Efficient Isolation and Ex Vivo Differentiation of Murine Satellite Cells from Healthy and Dystrophic Muscle

Satellite cells are the stem cells of adult skeletal muscles and confer skeletal muscle with remarkable regenerative ability. Under homeostatic conditions, satellite cells reside in a quiescent state in their niche along the basal lamina of muscle fibers. Upon receiving stimuli, satellite cells activate and engage in regenerative myogenesis to repair damaged fibers. Due to the impact of satellite cell differentiation on muscle physiology, studying their differentiation is relevant both within the context of healthy and diseased muscle. Due to the abundance of cell populations within skeletal muscle, the study of satellite cells is predicated on isolating highly pure populations. Fluorescence activated cell sorting (FACS) represents the gold standard for deriving highly pure satellite cell isolates but is costly and can reduce cell viability. In addition, proliferating satellite cells in vitro invariably transition to a homogeneous myoblast population that bestows a selective advantage on fast-dividing cells, reducing satellite cell heterogeneity. In this chapter, we describe our protocol for magnetic-activated cell sorting (MACS) of satellite cells. MACS preserves cell viability to a greater degree than FACS, and our approach allows for highly pure sorted populations of satellite cells. In addition, sorted cells can enter and progress through the myogenic program immediately upon plating, avoiding the need for lengthy expansion periods.