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.

抽象的 人肺的支气管网络是一个树状结构，由20多代二叉树组成 分枝;然而，这个复杂的网络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础知识差距，也是一个限制因素 开发再生疗法来对抗肺部疾病。虽然有几个看似合理的假设 对于这种模式机制如何运作，测试已证明超出了经典基因敲除的限制 由于复杂的信号串扰，我们无法进行实验和其他传统的逆向工程方法 现场。在这个提案中，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，以绘制设计图 分支模式机制的空间。在最先进的生物设计中心与团队一起工作 作为哺乳动物合成生物学和肺部发育方面的专家，我将采用“构建理解”的方法，其中 我构建了可以使用内源信号与离体组织进行通信的合成细胞群 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进行通信。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，我将能够分离出基本的 控制细胞之间的激活和抑制信号如何在更高层次上体现的设计原则 订单结构。此外，通过将特定的信号轴与其更大的发育背景分离，并通过 通过对细胞命运进行高分辨率、延时成像，我将处于独特的位置来询问组织模式- 具有前所未有的控制能力的机制。我假设，相互激活和抑制 两种细胞类型可以产生广泛的多细胞模式结果，具体取决于额外的条件 反馈循环和初始条件。为了检验这个假设，我将探讨形态学和拓扑学如何 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 该假设基于先前通过反应进行分支形态发生的计算模型的预测 扩散图案，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。 评估工程细胞的合成信号是否也可以成为产生再生的易于处理的方法 对于活动性肺组织，我将进一步研究 3D 体外模型，其中细胞间信号传导仅通过 合成形态发生素和受体。总而言之，这些研究将为如何 复杂的解剖结构可以用相对简单的信号方案进行编码并执行 局部细胞之间。对由此产生的分支模式的分析也有望激发一种新的Har- 合成细胞间信号传导以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。