Evaluating Human Pluripotent Stem Cell-Derived Neural Rosette Arrays as a Neural Tube Defect Risk Screening Platform

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Project Summary Neural tube defects (NTDs) are the second leading cause of congenital malformations affecting ~3000 births per year in the US alone and with significantly higher rates elsewhere. NTDs occur within the first month post- conception and are caused by failed closure of the neural tube, which is the developmental anlage of the entire central nervous system (CNS). The most prevalent NTDs are anencephaly and spina bifida. These defects are embryonic lethal or result in lifelong motor impairment with possible incontinence, respectively. While folic acid dietary supplementation has been clinically proven to reduce NTD risk in expecting mothers, the persistence of NTD occurrences despite proper diet suggest that multifactorial environmental and genetic factors are the primary etiological origins of remaining NTD cases. Furthermore, while rodent studies provide significant insights into NTD etiology and pathology, they do not provide the requisite throughput to efficiently screen environmental factors, i.e. chemical exposures, for associated NTD risks. Moreover, incongruence between rodent genetic models and clinical observations from human epidemiological and genome-wide association studies (GWAS) has limited development of precision medicine approaches for predicting and minimizing a patients’ NTD risk. Here, we propose to evaluate whether Neural Rosette Arrays (NRAs) can serve as a quantitative, high-throughput, human pluripotent stem cell (hPSC)-derived screening platform for assessing a chemical’s or genetic mutation’s NTD risk. While NRAs are not an exact recapitulation of in vivo primary neurulation, the bioengineered assay is the first to spatially and temporally standardize neural tube analog formation, i.e. singular neural rosette tissues, in a microarray format. Rosette tissues within NRAs possess the requisite cell phenotypes, tissue cytoarchitecture, and are derived using morphogenetic signaling pathways endogenous to the in vivo neural tube formation process. Thus, we hypothesize that the NRA platform will be an effective and efficient screening platform to detect NTD risk. Aim 1 test this hypothesis using a small chemical library screen to evaluate the NRA assay’s sensitivity and specificity. Aim 2 test this hypothesis using screens of hPSC lines genetically modified with NTD-associated genetic mutations and compared to the wild- type, isogenic control. If this high risk/high reward proposal is successful, then future research efforts would use this novel technology for systematic investigation of NTD multifactorial etiologies and clinical translation via forward screens of EPA/FDA-approved compound libraries and reverse screens using NTD patient induced pluripotent stem cell lines. In this manner, the NRA platform could have a transformative effect on NTD research, regulatory efforts to prevent commercial sale of NTD-causing agents, and future development of precision medicine approaches to minimize NTD risk in genetically predisposed patients.