Multilineage Differentiation for Formation of Innervated Skeletal Muscle Fibers from Healthy and Diseased Human Pluripotent Stem Cells

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.

Tissue-Engineered Skeletal Muscle Models to Study Muscle Function, Plasticity, and Disease

“…In particular, the NMJ pathophysiology in DMD is poorly studied and has the potential to be studied in hiPSC-derived tissues. The generation of multi-cellular 3D muscle tissues holds promise for studying complex tissue interactions in healthy and diseased states (Maffioletti et al, 2018;Mazaleyrat et al, 2020). Overall, the iPSCderived disease models have significant potential for generating personalized medicine platforms, disease specific drug screening, and studying pathogenic cellular or organ-organ crosstalk in a modular fashion.…”

Altered action potential waveform and shorter axonal initial segment in hiPSC-derived motor neurons with mutations inVRK1

“…However, maintaining hiPSC-MNs in culture for more than 30 days is very challenging, because of cells detaching from their support. On the other hand, it is well known that MNs and muscle cells have mutual stabilizing and supportive influence to each other(Maffioletti et al, 2018; Mazaleyrat et al, 2020; Yoshida et al, 2015), and therefore, we sought to increase the viability and functionality of our hiPSC-MNs by coculturing them with mouse myoblasts (C2C12) for 7-9 days. This allowed obtaining large multinucleated myotubes, and hiPSC-MNs extending long neurites alongside the myotubes ( Supplementary Figure 1 ).…”

Human engineered skeletal muscle of hypaxial origin from pluripotent stem cells with advanced function and regenerative capacity

“…The co-development of spinal motor neurons has recently been described in PSC-derived skeletal muscle 3D organoids (Faustino Martins et al, 2020; Mazaleyrat et al, 2020). Interestingly, the data from our model share several aspects of the development of neuromuscular junctions by Faustino Martins et al We observed efficient induction of neuromesodermal progenitor cells expressing SOX2 , Brachyury ( T ) and CDX2 as posterior axis “determinant” (Faustino Martins et al, 2020).…”

“…The derivation of skeletal muscle cells from PSC has been demonstrated previously either by transfection or transduction of myogenic transgenes (Albini et al, 2013; Darabi et al, 2012; Goudenege et al, 2012; Kim et al, 2017; Rao et al, 2018; Tedesco et al, 2012; Young et al, 2016) or directed, transgene-free differentiation under controlled growth factors or small molecules stimulation (Borchin et al, 2013; Caron et al, 2016; Chal et al, 2016; Chal et al, 2015; Choi et al, 2016; Shelton et al, 2016; Xi et al, 2017). Recently, more advanced skeletal muscle organoids have been introduced that recapitulate characteristic steps of embryonic neuromuscular co-development (Faustino Martins et al, 2020; Mazaleyrat et al, 2020).…”