Human iPSC-Derived Motor Neuron Innervation Enhances the Differentiation of Muscle Bundles Engineered with Benchtop Fabrication Techniques

Engineered skeletal muscle tissues are critical tools for disease modeling, drug screening, and regenerative medicine, but are limited by insufficient maturation. Because innervation is a critical regulator of skeletal muscle development and regeneration in vivo, motor neurons are hypothesized to improve the maturity of engineered skeletal muscle tissues. However, the impact of motor neurons on muscle phenotype when added prior to the onset of muscle differentiation is not clearly established. In this study, benchtop fabrication equipment was used to facilely fabricate chambers for engineering three-dimensional (3D) skeletal muscles bundles and measuring their contractile performance. Primary chick myoblasts were embedded in an extracellular matrix hydrogel solution and differentiated into engineered muscle bundles, with or without the addition of human induced pluripotent stem cell (hiPSC)-derived motor neurons. Muscle bundles differentiated with motor neurons had neurites distributed throughout their volume and a higher myogenic index compared to muscle bundles without motor neurons. Innervated muscle bundles also generated significantly higher twitch and tetanus forces in response to electrical field stimulation after 1 and 2 weeks of differentiation compared to noninnervated muscle bundles cultured with or without neurotrophic factors. Noninnervated muscle bundles also experienced a decline in rise and fall times as the culture progressed, whereas innervated muscle bundles and noninnervated muscle bundles with neurotrophic factors maintained more consistent rise and fall times. Innervated muscle bundles also expressed the highest levels of the genes for slow myosin light chain 3 (<i>MYL3</i>) and myoglobin (<i>MB</i>), which are associated with slow twitch fibers. These data suggest that motor neuron innervation enhances the structural and functional development of engineered skeletal muscle constructs and maintains them in a more oxidative phenotype.