Developmental Neurotox Assay Using Scalable Neupons and Astrocytes in High Content Imaging

The objective of this proposed project and the EPA Computational Toxicology program’s goals are aligned in assessing chemicals for potential risk to human health and the environment through the use of more representative multicellular human developmental neurotoxicology assays. ArunA Biomedical will develop a rapid, scalable, human pluripoten stem cell derived cell-based co-culture assay system to address a critical gap where animal developmental neurotoxicity testing for a single compound can be financially prohibitive and in excess of $1 million and time consuming (up to 1.5 yrs). ArunA will manufacture pluripotent stem cell derived neural cells using a patented systemto generate functional neurons and astrocyte cultures, more closely mimicking the developing human nervous system tan single cell type assays. §The innovation is a rapid enriched astrocyte differentiation process that does not require expensive growth factor cocktails nor cell sorting to obtain the astrocytes that can be used as co-planer/co-cultures with ArunA existing hN2™ neurons for high content imaging. This will be an improvement over an already robust monoculture system using hN2™ human neurons in cellular imaging neurotoxicant screens. Previously, the EPA used ArunA’s thaw and assay hN2™ neuron, demonstrating superior sensitivity over rodent primary cortical neurons when measuring neurite count, total length and average length for 3 or 6 known neurotoxicants. In no case were the hN2™ human neurons less sensitive than rodent primary in this six compound trainingset nor were there any false positives with the hN2™ neurons. Here we will build on these unique human cellular responses by adding scalable pluripotent stem cell sourced astrocytes to the hN2™ neuron assay. The importance of incorporating astrocytes in cellular neurotoxicological events by been demonstrated in vivo using rodents. For example, both Me-Hg and Pb acetate can affect neurons indirectly through compromising the developing astrocytes. This human co-culture cell assay should be more representative of neural tissue, achieving a level of biological organization for risk assessment and can be used in an adverse outcome pathway toxicology approach. §In Phase I, object 1, we will further refine a scalable and rapid means of generating enriched astrocytes form Arun based hNP1™ technology and cryopreserve them for use with hN2™ neurons, all within 48 hrs post thaw.In objective 2 assays, we will specifically control the neuron to astrocyte ratio, providing a flexible platform to more closely mimic ratios observed in the developing nervous system. In the last task these co-cultures will be exposed to increasing doses of Pb acetate given its know affects of astrocytes and in light of EPA published direct affects on hN2™ neurons. Together, commercialized kits and assay services eliminate unknown confounding factors from serum or other commonly used yet poorly defined biochemical compositions by controlled differentiation in defined serum free medium, further enhancing the utility of this system for screening neurotoxins. The anticipated differential affects of Pb acetate on hN2™ neuron;astrocyte compared to hN2™neurons alone will provide an initial indication of validity and unique role of astrocytes in a development neurotoxicity chemical screen. §The number of chemical in the neurotoxicity training set will be increased in Phase II and contemporary comparisons made to commercial animal and human primary cell sources of neuron and astrocyte products to further validate ArunA’s developmental neurotoxicity assay. It is important to realize that primary sourced cells are good sources of cells for early studies however these cells are rarely scalable for throughput assays. Finally by employing adult sources pluripotent stem cells (induced pluripotent cells) for more susceptible genetic and varied background in Phase II, we can develop products to help address a potential genetic influence on the developing human nervous system susceptibility to neurotoxicants. Once ArunA’s validated human neurite outgrowth assay is developed the value and savings to industry and government will be significant since a validated assayservices will augment or prioritize chemicals for expensive and time consuming animal developmental neurotoxicity assays. We anticipate sales and services to exceed $10 million for near term products and services developed through Phase I and Phase II funding and assay validation. IN addition, validated assays performed to ArunA will drive up use of related assays including but not limited to blood brain barrier assays and neural metabolomics studies. Toxicologists believe that complex tissue relevant, validated and informative human assays are needed in the foreseeable future (>5 yrs) and ArunA will be responsive to current and future EPA need and guidelines in this area.