Stuelsatz Pascal, Keire Paul, Yablonka-Reuveni Zipora
Department of Biological Structure, School of Medicine, University of Washington, Health Sciences Building, Room G520, 1959 NE Pacific Street, Box 357420, Seattle, WA, 98195, USA.
Methods Mol Biol. 2017;1556:51-102. doi: 10.1007/978-1-4939-6771-1_4.
Multinucleated myofibers, the functional contractile units of adult skeletal muscle, harbor mononuclear Pax7 myogenic progenitors on their surface between the myofiber basal lamina and plasmalemma. These progenitors, known as satellite cells, are the primary myogenic stem cells in adult muscle. This chapter describes our laboratory protocols for isolating, culturing, and immunostaining intact myofibers from mouse skeletal muscle as a means for studying satellite cell dynamics. The first protocol discusses myofiber isolation from the flexor digitorum brevis (FDB) muscle. These short myofibers are plated in dishes coated with PureCol collagen (formerly known as Vitrogen) and maintained in a mitogen-poor medium (± supplemental growth factors). Employing such conditions, satellite cells remain at the surface of the parent myofiber while synchronously undergoing a limited number of proliferative cycles and rapidly differentiate. The second protocol discusses the isolation of longer myofibers from the extensor digitorum longus (EDL) muscle. These EDL myofibers are routinely plated individually as adherent myofibers in wells coated with Matrigel and maintained in a mitogen-rich medium, conditions in which satellite cells migrate away from the parent myofiber, proliferate extensively, and generate numerous differentiating progeny. Alternatively, these EDL myofibers can be plated as non-adherent myofibers in uncoated wells and maintained in a mitogen-poor medium (± supplemental growth factors), conditions that retain satellite cell progeny at the myofiber niche similar to the FDB myofiber cultures. However, the adherent myofiber format is our preferred choice for monitoring satellite cells in freshly isolated (Time 0) myofibers. We conclude this chapter by promoting the Nestin-GFP transgenic mouse as an efficient tool for direct analysis of satellite cells in isolated myofibers. While satellite cells have been often detected by their expression of the Pax7 protein or the Myf5 knockin reporter (approaches that are also detailed herein), the Nestin-GFP reporter distinctively permits quantification of satellite cells in live myofibers, which enables linking initial Time 0 numbers and subsequent performance upon culturing. We additionally point out to the implementation of the Nestin-GFP transgene for monitoring other selective cell lineages as illustrated by GFP expression in capillaries, endothelial tubes and neuronal cells. Myofibers from other types of muscles, such as diaphragm, masseter, and extraocular, can also be isolated and analyzed using protocols described herein. Collectively, this chapter provides essential tools for studying satellite cells in their native position and their interplay with the parent myofiber.
多核肌纤维是成年骨骼肌的功能性收缩单位，在肌纤维基膜和质膜之间的表面上含有单核Pax7成肌祖细胞。这些祖细胞，即卫星细胞，是成年肌肉中的主要成肌干细胞。本章介绍了我们实验室从小鼠骨骼肌中分离、培养和免疫染色完整肌纤维的方法，作为研究卫星细胞动态的手段。第一个方法讨论了从趾短屈肌（FDB）中分离肌纤维。这些短肌纤维被接种在涂有PureCol胶原蛋白（以前称为Vitrogen）的培养皿中，并在低有丝分裂原培养基（±补充生长因子）中培养。在这种条件下，卫星细胞保留在亲代肌纤维表面，同时同步经历有限数量的增殖周期并迅速分化。第二个方法讨论了从趾长伸肌（EDL）中分离较长肌纤维。这些EDL肌纤维通常作为贴壁肌纤维单独接种在涂有基质胶的孔中，并在富含丝裂原的培养基中培养，在这种条件下卫星细胞从亲代肌纤维迁移离开，大量增殖并产生许多分化后代。或者，这些EDL肌纤维可以作为非贴壁肌纤维接种在未包被的孔中，并在低有丝分裂原培养基（±补充生长因子）中培养，这种条件下卫星细胞后代保留在肌纤维生态位中，类似于FDB肌纤维培养物。然而，贴壁肌纤维形式是我们监测新鲜分离（时间0）肌纤维中卫星细胞的首选。我们在本章结尾推荐Nestin-GFP转基因小鼠作为直接分析分离肌纤维中卫星细胞的有效工具。虽然卫星细胞经常通过其Pax7蛋白表达或Myf5基因敲入报告基因检测到（本文也详细介绍了这些方法），但Nestin-GFP报告基因独特地允许对活肌纤维中的卫星细胞进行定量，这使得能够将初始时间0的数量与培养后的后续表现联系起来。我们还指出了Nestin-GFP转基因在监测其他选择性细胞谱系中的应用，如毛细血管、内皮管和神经元细胞中的GFP表达所示。来自其他类型肌肉的肌纤维，如膈肌、咬肌和眼外肌，也可以使用本文所述方法进行分离和分析。总的来说，本章提供了研究卫星细胞在其原位及其与亲代肌纤维相互作用的重要工具。
