Engineering Human Organizer To Study Left-Right Symmetry Breaking

人体看起来左右对称，但内部器官的形状和位置是不同的 在两边。偏侧缺陷，如异构性(不对称性丧失)和异质性(协调性丧失 在各个器官中)在8000名活产婴儿中被观察到超过1/8000，有助于早产和 流产，并具有重要的临床意义。我们实验室在细胞LR的研究方面开创了先河 不对称使用新的体外微尺度装置，并在器官不对称建模方面拥有丰富的经验 例如心脏C-环。我们想把我们的研究扩展到研究整体的LR不对称体 计划，由胚胎发育中的一组选定的细胞决定，最初在非洲爪哇被鉴定为 斯佩曼-曼戈尔德组织中心。从那时起，许多研究探索了左右脑的功能 组织者(LRO)在各种脊椎动物中，特别是雏鸟、鱼和老鼠。出于道德方面的考虑和14- 人类胚胎体外培养的天数限制，研究人员研究人类形成的能力 组织者非常有限。因此，寻找人类组织者的仿生替代品将是非常有意义的 到基础科学和医疗保健。 最近在基础干细胞生物学和有机体工程方面的快速科学进步使我们有可能 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(HESCs)可以被诱导表达已知的组织者标记，包括类鹅 (GSC)，通过培养类胚体或在2D微图案化的圆上形成hESCs的图案。GSC 是LRO的关键组织者标记，已知在几个脊椎动物物种中保守。面临的主要挑战 现在是我们如何将细胞设计成高度有序和自然弯曲的平面细胞片的方法 极化，甚至与纤毛的特定位置，结构和运动，以便组织者可以履行其 对称破缺中的临界函数。作为一支训练有素的生物工程师和发育生物学家团队 凭借在干细胞生物学和LR不对称性方面的经验和专业知识，我们完全有能力解决这个问题。 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协议， 几何控制的3D水凝胶培养系统和稳定的发育梯度发生器 促进人类组织者的分化和结构形成的形态生成物。我们将进一步研究 细胞固有偏向，称为细胞手性，在平面细胞极性(PCP)信号中的作用及其 人类LRO形态发生的调控。 总体而言，拟议的研究及时解决了一个非常基本但令人着迷的问题，即 发育中的LR不对称。我们不仅将建立一个研究人类LRO的体外三维平台， 也揭示了PCP的生物物理机制和手性在实现LRO关键功能中的作用。它将会铺平 进一步开发致畸物质和产前药物筛查平台的方法。

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.