Adult heart muscle cells retain negligible proliferative capacity and this underlies the inability of the heart to replace muscle cells that are lost to injury, such as infarct, and underlies progression to heart failure. To date, no stem cell therapiy has produced significant cardiomyocyte replacement. Instead, transplanted cells, if they persist at all, produce endothelial cells or fibroblasts and the ameliorating effects on heart function that have been reported have been achieved by improving contractility, perfusion or other processes that are impaired in the failing heart. This proposal is to develop specific reagents and ultimately drugs to stimulate regeneration. Our approach integrates advances in stem cell biology, high-throughput (HT) biology, informatics and proteomics to identify small molecules, proteins and signal transduction pathways that control heart muscle formation from human embryonic stem cells (hESCs). High throughput assays will be developed and implemented to identify genes, signaling proteins, and small molecules that that control important steps in the differentiation, proliferation, and maturation of cardiac cells. Computer modeling and informatics will be used to identify and validate the signaling pathways that direct these critical processes. The discovery of small molecules and pathways will lead to protocols for 1) efficient directed differentiation of cardiomyogenic precursors from hESCs for research into transplantation and toxicology, 2) biotech reagents to stimulate cardiomyocyte renewal through directed differentiation of hESCs, and 3) cellular targets or lead compounds to develop drugs that stimulate regeneration from endogenous cells.
Statement of Benefit to California:
This proposal is a multidisciplinary collaboration among stem cell biologists, chemists, and engineers to address a critical problem that limits the widespread use of hESC for cardiology applications. Developing the multidisciplinary technology and overcoming the hurdles to application of hESCs to biotech and clinic will benefit California in many ways, including: Research to discover novel tools to stimulate heart muscle regeneration from hESCs is clinically important. Cardiovascular disease is the single largest cause of death in the U.S. and the assays we will develop and the reagents themselves will be useful tools to direct cardiomyocyte regeneration. This will speed the translation of hESCs to the clinic, specifically by stimulating production of cardiomyocytes and potentially by enhancing their integration and function after engraftment. Heart regeneration from hESCs probably uses similar cellular proteins and signaling pathways as regeneration of cardiomyocytes from other sources, thus, this research might be broadly applicable to heart muscle repair. Regeneration from endogenous cells remains controversial but these tools should be useful reagents to study and hopefully stimulate endogenous repair. Bringing the diverse people together (chemists, cell biologists, and engineers) to address a stem cell problem forges new links in the academic community that should be capable of opening new areas of research. These new areas of research will be a important legacy of the stem cell initiative and promises to invigorate academic research. The technology that we are developing applies the new discipline of chemical biology to stem cell biology, and the merger promises to spin off new areas of investigation and biotech products with the potential to benefit the practice of medicine and the local economy. Lastly, supporting the leading edge technology and the collaboration will build the California infrastructure of high throughput chemical library screening so that it can be focused on other areas of biomedical research, both stem cell and non-stem cell.
SYNOPSIS: The Principal Investigator (PI) proposes to develop novel small molecules to stimulate human embryonic stem cell (hESC) cardiomyogenesis. These will be used to study cellular signaling pathways and proteins controlling myocyte differentiation. Lead compounds and their targets will be used to develop drugs to stimulate regeneration from endogenous or transplanted cells. High throughput assays will be developed to identify small molecules that direct differentiation of cardiomyocytes from hESCs and proliferation of hESC-derived and native cardiomyocytes. Chemical optimization of small molecule hits will improve selectivity and activity via analogue synthesis, re-testing and structure activity relationship determination. Signaling pathways and targets will be identified via proteomic and inhibitor studies to interrogate signal transduction proteins modulated by the optimized compounds. Target confirmation will be biochemical using affinity-labeled lead compounds in direct binding studies. IMPACT & SIGNIFICANCE: Adult cardiomyocytes have little proliferative capacity. This is a cause of the failure of the heart to replace muscle cells lost during infarction, and is a cause of the evolution of congestive failure. To date, no stem cell therapies have produced significant cardiomyocyte replacement although there has been documentation of improved contractility, perfusion and ejection, more so in animal models than in human studies. Those transplanted cells that do persist often produce endothelial cells or fibroblasts. The impact of the plan is that it offers an organized approach to attempt optimization of the cells that are transplanted, and offers the opportunity as well to acquire knowledge about the processes of cell evolution into mature myocytes. Even if the project fails to achieve its major goal, the knowledge acquired can be an important way-station in the evolution of our use of hESCs. While chemical screens are often thought to be the domain of industry the high throughput screening done here may provide probes of use to the entire research community. Moreover the information acquired can have important application in diverse areas of cell biology: as such the project provides research tools applicable to a far larger pool of investigators. While the research is not hypothesis driven its impact may be equivalent to that of much hypothesis driven research. In fact the approach is one that is making ever more inroads into main-stream science as investigators realize the power provided by the new technologies to facilitate “brute-force” evaluation of large numbers of molecules and targets. There is currently no productive way of producing cardiomyocytes for cell-based therapies. It is not clear currently whether endogenous cardiomyocyte stem cells exist, and therefore one of the best alternatives remains direct specification of cardiomyocytes from hESCs. This represents a very high priority for hESC studies. This project aims at characterizing small molecular compounds that can guide hESCs toward cardiomyocyte differentiation. The compounds identified with this ability should enable the production of cardiomyocytes derived from hESCs for preclinical translation. The PI proposes