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Background Tetraspanins are a family of proteins known to assemble protein complexes at the cell membrane. They are thought to play diverse cellular functions in tissues by modifying protein-binding partners, thus bringing complexity and diversity in their regulatory networks. Previously, we identified the tetraspanin KAI/CD82 as a prospective marker for human muscle stem cells. CD82 expression appeared decreased in human Duchenne muscular dystrophy (DMD) muscle, suggesting a functional link to muscular dystrophy, yet whether this decrease is a consequence of dystrophic pathology or a compensatory mechanism in an attempt to rescue muscle from degeneration is currently unknown. Methods We studied the consequences of loss of CD82 expression in normal and dystrophic skeletal muscle and examined the dysregulation of downstream functions in mice aged up to 1 year. Results Expression of CD82 is important to sustain satellite cell activation, as in its absence there is decreased cell proliferation and less efficient repair of injured muscle. Loss of CD82 in dystrophic muscle leads to a worsened phenotype compared to control dystrophic mice, with decreased pulmonary function, myofiber size, and muscle strength. Mechanistically, decreased myofiber size in CD82−/− dystrophic mice is not due to altered PTEN/AKT signaling, although increased phosphorylation of mTOR at Ser2448 was observed. Conclusion Basal CD82 expression is important to dystrophic muscle, as its loss leads to significantly weakened myofibers and impaired muscle function, accompanied by decreased satellite cell activity that is unable to protect and repair myofiber damage.

The vascular endothelium is highly heterogeneous, with phenotypically and functionally distinct endothelial cell (EC) subtypes, partially driven by distinct metabolic programs. While this heterogeneity is important to maintain the multiple functions of the endothelium during tissue repair, it also contributes to challenges to therapeutically modulate ECs in pathological contexts. While the EC heterogeneity is far from being entirely unraveled, recent advances in single-cell technologies have greatly contributed to the identification of different EC subsets and cell states. In this chapter, we discuss new EC subsets captured by single-cell studies in health and disease, also focusing on metabolic EC signatures. We also highlight how such metabolic signatures could be exploited in therapeutic settings to promote or block angiogenesis.

The vascular endothelium is characterized by a remarkable level of plasticity, which is the driving force not only of physiological repair/remodeling of adult tissues but also of pathological angiogenesis. The resulting heterogeneity of endothelial cells (ECs) makes targeting the endothelium challenging, no less because many EC phenotypes are yet to be identified and functionally inventorized. Efforts to map the vasculature at the single-cell level have been instrumental to capture the diversity of EC types and states at a remarkable depth in both normal and pathological states. Here, we discuss new EC subtypes and functions emerging from recent single-cell studies in health and disease. Interestingly, such studies revealed distinct metabolic gene signatures in different EC phenotypes, which deserve further consideration for therapy. We highlight how this metabolic targeting strategy could potentially be used to promote (for tissue repair) or block (in tumor) angiogenesis in a tissue or even vascular bed-specific manner. ispartof: ANGIOGENESIS vol:24 issue:2 pages:311-326 ispartof: location:Germany status: published

Although long non-coding RNAs (lncRNAs) are non-protein-coding transcripts by definition, recent studies have shown that a fraction of putative small open reading frames within lncRNAs are translated. However, the biological significance of these hidden polypeptides is still unclear. Here we identify and functionally characterize a novel polypeptide encoded by the lncRNA LINC00961. This polypeptide is conserved between human and mouse, is localized to the late endosome/lysosome and interacts with the lysosomal v-ATPase to negatively regulate mTORC1 activation. This regulation of mTORC1 is specific to activation of mTORC1 by amino acid stimulation, rather than by growth factors. Hence, we termed this polypeptide â small regulatory polypeptide of amino acid response' (SPAR). We show that the SPAR-encoding lncRNA is highly expressed in a subset of tissues and use CRISPR/Cas9 engineering to develop a SPAR-polypeptide-specific knockout mouse while maintaining expression of the host lncRNA. We find that the SPAR-encoding lncRNA is downregulated in skeletal muscle upon acute injury, and using this in vivo model we establish that SPAR downregulation enables efficient activation of mTORC1 and promotes muscle regeneration. Our data provide a mechanism by which mTORC1 activation may be finely regulated in a tissue-specific manner in response to injury, and a paradigm by which lncRNAs encoding small polypeptides can modulate general biological pathways and processes to facilitate tissue-specific requirements, consistent with their restricted and highly regulated expression profile.

