Physical Regulation of Muscle Stem Cells in Bioengineered 3D Models

生物工程 3D 模型中肌肉干细胞的物理调节

• 批准号: 8166019
• 负责人: Penney Marie Gilbert
• 金额: $ 8.63万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8166019
• 关键词: Accidents; Adult; Advisory Committees; Aging; Architecture; Award; Bed rest; Behavior; Biocompatible Materials; Bioinformatics; Biological Assay; Biology; Biomechanics; Biomedical Engineering; Cachexia; Cell Culture Techniques; Cell Fate Control; Cell Line; Cell Shape; Cell Survival; Cell Therapy; Cells; Clinical; Critiques; Cues; Cytoskeleton; Data; Defect; Development; Disease; Elasticity; Encapsulated; Engineering; Ensure; Environment; Extracellular Matrix; Fiber; Focal Adhesions; Force of Gravity; Foundations; Funding; Future; Gene Expression; Genetic; Goals; HIV Infections; Healthcare Systems; Hereditary Disease; Hydrogels; K-Series Research Career Programs; Knowledge; Laboratories; Learning; Malignant Neoplasms; Mechanics; Mediating; Mentors; Methods; Microfabrication; Microscopy; Modeling; Molecular; Movement; Mus; Muscle; Muscle Fibers; Muscle satellite cell; Muscular Atrophy; Muscular Dystrophies; Natural regeneration; Nature; Pathway interactions; Patients; Pattern; Physical environment; Postdoctoral Fellow; Property; Public Health; Quality of life; RNA; RNA Sequences; Regulation; Research; Research Personnel; Rest; Risk; Role; Skeletal Muscle; Skeletal muscle injury; Stem cells; System; Testing; Therapeutic; Time; Time Management; TimeLine; Tissue Engineering; Tissues; Training; Universities; Work; Writing; adult stem cell; aged; base; career; career development; cell assembly; cell behavior; design; in vivo; injured; insight; interdisciplinary approach; multidisciplinary; muscle form; muscle regeneration; new therapeutic target; novel strategies; physical property; prospective; regenerative; repaired; sarcopenia; satellite cell; scaffold; self-renewal; skeletal; skeletal muscle wasting; skills; stem cell biology; stem cell fate; stem cell niche; stemness; success; therapeutic development; three-dimensional modeling; wasting

DESCRIPTION (provided by applicant): Skeletal muscle wasting that occurs during aging and numerous other pathological conditions including HIV infection and cancer is a serious public health concern and was a financial burden of more than $18.5 billion to the U.S. healthcare system in 20001. Currently there are no effective strategies to promote muscle regeneration in aged, injured, diseased or physical inactive skeletal muscle. The inherent capacity of muscle tissue to regenerate itself is mediated by resident skeletal muscle stem cells (MuSCs)2,3,4. Recent advances by our laboratory and others have identified strategies to prospectively isolate MuSCs from murine skeletal muscle5,6,7,8,9,10,11. Due to the relatively nascent nature of the MuSC field, we are only just beginning to understand the mechanisms underlying MuSC regulation. Elucidating MuSC regulation will be critical to harness the potential of this adult, tissue-specific stem cell and lend to the development of therapeutics to overcome muscle atrophy. In recent studies we demonstrated that substrate elasticity, a physical property of the MuSC niche, is a potent regulator of MuSC viability and self-renewal12,13. Using a biomaterials approach in conjunction with in vivo functional assays, we showed for the first time that 'stemness' is maintained by culturing MuSCs on substrates that match the softness of muscle tissue. In this Career Development Proposal, we aim to extend this finding to gain further insight into MuSC regulation by physical features of the in vivo niche. Specifically, we will use a multidisciplinary approach to explore MuSC fate modulation by substrate rigidity and niche architecture and will focus on revealing mechanistic insights. In Aim 1, we will elucidate the mechanisms of MuSC regulation imposed by 2D substrate elasticity. This aim will test the hypothesis that MuSCs possess 'rigidity sensing' systems and that these systems transduce information about the physical environment to modify gene expression and ultimately, cell fate. The goal of Aim 2 is to engineer a skeletal muscle tissue mimic using engineered extra-cellular matrix (eECM) to interrogate MuSC physical regulation in the context of a 3D environment. We hypothesize that MuSC regulation by substrate rigidity will be fundamentally different in a 3D setting as compared to our 2D observations and aim to decouple the effects of 'dimensionality' on MuSC viability, proliferation and fate (quiescence, activation, self-renewal, differentiation). Finally, in Aim 3 we will investigate the role of 3D niche architecture on MuSC fate. We will use microfabricated models to test the hypothesis that MuSC quiescence and activation are determined by 3D niche architecture. The knowledge gained from these Aims will lend to our fundamental understanding of niche composition and MuSC regulation. The ultimate goal of this work is to potentiate development of cell-based or systemically delivered therapeutics designed to promote skeletal muscle regeneration in specific contexts of muscle wasting. If funded, this Career Development Award (CDA) would afford me the opportunity to expand my background in skeletal muscle biology, gain necessary knowledge of biomaterials approaches, achieve the capacity to interpret bioinformatics data and acquire expertise in microscopy; skills critical to my future success as an independent researcher (see timeline). Formal and informal interactions with my Advisory Committee will assess my progress on the proposed Aims and provide critique and advice. Finally, professional skills (e.g. mentoring, research presentation, time management, writing, etc) vital to my long term success as an academic will be gained during the K99 mentored period through Stanford courses designed to prepare postdocs for the career transition and through career development advice acquired by my advisory committee and my sponsor, Dr. Helen Blau. Together, the proposed studies and career development training will ensure I achieve my long term career goal; to establish a successful and independently-funded laboratory at a major University and use bioengineering approaches to study the molecular mechanisms that control muscle stem cells in the resting tissue and during regeneration specifically in the context of a 3D tissue. PUBLIC HEALTH RELEVANCE: Skeletal muscle is critical to our day to day movement and loss of muscle mass and/or function due to genetic disorders, aging, cancer or physical inactivity impairs quality of life and increases the risk of mobility related accidents. Adult skeletal muscle contains stem cells that are responsible for the day to day repair of skeletal muscle injury. The research in this proposal used an interdisciplinary approach and is focused on identifying the mechanisms regulating muscle stem cells in the tissue and aims to harness this knowledge to develop novel strategies for promote skeletal muscle repair in patients.

