Basic Biology III
$1 593 000
In the United States, heart disease is the number one killer of adult men and women. Another 5 million survive with insufficient cardiac function. The demand by patients with end-stage heart failure exceeds the supply of suitable donor hearts, and so there has been considerable recent interest in cell-based therapies as an alternate means of regenerating injured hearts. There is an obvious medical need for restoration of myocardium. Self-repair is unquestionably insufficient to offset cell death in heart disease, and this has triggered considerable efforts to restore myocardium with cardiac cells from exogenous sources via transplantation, or by stimulating endogenous regeneration. Several reports show some degree of integration of exogenous myocardial cells in animal transplant models, and clinical trials have been performed using a variety of non-cardiac cells. Although some reports cite improvement in cardiac performance, others show equivocal results – improvement clearly requires more research. Thus, understanding how to produce the three main cardiac lineages will be a major advance in directing formation of these cell types for research and eventual clinical applications. Our approach integrates advances in stem cell biology, high-throughput (HT) biology, and informatics to identify key pathways that control heart muscle formation from human embryonic stem cells (hESCs). High throughput assays have been developed and implemented to identify genes, signaling proteins, and micro RNAs that control important steps in the differentiation, proliferation, and maturation of cardiovascular cells. Computer modeling and informatics will be used to identify and validate the pathways that direct these critical processes. The discovery of pathways will lead to protocols for efficient directed differentiation of cardiovascular lineages from hESCs for research into transplantation.
Statement of Benefit to California:
This proposal is a collaboration among stem cell biologists, bioinformaticians 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 clinic will benefit California in many ways, including: 1. Research to discover novel pathways 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 will be useful tools to direct cardiovascular regeneration. This will speed up the translation of hESCs to the clinic, specifically by stimulating production of cardiomyocytes and potentially by enhancing their integration and function after engraftment. 2. 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 insufficient to but these tools should be useful reagents to study and hopefully stimulate endogenous repair. 3. Bringing the people together (stem cell biologists, bioinformaticians) 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 an important legacy of the stem cell initiative and promises to invigorate academic research. 4. The identified active pathways during cardiovascular differentiation in this proposal are likely to provide promising therapeutical targets, which can spin off new areas of investigation and biotech products with the potential to benefit the practice of medicine and the local economy. 5. Lastly, supporting the leading edge technology such as of high throughput functional using ESCs reinforces California’s world leadership over the embryonic stem cell field, which both confers a strategical advantage and prestige from which the entire Californian scientific community and population benefit.
Project Synopsis: This proposal seeks to address how to improve restoration of the myocardium in heart disease. The aim is to identify pathways that regulate the lineage diversification of multipotent cardiovascular progenitors. The principal investigator (PI) plans to screen a mouse whole genome siRNA library in murine embryonic stem cell (mESC)-derived cardiac progenitors for effects on progenitor differentiation into cardiomyocytes, endothelial cells and vascular smooth muscle cells. A collection of miRNAs will be similarly screened using mESC and human (h)ESC. Next, the PI plans to utilize this data to build a hypothetical network of regulators involved in cardiovascular progenitor lineage diversification. Key hit targets will then be explored for potential pathways by which they could impact lineage diversification and validated by gain and loss of function studies in hESC and mESC and in Xenopus and mouse embryos. Significance and Innovation: - The project is not particularly innovative; recent reports have described regulators of many of the lineage fate decisions to be studied. Reviewers were therefore doubtful this work would have major impact on regenerative medicine. - The proposed work is descriptive and may not identify novel factors. Indeed, hits from pilot preliminary screens consisted of known pathways. Feasibility and Experimental Design: - Overall the proposed approach is overly broad and unfocused. - Preliminary data supports the applicant’s ability to perform screens and secondary functional assays in embryos. - Reviewers highlighted inadequate experimental detail. For example, there is little description of the differentiation technique, scale or progenitor yield. No alternative approaches were provided in the plan. - The application is poorly written and assembled; multiple errors and omissions were noted. Principal Investigator (PI) and Research Team: - The PI, while very well trained, appears to have few relevant publications and may not be adequately experienced to lead the proposed project. - The team integrates experienced personnel who provide complementary and critical expertise to the project including bioinformatics. Responsiveness to the RFA: - The proposal is focused on fate decisions of a pluripotent stem cell-derived progenitor. However, the included mESC studies are neither groundbreaking nor necessary and reduced responsiveness to the RFA.