br Material and methods br Acknowledgment The authors

Material and methods
Acknowledgment The authors would like to thank Mr. Chee Peng Ng (Institute of Molecular and Cell Biology, Singapore) for his help on the electron scanning microscope and Dr. William Birch (Institute of Materials Research and Engineering, Singapore) for his critical review of the manuscript. This work is funded generously by Agency for Science, Technology and Research (A*STAR) Singapore.
Introduction Skeletal muscle contains a population of stem buy LY2606368 to support postnatal muscle growth and regeneration. These cells, characterized as muscle satellite cells, adopt a position between the basal lamina and plasma membrane of muscle fibers and possess a self-renewing capacity to sustain the resident stem cell pool (Montarras et al., 2005). The characteristic PAX7 expression by satellite cells is important to maintain postnatal self-renewal and myogenic commitment until activation upon damage or during development occurs (Seale et al., 2000; McKinnell et al., 2008; Relaix et al., 2005; Oustanina et al., 2004). Here, cells start to proliferate and differentiate into myoblasts, a process coordinated by the muscle regulatory factors (MRFs) Myf5 and MyoD (Buckingham et al., 2003; Rudnicki et al., 1993; Tajbakhsh, 2005). The second differentiation step occurs after myoblast alignment, initiating myoblast fusion resulting in the formation of myotubes. The MRF Myogenin controls terminal differentiation into mature myofibers (Pownall et al., 2002; Alapat et al., 2009). Integrins function as receptors important for adhesion to extracellular matrix (ECM) proteins or to other cells. They are composed of α- (18 types) and β- (8 types) subunits that can form 24 distinct heterodimers of which the composition dictates ligand specificity (Hemler, 1999; Hynes, 2002; van der Flier and Sonnenberg, 2001). In particular the integrin β1 has a role in regulating muscle integrity and is essential for myoblast adhesion to the ECM (Menko and Boettiger, 1987). During muscle development various integrin subunits are expressed including α1, α4, α5, α6, α7, αV, β1 and β3. They play roles in mediating modulation of cell proliferation, differentiation, migration, polarity, and motility triggering calcium influx and are involved in apoptosis (Hynes, 2002; Danen and Sonnenberg, 2003; Gullberg et al., 1998). The integrin α7β1 is expressed on myoblasts localized in and around the myotendinous and neuromuscular junction of the muscle fiber and is required for muscle maintenance (Bao et al., 1993; Hayashi et al., 1998). In a study with quail embryonic muscle, α5 integrin in myoblasts was demonstrated to stimulate proliferation, whereas α6 integrin mediated differentiation (Sastry et al., 1996). This suggests distinct functions for α-integrins in regulating myogenesis from proliferation towards terminal differentiation. Functions of α6 integrin (α6ITG) have been studied using a GoH3 neutralizing antibody directed against α6ITG. It is suggested that α6β1 is involved in adhesion to the E8-cell-binding site of laminin in non-muscle cells, whereas in muscle cells, not α6β1, but a different β1 integrin-series binds laminin (von der Mark et al., 1991; Sonnenberg et al., 1990). Alpha 6 integrin is expressed during early mouse development at the stages of laminin containing basement formation, which remains during embryogenesis (Hierck et al., 1993). Here, delamination of Myf5 expressing muscle progenitor cells formed laminin-rich myotome mediated by α6β1 integrin expression (Bajanca et al., 2004, 2006). Previous results showed that sustained α6ITG expression of muscle stem cells cultured on Matrigel surface coating was correlated with a high myogenic differentiation in vitro, suggesting a role for α6ITG during muscle stem cell differentiation (Wilschut et al., 2010). Muscle tissue contains a heterogeneous population of muscle progenitor cells (Zammit et al., 2006; Kuang et al., 2007; Cerletti et al., 2008). Selection for specific progenitor cells can be important to enrich for homogeneous cell populations to study function and differentiation potentials. Indeed, in several studies integrins as well as other membrane associated biomarkers were used to select for specific stem cell populations (Cerletti et al., 2008; Webster et al., 1988; Sherwood et al., 2004; Tamaki et al., 2002; Zheng et al., 2007). In this respect, expression of the neuronal cell adhesion molecule (NCAM) by satellite cells has been used to mark a myogenic cell population (Capkovic et al., 2008; Mesires and Doumit, 2002).