Skeletal Muscle Regeneration from Differentiating Embryonic Stem Cells

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用于再生医学的特定细胞群的有力工具。虽然这个系统已经有二十多年的历史了，但是在我们最近的工作之前，对骨骼肌发育的研究除了记录肌肉标记基因的表达外，没有取得任何进展。该系统尚未用于研究新生中胚层骨骼肌谱系决定的分子机制。同样，尽管人们对胚胎干细胞治疗肌肉退行性疾病的潜在用途很感兴趣并大肆宣传，但具有增殖和再生潜力的胚胎干细胞来源的肌源性群体的产生尚未完成。这部分是由于EB内骨骼肌前体的罕见性，以及缺乏可靠的识别和分离标准。我们已经开发了一种胚胎干细胞系，其胚胎肌生成程序的主要调节因子Pax3的表达可以被强力霉素（dox）诱导。我们的研究结果表明，在EB发育过程中诱导Pax3增强了近轴中胚层，并在这部分细胞群中赋予了肌生成潜力。因此，通过PDGF1R（近轴中胚层标记）和Flk-1（侧板中胚层标记）的缺失，可以从异质EB培养物中分离出增殖性肌源性祖细胞的同质群体。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免疫抑制小鼠（肌内或全身）时，pax3诱导的细胞显示出骨骼肌再生的特殊潜力，分化为功能强大的成年肌纤维。这是首次证明治疗性骨骼肌祖细胞可以来自胚胎干细胞。在这里，我们提出了旨在进一步详细评估ES/ pax3来源的肌源性祖细胞的长期治疗潜力的研究，包括移植到更严重的肌肉萎缩症小鼠模型中，以及了解它们在ES/EB分化期间在近轴中胚层内产生的机制。公共卫生相关性：胚胎干细胞在治疗退行性疾病方面具有巨大的前景，然而迄今为止，由于胚胎干细胞难以分化为骨骼肌祖细胞，对其在治疗肌肉萎缩症方面的潜在用途的研究受到了阻碍。该应用程序建立在我们开发的从胚胎干细胞产生肌肉祖细胞的新方法之上。我们已经证明，这种祖细胞可以移植到正常的损伤和营养不良小鼠体内，在那里它们有助于肌纤维的再生，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es来源的细胞除了有助于纤维再生外，是否能够为肌肉干细胞库做出贡献，（目标2）测试它们是否能改善更严重的肌肉萎缩动物模型中的肌肉功能，（目标3）使用该系统来解剖胚胎最早肌肉祖细胞产生的分子事件。

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.