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 by
expressing 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.
