Basic Biology III
Stem cell-derived cardiomyocytes are promising for numerous in vitro and in vivo applications. Clinical applications include use in transplantation studies to treat heart disease, which remains a major cause of morbidity and mortality in developed countries despite advances in therapy. There are numerous applied and basic research applications, including their use directly in transplantation, their use in assays to discover drugs to treat heart disease, and the in vitro testing of drug candidates for adverse effects on cardiac physiology. Realization of the potential of stem cell-derived cardiomyocytes for these important applications is hampered by the electrical and mechanical immaturity of the cells. Immaturity makes them poor models of adult cardiomyocytes and poorly functioning in in vivo applications. Currently, it is not possible to direct functional maturation since the signals and genes that drive the process are largely unknown. Data are presented in this application showing the development of instrumentation and software for high throughput assessment of electrophysiological maturation. We also show, using this technology, that electrical and ion channel profile maturation of human embryonic stem cell and human induced pluripotent stem cell (hESC- and hiPSC)-derived cardiomyocytes can be driven by paracrine signaling from vascular endothelial cells and by Kruppel-like factors (KLFs) within the differentiating myocytes. The goal of this project is to identify and characterize the factors and genes that direct cardiomyocyte maturation. The findings will constitute a conceptual advance since little is known about the regulation of physiological maturation, and it will be highly significant for basic and applied research and regenerative medicine since it will enable the production of mature cardiomyocytes from hESCs/hiPSCs, and potentially from endogenous cardiac stem cells in vivo, thereby increasing the value of stem cell cardiogenesis for medical applications.
Statement of Benefit to California:
This proposal is a multidisciplinary collaboration among stem cell biologists and engineers to address a critical problem that limits the widespread use of stem cells (in particular hESCs and hiPSCs) for cardiology. Developing the multidisciplinary technology and overcoming the hurdles to application of stem cells will benefit California in many ways, including: 1. Research to discover novel tools to stimulate heart muscle regeneration from 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. 2. Heart regeneration from stem cells probably uses similar cellular proteins and signaling pathways as regeneration of cardiomyocytes from endogenous sources, thus, this research might lead to drugs that enhance natural repair of the heart. 3. Bringing the diverse people together (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 an important legacy of CIRM and promises to invigorate academic research. 4. 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.
Project Synopsis: The goal of this proposal is to define factors that promote maturation of human pluripotent stem cell derived cardiac myocytes (hPSC-CM). The program builds on preliminary data suggesting that endothelial cell-conditioned medium (EC-CM) and the transcription factor KLF-4 can promote maturation of hPSC-CM. First, the applicants plan to define the maturation parameters (electrical, structural and force generation) influenced by EC-CM and KLF4. Once these are identified, the applicants plan to isolate and identify the components in EC-CM that mediate these changes. In the last series of studies, the applicants will employ biochemical and molecular biology techniques to determine the gene targets and binding partners of KLF4 in maturation. The impact of EC-CM on KLF4 will be tested to determine if this factor could mediate EC-CMs effects. Significance and Innovation: - The application╒s strength lies in the significance of the problem addressed. Development of methods to derive mature cardiac myocytes from pluripotent stem cells could improve their utility for both drug toxicity screens and cell therapy applications. - Reviewers appreciated both the applicants╒ use of electrical properties to assess maturing cardiac myocytes and the innovative technology enabling high throughput measurement of this functional measure. Feasibility and Experimental Design: - The applicant team possesses all the expertise and technology required to execute the proposal. - Preliminary data suggesting a role for endothelial cell derived factors and KLF transcription factors in cardiac myocyte maturation provide the foundation for the proposed program. However, reviewers found the magnitude of maturation unconvincing evidence of maturation beyond the fetal stage, and noted it was smaller than that reported using prolonged culture. - The poorly detailed experimental plan dampened confidence in the proposal. Description of how hESC-CM progenitors will be obtained and enumeration of how many KLFs to be studied are absent. Further, description of multiple mechanistic studies were in adequate and methods of quantification including the number of cell lines tested, experiment numbers, and expected outcomes were unclear. - The scope of work proposed was excessive; it was noted that each aim, if detailed, would provide adequate work for an RO1. - The applicant proposes inappropriate criteria to assess cardiac myocyte maturation; these criteria will not distinguish adult from fetal cardiac myocytes as proposed. - The application does not provide convincing criteria linking data from this automated microscopy method and action potential rise time with the developmental progression of cardiac myocytes. - There is a risk that the conditioned medium de-convolution studies will fail to identify factors responsible for maturation. Principal Investigator (PI) and Research Team: - The PI is an established and productive cell biologist who is both experienced and well published in cardiomyogenesis. - S/he has assembled a team of accomplished scientists with expertise in expertise in characterizing calcium handling, myocyte force generation, isolation of bioactive molecules and imaging cytometry. However, these experts are listed at 0% effort. - The proposed number of bench staff at 3 FTE is inadequate to execute the project. Responsiveness to the RFA: - The proposal addresses the question of maturation of human pluripotent stem cell derived cardiac myocytes and is responsive to the RFA.
- This application scored below the initial scientific merit funding line, no programmatic reason to fund the application was proposed, and the GWG voted to place the application in Tier 3, Not Recommended for Funding.