Efficient and high yield isolation of myoblasts from skeletal muscle - ScienceDirect

Stem Cell Research

Aref Shahini¹ 

关键词: Skeletal muscle progenitors;    Myoblast isolation;    Satellite cells;    Myogenic differentiation;    FACS;    fluorescence activate cell sorting;    MACS;    magnetic-activate cell sorting;    EHS;    Engelbreth-Holm-Swarm;    TGF-β;    transforming growth factor beta;    EGF;    epidermal growth factor;    IGF1;    insulin-like growth factor;    PDGF;    platelet derived growth factor;    bFGF;    basic fibroblast growth factor;    CEE;    Chicken Embryo Extract;    PBS;    phosphate buffer saline;    PM;    proliferation medium;    DM;    Differentiation Medium;    FBS;    fetal bovine serum;    HS;    Horse Serum;   

DOI  :  10.1016/j.scr.2018.05.017

学科分类：生物科学（综合）

来源: Elsevier

【 摘 要 】

Skeletal muscle (SkM) regeneration relies on the activity of myogenic progenitors that reside beneath the basal lamina of myofibers. Here, we describe a protocol for the isolation of the SkM progenitors from young and old mice by exploiting their outgrowth potential from SkM explants on matrigel coated dishes in the presence of high serum, chicken embryo extract and basic fibroblast growth factor. Compared to other protocols, this method yields a higher number of myoblasts (10–20 million) by enabling the outgrowth of these cells from tissue fragments. The majority of outgrowth cells (~90%) were positive for myogenic markers such as α7-integrin, MyoD, and Desmin. The myogenic cell population could be purified to 98% with one round of pre-plating on collagen coated dishes, where differential attachment of fibroblasts and other non-myogenic progenitors separates them from myoblasts. Moreover, the combination of high serum medium and matrigel coating provided a proliferation advantage to myogenic cells, which expanded rapidly (~24 h population doubling), while non-myogenic cells diminished over time, thereby eliminating the need for further purification steps such as FACS sorting. Finally, myogenic progenitors gave rise to multinucleated myotubes that exhibited sarcomeres and spontaneous beating in the culture dish.

【 授权许可】

Unknown