Tools and Technologies II
Heart failure affects over five million Americans, with close to 700,000 new diagnoses each year. Because cells in the heart cannot regenerate, their permanent loss due to aging or disease compromises natural heart functions. Heart transplantation is typically the last resort for end-stage heart failure patients, but this treatment option has always been hampered by a shortage of donor organs. For example, only approximately 2000 patients received a heart transplant in the United States in 2008 and almost 3000 patients were on the waiting list as of 2009. The derivation of heart muscle cells (cardiomyocytes) from stem cells in the laboratory has attracted considerable interest for their potential to be used as an unlimited source of cardiomyocytes for transplantation to treat patients with heart disease, but the purity of these populations is low due to the presence of undifferentiated stem cells and other non-cardiomyocyte differentiated cell lineages. This is one major roadblock limiting the use of these cells for future cell-based therapies to treat patients because there is the risk that transplanting pluripotent stem cells may lead to tumor formation. Unfortunately, there are currently no clinically acceptable methods available to accurately separate stem cell derived cardiomyocytes from pluripotent cells and other derivatives to obtain high purity cardiomyocyte populations. The goal of this project is to develop an innovative optical technique for sorting stem cell derived cardiomyocytes based on the specific detection of myosin bundle found in cardiomyocytes but absent in stem cells. This protein organization generates a unique optical signal when excited with an intense laser beam. This signal will be the parameter by which cardiomyocytes will be separated from stem cells and other cell types. Moreover, this technique is particularly attractive for this application because it requires no labeling or genetic modification of the cells, which are both undesirable in a clinical setting. There are three primary research objectives in this project. The first objective is to characterize the optical properties of stem cell derived cardiomyocytes at different stages of their maturation to determine the accuracy of the signal to identify them. Different laser conditions will be used to determine the optimal excitation conditions to generate the strongest signal. The second objective is to develop a cell sorter instrument for sorting pure, large populations of stem cell derived cardiomyocytes by integrating the optical scheme with microfluidic devices and flow sorters. The third objective will determine the safety of the sorted heart cell populations by transplanting these cells into mice and monitoring for any tumor formation.
Statement of Benefit to California:
Heart disease is the leading cause of death in the United States. Heart failure – the ineffective pumping of the heart caused by the loss of function of heart muscle cells – affects over 5 million people, with over 500,000 new cases each year. Given that California is the most populous state in this country, this disease is also a major healthcare problem in this state. Heart transplantation is typically the last resort for end-stage heart failure patients, but this option is often hampered by a shortage of donor organs. Stem cells have received considerable interest for their ability to differentiate into heart muscle cells in culture, which could potentially be used as an unlimited source of cells for future cell-based transplantation procedures to treat heart failure. However, the low purity of these stem cell derived heart muscle cell populations is one of the major roadblocks impeding their use in novel stem cell heart therapies. In particular, undifferentiated stem cells need to be removed due to the potential safety risks in transplanting these cells into a patient. This research proposes to overcome this obstacle by developing a new optical method to purify these cell populations, which will help bring stem cell - based treatment of heart failure one step closer to reality. The ability to obtain purified stem cell derived heart muscle cell populations would also significantly benefit drug companies seeking to use these cells for studying their function and for cardiac safety pharmacology assays. This proposal is expected to benefit California and its citizens long-term by improving healthcare, reducing the economic burden of health care costs, and stimulating the biotechnology and biomedical instrumentation industry in California, thus leading to economic benefits to the state as well.
This proposal aims to develop a label-free method for purifying cardiac myocytes generated from human embryonic stem cells (hESC-CM). By using this technique, the applicants propose to resolve the translational bottleneck of teratoma risk faced by hESC-CM therapies. This technique exploits the optical properties of contractile protein bundles found in cardiac myocytes. The applicants propose to first characterize the optical signal generated by hESC-CM, optimize the excitation routines for distinguishing these cells from other cells, and demonstrate the specificity of their technique. Next, they will create a sorter based upon this optical signal, first using microfluidics and then by integrating the technology into a flow cytometer. In the last aim, they will test the ability of their approach to remove undifferentiated cells from differentiated cardiac myocyte preparations using an in vivo tumorigenicity assay. Reviewers universally appreciated the need to remove residual hESC from differentiated cell therapy products and they were impressed by the applicant’s novel and innovative approach. However, one reviewer highlighted that improved cardiac functional outcomes following increased cardiac myocyte purity remain theoretical. Another reviewer noted that since this approach depends upon a signal most intense in myocytes, its applicability to other therapeutic cell types will be limited. Reviewers also cautioned competing cardiac myocyte sorting technologies are already in development, and that these methods may obviate the need for the proposed technique. The panel expressed mixed opinions regarding the application’s preliminary data. While it demonstrated the ability of the optical technique to detect plated cardiac myocytes, reviewers were not convinced the proposed technology would enable purification of hESC-CM. Two reviewers highlighted the lack of demonstration that the technique can resolve these cells from others in the live single cell suspension format required for sorting, and felt this was a critical flaw in the proposal. These reviewers felt strongly that the analysis of suspension cells should be made a priority for this study. While reviewers were supportive of the concept of the proposed technology, they had critical feasibility concerns regarding its application to hESC-CM-based therapy and the proposed experimental plan. Two reviewers expressed concern that the throughput of the proposed sorting methods would prove inadequate for isolation of clinically relevant doses of hESC-CM. It was noted that the proposed separation would require more analysis and therefore more time per cell than conventional clinical flow sorting, which is already of borderline throughput for cardiac applications. Thus, even if successful, this technology is unlikely to solve a translational bottleneck. They also noted the applicant should compare the purity, yield and throughput of their technique to that of existing competing techniques. The last aim to demonstrate safety of the sorted hESC-CM by in vivo teratoma assay was criticized for lack of sufficient sensitivity and power to accurately predict teratoma formation. Reviewers considered hESC an inadequate control for the study and instead suggested testing pre-sorted and myocyte negative fractions to accurately determine the sensitivity of the assay. Finally, they found the testing of hiPSC-derived cardiac myocytes extraneous to the proposal’s goals. The Principal Investigator (PI) has assembled a strong multidisciplinary team with the appropriate expertise to perform the proposed studies. These collaborations are backed up by convincing letters of support. However, reviewers universally noted the program is inadequately staffed at the bench level to successfully execute the work. A key investigator’s 5% commitment further eroded confidence that the team could complete the work. The panel unanimously praised the excellent environment, which provides important engineering expertise. In summary, reviewers appreciated that the proposed novel technology exploits a unique feature of cardiac myocytes, and thus has the potential to remove residual undifferentiated hESC from cardiac myocyte preparations. However, they did not find the preliminary data in support of feasibility of the technology compelling, nor did they find the proposed approach capable of resolving a translational bottleneck. For these reasons, they did not recommend this application for funding.
- This application scored below the initial scientific merit funding line, no programmatic reason to fund the application was suggested, and the GWG voted to place the application in Tier 3, Not Recommended for Funding.