Groundbreaking Skeletal Muscle Organoid Combat Aging-Related Muscle Loss

With aging, many people notice a gradual decline in muscle strength, making lifting heavy objects and climbing stairs difficult. This can be an early sign of sarcopenia. Sarcopenia, an age-related loss of muscle mass and function, not only affects quality of life but also increases the risk of falls and fractures, posing a significant health threat to the elderly. However, the lack of an ideal human model has hindered research on the pathogenesis of sarcopenia and the development of treatments.

Recently, a study published in J Cachexia Sarcopenia Muscle, titled "Human Pluripotent Stem Cell-Derived Skeletal Muscle Organoid Model of Aging-Induced Sarcopenia", successfully developed a skeletal muscle organoid model based on human pluripotent stem cells, providing an important tool for in-depth research into sarcopenia and potential treatments.

Fig. 1. Efficient generation of hPSC‐derived 3D hSkMOs.

The research team generated three-dimensional human skeletal muscle organoids (hSkMOs) from human pluripotent stem cells (hPSCs) using an optimized differentiation protocol. These organoids exhibited remarkable structural maturity, reaching approximately 2 mm in diameter by day 100. The majority of cells differentiated into paraxial mesodermal progenitors, with T/BRA-positive cells accounting for 82.04% and TBX6-positive cells for 78.18%, while the proportion of neuromesodermal progenitors was relatively low.

In mature hSkMOs, myosin heavy chain (MyHC)-positive myofibers were twice as thick at day 100 as at day 50 and were surrounded by a continuous laminin basement membrane. PAX7-positive satellite cells were located beneath the basement membrane, consistent with the distribution of satellite cells in skeletal muscle in vivo. This structure provides an important foundation for muscle growth and repair. Approximately 10.5% of the organoids contained neural cells, including spinal motor neurons expressing choline acetyltransferase (ChAT), GFAP-positive glial cells, and S100B-positive Schwann cells. α-bungarotoxin labeling confirmed the formation of neuromuscular junctions (NMJs). Electron microscopy revealed synaptic vesicles, basement membranes, and myelin-like structures, indicating that hSkMOs possess functional neuromuscular connections.

Fig. 2. Structural characterization of mature muscle fibres in hSkMOs.

Single-nucleus RNA-sequencing further revealed the cellular diversity of hSkMOs, with myogenic progenitors/satellite cells accounting for 43.6%, myocytes 16.8%, myofibers 21.2%, and neurons 8.0%. Satellite cells were classified into three subtypes: quiescent, activated/proliferating, and differentiated. Neural cells included motor neurons, GABAergic interneurons, and glutamatergic interneurons, demonstrating the model's ability to mimic the complexity of the in vivo neuromuscular system.

Fig. 3. Single‐nucleus transcriptomic profiling reveals dynamic cellular heterogeneity in hSkMOs.

Functional studies revealed that hSkMOs exhibited spontaneous contractions, with significant calcium influx, and whole-cell patch-clamp recordings of spontaneous postsynaptic potentials in myofibers. Acetylcholine treatment increased contraction velocity, while tubocurarine significantly inhibited spontaneous contractions, confirming functional neuromuscular transmission in the organoids.

To mimic sarcopenia, the research team treated hSkMOs with tumor necrosis factor-α (TNF-α). Acute treatment (20 ng/mL, 2 days) significantly increased the phosphorylation levels of TNF-α/NF-κB pathway-related factors, such as NF-κB p65, IκBα, and AKT. The proportion of PAX7+/MYOD+ activated satellite cells increased sharply to 13.62% on day 0, then gradually declined. Concomitantly, myofiber cross-sectional area decreased temporarily, but recovered to near-control levels by day 7, demonstrating the organoids' intrinsic regenerative capacity.

Chronic TNF-α treatment, however, established a persistent sarcopenia-like phenotype, manifested by a significant reduction in myofiber volume and a significant decrease in the number of neuromuscular junctions. On this basis, testosterone treatment demonstrated significant improvements: compared to the TNF-α-only treatment group, combined testosterone treatment increased the proportion of PAX7+/MYOD+ activated satellite cells from 2.29% to 7.97%, the proportion of PAX7+/Ki67+ proliferating satellite cells from 2.07% to 7.03%, and the myofiber cross-sectional area from 644.7 μm² to 987.1 μm². The number of neuromuscular junctions remained close to that of the control group, and membrane potential responses were effectively preserved.

In summary, this study successfully established a human pluripotent stem cell-derived skeletal muscle organoid model that not only accurately recapitulates the structural maturity of skeletal muscle and its functional interactions with spinal cord-derived neurons, but also effectively mimics the pathological processes of aging-induced sarcopenia. The ameliorative effects of testosterone in this model provide strong evidence for hormonal therapeutic strategies for sarcopenia. The establishment of this model opens new avenues for in-depth understanding of the pathogenesis of sarcopenia and screening for potential therapeutic drugs. It holds the potential to help older adults better maintain muscle health, improve quality of life, and mitigate the rapid loss of muscle strength associated with aging.