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。干细胞生物学的最新进展创造了不同的机会-- 将人类多能干细胞转化为可扩展的3D组织，其中包含许多细胞和功能 胎儿器官的特征，我们称之为有机体。他们拥有巨大的潜力来回答长期存在的- 在人类发育的问题和有一天作为患者特定组织的可再生来源。 然而，由于依赖于自发组织，类有机物仍然只是大致概括了器官发生。 自组织和以不可预测的方式建立信号梯度。通用协议还 忽视周围发育器官的贡献，特别是来自其他生殖层的贡献，因为它们 缺乏血管系统，它们就不能伸缩。这项补充建议的目的正是为了在 当前的有机体范例；即通过从所有三个胚层生成有机体并通过模仿 体外血管生成，以增加其可扩展性。我们的重点是肠管的器官发生，胚胎 许多相邻器官形成的结构，其轴向模式强烈依赖于信号 三个细菌层之间的反馈。我们建议使用微流控技术来产生精确的信号颗粒-- 因此，可重复性地模拟体外肠管器官发生的信号和形态发生。 我们将产生由所有三层细菌组成的原始肠道管的精心组装的组件， 然后让它们暴露在高度受控的机械环境中的信号梯度中。结合现场直播- 细胞显微镜、CRISPR编辑和单细胞转录，我们的多有机化合物将揭示详细的高- 命运选择的层级细胞从多能性到区域分割的肠管，它们将允许我们 在信号、转录调控和组织形态发生之间建立关键联系。我们的工作 将阐明在很大程度上不为人所知的调控机制，这三个细菌层通过这些机制进行沟通 沿躯干轴线生成复杂且不对称的图案。

PROJECT SUMMARY Organogenesis is a process in which biochemical signals and mechanical cues transform embryonic germ layers into organs during fetal development. Recent advances in stem cell biology created opportunities to differenti- ating human pluripotent stem cells into expandable 3D tissues that contain many of the cellular and functional characteristics of fetal organs, which we call organoids. They hold a tremendous potential to answer longstand- ing questions of human development and one day to serve as a renewable source of patient-specific tissues. However, organoids still only approximatively recapitulate organogenesis as they rely on spontaneous tissue self-organization and the establishment of signaling gradients in unpredictable ways. Common protocols also neglect the contribution of surrounding developing organs, especially from other germ layers, and because they lack the vasculature, they are not scalable. This supplement proposal aims precisely to advance these gaps in the current organoid paradigm; namely, by generating organoids from all three germ layers and by mimicking in vitro angiogenesis to increase their scalability. We focus on the organogenesis of the gut tube, the embryonic structure on which many adjacent organs form, and whose axial patterning strongly depends on the signaling feedback between all three germ layers. We propose to employ microfluidics to generate precise signaling gra- dients so to reproducibly mimic both the signaling and the morphogenesis of gut tube organogenesis in vitro. We will generate carefully assembled components of the primitive gut tube comprising all three germ layers, then expose them to signaling gradients in a highly controlled mechanical environment. Combined with live- cell microscopy, CRISPR editing, and single cell transcriptomics, our multi-organoids will reveal a detailed hi- erarchy of fate choices cells make from pluripotency to regionally segmented 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 three germ layers communicate to generate complex and asymmetric patterns along the body axes.