Tools and Technologies 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 due to failure to adequately replace lost ventricular myocardium from ischemia-induced infarct. Consequently, stem cell-based therapies to replace lost or damaged ventricular myocardium hold great promise. However, pluripotent stem cells carry a risk of uncontrolled growth and subsequent tumors/teratomas. Furthermore, cell transplantation of heterogeneous populations of cardiac and non-cardiac tissue may result in an arrhythmic substrate in patients' hearts potentially leading to sudden cardiac death. Thus, creating protocols that allow for purification of homogeneous populations of differentiating cardiac subpopulations, in particular ventricular myocytes, is critical toward realizing the potential of stem cell therapy in heart failure patients. Toward this end, we have proposed to develop tools and technologies that address this rate-limiting issue by identifying surface biomarker codes that specifically define subpopulations of differentiating hPSC-derived cardiomyocytes. Moreover, we will use this code to develop protocols to FACS purify progenitor as well as mature hPSC-derived cardiomyocytes. These protocols will be applicable to cardiomyocytes derived from not only PSCs but also other cell populations including transdifferentiation of non-cardiac tissues and cardiac progenitor cells. As a result, this precise selection of specific populations of hPSC-derived cardiomyocytes will provide a source of human cardiomyocytes 1) to further study the development and expansion of differentiating hPSC-derived cardiac lineages; 2) to model cardiac diseases which affect specific cardiac cell types, including long QT syndrome, atrial/ventricular fibrillation, cardiac conduction blocks and cardiomyopathies; 3) to screen drugs for future cardiac treatments, and 4) to implement regenerative cell therapies for myocardial infarctions.
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." Though stem cell-based therapies to replace lost or damaged ventricular myocardium hold great promise, pluripotent stem cells carry a risk of uncontrolled growth and subsequent tumors/teratomas. Furthermore, cell transplantation of heterogeneous populations of cardiac and non-cardiac tissue may result in an arrhythmic substrate in patients' hearts potentially leading to sudden cardiac death. Thus, creating protocols that allow for isolation of pure populations of differentiating cardiac subpopulations, in particular ventricular myocytes, is critical toward realizing the potential of stem cell therapy in heart failure patients. Toward this end, we have proposed to create novel tools and strategies for the purification of specific cardiac populations for stem cell-based cardiac regenerative therapies.
The applicants seek to identify antibodies that recognize cell surface markers of either heart muscle cell progenitors or subtypes of mature heart muscle cells derived from human pluripotent stem cells (hPSC). They will then identify combinations of these antibodies that enable purification of these therapeutic cell types from mixed populations by flow sorting, thereby addressing two translational bottlenecks to cardiac cell replacement therapy, teratoma formation and arrhythmia risk. The investigators plan to achieve this by screening a library of existing antibodies for their interaction with heart muscle progenitors and specialized heart muscle types. They will then verify that the appropriate cells have been identified by comparing their expression profile to that of native human heart cell types and by performing functional tests. Finally, the group plans to transplant the purified cells into murine hearts to determine their engraftment, integration and tumor forming potential. Reviewers agreed that this proposal addresses an important translational bottleneck for both cardiac replacement therapy and in vitro cardiac toxicology models. If successful, the work would produce a significant impact. However the selected approach, while rational, is not innovative. Others have tried and failed to identify cardiac myocyte selective surface markers using commercially available antibodies. Therefore, reviewers were not convinced this approach would succeed. They would have also appreciated discussion of the rationale for pursuing surface labeling relative to alternative cardiac myocyte purification techniques, such as mitochondrial staining, as well as differentiation of this effort from prior failed attempts. Reviewers appreciated the preliminary data demonstrating the applicants’ expertise in cardiac differentiation, cardiac progenitor culture techniques, relevant cardiac myocyte assays and flow cytometry. However, evidence was lacking that flow sorting does not negatively impact the function of the sorted cells, raising concern about the utility of the proposed strategy. Reviewers agreed the research plan was logically developed, and a reviewer noted that the plans to analyze the purified populations were feasible. However, they expressed serious reservations regarding the proposed surface marker identification approach. The panel was unanimous that the selected antibody library was too small to accomplish this aim. Reviewers noted that the lack of diversity among the antibodies further limits the library’s utility and that some alternative plans were inadequate. In particular, the RNA expression based plan to identify surface epitopes for the generation of new antibodies was considered weak. Some reviewers found these studies inadequately detailed, while others noted that RNA based approaches do not reflect the posttranscriptional and posttranslational processing that contributes to cell surface protein expression and antigenicity. This was considered a critical flaw. Reviewers further cautioned that the comparison of hPSC-derived cardiac cells, which exhibit fairly immature phenotypes, to native tissue may not be informative unless tissue samples are obtained from a comparable developmental stage. Taken together, these feasibility concerns about the experimental approach eroded reviewer confidence that the applicants would achieve the aims of the proposal. Both the principal investigator (PI) and the Partner-PI are well established and have successful track records in the cardiac development and cardiac progenitor fields. The collaboration with the Partner-PI was considered a strength of the proposal; however, one reviewer noted some redundancy in efforts of the PI and Partner-PI laboratories. The PI has assembled a highly qualified team with all of the necessary skills (including electrophysiology and molecular biology) required to undertake the proposed research. The budget was deemed appropriate for the work. In summary, while this proposal from a strong team addresses two important translational bottlenecks to cardiac cell replacement therapy, the reviewers found the proposed experiments were neither novel nor likely to resolve the bottlenecks, and the application was not recommended for funding.