描述（由申请人提供）：衰老过程中发生的骨骼肌萎缩以及包括 HIV 感染和癌症在内的许多其他病理状况是一个严重的公共卫生问题，20001 年给美国医疗保健系统带来了超过 185 亿美元的经济负担。目前，没有有效的策略来促进衰老、受伤、患病或身体不活动的骨骼肌的肌肉再生。肌肉组织自身再生的固有能力是由常驻骨骼肌干细胞 (MuSC) 介导的2,3,4。我们实验室和其他实验室的最新进展已经确定了前瞻性地从小鼠骨骼肌中分离 MuSC 的策略5,6,7,8,9,10,11。由于 MuSC 领域相对新生，我们才刚刚开始了解 MuSC 调控的机制。阐明 MuSC 的调节对于利用这种成体组织特异性干细胞的潜力至关重要，并有助于开发克服肌肉萎缩的疗法。 在最近的研究中，我们证明基质弹性（MuSC 生态位的一种物理特性）是 MuSC 活力和自我更新的有效调节剂 12,13。通过使用生物材料方法与体内功能测定相结合，我们首次证明通过在与肌肉组织柔软度相匹配的基质上培养 MuSC 可以维持“干性”。在这份职业发展提案中，我们的目标是扩展这一发现，以进一步了解 MuSC 通过体内生态位的物理特征进行的调节。具体来说，我们将采用多学科方法来探索基质刚性和生态位结构对 MuSC 命运的调节，并将重点放在揭示机制见解上。在目标 1 中，我们将阐明 2D 基质弹性对 MuSC 的调节机制。这一目标将检验以下假设：MuSC 拥有“刚性传感”系统，并且这些系统可转换有关物理环境的信息以改变基因表达并最终改变细胞命运。目标 2 的目标是使用工程细胞外基质 (eECM) 设计骨骼肌组织模拟物，以在 3D 环境中探究 MuSC 的物理调节。我们假设，与我们的 2D 观察相比，在 3D 环境中，底物刚性对 MuSC 的调节将有根本不同，目的是解耦“维度”对 MuSC 活力、增殖和命运（静止、激活、自我更新、分化）的影响。最后，在目标 3 中，我们将研究 3D 利基架构对 MuSC 命运的作用。我们将使用微制造模型来检验 MuSC 静止和激活由 3D 生态位架构决定的假设。从这些目标中获得的知识将有助于我们对利基组成和 MuSC 监管的基本理解。这项工作的最终目标是加强基于细胞或系统传递的疗法的开发，旨在促进特定肌肉萎缩情况下的骨骼肌再生。 如果获得资助，该职业发展奖（CDA）将为我提供扩大骨骼肌生物学背景的机会，获得生物材料方法的必要知识，获得解释生物信息学数据的能力并获得显微镜方面的专业知识；对于我作为一名独立研究员未来的成功至关重要的技能（参见时间表）。与我的咨询委员会的正式和非正式互动将评估我在拟议目标方面的进展并提供批评和建议。最后，在 K99 指导期间，通过旨在为职业转型和职业发展准备博士后的斯坦福课程，我将获得对我作为一名学者的长期成功至关重要的专业技能（例如指导、研究报告、时间管理、写作等）我的咨询委员会和我的资助者海伦·布劳博士（Dr. Helen Blau）提供了建议。总之，拟议的学习和职业发展培训将确保我实现我的长期职业目标；在一所主要大学建立一个成功且独立资助的实验室，并使用生物工程方法来研究控制静息组织中的肌肉干细胞以及特别是在 3D 组织背景下的再生过程中的分子机制。 公众健康相关性：骨骼肌对于我们的日常运动至关重要，由于遗传性疾病、衰老、癌症或缺乏身体活动而导致的肌肉质量和/或功能丧失会损害生活质量并增加与行动相关的事故风险。成人骨骼肌含有干细胞，负责骨骼肌损伤的日常修复。该提案中的研究采用了跨学科方法，重点是确定组织中调节肌肉干细胞的机制，旨在利用这些知识来开发促进患者骨骼肌修复的新策略。