An assay suite featuring hiPSC-derived neurons and support cells, to test toxicants and industrial chemicals for developmental neurotoxic effects - This “direct to Phase II” project, proposed in response to RFA-ES-23-008 “Novel Approaches for Developmental Neurotoxicity Testing (R43/R44)”, will develop a suite of in vitro assays to test compounds for potential developmental neurotoxic (DNT) effects. Neurodevelopmental disorders (NDs, including autism spectrum disorder, intellectual disabilities, attention-deficit/hyperactivity and others) affect at least 15% of people in the USA, appear in childhood/early adulthood, and cannot be cured. NDs originate from dysregulation of brain development during early life (fetal, neonatal, and childhood), during which the developing CNS is particularly vulnerable to environmental toxicants and many of these chemicals have been linked to NDs (e.g., mercury, pesticides, air pollutants). In the past, such compounds have been investigated for ND effects, via epidemiological studies, only after they cause neurotoxicity to adults; this approach has tragic, global, consequences, since decades of research are required to establish the links between adult neurotoxicity and DNT, during which thousands of children become impaired through exposure to the culprit toxicant. Our project will develop standardized, high throughput, assays to test chemicals for such effects. The DevNeuroToxAssay (DNTA) suite will include assays to test compounds for effects on neurogenesis, a key processes in early brain formation (DNTA-Neurogenesis), on neuronal circuit formation/activity, relevant to neuronal circuits within the brain (DNTA-Neurocircuitry), on development of 3-D neural organoids, (DNTA-Neural Organoids), and on development and function of the blood-brain barrier, a structure that protects the brain from toxicants and pathogens (DNTA-BBB). The DNTA assays will feature neurons, glial, and support cells derived from human induced pluripotent stem cells, cultured in either 2-D or 3-D configurations. Automated digital microscopy will be used to quantify effects of chemicals on biomarkers and structures (high content analysis, HCA); additionally, effects of compounds on biological neuronal circuits will be quantified via collection/analysis of digital movies (frame rates up to 100 frames per second, using Kinetic Image Cytometry, [KIC]). Compounds from the National Toxicology Program DNT chemical library will be used to develop, validate, and demonstrate the utility of the DNTAs. Since the DNTAs will feature human cells, the results obtained will be more predictive of human outcomes vs. tests conducted on animals or animal cells. Vala Sciences Inc. has recently developed similar assays to test candidate anti-HIV therapeutics for neurotoxicity, and this previous experience will expedite rapid development of the DNTAs.
