Human Myoblasts and Muscle-Derived SP Cells

Skeletal muscle cells can be used in vitro for the study of myogenesis, as well as in vivo as gene-delivery vehicles for the therapy of muscle and nonmuscle diseases. These skeletal muscle cells are derived from muscle satellite cells that lie between the basal lamina and the sarcolemma of differentiated muscle fibers (1 ). Normally quiescent after the period of muscle development and growth during fetal life and the early postnatal period, these cells are induced to proliferate upon muscle damage and fuse with existing muscle fibers. Satellite cells isolated and grown in vitro are called myoblasts. Myoblasts proliferate in mitogen-rich media, but upon reaching high cell density followed by exposure to mitogen-poor media, are induced to differentiate and become postmitotic. Muscle differentiation is characterized by the fusion of myoblasts to form multinucleated myotubes that express differentiationspecific proteins. In this chapter, methods are given for the isolation of myoblasts from human muscle tissue using two different techniques: (a) flow cytometry (2 ) and (b) cell cloning (3 ,4 ). Recent reports have also highlighted the existence of highly primitive cells within mouse skeletal muscle, whose relationship with satellite cells is still under study (5 -7 ). These primitive cells have been purified using different methods and techniques, including the preplating technique (8 -10 ) and the fluorescence-activated cell sorter (FACS) (11 -14 ). Depending on the isolation technique, these cells have been named differently. Muscle SP cells have been isolated from mouse skeletal muscle by staining the dissociated primary muscle cells with the vital DNA dye Hoechst 33342, followed by FACS purification (11 -14 ). Mouse muscle SP cells have demonstrated hematopoietic and myogenic differentiation potential both in vitro and in vivo (11 -14 ) and these studies are being extended to human-derived SP cells. Methods to isolate human muscle SP cells from fetal and from adult skeletal muscle are also given. The methods in this chapter are applicable to muscle tissue from both fetal and postnatal donor as well as from normal and diseased individuals.