Heart disease is the leading killer of adults in the Western world. In addition, congenital heart malformations are the most common birth defects, occurring in nearly 1% of the population worldwide. A major goal of regenerative medicine is to repair damaged heart tissue. Our research is focused on developing new methods to stimulate human embryonic stem cells (hESCs) to form specific tissues to repair diseased heart tissue. There is growing optimism that cell-based therapies can repair damaged hearts. Although heart regeneration is minimal in humans, many animals show remarkable heart regeneration in response to a wide variety of injuries. Several clinical researchers have begun human clinical trials of cell preparations from muscle and bone marrow. The first small clinical trials yielded mixed results, and larger controlled trials are still in progress. The therapeutic action (if any) of these early attempts at cell therapy is unknown, and most investigators agree that more basic research is needed so that rational approaches to therapy can be devised. Most theories of heart regeneration focus on early cardiac progenitor cells, which are also the focus of our research plan. To devise new approaches to repair damaged heart tissue, we must learn more about the molecular “wiring” that is required to change hESCs into heart cells. We are using hESCs to examine how G-protein-coupled receptor (GPCR) signaling influences key cell developmental decisions required for proper heart formation. GPCRs, the largest class of cell-surface receptors, are ideal therapeutic targets. Although some GPCRs are the focus of intense pharmacological research, their potential for additional uses has not been explored fully. Specific drugs are lacking for >80% of GPCRs. Nevertheless, we can use genetic tools to “turn on” and “turn off” specific receptor signals at key times in development. These studies will reveal the GPCRs that would be most ideal for developing new drugs for regenerative medicine. The goal of our studies is to eventually improve the overall health of Californians. Heart disease remains a major cause of death and disability, resulting in billions of dollars in health care costs and lost days at work. Cardiac regeneration could dramatically improve recovery from heart disease such as heart attacks. This would improve the quality of life and increase productivity for millions of people worldwide. Additionally, this study will contribute to building the California economy by producing new technologies for the biotechnology industry.
Statement of Benefit to California:
Heart disease is the leading killer of adults in the Western world. In addition, congenital heart malformations are the most common birth defects, occurring in nearly 1% of the population worldwide. A major goal of regenerative medicine is to repair damaged cardiac tissue. Our research is focused on developing new methods to differentiate human embryonic stem cells (hESCs) into specific cell types for cardiac regeneration. Our research could benefit the California economy by creating jobs in the biomedical industry by developing new technology. Ultimately, this study could help reduce heart disease, thereby increasing the productivity and enhancing the quality of life of Californians. The results of our studies will help develop new technology that could contribute to the California biotechnology industry. Our studies will create multiple lines of hESCs that have genetic markers that turn on at specific time points. These cell lines could be valuable for biotechnology companies and researchers who are screening for drug compounds that will cause these developmental changes. Furthermore, we are working closely with California companies to develop new microscopes and analysis software that could be the basis for new product lines or new businesses. If therapies do come to fruition, we anticipate that California medical centers will be leading the way. The most important contribution of this study will be to improve the health of Californians. Heart disease is a major cause of mortality and morbidity, resulting in billions of dollars in health care costs and lost days at work. Heart failure—the loss of cardiac pumping ability—results in a chronic debilitating disease that lasts for years. Cardiac regeneration could dramatically reduce heart disease worldwide. Our goal is to contribute research that would ultimately improve the quality of life and increase productivity for millions of people who suffer from heart disease. Our studies will also help understand how currently available medications can affect normal human hearts. In addition to identifying potential heart-related side effects in current medications, we may also find new uses for medications in preventing heart disease or repairing damaged tissue. As we continue our efforts in medical research, we hope to one day unlock the secrets of heart development and repair. This knowledge will help medical researchers develop beneficial therapies beyond what is currently available and potentially improve the quality of life and life expectancy of patients who suffer from heart disease.
