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Abstract
Induced pluripotent stem cells (iPSCs) obtained by reprogramming primary somatic cells have revolutionized the fields of cell biology and disease modeling. However, the number protocols for generating mature muscle fibers with sarcolemmal organization using iPSCs remain limited, and partly mimic the complexity of mature skeletal muscle. Methods: We used a novel combination of small molecules added in a precise sequence for the simultaneous codifferentiation of human iPSCs into skeletal muscle cells and motor neurons. Results: We show that the presence of both cell types reduces the production time for millimeter-long multinucleated muscle fibers with sarcolemmal organization. Muscle fiber contractions are visible in 19–21 days, and can be maintained over long period thanks to the production of innervated multinucleated mature skeletal muscle fibers with autonomous cell regeneration of PAX7-positive cells and extracellular matrix synthesis. The sequential addition of specific molecules recapitulates key steps of human peripheral neurogenesis and myogenesis. Furthermore, this organoid-like culture can be used for functional evaluation and drug screening. Conclusion: Our protocol, which is applicable to hiPSCs from healthy individuals, was validated in Duchenne Muscular Dystrophy, Myotonic Dystrophy, Facio-Scapulo-Humeral Dystrophy and type 2A Limb-Girdle Muscular Dystrophy, opening new paths for the exploration of muscle differentiation, disease modeling and drug discovery.
Highlights
Based on the use of small molecules that mimic the activation or repression of the signaling cascades required for in vivo myogenesis, and neurogenesis, we sought to optimize conditions to produce innervated mature muscle fibers; to this end, hiPSCs were collected by mechanical dissociation
Small hiPSC clumps were plated on Matrigel-coated dishes in the presence of Thiazovivin in differentiation medium (DM) supplemented with ITS-A, LDN193189 (LDN), a potent BMP pathway inhibitor, and CHIR99021, a GSK3 inhibitor (Figure 1A)
Compared to our two control cells and the two clones analyzed for DM1 and LGMD2A, we observed that the fiber section size was significantly reduced in Duchenne Muscular Dystrophy (DMD) cells, and wasvariable as well as slightly increased in FSHD2 cells (Figure 5C)
Summary
Since the discovery in Human of somatic cell reprogramming into pluripotent stem cells (hiPSCs), protocols aimed at modeling skeletal muscle differentiation have been lagging behind those of other cell lineages [1]. Skeletal myogenesis involves tightly controlled spatial and temporal cues, and most of the current strategies take advantage of the cascade of events of somitogenesis during embryogenesis. In this context, available protocols, the vast majority of which are only focused on the production of myotubes, are either based on the induced expression of exogenous myogenic genes (PAX3, PAX7, MYOD1) [2,3,4] or the use of small molecules [5,6,7,8,9,10] with variable degrees of efficiency [2,11,12,13,14]. Differentiated cells are functional and respond to different drugs, opening new perspectives for the modeling of neuromuscular disorders and drug discovery
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