Optimization in the Identification, Selection and Induction of Maturation of Subtypes of Cardiomyocytes derived from Human Embryonic Stem Cells
Tools and Technologies I
Statement of Benefit to California:
This application focuses on the development of new and improved techniques for deriving homogenous, mature, and functionally relevant cardiomyocyte populations from human embryonic stem cells (hESCs). The applicant proposes to generate transgenic hESC lines expressing a fluorescent protein under the control of promoters or enhancers which are associated with specific cardiomyocyte (CM) subtypes. The analysis of temporal expression and persistence of these reporters will allow identification of key steps and stages of CM subtype differentiation. This information, together with the generation of antibiotic-resistant hESC lines, will facilitate isolation of cardiomyocyte subtype populations. Finally, the resulting cells will be characterized by analysis of their electrophysiological properties, and the role of extra-cardiac cells in the differentiation and maturation process will be investigated. The reviewers agreed that this straightforward but unique effort could be of great impact, particularly in the field of cardiovascular stem cell research. This proposal directly addresses key roadblocks in this area, including the difficulty in deriving sufficient numbers of homogenous, pure, specific cardiomyocyte populations with mature functionalities. These studies could offer new insights as to what defines functional maturity as well as a powerful strategy for isolating and scaling up the production of these cells. In contrast to current methodologies, the proposed strategies are not labor intensive and would likely not compromise cell viability. Finally, while potentially useful for regenerative medicine, the resulting cells also could be extremely valuable for future drug screening efforts. In terms of feasibility, the reviewers were confident in the qualifications of the research team. The proposal was well written and conceived, and the applicant provided appropriate discussion of expected results, potential hurdles, and alternative methods that might become necessary. Furthermore, the preliminary data made a strong case for the overall feasibility of the selection scheme. Reviewers expressed some concern that the project was overly ambitious and that some plan details (e.g. the use of single promoter expression strategies and the heterogeneity of embryoid bodies) would prove problematic. However, the significant preliminary data and the overall simplicity and elegance of the approach, convinced reviewers of its potential to substantially impact the field. The investigators are very well qualified to pursue the objectives of this proposal. The principal investigator has an excellent track record. The collaborators bring exceptional expertise in all relevant areas, including stem cell biology, cardiac differentiation, antibiotic selection, electrophysiological profiling, and lentiviral gene targeting. The budget was judged to be fairly appropriate, although proposed travel expenses and consultant fees appeared somewhat excessive. Overall, this is a well written application that addresses several key roadblocks in stem cell biology. Despite some minor flaws, the reviewers were optimistic that this simple but elegant approach would prove useful and productive.