Three-dimensional tissue-engineered human skeletal muscle model of satellite cell quiescence

In vivo, satellite cells (SCs) are essential for skeletal muscle repair. However, in vitro investigation of SC function is challenged by isolation-induced SC activation, loss of the native quiescent state, and differentiation to myoblasts. This study applies tissue-engineered human skeletal muscle to track myoblast deactivation to 3D-SCs, which bear a quiescent phenotype, as well as examine melittin-induced injury response. CD56+ sorted cells from human muscle were expanded for 5 passages (2D) and used to generate 3D engineered muscle (myobundles). After 4 days of 3D growth conditions, myobundles were differentiated for 3 days (d3), 9 days (d9), or melittin-injured at d7 and sampled 2 days post injury (i2) and 5 days post injury (i5). In the myobundle samples (d3,d9,i2,i5), cells were dissociated from the myobundles before being subjected to single cell RNA-sequencing (scRNA-seq) using the 10x Genomics Chromium technology.