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 人类肺的支气管网是一个树状结构，由超过20代的二分体组成 分支；然而，这个复杂的网络是如何形成模式的信号基础仍然是一个永恒的谜。 这不仅代表着发育生物学的一个基本知识鸿沟，也是一个限制因素。 用于开发再生疗法来对抗肺部疾病。虽然有几个看似合理的假设，如 对于这种模式机制是如何运作的，测试它们已经证明超出了经典基因敲击的限制- OUT实验和其他传统的反向工程方法，因为在 SITE。在这个提案中，我将统一一个肺发育的经典实验模型(无间充质培养 远端肺上皮)使用最先进的合成细胞信号工具，以便绘制设计图 树枝图案机构的空间。在一家最先进的生物设计中心与一个 哺乳动物合成生物学和肺发育方面的专家，我将采用“构建到理解”的方法， 我构建的人造细胞群体可以通过内源性信号与体外组织进行交流 或者使用与自然界中发现的任何信号通路垂直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概括激活/抑制反馈周期，这被认为是在 肺分支形态发生。通过操纵细胞间的通信，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高等- 订单结构。此外，通过将特定的信号轴与其更大的发展背景分离，并通过 对细胞命运进行高分辨率、延时成像，我将处于独一无二的位置来询问组织模式- 具有前所未有的可控性的ING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图案化结果，这取决于 反馈回路和初始条件。为了验证这一假设，我将探索形态和拓扑如何- 可以通过控制多细胞模式之间的信号相互作用来调整多细胞模式的序列。我最重要的是 假说是基于先前通过反应分枝形态发生的计算模型的预测- 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。 为了评估工程细胞合成信号是否也可能是产生再生的一种容易处理的方法- 肺组织，我将进一步询问3D体外模型，在该模型中细胞-细胞信号仅通过 人工合成的成形剂和受体。综上所述，这些研究将提供关于如何 复杂的解剖结构可以以相对简单的信令方案进行编码，这些信令方案被执行 细胞之间的局部。对由此产生的分支模式的分析也有望激发出一种新的哈尔-哈尔模式。 奈辛合成细胞-细胞信号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结构。