Engineering Human Organizer To Study Left-Right Symmetry Breaking

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The human body appears left-right (LR) symmetric, but the shape and positioning of internal organs are distinct at two sides. Defects in laterality such as isomerism (loss of asymmetry), and heterotaxia (a loss of concordance among the individual organs) are observed in more than 1 in 8000 live births, contribute to pre-term births and miscarriage, and have significant clinical implications. Our lab has pioneered in the research of the cellular LR asymmetry using novel in vitro microscale devices and has extensive experience in modeling organ asymmetries such as cardiac c-looping. We would like to extend our research into studying the overall LR asymmetric body plan, which is determined by a select group of cells in embryonic development, first identified in Xenopus as the Spemann-Mangold organizing center. Since then, many studies have explored the functionality of a left-right organizer (LRO) in various vertebrates, in particular, chick, fish, and mouse. Due to ethical concerns and the 14- day restriction of culturing human embryos in vitro, the ability of researchers to study the formation of a human organizer is very limited. Therefore, finding a biomimetic surrogate of the human organizer will be of great interest to basic science and health care. Recent rapid scientific advances in basic stem cell biology and organoid engineering have made it possible to engineer a human organizer for studying LR symmetry breaking. Scientists have demonstrated that human embryonic stem cells (hESCs) can be induced to express known organizer markers, including the Goosecoid (GSC), with either the culture of embryoid bodies or the patterning of hESCs on 2D micropatterned circles. GSC is a key organizer marker of LRO known to be conserved across several vertebrate species. The major challenge now is how we can engineer the cells into highly organized and naturally curved cell sheets with planar polarization and even with specific localization, structure, and motion of cilia so that the organizer can fulfill its critical function in symmetry breaking. As a team of well-trained bioengineers and development biologists with experience and expertise in stem cell biology and LR asymmetry, we are well-equipped to address this problem. We propose to develop a novel in vitro human organizer model that will utilize organizer differentiation protocols, a geometrically-control 3D hydrogel culture system, and a stable gradient generator for developmental morphogens to facilitate the differentiation and structural formation of a human organizer. We will further study the role of the cellular intrinsic bias, termed cell chirality, in planar cellular polarity (PCP) signaling and its regulation of the human LRO morphogenesis. Overall, the proposed study is timely in addressing a very fundamental yet fascinating question regarding the developmental LR asymmetry. We will not only establish an in vitro 3D platform for studying the human LRO, but also reveal biophysical mechanisms of PCP and chirality in realizing the critical function of LRO. It will pave the way towards the further development of screening platforms for teratogens and prenatal drugs.