Regulation of adult stem cell quiescence and its functions in the maintenance of tissue integrity.

Adult stem cells are important for mammalian tissues, where they act as a cell reserve that supports normal tissue turnover and can mount a regenerative response following acute injuries. Quiescent stem cells are well established in certain tissues, such as skeletal muscle, brain, and bone marrow. The quiescent state is actively controlled and is essential for long-term maintenance of stem cell pools. In this Review, we discuss the importance of maintaining a functional pool of quiescent adult stem cells, including haematopoietic stem cells, skeletal muscle stem cells, neural stem cells, hair follicle stem cells, and mesenchymal stem cells such as fibro-adipogenic progenitors, to ensure tissue maintenance and repair. We discuss the molecular mechanisms that regulate the entry into, maintenance of, and exit from the quiescent state in mice. Recent studies revealed that quiescent stem cells have a discordance between RNA and protein levels, indicating the importance of post-transcriptional mechanisms, such as alternative polyadenylation, alternative splicing, and translation repression, in the control of stem cell quiescence. Understanding how these mechanisms guide stem cell function during homeostasis and regeneration has important implications for regenerative medicine.

Ageing and rejuvenation of tissue stem cells and their niches.

Most adult organs contain regenerative stem cells, often organized in specific niches. Stem cell function is critical for tissue homeostasis and repair upon injury, and it is dependent on interactions with the niche. During ageing, stem cells decline in their regenerative potential and ability to give rise to differentiated cells in the tissue, which is associated with a deterioration of tissue integrity and health. Ageing-associated changes in regenerative tissue regions include defects in maintenance of stem cell quiescence, differentiation ability and bias, clonal expansion and infiltration of immune cells in the niche. In this Review, we discuss cellular and molecular mechanisms underlying ageing in the regenerative regions of different tissues as well as potential rejuvenation strategies. We focus primarily on brain, muscle and blood tissues, but also provide examples from other tissues, such as skin and intestine. We describe the complex interactions between different cell types, non-cell-autonomous mechanisms between ageing niches and stem cells, and the influence of systemic factors. We also compare different interventions for the rejuvenation of old regenerative regions. Future outlooks in the field of stem cell ageing are discussed, including strategies to counter ageing and age-dependent disease.

Geroscience: linking aging to chronic disease.

Mammalian aging can be delayed with genetic, dietary, and pharmacologic approaches. Given that the elderly population is dramatically increasing and that aging is the greatest risk factor for a majority of chronic diseases driving both morbidity and mortality, it is critical to expand geroscience research directed at extending human healthspan.

H3K4me3 breadth is linked to cell identity and transcriptional consistency.

Trimethylation of histone H3 at lysine 4 (H3K4me3) is a chromatin modification known to mark the transcription start sites of active genes. Here, we show that H3K4me3 domains that spread more broadly over genes in a given cell type preferentially mark genes that are essential for the identity and function of that cell type. Using the broadest H3K4me3 domains as a discovery tool in neural progenitor cells, we identify novel regulators of these cells. Machine learning models reveal that the broadest H3K4me3 domains represent a distinct entity, characterized by increased marks of elongation. The broadest H3K4me3 domains also have more paused polymerase at their promoters, suggesting a unique transcriptional output. Indeed, genes marked by the broadest H3K4me3 domains exhibit enhanced transcriptional consistency and [corrected] increased transcriptional levels, and perturbation of H3K4me3 breadth leads to changes in transcriptional consistency. Thus, H3K4me3 breadth contains information that could ensure transcriptional precision at key cell identity/function genes.

Type 2 innate signals stimulate fibro/adipogenic progenitors to facilitate muscle regeneration.

In vertebrates, activation of innate immunity is an early response to injury, implicating it in the regenerative process. However, the mechanisms by which innate signals might regulate stem cell functionality are unknown. Here, we demonstrate that type 2 innate immunity is required for regeneration of skeletal muscle after injury. Muscle damage results in rapid recruitment of eosinophils, which secrete IL-4 to activate the regenerative actions of muscle resident fibro/adipocyte progenitors (FAPs). In FAPs, IL-4/IL-13 signaling serves as a key switch to control their fate and functions. Activation of IL-4/IL-13 signaling promotes proliferation of FAPs to support myogenesis while inhibiting their differentiation into adipocytes. Surprisingly, type 2 cytokine signaling is also required in FAPs, but not in myeloid cells, for rapid clearance of necrotic debris, a process that is necessary for timely and complete regeneration of tissues.

Aging, rejuvenation, and epigenetic reprogramming: resetting the aging clock.

The underlying cause of aging remains one of the central mysteries of biology. Recent studies in several different systems suggest that not only may the rate of aging be modified by environmental and genetic factors, but also that the aging clock can be reversed, restoring characteristics of youthfulness to aged cells and tissues. This Review focuses on the emerging biology of rejuvenation through the lens of epigenetic reprogramming. By defining youthfulness and senescence as epigenetic states, a framework for asking new questions about the aging process emerges.

Exercise plasma boosts memory and dampens brain inflammation via clusterin.

Physical exercise is generally beneficial to all aspects of human and animal health, slowing cognitive ageing and neurodegeneration1. The cognitive benefits of physical exercise are tied to an increased plasticity and reduced inflammation within the hippocampus2-4, yet little is known about the factors and mechanisms that mediate these effects. Here we show that 'runner plasma', collected from voluntarily running mice and infused into sedentary mice, reduces baseline neuroinflammatory gene expression and experimentally induced brain inflammation. Plasma proteomic analysis revealed a concerted increase in complement cascade inhibitors including clusterin (CLU). Intravenously injected CLU binds to brain endothelial cells and reduces neuroinflammatory gene expression in a mouse model of acute brain inflammation and a mouse model of Alzheimer's disease. Patients with cognitive impairment who participated in structured exercise for 6 months had higher plasma levels of CLU. These findings demonstrate the existence of anti-inflammatory exercise factors that are transferrable, target the cerebrovasculature and benefit the brain, and are present in humans who engage in exercise.

