Optical dissection of human embryonic germ layer patterning mechanisms using microengineered stem cell models

项目摘要 在发育的前两周，人类胚胎两次打破对称， 将一个均匀的细胞球转化为高度图案化的、空间组织化的组织。虽然几十年的优雅 非人类模式生物的遗传学和生物化学已经揭示了许多重要的信号传导， 蛋白质和途径的胚胎图案在这个早期阶段，缺乏工具，直接操纵 时间和空间上的信号，以及对人类胚胎的限制，限制了我们的能力， 理解这些早期模式事件是如何在人类中产生的。因此，我的实验室致力于开发 工程化策略以（1）理解什么样的信号传导活性模式编码细胞外信息， 以及（2）确定如何在组织水平上解码这些模式以驱动高保真集体细胞命运 人类胚胎干细胞中的决定。 这项提议汇集了干细胞和分子工程方面的许多最新进展， 时空信号和胚胎大小如何指导组织命运模式。我们利用2D技术的进步 微图案化，3D生物打印，发育信号通路的细胞光遗传学控制，以及 基于CRISPR的细胞信号传导通路报告基因。这些技术共同为我们提供了前所未有的 控制和可视化人类原肠胚形成的微工程模型，从而使我们能够 研究环境信息传递的原理和怀孕的潜在机制 丧失和发育异常的发生率高得惊人（据估计为10 - 20%）， 早期人类胚胎此外，我们的平台不会面临限制我们的业务发展的道德障碍。 人类胚胎研究，使我们能够确定如何物理和信息承载参数的 胚胎在人类原肠胚形成期间导致胚层的定型模式。在本提案中，我们将重点 通过剖析时空信号的作用， 和Wnt途径在2D中对胚层命运定位的变化（目的1）;检查Erk的作用 在发育过程中，细胞的定位，动力学和协调信号 中胚层/滋养外胚层边界（目标2）;并在3D中检查细胞数量和组织大小的影响 人上胚层和羊膜囊的原肠胚形成模型（目的3）。

Project Summary In the first two weeks of development, the human embryo breaks symmetry twice, transforming itself from a uniform ball of cells into a highly-patterned, spatially organized set of tissues. Although decades of elegant genetics and biochemistry in non-human model organisms have uncovered many of the essential signaling proteins and pathways for embryonic patterning at this early stage, a lack of tools for directly manipulating the signaling in time and space, as well as limitations to working with human embryos, have limited our ability to understand how these early patterning events arise in humans. Thus, my laboratory seeks to develop engineering strategies to (1) understand what patterns of signaling activity encode extracellular information, and (2) determine how these patterns are decoded at the tissue level to drive high fidelity collective cell fate decisions in human embryonic stem cells. This proposal brings together many recent advances in stem cell and molecular engineering to decode how spatiotemporal signaling and embryo size instruct tissue fate patterning. We leverage advances in 2D micropatterning, 3D bioprinting, cellular optogenetic control over developmental signaling pathways, and CRISPR-based reporters of cell signaling pathways. Together, these technologies give us unprecedented control over and visualization of microengineered models of human gastrulation, thereby enabling us to investigate the principles of environmental information transmission and potential mechanisms of pregnancy loss and developmental anomalies that arise with a surprisingly high frequency (10-20% by some estimates) in the early human embryo. In addition, our platform does not face the same ethical barriers that have limited human embryo research, allowing us to ascertain how physical and information-bearing parameters of the embryo lead to stereotyped patterning of the germ layers during human gastrulation. In this proposal, we focus on the role of canonical developmental signaling pathways by dissecting the effects of spatiotemporal signaling and variance of the Wnt pathway on germ layer fate positioning in 2D (Aim 1); examining the role of Erk signaling on positioning, dynamics, and coordination of cells during the development of the mesoderm/trophectoderm boundary (Aim 2); and examining the effect of cell number and tissue size in 3D gastrulating models of the human epiblast and amniotic sac (Aim 3).