Skeletal muscle is the largest tissue in the body and loss of its function or its regenerative properties results in debilitating musculoskeletal disorders. Understanding the mechanisms that drive skeletal muscle formation will not only help to unravel the molecular basis of skeletal muscle diseases, but also provide a roadmap for recapitulating skeletal myogenesis in vitro from pluripotent stem cells (PSCs). PSCs have become an important tool for probing developmental questions, while differentiated cell types allow the development of novel therapeutic strategies. In this Review, we provide a comprehensive overview of skeletal myogenesis from the earliest premyogenic progenitor stage to terminally differentiated myofibers, and discuss how this knowledge has been applied to differentiate PSCs into muscle fibers and their progenitors in vitro.

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Making muscle: skeletal myogenesis in vivo and in vitro.

Gastruloids are three-dimensional aggregates of embryonic stem cells that display key features of mammalian development after implantation, including germ-layer specification and axial organization1-3. To date, the expression pattern of only a small number of genes in gastruloids has been explored with microscopy, and the extent to which genome-wide expression patterns in gastruloids mimic those in embryos is unclear. Here we compare mouse gastruloids with mouse embryos using single-cell RNA sequencing and spatial transcriptomics. We identify various embryonic cell types that were not previously known to be present in gastruloids, and show that key regulators of somitogenesis are expressed similarly between embryos and gastruloids. Using live imaging, we show that the somitogenesis clock is active in gastruloids and has dynamics that resemble those in vivo. Because gastruloids can be grown in large quantities, we performed a small screen that revealed how reduced FGF signalling induces a short-tail phenotype in embryos. Finally, we demonstrate that embedding in Matrigel induces gastruloids to generate somites with the correct rostral-caudal patterning, which appear sequentially in an anterior-to-posterior direction over time. This study thus shows the power of gastruloids as a model system for exploring development and somitogenesis in vitro in a high-throughput manner.

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Single-cell and spatial transcriptomics reveal somitogenesis in gastruloids

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.

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Engineering human pluripotent stem cells into a functional skeletal muscle tissue.

Generation of Functional Human 3D Cortico-Motor Assembloids

Three-dimensional Human iPSC-derived Artificial Skeletal Muscles Model Muscular Dystrophies and Enable Multilineage Tissue Engineering

Key cell types including skeletal muscle have proven difficult to differentiate in
vitro from pluripotent cells. During embryonic development, skeletal muscles
arise from somites, which derive from the presomitic mesoderm (PSM). Based on
our understanding of PSM development, we established serum-free conditions
allowing efficient differentiation of monolayer cultures of mouse embryonic stem
(ES) cells into PSM-like cells without introduction of exogenous genetic material
or cell sorting. We show that primary and secondary skeletal myogenesis can be
recapitulated in vitro from these PSM-like cells. Our strategy allowed for the
production of striated contractile fibers from mouse and human pluripotent cells
in vitro with an efficiency comparing with current cardiomyocytes differentiation
protocols. We also differentiated ES cells into Pax7-positive cells with satellite
cell characteristics, including the ability to generate dystrophin-positive fibers
when grafted into muscles from dystrophin-deficient mdx mice. We show that
differentiated ES cells derived from mdx mice exhibit a striking branched
phenotype resembling that described in vivo, thus providing an attractive model
to study the origin of the pathological defects associated with Duchenne
Muscular Dystrophy.

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Differentiation of pluripotent stem cells to muscle fiber to model Duchenne muscular dystrophy.

Progress toward finding a cure for muscle diseases has been slow because of the absence of relevant cellular models and the lack of a reliable source of muscle progenitors for biomedical investigation. Here we report an optimized serum-free differentiation protocol to efficiently produce striated, millimeter-long muscle fibers together with satellite-like cells from human pluripotent stem cells (hPSCs) in vitro. By mimicking key signaling events leading to muscle formation in the embryo, in particular the dual modulation of Wnt and bone morphogenetic protein (BMP) pathway signaling, this directed differentiation protocol avoids the requirement for genetic modifications or cell sorting. Robust myogenesis can be achieved in vitro within 1 month by personnel experienced in hPSC culture. The differentiating culture can be subcultured to produce large amounts of myogenic progenitors amenable to numerous downstream applications. Beyond the study of myogenesis, this differentiation method offers an attractive platform for the development of relevant in vitro models of muscle dystrophies and drug screening strategies, as well as providing a source of cells for tissue engineering and cell therapy approaches.

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Marie Hestin

Papers

Differentiation of pluripotent stem cells to muscle fiber to model Duchenne muscular dystrophy.

Generation of human muscle fibers and satellite-like cells from human pluripotent stem cells in vitro.