Spheroid-based generation of 3D myogenic tissues using transgene- and sorting-free differentiation of hiPSCs

Human induced pluripotent stem cells (hiPSCs) represent a clinically relevant cell source for skeletal muscle tissue engineering, yet transgene-free maturation remains challenging. Moreover, the potential of spheroids as building blocks for muscle engineering remains poorly defined. This study evaluated whether hiPSC-derived myogenic spheroids enhance biofabrication outcomes in an in vitro bio-artificial muscle (BAM) system compared to a conventional single cell approach. Methods. CD56+ hiPSC-derived myoblasts were generated using a transgene-free protocol and differentiated toward 2D myotubes. In 3D, myoblasts were aggregated into 140-200 µm spheroids. Spheroids or single cells were encapsulated in a fibrin-based BAM, kept under tension using metal pins. Non-encapsulated spheroids and BAMs were cultured up to 15 days and evaluated for myosin heavy chain (MYH)+ organization, transcriptional profile, and contractile function. Results. In 2D, MYH+ myotubes showing multinucleation and, occasionally, sarcomere striation were formed. Spheroids were successfully generated, increased in size and circularity, and showed an enhancing MYH positivity, particularly during the proliferation phase, and displayed a fetal/perinatal myogenic state. In BAMs, spheroids promoted MYH positivity and established larger continuous MYH+ structures. Transcriptional profiles of spheroid and single cell BAMs were broadly similar. Tetanic forces were generally higher in spheroid BAMs; however, this difference did not reach statistical significance. Conclusion. hiPSC-derived myogenic spheroids advance MYH expression, but terminal maturation is constrained, likely due to their intrinsically isotropic nature. Within BAMs, spheroids enhance MYH+ myotube formation, but do not overcome limitations in myoblast fusion. Incomplete spheroid outgrowth and poor control over their spatial distribution are important challenges for their integration. Future work should focus on improving hiPSC differentiation efficiency, implementing spheroids in earlier differentiation stages, and optimizing spheroid outgrowth and positioning, alongside incorporating remaining muscle components as fibroblasts and vascular cells. This will refine whether spheroid-based assembly can add value in specific biofabrication contexts. .