br Results br Discussion In muscle tissue satellite cells

Results
Discussion In muscle tissue, satellite all trans retinoic acid are heterogeneous as demonstrated by a distinction in α6ITG expression (Fig. 1). Laminin is expressed along the muscle fiber within the extracellular matrix enclosing the muscle fiber, where the laminin ligands cause α6/β1 integrin heterodimers receptor binding (Sonnenberg et al., 1990; Hall et al., 1990; Aumailley et al., 1990; Le Bellego et al., 2002). The localization of α6ITG integrin expressing satellite cells with laminin-rich extracellular matrix enclosing the muscle fiber emphasizes this important ligand binding interaction (Fig. 1H). Previously, we observed that increased levels of α6ITG expression in primary muscle stem cells were correlated with a better myogenic differentiation capacity (Wilschut et al., 2010). Therefore, we further investigated the role of α6ITG during myogenesis by selecting and sorting for NCAM expressing cells with or without α6ITG expression from isolated primary muscle stem cell culture. NCAM is involved in myoblast fusion and is expressed by satellite cells that are associated with myogenic cell commitment (Capkovic et al., 2008; Charlton et al., 2000). Thereby we could distinguish between myogenic and non-myogenic cells (Webster et al., 1988; Capkovic et al., 2008; Mesires and Doumit, 2002; Blanton et al., 1999; Krauss et al., 2005; Covault and Sanes, 1986). Three cell populations were obtained; muscle stem cells positive for α6ITG expression, muscle stem cells negative for α6ITG expression and the non-myogenic cells expressing neither NCAM nor α6ITG (Fig. 2). Here, the α6ITG positive myogenic stem cells showed a robust myogenic differentiation capacity as indicated by the expression of MYOGENIN, DESMIN and MYHC. Interestingly, it was shown that expression of α6ITG expression is required for myogenic differentiation into myotubes. The α6ITG− cells showed cell elongation and alignment, but no myoblast fusion (Fig. 3). The myogenic commitment of the cell populations was further examined by determination of mRNA expression levels. PAX7 expression was similar between the two myogenic NCAM+ cell populations after isolation indicating that they both related to satellite cells. Interestingly, both α6ITG+ and α6ITG− cells expressed the MRFs MYF5 and DESMIN, indicating the myogenic commitment of both cell types. It has been shown that in murine muscle tissue, myogenic progenitor cells are subdivided in Pax7 expressing satellite cells lacking Myf5 and committed satellite cells co-expressing Myf5 (Kuang et al., 2007). The hierarchy is based on the apical–basal position of the satellite cells towards the muscle fiber. Satellite cells identified as Pax7+/ Myf5− with an orientation towards the basal lamina (basal-position) were demonstrated to express α7β1 integrins. Satellite cells with an apical orientated position towards the plasma membrane of the myofiber expressed Myf5 (Kuang et al., 2007). These Myf5+ cells were indicated as a committed phenotype due to the loss of contact with the basal lamina and extracellular environment or niche, which is important in sustaining stem cell identity. Interestingly, in our experiments the α6ITG− cells expressed significantly higher levels of A7ITG and lower levels of MYF5 compared to α6ITG+ cells. This suggests that α6ITG- cells could have a basal orientated position and not yet directed into the myogenic program. This indicates that these cells are uncommitted early muscle precursor cells whereas the α6ITG+ cells could be the myogenic initiated myogenic progenitor cells. The non-myogenic cells lacking NCAM expression showed significant higher levels of A5ITG, which is the receptor for fibronectin in fibroblasts (Huveneers et al., 2008). Examination of the proliferation capacity of the two populations revealed that α6ITG− cells proliferated at a higher rate than α6ITG+ cells. The α6ITG+ cells stopped proliferation and formed myotubes by cell fusion, while α6ITG− cells continued cell proliferation until confluence was reached (Fig. 4). The high proliferation capacity of α6ITG− cells is consistent with the involvement of α6 integrin in the negative regulation of cell growth (Sastry et al., 1996). The differentiation capacity revealed a diminished myogenic commitment by cells lacking α6ITG expression compared to the α6ITG positive cells. Whether the α6ITG negative cells will become less proliferative and more myogenic when α6ITG is introduced into these cells for instance by overexpression, or whether the α6ITG negative cells have already entered different differentiation pathways is currently unknown.