ABSTRACTBrown adipocytes represent a specialized type of mammalian adipocytes able to uncouple nutrient catabolism from ATP generation to dissipate energy as heat. They play an important role in mammals, allowing non-shivering thermogenesis to regulate body temperature in response to cold exposure. In humans, the brown fat tissue is composed of small discrete depots found mostly throughout the neck and trunk region. Increasing brown fat activity either with drug treatment or cell therapy is considered a potential approach for the treatment of metabolic syndrome and obesity. The recent development of in vitro differentiation strategies relying on human pluripotent stem cells (hPSCs) offers the possibility to produce unlimited amounts of brown adipocytes. A strategy efficiently applied to several tissues is to recapitulate step by step the development of the tissue of interest by exposing hPSCs to the signaling cues used during normal embryonic development. However, this strategy has proven difficult to implement for brown fat as the development of this tissue is poorly understood. Here, we first used single cell RNA sequencing to characterize the development of interscapular brown fat in mouse. Our analysis identified a previously unrecognized population of brown adipocytes precursors characterized by expression of the transcription factor GATA6. We show that this precursor population can be efficiently generated from paraxial mesoderm precursors differentiated in vitro from hPSCs by modulating the signaling pathways identified in our transcriptomic analysis. These precursors can in turn be efficiently converted into functional brown adipocytes which can respond to adrenergic stimuli by increasing their metabolism resulting in heat production.

Embryonic stem cells (ESCs) can adopt lineage-specific gene-expression programs by stepwise exposure to defined factors, resulting in the generation of functional cell types. Bulk and single-cell-based assays were employed to catalog gene expression, histone modifications, chromatin conformation, and accessibility transitions in ESC populations and individual cells acquiring a presomitic mesoderm fate and undergoing further specification toward myogenic and neurogenic lineages. These assays identified cis-regulatory regions and transcription factors presiding over gene-expression programs occurring at defined ESC transitions and revealed the presence of heterogeneous cell populations within discrete ESC developmental stages. The datasets were employed to identify previously unappreciated genomic elements directing the initial activation of Pax7 and myogenic and neurogenic gene-expression programs. This study provides a resource for the discovery of genomic and transcriptional features of pluripotent, mesoderm-induced ESCs and ESC-derived cell lineages.

Duchenne muscular dystrophy (DMD) is a devastating genetic disease leading to degeneration of skeletal muscles and premature death. How dystrophin absence leads to muscle wasting remains unclear. Here, we describe an optimized protocol to differentiate human induced pluripotent stem cells (iPSC) to a late myogenic stage. This allows us to recapitulate classical DMD phenotypes (mislocalization of proteins of the dystrophin-associated glycoprotein complex, increased fusion, myofiber branching, force contraction defects, and calcium hyperactivation) in isogenic DMD-mutant iPSC lines in vitro. Treatment of the myogenic cultures with prednisolone (the standard of care for DMD) can dramatically rescue force contraction, fusion, and branching defects in DMD iPSC lines. This argues that prednisolone acts directly on myofibers, challenging the largely prevalent view that its beneficial effects are caused by antiinflammatory properties. Our work introduces a human in vitro model to study the onset of DMD pathology and test novel therapeutic approaches.

The heterochronic genes Lin28a/b and let-7 regulate invertebrate development, but their functions in patterning the mammalian body plan remain unexplored. Here, we describe how Lin28/let-7 influence caudal vertebrae number during body axis formation. We found that FoxD1-driven overexpression of Lin28a strikingly increased caudal vertebrae number and tail bud cell proliferation, whereas its knockout did the opposite. Lin28a overexpression downregulated the neural marker Sox2, causing a pro-mesodermal phenotype with a decreased proportion of neural tissue relative to nascent mesoderm. Manipulating Lin28a and let-7 led to opposite effects, and manipulating Lin28a's paralog, LIN28B caused similar yet distinct phenotypes. These findings suggest that Lin28/let-7 play a role in the regulation of tail length through heterochrony of the body plan. We propose that the Lin28/let-7 pathway controls the pool of caudal progenitors during tail development, promoting their self-renewal and balancing neural versus mesodermal cell fate decisions.

