The concept of therapeutic implantation of autologous cells capable of replicating and differentiating into specialized progeny has generated great interest. This approach is being intensively investigated especially in cardiovascular medicine for the regeneration of the infarcted heart . Skeletal muscle has an outstanding ability to respond to the requirements of growth, remodeling, physical activity, and injury. Since adult myofibers are terminally differentiated, the regeneration of skeletal muscle is mostly dependent on a population of resident cells termed satellite cells (SC). These cells lie beneath the basal lamina, but above the plasma membrane of myofibers . When physiologically required, SC become activated, proliferate, differentiate and eventually fuse into myotubes that mature into myofibers . The process of skeletal muscle regeneration is influenced by the nature of injury (e.g., traumatic, chemical, ischemic), the composition of the extracellular matrix, the availability of growth factors produced by inflammatory cells, vascular cells, and motor neurons . SC have a smaller nuclear size compared with the adjacent nucleus of the myotube, and an increased amount of nuclear heterochromatin compared with that of the myonucleus . Quiescent SC show little transcriptional activity. Following activation, they appear as a swelling on the myofiber with cytoplasmic processes that extend from one or both poles of the cell. In adult skeletal muscles, SC have been estimated to represent 1% to 12% of cells in adult muscle. Activation of SC during muscle regeneration requires upregulation of Myf5 and MyoD, transcriptional activators of the myogenic regulatory factor family (MRF). Expression of these factors determines the transition of myogenic cells into myoblasts . Expression of Pax3, Pax7, Sox15, MNF, cyclooxygenases and other regulatory factors is also needed for SC activation [5–7]. Expression of the late MRFs (myogenin and MRF4) precedes the production of muscle-specific proteins (e.g., myosin heavy chain and muscle creatine kinase). Expression of cytoskeletal (desmin) and surface markers (M-cadherin, Syndecan-3 and -4, VCAM-1, CD56, Glycoprotein Leu-19, CD34) as evidenced by immunostaining is often exploited to follow myogenic differentiation [3, 8, 9].
Over the past decade, experimental evidence has been accumulating that the implant of cultured myoblasts can represent an effective approach for repairing damaged myocardium [1, 10–12]. The functional benefit of intramyocardially transplanted autologous or heterologous skeletal myoblasts has been established in different animal models (rat, hamster, sheep) in which heart damage was determined primarily by ischemia [13, 14]. In humans, autologous skeletal myoblast implantation has been practiced only in cases of left ventricular dysfunction produced by ischemic damage [15, 16]. Recently, the implant of autologous myoblasts has been proposed also in the setting of dilated cardiomyopathy , urinary incontinence [18, 19], traumatic muscle damage , congenital muscle disease . As compared to other attractive cell types, skeletal myoblasts have several clinically appealing properties, including autologous origin, ease of procurement, the ability of sufficient expansion in vitro, and the ability of adequate differentiation upon in vivo implant.
The present study was initiated to determine if currently available cell culture methods were suitable for obtaining sufficient numbers of differentiated cells from muscle biopsies of human donors aged over 40 years. The latter condition was set since, with the exception of traumatic injury, most of the above listed clinical conditions arise in patients older than 50 years. In this context, it should be borne in mind that, for cellular therapy, substantial numbers of implantable cells (i.e., ≥108) are needed within 1–2 months of disease onset. In addition, cells expanded in vitro need to be adequately differentiated along myogenic lineages, free of demonstrable genetic alterations, and free of contaminant macromolecules  and microorganisms.