Design principles for engineering and quantitatively interrogating artificial tissues

发现挑战：控制组织组装和模式的潜在设计规则尚未得到很好的理解，这是发育生物学中一个长期存在的难题。我的研究项目的长期目标是通过体外工程和硅系统来理解组织模式和形态发生，从而能够系统地操纵和量化这些基本的生物过程。**我之前的DG培训了24 HQP，并开发了控制组织模式和量化群体细胞迁移的工具。利用这些独特的工具，本提案侧重于理解组织组装和图案过程的两个关键问题：*1。在几何或化学线索存在的情况下，什么规则决定了集体细胞迁移行为？我们能否将生物传感器整合到工程组织中，在复杂的异质微环境中定量监测化学信号？**目的1：量化暴露在宏观几何重组下的单上皮片的协调和细胞形状变化*方法：在胚胎中，简单上皮片被广泛重组，以响应组织几何结构宏观变化产生的生物物理力。利用底物微图像化、实时成像和定量细胞跟踪，我们将量化上皮细胞、成纤维细胞和多能基质细胞（MSCs）单层在宏观薄片几何变化下重组过程中的细胞形状和细胞间协调，并开发这一过程的硅模型。我们将通过操纵钙粘蛋白和肌球蛋白水平来探讨连接强度和细胞张力对这一过程的影响。**目的2：在3D中量化氧和化学信号梯度对细胞重组的影响*方法：组织是含有氧和化学信号梯度的异质结构。在异质三维结构中控制细胞重组的规则还没有被很好地理解。我们开发了一种独特的分层组织组装策略，可以通过分层去堆叠轻松地拆卸组织和从特定组织位置分离细胞。使用这个工具，我们将探索MSCs在密集的3D层状结构中的重组。具体来说，我们将量化细胞密度和氧和视黄酸信号梯度对细胞重组行为的影响。**目的3：将生物传感器整合到3D组织模型中，以报告来自局部微环境的信号提示的空间变化。*方法：细胞通讯是组织结构协调行为和自组装的基础。因此，为了更好地理解组织形态发生，可视化和量化细胞结构重组过程中信号环境的变化将是有用的。利用我们的分层组织策略，结合基于蛋白质的生物传感器，我们将开发出在含有MSCs的体外组织中三维空间监测维甲酸的策略。**新颖性和意义：我的研究项目的新颖性是应用工程中的实验和建模工具，来操纵和询问复杂组织组装的规则。这里描述的具体目标将产生一套新的定量规则，控制组织组装和多维细胞间通讯。我们独特的体外和计算机方法将提供补充动物模型的平台，以更好地了解组织发育的基本机制。我们的工作还将产生新的数据采集和分析方法，以定量地询问空间异构复杂系统。

DISCOVERY CHALLENGE: The underlying design rules that govern tissue assembly and patterning are not well understood and represent a long-standing puzzle in developmental biology. The long-term objective of my research program is to understand tissue patterning and morphogenesis by engineering in vitro and in silico systems to enable systematic manipulation and quantification of these fundamental biological processes.**My previous DG trained 24 HQP and developed tools to control tissue patterning and quantify group cell migration. Exploiting these unique tools, this proposal focuses on two key questions critical to understanding the process of tissue assembly and patterning: *1. What rules dictate collective cell migration behaviours in the presence of geometric or chemical cues?*2. Can we incorporate biosensors into engineered tissues to quantitatively monitor chemical signals in complex heterogeneous microenvironments?**OBJECTIVE 1: Quantify coordination and cell shape changes in sheets exposed to macroscopic geometric re-organization *APPROACH: In the embryo simple epithelial sheets are re-structured extensively, in response to biophysical forces generated from macroscopic changes in tissue geometry. Using substrate micropatterning, live imaging and quantitative cell tracking we will quantify cell shape and cell-cell coordination during re-organization of epithelial, fibroblast and multipotent stromal cell (MSCs) monolayers in response to changes in macroscopic sheet geometry and develop an in silico model of this process. We will probe the impact of junction strength and cellular tension on this process by manipulating cadherin and myosin levels. **OBJECTIVE 2: Quantify the impact of oxygen and chemical signalling gradients on cell re-organization in 3D*APPROACH: Tissues are heterogeneous structures containing gradients of oxygen and chemical signals. The rules that govern cell re-organization in heterogeneous 3D structures are not well understood. We have developed a unique layered tissue assembly strategy that allows easy tissue disassembly and separation of cells from specific tissue locations by layer de-stacking. Using this tool we will explore re-organization of MSCs in dense 3D layered structures. Specifically we will quantify the impact of cell density and the presence of oxygen and retinoic acid signalling gradients on cellular re-organization behaviour.**OBJECTIVE 3: Incorporate biosensors into 3D tissue models to report spatial variations in signalling cues from the local microenvironment. *APPROACH: Cellular communication underlies coordinated behaviour and self-assembly of tissue structures. To better understand tissue morphogenesis it would therefore be useful to visualize and quantify variations in the signalling environment in re-organizing cellular structures. Using our layered tissue strategy, combined with protein-based biosensors, we will develop strategies to spatially monitor retinoic acid in 3D in vitro tissues containing MSCs. **NOVELTY AND SIGNIFICANCE: The novelty of my research program is the application of experimental and modelling tools from engineering, to manipulate and interrogate the rules that dictate assembly of complex tissues. The specific objectives described here will generate a set of novel quantitative rules that govern tissue assembly and multi-dimensional intercellular communication. Our unique in vitro and in silico approaches will provide platforms that complement animal models to better understand the fundamental mechanisms that underlie tissue development. Our work will also generate novel data acquisition and analytical methods to quantitatively interrogate spatially heterogeneous complex systems.