G.T. Knight, B.F. Lundin, N. Fedorchak, N.R. Iyer, R. Willet, and R.S. Ashton
Wisconsin Institute for Discovery,
United States
Keywords: hPSC, neural organoid, developmental neurotox, neural tube defect
Summary:
Human pluripotent stem cell (hPSC)-derived neural organoids, which model central nervous system (CNS) development and function, are powerful in vitro tools for investigating human neural physiology and disease. However, their spontaneous and inconsistent emergence during derivation limits their reproducibility and scalability for commercial application. We hypothesized that inconsistencies in neural organoid structure and composition are caused by the absence of biophysical and biochemical cues normally present within the developing human embryo. Thus, we bioengineered a microarray culture platform that enables facile spatiotemporal control of microenvironmental cues to standardize neural organoid morphogenesis, i.e., the Rosette ArrayTM. Currently, this platform enables efficient, reproducible, and high-throughput derivation and analysis of hPSC-derived neural organoids that mimic the earliest stage of CNS development, i.e., neural tube formation. Importantly, the Rosette Array represents a technology basis that can be expanded to model and screen numerous facets of human CNS development and disorders for commercial toxicology and drug discovery applications. Here, we present validation of the Rosette Array platform for use as a developmental neurotoxicity (DNT) screening tool with specific focus on identifying teratogenic effects, i.e., assessing neural tube defect (NTD) risk—the second most prevalent birth defect. Through screening a library of pharmaceutical, agrochemical, and industrial compounds, many of which are known to cause NTDs, we have established a high-throughput pipeline to identify compounds that perturb microarrayed rosette tissue formation and provide insights into their mode of action. Uniquely, the Rosette Array platform can screen for teratogenic effects across developing CNS brain and spinal regions. It is compatible with the solvent dimethylsulfoxide (DMSO) up to 0.1%, and it is conducted in a 96-well plate over 7-days with direct seeding of cryopreserved cell banks. Assay quantification is provided by automated image analysis of immunocytochemistry results captured via standard microscopy. Also, it can incorporate simulated human metabolism of screened chemical compounds. In summary, the Rosette Array enables high-throughput screening of factors that perturb human CNS development and discovery of drugs that mitigate patient-specific NTD risk. Thus, it can significantly streamline, increase the human relevance, and reduce the cost of traditional in vitro DNT assay batteries and drug discovery pipelines for neurological disorders.