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

Kilian Mazaleyrat (1) , Cherif Badja (1) , Natacha Broucqsault (1) , Raphaël Chevalier (1) , Camille Laberthonnière (1) , Camille Dion (1) , Lyla Baldasseroni (1) , Claire El-Yazidi (1) , Morgane Thomas (1) , Richard Bachelier (2) , Alexandre Altié (2) , Karine Nguyen (1, 3) , Nicolas Levy (1, 3) , Jérôme Robin-Ducellier (1) , Frédérique Magdinier (1)

1 MMG - Marseille medical genetics - Centre de génétique médicale de Marseille
2 C2VN - Centre recherche en CardioVasculaire et Nutrition = Center for CardioVascular and Nutrition research
3 Département de génétique médicale [Hôpital de la Timone - APHM]

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.

Mots clés: human induced pluripotent cells; differentiation; myoblasts; myotubes; motor neurons; satellite cells; Z-lines; contraction; muscular dystrophy; Facio-Scapulo-Humeral Dystrophy; Myotonic Dystrophy; Duchenne Muscular Dystrophy; Limb girdle muscular dystrophy

Domaines: Biologie cellulaire

Kilian Mazaleyrat, Cherif Badja, Natacha Broucqsault, Raphaël Chevalier, Camille Laberthonnière, et al.. Multilineage Differentiation for Formation of Innervated Skeletal Muscle Fibers from Healthy and Diseased Human Pluripotent Stem Cells. Cells, 2020, 9 (6), pp.1531. ⟨10.3390/cells9061531⟩. ⟨hal-03080368⟩