Ex Vivo Myogenesis of Human Adult Masseter NCAM/CD56+ cells

Objectives: We have previously identified a population of NCAM+ve cells (the myogenic fraction) isolated from human adult masseter muscle using magnetic activated cell sorting (MACS) to study jaw muscle regeneration (Sinanan et al 2004). This study describes the growth dynamics of these cells and optimization of their myogenic differentiation. Methods: Heterogenous cultures derived from masseter muscle biopsies were immunomagnetically labeled with CD56 microbeads and fractionated by dual-pass positive selection MACS. In addition to co-culture with unsorted parent cells or the CD56-ve fraction generated during cell sorting, multiple cytokine-augmented media were tested to optimize the survival, proliferation and myogenic differentiation of the cells; cultures were analyzed by immunoflurocytochemistry. Results: Optimal proliferation of myogenic masseter muscle cultures required serum-free growth media supplemented with insulin-like growth factor-1 (IGF-1), hepatocyte growth factor (HGF), heregulin-beta-1 (HRG-β1) and fibroblast growth factors (FGF 2,4,6). Myogenic differentiation was encouraged by either adjacent co-cultures (of unsorted masseter muscle cells or CD56-ve cells) or by myogenic inducers (IGF-1 and HRG-β1), the latter resulting in frequent formation of force-generating polynuclear (>20 nuclei) myosacs. A molecular profile for mononuclear cells (MC) and myotubes (MT) was also revealed: CD34, vascular cell adhesion molecule-1, α-smooth muscle actin, integrin α2/CD48b, MyoD1 (few MC); myf5, Thy1/CD90, integrins (αv/CD51, β3/CD61, β5), syndecan-3 and heregulin receptor-2 (HER 2) (MC); NCAM/CD56, desmin, m-cadherin, integrins (α7, β1/CD29), HGF-R/c-Met, FGF-R (1,4), HER (3,4), vascular endothelial growth factor receptor-2, syndecan-4 and myogenin (MC and MT) ; nerve growth factor receptor and α-saromeric actin (few MC, all MT); myf-6 and α-saromeric actinin (MT). Conclusions: These results indicate that MACS-selected human adult muscle-derived CD56+ cells are functional myogenic precursors but also co-express lineage markers of several non-myogenic muscle-associated cells; their survival, proliferation and myogenic differentiation are regulated by both autocrine and paracrine circuits, probably involving IGF-1, FGF, HRG-β1 and HGF.