Evaluating teratogenic risk of anti-seizure medications in single-rosette brain organoids

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Project Abstract Neural tube defects (NTDs) are common malformations of the nervous system that occur during pregnancy. NTDs are caused by genetic variants or maternal environmental exposures, and they often lead to severe physical and/or cognitive disabilities. Pharmaceuticals can sometimes lead to NTDs in human embryos despite screening in rodent models due to human-specific toxicity. Many anti-seizure medications (ASMs), drugs used in the management of epilepsy increase the risk of NTDs during pregnancy but are typically not discontinued because of danger to the mother and fetus from seizures. Therefore, it is important to compare the neuroteratogenic risk of these compounds to choose the most appropriate ASM during pregnancy. Having a human-specific model of early neurodevelopment should increase this screening predictiveness. Attempts to utilize human brain organoid technology to this end are limited due to structural heterogeneity and intra- organoid variability. Our recent development of reproducible self-organizing single rosette spheroids (SOSRS) from human induced pluripotent stem cells has allowed us to treat SOSRS with known neuroteratogens and observe distinct structural changes consistent with NTDs. The goal in Aim 1 is to compare the structural consequences of all 20 commonly used ASMs in SOSRS at multiple concentrations to determine a concentration dependent risk for each. In Aim 2, we will perform comparative transcriptomics of SOSRS treated with 5 mechanistically distinct neuroteratogens. Transcriptomic changes shared by all 5 will be considered a neuroteratogenic biomarker that will then be applied by targeted NGS to RNA samples from SOSRS treated with each of the commonly used ASMs to provide further risk comparison. In Aim 3, we will adapt the SOSRS protocol to generate lumbar spinal cord SOSRS in order to compare teratogenicity of valproic acid between rostral (cortical SOSRS) and caudal (lumbar SOSRS) neural tube models. Our study may also shed light on the undefined mechanisms by which ASMs cause NTDs. The platform and biomarkers developed by this study could also allow for screening novel pharmaceuticals for NTD risk in a human-specific system in the future.