Engineering Human Organizer To Study Left-Right Symmetry Breaking

人体呈现左右对称，但内脏器官的形状和位置是不同的 在两边。偏侧性缺陷，如异构（失去不对称性）和异位（失去一致性 每8 000名活产婴儿中就有1名以上的婴儿患有先天性心脏病（在单个器官中），这导致早产， 流产，并具有重要的临床意义。我们实验室在细胞LR的研究方面处于领先地位 使用新的体外微尺度装置来测量器官不对称性，并在器官不对称性建模方面具有丰富的经验 例如心脏C环。我们希望将我们的研究扩展到研究整个LR非对称体 计划，这是由一组选定的细胞在胚胎发育，首先确定在非洲爪蟾作为 Spemann-Mangold组织中心。从那时起，许多研究探索了左-右的功能， 组织者（LRO）在各种脊椎动物，特别是鸡，鱼和小鼠。出于道德考虑和14- 在体外培养人类胚胎的时间限制，研究人员研究人类胚胎形成的能力， 组织者非常有限。因此，寻找一种仿生的人类组织者的替代品将引起人们极大的兴趣 基础科学和医疗保健。 最近在基础干细胞生物学和类器官工程方面的快速科学进展使得有可能 设计一个人类组织者来研究LR对称性破缺。科学家已经证明，人类 胚胎干细胞（hESC）可以被诱导表达已知的组织者标记物，包括Goosecoid (GSC)用胚状体的培养或在2D微图案化的圆上图案化hESC。GSC 是已知在几种脊椎动物物种中保守的LRO的关键组织者标记。的主要挑战 现在是我们如何将细胞设计成高度有序的自然弯曲的细胞片， 极化，甚至与特定的定位，结构和运动的纤毛，使组织者可以履行其 对称破缺的临界函数作为一个训练有素的生物工程师和开发生物学家团队， 凭借在干细胞生物学和LR不对称方面的丰富经验和专业知识，我们有能力解决这一问题。 我们建议开发一种新的体外人类组织者模型，该模型将利用组织者分化方案， 几何控制的3D水凝胶培养系统，以及用于发育的稳定梯度发生器， 形态发生素，以促进人类组织者的分化和结构形成。我们将进一步研究 细胞固有的偏向，称为细胞手性，在平面细胞极性（PCP）信号传导中的作用及其 调节人类LRO形态发生。 总的来说，拟议的研究是及时解决一个非常基本但迷人的问题， 发育性LR不对称。我们不仅将建立一个研究人体LRO的体外3D平台， 而且揭示了PCP和手性在实现LRO关键功能中的生物物理机制。它将铺路 进一步发展致畸剂和产前药物筛选平台的途径。

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.