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.
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.