Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages
Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages
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DOI：
10.3791/51354
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发表时间：
2014-04-01
影响因子：
1.2
通讯作者：
Biga, Peggy R.
中科院分区：
文献类型：
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作者：
Froehlich, Jacob Michael;Seiliez, Iban;Biga, Peggy R.
由于在体内研究肌源性程序的固有困难和时间，原代培养系统来源于骨骼肌的常驻成体干细胞，肌源性前体细胞（MPC），已被证明是不可或缺的，我们了解哺乳动物骨骼肌的发育和生长。特别是在脊椎动物的基础类群，然而，数据是有限的，描述控制自我更新，增殖和分化的MPC的分子机制。特别令人感兴趣的是潜在的机制，基础脊椎动物的能力，经历相当大的postlarval骨骼肌纤维增生（即硬骨鱼）和完全再生后的附属物损失（即有尾目两栖动物）。此外，使用培养的成肌细胞可以帮助理解再生和再生肌的程序和它们之间的差异。为此，我们详细描述了一个强大的和有效的协议（和其中的变化），用于分离和维护MPC及其后代，成肌细胞和未成熟的肌管，在细胞培养作为一个平台，了解肌源性程序的演变，开始与更多的基础脊椎动物。利用模式生物的斑马鱼（Danio rerio）的状态，我们报告了该协议的应用程序的鲤科鱼类分支Danioninae的小鱼。在串联，该协议可以用来实现更广泛的比较方法，通过分离MPC从墨西哥美西螈（Ambystomamexicanum），甚至实验室啮齿动物。该方案现在广泛用于研究几种鱼类的肌发生，包括虹鳟鱼、鲑鱼和鲷（1-4）。
Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e. teleost fish) and full regeneration following appendage loss (i.e. urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream(1-4).