Cell Chirality Based In Vitro Models For Embryonic Development and Abnormalities

描述（由申请人提供）：手性，也称为手性或左右（LR）不对称性，是多细胞生物体发育中的保守特征，并且可以在攀援植物的生长、蜗牛壳的螺旋和内部结构的定位中看到。 人体内的器官。对于人类，在8000例活产中观察到超过1例的偏侧性缺陷，如异构（不对称性丧失）和异位（单个器官之间的一致性丧失），并具有显著的临床意义。在脊椎动物中，上皮手性形态发生是重要的，在建立LR不对称的身体计划，从早期的节点流在腹节点到后来的心脏c-循环和肠道不对称旋转。传统上，LR不对称性是用动物胚胎在体内研究的，这通常是非常具有挑战性的。由于明显的伦理问题，直接操纵人类胚胎受到限制。最近，我们概括了上皮细胞的手性形态发生的微图案化的表面。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，新的细胞手性为基础的高通量平台和更好地了解上皮细胞手性的分子机制，可以大大促进LR不对称性在发育生物学的研究。我们建议使用胚胎干细胞培养，微加工，活细胞成像，分子测定，牵引力测量和高通量筛选的工具，阐明上皮手性形态发生的生物物理和生化机制的组合。我们的目标是建立多尺度的LR不对称性在发展中的体外模型，并确定重要的信号通路和细胞骨架蛋白，影响上皮细胞手性。具体目标1（SA 1）：建立和优化多尺度体外模型，用于研究发育中的LR不对称性。我们建议通过将基底硬度和配体类型与天然胚胎组织匹配来改进我们的2D多细胞模型，并扩展到单细胞水平和3D管状细胞片的模型。具体目标2（SA 2）：确定干细胞分化、生长因子、对LR不对称性重要的药物的作用和机制。我们的目标是建立一个模式化的上皮细胞手性和发展LR不对称之间的联系。具体目标3（SA 3）：确定模式化细胞手性中的细胞机械机制。这将使我们能够确定最终的细胞机制， 手性形态发生的出现。总的来说，如果我们成功的话，这些研究将建立新的范式转换系统，以高通量的方式测量细胞手性，研究发育和疾病中的LR不对称性，并筛选导致出生缺陷的遗传和生化因素。此外，这项拟议的研究是变革性的，并可能开辟一个新的研究领域：细胞手性，一个基本的细胞属性定义的细胞的第三轴。

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.