A developmental engineering toolbox for large-scale tissue engineering

项目摘要 在复杂的、分层组织的组织如乳腺、肺和前列腺中的许多疾病已经被发现。 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 肾脏疾病是惊人的普遍。大约三分之一的出生缺陷与 肾脏和泌尿道发育的问题，但研究人员几乎没有选择捕捉完整的 这个器官系统在体外的功能复杂性。这是因为目前的肾脏模型 无论是2D，单细胞型近似，或具有更多细胞多样性的类器官模型，但几乎没有 对于肾脏功能至关重要的远程空间结构。 休斯实验室的目标是解决两个关键的工程障碍，以发展更好的， 体外人体组织模型。首先，我们的目标是标准化并大大提高类器官的产量- 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，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”， 通过模仿其目标组织的结构的发育转变来引导。这些转变 将扁平的组织支架变形为最终的组织形式，以实现确定的形状、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， 肾 对于第一个目标，我们建议重新设计我们的细胞DNA“维可牢”细胞和类器官模式 技术.这项技术使我们能够以单细胞分辨率精确地对多种细胞类型进行图案化， 尽管其与有机型凝胶层的界面结合，但其通量目前受到限制。我们将应用一种新的模式 细胞粘附性ssDNA链可以同时在数百万个位置形成图案的方法是一个关键 全基因组类器官筛选所必需的。其次，我们提出了高通量多能干细胞 减少类器官间变异的模式化和培养技术，使全基因组CRISPR- 基于疾病遗传风险因素的筛查，使用肾脏类器官作为原型系统。到 第二个目标，我们建立在我们最近的动态组织支架的描述，以定位类器官， 3D使用自主折叠的凝胶，通过动态重塑的ECM束耦合它们的小生境。 使用这些厘米级的3D类器官图案化能力，我们设想了一个类比， 肾集合管网络的分支模式和“闪光”折纸模式的边缘网络。通过 控制这些模式的形态发生，我们寻求工程师的逐步形成， 局部自组织组织小生境之间的连续集合管网络。而不是直接 构建最终的组织，但不成熟的形式，我们相信，通过引导构建分层组织， 形态发生为组织稳态和疾病的建模提供了变革性的机会。

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.