Skeletal Muscle Satellite Cells: Background and Methods for Isolation and Analysis in a Primary Culture System

Abstract

Summary Repair of adult skeletal muscle depends on satellite cells, myogenic stem cells located between the basal lamina and the plasmalemma of the myofiber. Standardized protocols for the isolation and culture of satellite cells are key tools for understanding cell autonomous and extrinsic factors that regulate their performance. Knowledge gained from such studies can contribute important insights to developing strategies for the improvement of muscle repair following trauma and in muscle wasting disorders. This chapter provides an introduction to satellite cell biology and further describes the basic protocol used in our laboratory to isolate and culture satellite cells from adult skeletal muscle. The cell culture conditions detailed herein support proliferation and differentiation of satellite cell progeny and the development of reserve cells, which are thought to reflect the in vivo self-renewal ability of satellite cells. Additionally, this chapter describes our standard immunostaining protocol that allows the characterization of satellite cell progeny by the temporal expression of characteristic transcription factors and structural proteins associated with different stages of myogenic progression. While emphasis is given here to the isolation and characterization of satellite cells from mouse hindlimb muscles, the protocols are suitable for other muscle types (such as diaphragm and extraocular muscles) and for muscles from other species, including chicken and rat. Altogether, the basic protocols described are straightforward and facilitate the study of diverse aspects of skeletal muscle stem cells.

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

“…Since more than 90% of the isolated cells express Pax7, no further purification is required (Figures 6-8). This avoids extra purification steps in other methods such as pre-plating on uncoated dishes 14,37,38 , fractionation on Percoll 39,40 , or fluorescent-or magnetic cell sorting 41,43 . For trituration it is essential to induce shear between the tissue fragments and the opening of the pipette tip as this allows the mechanical release of the SCs.…”

“…They then proliferate, differentiate, and fuse to form new myofibers or repair damaged ones 13 . Quiescent SCs express the transcription factor Pax7 14,15 , while their progeny, the proliferating myoblasts, additionally express the myogenic determination factor 1 (MyoD) 16 . Differentiating myoblasts start to express myogenin (MyoG) 17 .…”

“…In general, SCs can be obtained after dissociation of muscle tissue 14 . Mincing, enzymatic digestion, and trituration are generally required to release SCs from their niche.…”

“…Mincing, enzymatic digestion, and trituration are generally required to release SCs from their niche. SCs can be purified by pre-plating on uncoated dishes 14,37,38 , fractionation on Percoll 39,40 , or fluorescent-or magnetic cell sorting [41][42][43] . Here we present a new economic and rapid protocol for the isolation of satellite cells from branchiomeric head muscles of young adult rats.…”

“…Here we present a new economic and rapid protocol for the isolation of satellite cells from branchiomeric head muscles of young adult rats. This protocol is based on a previous manuscript 14 and specifically adapted for small tissue samples. , and 4 th branchial arches are described.…”