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: 10469664
• 负责人: Christopher Matthew Madl
• 金额: $ 12.02万
• 依托单位: STANFORD UNIVERSITY
• 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 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10469664
• 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; Lead; 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; Play; Property; Protein Engineering; Reaction; Regenerative capacity; 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; base; 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.

项目摘要 尽管纤维化在由衰老和疾病引起的组织功能障碍中的作用无处不在， 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 允许定量、动态调节基质力学和组成的体外细胞培养基质， 必要然而，现有的动态水凝胶培养平台通常依赖于可能被生物降解的化学物质。 对细胞有毒或同时改变多个参数，使得难以指定因果关系 基质性质改变与细胞命运改变之间的关系。纤维化硬化发生在广泛的 包括骨骼肌。沿着纤维化的增加，骨骼肌的再生功能 随着年龄的增长而减少。肌肉干细胞（Muscle Stem Cells，MuSCs）是维持和修复肌肉的重要细胞。 在整个生命过程中，已知是急性机械敏感性的，当培养在 刚性基材。因此，硬化的纤维化微环境可能有助于减少再生性纤维化。 老年人的能力。本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，并利用该模型来确定MuSC的分子机制 机械感测与衰老中的MuSC功能障碍有关。本建议的指导阶段将 提供先进的技术培训，在老化生物学，转基因小鼠模型，细胞牵引力 测量和生物信息学的机器学习方法。这项培训将使独立的 研究计划利用动态生物材料去卷积复杂的相互作用的机械 力量，基质生物化学和细胞-细胞信号，决定了衰老和疾病的进展。额外 在科学写作、语法和研究管理方面的结构化培训将有助于向 独立性，由来自斯坦福大学医学院和工程学院的教师委员会支持。 Aim 1将优化一种合成水凝胶系统，该系统使用近红外光和生物正交反应， 动态地使凝胶变性，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将模型 体外肌肉老化，使用具有老化特征的ECM组分改性的动态硬化凝胶。 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，降低衰老中MuSC的再生潜力。Aim 3将开发新的 用于三维细胞培养的材料，具有粘弹性的动态调节，以建立第一个人体模型， 肌肉“在培养皿中老化”该项目旨在确定新的治疗靶点，以改善肌肉功能， 衰老和开发工程平台，以研究许多遗传性疾病和不同组织的衰老。

项目成果

Machine learning-based classification of dual fluorescence signals reveals muscle stem cell fate transitions in response to regenerative niche factors.

• DOI: 10.1038/s41536-023-00277-4
• 发表时间: 2023-01-14
• 期刊: NPJ REGENERATIVE MEDICINE
• 影响因子: 7.2
• 作者: Togninalli, Matteo;Ho, Andrew T. V.;Madl, Christopher M. M.;Holbrook, Colin A. A.;Wang, Yu Xin;Magnusson, Klas E. G.;Kirillova, Anna;Chang, Andrew;Blau, Helen M. M.
• 通讯作者: Blau, Helen M. M.