Cell Chirality Based In Vitro Models For Embryonic Development and Abnormalities

基于细胞手性的胚胎发育和异常体外模型

• 批准号: 8757997
• 负责人: Leo Q. Wan
• 金额: $ 243万
• 依托单位: RENSSELAER POLYTECHNIC INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8757997
• 关键词: Affect; Animals; Biochemical; Biological Assay; Cell Culture Techniques; Cells; Clinical; Congenital Abnormality; Cytoskeletal Proteins; Defect; Development; Developmental Biology; Differentiation and Growth; Disease; Embryo; Embryonic Development; Epithelial; Epithelial Cells; Ethics; Genetic Screening; Growth; Growth Factor; Handedness; Heart; Helix (Snails); Human; Human body; In Vitro; Individual; Isomerism; Left; Life; Ligands; Link; Live Birth; Measurement; Measures; Modeling; Molecular; Morphogenesis; Nodal; Organ; Organism; Pattern; Pharmaceutical Preparations; Plants; Positioning Attribute; Property; Research; Rotation; Signal Pathway; Surface; System; Time; Traction; Tubular formation; Vertebrates; base; cellular imaging; embryo tissue; embryonic stem cell; high throughput screening; improved; in vitro Model; in vivo; novel; public health relevance; stem cell differentiation; tool

DESCRIPTION (provided by applicant): Chirality, also known as handedness or left-right (LR) asymmetry, is a conserved feature in the development of multi-cellular organisms, and can be seen in the growth of climbing plants, the helices of snail shells, and the positioning of internal organs in the human body. For human, 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, and have significant clinical implications. In vertebrates, epithelial chiral morphogenesis is important in establishing the LR asymmetric body plan, from the early nodal flow at the ventral node to the later heart c-looping and gut asymmetric rotation. Traditionally, LR asymmetry is studied with animal embryos in vivo, which is often very challenging. The direct manipulation of human embryos is restricted because of the obvious ethical concerns. Recently, we have recapitulated epithelial chiral morphogenesis on micropatterned surfaces. Now we want to further develop in vitro systems for studying embryonic LR axis development. Our rationale is that novel cell chirality based high-throughput platforms and a better understanding of molecular mechanisms of epithelial cell chirality can greatly facilitate the LR asymmetry research in developmental biology. We propose to use a combination of embryonic stem cell culture, micro-fabrication, live cell imaging, molecular assay, traction force measurement, and high-throughput screening as tools to elucidate the underlying biophysical and biochemical mechanisms for epithelial chiral morphogenesis. Our objectives are to establish multiscale in vitro models for LR asymmetry in development and to identify important signaling pathways and cytoskeletal proteins that affect epithelial cell chirality. Specific Aim 1 (SA1): Establish and optimize multiscale in vitro models for studying LR asymmetry in development. We propose to improve our 2D multicellular model by matching substrate stiffness and ligand type with that of native embryonic tissue and to extend to models at single cell level and 3D tubular cell sheet. Specific Aim 2 (SA2): Determine effects and mechanisms of stem cell differentiation, growth factors, drugs that are important for LR asymmetry. We aim to establish a link between patterned epithelial cell chirality and developmental LR asymmetry. Specific Aim 3 (SA3): Determine cellular machinery mechanisms in patterned cell chirality. This will allow us to identify the ultimate cellular machinery for the emergence of chiral morphogenesis. Overall, if we are successful, these studies will establish novel, paradigm-shifting systems for measuring cell chirality in a high throughput fashion for studying LR asymmetry in development and disease, and screening genetic and biochemical factors that cause birth defects. In addition, this proposed research is transformative, and potentially open a new field of research: cell chirality, a fundamental cellular property defining the third axis of the cell.

描述（由申请人提供）：手性，也称为惯用性或左右（LR）不对称性，是多细胞生物的发展中的保守特征，可以在攀岩植物的生长，蜗牛壳的螺旋螺旋和内部的位置中看到 人体中的器官。对于人类，在8000多个活产生中有1个以上，观察到横向性缺陷，例如异构主义（不对称丧失）和异性疾病（单个器官之间的一致性丧失），并具有显着的临床意义。在脊椎动物中，上皮性手性形态发生对于建立LR不对称的身体计划很重要，从腹侧节点的早期淋巴结到后期的心脏C循环和肠道不对称旋转。传统上，LR不对称性是用体内动物胚胎研究的，这通常非常具有挑战性。由于明显的道德问题，对人类胚胎的直接操纵受到限制。最近，我们概括了微图表上的上皮性手性形态发生。现在，我们希望进一步开发用于研究胚胎LR轴发育的体外系统。我们的理由是，基于细胞手性的新型高通量平台以及对上皮细胞手性的分子机制的更好理解可以极大地促进发育生物学中的LR不对称研究。我们建议将胚胎干细胞培养，微作用，活细胞成像，分子测定，牵引力测量和高通量筛选的组合用作工具，以阐明上皮性脊髓形态发生的基本生物物理和生物化学机制。我们的目标是建立发育中LR不对称性的多尺度体外模型，并确定影响上皮细胞手性的重要信号通路和细胞骨架蛋白。特定目标1（SA1）：建立和优化用于研究开发中LR不对称的多尺度模型。我们建议通过将底物刚度和配体类型与天然胚胎组织匹配，并在单细胞水平和3D管状细胞板上延伸到模型，以改善2D多细胞模型。特定目标2（SA2）：确定干细胞分化的效果和机制，生长因子，对LR不对称重要的药物。我们旨在在图案上的上皮细胞手性和发育性LR不对称性之间建立联系。特定目标3（SA3）：确定图案细胞手性的细胞机制机制。这将使我们能够确定最终的蜂窝机械 手性形态发生的出现。总体而言，如果我们成功，这些研究将建立新颖的，范式转移的系统，以高通量的方式测量细胞手性，以研究发育和疾病中的LR不对称性，并筛选导致先天缺陷的遗传和生化因素。此外，这项提出的研究具有变革性，并有可能打开一个新的研究领域：细胞手性，这是定义细胞第三轴的基本细胞特性。