia or infarction, and is the leading cause of mortality in California, the United States and other developed countries. A potential therapy to repair the heart consists of transplant of cardiac cells which will replace the damaged or diseased heart. Successful transplants will require defining the best source of cells to use for transplant, and the kinds of cells to use , for example, cardiac muscle cells, or cells which will help to build new blood vessels. We will also need to be able to provide enough cells, which will require standardized protocols for cell amplification and purification. Another important factor will be techniques which help the cells to survive after being transplanted. To define the optimal cell type, and how to amplify them, it is important to have a model system for human cardiovascular cells. Human embryonic stem cells (hESCs) are an ideal model because they can be amplified to large numbers, and they have the potential to become a number of different cardiac cell types. Not only are hESCs an ideal experimental model, they may themselves be able to provide cardiac cells for heart repair. Building functional heart tissue requires interaction between a number of different kinds of cardiac cells, including muscle cells and blood vessel cells. To investigate the potential of these different cell populations for cardiac repair, it will be important to be able to identify them and purify them from hESC cultures. This will require generating some model hESC lines which will make it easier to identify certain cardiac cell populations, and to work out conditions for growing large numbers of them. To generate these model hESC lines, we need to develop technologies so that we can specifically "knockin" some reporter genes (usually genes which make the cells a particular fluorescent color) to mark specific cardiac cell populations. These reporters are expressed when hESCs differentiate to specific cell types. Fuorescently marked cells can then be easily "seen" with a fluorescent microscope, and purified, based on sorting methods which recognize the fluorescent marker. The diseased heart is a relatively hostile environment for transplanted cells, so they do not survive very well. It is necessary to create a more friendly environment for cell transplants, by adding factors which improve survival, and by providing a more friendly environment using biomaterials to deliver the cells. Our proposed studies will try to improve several technologies required to repair diseased heart with cell transplants. We will optimize technologies to generate specific reporter hESC lines. We will also define specific growth conditions which will amplify specific cardiac populations, and that can be scaled up. We will also investigate methods for transplanting cells into infarcted heart in rat model systems, to optimize cell delivery and cell survival.
Statement of Benefit to California:
Heart failure can result from either myocardial ischemia or infarction, and is the leading cause of mortality in California, the United States and other developed countries. One potential therapy for heart failure is replacement of damaged cardiac tissue with healthy tissue, by cell transplant. Our experiments will develop several technologies that will be required to investigate the potential of different cardiac cell types for use in cell transplant, to amplify appropriate donor cell populations,and also to improve their survival in the diseased heart.
This application is focused on developing improved methods for isolating, culturing, and delivering therapeutic cardiomyocytes to the injured or diseased heart. To achieve these ends, the Principal Investigator (PI) proposes to genetically engineer human stem cell lines to express fluorescent biomarkers under the control of promoters that are specific for the cardiomyocyte lineage. Using these biomarkers, the PI proposes to develop scalable culture conditions for optimal cardiovascular differentiation. Finally, the derived cardiomyocytes will be combined with a scaffold and survival cocktail and delivered directly into infarcted rat myocardium. Histological and functional assessments will be performed to assess the validity of these methods. While acknowledging the need for improved tools for cardiac differentiation and delivery, the reviewers judged the impact of the proposed technology to be limited. They commented that much of the proposed work was not innovative, and proposed only incremental advances over previous work. Finally, one reviewer commented that the unmet technology need that the proposal addresses was unclear as the proposal was difficult to follow. Reviewers agreed that the research team is very well qualified, and the panel recognized the importance of performing this work in non-federally approved lines, enabling clonal analysis. However, all reviewers doubted that this overly ambitious project could be completed in two years. One reviewer noted that simply achieving the homologous recombination objectives would be a major contribution to the field. This reviewer also noted that other proposals focused on homologous recombination proposed only those experiments in the allotted two-year timeframe. In addition to unrealistic timelines, the proposal was poorly written and difficult to read, and the rationale and justification for many experiments were not provided. For example, it was unclear as to why the PI chose to examine one parameter as opposed to others, as a critical factor for gene targeting efficiency. The reviewers were disappointed with the preliminary data, which were largely redundant with published results. Concerns were raised over the proposed scaffold strategy, as this delivery vehicle is notoriously variable by lot. No alternative scaffolds were proposed. Such undefined, uncontrollable variation could severely confound the interpretation of the proposed experiments. Finally, one reviewer expressed concern that the chosen myocardial infarct model might limit the significance of the results. The research team was judged to be extremely well qualified to conduct the described research. The PI is an established, talented scientist with many important contributions to the field of cardiac development. The co-investigator has extensive experience in molecular biology and genetic modification of hESC. The remaining team members have expertise in the appropriate areas, including use of scaffold delivery vehicles and cardiac infarct models. The research environment is outstanding. Overall, although the research team was felt to be talented, reviewers could not overcome concerns of feasibility with this over-ambitious proposal. Additionally, the reviewers were unconvinced of its potential to significantly advance the field of stem cell biology.