Recapitulating the Development of Neuromuscular and Muscle-Tendon Interfaces Using Human Pluripotent Stem Cells

Abstract

Skeletal muscle tissues can be derived from human pluripotent stem cells (hiPSCs) by directed reprogramming or through stepwise differentiation. The latter employs defined combinations of small molecules and growth factors to modulate key signaling pathways that control muscle development. This strategy provides a powerful platform to dissect myogenesis in a controlled environment. Moreover, stepwise differentiation does not require genetic manipulation and therefore represents a promising candidate for cell-based therapies. However, a major challenge is that hiPSC-derived skeletal muscles remain in an embryonic-to-fetal stage of maturity. In vivo, nascent myofibers begin to interact with motor neurons and tendons during this developmental window, establishing neuromuscular and myotendinous junctions. Importantly, fetal myofiber formation is dependent on the electrical activity of motoneurons, while connective tissue and tendons provide essential cues for skeletal muscle patterning. We hypothesized that incorporating motor neurons and tendons in hiPSC-derived skeletal muscles would better recapitulate fetal myogenesis and promote myofiber growth and maturation. To test this, we developed novel protocols to generate hiPSC-derived ventral spinal cord neuron aggregates enriched in motor neurons, as well as connective tissue aggregates enriched in tenogenic cells. Co-culture of ventral spinal cord neurons with skeletal muscle led to the successful establishment of neuromuscular junctions, hypertrophy of the myofibers, and increased proliferation of their progenitors. Co-culture of tenogenic aggregates with skeletal muscle resulted in the formation of a muscle-tendon interface enriched in muscle progenitors. To further increase the physiological relevance, we engineered a 3D culture system that enables the spatially organized co-culture of skeletal muscle with motor neurons and tendons. Together, these approaches establish a set of novel platforms to model cell-cell interactions between skeletal muscle, motoneurons, and tendons, an essential step towards understanding the coordinate assembly of the musculoskeletal system and advancing tissue engineering therapies.