Abstract
SummaryAlthough skeletal muscle cells can be generated from human induced pluripotent stem cells (iPSCs), transgene-free protocols include only limited options for their purification and expansion. In this study, we found that fluorescence-activated cell sorting-purified myogenic progenitors generated from healthy controls and Pompe disease iPSCs can be robustly expanded as much as 5 × 1011-fold. At all steps during expansion, cells could be cryopreserved or differentiated into myotubes with a high fusion index. In vitro, cells were amenable to maturation into striated and contractile myofibers. Insertion of acid α-glucosidase cDNA into the AAVS1 locus in iPSCs using CRISPR/Cas9 prevented glycogen accumulation in myotubes generated from a patient with classic infantile Pompe disease. In vivo, the expression of human-specific nuclear and sarcolemmar antigens indicated that myogenic progenitors engraft into murine muscle to form human myofibers. This protocol is useful for modeling of skeletal muscle disorders and for using patient-derived, gene-corrected cells to develop cell-based strategies.
Highlights
Over 700 human genetic disorders are known that affect skeletal muscle (Kaplan and Hamroun, 2015), very few therapies are available
Muscle regeneration is mediated by satellite cells (SCs) (Lepper et al, 2011; Murphy et al, 2011; Sambasivan et al, 2011); i.e., adult stem cells located between the sarcolemma and the plasma membrane (Mauro, 1961) that are quiescent in healthy, uninjured muscle
It has not proved easy to date to establish robust in vitro disease models for skeletal muscle disorders, to efficiently restore gene function in skeletal muscle cells, and to develop cell-based therapeutic strategies based on muscle regeneration
Summary
Over 700 human genetic disorders are known that affect skeletal muscle (Kaplan and Hamroun, 2015), very few therapies are available. Skeletal muscle has a high capacity for regeneration after injury (Baghdadi and Tajbakhsh, 2017; Bursac et al, 2015; Dumont et al, 2015). SCs are considered useful for in vitro disease modeling to investigate molecular mechanisms of disease, test drugs, or develop cell-based therapies. To decipher molecular mechanisms of disease, it is important to generate isogenic controls, given the high variability of gene expression and functional parameters between individuals (Hockemeyer and Jaenisch, 2016; Soldner et al, 2011). It has not proved easy to date to establish robust in vitro disease models for skeletal muscle disorders, to efficiently restore gene function in skeletal muscle cells, and to develop cell-based therapeutic strategies based on muscle regeneration
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