Defining the heterogeneity of skeletal muscle‐derived side and main population cells isolated immediately ex vivo

Abstract

Myoblast transfer therapy for Duchenne muscular dystrophy (DMD) largely fails due to cell death and inability of transplanted cells to engraft in diseased muscles. One method attempting to enrich for cell subpopulations is the Hoechst 33342 dye exclusion assay, yielding a side population (SP) thought to be progenitor enriched and a main population (MP). However, in vitro and transplant studies yielded inconsistent results relative to downstream progeny. Cell surface markers expressed by skeletal muscle-derived MP and SP cells have not been fully characterized directly ex vivo. Using flow cytometry, MP and SP cells were characterized based on their expression of several well-accepted progenitor cell antigens. Both the MP and SP populations are heterogeneous and overlapping in the cells they contain. The percentages of cells in each population vary with species and specific muscle examined. MP and SP populations contain both satellite and multipotent progenitor cells, based on expression of CD34, Sca-1, Pax7, and M-cadherin. Thus, isolation using this procedure cannot be used to predict downstream differentiation outcomes, and explains the conflicting literature on these cells. Hoechst dye also results in significant mortality of sorted cells. As defined subpopulations are easily obtained using flow cytometry, sorting immediately ex vivo based on accepted myogenic precursor cell markers will yield superior results in terms of cell homogeneity for transplantation therapy.Skeletal muscle possesses a remarkable ability to remodel and repair after injury and in disease. The repair process is mainly accomplished through activation of satellite cells along the myofibers, which differentiate and fuse into damaged areas. Although the satellite cell was described nearly 50 years ago (Mauro, 1961), the genotype and maintenance of these regenerative cells are not well understood. In dystrophic and sarcopenic muscles, the satellite cell pool becomes exhausted leading to severe and permanent muscle degeneration Renault et al., 2000). Many investigations have sought a method of replacing the satellite cell population through transplantation. Adult-derived myoblasts have been investigated for their therapeutic potential in the treatment of dystrophies, but unfortunately, this approach has been largely unsuccessful due to relatively rapid death of the majority of the transplanted cells Miller et al., 1997;Skuk et al., 2002). In part, this is due to immune rejection of the transplanted cells (Watt, 1990;Rando and Blau, 1994). However, immune system rejection only partly explains the failure of myoblast therapy, as even transplantation between monozygotic twin girl carriers of the Duchenne muscular dystrophy (DMD) gene does not result in long-term survival of the transplanted cells (Tremblay et al., 1993). In addition, transplanted myoblasts do not disperse very well within treated muscles (Lipton and Schultz, 1979;Morgan et al., 1987; Huard et al., 1994), and their expansion in vitro is limited by replicative senescence (K...

Regeneration of skeletal muscle

“…These include myogenic progenitor cells characterized as CD56+, CD34-, CD144-, CD45-, and CD146-; myo-endothelial cells characterized as CD56+, CD34+, CD144+, CD45-, and CD146-; perivascular progenitor cells characterized as CD56-, CD34-, CD144-, CD45-and CD146+; and a muscle-derived side population that has similar properties to hematopoietic stem cells in the bone marrow (Quintero et al 2009;Ten Broek et al 2010;Crisan et al 2009;Deasy et al 2004;Huard 2008;Jankowski et al 2002;Kallestad and McLoon 2010;Lecourt et al 2010;Peault et al 2007;Qu-Petersen et al 2002;Usas and Huard 2007;Wu et al 2010). MDSCs, once activated, are capable of differentiating along myogenic, osteogenic, chondrogenic and adipogenic lineages in vitro similar to other mesenchymal progenitor cells (Gates et al 2008).…”

MicroRNA-1 and MicroRNA-206 Improve Differentiation Potential of Human Satellite Cells: A Novel Approach for Tissue Engineering of Skeletal Muscle

“…Due to donor variation, the efficiency of tissue engineering of skeletal muscle will vary between individual patients. 13,14 Therefore, novel approaches to improve myogenesis are mandatory to augment tissue engineering of human skeletal muscle. The process of myogenesis is strongly regulated by epigenetic factors, in particular by microRNAs.…”

Rapid isolation of muscle-derived stem cells by discontinuous Percoll density gradient centrifugation

“…Because muscle-derived cells are a mixture of subpopulations from different lineages and different developmental stages, it may not be possible to predict their fates based solely on molecular marker expression (Kallestad and McLoon 2010). However, from the perspective of clinical applications, the heterogeneity of the cells may not be critically important.…”

“…However, identification of MDSCs can be ambiguous, because distinct cell populations may share identical molecular markers (Kallestad and McLoon 2010). Furthermore, cell surface markers used to define muscle-derived stem and progenitor cells are variable and change under cell culture conditions (Machida et al 2004).…”