Skeletal Muscle Regeneration from Differentiating Embryonic Stem Cells

分化胚胎干细胞的骨骼肌再生

• 批准号: 7692925
• 负责人: Rita C. R. Perlingeiro
• 金额: $ 33.22万
• 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/7692925
• 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; 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; 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; public health relevance; 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）：在体外胚胎体（EB）形成期间，胚胎茎（ES）细胞分化为多个谱系。这使ES/EB系统成为研究早期胚胎发育途径并为再生医学生成特定细胞群体的强大工具。尽管该系统已经使用了二十年了，但有关骨骼肌发育的研究，直到我们最近的工作一直远不止于记录肌肉标记基因表达。该系统尚未用于研究起作用中胚层作用的骨骼肌谱系测定的分子机制。同样，尽管对ES细胞的潜在用途具有兴趣和炒作，但尚未实现ES衍生的具有增殖和再生潜力的ES衍生的肌源种群的产生。这部分是由于EB内的骨骼肌前体的稀有性以及缺乏可靠的识别和隔离标准。我们已经开发了ES细胞系，其中Pax3的表达是胚胎肌原源程序的主要调节剂，可以由强力霉素（DOX）诱导。我们的结果表明，EB发育过程中PAX3的诱导增强了近端中胚层，并且在该馏分中具有肌源性潜力。因此，可以通过排序pdgf1r（一种偏轴中的中胚层标记物）和不存在FLK-1（侧板中胚层标记物），从异质的EB培养物中分离出增殖的肌祖祖细胞的同质群体。 DOX戒断后，这些细胞在体外分化为肌肉。当移植到心脏毒素造成的免疫缺陷或肌营养不良蛋白缺陷的免疫抑制小鼠（肌内或全身性）中时，PAX3诱导的细胞表现出骨骼肌再生的特殊潜力，可与功能强大的成人肌纤维区分开。这是第一次证明治疗性骨骼肌祖细胞可以源自ES细胞。在这里，我们提出了旨在详细评估ES/PAX3衍生的肌源祖细胞的长期治疗潜力的研究，包括将移植到更严重的肌肉营养不良的小鼠模型中，并了解ES/EB分化过程中甲状腺素性中胚层内产生的机制。公共卫生相关性：胚胎干细胞对治疗退化性疾病的巨大希望，但是迄今为止，由于将ES细胞分化为骨骼肌祖细胞的困难，已经阻碍了其对肌营养不良症治疗的潜在使用的研究。该应用以一种新的方法为基础，我们已经开发出来从ES细胞中产生肌肉祖细胞。我们已经表明，这种祖细胞可以移植到正常受伤和营养不良的小鼠中，在那里它们有助于肌肉纤维再生，并在受伤后改善肌肉功能。 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.