RESUMEN
Skeletal muscle is the largest tissue of the body and performs multiple functions, from locomotion to body temperature control. Its functionality and recovery from injuries depend on a multitude of cell types and on molecular signals between the core muscle cells (myofibers, muscle stem cells) and their niche. Most experimental settings do not preserve this complex physiological microenvironment, and neither do they allow the ex vivo study of muscle stem cells in quiescence, a cell state that is crucial for them. Here, a protocol is outlined for the ex vivo culture of muscle stem cells with cellular components of their niche. Through the mechanical and enzymatic breakdown of muscles, a mixture of cell types is obtained, which is put in 2D culture. Immunostaining shows that within 1 week, multiple niche cells are present in culture alongside myofibers and, importantly, Pax7-positive cells that display the characteristics of quiescent muscle stem cells. These unique properties make this protocol a powerful tool for cell amplification and the generation of quiescent-like stem cells that can be used to address fundamental and translational questions.
Asunto(s)
Músculo Esquelético , Células Satélite del Músculo Esquelético , Ratones , Animales , Diferenciación Celular , División Celular , Células Madre , Nicho de Células Madre/fisiologíaRESUMEN
Short-term fasting is beneficial for the regeneration of multiple tissue types. However, the effects of fasting on muscle regeneration are largely unknown. Here, we report that fasting slows muscle repair both immediately after the conclusion of fasting as well as after multiple days of refeeding. We show that ketosis, either endogenously produced during fasting or a ketogenic diet or exogenously administered, promotes a deep quiescent state in muscle stem cells (MuSCs). Although deep quiescent MuSCs are less poised to activate, slowing muscle regeneration, they have markedly improved survival when facing sources of cellular stress. Furthermore, we show that ketone bodies, specifically ß-hydroxybutyrate, directly promote MuSC deep quiescence via a nonmetabolic mechanism. We show that ß-hydroxybutyrate functions as an HDAC inhibitor within MuSCs, leading to acetylation and activation of an HDAC1 target protein p53. Finally, we demonstrate that p53 activation contributes to the deep quiescence and enhanced resilience observed during fasting.
Asunto(s)
Ayuno , Proteína p53 Supresora de Tumor , Ácido 3-Hidroxibutírico , Ayuno/fisiología , Músculos , MioblastosRESUMEN
Single-nucleus RNA sequencing allows the profiling of gene expression in isolated nuclei. Here, we describe a step-by-step protocol optimized for adult mouse skeletal muscles. This protocol provides two main advantages compared to the widely used single-cell protocol. First, it allows us to sequence the myonuclei of the multinucleated myofibers. Second, it circumvents the cell-dissociation-induced transcriptional modifications. For complete details on the use and execution of this protocol, please refer to Dos Santos et al. (2020) and Machado, Geara et al. (2021).
Asunto(s)
Núcleo Celular/genética , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Animales , Técnicas de Cultivo de Célula/métodos , Separación Celular/métodos , Ratones , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citología , ARN/química , ARN/aislamiento & purificación , Células Satélite del Músculo Esquelético/metabolismoRESUMEN
The rapid progress of single-cell RNA-sequencing (scRNA-seq) at large scales has led to what seemed impossible until recently: the generation of comprehensive transcriptional maps of nearly all cells in multicellular tissues. We pinpoint three key elements as being critical to the production of these maps: scalability, spatial information, and accuracy of the transcriptome of the individual cells. Here, we discuss the ramifications of traditional cell-isolation protocols when capturing the transcriptional signature of cells as they exist in their native tissue context, the methods that have been developed to avoid these distortions, and the biological processes that have unraveled on account of these upgraded methodological approaches.
