Synthetic morphogenesis to recapitulate multicellular airway branching patterns

合成形态发生来概括多细胞气道分支模式

• 批准号: 10606897
• 负责人: Ian S Kinstlinger
• 金额: $ 6.95万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10606897
• 关键词: 3-Dimensional; Anatomy; Architecture; Area; Biochemical; Biological; Biological Models; Cell Communication; Cell Proliferation; Cell model; Cells; Cellular Morphology; Cellular Structures; Clinical Treatment; Coculture Techniques; Communication; Complex; Comprehension; Computer Models; Coupled; Cues; Development; Developmental Biology; Devices; Diffuse Pattern; Diffusion; Distal; Embryo; Engineering; Environment; Epithelium; Experimental Designs; Experimental Models; Feedback; Fractals; Functional Regeneration; Gene Expression; Generations; Goals; Higher Order Chromatin Structure; Human; In Situ; Investigation; Kidney; Knowledge; Light; Lung; Lung diseases; Mammary gland; Maps; Mathematics; Measures; Mesenchymal; Mesenchyme; Microfluidics; Microscopy; Modeling; Morphogenesis; Morphology; Mus; Nature; Outcome; Pancreas; Paracrine Communication; Pathway interactions; Pattern; Physiological; Population; Positioning Attribute; Process; Program Development; Reaction; Regenerative Medicine; Reporter; Repression; Resolution; Reverse engineering; Scheme; Signal Pathway; Signal Repression; Signal Transduction; Stereotyping; Structure; Structure of parenchyma of lung; Synthetic Genes; Testing; Therapeutic; Tissue Engineering; Tissues; Training; Trees; Work; biliary tract; cell type; cellular imaging; design; experience; experimental study; in vitro Model; inducible gene expression; insight; intercellular communication; interest; knockout gene; lung development; lung regeneration; migration; morphogens; network architecture; novel; optogenetics; progenitor; programs; receptor; regenerative; regenerative therapy; response; self assembly; small molecule; spatiotemporal; stem cells; synthetic biology; synthetic construct; tool

Abstract The bronchial network of the human lung is a tree-like structure comprising over 20 generations of dichotomous branching; yet, the signaling basis for how this elaborate network is patterned has remained an enduring mystery. This represents not only a fundamental knowledge gap in developmental biology, but also a limiting factor for developing regenerative therapies to counter lung disease. While there are several plausible hypotheses as to how this patterning mechanism could operate, testing them has proven beyond the limits of classical gene knock- out experiments and other traditional reverse engineering approaches due to the complex signaling crosstalk found in situ. In this proposal, I will unify a classic experimental model in lung development (mesenchyme-free culture of distal lung epithelium) with state-of-the-art synthetic cell-cell signaling tools in order to map the design space for branch-patterning mechanisms. Working in a state-of-the-art Biological Design Center with a team of experts in mammalian synthetic biology and lung development, I will employ a “build-to-understand” approach wherein I construct synthetic cell populations that can either communicate with ex vivo tissues using endogenous signaling networks, or communicate with other synthetic cells using signaling pathways orthogonal to any found in nature. I will use these engineered cells to recapitulate an activation/repression feedback cycle which is thought to be vital in lung branching morphogenesis. By manipulating cell-cell communication, I will be able to isolate the fundamental design principles that govern how activation and repression signals between cells can manifest in higher- order structures. Furthermore, by decoupling specific signaling axes from their larger developmental context, and by performing high-resolution, time-lapse imaging of cell fate, I will be uniquely positioned to interrogate tissue pattern- ing mechanisms with unprecedented control. I hypothesize that reciprocal activation and repression between two cell types can give rise to a broad range of multicellular patterning outcomes depending on additional feedback loops and initial conditions. To test this hypothesis, I will explore the how the morphology and topol- ogy of multicellular patterns can be tuned by manipulating the signaling interactions between them. My overarching hypothesis is based on the predictions of previous computational models of branching morphogenesis via reaction- diffusion patterning, so I will use those predictions, and this theoretical framework, to guide my experimental designs. To assess whether synthetic signaling by engineered cells could also be a tractable approach for generating regen- erative lung tissue, I will further interrogate a 3D in vitro model where cell-cell signaling occurs exclusively through synthetic morphogens and receptors. Taken together, these studies will provide fundamental insights into how complex anatomical structures can be encoded in relatively simple signaling schemes which are executed locally between cells. Analysis of the resulting branch patterns is also expected to inspire a new paradigm for har- nessing synthetic cell-cell signaling to guide and direct the morphogenesis of therapeutically relevant cell types into tissue-specific architectures for regenerative medicine.

摘要 人肺的支气管网络是树状结构，包括超过20代的二分支气管。 然而，这个精心设计的网络是如何形成的信号基础仍然是一个持久的谜。 这不仅代表了发育生物学的基本知识差距，也是一个限制因素 用于开发再生疗法来对抗肺部疾病。虽然有几个合理的假设， 这种模式机制如何运作，测试它们已经证明超越了经典基因敲除的限制- 由于存在复杂的信令串扰， 原地。在这个建议中，我将统一肺发育的经典实验模型（无间充质培养 远端肺上皮）与最先进的合成细胞-细胞信号传导工具，以绘制设计 用于分支图案化机构的空间。在最先进的生物设计中心工作， 作为哺乳动物合成生物学和肺发育方面的专家，我将采用一种“构建理解”的方法， 我构建了合成细胞群，它们可以通过内源性信号与离体组织进行交流， 网络，或使用与自然界中发现的任何信号通路正交的信号通路与其他合成细胞通信。我 将使用这些工程细胞来重现激活/抑制反馈循环，这被认为是至关重要的， 肺分支形态发生通过操纵细胞间的通讯，我将能够分离出 设计原则，支配细胞之间的激活和抑制信号如何在更高层次上表现出来， 秩序结构。此外，通过将特定的信号传导轴与其更大的发育背景脱钩， 对细胞命运进行高分辨率的延时成像，我将处于独特的位置来询问组织模式- 以前所未有的控制机制。我假设，相互激活和抑制之间 两种细胞类型可以产生广泛的多细胞模式化结果，这取决于另外的细胞类型。 反馈回路和初始条件。为了验证这一假设，我将探讨形态和拓扑- 多细胞模式的逻辑可以通过操纵它们之间的信号相互作用来调整。我的首要任务 假设是基于以前的计算模型的预测分支形态发生通过反应- 扩散模式，所以我将使用这些预测，和这个理论框架，来指导我的实验设计。 为了评估工程细胞的合成信号传导是否也是产生再生的一种容易处理的方法， 对于再生性肺组织，我将进一步询问3D体外模型，其中细胞-细胞信号传导仅通过 合成形态发生素和受体。总之，这些研究将提供基本的见解，如何 复杂的解剖结构可以用相对简单的信号方案编码 在细胞之间。对由此产生的分支模式的分析也有望激发一种新的模式， 利用合成的细胞-细胞信号传导来引导和指导治疗相关细胞类型的形态发生， 再生医学的组织特异性架构。