Skeletal Muscle Regeneration from Differentiating Embryonic Stem Cells

分化胚胎干细胞的骨骼肌再生

• 批准号: 8322195
• 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 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8322195
• 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）： 胚胎干细胞在体外形成胚状体的过程中分化为多个谱系。这使得ES/EB系统成为研究早期胚胎发育途径和产生再生医学特定细胞群的有力工具。虽然这个系统已经有20多年的历史了，但在我们最近的工作之前，骨骼肌发育的研究进展并不超过记录肌肉标记基因的表达。该系统尚未用于研究骨骼肌谱系决定作用于新生中胚层的分子机制。类似地，尽管对ES细胞治疗肌肉变性疾病的潜在用途感兴趣并大肆宣传，但尚未实现具有增殖和再生潜力的ES衍生的肌源性群体的产生。这部分是由于EB内骨骼肌前体的稀有性，以及缺乏可靠的鉴定和分离标准。我们已经开发了一个ES细胞系，其中表达的Pax 3，胚胎肌原性程序的主调节器，可以诱导强力霉素（dox）。我们的研究结果表明，诱导Pax 3在EB发展过程中增强近轴中胚层，并赋予肌细胞群在这一部分的潜力。因此，可以通过对PDGF 1 R（一种近轴中胚层标记物）和Flk-1（一种侧板中胚层标记物）的缺失进行分选，从异质EB培养物中分离增殖的肌源性祖细胞的同质群体。在dox停药后，这些细胞在体外分化为肌肉。当移植到心脏毒素损伤的免疫缺陷或肌营养不良蛋白缺陷的免疫抑制小鼠（肌内或全身），Pax 3诱导的细胞表现出骨骼肌再生的特殊潜力，分化成功能性的成年肌纤维。这是第一次证明治疗性骨骼肌祖细胞可以来自ES细胞。在这里，我们提出的研究旨在进一步详细评估ES/Pax 3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症小鼠模型，以及了解其在ES/EB分化过程中在近轴中胚层内产生的机制。公共卫生相关性：胚胎干细胞在退行性疾病的治疗方面有着巨大的前景，然而迄今为止，关于其在肌营养不良症治疗中的潜在用途的研究受到了ES细胞分化为骨骼肌祖细胞的困难的阻碍。该应用建立在我们开发的从ES细胞产生肌肉祖细胞的新方法上。我们已经证明，这种祖细胞可以移植到正常的受伤和营养不良的小鼠，在那里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（目的1）测试这些ES衍生的细胞除了有助于再生纤维之外，是否能够有助于肌肉干细胞库，（目的2）测试它们是否在更严重的肌营养不良症动物模型中改善肌肉功能，和（目的3）使用该系统来剖析胚胎最早的肌肉祖细胞的产生中涉及的分子事件。