Activators of Muscle Genes

描述（由申请人提供）：肌肉干细胞（musc）在损伤和移植后对肌肉组织再生有广泛的贡献。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萎缩状况，如肌肉减少症和恶病质。由于这些细胞在成人组织中的罕见性，以及在培养中无法将其繁殖到治疗数量而不丧失其干细胞特性，因此musc的治疗效用受到限制。肌干细胞在培养过程中很容易产生大量的肌源性祖细胞，称为成肌细胞，但这些细胞在移植后的再生潜力极其有限，这一点在以成肌细胞为基础的治疗杜氏肌营养不良症（DMD）的临床试验中失败了。无法产生治疗水平的MuSC部分是由于缺乏对调节维持或诱导MuSC表型的分子机制的理解。由于MuSC的分离和表征方法在过去几年中才得到充分验证，因此这种知识的缺乏并不奇怪。在这里，我们建议通过核重编程从更丰富的体细胞中产生musc。在Aim 1中，存在于肌肉组织中的两种丰富且可培养扩增的人类体细胞类型，即成肌细胞和前脂肪细胞，将通过与小鼠musc细胞融合形成非分裂的双种异核体，重新编程为肌肉干细胞表型。异核体将允许阐明重编程到MuSC命运的最早步骤，并确定调节表型的逆转（成肌细胞到MuSC）或转化（前脂肪细胞到MuSC）的机制。在目标2中，将测试在异核体和musc微阵列（编码转录因子和表观遗传调节因子）中鉴定的关键重编程基因的遗传操作，以确定其指导成肌细胞和前脂肪细胞重编程为功能性musc的潜力。重编程表型将评估体外MuSC基因表达和表观遗传谱，并最终评估移植后的体内功能。在Aim 3中，将比较从年轻小鼠和老年小鼠分离的细胞中成肌细胞到musc和前脂肪细胞到musc的重编程，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来研究MuSCs的维持和生成的拟议研究受益于我们实验室最近的几项进展：(a)开发新技术来评估对区分双种异核体中每种细胞类型的核重编程贡献至关重要的全局转录和表观遗传变化，(b)在体外维持肌肉干细胞的基于水凝胶的细胞培养底物，以及(c)移植后体内MuSC功能的无创成像分析。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。

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.