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.
Public Health Relevance: This proposal will develop a novel flow cytometric based assay to predict embryotoxic compounds using undifferentiated human embryonic stem cells.
Thesaurus Terms: Address; Adverse Effects; Anaplastic; Animal Model; Animal Models And Related Studies; Antibodies; Antioncogene Protein P53; Apoptosis; Apoptosis Pathway; Apoptotic; Assay; Bioassay; Biologic Assays; Biological; Biological Assay; Cardiac Myocytes; Cardiocyte; Cell Communication And Signaling; Cell Culture Techniques; Cell Death, Programmed; Cell Line; Cell Lines, Strains; Cell Signaling; Cell Membrane; Cell Model; Cellline; Cells; Cellular Tumor Antigen P53; Cellular Model; Chemicals; Clinical; Complex; Conditioned Culture Media; Conditioned Medium; Contracting Opportunities; Contracts; Culture Media; Culture Media, Conditioned; Cytofluorometry, Flow; Cytoplasmic Membrane; Dna; Dna Damage; Dna Injury; Deoxyribonucleic Acid; Development; Drug Delivery; Drug Delivery Systems; Drug Evaluation, Preclinical; Drug Industry; Drug Screening; Drug Targeting; Drug Targetings; Es Cell; Embryo; Embryo Development; Embryogenesis; Embryonic; Embryonic Development; Esteroproteases; Evaluation Studies, Drug, Pre-Clinical; Evaluation Studies, Drug, Preclinical; Extravasation; Flr; Failure (Biologic Function); Financial Cost; Flow Cytofluorometries; Flow Cytometry; Flow Microfluorimetry; Genes, P53; Genetic Alteration; Genetic Change; Genetic Defect; Genome Stability; Heart Myocyte; High Throughput Assay; Human; Human Development; Human, General; Immunoassay; In Vitro; Industry, Pharmaceutic; Intermediary Metabolism; Intracellular Communication And Signaling; Leakage; Lytotoxicity; Metbl; Mammals, Mice; Man (Taxonomy); Man, Modern; Masks; Measurable; Metabolic Processes; Metabolism; Methods And Techniques; Methods, Other; Mice; Microfluorometry, Flow; Murine; Mus; Muscle Cells, Cardiac; Muscle Cells, Heart; Mutation; Myocytes, Cardiac; Nerve Cells; Nerve Unit; Neural Cell; Neurocyte; Neurons; Oncogenic; Oncoprotein P53; Organ; P53; Peptidases; Peptide Hydrolases; Pharmaceutical Agent; Pharmaceutical Industry; Pharmaceuticals; Pharmacologic Substance; Pharmacological Substance; Phase; Phosphoprotein P53; Phosphoprotein Pp53; Plasma Membrane; Preclinical Drug Evaluation; Process; Proteases; Protein Tp53; Protein P53; Proteinases; Proteins; Proteolytic Enzymes; Protocol; Protocols Documentation; Publishing; Relative; Relative (Related Person); Reporter; Role; Signal Transduction; Signal Transduction Systems; Signaling; Spillage; Stability, Genomic; Staging; Stress; System; System, Loinc Axis 4; Tp53; Tp53 Gene; Trp53; Techniques; Technology; Testing; Time; Toxic Effect; Toxicities; Treatment Side Effects; Tumor Protein P53; Tumor Protein P53 Gene; Undifferentiated; Validation; Base; Biological Signal Transduction; Cardiomyocyte; Cell Preparation; Cell Type; Cost; Cultured Cell Line; Cytotoxic; Cytotoxicity; Design; Designing; Drug Discovery; Embryonic Stem Cell; Failure; Flow Cytophotometry; Gene Product; Genome Mutation; Genotoxicity; Growth Media; Hesc; High Throughput Screening; Human Es Cell; Human Esc; Human Embryonic Stem Cell; Immortalized Cell; In Vitro Model; In Vivo; Model Organism; Neuronal; Novel; P53 Antigen; P53 Tumor Suppressor; Plasmalemma; Pluripotency; Pre-Clinical; Preclinical; Protein Expression; Public Health Relevance; Response; Side Effect; Skills; Social Role; Stem; Stem Cell Of Embryonic Origin; Therapy Adverse Effect; Treatment Adverse Effect
