Leibniz Institute on Aging, Fritz-Lipmann-Institute.
Leibniz Institute on Aging, Fritz-Lipmann-Institute;
J Vis Exp. 2021 Feb 15(168). doi: 10.3791/62257.
Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.
成人骨骼肌组织中存在着一种干细胞群体，对于其再生能力至关重要。在肌肉损伤后，肌肉干细胞离开其静止状态并激活成肌细胞程序，最终导致受损组织的修复，同时补充肌肉干细胞池。各种因素影响肌肉干细胞的活性，其中包括内在刺激，还有来自肌肉干细胞微环境（干细胞龛）的信号。分离和培养带有相关肌肉干细胞的单个肌纤维可以保留干细胞与其龛之间的大部分相互作用，因此是体外研究肌肉干细胞功能的最接近的可能性。这里提供了一种从小鼠 EDL（伸趾长肌）肌肉中分离、培养、siRNA 转染和免疫染色其各自肌纤维上的肌肉干细胞的方案。这里概述的实验条件允许在体外研究和操作肌肉干细胞，包括在无需进行体内动物实验的情况下研究成肌活性。
