Design principles for engineering and quantitatively interrogating artificial tissues

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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.