Activators of Muscle Genes

肌肉基因激活剂

• 批准号: 7888593
• 负责人: Helen M Blau
• 金额: $ 44.71万
• 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/7888593
• 关键词: Adhesions; Adipocytes; Adult; Age; Aging; Bioinformatics; Biological Assay; Biology of Aging; Bioluminescence; Cachexia; Candidate Disease Gene; Cell Culture Techniques; Cell Maintenance; 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; 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 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; 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; tissue regeneration; transcription factor; 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. PUBLIC HEALTH RELEVANCE: A rare but highly functional population of specialized cells, muscle stem cells, is essential to effective skeletal muscle regeneration. Transplantation of muscle stem cells has the potential to treat numerous muscle diseases but is currently impractical due to the limited quantities of these cells that can be isolated from human muscle tissue. We propose to generate clinically sufficient quantities of muscle stem cells from more abundant mature muscle and fat cells through nuclear and cellular reprogramming involving the manipulation of expression of genes associated with muscle stem cell generation.

描述（由申请人提供）：肌肉干细胞（MuSC）在损伤和移植后广泛促进肌肉组织再生。因此，MuSC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消耗状况，如肌肉减少症和恶病质。MuSC的治疗效用受限于这些细胞在成体组织中的稀有性，并且不能在培养物中将它们繁殖到治疗数量而不损失它们的干细胞特性。MuSC容易在培养物中产生丰富的肌源性祖细胞，称为成肌细胞，但这些细胞在移植后具有极其有限的再生潜力，如在用于治疗杜氏肌营养不良症（DMD）的基于成肌细胞的临床试验中的失败所证明的。不能产生治疗水平的MuSC部分是由于缺乏对调节MuSC表型的维持或诱导的分子机制的理解。由于MuSC分离和表征方法仅在过去几年中得到充分验证，因此知识的缺乏并不奇怪。在这里，我们提出通过核重编程从更丰富的体细胞产生MuSCs。在目的1中，存在于肌肉组织中的两种丰富的且可培养扩增的人类体细胞类型（成肌细胞和前脂肪细胞）将通过与小鼠MuSC的细胞融合而重编程为肌肉干细胞表型以形成非分裂的双物种异核体。异核体将允许阐明重编程为MuSC命运的最早步骤，并鉴定调节表型逆转（成肌细胞至MuSC）或转化（前脂肪细胞至MuSC）的机制。在目标2中，将测试在异核体和MuSC微阵列中鉴定的关键重编程基因（编码转录因子和表观遗传调节因子）的遗传操作，以确定其将成肌细胞和前脂肪细胞重编程为功能性MuSC的潜力。将评估重编程表型的体外MuSC基因表达和表观遗传特征，并最终评估移植后的体内功能。在目标3中，将比较从年轻和老年小鼠分离的细胞的成肌细胞至MuSC和前脂肪细胞至MuSC的重编程，以更好地理解年龄的临床相关参数的影响。使用成肌细胞和前脂肪细胞研究MuSC的维持和生成的拟议研究受益于我们实验室的几项最新进展：（a）开发新技术以评估对于区分双物种异核体中每种细胞类型的核重编程贡献至关重要的整体转录和表观遗传变化，（B）基于水凝胶的细胞培养基质，其在体外维持肌肉干细胞，和（c）移植后体内MuSC功能的非侵入性成像测定。所获得的分子见解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。 公共卫生相关性：肌肉干细胞是一种罕见但功能性很强的特化细胞群，对有效的骨骼肌再生至关重要。肌肉干细胞的移植具有治疗许多肌肉疾病的潜力，但由于可以从人类肌肉组织中分离的这些细胞的数量有限，目前是不切实际的。我们建议从更丰富的成熟肌肉和脂肪细胞中产生临床上足够数量的肌肉干细胞，通过核和细胞重编程，涉及操纵与肌肉干细胞生成相关的基因的表达。