Engineering Human Organizer To Study Left-Right Symmetry Breaking

工程人类组织者研究左右对称性破缺

• 批准号: 10667938
• 负责人: Leo Q. Wan
• 金额: $ 23.17万
• 依托单位: RENSSELAER POLYTECHNIC INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-04 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10667938
• 关键词: 3-Dimensional; Address; Adopted; Animals; Basic Science; Biomedical Engineering; Biomimetics; Biophysical Process; Cardiac; Cell Differentiation process; Cell Polarity; Cells; Chick; Cilia; Clinical; Congenital Abnormality; Defect; Development; Devices; Diffusion; Embryo; Embryonic Development; Engineering; Ethics; Extracellular Matrix; Fishes; Handedness; Healthcare; Human; Human Engineering; Human body; Hydrogels; In Vitro; Individual; Isomerism; Left; Live Birth; Mechanics; Modeling; Morphogenesis; Morphology; Motion; Mus; Organ; Organ Model; Organoids; Pattern; Pharmaceutical Preparations; Play; Positioning Attribute; Premature Birth; Process; Regulation; Research; Research Personnel; Role; Scientific Advances and Accomplishments; Scientist; Shapes; Side; Signal Transduction; Spontaneous abortion; Structure; System; Techniques; Teratogens; Tissue Engineering; Training; Vertebrates; Xenopus; differentiation protocol; experience; fascinate; human embryonic stem cell; interest; morphogens; novel; organ on a chip; planar cell polarity; prenatal; screening; stem cell biology; stem cell fate; stem cells; three-dimensional modeling

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.

人体呈现左右对称，但内脏器官的形状和位置不同 在两侧。偏侧性缺陷，例如异构（不对称性丧失）和异位性（一致性丧失） 在超过八千分之一的活产中观察到这种现象，导致早产和 流产，具有重要的临床意义。我们实验室在细胞LR研究方面处于领先地位 使用新颖的体外微型设备研究不对称性，并在模拟器官不对称性方面拥有丰富的经验 例如心脏c环。我们希望将我们的研究扩展到整体 LR 不对称身体的研究 计划是由胚胎发育中的一组选定的细胞决定的，首先在非洲爪蟾中被识别为 斯佩曼-曼戈尔德组织中心。从那时起，许多研究都探索了左右手的功能 各种脊椎动物，特别是鸡、鱼和小鼠中的组织者（LRO）。由于道德问题和 14- 体外培养人类胚胎的天数限制，研究人员研究人类形成的能力 组织者的能力非常有限。因此，寻找人类组织者的仿生替代品将引起人们极大的兴趣 基础科学和医疗保健。 最近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 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，包括 Goosecoid (GSC)，可以是胚状体的培养，也可以是 hESC 在 2D 微图案圆圈上的图案化。 GSC 是已知在多个脊椎动物物种中保守的 LRO 的关键组织者标记。主要挑战 现在我们如何将细胞设计成高度组织且自然弯曲的细胞片，并具有平面 极化，甚至具有纤毛的特定定位、结构和运动，以便组织者能够实现其 对称性破缺的关键函数。作为一支由训练有素的生物工程师和发育生物学家组成的团队 凭借在干细胞生物学和 LR 不对称方面的经验和专业知识，我们有能力解决这个问题。 我们建议开发一种新型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 3D 水凝胶培养系统和用于发育的稳定梯度发生器 形态发生素促进人类组织者的分化和结构形成。我们将进一步研究 细胞内在偏差（称为细胞手性）在平面细胞极性（PCP）信号传导中的作用及其 人类 LRO 形态发生的调节。 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： 发育性LR不对称。我们不仅会建立一个体外3D平台来研究人类LRO， 还揭示了 PCP 和手性在实现 LRO 关键功能中的生物物理机制。它将铺平 进一步开发致畸剂和产前药物筛查平台的途径。