MICROPATTERNED HUMAN EMBRYONIC STEM CELL CULTURES FOR MODELLING NEURAL TUBE DEFECTS
Geetika Sahni1, Yi-Chin Toh1,2
1National University of Singapore, Singapore; 2Singapore Institute for Neurotechnology, Singapore
Neural tube defects (NTDs) are one of the most common human birth defects and arise from inappropriate differentiation and morphogenic cell movements of the neuroepithelium sheet when it undergoes apical constriction and folds into the neural tube. Although several factors, including folate and genetic mutations have been implicated in the neural tube closure, it is still unclear how they affect the neural tubulogenesis, in part due to the lackof a human-relevant neural developmental model. Human embryonic stem cells (hESC) display innate capability to undergo differentiation and self-organization processes that is similar to neural tube formation but current 2D monolayer and 3D embryoid body cultures cannot spatially control the resultant neuroepithelial tissue structures that can be used as a quantiative platform to study NTDs. Here, we report a novel method to generate a spatially patterned hESC-based 3D neuroepithelial (NE) model, which can be used as a human-relevant phenotypic assay to study the effects of different genes and environmental factors involved in NTDs. We combined cell micropatterning and a multi-step induction protocol to achieve spatio-temporal control over hESC differentiation and organization into a uniform 3D annular NE structure, displaying positive expression of neural epithelial markers, Sox2, Nestin and N-cadherin. The formation of the micropatterned 3D NE was observed to capture similar aspects of in-vivo neural folding morphogenic processes, as indicated by positive expression of Nestin. In addition, a folic acid antagonist, Valproic acid, could elicit a dose-dependent disruption on the formation of the micropatterned 3D NE structure. This highlights the potential of using this model in screening NTD risk factors and quantitatively studying environmental and genetic factors involved in NTDs in a human relevant manner.