Skeletal Muscle Regeneration from Differentiating Pluripotent Stem Cells

描述（由申请人提供）：多能干细胞（ES和iPS细胞）具有自我更新和体外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有力工具，并为再生医学和疾病研究产生特定的细胞群。在R 01 AR 055299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细胞中获得骨骼肌细胞的方法。直到我们的工作，在这方面的研究是罕见的，因为骨骼肌分化是非常低效的体外分化。我们推断 因为体外系统不能重演神经管或脊索的发育，而且确实不能产生类似体节的结构，所以不存在适当地形成近轴中胚层的诱导信号。我们已经克服了这个障碍，激活生肌程序，通过短暂诱导Pax 3或Pax 7在早期中胚层发育。这种方法允许在体外产生大量的早期胚胎骨骼肌祖细胞。将这些细胞移植到营养不良小鼠中导致肌纤维和卫星细胞植入，伴随着肌肉力量产生的改善。此更新应用程序建立在我们在过去5年中为该领域带来的进步的基础上，并从这些发展到3个领域：ii）研究人多能供体衍生的卫星细胞的长期再生能力以及它们与体外产生的肌原性祖细胞相比的转录谱，和iii）使用来自患者特异性多能细胞的肌管以生物化学和功能性地模拟DMD/BMD病理及其通过肌营养不良蛋白小基因的治疗性递送的逆转。

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.