Expanding the throughput of real-time toxicological screening of cardiac differentiation by expressing a synthetic luciferase/luciferin genetic pathway in iPSCs

Expanding the throughput of real-time toxicological screening of cardiac differentiation byexpressing a synthetic luciferase/luciferin genetic pathway in iPSCs Project Summary This Small Business Innovation Research (SBIR) Phase II project proposes to develop and validate autonomously bioluminescent induced pluripotent stem cells (iPSCs) for use in continuously data producing, reagent-free, and real-time toxicological screening assays. These tools will address the National Institute of Environmental Health Sciences (NIEHS) request for novel high-throughput assays to evaluate the effects of chemical compounds on the differentiation of pluripotent stem cells, as their ability to differentiate along well-defined lineage pathways offers a powerful approach to understanding how chemical perturbations disrupt metabolic and regulatory functions along those pathways. The autobioluminescent iPSC-based assay systems developed here will significantly contribute towards NIEHS’s mission to ‘discover how the environment affects people in order to promote healthier lives’ by expanding the knowledgebase of chemical exposure toxicological effects. This is especially important given that the commercial marketplace maintains an inventory of tens of thousands of chemicals, the majority of which have poorly understood human health impacts, and currently require animal- based testing approaches that are expensive, time consuming, and ethically contentious to determine their effects. As an alternative, stem cell-based assays such as those developed here can mimic human disease states more reliably than animal models while providing valuable information towards understanding how chemical exposures influence cancer risks, developmental defects, and other adverse health outcomes. A significant impetus therefore exists to integrate stem cells into chemical screening programs such as Tox21 and ToxCast, but under the mandate that they function under high-throughput conditions. While this goal is not obtainable using existing bioluminescent reporter technologies such as firefly luciferase that must be provided with a chemical substrate to activate its light emission responses, resulting in only marginally informative single time point snapshots of potential toxicological interactions, 490 BioTech’s synthetic luciferase technology rather enables reporter cells to emit light continuously and in real-time, thereby providing an uninterrupted stream of visual data over the lifetime of the cell as it interacts and reacts to chemical perturbations. The goal of this research effort is to develop improved assays based upon synthetic-luciferase-expressing iPSCs and validate their ability to report the impact of chemical exposure to cellular health and development in real time. To accomplish these goals, our specific aims will focus on assay development and optimization, validation against a chemical subset of the Tox21 10K library, and benchmark comparisons against existing commercial assay systems.Project Narrative Humans interact with a vast landscape of chemicals that, despite the efforts of numerous U.S. and international multiagency sponsored programs to test for their toxic effects and ensure consumer safety, often have poorly understood impacts on our health. Because the primary limitation preventing these agencies from better characterizing chemical health effects is a lack of testing methods that can rapidly provide data with sufficient informational content and direct relevance to human health impacts, 490 BioTech is creating and validating a next-generation testing system that uses human stem cell lines capable of autonomously emitting bioluminescent light in response to chemical exposure events. This new testing system will enable chemical toxicity screening to occur throughout the lifetime of the cell and produce an expanded informational database of toxicological effects with direct relevance to human health.