A developmental engineering toolbox for large-scale tissue engineering

项目总结 在复杂的、层次分明的组织中，如乳腺、肺和前列腺中的许多疾病已经被 很难解决，因为它们是复杂的多细胞动力学的产物。例如，先天性 肾脏疾病非常常见。大约三分之一的出生缺陷与 肾脏和尿路发育的问题，但研究人员几乎没有选择来捕捉完整的 这个体外器官系统的功能复杂性。这是因为目前的肾脏模型 或者是二维单细胞类型的近似模型，或者是具有更多细胞多样性但几乎没有 对肾脏功能至关重要的远程空间结构。 休斯实验室旨在解决开发Better In的两个关键工程障碍 体外人体组织模型。首先，我们的目标是标准化并大幅提高有机化合物的产量- 基于与人类疾病相关的表型筛选。第二，我们的目标是将一种全新的哲学带到 组织工程学，其中组织支架不是以最终形式构建的，而是作为未成熟的“种子”而构建的 被引导通过结构上的发育转变，模仿它们的目标组织。这些转变 将扁平组织支架变形为最终的组织形式，以实现定义的形状、细胞分布和ECM 3D中的压缩和对齐模式，建立了一种构建分层组织的新方法，如 肾脏。 对于第一个目标，我们建议重新设计我们的细胞dna“尼龙搭扣”细胞和类器官图案。 技术这项技术使我们能够以单细胞分辨率精确地将多种细胞类型 与有机类型凝胶层的界面，但其吞吐量目前是有限的。我们将应用感光效果 一种细胞粘附型单链DNA可以在数百万个位置同时构图的方法，这是一个关键 是全基因组有机筛选的必需品。其次，我们提出了高通量的多能干细胞。 减少有机体间变异的构图和培养技术，使全基因组CRISPR- 基于疾病遗传危险因素的筛查，以肾脏器官类物质为原型系统。发送到 第二个目标，我们建立在我们最近描述的动态组织支架的基础上来定位有机物 3D使用自主折叠的凝胶，通过动态重塑的ECM将它们的壁龛耦合在一起。 使用这些厘米级的3D有机图案能力，我们设想了一种类似于 肾脏集合管网络的分枝图案和边缘网络的“闪光”折纸图案。通过 控制这些图案的形态发生，我们试图设计一个渐进的形成 局部自组织壁龛之间毗连的收集管道网络。而不是直接 在最终但不成熟的形式下构建组织，我们相信在指导下构建分层组织 形态发生学为模拟组织动态平衡和疾病提供了一个变革性的机会。

PROJECT SUMMARY Many diseases in complex, hierarchically organized tissues such as the breast, lung, and prostate have been difficult to address, because they are a product of complex multicellular dynamics. For example, congenital diseases of the kidney are staggeringly common. Around a third of all birth defects are associated with problems in kidney and urinary tract development, but researchers have few options for capturing the full functional complexity of this organ system outside of the body. This is because current kidney models are either 2D, single cell-type approximations, or are organoid models with more cellular diversity, but with little of the long-range spatial structure that is crucial for kidney function. The Hughes lab aims to solve two critical engineering barriers to the development of better in vitro human tissue models. First, we aim to standardize and vastly increase the throughput of organoid- based phenotypic screens related to human disease. Second, we aim to bring an entirely new philosophy to tissue engineering, in which tissue scaffolds are not built in final form, but rather as immature “seeds” that are guided through developmental transitions in structure that mimic those of their target tissue. These transitions morph flat tissue scaffolds into final tissue forms that achieve defined shapes, cell distributions, and ECM compaction and alignment patterns in 3D that establish a new way of building hierarchical tissues like the kidney. To the first aim, we propose to re-engineer our cell DNA “velcro” cell and organoid patterning technology. This technology allows us to precisely pattern multiple cell types with single-cell resolution at the interface with organotypic gel layers, yet its throughput is currently limited. We will apply a photopatterning approach in which cell-adhesive ssDNA strands can be patterned in millions of locations simultaneously, a key requisite for whole-genome organoid screens. Secondly, we propose high-throughput pluripotent stem cell patterning and culture technologies that reduce inter-organoid variation, to enable whole genome CRISPR- based screening for genetic risk factors of disease, using kidney organoids as a prototypical system. To the second aim, we build upon our recent description of dynamic tissue scaffolds to position organoids in 3D using autonomously folding gels that couple their niches through tracts of dynamically remodeled ECM. Using these centimeter-scale, 3D organoid patterning capabilities, we envision an analogy between the branching pattern of the kidney collecting duct network and the edge networks of “flasher” origami patterns. By controlling the morphogenesis of these patterns, we seek to engineer the progressive formation of a contiguous collecting duct network between locally self-organizing tissue niches. Rather than directly building tissues in a final, yet immature form, we believe that building hierarchical tissues by guided morphogenesis presents a transformative opportunity for modeling tissue homeostasis and disease.