Background <p>Pericytes hold potential as therapeutic cells in skeletal muscle regeneration, particularly for neuromuscular disorders. While preclinical and clinical studies have studied the role of pericytes in muscle repair, robust evidence of myogenesis remains limited due to insufficient understanding of pericyte survival, distribution, and fusion capacity in vivo. Using an optimized multiscale imaging approach, we assessed presence, survival, localization and fusion capacity of primary human pericytes following intramuscular injection in an acute muscle injury mouse model.</p> Methods <p>Cardiotoxin was administered to the tibialis anterior muscle of <i>Rag2</i>^−/− immunodeficient mice to induce muscle regeneration, followed by intramuscular delivery of primary human pericytes. One month post-treatment, whole muscles were cleared using an ethanol–ethyl cinnamate protocol and immunolabeled with a human-specific lamin A/C antibody. Light sheet fluorescence microscopy enabled 3D visualization of transplanted pericyte distribution, guiding the selection of samples for cryosectioning and confocal laser scanning microscopy using laminin as a tissue marker.</p> Results <p>By combining 3D whole muscle visualization with in-depth confocal laser scanning microscopy, we showed that transplanted human pericytes contributed to muscle regeneration in vivo. Variability in human nuclei traceability after intramuscular delivery highlighted the procedural complexity.</p> Discussion <p>Our findings provide new insights into pericyte behavior and support their potential in cell-based therapies for neuromuscular disease. Methodologically, this versatile multiscale imaging pipeline offers broad applicability for tracking cell fate and enhancing the preclinical evaluation of regenerative interventions.</p> Clinical trial number <p>Not applicable.</p>