Generation of human neuromusculoskeletal tri-tissue organoids [snRNA-seq]

The human body function requires the crosstalk between the central nervous system and its innervated peripheral targets. One such essential crosstalk involves the connections between neural, muscular, and skeletal tissues, which have not yet been modeled with human cells. Here, we describe the generation of three-dimensional, neuromusculoskeletal tri-tissue organoids (hNMSOs) from human pluripotent stem cells. Staining, single-nucleus RNA-sequencing, and spatial transcriptome profiling revealed the co-emergence and spatially confined organization of neural, muscular, and skeletal lineages relevant to human tissues within individual organoids. The neural, muscular, and skeletal regions of hNMSOs exhibited maturation and established functional connections during development, and skeletal support benefited skeletal muscles' structural and functional development. Modeling with hNMSOs also unveiled the neuromuscular alterations following pathological skeletal degeneration. Together, our study provides an experimental model for future studies of human neuromusculoskeletal crosstalk and disease. Overall design: Single nucleus RNA sequencing from day 38 and day 75 of hNMSOs.