Applications of microengineered platforms to study human laterality disorders

Abstract

During the early stages of pregnancy, exposure to certain pharmaceutical drugs can potentially lead to human laterality disorders (HLD). These disorders are characterized by alterations in the shape and positioning of tissues and organs within the body along the left-right (LR) axis. Lateralization of tissues and organs through embryonic LR symmetry breaking is a well-conserved and fundamental property of organogenesis. Exposure to teratogenic drugs (e.g., thalidomide, lithium, lead) during the first trimester of pregnancy has profound effects on the final lateralization of organs. The molecular mechanisms underlying HLD and the effects of drugs during pregnancy are still understudied. Recent studies have demonstrated that LR asymmetry at the cellular level, termed cellular chirality, may play a significant role during embryonic LR symmetry breaking. The goal of this dissertation is to identify key developmental signaling pathways that directly affect embryonic LR symmetry breaking and their molecular mechanisms. Current studies in embryonic development rely on pre-gastrulating animal embryos to identify teratogenic drugs and the disturbance of key developmental signaling pathways, which are often inapplicable to human development due to remarkable interspecies differences. Herein, we study the chirality of human embryonic stem cells (hESCs) using a 3D microengineered platform to screen drugs affecting several developmental signaling pathways. To mimic the embryonic LR asymmetry during development, hESCs were embedded within a Matrigel bilayer of different concentrations, exposed to different doses of small-molecule drugs within various signaling pathways, and their LR chiral bias (clockwise versus counterclockwise) were assessed. The canonical WNT signaling pathway (β-CATENIN-dependent) was identified as a key mediator of cellular chirality. Transient modulation of WNT pathway via shRNA targeted against β-CATENIN exhibited a dominant counterclockwise bias as WNT inhibitors. Our results provide evidence that disruption of the canonical WNT pathway affects intrinsic cellular chirality and suggest that drugs within this pathway may have adverse effects during pregnancy. Taken together, this work suggests that cellular chirality regulated by developmental signaling pathways such as the WNT pathway may alter the embryonic LR asymmetry during human development.