Asunto(s)
Artefactos , Análisis de la Célula Individual , Perfilación de la Expresión Génica/métodos , Humanos , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , TranscriptomaRESUMEN
The epigenetic mechanisms coordinating the maintenance of adult cellular lineages and the inhibition of alternative cell fates remain poorly understood. Here we show that targeted ablation of the histone chaperone HIRA in myogenic cells leads to extensive transcriptional modifications, consistent with a role in maintaining skeletal muscle cellular identity. We demonstrate that conditional ablation of HIRA in muscle stem cells of adult mice compromises their capacity to regenerate and self-renew, leading to tissue repair failure. Chromatin analysis of Hira-deficient cells show a significant reduction of histone variant H3.3 deposition and H3K27ac modification at regulatory regions of muscle genes. Additionally, we find that genes from alternative lineages are ectopically expressed in Hira-mutant cells via MLL1/MLL2-mediated increase of H3K4me3 mark at silent promoter regions. Therefore, we conclude that HIRA sustains the chromatin landscape governing muscle cell lineage identity via incorporation of H3.3 at muscle gene regulatory regions, while preventing the expression of alternative lineage genes.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Linaje de la Célula , Chaperonas de Histonas/metabolismo , Músculo Esquelético/patología , Factores de Transcripción/metabolismo , Acetilación , Animales , Proteínas de Ciclo Celular/deficiencia , Línea Celular , Linaje de la Célula/genética , Sitios Genéticos , Chaperonas de Histonas/deficiencia , Histonas/metabolismo , Lisina/metabolismo , Ratones , Desarrollo de Músculos/genética , Músculo Esquelético/lesiones , Músculo Esquelético/fisiopatología , Regeneración , Secuencias Reguladoras de Ácidos Nucleicos/genética , Células Satélite del Músculo Esquelético/metabolismo , Factores de Transcripción/deficienciaRESUMEN
Tissue damage dramatically alters how cells interact with their microenvironment. These changes in turn dictate cellular responses, such as stem cell activation, yet early cellular responses in vivo remain ill defined. We generated single-cell and nucleus atlases from intact, dissociated, and injured muscle and liver and identified a common stress response signature shared by multiple cell types across these organs. This prevalent stress response was detected in published datasets across a range of tissues, demonstrating high conservation but also a significant degree of data distortion in single-cell reference atlases. Using quiescent muscle stem cells as a paradigm of cell activation following injury, we captured early cell activation following muscle injury and found that an essential ERK1/2 primary proliferation signal precedes initiation of the Notch-regulated myogenic program. This study defines initial events in response to tissue perturbation and identifies a broadly conserved transcriptional stress response that acts in parallel with cell-specific adaptive alterations.
Asunto(s)
Células Satélite del Músculo Esquelético , Proliferación Celular , Desarrollo de Músculos , Músculos , Células MadreRESUMEN
Duchenne muscular dystrophy (DMD) is a rare, muscle degenerative disease resulting from the absence of the dystrophin protein. DMD is characterized by progressive loss of muscle fibers, muscle weakness, and eventually loss of ambulation and premature death. Currently, there is no cure for DMD and improved methods of disease monitoring are crucial for the development of novel treatments. In this study, we describe a new method of assessing disease progression noninvasively in the mdx model of DMD. The reporter mice, which we term the dystrophic Degeneration Reporter strains, contain an inducible CRE-responsive luciferase reporter active in mature myofibers. In these mice, muscle degeneration is reflected in changes in the level of luciferase expression, which can be monitored using noninvasive, bioluminescence imaging. We monitored the natural history and disease progression in these dystrophic report mice and found that decreases in luciferase signals directly correlated with muscle degeneration. We further demonstrated that this reporter strain, as well as a previously reported Regeneration Reporter strain, successfully reveals the effectiveness of a gene therapy treatment following systemic administration of a recombinant adeno-associated virus-6 (rAAV-6) encoding a microdystrophin construct. Our data demonstrate the value of these noninvasive imaging modalities for monitoring disease progression and response to therapy in mouse models of muscular dystrophy.