Oxidation-reduction chemistry is fundamental to the metabolism of all living organisms, and hence quantifying the principal redox players is important for a comprehensive understanding of cell metabolism in normal and pathological states. In mammalian cells, this is accomplished by measuring oxygen partial pressure (pO2) in parallel with free and enzyme-bound reduced nicotinamide adenine dinucleotide (phosphate) [H] (NAD(P)H) and flavin adenine dinucleotide (FAD, a proxy for NAD+). Previous optical methods for these measurements had accompanying problems of cytotoxicity, slow speed, population averaging, and inability to measure all redox parameters simultaneously. Herein we present a Förster resonance energy transfer (FRET)-based oxygen sensor, Myoglobin-mCherry, compatible with fluorescence lifetime imaging (FLIM)-based measurement of nicotinamide coenzyme state. This offers a contemporaneous reading of metabolic activity through real-time, non-invasive, cell-by-cell intracellular pO2 and coenzyme status monitoring in living cells. Additionally, this method reveals intracellular spatial heterogeneity and cell-to-cell variation in oxygenation and coenzyme states., Graphical abstract Image 1, Highlights • A new method for concurrent oxygen and metabolic cofactor imaging. • This method quantifies the relationship between metabolism and changing oxygenation. • Optical and fluorescence lifetime redox ratios versus intracellular oxygenation can infer shifts in metabolic behavior. • Tandem oxygen and metabolic cofactor imaging shows intracellular heterogeneity. • Response to changing oxygenation and metabolism between cells and cell lines is heterogeneous.

On the basis of emerging evidence from patients with COVID-19, we postulate that endothelial cells are essential contributors to the initiation and propagation of severe COVID-19. Here, we discuss current insights into the link between endothelial cells, viral infection and inflammatory changes and propose novel therapeutic strategies., Here, Carmeliet and colleagues discuss the role of endothelial cells in inflammation and viral infection and propose novel therapeutic strategies for COVID-19.

SummaryBrown adipose tissue (BAT) is an energy-dispensing thermogenic tissue that plays an important role in balancing energy metabolism. Lineage-tracing experiments indicate that brown adipocytes are derived from myogenic progenitors during embryonic development. However, adult skeletal muscle stem cells (satellite cells) have long been considered uniformly determined toward the myogenic lineage. Here, we report that adult satellite cells give rise to brown adipocytes and that microRNA-133 regulates the choice between myogenic and brown adipose determination by targeting the 3′UTR of Prdm16. Antagonism of microRNA-133 during muscle regeneration increases uncoupled respiration, glucose uptake, and thermogenesis in local treated muscle and augments whole-body energy expenditure, improves glucose tolerance, and impedes the development of diet-induced obesity. Finally, we demonstrate that miR-133 levels are downregulated in mice exposed to cold, resulting in de novo generation of satellite cell-derived brown adipocytes. Therefore, microRNA-133 represents an important therapeutic target for the treatment of obesity.

The expansive dimension of non-coding genes is by now a well-recognized feature of eukaryotes genomes. Over the past decades, in vitro functional studies and in vivo manipulation of non-coding genes through Genetically Engineered Mouse Models (GEMMs) have provided compelling evidence that almost every biological phenomenon is regulated, at some level, by non-coding RNA transcripts or by coding RNAs with non-coding functions. In this opinion article, we will discuss how recent discoveries in the field of non-coding RNAs are contributing to advance our understanding of evolution and organismal complexity and its relevance to human diseases.