The cardiac action potential (AP) is vital for understanding healthy and diseased cardiac biology and drug safety testing. However, techniques for high throughput cardiac AP measurements have been limited. Here, we introduce a novel technique for reliably increasing the coupling of cardiomyocyte syncytium to planar multiwell microelectrode arrays, resulting in a stable, label-free local extracellular action potential (LEAP). We characterized the reliability and stability of LEAP, its relationship to the field potential, and its efficacy for quantifying AP morphology of human induced pluripotent stem cell derived and primary rodent cardiomyocytes. Rise time, action potential duration, beat period, and triangulation were used to quantify compound responses and AP morphology changes induced by genetic modification. LEAP is the first high throughput, non-invasive, label-free, stable method to capture AP morphology from an intact cardiomyocyte syncytium. LEAP can accelerate our understanding of stem cell models, while improving the automation and accuracy of drug testing.

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.

The skeletal muscle lineage derives from the embryonic paraxial mesoderm (PM) which also gives rise to the axial skeleton, the dermis of the back, brown fat, meninges, and endothelial cells. Direct conversion was pioneered in skeletal muscle with overexpression of the transcription factor MyoD which can convert fibroblasts to a muscle fate. In contrast, directed differentiation of skeletal muscle from pluripotent cells (PC) in vitro has proven to be very difficult compared to other lineages and has only been achieved recently. Experimental strategies recapitulating myogenesis in vitro from mouse and human PC (ES/iPS) have now been reported and all rely on early activation of Wnt signaling at the epiblast stage. This leads to induction of neuromesodermal progenitors that can subsequently be induced to a PM fate and to skeletal muscle. These protocols can efficiently produce fetal muscle fibers and immature satellite cells. These new in vitro systems now open the possibility to better understand human myogenesis and to develop in vitro disease models as well as cell therapy approaches.

Mammalian embryos transiently exhibit aerobic glycolysis (Warburg effect), a metabolic adaptation also observed in cancer cells. The role of this particular type of metabolism during vertebrate organogenesis is currently unknown. Here, we provide evidence for spatiotemporal regulation of glycolysis in the posterior region of mouse and chicken embryos. We show that a posterior glycolytic gradient is established in response to graded transcription of glycolytic enzymes downstream of fibroblast growth factor (FGF) signaling. We demonstrate that glycolysis controls posterior elongation of the embryonic axis by regulating cell motility in the presomitic mesoderm and by controlling specification of the paraxial mesoderm fate in the tail bud. Our results suggest that glycolysis in the tail bud coordinates Wnt and FGF signaling to promote elongation of the embryonic axis.

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.

Vertebrate segmentation is characterized by the periodic formation of epithelial somites from the mesenchymal presomitic mesoderm (PSM). How the rhythmic signaling pulse delivered by the segmentation clock is translated into the periodic morphogenesis of somites remains poorly understood. Here, we focused on the role of paraxial protocadherin (PAPC/Pcdh8) in this process. We showed that in chicken and mouse embryos, PAPC expression is tightly regulated by the clock and wavefront system in the posterior PSM. We observed that PAPC exhibits a striking complementary pattern to N-cadherin (CDH2), marking the interface of the future somite boundary in the anterior PSM. Gain and loss of function of PAPC in chicken embryos disrupted somite segmentation by altering the CDH2-dependent epithelialization of PSM cells. Our data suggest that clathrin-mediated endocytosis is increased in PAPC-expressing cells, subsequently affecting CDH2 internalization in the anterior compartment of the future somite. This in turn generates a differential adhesion interface, allowing formation of the acellular fissure that defines the somite boundary. Thus, periodic expression of PAPC in the anterior PSM triggers rhythmic endocytosis of CDH2, allowing for segmental de-adhesion and individualization of somites.

Mammalian embryos transiently exhibit aerobic glycolysis (Warburg effect), a metabolic adaptation also observed in cancer cells. The role of this particular type of metabolism during vertebrate organogenesis is currently unknown. Here, we provide evidence for spatio-temporal regulation of aerobic glycolysis in the posterior region of mouse and chicken embryos. We show that a posterior glycolytic gradient is established in response to graded transcription of glycolytic enzymes downstream of FGF signaling. We demonstrate that glycolysis controls posterior elongation of the embryonic axis by regulating cell motility in the presomitic mesoderm and by controlling specification of the paraxial mesoderm fate in the tail bud. Our results suggest that Warburg metabolism in the tail bud coordinates Wnt and FGF signaling to promote elongation of the embryonic axis.