three specific aims around this goal. Aim 1 is to identify molecules that control key steps in stem cell cardiogenesis. First, those will be scored on the basis of compounds that are able to activate signaling pathways known to promote cardiogenesis. Second, those that direct hESC line differentiation to transition stages of cardiogenesis, and finally those that generation cardiomycyte cell cycle entry. Aim 2 will provide a repertoire of analogues designed around the basic structure of active compounds. Aim 3 will use the compounds as probes to identify proteins and pathways affected by the reagents. This proposal has potentially very high impact from both a scientific and clinical perspective. It incorporates chemical screening approaches to discover agents that would promote the renewal and directed differentiation of cardiac progenitors and their differentiated progeny from hESC systems. QUALITY OF RESEARCH PLAN: This is a proposal of very high quality written by a world specialist in the field. The PI demonstrates his/her complete mastery of the field, not only at the embryological or cellular level, but also in the detailed molecular components involved in specification of the cardiac field during embryogenesis. The team is an outstanding one that should be able to not only perform the research but deal with any confounding issues that arise. The study is highly innovative, and could have a major impact on the entire field of hESC model systems, as the chemical biology approach taken could be generalized to many other cell types of interest. The infrastructure for this type of work is considerable, and San Diego in general, with parallel efforts at Scripps, and the Burnham in particular, are well positioned to play a leadership role in this endeavor. Preliminary data exists documenting the feasibility of the approach. The interdisciplinary, team based approach that is put forward is exemplary, as the effective use of hESC based systems will require multiple areas of expertise to be focused simultaneously on a core set of compelling questions. The specific aims offer a logical, orderly approach to achieving their overall goal. With regard to their first aim of identifying molecules controlling key steps in hESC cardiogenesis, they will employ three automated screens. One will be a focused screen of small molecule-modulators of pathways recognized as regulating ESC cardiogenesis; one will be a phenotypic screen to identify small molecule stimulators or progression through discrete stages of differentiation; and one will screen for inducers of terminally differentiated cardiomyocyte reentry into the cell cycle. The investigators are experienced in these techniques and the processing of the volumes of information that are generated. The second aim uses an iterative analysis of design, synthesis and testing to optimize the lead compounds. The third aim identifies proteins and pathways affected by these active compounds. The preliminary data suggest that even if all the goals of the research are not attainable, much valuable information may be obtained. The robotic, high-throughput screening to be used is an ideal tool given the fact that our limited knowledge of hESCs makes them largely a tabula rasa to us. STRENGTHS: The proposal opens a different doorway than that used by most to study hESCs and apply their biology to the betterment of medical care. Admittedly the medical care aspect is distant from the research being performed, but the approach the investigators use to identify small molecules that can be used as probes to understand pathways and eventually as means to optimize cell development and survival cannot help but develop new research tools and new drugs. The research plan is ambitious but should be do-able, and the team is well-suited to the performance of the studies. The strength of the proposal remains on the quality of the PI, the environment the proposal is to occur, and the investment of time energy and money that the Burnham Institute has provided for these types of high throughput screens aimed at differentiation. The PI proposes use of more than 55,000 independent compounds representing small synthetics as well as natural products. In addition, another combinatorial chemistry collection of 6 million small synthetics will be used as mixtures. Therefore, the quality of the personnel, environment, breadth fo the project, and the availability of a large subsect of chemical compounds with the leadership of the Burnham Institute represents the key strengths of the proposal. In summary, the strengths are: (1)the innovation, and potential impact of the study; (2)the quality of the investigators; (3)the quality of the preliminary data and the pre-existing infrastructure at the Burnham, and (4) the high likelihood of meaningful, important insights and reagents that will emanate from the studies. WEAKNESSES: While they will attempt to direct differentiation and proliferation of cardiomyogenic precursors, promote maturation/survival on transplantation and evaluate functional integration, this aspect of the research is not well spelled out. In part this is understandable, as much remains to be done (in fact the lion’s share of the research) before this step is reached. In addition, the quality of the chemical libraries at the outset may be rate limiting. Finally, the ability to move from a specific chemical probe to defined effects of specific biochemical pathways will ultimately require development of high affinity chemical probes and eventual affinity purification of the specific protein target DISCUSSION: This is an ambitious proposal, but the PI has really built an outstanding interdisciplinary team with chemists, physiologists,etc and the team-based approach is exemplary. The PI is an embryologist who has successfully done small molecule screens before with experience in running screens in tadpoles. The proposal is strong, specific aims logical and the approach orderly. One reviewer felt that this was not hypothesis driven as much as brute force research, but sometimes brute force research is needed The 3 different automated screens are mutually reinforcing, and there is an iterative analysis to optimize the leads. Preliminary data provides evidence that the PI will gain valuable information even if not all research goals are met. Another reviewer said that this was the best of his/her pile due to its innovation and is clearly not be fundable by the NIH. Few proposals have team-based approaches; this one has an exemplary multidisciplinary team. The San Diego collaborations and region is strong in this area (small molecule screening) and in a good position to play a role in chemical screens. The research plan is ambitious and some parts of the plan were lacking. Weaknesses were not significant. The reivewers recommend focusing on the core work, not on the back end of trying to repair infarct in animal models because you need to lay groundwork first. Everyone feels that they have to go into animals when often the animal work is premature.