Polysialylated NCAM distinguishes differentiated myoblasts from proliferating myoblasts prior to fusion

The differentiation of skeletal muscle myoblasts is a crucial step in muscle regeneration and hypertrophy. However, the phenotypic changes that occur in myoblasts just before myotube formation have remained poorly understood. In this study, we established a novel flow cytometric method to examine these changes. We identified two distinct populations in human desmin-positive myoblasts based on immunological NCAM (neural cell adhesion molecule) staining intensity after paraformaldehyde fixation. Our results indicate that these populations correspond to differentiated and proliferating myoblast phenotypes. Further analyses revealed that antigenicity for a polysialylated-NCAM (PSA-NCAM) antibody is strongly correlated with this fixation-resistant antigenicity of NCAM, suggesting that polysialylation is associated with myogenic differentiation. Using magnetic-activated cell sorting (MACS), we separated PSA-NCAM positive and negative myoblast fractions and demonstrated that PSA-NCAM positive cells show higher differentiation marker expression and faster myotube formation. These findings suggest that PSA-NCAM can serve as a valuable surface marker to distinguish between proliferating and differentiated myoblasts. Our method provides new insights into the phenotypic heterogeneity of myoblasts and offers an approach to studying the early stages of muscle cell differentiation before myotube formation.