Cell Chirality Based In Vitro Models For Embryonic Development and Abnormalities

描述（由申请人提供）：手性，也称为旋手性或左右（LR）不对称性，是多细胞生物发育中的保守特征，可以在攀缘植物的生长、蜗牛壳的螺旋以及内部结构的定位中看到。 人体的器官。对于人类来说，在超过八千分之一的活产儿中观察到异构性（不对称性丧失）和异位性（个体器官之间一致性丧失）等偏侧性缺陷，并且具有重大的临床意义。在脊椎动物中，上皮手性形态发生对于建立 LR 不对称身体计划非常重要，从腹侧节点的早期节点流到后来的心脏 C 环和肠道不对称旋转。传统上，LR 不对称性是通过体内动物胚胎来研究的，这通常非常具有挑战性。由于明显的伦理问题，对人类胚胎的直接操作受到限制。最近，我们概括了微图案表面上的上皮手性形态发生。现在我们想要进一步开发体外系统来研究胚胎 LR 轴发育。我们的理由是，基于新型细胞手性的高通量平台以及对上皮细胞手性分子机制的更好理解可以极大地促进发育生物学中的LR不对称性研究。我们建议结合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作为工具来阐明上皮手性形态发生的潜在生物物理和生化机制。我们的目标是建立发育过程中 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。具体目标 1 (SA1)：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。我们建议通过将基质刚度和配体类型与天然胚胎组织相匹配来改进我们的 2D 多细胞模型，并将其扩展到单细胞水平和 3D 管状细胞片层模型。具体目标 2 (SA2)：确定对 LR 不对称重要的干细胞分化、生长因子、药物的影响和机制。我们的目标是建立图案化上皮细胞手性与发育 LR 不对称性之间的联系。具体目标 3 (SA3)：确定图案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，这些研究将建立新颖的、范式转换的系统，以高通量方式测量细胞手性，以研究发育和疾病中的 LR 不对称性，并筛选导致出生缺陷的遗传和生化因素。此外，这项拟议的研究具有变革性，并有可能开辟一个新的研究领域：细胞手性，一种定义细胞第三轴的基本细胞特性。

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.