Protocol for Isolation and Characterization of In Situ Fixed Quiescent Muscle Stem Cells.
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ABSTRACT:
SUBMITTER: Yue L
PROVIDER: S-EPMC7757111 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Muscle stem cells (MuSCs) persist in a quiescent state and activate in response to specific stimuli. The quiescent state is both actively maintained and dynamically regulated. However, analyses of quiescence have come primarily from cells removed from their niche. Although these cells are still quiescent, biochemical changes certainly occur during the isolation process. Here, we analyze the transcriptome of MuSCs in vivo utilizing MuSC-specific labeling of RNA. Notably, labeling transcripts during the isolation procedure revealed very active transcription of specific subsets of genes. However, using the transcription inhibitor ?-amanitin, we show that the ex vivo transcriptome remains largely reflective of the in vivo transcriptome. Together, these data provide perspective on the molecular regulation of the quiescent state at the transcriptional level, demonstrate the utility of these tools for probing transcriptional dynamics in vivo, and provide an invaluable resource for understanding stem cell state transitions.
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Project description:Quiescence regulation is essential for adult stem cell maintenance and sustained regeneration. Our studies uncovered that physiological changes in mitochondrial shape regulate the quiescent state of adult muscle stem cells (MuSCs). We show that MuSC mitochondria rapidly fragment upon an activation stimulus, via systemic HGF/mTOR, to drive the exit from deep quiescence. Deletion of the mitochondrial fusion protein OPA1 and mitochondrial fragmentation transitions MuSCs into G-alert quiescence, causing premature activation and depletion upon a stimulus. OPA1 loss activates a glutathione (GSH)-redox signaling pathway promoting cell-cycle progression, myogenic gene expression, and commitment. MuSCs with chronic OPA1 loss, leading to mitochondrial dysfunction, continue to reside in G-alert but acquire severe cell-cycle defects. Additionally, we provide evidence that OPA1 decline and impaired mitochondrial dynamics contribute to age-related MuSC dysfunction. These findings reveal a fundamental role for OPA1 and mitochondrial dynamics in establishing the quiescent state and activation potential of adult stem cells.
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Project description:Muscle stem cells (MuSCs) are involved in homeostatic maintenance of skeletal muscle and play a central role in muscle regeneration in response to injury. Thus, understanding MuSC autonomous properties is of fundamental importance for studies of muscle degenerative diseases and muscle plasticity. Rat, as an animal model, has been widely used in the skeletal muscle field, however rat MuSC isolation through fluorescence-activated cell sorting has never been described. This work validates a protocol for effective MuSC isolation from rat skeletal muscles. Tibialis anterior was harvested from female rats and digested for isolation of MuSCs. Three protocols, employing different cell surface markers (CD106, CD56, and CD29), were compared for their ability to isolate a highly enriched MuSC population. Cells isolated using only CD106 as a positive marker showed high expression of Pax7, ability to progress through myogenic lineage while in culture, and complete differentiation in serum-deprived conditions. The protocol was further validated in gastrocnemius, diaphragm, and the individual components of the pelvic floor muscle complex (coccygeus, iliocaudalis, and pubocaudalis), proving to be reproducible. CD106 is an efficient marker for reliable isolation of MuSCs from a variety of rat skeletal muscles.
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Project description:Tissue stem cell niches are regulated by their mechanical environment, notably the extracellular matrix (ECM). Skeletal muscles consist of bundled myofibers for force transmission. Within this macroscopic architecture, quiescent Pax7-expressing (Pax7+) muscle stem cells (MuSCs) are compressed between ECM basally and myofiber apically. Muscle injury causes MuSCs to lose apical compression from the myofiber and re-enter the cell cycle for regeneration. While ECM elasticities have been shown to affect MuSC's renewal, the significance of apical compression remains unknown. To investigate the role of apical compression, we simulate the MuSCs' in vivo mechanical environment by applying physical compression to MuSCs' apical surface. We demonstrate that compression drives activated MuSCs back to a quiescent stem cell state, regardless of basal elasticities and chemistries. By mathematical modeling and cell tension manipulation, we conclude that low overall tension combined with high axial tension generated by compression leads to MuSCs' stemness and quiescence. Unexpectedly, we discovered that apical compression results in up-regulation of Notch downstream genes, accompanied by the increased levels of nuclear Notch1&3 in a Delta ligand (Dll) and ADAM10/17 independent manner. Our results fill a knowledge gap on the role of apical compression for MuSC fate and have implications to stem cells in other tissues.
