Engineering Functional Skeletal Muscle Tissues from Human Induced Pluripotent Stem Cells

In vitro cell culture models of human skeletal muscle offer a valuable platform for studies of pathology and discovery of new therapeutics. However, traditional two-dimensional cell culture models suffer from limited durability and challenges with assessment of skeletal muscle contractile function. Furthermore, finite proliferation potential and scarce availability of patient-derived primary myoblasts limit experimental throughput when using these cells. Here, we describe a method for using human induced pluripotent stem cells (hiPSCs) to generate functional three-dimensional skeletal muscle tissues. Briefly, transient overexpression of Pax7 in hiPSC-derived paraxial mesoderm cells permits efficient formation of myogenic progenitor cells (iMPCs), which are then combined with hydrogel matrix to form cylindrically shaped skeletal muscle (iSKM) "myobundles." After 2 weeks of differentiation, iSKM myobundles contain aligned and densely packed multinucleated myotubes that, in response to electrical stimulation, exhibit robust calcium transients and positive force-frequency and force-length relationships. The methods detailed here have been successfully utilized to model and study limb-girdle muscular dystrophy type R2/2B and can be applied for studies of skeletal muscle regeneration and testing of candidate gene and cell therapies for disease. Collectively, iSKM myobundle engineering is a powerful in vitro methodology expected to significantly advance human skeletal muscle research and therapy.