Duchenne muscular dystrophy (DMD) is the most common type of muscular dystrophy. DMD is a very severe disorder presenting with rapidly progressing muscle wasting and premature death. No current treatment is available and drugs improving the course of the disease are limited and with various side effects. The aim of this work is to develop a drug screening assay to find new drug candidates for DMD. A high throughput screening test was developed using a unique in vitro differentiation protocol recapitulating key signaling events in skeletal muscle development. This protocol allows the production of fully differentiated myotubes associated with satellite-like cells expressing the Pax7 marker, starting from human induced pluripotent stem cells (hiPSCs) carrying two common DMD mutations. Practically, a population of muscle progenitors enriched in Pax7+ satellite-like cells is placed in 384 well-plates in a medium allowing their spontaneous differentiation into myotubes and exposed to the test compounds. The readout selected is the surface of the wells covered by myotubes, where increased myotube area reflects an increased commitment of Pax7+ cells towards myotube formation. This has the potential to translate into a therapeutic benefit in patients as recent data supports the existence of a defect in satellite cell commitment towards muscle fiber generation in DMD boys. Several hits coming out of the first screening campaign (5'000 compounds) were already known in the literature to have an effect on muscle function in mdx mice and/or DMD patients, thus validating the predictivity of the HTS assay. Furthermore, a selection of hits was tested in vivo in the mdx mouse model with or without combination with the standard of care, and all were found to improve skeletal muscle function and resistance to muscle damage. This approach should lead to the identification of promising drug candidates for DMD patients.

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.

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.

One of the most striking features of the human spine is its periodic organization. This so-called “segmental” arrangement of the vertebrae along the anteroposterior body axis is established during embryonic development. Structures called somites, which contain the precursors of the vertebrae, form in a rhythmic fashion at the posterior end of the embryo during the process of somitogenesis. Somites are sequentially added to the growing axis, thus establishing the characteristic periodic pattern of the future vertebral column. The primary segmentation of the vertebrate embryo displayed by somitic organization also underlies much of the segmental organization of the body, including muscles, nerves, and blood vessels. In amniotes, somites are the major component of the paraxial mesoderm that form bilaterally along the nerve cord as a result of primitive streak and tail bud regression during body axis formation. Somites bud off from the anterior presomitic mesoderm (PSM) as epithelial spheres surrounding a core of mesenchymal cells called the somitocoele. The dorsal portion of the somite remains epithelial and forms the dermomyotome, which differentiates into muscle and dermis while its ventral moiety undergoes an epithelio-mesenchymal transition, leading to the formation of the sclerotome. The sclerotome gives rise to the skeletal elements of the vertebral column: the vertebrae, ribs, intervertebral disks, and tendons. Most of our understanding of amniote somitogenesis at the morphogenetic and molecular levels results from studies involving the chicken ( Gallus gallus ) and the mouse ( Mus musculus ). In this chapter, we essentially focus on the patterning and development of the spine in...

The segmented body plan of vertebrate embryos arises through segmentation of the paraxial mesoderm to form somites. The tight temporal and spatial control underlying this process of somitogenesis is regulated by the segmentation clock and the FGF signaling wavefront. Here, we report the cyclic mRNA expression of Snail 1 and Snail 2 in the mouse and chick presomitic mesoderm (PSM), respectively. Whereas Snail genes' oscillations are independent of NOTCH signaling, we show that they require WNT and FGF signaling. Overexpressing Snail 2 in the chick embryo prevents cyclic Lfng and Meso 1 expression in the PSM and disrupts somite formation. Moreover, cells mis-expressing Snail 2 fail to express Paraxis, remain mesenchymal, and are thereby inhibited from undergoing the epithelialization event that culminates in the formation of the epithelial somite. Thus, Snail genes define a class of cyclic genes that coordinate segmentation and PSM morphogenesis.