Differentiation of the human PAX7-positive myogenic precursors/satellite cell lineage in vitro.

Satellite cells (SC) are muscle stem cells that can regenerate adult muscles upon injury. Most SC originate from PAX7+ myogenic precursors set aside during development. Although myogenesis has been studied in mouse and chicken embryos, little is known about human muscle development. Here, we report the generation of human induced pluripotent stem cell (iPSC) reporter lines in which fluorescent proteins have been introduced into the PAX7 and MYOG loci. We use single cell RNA sequencing to analyze the developmental trajectory of the iPSC-derived PAX7+ myogenic precursors. We show that the PAX7+ cells generated in culture can produce myofibers and self-renew in vitro and in vivo Together, we demonstrate that cells exhibiting characteristics of human fetal satellite cells can be produced in vitro from iPSC, opening interesting avenues for muscular dystrophy cell therapy. This work provides significant insights into the development of the human myogenic lineage.

Recapitulating early development of mouse musculoskeletal precursors of the paraxial mesoderm in vitro.

Body skeletal muscles derive from the paraxial mesoderm, which forms in the posterior region of the embryo. Using microarrays, we characterize novel mouse presomitic mesoderm (PSM) markers and show that, unlike the abrupt transcriptome reorganization of the PSM, neural tube differentiation is accompanied by progressive transcriptome changes. The early paraxial mesoderm differentiation stages can be efficiently recapitulated in vitro using mouse and human pluripotent stem cells. While Wnt activation alone can induce posterior PSM markers, acquisition of a committed PSM fate and efficient differentiation into anterior PSM Pax3+ identity further requires BMP inhibition to prevent progenitors from drifting to a lateral plate mesoderm fate. When transplanted into injured adult muscle, these precursors generated large numbers of immature muscle fibers. Furthermore, exposing these mouse PSM-like cells to a brief FGF inhibition step followed by culture in horse serum-containing medium allows efficient recapitulation of the myogenic program to generate myotubes and associated Pax7+ cells. This protocol results in improved in vitro differentiation and maturation of mouse muscle fibers over serum-free protocols and enables the study of myogenic cell fusion and satellite cell differentiation.

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

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.

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

During embryonic development, skeletal muscles arise from somites, which derive from the presomitic mesoderm (PSM). Using PSM development as a guide, we establish conditions for the differentiation of monolayer cultures of mouse embryonic stem (ES) cells into PSM-like cells without the introduction of transgenes or cell sorting. We show that primary and secondary skeletal myogenesis can be recapitulated in vitro from the PSM-like cells, providing an efficient, serum-free protocol for the generation of striated, contractile fibers from mouse and human pluripotent cells. The mouse ES cells also differentiate into Pax7(+) cells with satellite cell characteristics, including the ability to form dystrophin(+) fibers when grafted into muscles of dystrophin-deficient mdx mice, a model of Duchenne muscular dystrophy (DMD). Fibers derived from ES cells of mdx mice exhibit an abnormal branched phenotype resembling that described in vivo, thus providing an attractive model to study the origin of the pathological defects associated with DMD.