Design principles for engineering and quantitatively interrogating artificial tissues

发现挑战：管理组织组装和图案形成的潜在设计规则还没有被很好地理解，这是发育生物学中一个长期存在的难题。我的研究计划的长期目标是通过在体外和硅胶系统中通过工程来了解组织模式和形态发生，以便能够系统地操作和量化这些基本的生物过程。我之前的DG培训了24名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，这项建议集中在两个关键问题上，对理解组织组装和图案化过程至关重要：1.在几何或化学线索存在的情况下，什么规则规定了集体细胞的迁移行为？2.我们能否将生物传感器引入工程组织中，以定量监测复杂的异质微环境中的化学信号？目标1：量化暴露在宏观几何重组方法下的薄片中的配位和细胞形状变化：在胚胎中，简单的上皮片被广泛地重组，以响应组织几何的宏观变化产生的生物物理力。利用底物微图案化、实时成像和定量细胞跟踪，我们将量化上皮细胞、成纤维细胞和多潜能基质细胞(MSCs)单分子层重组过程中的细胞形状和细胞-细胞协调，以响应宏观片层几何结构的变化，并开发这一过程的计算机模型。我们将通过控制钙粘附素和肌球蛋白的水平来探讨连接强度和细胞张力对这一过程的影响。目的：量化氧和化学信号梯度对3DAPPROACH细胞重组的影响：组织是包含氧和化学信号梯度的异质结构。在异质3D结构中管理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，允许通过层叠来轻松地拆解组织并将细胞从特定组织位置分离出来。使用这个工具，我们将探索MSCs在致密的3D分层结构中的重组。具体地说，我们将量化细胞密度以及氧气和维甲酸信号梯度对细胞重组行为的影响。目标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线索的空间变化。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，在重组细胞结构的过程中，可视化和量化信号环境中的变化将是有用的。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3D体外组织中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了工程学的实验和建模工具，来操纵和询问指示复杂组织组装的规则。这里描述的具体目标将产生一套管理组织组装和多维细胞间通信的新的量化规则。我们独特的体外和硅胶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，以定量询问空间不同的复杂系统。

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 3DAPPROACH: 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.