Background Mini-invasive techniques for gastroesophageal reflux disease (GERD), such as endoscopic injections of inert materials, have been introduced in recent years. However, results are still preliminary. Cell injection has emerged as an alternative strategy in both vesicoureteral reflux and incontinence. Here we report, for the first time, the injection of muscle precursor cells (MPCs) in the gastroesophageal junction (GEJ). Materials and methods MPCs were derived from expanded satellite cells isolated from skeletal muscle fibers of green fluorescent protein (GFP) positive mice. Via laparotomy, GFP-negative mice were subjected to cryoinjury of GEJ followed by injection of MPCs (experimental animals), bone marrow derived cells, or saline (controls). Results Immunofluorescence analyses of experimental GEJs demonstrated coexpression of GFP and desmin in grafted cells. GFP+ muscle neofibers were evident at 4 wk after injection. Coexpression of GFP and smooth muscle actin was also observed at 2 wk. Conclusions Satellite cells could be easily harvested, expanded in culture, and used as injectable substance in the GEJ. These results could be the background for the development of a new injection technique for GERD treatment, which might combine bulging and functional actions.

Dystrophin is expressed in differentiated myofibers, in which it is required for sarcolemmal integrity, and loss-of-function mutations in the gene that encodes it result in Duchenne muscular dystrophy (DMD), a disease characterized by progressive and severe skeletal muscle degeneration. Here we found that dystrophin is also highly expressed in activated muscle stem cells (also known as satellite cells), in which it associates with the serine-threonine kinase Mark2 (also known as Par1b), an important regulator of cell polarity. In the absence of dystrophin, expression of Mark2 protein is downregulated, resulting in the inability to localize the cell polarity regulator Pard3 to the opposite side of the cell. Consequently, the number of asymmetric divisions is strikingly reduced in dystrophin-deficient satellite cells, which also display a loss of polarity, abnormal division patterns (including centrosome amplification), impaired mitotic spindle orientation and prolonged cell divisions. Altogether, these intrinsic defects strongly reduce the generation of myogenic progenitors that are needed for proper muscle regeneration. Therefore, we conclude that dystrophin has an essential role in the regulation of satellite cell polarity and asymmetric division. Our findings indicate that muscle wasting in DMD not only is caused by myofiber fragility, but also is exacerbated by impaired regeneration owing to intrinsic satellite cell dysfunction.

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved (1). In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation (2, 3). The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions (3-6). The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation (7). Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles (8, 9) so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium (1-3). This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process (1-3). The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Satellite cells are a heterogeneous population of muscle progenitors with stem cell properties responsible for the regeneration of adult skeletal muscle. Increasing interest in the therapeutic potential of satellite cells has challenged researchers with the need to purify a homogenous population of muscle progenitors. Here we provide a detailed protocol for the isolation of a pure population of satellite cells using fluorescence activated cell sorting. We give specific guidelines to ameliorate the reproducibility of the satellite cell isolation protocol with the goal to standardize procedures across labs. This protocol identifies satellite cells within adult skeletal muscle as an enriched population of Integrin α7(+)/CD34(+) double positive cells and CD45, CD31, CD11b, and Sca1 negative (Lin(-)) cells (Integrin α7(+)/CD34(+)/Lin(-)). Functional assay shows that Integrin α7(+)/CD34(+)/Lin(-) satellite cells possess high myogenic potential and ability to regenerate muscle depleted satellite cells upon transplantation.

