Muscle Organoids Frontier: Construction Strategies and Applications of Multi-lineage Muscle Organoids and Multi-organoid Systems.

In recent years, multi-lineage muscle organoids have rapidly emerged as transformative research tools, providing high-fidelity models for deciphering skeletal muscle development and the pathological mechanisms of muscle diseases such as sarcopenia, amyotrophic lateral sclerosis, and myasthenia gravis. Primarily derived from pluripotent stem cells, these organoids integrate multiple functional lineages, such as skeletal muscle, neural, vascular, and skeletal components, thereby addressing the limitations of conventional single-tissue muscle models. In parallel, multi-organoid systems, such as assembloids and organ-on-a-chip platforms, further integrate distinct organoids (e.g., muscle, skeletal tissue, tendon, and hindbrain/cervical spinal cord) to reconstruct inter-organ communication and systemic physiology under physiologically relevant conditions. By recapitulating in vivo-like multi-tissue interactions, they serve as critical platforms for drug screening and therapeutic development. This review summarizes recent advances in multi-lineage muscle organoids and multi-organoid systems, outlining their cellular sources, construction strategies, and major applications in basic research, disease modeling, and drug discovery, and discusses current challenges and future directions to enhance their potential in muscle research. Graphical Abstract: