The mini-IDLE 3D biomimetic culture assay enables interrogation of mechanisms governing muscle stem cell quiescence and niche repopulation.
The mini-IDLE 3D biomimetic culture assay enables interrogation of mechanisms governing muscle stem cell quiescence and niche repopulation.
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DOI：
10.7554/elife.81738
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发表时间：
2022-12-20
期刊：
影响因子：
7.7
通讯作者：
Gilbert, Penney M.
中科院分区：
文献类型：
--
作者：
Jacques, Erik;Kuang, Yinni;Kann, Allison P.;Le Grand, Fabien;Krauss, Robert S.;Gilbert, Penney M.
成人骨骼肌含有大量的肌肉干细胞(MuSCs)，这些干细胞在组织损伤后需要修复。在青年时期，在损伤解决后，MSC会回到一种可逆的细胞周期停滞状态，称为“静止”。相反，老化肌肉中的一些MuSCs仍然处于半激活状态，导致对损伤的过早反应，从而导致不完全修复和最终的干细胞枯竭。调节MUSC静止和激活之间的这种平衡可能是随着年龄增长恢复组织动态平衡的关键，但人们对此还不完全了解。为了填补这一空白，我们开发了一种简单易处理的体外方法，快速灭活新鲜从年轻小鼠骨骼肌分离的MuSCs，并将其恢复到静止状态至少1周，我们将其命名为mini-IDLE(体外利用失活和休眠)。这是通过将MuSCs引入由一张薄薄的小鼠肌管组成的3D生物利基中实现的，我们证明这提供了诱导静止所需的最低限度的线索。对于不同的起始数量的MuSCs，该分析揭示了细胞的异质性和种群水平的适应，它们聚集在一个共同的生态位重新种群密度上；以前只在体内观察到的行为。与静止相关的特征包括Pax7+CalcR+DDX6+MyoD-c-fos-Signature，静止样形态，以及两极化的生态位标记。利用高含量的生物成像管道，我们展示了形态和细胞命运特征之间的关系，从而可能进行基于形态的实时筛选。当使用来自老化肌肉的MuSCs时，它们表现出异常的增殖活性和延迟失活动力学，以及我们显示的其他与静止相关的缺陷，这些缺陷可以通过Wortmannin治疗部分恢复。因此，该分析提供了一个前所未有的机会，可以系统地研究长期存在的问题，如MUSC群体中池大小的调节和功能异质性，并揭示年轻人和老年人的静止调节因子。当我们的肌肉受伤时，组织中的干细胞被激活，开始修复过程。然而，当没有损伤时，这些细胞往往处于一种被称为静止的保护性休眠状态。如果没有保持静止，当肌肉受损时，干细胞就不能正常修复。这发生在老年，当部分细胞保持半激活状态，并变得枯竭。然而，研究人员仍然没有完全了解静默是如何被调节的。这在一定程度上是因为为了研究静止状态，必须使用活体动物，因为当肌肉干细胞从肌肉组织中移除时，它们会立即脱离静止状态。为了克服这一实验限制，雅克等人。开发了一种新的方法来研究肌肉干细胞，方法是将小鼠的肌肉干细胞移植到实验室培养的三维工程肌肉组织中。这种组织是通过渗透到茶包纸的毛孔中的肌肉前体细胞形成的，然后这些细胞相互融合，形成包含三层收缩肌肉细胞的薄肌肉。将年轻的健康动物的肌肉干细胞引入这种经过工程的肌肉组织中，使它们能够恢复到静止的状态，并保持至少一周。如果将来自较老动物的细胞放置在工程肌肉组织中后进行化学处理，也可以使它们恢复休眠。这种方法以一种微型化的方式工作，每个工程组织只需要不到从每只小鼠身上收集的肌肉干细胞的1%。与目前使用活体动物的方法相比，这允许进行100倍的实验。该系统可以帮助研究人员研究遗传和化学因素对肌肉干细胞静止的影响。对这一领域的进一步了解可能会导致恢复旧肌肉组织愈合能力的治疗方法。
Adult skeletal muscle harbours a population of muscle stem cells (MuSCs) that are required for repair after tissue injury. In youth, MuSCs return to a reversible state of cell-cycle arrest termed ‘quiescence’ after injury resolution. Conversely, some MuSCs in aged muscle remain semi-activated, causing a premature response to injuries that results in incomplete repair and eventual stem cell depletion. Regulating this balance between MuSC quiescence and activation may hold the key to restoring tissue homeostasis with age, but is incompletely understood. To fill this gap, we developed a simple and tractable in vitro method, to rapidly inactivate MuSCs freshly isolated from young murine skeletal muscle, and return them to a quiescent-like state for at least 1-week, which we name mini-IDLE (Inactivation and Dormancy LEveraged in vitro). This was achieved by introducing MuSCs into a 3D bioartificial niche comprised of a thin sheet of mouse myotubes, which we demonstrate provides the minimal cues necessary to induce quiescence. With different starting numbers of MuSCs, the assay revealed cellular heterogeneity and population-level adaptations that converged on a common niche repopulation density; behaviours previously observed only in vivo. Quiescence-associated hallmarks included a Pax7+CalcR+DDX6+MyoD-c-FOS- signature, quiescent-like morphologies, and polarized niche markers. Leveraging high-content bioimaging pipelines, we demonstrate a relationship between morphology and cell fate signatures for possible real-time morphology-based screening. When using MuSCs from aged muscle, they displayed aberrant proliferative activities and delayed inactivation kinetics, among other quiescence-associated defects that we show are partially rescued by wortmannin treatment. Thus, the assay offers an unprecedented opportunity to systematically investigate long-standing queries in areas such as regulation of pool size and functional heterogeneity within the MuSC population, and to uncover quiescence regulators in youth and age. When our muscles are injured, stem cells in the tissue are activated to start the repair process. However, when there is no damage, these cells tend to stay in a protective, dormant state known as quiescence. If quiescence is not maintained, the stem cells cannot properly repair when the muscle is damaged. This happens in old age, when a proportion of the cells remain semi-activated, and become depleted. However, researchers still do not fully understand how quiescence is regulated. This is partly because in order to study quiescence, live animals must be used, because muscle stem cells immediately come out of quiescence when they are removed from muscle tissue. To overcome this experimental limitation, Jacques et al. developed a new method to study muscle stem cells by transferring them from mice into three-dimensional engineered muscle tissue grown in the lab. This tissue is made by infiltrating the pores of teabag paper with muscle progenitor cells, which then fuse with one another to make a thin muscle that contains three layers of contractile muscle cells. Introducing muscle stem cells from young healthy animals into this engineered muscle tissue allowed them to return to a quiescent-like state and to remain in that state for at least a week. Cells from older animals could also be returned to dormancy if they were chemically treated after placing them in the engineered muscle tissue. The approach works in a miniaturized fashion, with each engineered tissue requiring less than one per cent of the muscle stem cells collected from each mouse. This allows 100 times as many experiments compared to the current methods using live animals. This system could help researchers to study the genetic and chemical influences on muscle stem cell quiescence. Further understanding in this area could lead to treatments that restore healing abilities in older muscle tissue.