Adult skeletal muscle regenerates in response to traumatic injuries or degenerative conditions. This property is afforded mainly by satellite cells (SCs), a heterogeneous population of resident committed myogenic progenitors and noncommitted stem cells (Sherwood et al. 2004; Collins et al. 2005; Montarras et al. 2005; Kuang et al. 2007). In the mouse, postnatal SCs are mitotically active for the initial 2 wk after birth. After this period, they enter quiescence and their number declines. However, following muscle injury or degeneration, adult SCs undergo intense proliferation and efficiently differentiate. To replenish the reservoir, a subset of dividing SCs returns to the niche following a process of asymmetric and symmetric cell division (Shinin et al. 2006; Conboy et al. 2007; Kuang et al. 2007; Shea et al. 2010). Approximately 10% of noncommitted Pax7+/Myf5− SCs can asymmetrically generate a self-renewing, noncommitted Pax7+/Myf5− cell and a committed Pax7+/Myf5+ daughter cell in vivo. The noncommitted Pax7+/Myf5− cell returns to the niche to maintain the SC reservoir, while the committed Pax7+/Myf5+ SC undergoes several rounds of cell division and the ensuing cells eventually differentiate into pre-existing or newly formed myofibers (Kuang et al. 2008). Polycomb group (PcG) proteins regulate differentiation of totipotent embryonic stem (ES) cells and maintenance of multipotent and progenitor stem cell populations (Sauvageau and Sauvageau 2010). The Polycomb-repressive complex 2 (PRC2) subunit EZH2 methylates histone H3 Lys 27 (H3K27me3), establishing an epigenetic mark that identifies repressed chromatin regions. Ablation of PRC2 members in ES cells impairs their differentiation (Pasini et al. 2007; Chamberlain et al. 2008; Shen et al. 2008) and results in unscheduled expression of mixed cell lineage genes (Boyer et al. 2006; Lee et al. 2006). While PcG establishes and maintains positional patterning of the body axis through regulation of Hox genes in both Drosophila and mammals, its role in conferring cell identity by repressing inappropriate cell lineage-specific transcription in animal development has not been demonstrated. Indeed, derepression of mixed cell lineage genes does not occur in epidermal, neuronal, or pancreatic cells of Ezh2 conditional null mice (Chen et al. 2009; Ezhkova et al. 2009; Hirabayashi et al. 2009). We generated mice in which Ezh2 was conditionally ablated in SCs (Ezh2 muscle knockout, Ezh2mKO). While EZH2 was dispensable for fetal muscle development, it was required for postnatal muscle growth and adult muscle regeneration, ensuring appropriate homeostasis of the SC pool. Unlike other progenitor cells, reduced H3K27me3 in Ezh2mKO SCs was accompanied by RNA polymerase II (PolII) recruitment and transcriptional activation of genes normally repressed in SCs and expressed in other cell lineages, including cardiac progenitors, retinal cones, neurons, and chondrocytes. Thus, our findings indicate that EZH2, which regulates proliferation and maintains transcriptional identity of adult muscle stem cells, is an important molecular component of adult skeletal myogenesis.

The in vitro development of human myotubes carrying genetic diseases, such as Duchenne Muscular Dystrophy, will open new perspectives in the identification of innovative therapeutic strategies. Through the proper design of the substrate, we guided the differentiation of human healthy and dystrophic myoblasts into myotubes exhibiting marked functional differentiation and highly defined sarcomeric organization. A thin film of photo cross-linkable elastic poly-acrylamide hydrogel with physiological-like and tunable mechanical properties (elastic moduli, E: 12, 15, 18 and 21 kPa) was used as substrate. The functionalization of its surface by micro-patterning in parallel lanes (75 microm wide, 100 microm spaced) of three adhesion proteins (laminin, fibronectin and matrigel) was meant to maximize human myoblasts fusion. Myotubes formed onto the hydrogel showed a remarkable sarcomere formation, with the highest percentage (60.0% +/- 3.8) of myotubes exhibiting sarcomeric organization, of myosin heavy chain II and alpha-actinin, after 7 days of culture onto an elastic (15 kPa) hydrogel and a matrigel patterning. In addition, healthy myotubes cultured in these conditions showed a significant membrane-localized dystrophin expression. In this study, the culture substrate has been adapted to human myoblasts differentiation, through an easy and rapid methodology, and has led to the development of in vitro human functional skeletal muscle myotubes useful for clinical purposes and in vitro physiological study, where to carry out a broad range of studies on human muscle physiopathology.

