Dissecting the molecular pathways required for the generation and maintenance of induced myogenic progenitors (iMPCs) and assessing their potential for gene therapy

骨骼肌是一种再生组织，主要由负责收缩和运动的分化肌纤维组成。此外，肌肉中含有一种罕见的被称为卫星细胞的干细胞群体，这些干细胞在动态平衡条件下处于静止状态，但在组织损伤时会被激活。一旦被激活，卫星细胞就会增殖，形成未固定的成肌细胞，然后分化并与其他成肌细胞融合，再生受损的组织。值得注意的是，当单个卫星细胞移植到受损肌肉中时，它们有能力产生肌纤维并补充卫星细胞的壁龛。卫星细胞为肌肉组织的生成提供了巨大的希望。虽然它们可以在培养中使用生长因子和/或小化合物在一段有限的时间内被移植，但它们不能按临床使用所需的数量进行扩展。最近，宿主实验室证实了皮肤成纤维细胞在体外和体内转分化为肌源性干细胞，在培养中增殖并产生肌纤维。然而，目前还不清楚这些诱导的肌源性祖细胞(IMPC)在多大程度上类似于卫星细胞。此外，成纤维细胞向间充质干细胞“转分化”的遗传基础仍然难以捉摸。破译这些分子机制是理解肌肉干细胞生成以外的细胞命运转变的关键，对于评估iMPC在基因治疗方法方面的潜力也是重要的。·目标1.iMPC亚群在分子和功能上是否等同于卫星细胞？第一个目标是利用卫星细胞报告小鼠比较纯化的iMPC和卫星细胞之间的基因表达和染色质可及性特征。·目的2.转录因子和小分子如何协同作用从机制上改变表观遗传、基因表达和分化状态？仅有转录因子MyoD的异位表达是不够的，而MyoD的表达和化学鸡尾酒的处理足以建立肌源性干/祖细胞样状态。因此，我们的目标是了解皮肤成纤维细胞暴露在特定化学物质下如何促进染色质状态，从而允许MyoD结合，从而实现类似祖细胞的程序。·目标3：为iMPC在基因和细胞治疗中的应用建立原则证据我们假设所建立的iMPC培养系统可能有助于体外研究病理性肌发生。IMPC应该促进疾病相关突变的引入或纠正，这具有治疗意义。因此，这一目标的目的是收集证据，证明(I)iMPC可以从营养不良的小鼠模型中分离出来，用于研究疾病表型，(Ii)iMPC可以接受基因纠正，以及(Iii)纠正后的iMPC在移植后分化为健康的肌纤维。

Skeletal muscle is a regenerative tissue that is mostly comprised of differentiated myofibers responsible for contraction and thus movement. In addition,muscle contains a rare population of stem cells termed satellite cells, which are in a quiescent state under homeostatic conditions but undergo activation upon tissue injury. Once activated, satellite cells proliferate and form uncommitted myoblasts, which then differentiate and fuse with other myoblasts to regenerate damaged tissue. Remarkably, individual satellite cells have the capacity to produce myofibers and replenish the satellite cell niche when transplanted into damaged muscle. Satellite cells hold a great promise for muscle tissue generation. Although they can be explanted in culture for a limited period of time using growth factors and/or small compounds they cannot be expanded at numbers required for clinical use. Recently, the host lab has demonstrated the transdifferentiation of skin fibroblasts into myogenic stem-¬like cells that proliferate in culture and produce myofibers in vitro and in vivo. However, it is unclear to what extent these induced myogenic progenitor cells (iMPCs) resemble satellite cells. Also, the genetic that the “transdifferentiation” of fibroblasts to iMPCs underlie, remain elusive. Decoding these molecular mechanisms is key for understanding cell fate transitions beyond muscle stem cell generation and will be important for evaluating the iMPC’s potential for gene therapy approaches. •Objective 1. Are iMPC subsets molecularly and functionally equivalent to satellite cells? The first aim will be to compare gene expression and chromatin accessibility signatures between purified iMPCs and satellite cells using satellite cell reporter mice.•Objective 2. How do transcription factors and small molecules synergize to alter the epigenetic, gene expression and differentiation state mechanistically? Ectopic expression of the transcription factor MyoD alone is insufficient, while the expression of MyoD and treatment with a chemical cocktail is sufficient to establish a myogenic stem/progenitor-like state. We, therefore, aim to understand how exposure of skin fibroblasts to certain chemicals facilitates a chromatin state, that allows MyoD binding and thus a progenitor-¬like program.•Objective 3: Establish proof-of-principle evidence for the utility of iMPCs in gene and cell therapyWe hypothesize that the established iMPC culture system could be useful to study pathological myogenesis ex vivo. iMPCs should facilitate the introduction or correction of disease-associated mutations, which has therapeutic relevance. The goal of this aim is therefore to gather evidence that (i) iMPCs can be derived from dystrophic mouse models to study disease phenotypes, (ii) iMPCs are amenable to gene correction and (iii) corrected iMPCs differentiate into healthy myofibers upon transplantation.