Funding opportunities

EXECUTIVE SUMMARY The primary objective of this proposal is to promote first the proliferation and then the maturation of ventricular cardiomyocytes derived from human embryonic stem cells (hESCs) using co-culture with cardiac fibroblasts isolated from mouse embryos or adults. Upon reaching both of these goals in the two specific aims, putative microRNA (miRNA) candidates that may regulate cell cycle and cardiomyocyte maturation will be identified and functionally assessed. In Aim 1, the applicant proposes to investigate mechanisms promoting prolonged proliferation of hESC-derived early-differentiated ventricular cardiomyocytes. In Aim 2, conditions that promote progressive maturation of hESC-derived early-differentiated ventricular cardiomyocytes to an adult ventricular phenotype will be identified. Reviewers believed the significance of the proposal lies in its potential to improve cardiomyocyte differentiation from pluripotent stem cells and therefore may impact future development of stem cell-based therapies for myocardial repair. Reviewers also appreciated the mechanistic focus of the novel hypothesis that miRNAs play a role in the response of hESC-derived ventricular cardiomyocytes to signals from cardiac fibroblasts. However, the proposal did not contain any preliminary data relating to miRNAs, which reviewers felt severely weakened the rationale. Additional weaknesses of the preliminary data were identified; critical data, such as the purity of the cardiomyocyte population and the percentage of cells surviving selection, the purity of the fibroblast cultures and the percentage of cells expressing the various markers were not provided. Furthermore, a number of phenomena were presented only with descriptive terms such as “more” and “less”, thus lacking quantitative analysis, and other critical data were mentioned, but not shown. A reviewer criticized that key citations to relevant literature regarding proliferation studies of hESC-derived cardiomyocytes were noticeably absent from the discussion and list of references. The preliminary data would have been strengthened by the inclusion of information about gene expression in normal heart transitioning through development and a discussion of other environmental signals that influence cardiomyocyte proliferation versus maturation. With regard to experimental design, the specific aims were judged to be reasonable, but they lacked any mention of statistical analysis methods to be employed, thus limiting the ability to obtain definitive and sound conclusions. A reviewer recommended the proposal would be strengthened if conditioned media or transwell studies were included to address how fibroblasts affect cardiomyocyte behavior, and perhaps fibroblasts isolated from mutant mice could be used to dissect the progression of proliferative and maturational signals from fibroblasts to cardiomyocytes. The applicant is a recently appointed assistant professor, has been productive and has expertise in cardiac development in a model organism, but lacks stem cell biology experience. However, the assembled team is strong and has the necessary expertise to accomplish the goals. A reviewer pointed to some weaknesses in the budget justification, for example, the travel budget included graduate students, but none were listed as key personnel, and the personnel justification contained no details. In summary, although the proposal has the potential to impact our understanding of stem cell behavior and the ability to derive functional cardiomyocytes for therapeutic purposes, reviewers were not enthusiastic because of its weak rationale and insufficient support from the preliminary data.