Objective: Autologous cell transplantation has been proposed as a possible therapeutic approach for Duchenne dystrophy. In this approach, patients' muscle precursor cells (mpcs) obtained from muscle biopsies would be expanded ex vivo, genetically modified to restore dystrophin expression and then re-implanted in the original donor. Such strategy would have the advantage of bypassing the immune response problem, but on the other hand, it would require a large number of cells because of the poor viability and mobility of transplanted myoblasts. Besides, extensive multiplication of mpcs is difficult and can affect their myogenic ability. Given the key role of inflammation in muscle regeneration, we set out to verify if factors secreted by inflammatory cells could be used to improve in vitro expansion of DMD-mpcs.Methods: We have previously shown that a murine macrophage conditioned medium (mMCM) could increase the in vitro proliferation rate of rat and mouse mpcs. Here we tested the effect of mMCM on cultures of human, dystrophin-deficient mpcs (DMD-mpcs).Results: In the presence of mMCM, DMD-mpcs displayed an increased proliferation rate, while at the same time, maintaining their myogenicity after many in vitro passages. Expanded cells were also injected in muscles of immuno-deficient mice, showing that they were also able to participate in muscle regeneration within recipient muscles.Discussion: Using macrophagic factors, we were able to increase the amount of DMD-mpcs obtainable after 38 days of culture by >7x10(3) -fold. These findings indicate that macrophagic factors hold great potential for future use in cell transplantation protocol.

Skeletal muscle regeneration relies on satellite cells, a population of myogenic precursors. Inflammation also plays a determinant role in the process, as upon injury, macrophages are attracted by the damaged myofibers and the activated satellite cells and act as key elements of dynamic muscle supportive stroma. Yet, it is not known how macrophages interact with the more profound stem cells of the satellite cell niche. Here we show that in the presence of a murine macrophage conditioned medium (mMCM) a subpopulation of multipotent cells could be selected and expanded from adult rat muscle. These cells were small, round, poorly adhesive, slow-growing and showed mesenchymal differentiation plasticity. At the same time, mMCM showed clear myogenic capabilities, as experiments with satellite cells mechanically isolated from suspensions of single myofibers showed that the macrophagic factors inhibited their tendency to shift towards adipogenesis. In vivo, intramuscular administrations of concentrated mMCM in a rat model of extensive surgical ablation dramatically improved muscle regeneration. Altogether, these findings suggest that macrophagic factors could be of great help in developing therapeutic protocols with myogenic stem cells.

SummaryPax7 is a nodal transcription factor that is essential for regulating the maintenance, expansion, and myogenic identity of satellite cells during both neonatal and adult myogenesis. Deletion of Pax7 results in loss of satellite cells and impaired muscle regeneration. Here, we show that ectopic expression of the constitutively active intracellular domain of Notch1 (NICD1) rescues the loss of Pax7-deficient satellite cells and restores their proliferative potential. Strikingly NICD1-expressing satellite cells do not undergo myogenic differentiation and instead acquire a brown adipogenic fate both in vivo and in vitro. NICD-expressing Pax7−/− satellite cells fail to upregulate MyoD and instead express the brown adipogenic marker PRDM16. Overall, these results show that Notch1 activation compensates for the loss of Pax7 in the quiescent state and acts as a molecular switch to promote brown adipogenesis in adult skeletal muscle.

On the basis of emerging evidence from patients with COVID-19, we postulate that endothelial cells are essential contributors to the initiation and propagation of severe COVID-19. Here, we discuss current insights into the link between endothelial cells, viral infection and inflammatory changes and propose novel therapeutic strategies. Here, Carmeliet and colleagues discuss the role of endothelial cells in inflammation and viral infection and propose novel therapeutic strategies for COVID-19.

The use of alternative polyadenylation sites is emerging as an important regulator of gene expression. In this issue of Cell Stem Cell, Boutet et al. (2012) report that alternative 3′UTRs of the Pax3 transcript restrict its expression to axial satellite cells through miR-mediated targeting of one of the isoforms.