Cellular alignment and fusion: Quantifying the effect of macrophages and fibroblasts on myoblast terminal differentiation.

Successful skeletal muscle wound repair requires the alignment and fusion of myoblasts to generate multinucleated myofibers. In vitro, the accurate quantification of cellular alignment remains a challenge. Here we present the application of ImageJ and ct-FIRE to quantify muscle cell orientation by means of an alignment index (AI). Our optimised method, which does not require programming skills, allows the alignment of myoblasts in vitro to be determined independently of a predefined reference point. Using this method, we demonstrate that co-culture of myoblasts with macrophages, but not fibroblasts, promotes myoblast alignment in a cell density-dependent manner. Interestingly, myoblast fusion was significantly decreased in response to co-culture with macrophages, while the effect of fibroblasts on fusion was density-dependent. At lower numbers, fibroblasts significantly increased myoblast fusion, whereas at higher numbers a significant decrease was observed. Finally, triple co-culture revealed that the effect of macrophages on myoblast alignment and fusion is unaltered by the additional presence of fibroblasts. Application of our optimised method has therefore revealed quantitative differences in the roles of macrophages versus fibroblasts during alignment and fusion: while successful myoblast alignment is promoted by increasing macrophage numbers, regenerative fusion coincides with a decreasing macrophage population and initial rise in fibroblast numbers.