Elucidating Effects of Fibrosis on Aged Stem Cells with Dynamic Biomaterials

用动态生物材料阐明纤维化对衰老干细胞的影响

• 批准号: 10740968
• 负责人: Christopher Matthew Madl
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-15 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10740968
• 关键词: 3-Dimensional; Acute; Adhesives; Aging; Attention; Biochemical; Biochemistry; Biocompatible Materials; Bioinformatics; Biological Models; Biology of Aging; Biophysics; Cell Culture Techniques; Cells; Cessation of life; Characteristics; Chemistry; Chronic; Complex; Coupled; Cues; Data; Defect; Deposition; Development; Disease; Engineering; Engraftment; Environment; Exhibits; Extracellular Matrix; Extracellular Matrix Proteins; Faculty; Failure; Fibrosis; Fluorescence Resonance Energy Transfer; Functional disorder; Gel; Generations; Goals; Health; Heritability; Histology; Human; Hydrogels; Impairment; In Vitro; Individual; Inflammation; Life; Light; Machine Learning; Measurement; Measures; Mechanics; Mentors; Modeling; Molecular; Molecular Target; Mus; Muscle; Muscle function; Muscle satellite cell; Muscular Atrophy; Natural regeneration; Organ failure; Pathogenesis; Pathologic; Pharmaceutical Preparations; Phase; Physiological; Property; Protein Engineering; Reaction; Regenerative capacity; Rejuvenation; Research; Research Proposals; Role; Skeletal Muscle; Stromal Cells; Structure; System; Time; Tissue Model; Tissues; Traction; Training; Transgenic Mice; Transplantation; Writing; adult stem cell; aged; aging population; bioluminescence imaging; crosslink; force sensor; genetic approach; human model; improved; in vitro Model; in vivo; induced pluripotent stem cell; insight; intercellular communication; mechanical force; mechanical properties; mechanotransduction; medical schools; mouse model; muscle aging; new therapeutic target; novel; novel therapeutics; p38 Mitogen Activated Protein Kinase; programs; regeneration function; regeneration potential; repaired; response; rho GTP-Binding Proteins; single-cell RNA sequencing; small molecule inhibitor; stem cell function; stem cell niche; stem cell population; stem cells; three dimensional cell culture; three-dimensional modeling; tissue regeneration; tissue repair; transcriptomics; viscoelasticity; wound healing

PROJECT SUMMARY Despite the ubiquitous role of fibrosis in tissue dysfunction arising from aging and disease, no representative in vitro model of the fibrotic microenvironment exists. Fibrosis is characterized by excess extracellular matrix (ECM) deposition that stiffens the cellular microenvironment. Therefore, to model fibrosis in vitro, cell culture substrates that permit quantitative, dynamic tuning of matrix mechanics and composition are necessary. However, existing dynamic hydrogel culture platforms generally rely on chemistries that may be toxic to cells or that simultaneously change multiple parameters, making it difficult to assign causal relationships between altered matrix properties and cell fate changes. Fibrotic stiffening occurs in a wide range of tissues, including skeletal muscle. Along with increased fibrosis, the regenerative function of skeletal muscle decreases with aging. Muscle stem cells (MuSCs) are responsible for maintaining and repairing muscle throughout life and are known to be acutely mechanosensitive, losing their stem cell potential when cultured on stiff substrates. Thus, the stiffened, fibrotic microenvironment may contribute to the diminished regenerative capacity of aged MuSCs. The goal of this project is to develop an in vitro model of tissue fibrosis based on dynamic hydrogel biomaterials and to employ this model to identify molecular mechanisms of MuSC mechanosensing that are implicated in MuSC dysfunction in aging. The mentored phase of this proposal will provide advanced technical training in aging biology, transgenic mouse models, cellular traction force measurement, and machine learning approaches for bioinformatics. This training will enable an independent research program leveraging dynamic biomaterials to deconvolve the complex interactions of mechanical forces, matrix biochemistry, and cell-cell signaling that dictate the progression of aging and disease. Additional structured training in scientific writing, grantsmanship, and research management will facilitate the transition to independence, supported by a committee of faculty from the Stanford Schools of Medicine and Engineering. Aim 1 will optimize a synthetic hydrogel system that uses near-infrared light and bioorthogonal reactions to dynamically stiffen the gels, mimicking fibrosis. These hydrogels will be used to elucidate mechanisms of mechanosensing in MuSCs, using FRET-based force sensors and transgenic mouse models. Aim 2 will model muscle aging in vitro, using dynamically stiffening gels modified with ECM components characteristic of aging. Single cell RNA sequencing and machine learning bioinformatics approaches will identify unique mechanically regulated drivers of cell fate that reduce MuSC regenerative potential in aging. Aim 3 will develop novel materials for 3D cell culture with dynamic tuning of viscoelastic properties to establish the first human model of muscle “aging in a dish.” This project stands to identify new therapeutic targets to improve muscle function with aging and to develop engineered platforms to study numerous heritable diseases and aging in diverse tissues.

项目概要 尽管纤维化在衰老和疾病引起的组织功能障碍中发挥着普遍的作用，但没有 存在纤维化微环境的代表性体外模型。纤维化的特点是过度 细胞外基质（ECM）沉积使细胞微环境变硬。因此，为了模拟纤维化 在体外，允许定量、动态调整基质力学和成分的细胞培养基质是 必要的。然而，现有的动态水凝胶培养平台通常依赖于化学物质，这些化学物质可能 对细胞有毒或同时改变多个参数，使得很难确定因果关系 改变的基质特性和细胞命运变化之间的关系。纤维化硬化的发生范围很广 组织，包括骨骼肌。随着纤维化的增加，骨骼肌的再生功能 随着年龄的增长而减少。肌肉干细胞（MuSC）负责维持和修复肌肉 在整个生命周期中，已知具有敏锐的机械敏感性，在培养时会失去干细胞潜力 坚硬的基材。因此，僵化的、纤维化的微环境可能导致再生能力的减弱。 老化 MuSC 的容量。该项目的目标是开发一种基于组织纤维化的体外模型 动态水凝胶生物材料并利用该模型来识别 MuSC 的分子机制 机械传感与衰老过程中 MuSC 功能障碍有关。该提案的指导阶段将 提供衰老生物学、转基因小鼠模型、细胞牵引力方面的先进技术培训 生物信息学的测量和机器学习方法。该培训将使独立 利用动态生物材料来解构机械的复杂相互作用的研究计划 力、基质生物化学和细胞信号传导决定衰老和疾病的进展。额外的 科学写作、资助和研究管理方面的结构化培训将促进向 独立性，得到斯坦福大学医学与工程学院教师委员会的支持。 目标 1 将优化合成水凝胶系统，该系统使用近红外光和生物正交反应来 动态地使凝胶变硬，模拟纤维化。这些水凝胶将用于阐明 使用基于 FRET 的力传感器和转基因小鼠模型在 MuSC 中进行机械传感。目标 2 将建模 使用经 ECM 成分修饰的动态硬化凝胶进行体外肌肉老化特征的老化。 单细胞 RNA 测序和机器学习生物信息学方法将识别独特的机械 调节细胞命运的驱动因素，降低衰老过程中 MuSC 的再生潜力。目标3将开发新颖 用于 3D 细胞培养的材料，可动态调整粘弹性，以建立第一个人体模型 肌肉“在盘子里老化”。该项目旨在确定新的治疗靶点，以改善肌肉功能 衰老并开发工程平台来研究多种遗传性疾病和不同组织中的衰老。