Developing organoid model to study active folding in a human genetic context

开发类器官模型来研究人类遗传背景下的主动折叠

基本信息

批准号：
9810045
负责人：
Sebastian J Streichan
金额：
$ 19.38万
依托单位：
UNIVERSITY OF CALIFORNIA SANTA BARBARA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-15 至 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9810045
关键词：
3-Dimensional Actomyosin Anatomy Anencephaly Animal Model Architecture Atlases Behavior Biological Models Brain Cell Culture Techniques Cells Cellular Structures Characteristics Complex Controlled Environment Cytoskeleton Data Analyses Defect Development Developmental Biology Dimensions Disease Embryonic Development Epithelium Extracellular Space Failure Foundations Gene Expression Profile Genetic Human Human Development Human Genetics Image In Vitro Investigation Kinetics Light Live Birth Lung Mechanics Methods Microfluidics Modeling Molecular Natural regeneration Neural Tube Closure Neural tube Neurons New Territories Nonmuscle Myosin Type IIA Organ Organism Organogenesis Organoids Pattern Pharmaceutical Preparations Physics Positioning Attribute Process Protocols documentation Reproducibility Role SHH gene Severities Shapes Signal Transduction Spinal Dysraphism Stem cells Structure Technology Tissues Tongue Tube Vertebral column base behavior measurement cell behavior cell type convergent extension design flexibility human model imaging approach in vivo insight matrigel morphogens neural model neuroepithelium novel overexpression prevent programs quantitative imaging relating to nervous system small molecule smoothened signaling pathway stillbirth transcription factor

项目摘要

ABSTRACT Organ shape is critical for the organism to function properly. For the example neural tube, early formation defects may have devastating consequences to the body. A fundamental building principal in embryogenesis is first organizing cells into simple structures, such as a closed sheet surrounding a lumen. Cells then undergo well-concerted programs to reconfigure tissue shape, or fold out of the plane of the sheet. In plane flows elongate one axis while shortening another during convergent extension. Folding in contrast dramatically changes tissue form, to endow the organ with its characteristic shape. In neural tube closure, an initially flat sheet of cells undergoes two sequential bending steps to fold up into a cylindrical tube surrounding a single lumen. A large body of work uncovered fundamental insights on fate determination. Yet, mechanisms controlling shape, particularly folding in the context of early human development, remain poorly understood. At the cellular level genetic patterning coordinates behaviors over long distances to instruct active forces that underlie folding. Before distilling a physical picture of how forces fold tissue, we need to identify behaviors leading to folding, and characterize coordination. Organoids harbor promise for studying early development, disease and regeneration in tightly controlled environments and human genetic background. However, progress is hampered by technical limitations, due to irregular patterning, and shape. This proposal seeks to develop a new strategy for designing robust models to study basic mechanisms of human organogenesis. Using micro patterning we successfully generated highly reproducible organoids with set size, shape, and formation kinetics. Our approach features flat rectangular shaped sheets that spontaneously fold out of the plane, and seamlessly close along the short dimension, generating a tube. The resulting platform is high throughput, compatible with live imaging, and well suited for quantitative data analysis strategies. This setup recapitulates major steps during neural tube closure in animal model systems, such as hinge formation and a zippering mechanism with actomyosin cable accompanying closure. Here we aim to adapt existing protocols to our platform to 1) induce formation of neuroepithelium and more closely model neural tube formation. We then aim to 2) assemble a quantitative atlas of cell behaviors observed during closure. Finally, we aim to 3) enter new territory and generate anatomical axes spanning the organoid tube using microfluidics. Our approach opens up a new path towards controlled models of human organogenesis, using the neural tube as example.

抽象的器官形状对于生物体的正常运作至关重要。以神经管为例，早期形成缺陷可能会给身体带来毁灭性的后果。胚胎发生的基本构建原理是首先将细胞组织成简单的结构，例如围绕管腔的封闭片。然后细胞经历协调一致的程序可以重新配置组织形状，或折叠出纸张平面。平面内流动在会聚伸展过程中拉长一根轴，同时缩短另一根轴。折叠形成鲜明对比改变组织形态，赋予器官其特征形状。在神经管闭合中，最初平坦细胞片经过两个连续的弯曲步骤，折叠成围绕单个细胞的圆柱形管流明。大量的工作揭示了关于命运决定的基本见解。然而，机制控制形状，特别是在早期人类发展的背景下的折叠，仍然知之甚少。在细胞水平上，遗传模式协调长距离的行为，以指导主动力量位于折叠之下。在提取力如何折叠组织的物理图像之前，我们需要识别行为导致折叠，并表征协调性。类器官有望研究早期发育，在严格控制的环境和人类遗传背景下的疾病和再生。然而，由于图案和形状不规则，技术限制阻碍了进展。该提案旨在制定新的策略来设计稳健的模型来研究人类器官发生的基本机制。使用微图案，我们成功地生成了具有设定大小、形状和形状的高度可重复的类器官。形成动力学。我们的方法采用扁平矩形片材，可自发折叠出平面，并沿着短尺寸无缝闭合，形成管子。由此产生的平台很高吞吐量，与实时成像兼容，非常适合定量数据分析策略。这个设置概括了动物模型系统中神经管闭合过程中的主要步骤，例如铰链形成和拉链机构带有肌动球蛋白电缆并附有闭合件。在这里，我们的目标是使现有协议适应我们的平台1）诱导神经上皮的形成并更接近地模拟神经管的形成。我们然后目标是 2) 构建闭合过程中观察到的细胞行为的定量图谱。最后，我们的目标是 3) 输入新领域并使用微流体生成跨越类器官管的解剖轴。我们的方法以神经管为例，为人类器官发生的受控模型开辟了一条新途径。