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

DESCRIPTION provided by applicant Expanding the throughput of real time toxicological screening of cardiac differentiation by expressing a synthetic luciferase luciferin genetic pathway in iPSCs Project Summary This Small Business Innovation Research SBIR Phase I project proposes to develop autonomously bioluminescent induced pluripotent stem cells iPSCs for continuous reagent free and real time toxicological screening to 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 iPSCs developed here will significantly contribute towards NIEHSandapos s mission to andapos discover how the environment affects people in order to promote healthier livesandapos by expanding the knowledge base 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 are poorly understood in terms of their risks and hazards to human health and currently require animal based testing approaches that are expensive time consuming and ethically contentious to determine their human health effects As an alternative stem cell based assays such as the one developed here may 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 Therefore there exists significant impetus for the integration of stem cells in chemical screening programs such as Tox 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 their light emission responses resulting in only marginally informative single time point snapshots of potential toxicological interactions BioTechandapos s synthetic luciferase technology 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 o express our synthetic luciferase system in iPSCs and demonstrate real time continuous visualization of iPSC to cardiomyocyte differentiation under chemical toxicity exposure pressures Our specific aims will focus on assay development and optimization benchmark comparisons against existing commercial assay systems and assay validation against a chemical subset of the Tox K library PUBLIC HEALTH RELEVANCE Humans interact with a vast landscape of chemicals on a daily basis yet the health effects of many of these chemicals are poorly understood Numerous U S and international multiagency sponsored programs have been established to test chemicals for toxicity effects to better ensure consumer safety but the process of testing tens of thousands of chemicals is challenging and is currently limited by assays that do not provide data fast enough nor with sufficient informational content that directly relates to human health impacts To assist in creating assays that will better predict chemical risks and hazards BioTech proposes to create human stem cell lines capable of autonomously emitting bioluminescent light in response to chemical exposure events thereby enabling chemical toxicity screening to occur throughout the lifetime of the cell to yield an expanded informational database of toxicological effects with direct relevance to human health