Stem cell therapies hold great promise for repairing tissues damaged due to disease or injury. However, one of the major obstacles in translating stem cell biology into tissue replacement therapy will be the establishment of effective purification methods which specifically isolate the desired cells for implantation and exclude those which may have adverse effects on the performance of the implanted graft or the health of the patient. Previous stem cell tissue replacement studies in the heart have failed, largely due to poor electromechanical coupling of implanted cells with the heart. We are developing a new technology for cell purification which will enable us to specifically isolate functional heart cells from mixed stem cell populations. Rather than looking for a protein or genetic marker, as is conventionally done, our technology electrically stimulates flowing cells and examines their response. Muscle cells, such as those found in the heart, will twitch upon electrical stimulation, and this twitch can be measured with a sensitive electrode. As this technique does not introduce any fluorescent labeling molecules or genetic modifications to the cell, it is much safer than traditional cell purification methods. Furthermore, since this functional test is closely related to the task that cells must perform in the heart (namely, contract in an organized, controllable manner), it is expected that cells purified in this way will form better tissue grafts. This proposal represents the first attempt to sort cells based on a dynamic, functional response to stimulus. As many of the cell types relevant for regenerative medicine are electrically-excitable (e.g. heart cells, brain cells, and blood vessel cells), this technology is also applicable to a variety of other neurodegenerative and cardiovascular therapies. The proposed system utilizes a microfluidic device with integrated electrodes for electrical stimulation and recording of extracellular field potential signals from suspended cells in flow. By combining hundreds or thousands of these tiny microdevices on a single chip, we can achieve throughputs relevant for clinical applications.
Statement of Benefit to California:
Cardiovascular disease (CVD) affects more than 1.7 million Californians and 71 million Americans. The societal and financial impacts are tremendous, with CVD accounting annually for an estimated $8 billion in CA and nearly $400 billion in US health care costs. In the case of chronic illnesses such as CVD, the state and national health care systems may not be able to meet the needs of patients or control spiraling costs, unless the focus of therapy switches away from maintenance and toward cures. CVD is typically caused by a loss in heart muscle. Once this heart muscle is gone, the body is not able to replace it. With the discovery of induced pluripotent stem cells, we may one day be able to create replacement heart tissue grafts derived from a patient's own cells, free of the ethical controversies and immune rejection issues associated with human embryonic stem cells.
We believe that the objectives of our research will benefit the people of California by addressing a specific bottleneck in the translation of stem cell biology to clinical tissue replacement therapies for the heart and other organs. The development of these tissue replacement therapies will require radically new methods for the cultivation and purification of stem cell-derived heart cells, and we believe that purification based on a functional assessment has the potential to produce highly pure populations in a safe, effective manner.
With the passage of Proposition 71 in 2004 and the creation of the California Institute for Regenerative Medicine (CIRM), California has positioned itself to remain at the forefront of stem cell research. We believe that the results of our work will lead to marketable tools and technologies which will generate royalties, patents, and licensing revenues for the state economy. Furthermore, the development of tools which enable cures for diseases such as CVD would improve the California health care system by reducing the long-term health care cost burden of these diseases. Our previous work has shown a commitment to developing practical technologies suitable for the marketplace, including the establishment of four companies from alumni of our research lab.
We have assembled a multi-disciplinary team to attack the objectives of our proposed research with expertise in microfluidic device development, electronic instrumentation, stem cell biology, and regenerative medicine. At the same time, we will train and mentor a new generation of bright students and junior scientists in the areas of technology development, regenerative medicine, and iPSC biology. This will ensure that an essential knowledge base will be preserved and passed on to both investigators and patients within and beyond California.
The goal of this application is to develop a non-genetic, label-free technology to sort cells using their responses to electrical stimulation, in order to achieve sufficient purity for cardiac cell replacement therapies. First, the applicant proposes to engineer a microfluidic cell sorter and associated instrumentation for separating individual cells based on their electrical excitability. Next, the applicant will demonstrate cell sorting of single induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), and confirm their cardiac myocyte subtypes using standard assays. Finally, the applicant will scale up the cell sorting technology to achieve throughputs in the range of 1 million cells/hour.
Reviewers agreed that heterogeneity of cell types in iPSC-CM preparations poses a safety bottleneck to the translation of cardiac regenerative therapies, and they appreciated the innovative approach to sort iPSC-CM based on electrical activity, a functional parameter intrinsic to each cardiac myocyte subtype. However, the panel was uncertain of the potential impact of the proposed technology. For example, sorting typically requires sacrificing yield to achieve high purity, and the applicants do not propose to compare yield and purity achieved with their methods to existing techniques such as fluorescence-activated cell sorting (FACS). The proposed >99% purity target is inadequate to resolve the risk related to teratomas in clinical applications. Additionally, the applicant’s target throughput of 1 million (1e6) cells/hour was judged inadequate to support sorting of the estimated therapeutic iPSC-CM dose of 1e9 cells/patient for clinical applications.
Although aims were logically organized, reviewers raised several concerns regarding proposal’s feasibility to achieve a system appropriate for translational applications. While data is presented to support the applicant’s ability to construct a sorter based on electrical properties of cells, to differentiate iPSC to CM, and to distinguish undifferentiated iPSC clusters iPSC-CM clusters using their device, the application provides no statistical information to confirm the significance or reproducibility of this data. Importantly, the preliminary data do not demonstrate the applicant’s ability to sort single cell suspensions containing iPSC-CM in a mixture of other cell types, which was highlighted as a critical inadequacy of the proposal. For example, one reviewer questioned the applicant’s ability to accurately predict throughput of the proposed device in the absence of this single cell data, given that the signal from single cells may necessitate slower flow rates and thereby further limit throughput. Although multiplexing hundreds or even thousands of sorting channels simultaneously is proposed, the technical challenges of this process were judged significant by reviewers, and were not addressed in the application.
The Principal Investigator (PI) is well-established with a very good track record in microfluidics. S/he has teamed with experts in stem cell biology and cardiac differentiation to bring in complimentary expertise. The requested budget is reasonable.
In summary, the applicant proposes to develop a novel microfluidic cell sorter that separates cells based on intrinsic electrical behaviors. Although the PI has appropriate expertise and the concept is innovative, the reviewers judged both the proposed cell purity and throughput of the technology inadequate to resolve the targeted translational bottleneck. Thus, the application was not recommended for funding.