Rational: Myotonic dystrophy type 1 (DM1) is the most common adult-onset muscular dystrophy. Despite identifying the causal mutation three decades ago, our incomplete understanding of the underlying pathogenic mechanisms hinders progress in treatment development. This underscores the need for innovative strategies to develop new medicines with more predictive translational models. Recent advances in three-dimensional cell culture techniques using human pluripotent stem cells (hPSCs) have enabled the development of functional human skeletal muscle constructs.

Methods: We implemented a transgene-free protocol to differentiate three distinct hPSC lines into a highly homogeneous and expandable population of myogenic progenitor cells. Myotubes derived from these myogenic progenitor cells in 2D culture exhibited a high fusion index, well-organized sarcomeric structures and spontaneous contractions. Notably, a small fraction of cells expressed Pax7 adjacent to muscle fibers. A cardiotoxin (CTX) injury assay further revealed the functionality of these satellite-like cells for muscle regeneration. We next formed functional skeletal muscle constructs by embedding myogenic progenitors within 3D hydrogel scaffolds using culturing platforms with anchoring pillars.

Results: Engineered myobundles reproducibly exhibited the formation of cross-striated myotubes capable of generating active twitch in response to acetylcholine or electrical stimulation after 7 days of differentiation. Myobundle contractions correlated with strong calcium transients after exposure to acetylcholine. We applied our 3D skeletal muscle model to hPSCs carrying mutations in the DMPK gene to model DM1. We validated the pathological relevance of these models by observing known molecular hallmarks of the disease, such as the presence of nuclear RNA foci and splicing defects. We also evidenced functional defects with decreased contractile force at 7 and 14 days of differentiation, mirroring the muscle weakness seen in DM1 patients.

Conclusion: Our 3D skeletal muscle model opens new avenues for in-depth exploration of pathophysiological mechanisms in DM1. It also provides a unique opportunity to assess novel therapeutic strategies using functional readouts, irrespective of molecular targets.