Physiological drug screening represent an unprecedented opportunity for the application of human stem cell(hESC) and human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. Drug companies conduct large screening campaigns for cardiac active compounds utilizing in vitro assays that measure enzymatic activities or biological models constituted by engineered tumor cell culture systems. The poor biological relevance of these models is one of the main causes of the high rate of failure of new drug candidates during the following phases of preclinical and clinical testing. The reason of these choices should be individuated in the impossibility of obtaining the large primary cell number and in the difficulty of designing physiological assays that can be implemented in high-throughput (HT) screening, especially for excitable cells. Automatic whole cell manual patch-clamp systems, for example, are available but cannot be readily scaled for high throughput. Use of human stem cell-derived cardiomyocytes bring more physiological relevance, and this would increase the effectiveness of drug screening for compounds active on cardiomyocytes, but the large number of cells necessary for primary screening campaigns is still a limitation to using such cells for testing chemical libraries consisting of hundreds of thousands of compounds. This proposal will overcome these limitations designing tools and assays that will allow fast reading in human stem cell (hSC)-derived cardiomyocytes of physiological parameters relevant to cardiac contractility in a HT format. Stable cell lines expressing the light triggered protein channel Channelrhodopsin 2 (ChR2) will be generated and cocultured with hSC-derived cardiomyocytes to induce action potential in cardiac cells by light exposure. Fluorescent probes will be utilized to measure action potential and calcium flux in cardiomyocytes during the stimulation. While this approach will overcome the necessity of electrode coupling, allowing fast and automatic excitability measurement, several approaches will be developed to reduce the number of cells necessary for testing. The final objective is to develop a cell chip where cardiomyocytes and ChR2 expressing cells are spotted along a microscope glass slide to allow the contemporary testing of thousand of compounds in a short time. Libraries, constituted by chemical drugs and small RNAs, will be screened to validate the platform and discover new agents affecting cardiac functionality . The approach can be easily extended to other cell types and physiological parameters in the near future.
Statement of Benefit to California:
This proposal is a multidisciplinary collaboration among scientists of different backgrounds to address a critical problem that limits the identification of new drugs during the discovery process. Moreover, it can be used to study many physiological-relevant parameters in human stem cell-derived cardiomyocytes. It can also be extended to other excitable cell types. The research will benefit California in many ways, including:
1. The new technology will find application in screening of large library of chemicals compounds by pharmaceutical industries, using high physiological assays to identify new drugs that affect heart activity, increasing the availability of therapies for conditions like heart failure, arrhythmia and other cardiac pathologies.
2. Several of the screenings proposed to validate the platform will define lead compounds and targets that will be make accessible and can be further developed to generate new drugs and therapies.
3. One of the screening proposed will increase human stem cell-derived cardiomyocyte maturation, improving the availability of regenerative medicine therapies for the heart in the near future.
4. The assays that will be developed can be easily adapted to other excitable cells of great interest for regenerative medicine, like skeletal muscle cells, neurons and pancreatic beta cells, increasing the tools for research in these other areas.
5. Bringing the diverse people together (engineers, stem cell biologists, electrophysiologists and chemists) to address a stem cell problem and a high throughput screening 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 will invigorate academic research.
This proposal is focused on the development of tools for high-throughput drug screening of human cardiomyocytes (CMs) derived from human embryonic or induced pluripotent stem cells (hESCs or iPSCs). The translational bottlenecks addressed are the limited availability of human CMs and the difficulty in screening them using physiological assays in a high-throughput manner. The applicant proposes four Specific Aims: (1) to develop a stable cell line that expresses a light-activated channel and can electrically couple to CMs in culture to allow physiological stimulation of CMs by light pulses; (2) to optimize this co-culture system and validate its ability to differentiate between cardiotoxic and safe drugs; (3) to develop the capability to measure conduction velocity in these cultures; and (4) to develop tools for high-throughput screening, including scaling down to small wells and volumes and performing several pilot chemical screens.
Reviewers agreed that this is a highly innovative proposal that addresses a significant translational bottleneck. If successful in developing a high-throughput human CM-based screening platform that requires fewer cells, it could have a major impact on drug development. However, reviewers were not convinced of the scientific rationale for approaches central to the proposal. They did not find adequate justification for the technological focus of the proposal, a light-activated switch for CM activity, as opposed to electrical stimulation. Nor was the importance of Aim 3, to develop the capacity to measure conduction velocity, adequately explained.
The panel found the research plan too broad in scope to be completed during the award period and raised several feasibility concerns. They noted that one of the fundamental bottlenecks in this field is the inability to generate pure populations of mature CMs from hESCs or iPSCs. This bottleneck is not addressed until the final part of the final aim of this proposal, in which the applicant proposes to screen for compounds to facilitate CM maturation. Reviewers cautioned that a heterogeneous population of immature CMs, likely comprised of ventricular, atrial and pacemaker subtypes, could limit their utility for safety screening. Panelists also noted that co-culture could affect the derived CM phenotype, maturation and function.
In addition to concerns regarding the cellular model, reviewers expressed doubts about the proposed readouts. They did not find calcium imaging a satisfactory proxy for direct recording of action potentials. Calcium imaging is indirect at best, particularly when screening drugs for their tendency to increase action potential duration and potentially cause arrhythmia. While the applicant also proposes to use the co-culture system to measure voltage using various dyes, the application does not contain preliminary data employing these techniques. In addition, reviewers raised questions about the ability of the system to monitor cells at sufficiently high temporal resolution to capture action potential prolongation, as well as its ability to record events in large numbers of CMs in parallel. Finally, they noted that very few pitfalls or potential limitations are included in the research plan. For example, the group would have appreciated a discussion regarding the number of CMs required to develop this technology, both before and after scale down, and a description of how these needs would be met.
The reviewers described the Principal Investigator (PI) as a relatively inexperienced independent investigator with a limited track record. They questioned whether the PI is prepared to direct such a large, complex program involving a large team of investigators. The reviewers appreciated the strong team of collaborating senior investigators and their reasonable commitments of effort. However, they noted that there are at least five senior investigators involved in the project, but only one technician and one post-doctoral fellow, which may not be sufficient technical staff to carry out the proposed research.
Overall, reviewers appreciated the innovative aspects of this proposal and the potential impact of high-throughput CM screening on drug development. However, questions about the scientific rationale for certain aspects of the proposal, the feasibility of the research plan, and the qualifications of the PI significantly lowered their enthusaism.