Isolation, culture, and transplantation of muscle satellite cells.
Isolation, culture, and transplantation of muscle satellite cells.
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DOI：
10.3791/50846
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发表时间：
2014-04-08
期刊：
影响因子：
--
通讯作者：
Asakura A
中科院分区：
文献类型：
--
作者：
Motohashi N;Asakura Y;Asakura A
肌肉卫星细胞是出生后骨骼肌发育和再生所需的干细胞群体，占肌纤维层下核的2-5%。在成年肌肉中，卫星细胞通常处于有丝分裂静止状态。然而，损伤后，卫星细胞启动细胞增殖，产生成肌细胞及其后代，以调节肌肉的再生。卫星细胞来源的成肌细胞移植已被广泛研究为一种可能的治疗多种再生疾病的方法，包括肌肉营养不良、心力衰竭和泌尿系统功能障碍。成肌细胞移植到营养不良的骨骼肌、心肌梗死和功能障碍的尿管中，表明移植的成肌细胞可以在宿主组织中分化为肌纤维，并在这些疾病中表现出部分功能改善。因此，发展高效的骨骼肌静止卫星细胞的纯化方法，建立卫星细胞来源的成肌细胞培养和成肌细胞移植方法，对于了解卫星细胞自我更新、激活和分化的分子机制是必不可少的。此外，基于细胞的治疗肌营养不良症和其他再生疾病的发展也依赖于这些因素。然而，目前预期的静止卫星细胞的纯化方法需要使用昂贵的荧光激活细胞分选(FACS)机器。在这里，我们提出了一种快速、经济、可靠地从成年小鼠骨骼肌中分离静止卫星细胞的新方法，该方法是通过酶解结合磁激活细胞分选(MACS)来实现的。在分离出静止的纯卫星细胞后，这些细胞可以在几代后培养成大量的成肌细胞。这些新鲜分离的静止卫星细胞或体外扩增的成肌细胞可以移植到心脏毒素(CTX)诱导的再生小鼠骨骼肌中，以检测供体来源的细胞对再生肌肉纤维的贡献，以及移植到卫星细胞室，以检测自我更新活性。
Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2–5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors. However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.
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影响因子：
7.8
作者：
Asakura, Atsushi;Seale, Patrick;Girgis-Gabardo, Adele;Rudnicki, Michael A
通讯作者：
Rudnicki, Michael A
影响因子：
23.9
作者：
Tanaka KK;Hall JK;Troy AA;Cornelison DD;Majka SM;Olwin BB
通讯作者：
Olwin BB
影响因子：
64.8
作者：
Sacco, Alessandra;Doyonnas, Regis;Kraft, Peggy;Vitorovic, Stefan;Blau, Helen M.
通讯作者：
Blau, Helen M.
影响因子：
4.9
作者：
Farina, Nicholas H.;Hausburg, Melissa;Olwin, Bradley B.
通讯作者：
Olwin, Bradley B.
DOI：
10.1007/978-1-60761-063-2_11
发表时间：
2010-01-01
期刊：
PROTOCOLS FOR ADULT STEM CELLS
影响因子：
--
作者：
Conboy, Michael J.;Cerletti, Massimiliano;Conboy, Irina M.
通讯作者：
Conboy, Irina M.