Understanding Bioenergetics of Myogenic Differentiation of Human Mesenchymal Stem Cells Using Label‐Free Imaging

Mesenchymal stem cells are essential for tissue repair, making them a prime focus for regenerative medicine and cell‐based therapies. However, their full therapeutic potential for treating muscle disorders remains constrained by an incomplete understanding of the mechanisms underlying myogenic differentiation. We characterized the metabolic reprogramming that occurs during the differentiation of human mesenchymal stem cells into the myogenic lineage. We employed wide‐field fluorescence microscopy to image endogenous NADH and FAD fluorescence, quantifying the redox ratios (FAD/(NADH + FAD)) to assess changes in cellular metabolism. This revealed distinct shifts in the redox state accompanying the progression of differentiation, indicating a significant transition toward a glycolytic pathway in differentiated myotubes. These findings provide quantitative insights into the metabolic alterations underpinning myogenic differentiation and help in the optimization of stem cell–based approaches for muscle regeneration.