Asunto(s)
Dependovirus , Distrofina , Terapia Genética , Fibras Musculares Esqueléticas , Distrofia Muscular de Duchenne , Transducción Genética , Animales , Distrofina/biosíntesis , Distrofina/genética , Humanos , Ratones , Ratones Transgénicos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/fisiopatología , Distrofia Muscular de Duchenne/terapiaRESUMEN
The cell microenvironment, which is critical for stem cell maintenance, contains both cellular and non-cellular components, including secreted growth factors and the extracellular matrix1-3. Although Notch and other signalling pathways have previously been reported to regulate quiescence of stem cells4-9, the composition and source of molecules that maintain the stem cell niche remain largely unknown. Here we show that adult muscle satellite (stem) cells in mice produce extracellular matrix collagens to maintain quiescence in a cell-autonomous manner. Using chromatin immunoprecipitation followed by sequencing, we identified NOTCH1/RBPJ-bound regulatory elements adjacent to specific collagen genes, the expression of which is deregulated in Notch-mutant mice. Moreover, we show that Collagen V (COLV) produced by satellite cells is a critical component of the quiescent niche, as depletion of COLV by conditional deletion of the Col5a1 gene leads to anomalous cell cycle entry and gradual diminution of the stem cell pool. Notably, the interaction of COLV with satellite cells is mediated by the Calcitonin receptor, for which COLV acts as a surrogate local ligand. Systemic administration of a calcitonin derivative is sufficient to rescue the quiescence and self-renewal defects found in COLV-null satellite cells. This study reveals a Notch-COLV-Calcitonin receptor signalling cascade that maintains satellite cells in a quiescent state in a cell-autonomous fashion, and raises the possibility that similar reciprocal mechanisms act in diverse stem cell populations.
Asunto(s)
Proteína Similar al Receptor de Calcitonina/metabolismo , Colágeno/metabolismo , Músculo Esquelético/citología , Receptores Notch/metabolismo , Células Satélite del Músculo Esquelético/citología , Células Satélite del Músculo Esquelético/metabolismo , Transducción de Señal , Nicho de Células Madre , Animales , Diferenciación Celular , Proliferación Celular , Autorrenovación de las Células , Colágeno/genética , Regulación de la Expresión Génica , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Ratones , Transcripción GenéticaRESUMEN
State of the art techniques have been developed to isolate and analyze cells from various tissues, aiming to capture their in vivo state. However, the majority of cell isolation protocols involve lengthy mechanical and enzymatic dissociation steps followed by flow cytometry, exposing cells to stress and disrupting their physiological niche. Focusing on adult skeletal muscle stem cells, we have developed a protocol that circumvents the impact of isolation procedures and captures cells in their native quiescent state. We show that current isolation protocols induce major transcriptional changes accompanied by specific histone modifications while having negligible effects on DNA methylation. In addition to proposing a protocol to avoid isolation-induced artifacts, our study reveals previously undetected quiescence and early activation genes of potential biological interest.
Asunto(s)
Código de Histonas , Mioblastos Esqueléticos/metabolismo , Cultivo Primario de Células/métodos , Animales , Células Cultivadas , Metilación de ADN , Femenino , Ratones , Mioblastos Esqueléticos/citología , Cultivo Primario de Células/normas , Fijación del Tejido/métodos , TranscriptomaRESUMEN
In this report, Boonsanay and colleagues describe a novel mechanism of maintenance of skeletal muscle stem cells [also known as satellite cells (SCs)] quiescence via the di-methyltransferase Suv4-20h1, regulator of heterochromatin formation. Conditional ablation of Suv4-20h1 in SCs leads notably to the loss of the histone modification H4K20me2 on the distal regulatory element of Myod combined with a relocation of the Myod locus toward a central position in the nucleus. This switch in nuclear compartment is correlated with decreased facultative H3K27me3 associated heterochromatin, and an increase in spontaneously activated MYOD-expressing SCs in homeostatic muscles. Consequently, Suv4-20h1 knock-out SCs demonstrate compromised stem cell potential, as they fail to efficiently self-renew and replenish the SC pool upon muscle injury. Strikingly, restoring MYOD expression alone rescues the levels of facultative chromatin and reverses the loss-of-quiescence phenotype.