Synthetic organogenesis: new paradigms in reconstituting human organ development in vitro

项目总结 器官发生是生化信号和机械信号改变胚胎胚层的过程。 在胎儿发育过程中进入器官。随着干细胞生物学的最新进展，有可能 将人类多能干细胞分化为可扩展的3D组织，其中包含许多细胞和 胎儿器官的功能特征。这些所谓的有机化合物具有巨大的潜力来回答 人类器官发生的长期问题，并有一天成为患者特有的可再生来源 人体组织。然而，人类类器官仍然只是大致概括了它们所依赖的器官发生。 关于自发组织自组织和以不可预测的方式建立信号梯度。 此外，现有的有机体方案侧重于单个器官，而不关注形态发生。我们是- 我认为，要在该领域取得有意义的进展需要1)，对耦合有更深的理解 在信号网络、组织特异性转录特征和组织形态发生之间，以及2)，发展- 控制时空信号以准确模拟器官发生的新的跨学科工具 体外培养。这项提议的目的正是为了推进我们知识中的这些差距。我们主要关注生物的器官发生 肠管，许多相邻器官在其上形成的胚胎结构，从胸腺、肺到结肠。 摆脱目前严重依赖自组织和建立的有机体范式-- 对于通常不受控制的内部梯度，我们将结合组织微图案和微流体来产生- 建立精确的信号梯度，以重复性地模拟肠管的信号和形态发生 体外器官发生。结合活细胞显微镜、CRISPR编辑和单细胞转录我们的 有机化合物将揭示细胞从多能性到区域专门化的命运选择的详细层次 肠管中的组织，它们将允许我们在信号、转录和 调节和组织形态发生。我们的工作将揭开大部分未知的调控机制的面纱。 通过它，复杂的信号通路相互作用，沿着身体轴线创造不对称的模式。对于 第一次，我们的方法将概括由一个子宫内膜形成多个相邻的肠管祖细胞。 苔藓胚芽层，为了解发育生物学中的一个长期问题提供了一个独特的窗口： 连续的信号是如何沿着身体轴线产生离散的分离器官的？最后，通过与 CRISPR专家们，我们将创造一条绕过传统形态因子驱动的外源基因分化的管道。 而是使用可诱导的基因电路来模拟命运的决定。这一新颖的系统将具有 在飞行中产生高度精确的人体组织的能力，这种方法可以被称为合成器官- 起源。我们提出的工作将为研究早期人类长期难以捉摸的分子机制开辟道路 并开始弥合有机类生物学和再生医学之间的关键鸿沟。

PROJECT SUMMARY Organogenesis is a process in which biochemical signals and mechanical cues transform embryonic germ layers into organs during fetal development. With recent advancements in stem cell biology, it has become possible to differentiate human pluripotent stem cells into expandable 3D tissues that contain many of the cellular and functional characteristics of fetal organs. These so-called organoids hold a tremendous potential to answer longstanding questions of human organogenesis and to one day serve as a renewable source of patient-specific human tissues. However, human organoids still only approximatively recapitulate organogenesis as they rely on spontaneous tissue self-organization and the establishment of signaling gradients in unpredictable ways. Furthermore, available organoid protocols focus on single organs and do not focus on morphogenesis. We be- lieve that making a meaningful progress in the field demands 1), gaining a deeper understanding of the coupling between signaling networks, tissue-specific transcriptional signatures, and tissue morphogenesis and 2), devel- oping novel cross-disciplinary tools that control spatiotemporal signaling to accurately mimic organogenesis in vitro. This proposal aims precisely to advance these gaps in our knowledge. We focus on the organogenesis of the gut tube, the embryonic structure on which many adjacent organs form, from thymus and lungs to the colon. Breaking away from the current organoid paradigms, which heavily rely on self-organization and the establish- ment of often uncontrolled internal gradients, we will combine tissue micropatterning and microfluidics to gen- erate precise signaling gradients so to reproducibly mimic both the signaling and the morphogenesis of gut tube organogenesis in vitro. Combined with live-cell microscopy, CRISPR editing, and single cell transcriptomics our organoids will reveal a detailed hierarchy of fate choices cells make from pluripotency to regionally specialized tissues in the gut tube, and they will allow us to make crucial connections between signaling, transcriptional regulation, and tissue morphogenesis. Our work will shed light on the largely unknown regulatory mechanisms by which the complex signaling pathways interact to create asymmetric patterns along the body axes. For the first time, our approach will recapitulate the formation of multiple adjacent gut tube progenitors from an em- bryonic germ layer, providing a unique window into one of longstanding questions in developmental biology: how do continuous signals create discretely separated organs along body axes? Finally, in collaboration with CRISPR experts, we will generate a pipeline that bypasses traditional morphogen-driven differentiation of plu- ripotent cells, but instead uses inducible genetic circuits to mimic fate decisions. This novel system will have the capacity to generate highly precise human tissues on the fly, in an approach that can be termed synthetic organ- ogenesis. Our proposed work will open routes to studying long elusive molecular mechanisms of early human organogenesis and start bridging the crucial gap between organoid biology and regenerative medicine.