SYNOPSIS: The goal of this proposal is to use hESCs to determine how G protein-coupled receptor(GPCR)- signaling influences cell fate and cardiomyocyte development. The investigators propose to induce differentiation of cardiac progenitor cells to effect myocardial repair. This requires hESC lines that can be dissociated into single cells, clonally selected and expanded. The aims are: 1: Define the repertoire of GPCRs and related signaling genes expressed in hESC-derived cardiovascular tissues. (A) Create BAC reporter hESC lines to map cardiomyocyte development. (B) Use the marker cell lines to identify mesoderm, cardiac progenitors, and early cardiomyocytes and temporal expression of GPCR and related genes; 2: Determine effects of GPCR signals on cardiac development; 3: Use genetic tools to mimic GPCR drug actions on cells in Aim 2; (A) Use RNAi to inactivate selected GPCRs and downstream components. (B) Activate each major GPCR pathway using engineered synthetic GPCRs. IMPACT AND SIGNIFICANCE: Reviewer 1: This is a potentially high impact and high risk application. The significance is as follows: if the applicant can adequately map the steps required to follow GPCR signaling from membrane receptor to effector (a huge task in its own right) and understand the roles of pathways and way stations on normal myocyte growth and development (also a major task, as the function of GPCRs changes with both development and context), this should facilitate attempts to clone large quantities of myocytes or myocyte precursors that would take the idea of myocardial repair from a pipe-dream to a potential reality. To date, attempts at repair/regeneration have largely been limited to the latter and have relied heavily on recruitment of native precursor cells, perhaps via paracrine effects of the injected hMSCs or other bone-marrow derived elements. Efforts at repair have been limited by the inability to obtain sufficient numbers of cells to replace dead/dying myocardium. Even if the applicants cannot follow this research through to its anticipated outcome, the knowledge gained will be applicable to our understanding of GPCR pathways, to the optimization of existing drugs that modify components of these pathways and to the development of new drugs. In addition the bacterial artificial chromosome reporter hESC lines to be developed will be of value in high-throughput screening of small molecules. Although understanding the role of G-protein-coupled-receptors (GPCRs) in general and their possible causal role in specification of cardiac progenitors in humans represents an important endeavor, several elements dampened the enthusiasm of some reviewers. First, as acknowledge by the applicant, GPCRs have a variety of functions, from embryogenesis all the way to adulthood. Some are related to cardiac development and some are not. Second, studying the role of GPCRs in hESCs may not be the best or fastest way to understand the role of cardiogenesis, as development of hESCs is very slow and the technology for gene targeting is incompletely developed, as even a simple stable integration is still extremely challenging. The more productive approach would be to address the role of GPCRs in a model system to narrow the few present in the right time and place to be involved in these events, and then to use them in a study with hESCs. QUALITY OF THE RESEARCH PLAN: The research plan has been carefully constructed to test the hypothesis that GPCR-mediated signaling affects hESC differentiation toward cardiac myocytes. Given that GPCRs are expressed in hESCs (shown in preliminary data) and the involvement of GPCRs in many cell proliferation and differentiation events, it is likely that these receptors will regulate hESC differentiation. The combination of pharmacological inhibitors, RNAi, and RASSLs and different time points during differentiation will clearly identify which GPCRs promote or inhibit differentiation along the cardiac myocyte lineage. The majority of the preliminary data have been obtained using murine ESCs. Thus, there may be some technical difficulties encountered in utilizing the BAC system, RNAi, and expressing RASSLs in hESCs, but the proposed experiments seem reasonable for the project timeframe. STRENGTHS: The proposal will be carried out by a team of investigators who are fully qualified in a supportive environment. The research plan is highly ambitious with innovative ideas and directions. The research program has potential for high impact with respect to myocardial repair and there is a well-developed experimental plan to test the hypothesis that GPCRs reglate differentiation of hESCs toward cardiac myocytes. The investigator has the relevant expertise and good preliminary data. WEAKNESSES: Because the is highly ambitious, it may not be brought to fruition (or even near to fruition) within the period planned. Given the numbers of GPCRs extant and their linkages and interactions, it is difficult to understand how the investigators will achieve focus on those which are most important. It is precisely the failure to provide any understanding of the prioritization of experiments, of pathways, and of molecules that provides the greatest concern here. It is unfortunate that some preliminary data providing at least a template for achieving some milestones within the period of investigation were not provided. Characterizing cellular response to external signals at different stages of development is critical toward understanding how hESCs become cardiac myocytes. However, the reliance on expression of a single marker (reporter) may be problematic as it will likely result in heterogeneous population. Significant characterization of the sorted cells will be required to determine the nature of the starting population exposed to changes in GPCR signaling. It is also not clear whether the sorted cells will be able to efficiently mature to cardiac myocytes. During cardiogenesis, inductive interactions from non-mesodermal cell types are required. Thus, relatively pure mesodermal populations may not develop as well as cells in EBs. It is possible, however, that the investigators will discover GPCRs that allow pure populations to efficiently differentiate toward cardiac myocytes. The proposal focuses on the roles of GPCRs in early differentiation events, up to NKX2.5 expression. One of the major problems facing use of hESC-derived cardiac myocytes in cell-based therapies is they have not been shown to effectively generate mature cardiac myocytes with adult phenotypes (e.g. action potentials). While the effects of GPCR signaling on early differentiation events is important and interesting, the applicant should not neglect the effects of this signaling on the mature cardiac myocytes. Monitoring and functional characterization of cells exposed to altered GPCR signaling beyond what is proposed would be warranted. While the proposed experiments will determine which individual GPCRs regulate hESC differentiation toward cardiac myocytes, they will provide little insight into the pathways downstream of GPCRs, and how multiple signals coordinate to affect differentiation. DISCUSSION: The discussion centered on trying to balancing the concern about the lack of focus and of a clearly prioritized experimental plan against the possible importance of results that may be obtained.