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(申请人提供)：胚胎干细胞(ES)在体外形成类胚体(EB)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为再生医学产生特定细胞群的强大工具。虽然这个系统已经有20多年的历史了，但直到我们最近的工作之前，对骨骼肌发育的研究只是记录了肌肉标记基因的表达。该系统还没有被用于研究作用于新生中胚层的骨骼肌谱系确定的分子机制。同样，尽管人们对ES细胞用于治疗肌肉退行性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来源的肌源性群体的生成尚未完成。这在一定程度上是由于EB内骨骼肌前体的稀有，以及缺乏可靠的鉴定和分离标准。我们已经建立了一种ES细胞系，在该细胞系中，Pax3的表达可以被多西环素(Dox)诱导，Pax3是胚胎肌肉生成程序的主要调节因子。我们的结果表明，在EB发育过程中，Pax3的诱导增强了近轴中胚层，并且在这一部分中的一个细胞群体被赋予了肌源性潜能。因此，通过分选PDGF1R(轴旁中胚层标志物)和缺失Flk-1(侧板中胚层标志物)，可以从异质EB培养物中分离出均一群增殖的肌源性祖细胞。在DOX停用后，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dystrophin缺陷免疫抑制的小鼠(肌肉内或全身)时，Pax3诱导的细胞显示出非凡的骨骼肌再生潜力，分化为功能强大的成人肌纤维。这是第一次证明治疗性骨骼肌祖细胞可以从ES细胞分化而来。在这里，我们提出的研究旨在更详细地评估ES/Pax3来源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，以及了解它们在ES/EB分化过程中在近轴中胚层内生成的机制。公共卫生相关性：胚胎干细胞在治疗退行性疾病方面大有可为，然而到目前为止，由于难以将ES细胞分化为骨骼肌祖细胞，有关其在肌肉营养不良治疗中的潜在应用的研究一直受到阻碍。这一应用建立在我们开发的一种新方法之上，该方法可以从ES细胞生成肌肉祖细胞。我们已经证明，这些祖细胞可以移植到正常受损和营养不良的小鼠体内，在那里它们有助于肌肉纤维的再生，并改善损伤后的肌肉功能。在这些研究中，我们将(目标1)测试这些胚胎干细胞，除了有助于再生纤维，是否能够促进肌肉干细胞池，(目标2)测试它们是否能改善更严重的肌营养不良动物模型的肌肉功能，以及(目标3)使用该系统剖析与胚胎最早的肌肉前体细胞生成有关的分子事件。