In situ spatial transcriptomic analysis of human skeletal muscle using the Xenium platform

Background: Traditional transcriptomic studies struggle to capture this heterogeneity of skeletal muscle, particularly at the fibre type-specific level. This study aimed to evaluate capability of the recently developed Xenium platform for conducting detailed spatial transcriptomic analysis of skeletal muscle histological sections. Methods: Human vastus lateralis muscle samples from two individuals were analysed using the Xenium platform and Human Multi-Tissue and Cancer Panel which targets 377 genes. Successive tissue sections were additionally stained for specific Myosin Heavy Chain isoforms to differentiate between type-1 and type-2 muscle fibres. Muscle fibres were manually segmented which allowed for the comparison of transcript density between muscle fibre types and specific subcellular regions. Results: Manual segmentation was crucial for accurate analysis as the automatic algorithms in the Xenium platform were inadequate for the precise segmentation of muscle fibres. The analysis revealed that transcript density was higher in type-1 compared to type-2 fibres, particularly in the nuclear and perinuclear regions where it was higher than in cytosolic region. Additionally, 191 out of 377 genes were differentially expressed between muscle fibres and the perimysium. Comparing the fibre types, specific genes such as PROX1, S100A1, LGR5, ACTA2 and LPL exhibited higher expression in type-1 fibres, while PEBP4, CAVIN1, GATM andPVALB showed higher expression in type-2 fibres. Conclusions: We demonstrated that the Xenium platform is capable of high-resolution spatial in situ transcriptomic analysis of skeletal muscle histological sections. Manual segmentation of muscle fibres, although very labour-intensive, is currently required for successful differentiation of transcriptomic profiles between fibre types.