An in vitro human skeletal muscle model: coculture of myotubes, neuron-like cells, and the capillary network

Abstract

This study reports the generation of a new human muscle tissue equivalent from skeletal muscle-derived stem cells and human umbilical vein endothelial cells (HUVECs). Skeletal muscle stem cells were isolated by the preplate technique and differentiated into neuron-like cells that were positive for neuronal beta-tubulin3 and nestin and negative for the astrocyte marker glial fibrillary acidic protein (GFAP). Coculture of skeletal muscle stem cells with the HUVECs under optimized fetal bovine serum and media conditions resulted in formation of a capillary network among the multinucleated myotubes. The neuron-like cells derived from the human skeletal muscle stem cells were seeded onto vascularized myotubes to obtain the neuromuscular junctions in the coculture. At the end of 24 h of coculture, the neuron-like cells were found to be in association with the myotubes. This model represents a novel complex in vitro human skeletal muscle model containing advanced capillary networks and interacting myotubes and neurons, and it can be used for in vitro drug testing or for skeletal muscle regeneration either through application of cellular therapy or cell-laden tissue-engineered muscle constructs.