Human iPSC-derived motor neuron innervation enhances the differentiation of muscle bundles engineered with benchtop fabrication techniques.
Publication Year:
2024
PubMed ID:
39677637
Funding Grants:
Public Summary:
Scientists are working to create lab-grown skeletal muscle tissues that can be used for studying disease, testing drugs, and developing new treatments. One major challenge is that these engineered muscles often don’t mature enough to behave like real muscle. Because real muscles develop and function with help from motor neurons—the nerve cells that control movement—researchers tested whether adding motor neurons earlier in the engineering process would improve muscle development. In this study, muscle cells grown with human stem-cell–derived motor neurons became more mature, showed better structure, and produced stronger contractions. These nerve-supported muscles also maintained healthier function over time and expressed genes linked to high-endurance muscle fibers. Overall, adding motor neurons helps engineered muscle tissues grow stronger and more realistically, improving their usefulness for research and therapy development.
Scientific Abstract:
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. Although motor neurons have been added to pre-engineered muscle constructs, the impact of motor neurons added prior to the onset of muscle differentiation has not been evaluated. In this study, benchtop fabrication equipment was used to facilely fabricate chambers for engineering 3-dimensional (3-D) 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 one and two weeks of differentiation compared to non-innervated muscle bundles cultured with or without neurotrophic factors. Non-innervated muscle bundles also experienced a decline in rise and fall times as the culture progressed, whereas innervated muscle bundles and non-innervated 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 (MYL3) and myoglobin (MB), 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.
