Skeletal Muscle Regeneration from Differentiating Pluripotent Stem Cells

描述(申请人提供)：多能干细胞(ES和iPS细胞)在体外具有自我更新和分化为多个谱系的能力。这使得这些细胞成为研究早期胚胎发育途径的强大工具，并为再生医学和疾病研究产生特定的细胞群。在R01 AR055299的支持下，我们的研究小组开创了从小鼠和人类多能ES和iPS细胞中分离骨骼肌源性细胞的方法。在我们的工作之前，这方面的研究很少，因为骨骼肌源性分化在体外分化过程中效率极低。我们推论说 因为在体外系统不会概括神经管或脊索的发育，也不会产生类似体节的结构，所以缺乏正确形成轴旁中胚层模式的诱导信号。我们已经克服了这一障碍，通过在中胚层发育早期瞬时诱导Pax3或Pax7来激活肌原程序。这种方法允许在体外产生大量早期胚胎骨骼肌生肌祖细胞。将这些细胞移植到营养不良的小鼠体内，会导致肌纤维和卫星细胞的植入，并伴随着肌力生成的改善。这一新的应用建立在我们在过去5年中为该领域带来的进展的基础上，并从这些方面演变为3个领域：i)了解Pax3对胚胎成肌计划的分子调控，ii)研究人类多能性供体衍生卫星细胞的长期再生能力及其转录图谱，并将其与体外产生的成肌前体细胞进行比较，iii)使用患者特定的多能性细胞的肌管来从生化和功能上模拟DMD/BMD病理，并通过治疗性地交付dystrophin微型基因来逆转其。

DESCRIPTION (provided by applicant): Pluripotent stem cells (ES and iPS cells) have the ability to self-renew and to differentiate into multiple lineages in vitro. This makes these cells powerful tool to study early embryonic developmental pathways and to generate specific cell populations for regenerative medicine and disease investigation. Supported by R01 AR055299, our research group has pioneered methods to derive skeletal myogenic cells from mouse and human pluripotent ES and iPS cells. Until our work, studies in this area were rare since skeletal myogenic differentiation is extremely inefficient during in vitro differentiation. We reasoned that because in vitro systems do not recapitulate neural tube or notochord development, and indeed do not produce structures resembling somites, that the inducing signals that properly pattern paraxial mesoderm were absent. We have overcome this roadblock by activating the myogenic program through transient induction of Pax3 or Pax7 during early mesoderm development. This approach allows for the in vitro generation of large quantities of early embryonic skeletal myogenic progenitors. Transplantation of these cells into dystrophic mice results in myofiber and satellite cell engraftment that is accompanied by improvement in muscle force generation. This renewal application builds on the advances we have brought to the field over the past 5 years and evolves from these into 3 areas: i) understanding the molecular regulation of the embryonic myogenic program by Pax3, ii) investigating the long-term regenerative capacity of human pluripotent donor-derived satellite cells as well as their transcriptional profiling in comparison to in vitro generated myogenic progenitors, and iii) using myotubes from patient-specific pluripotent cells to biochemically and functionally model DMD/BMD pathology and its reversal by therapeutic delivery of dystrophin mini-genes.