Abstract
The generation of functional skeletal muscle tissues from human pluripotent stem cells (hPSCs) has not been reported. Here, we derive induced myogenic progenitor cells (iMPCs) via transient overexpression of Pax7 in paraxial mesoderm cells differentiated from hPSCs. In 2D culture, iMPCs readily differentiate into spontaneously contracting multinucleated myotubes and a pool of satellite-like cells endogenously expressing Pax7. Under optimized 3D culture conditions, iMPCs derived from multiple hPSC lines reproducibly form functional skeletal muscle tissues (iSKM bundles) containing aligned multi-nucleated myotubes that exhibit positive force–frequency relationship and robust calcium transients in response to electrical or acetylcholine stimulation. During 1-month culture, the iSKM bundles undergo increased structural and molecular maturation, hypertrophy, and force generation. When implanted into dorsal window chamber or hindlimb muscle in immunocompromised mice, the iSKM bundles survive, progressively vascularize, and maintain functionality. iSKM bundles hold promise as a microphysiological platform for human muscle disease modeling and drug development.
Highlights
The generation of functional skeletal muscle tissues from human pluripotent stem cells has not been reported
Two human pluripotent stem cells (hPSCs) lines were used for detailed characterization of myogenic differentiation, an H9 hES cell line and a transgene-free hiPS cell line (TRiPS) from the Duke iPSC Shared Resource Facility (Supplementary Fig. 1)
Using four distinct hPSC sources, we developed a reproducible method to generate expandable myogenic progenitors, termed induced myogenic progenitor cells (iMPCs), capable of highly efficient differentiation into multinucleated myotubes in 2D cell culture
Summary
The generation of functional skeletal muscle tissues from human pluripotent stem cells (hPSCs) has not been reported. Large-scale physiological and drug screening studies would require a readily available and expandable source of muscle progenitor cells[5] as well as 3D culture conditions leading to formation of biomimetic muscle tissues capable of electrically and chemically induced force generation. We recently reported the first engineering of functional 3D muscle tissues (“myobundles”) made from primary human myoblasts that displayed physiological force generation and calcium (Ca2+) transients in response to electrical and biochemical stimulation[6]. These biomimetic muscle equivalents responded like native muscle to drugs that promote or decrease muscle function demonstrating the utility as a drug-screening tool.
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