VALIDATION OF A HUMAN CD34+ STEM CELL TOXICITY BIOASSAY

DESCRIPTION (provided by applicant): This SBIR application is submitted in response to the objectives of the NIEHS Predictive Test Systems for Safety Evaluation Program by developing, standardizing, and validating sensitive and specific new and novel test s or batteries of tests . Primary human CD34+ bone marrow stem cells will be used as a novel biological system to identify predictive biomarkers of altered immune system differentiation, cellular toxicity, and genotoxicity. Human CD34+ bone marrow stem cel ls are pluripotent cells that possess the potential of self-renewal, proliferation, and differentiation toward different lineages of blood cells. Human CD34+ cells can be directed to differentiate in vitro and expand along specific cell lineages of immune cells. This lineage-specific differentiation of CD34+ cells is a complex biological process that includes the combinatorial and coordinated expression of genetic pathways that drive cellular proliferation and the acquisition of specialized cell functions. A screening assay based on human primary cells would be useful to assess the predictive power, or in vitro in vivo correlation, of high-throughput screens based on tumor cell lines. As cell lines derived from tumors often lack many critical genes that regu late cellular responses to stressors, the results of environmental agents tested in tumor cell line-based assays may have limited relevance to human health and disease, and there is a need for a human primary cell- based screening assay. This Phase I SBIR has three specific aims: Miniaturize human TK6 cell assay as a prototype assay to establish FCM-based measures of cytotoxicity, apoptosis, cell cycle arrest, oxidative DNA damage and DNA double strand breaks, adapt biological endpoints established in human TK6 cells with human CD34+ stem cells, establish cellular and molecular biomarkers of human CD34+ stem cell differentiation along specific lineages, assess impact of toxicants on CD 34+ health status and differentiation. Since bone marrow stem cells play a pivotal role in the function of the hematopoietic and immune systems and are the putative target cell population of concern for a host human cancers and diseases, the results obtained from these systems are biologically relevant to human disease and can be extrapolated to humans. The human CD34+ stem cell multiplex assay proposed here is ideally suited as platform to assess toxicity for environmental agents and pre-clinical drug candidates, as well a follow-up test system for high-throughput testing initi atives. PUBLIC HEALTH RELEVANCE: Stem cells have the unique ability among all of the cells of the human body of self- renewal, that is, they can remain in a primitive unspecialized state. Under the right conditions, they can give rise to specialized cells of the body (differentiation) like the heart, liver, or pancreas. CD34+ stem cells are the stem cell of bone marrow that differentiates into all of the cells in the blood (white and red blood cells). Therefore, these cells present a unique model system to understand and assess the effects of environmental agents and new drug candidates to predict or anticipate toxicity in humans.