Toxicological risk assessment lacks direct estimation of the magnitude and variability of human responses to environmental toxicants. Specifically, there is very little data on the effects of low dose toxicants on human developmental processes, including cellular differentiation. That is because the current basis of toxicological assessment, animals and immortalized cell lines, cannot adequately model the impact of low level toxicant exposure on human populations. An alternative detection platform should enable incorporation of human variability in screening of a large number of toxicants in a high-throughput manner. To establish such a platform, we propose using human donor-specific cells that can be (a) isolated in a non-invasive manner, (b) easily expanded in culture, and (c) cryopreserved without loss of viability. Our preliminary data suggest that human endothelial progenitor cells, or endothelial colony forming cells (ECFCs), a population of CD31+/CD34+ pluripotent cells found in circulating blood, would fulfill these requirements. ECFCs are highly proliferative in vitro and ca differentiate into mature endothelial cells (ECs). The overall goal of this research is to evaluate the effect of chemical toxicants on viability, proliferation, and differentiation of ECFCs derived from different individuals. Prior to our recent and successful completion of a Phase I program, we established cell culture parameters for ECFCs using a proprietary cell growth media supplement and completed initial toxicological risk assessment studies using low-dose ionizing radiation (LDIR). These studies confirmed the utility of ECFCs in toxicological risk assessment screening as well as their ability to broadly represent varying responses expected in human populations. Phase I studies successfully expanded these findings by performing ECFC toxicology assays using Bisphenol A (BPA), perfluorooctanoic acid (PFOA) and cadmium (Cd). ECFCs were found to be sensitive to BPA and Cd and less sensitive to PFOA, exhibiting donor-specific variability in proliferation rates. BPA and Cd also induced reactive oxygen species (ROS) production in a donor-specific manner. ECFCs were shown to experience oxidative stress and cell cycle arrest after exposure to toxicants. Elevated ROS production was shown to correlate with cell death after longer incubation times with toxicants. We found that the toxicants we analyzed also affected expression of endothelial-specific cell markers at a protein level in a newly developed differentiation assay. Phase II will focus on the continued development of this screening platform toward a validated quantitative high content imaging-based screen. Commercialization of the platform as a service will also be pursued as well as the production of kits for ECFC culture and subsequent toxicological analysis. Finally, the screen will be expanded to include toxicant-induced effects on ECFC differentiation into mature endothelial cells.
Toxicological assays currently rely on a range of cell lines and in vivo models, as well as assays for viability and proliferation. Here, employing a primary human cell type (endothelial colony forming cells; ECFCs), Brite Bio and Creative Scientist have collaborated to develop a quantitative, high content imaging- based platform not only suited to environmental agent-induced cell death, but also differentiation disruption. Data derived from this screen is likely to be incorporated into toxicology forecasting algorithms, providing novel insights into differentiation disruption in a cell type critical to vascular development and maintenance.
