Development of in vitro human spinal neural tube model to study genetic and environmental etiology of Spina Bifida.

PROJECT SUMMARY Spina bifida (SB) is a viable type of neural tube defect (NTD), the second most prevalent birth defect in humans. The genetic and environmental factors combinatorically contribute to the etiology of SB. Neuromesodermal progenitors (NMPs) are the precursors of spinal neural tubes (SNT), yet how genetic modification and teratogen affect NMP-mediated SNT formation is largely unknown. Model organisms have been widely used to study the effects of genetic and environmental factors on SB. Still, they have limitations in fully recapitulating human NTDs due to their different genetic backgrounds and subsequent drug sensitivity. Significant progress on in vitro 3D SNT organoids has been made, yet they did not fully recapitulate the in vivo spatiotemporal microenvironment to study the gene-environment interaction. Here, we will investigate the complex interplay between the genetic predisposition and teratogen in NMP-mediated human SNT formation using human induced pluripotent stem cells (hiPSCs) that recreate three-dimensional (3D) SNT organoids to fill the current scientific gaps. We will further develop a bioengineered Spinal Neural Tube-on-a-chip (SNT Chip) model to recapitulate the spatiotemporal microenvironment by controlling microfluidic modules to fulfill the technical gaps. Our preliminary study supported that GPR161 has a strong genetic association with human and mouse SB and NMPs are involved in GPR161-mediated SNT formation in mice. We will utilize GPR161 KO iPSCs-induced 3D SNT organoids as a human-relevant SB model to investigate how GPR161 genetic modification and its interaction with teratogens (Vismodegib and Valproic acid) affect NMP-mediated human SNT formation in both the static 3D human SNT organoids and an SNT-Chip. This will verify how the spatiotemporal modulations with biomechanical cues regulate the human SNT formation and the subsequent cellular and molecular profiles of human SNTs compared to the static 3D organoid cultures. We anticipate enhancing the scientific knowledge of gene- environmental interactions on human SNT formation and advancing the in vitro modeling of a human SNT system with in vivo-relevant microenvironmental milieu.