Basic Biology II
Nearly 5 million people in the US are afflicted with heart failure with an additional 550,000 new cases diagnosed each year. Despite current treatment regimens, heart failure still remains the leading cause of morbidity and mortality in the US and developed world because of the inability to adequately replace lost ventricular myocardium from myocardial infarctions/ "heart attacks." Thus, cell-based therapies to replace lost or damaged ventricular myocardium hold great potential. In addition, potential therapies for heart failure and other cardiac diseases would also benefit from an experimental human ventricular cardiomyocyte model. Progenitor cells which can differentiate into various myocardial cell types and include human embryonic stem cells (hESCs), human induced pluripotent stem cells (hiPSCs), or adult human stem cells, can potentially address both of these therapeutic needs. Although progenitor cells can be differentiated into immature myocardial cell types, the failure to expand sufficiently and fully differentiate them into mature and functional ventricular cardiomyocytes has remained a major bottleneck in realizing the potential of human pluripotent stem cell (hPSC)-derived cardiomyocytes for human cardiac regenerative repair and in vitro modeling of adult human cardiac diseases. To overcome limiting numbers of progenitor cell derived cardiomyocytes, a major thrust of investigation has been directed toward increasing the efficiency with which a pluripotent cell adopts mature cardiomyocyte cell fates. In this proposal, we will perform complementary studies to enhance the yield of mature and functional ventricular cardiomyocytes by defining factors which promote their expansion. Additionally, we will also identify factors which promote maturation of early differentiated ventricular cardiomyocytes. Because of its great therapeutic potential, we will focus on defining microRNAs which can promote either proliferation or maturation. Toward this end, we have developed a novel system that allows us to specifically monitor in real-time hESC-derived cardiomyocytes as they proliferate and mature into functional ventricular cardiomyocytes after treatment with identified microRNAs . Overall, understanding these basic mechanisms which lead to expansion of early differentiated ventricular heart cells and their maturation will ultimately provide novel approaches towards creating a safer and more functional source of ventricular myocardial replacement for injured ventricles in heart failure patients. Additionally, although we are utilizing hESCs as our model system, it is likely that these basic mechanisms that we identify will also be applicable to cardiomyocytes derived from other potential cellular sources such as hiPSCs or other progenitor cell populations.
Statement of Benefit to California:
Nearly 5 million people in the US are afflicted with heart failure with an additional 550,000 new cases diagnosed each year. Despite current treatment regimens, heart failure still remains the leading cause of morbidity and mortality in California, US and developed world because of the inability to adequately replace lost ventricular heart cells from myocardial infarctions or "heart attacks." Thus, the goal of our experiments is to address this major roadblock by understanding the molecules that are required to instruct human stem cells to expand and become mature and functional ventricular heart cells. These studies have the real potential to not only revolutionize our understanding of creating ventricular heart cells but also provide a safer and more functional source of heart tissue replacement for the many citizens of the State of California who suffer from heart failure.
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.
- Ali Brivanlou