New substances are routinely screened by pharmaceutical companies for their biological toxicity but failure to predict the toxicity of compounds to humans is a major financial cost for the pharmaceutical industry. In vivo multigeneration studies are expensive, laborious, and time consuming. Alternatively, in vitro cellular models can be completed in substantially less time and cost. The predictive potential of in vitro cellular model assays in drug screening is largely influenced by the cell types and the endpoint in the assays. Primary cells are limited in their proliferation capacity and the immortalized cell lines do not represent the in vivo counterparts. Here we propose to develop a novel, simple, low cost bead-based flow cytometric analytical assay with potentially high predictive in vitro embryotoxicity assessment using human embryonic stem cells. In order to assess the embryotoxic effects of chemicals, assays need to be developed to detect the early chemically-induced stress conditions. When placed in cytotoxic or genotoxic culture conditions, undifferentiated pluripotent ES cells respond by either activating apoptosis processes, or by inducing differentiation process. These survival processes protect hESC against the propagation of cells that carry damaged DNA with potentially oncogenic mutations. Murine ES cells maintain their pluripotency and genomic stability longer than hESC under suboptimum conditions. This indicates that hES cells are a better choice than mES cells for a more sensitive in vitro toxicity system to environmental-induced stress conditions for spontaneous differentiation and apoptosis. The protein with a central role in controlling undifferentiated hESC to spontaneous apoptosis and differentiation under culture-induced stress condition is the p53 protein. P53 was shown to express strongly in both early and late apoptotic hESC. In Phase I of this proposal, we will develop an analytical bead-based flow cytometry immunoassay to capture and quantify the chemically-induced p53 accumulation in undifferentiated hESCs culture medium in response to known embryotoxic substances. The Relative Embryotixic Potency (REP) values of known embryotxic compounds with high-, medium-, and no-embryotoxicity effects will be calculated using p53 expression and compared with the published REP values for validation. In Phase II we will apply this technology to screen a broader spectrum of chemicals for embryotoxicty using undifferentiated hESCs and specific hESC-derived differentiated cells. Ultimately, the technique will be adapted to a high- throughput screening platform.
This proposal will develop a novel flow cytometric based assay to predict embryotoxic compounds using undifferentiated human embryonic stem cells.