Skeletal Muscle Regeneration from Differentiating Embryonic Stem Cells

分化胚胎干细胞的骨骼肌再生

• 批准号: 8126379
• 负责人: Rita C. R. Perlingeiro
• 金额: $ 31.57万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-25 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8126379
• 关键词: Adult; Affect; Animal Model; Cells; Degenerative Disorder; Development; Direct Lytic Factors; Disease; Doxycycline; Dystrophin; ES Cell Line; Embryo; Event; Fiber; Gene Expression; Gene Targeting; Generations; Health; In Vitro; Injury; Intramuscular; Lateral; Longevity; Mesoderm; Methods; Modeling; Molecular; Mononuclear; Motor; Mus; Muscle; Muscle Development; Muscle Fibers; Muscle function; Muscle satellite cell; Muscular Dystrophies; Mutant Strains Mice; Natural regeneration; Paraxial Mesoderm; Pathway interactions; Physiological; Platelet-Derived Growth Factor; Population; Proliferating; Regenerative Medicine; Scheme; Seeds; Skeletal Muscle; Sorting - Cell Movement; Stem cells; System; Testing; Therapeutic; Time; Transplantation; Utrophin; Withdrawal; Work; body system; chromatin immunoprecipitation; embryonic stem cell; immunosuppressed; improved; in vivo; injured; interest; knock-down; muscle degeneration; muscle regeneration; muscular dystrophy mouse model; novel; progenitor; programs; regenerative; satellite cell; stem; tool

DESCRIPTION (provided by applicant): Embryonic stem (ES) cells differentiate into multiple lineages during in vitro embryoid body (EB) formation. This makes the ES/EB system a powerful tool to study early embryonic developmental pathways and to generate specific cell populations for regenerative medicine. Although this system has been available for more than two decades, studies on skeletal muscle development, until our recent work, had progressed no farther than documenting muscle marker gene expression. The system had not been used to investigate the molecular mechanisms of skeletal muscle lineage determination acting in nascent mesoderm. Similarly, in spite of the interest in and hype surrounding the potential use of ES cells to treat diseases of muscle degeneration, the generation of an ES-derived myogenic population with proliferative and regenerative potential had not been accomplished. This is in part due to the rarity of skeletal muscle precursors within the EB, as well as the lack of reliable identification and isolation criteria. We have developed an ES cell line in which expression of Pax3, the master regulator of the embryonic myogenic program, can be induced by doxycycline (dox). Our results show that induction of Pax3 during EB development enhances paraxial mesoderm, and a cell population endowed with myogenic potential within this fraction. Accordingly, a homogenous population of proliferating myogenic progenitors can be isolated from heterogeneous EB cultures by sorting for PDGF1R, a paraxial mesoderm marker, and absence of Flk-1, a lateral plate mesoderm marker. Upon dox withdrawal, these cells differentiate into muscle in vitro. When transplanted into cardiotoxin-injured immunodeficient or dystrophin-deficient immunosuppressed mice (intramuscular or systemic), Pax3-induced cells demonstrate an exceptional potential for skeletal muscle regeneration, differentiating robustly into functional adult myofibers. This is the first demonstration that therapeutic skeletal muscle progenitors can be derived from ES cells. Here we propose studies aimed at assessing in further detail the long-term therapeutic potential of ES/Pax3-derived myogenic progenitors, including the transplantation into more severe mouse models of muscular dystrophy as well as understanding the mechanism underlying their generation within paraxial mesoderm during ES/EB differentiation. PUBLIC HEALTH RELEVANCE: Embryonic stem cells hold great promise for the treatment of degenerative diseases, however to date studies on their potential use in the treatment of muscular dystrophies have been hampered by the difficulty of differentiating ES cells into skeletal muscle progenitors. This application builds on a novel method we have developed to generate muscle progenitors from ES cells. We have shown that such progenitors can be transplanted into normal injured and dystrophic mice, where they contribute to muscle fiber regeneration, and improve muscle function after injury. In these studies, we will (Aim 1) test whether these ES-derived cells, in addition to contributing to regenerating fibers, are capable of contributing to the muscle stem cell pool, (Aim 2) test whether they improve muscle function in more severe animal models of muscular dystrophy, and (Aim 3) use this system to dissect the molecular events involved in the generation of the earliest muscle progenitor cells of the embryo.

描述（由申请人提供）：胚胎干（ES）细胞在体外胚状体（EB）形成过程中分化成多个谱系。这使得 ES/EB 系统成为研究早期胚胎发育途径和生成再生医学特定细胞群的强大工具。尽管该系统已经存在了二十多年，但直到我们最近的工作之前，关于骨骼肌发育的研究仅取得了记录肌肉标记基因表达的进展。该系统尚未用于研究作用于新生中胚层的骨骼肌谱系测定的分子机制。同样，尽管人们对 ES 细胞治疗肌肉退化疾病的潜在用途感兴趣并大肆宣传，但具有增殖和再生潜力的 ES 衍生肌原细胞群的产生尚未实现。这部分是由于 EB 内骨骼肌前体的稀有性，以及缺乏可靠的识别和分离标准。我们开发了一种 ES 细胞系，其中 Pax3（胚胎生肌程序的主要调节因子）的表达可以由多西环素 (dox) 诱导。我们的结果表明，EB 发育过程中 Pax3 的诱导增强了近轴中胚层，并且该部分中的细胞群被赋予了生肌潜力。因此，通过分选 PDGF1R（一种近轴中胚层标记物）和不存在 Flk-1（一种侧板中胚层标记物），可以从异质 EB 培养物中分离出同质的增殖肌源性祖细胞群。撤除阿霉素后，这些细胞在体外分化成肌肉。当移植到心脏毒素损伤的免疫缺陷或抗肌营养不良蛋白缺陷的免疫抑制小鼠（肌内或全身）时，Pax3诱导的细胞表现出骨骼肌再生的非凡潜力，可强力分化为功能性成年肌纤维。这是首次证明治疗性骨骼肌祖细胞可以源自 ES 细胞。在这里，我们提出的研究旨在进一步详细评估 ES/Pax3 衍生的肌源性祖细胞的长期治疗潜力，包括移植到更严重的肌营养不良小鼠模型中，以及了解 ES/EB 分化过程中它们在轴旁中胚层内生成的潜在机制。公共健康相关性：胚胎干细胞在治疗退行性疾病方面具有广阔的前景，但迄今为止，由于难以将 ES 细胞分化为骨骼肌祖细胞，因此对其在治疗肌营养不良症中的潜在用途的研究受到阻碍。该应用建立在我们开发的一种从 ES 细胞生成肌肉祖细胞的新方法的基础上。我们已经证明，此类祖细胞可以移植到正常受伤和营养不良的小鼠体内，它们有助于肌肉纤维再生，并改善受伤后的肌肉功能。在这些研究中，我们将（目标 1）测试这些 ES 衍生细胞除了有助于纤维再生之外，是否能够对肌肉干细胞库做出贡献，（目标 2）测试它们是否可以改善更严重的肌营养不良动物模型中的肌肉功能，以及（目标 3）使用该系统来剖析参与胚胎最早肌肉祖细胞生成的分子事件。