Activators of Muscle Genes

肌肉基因激活剂

• 批准号: 8456997
• 负责人: Helen M Blau
• 金额: $ 45.54万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-06-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8456997
• 关键词: Adhesions; Adipocytes; Adult; Age; Aging; Bioinformatics; Biological Assay; Biology of Aging; Cachexia; Candidate Disease Gene; Cell Culture Techniques; Cell Maintenance; Cell Separation; Cell Therapy; Cell fusion; Cell physiology; Cell surface; Cells; Clinical; Clinical Treatment; Clinical Trials; Complex; DNA; Development; Duchenne muscular dystrophy; Early identification; Epigenetic Process; Failure; Flow Cytometry; Fluorescence Microscopy; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Generations; Genes; Goals; Histones; Home environment; Human; Hydrogels; Image; In Vitro; Individual; Injury; Interphase Cell; Intrinsic factor; Knowledge; Laboratories; Ligands; Luciferases; Maintenance; Methods; Methylation; Modification; Molecular; Mus; Muscle; Muscle Cells; Muscle satellite cell; Mutation; Myoblasts; Myopathy; Nuclear; Phenotype; Polycomb; Population; Property; Proteins; Protocols documentation; Regenerative Medicine; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Role; Skeletal Muscle; Somatic Cell; Specific qualifier value; Stem cells; Subfamily lentivirinae; Techniques; Testing; Therapeutic; Therapeutic Uses; Time; Tissues; Transgenic Organisms; Transplantation; Wing; age effect; aged; base; bioluminescence imaging; bisulfite; cell type; clinically relevant; demethylation; genetic manipulation; genome-wide; heterokaryon; in vivo; insight; loss of function; member; muscle regeneration; novel; nuclear reprogramming; overexpression; professor; progenitor; promoter; public health relevance; regenerative; research study; sarcopenia; satellite cell; self-renewal; stem cell fate; stem cell fate specification; tissue regeneration; transcription factor; transcriptome sequencing; wasting

DESCRIPTION (provided by applicant): Muscle stem cells (MuSCs) contribute extensively to muscle tissue regeneration following injury and transplantation. Consequently, MuSCs offer much promise for treating skeletal muscle diseases, including genetic defects and muscle wasting conditions such as sarcopenia and cachexia. The therapeutic utility of MuSCs is limited by the rarity of these cells in adult tissues and the inability to propagate them in culture to therapeutic numbers without loss of their stem cell properties. MuSCs readily give rise in culture to abundant myogenic progenitors called myoblasts, but these cells have extremely limited regenerative potential upon transplantation, as evidenced by failures in myoblast-based clinical trials for the treatment of Duchenne Muscular Dystrophy (DMD). The inability to generate therapeutic levels of MuSCs is due, in part, to a lack of understanding of the molecular mechanisms that regulate the maintenance or induction of the MuSC phenotype. Since MuSC isolation and characterization methods have only been fully validated within the last few years, this paucity of knowledge is not surprising. Here we propose to generate MuSCs from more abundant somatic cells by nuclear reprogramming. In Aim 1, two abundant and culture- expandable human somatic cell types present in muscle tissue, myoblasts and pre-adipocytes, will be reprogrammed to a muscle stem cell phenotype by cell fusion with mouse MuSCs to form non-dividing bi- species heterokaryons. Heterokaryons will allow elucidation of the earliest steps in reprogramming to a MuSC fate and identification of the mechanisms regulating the reversion (myoblast-to-MuSC) or conversion (pre-adipocyte-to-MuSC) of a phenotype. In Aim 2, genetic manipulations of critical reprogramming genes identified in heterokaryons and microarrays of MuSCs (encoding transcription factors and epigenetic regulators) will be tested for their potential to direct reprogramming of myoblasts and pre-adipocytes to functional MuSCs. Reprogrammed phenotypes will be assessed for in vitro MuSC gene expression and epigenetic profiles, and, ultimately, in vivo function following transplantation. In Aim 3, myoblast-to-MuSC and pre-adipocyte-to-MuSC reprogramming will be compared for cells isolated from young and old mice to provide greater understanding of the effects of clinically relevant parameters of age. The proposed studies to investigate maintenance and generation of MuSCs using myoblasts and pre-adipocytes benefit from several recent advances in our laboratory: (a) development of novel techniques to assess global transcriptional and epigenetic changes essential to distinguishing nuclear reprogramming contributions of each cell type in bi-species heterokaryons, (b) a hydrogel-based cell culture substrate that maintains muscle stem cells in vitro, and (c) a noninvasive imaging assay of in vivo MuSC function following transplantation. The molecular insights gained will increase the clinical utility of muscle stem cells and increase our understanding of muscle biology and aging.

描述(由申请人提供)：肌肉干细胞(MuSCs)对损伤和移植后的肌肉组织再生有广泛的贡献。因此，骨髓间充质干细胞为治疗骨骼肌疾病提供了很大的希望，包括遗传缺陷和肌肉萎缩情况，如石棺减少症和恶病质。MuSCs的治疗作用受到这些细胞在成人组织中的稀有性以及无法在不损失其干细胞特性的情况下在培养中繁殖到治疗数量的限制。MSC在培养过程中很容易产生大量的成肌细胞，但这些细胞在移植后再生潜力极其有限，基于成肌细胞的临床试验治疗Duchenne肌营养不良症(DMD)的失败证明了这一点。之所以不能产生治疗水平的MUSCs，部分原因是缺乏对维持或诱导MUSC表型的分子机制的了解。由于MUSC的分离和表征方法在最近几年才得到充分验证，这种知识的匮乏并不令人惊讶。在这里，我们建议通过核重新编程从更丰富的体细胞中产生MSC。在目标1中，肌肉组织中存在的两种丰富的、可培养扩增的人体体细胞类型，即成肌细胞和前脂肪细胞，将通过与小鼠骨髓间充质干细胞的融合重新编程为肌肉干细胞表型，形成不分裂的双物种异核体。异核体将有助于阐明重新编程为MUSC命运的最早步骤，并鉴定调节表型逆转(成肌细胞到MUSC)或转换(前脂肪细胞到MUSC)的机制。在目标2中，将测试在异核体和MuSCs微阵列(编码转录因子和表观遗传调节因子)中确定的关键重编程基因的遗传操作，以确定它们是否有可能将成肌细胞和前脂肪细胞重编程为功能性MuSCs。重新编程的表型将在体外评估MUSC基因的表达和表观遗传学特征，并最终评估移植后的体内功能。在目标3中，将对从幼鼠和老年鼠分离的细胞进行成肌细胞到MUSC和前脂肪细胞到MUSC的重新编程，以更好地了解临床相关年龄参数的影响。利用成肌细胞和前脂肪细胞研究MUSCs的维持和生成得益于我们实验室最近的几项进展：(A)开发新的技术来评估全球转录和表观遗传学变化，这对于区分双物种异核体中每种细胞类型的核重新编程贡献至关重要，(B)在体外维持肌肉干细胞的水凝胶细胞培养底物，以及(C)移植后体内MUSC功能的非侵入性成像分析。所获得的分子洞察力将增加肌肉干细胞的临床应用，并增加我们对肌肉生物学和衰老的理解。