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Project description:Satellite cells (SCs) reside in a dormant state during tissue homeostasis. The specific paracrine agents and niche cells that maintain SC quiescence remain unknown. We find that Wnt4 produced by the muscle fiber maintains SC quiescence through RhoA. Using cell-specific inducible genetics, we find that a Wnt4-Rho signaling axis constrains SC numbers and activation during tissue homeostasis in adult mice. Wnt4 activates Rho in quiescent SCs to maintain mechanical strain, restrict movement in the niche, and repress YAP. The induction of YAP upon disruption of RhoA is essential for SC activation under homeostasis. In the context of injury, the loss of Wnt4 from the niche accelerates SC activation and muscle repair, whereas overexpression of Wnt4 transitions SCs into a deeper state of quiescence and delays muscle repair. In conclusion, the SC pool undergoes dynamic transitions during early activation with changes in mechano-properties and cytoskeleton signaling preceding cell-cycle entry.
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Project description:Aging impairs tissue repair. This is pronounced in skeletal muscle, whose regeneration by muscle stem cells (MuSCs) is robust in young adult animals but inefficient in older organisms. Despite this functional decline, old MuSCs are amenable to rejuvenation through strategies that improve the systemic milieu, such as heterochronic parabiosis. One such strategy, exercise, has long been appreciated for its benefits on healthspan, but its effects on aged stem cell function in the context of tissue regeneration are incompletely understood. Here we show that exercise in the form of voluntary wheel running accelerates muscle repair in old animals and improves old MuSC function. Through transcriptional profiling and genetic studies, we discovered that the restoration of old MuSC activation ability hinges on restoration of Cyclin D1, whose expression declines with age in MuSCs. Pharmacologic studies revealed that Cyclin D1 maintains MuSC activation capacity by repressing TGFβ signaling. Taken together, these studies demonstrate that voluntary exercise is a practicable intervention for old MuSC rejuvenation. Furthermore, this work highlights the distinct role of Cyclin D1 in stem cell quiescence.
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Project description:The ability to isolate prostate stem cells is essential to explore their role in prostate development and disease. In vitro prostate colony- and sphere-forming assays were used to quantitatively measure murine prostate stem/progenitor cell enrichment and self-renewal. Cell surface markers were screened for their ability to positively or negatively enrich for cells with enhanced growth potential in these assays. Immunohistochemical and FACS analyses demonstrate that specific cell surface markers can be used to discriminate prostate stromal (CD34(+)), luminal epithelial (CD24(+)CD49f(-)), basal epithelial (CD24(+)CD49f(+)), hematopoietic (CD45(+), Ter119(+)), and endothelial (CD31(+)) lineages. Sorting for cells with a CD45(-)CD31(-)Ter119(-)Sca-1(+)CD49f(+) antigenic profile results in a 60-fold enrichment for colony- and sphere-forming cells. These cells can self-renew and expand to form spheres for many generations and can differentiate to produce prostatic tubule structures containing both basal and luminal cells in vivo. These cells also localize to the basal cell layer within the region of the gland that is proximal to the urethra, which has been identified as the prostate stem cell niche. Prostate stem cells can be isolated to a purity of up to 1 in 35 by using this antigenic profile. The remarkable similarity in cell surface profile between prostate and mammary gland stem cells suggests these markers may be conserved among epithelial stem cell populations.
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Project description: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 β-hydroxybutyrate, directly promote MuSC deep quiescence via a nonmetabolic mechanism. We show that β-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.
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