Division-Independent Differentiation of Muscle Stem Cells During a Growth Stimulus.

Adult muscle stem cells (MuSCs) are known to replicate upon activation before differentiating and fusing to regenerate myofibers. It is unclear whether MuSC differentiation is intrinsically linked to cell division, which has implications for stem cell population maintenance. We use single-cell RNA-sequencing to identify transcriptionally diverse subpopulations of MuSCs after 5 days of a growth stimulus in adult muscle. Trajectory inference in combination with a novel mouse model for tracking MuSC-derived myonuclei and in vivo labeling of DNA replication revealed an MuSC population that exhibited division-independent differentiation and fusion. These findings demonstrate that in response to a growth stimulus in the presence of intact myofibers, MuSC division is not obligatory.

Molecular regulation of stem cell quiescence.

Subsets of mammalian adult stem cells reside in the quiescent state for prolonged periods of time. This state, which is reversible, has long been viewed as dormant and with minimal basal activity. Recent advances in adult stem cell isolation have provided insights into the epigenetic, transcriptional and post-transcriptional control of quiescence and suggest that quiescence is an actively maintained state in which signalling pathways are involved in maintaining a poised state that allows rapid activation. Deciphering the molecular mechanisms regulating adult stem cell quiescence will increase our understanding of tissue regeneration mechanisms and how they are dysregulated in pathological conditions and in ageing.

ATR activity controls stem cell quiescence via the cyclin F-SCF complex.

A key property of adult stem cells is their ability to persist in a quiescent state for prolonged periods of time. The quiescent state is thought to contribute to stem cell resilience by limiting accumulation of DNA replication­associated mutations. Moreover, cellular stress response factors are thought to play a role in maintaining quiescence and stem cell integrity. We utilized muscle stem cells (MuSCs) as a model of quiescent stem cells and find that the replication stress response protein, ATR (Ataxia Telangiectasia and Rad3-Related), is abundant and active in quiescent but not activated MuSCs. Concurrently, MuSCs display punctate RPA (replication protein A) and R-loop foci, both key triggers for ATR activation. To discern the role of ATR in MuSCs, we generated MuSC-specific ATR conditional knockout (ATRcKO) mice. Surprisingly, ATR ablation results in increased MuSC quiescence exit. Phosphoproteomic analysis of ATRcKO MuSCs reveals enrichment of phosphorylated cyclin F, a key component of the Skp1­Cul1­F-box protein (SCF) ubiquitin ligase complex and regulator of key cell-cycle transition factors, such as the E2F family of transcription factors. Knocking down cyclin F or inhibiting the SCF complex results in E2F1 accumulation and in MuSCs exiting quiescence, similar to ATR-deficient MuSCs. The loss of ATR could be counteracted by inhibiting casein kinase 2 (CK2), the kinase responsible for phosphorylating cyclin F. We propose a model in which MuSCs express cell-cycle progression factors but ATR, in coordination with the cyclin F­SCF complex, represses premature stem cell quiescence exit via ubiquitin­proteasome degradation of these factors.

Hairless regulates heterochromatin maintenance and muscle stem cell function as a histone demethylase antagonist.

Skeletal muscle possesses remarkable regenerative ability because of the resident muscle stem cells (MuSCs). A prominent feature of quiescent MuSCs is a high content of heterochromatin. However, little is known about the mechanisms by which heterochromatin is maintained in MuSCs. By comparing gene-expression profiles from quiescent and activated MuSCs, we found that the mammalian Hairless (Hr) gene is expressed in quiescent MuSCs and rapidly down-regulated upon MuSC activation. Using a mouse model in which Hr can be specifically ablated in MuSCs, we demonstrate that Hr expression is critical for MuSC function and muscle regeneration. In MuSCs, loss of Hr results in reduced trimethylated Histone 3 Lysine 9 (H3K9me3) levels, reduced heterochromatin, increased susceptibility to genotoxic stress, and the accumulation of DNA damage. Deletion of Hr leads to an acceleration of the age-related decline in MuSC numbers. We have also demonstrated that despite the fact that Hr is homologous to a family of histone demethylases and binds to di- and trimethylated H3K9, the expression of Hr does not lead to H3K9 demethylation. In contrast, we show that the expression of Hr leads to the inhibition of the H3K9 demethylase Jmjd1a and an increase in H3K9 methylation. Taking these data together, our study has established that Hr is a H3K9 demethylase antagonist specifically expressed in quiescent MuSCs.

Functional redundancy of type I and type II receptors in the regulation of skeletal muscle growth by myostatin and activin A.

Myostatin (MSTN) is a transforming growth factor-ß (TGF-ß) family member that normally acts to limit muscle growth. The function of MSTN is partially redundant with that of another TGF-ß family member, activin A. MSTN and activin A are capable of signaling through a complex of type II and type I receptors. Here, we investigated the roles of two type II receptors (ACVR2 and ACVR2B) and two type I receptors (ALK4 and ALK5) in the regulation of muscle mass by these ligands by genetically targeting these receptors either alone or in combination specifically in myofibers in mice. We show that targeting signaling in myofibers is sufficient to cause significant increases in muscle mass, showing that myofibers are the direct target for signaling by these ligands in the regulation of muscle growth. Moreover, we show that there is functional redundancy between the two type II receptors as well as between the two type I receptors and that all four type II/type I receptor combinations are utilized in vivo. Targeting signaling specifically in myofibers also led to reductions in overall body fat content and improved glucose metabolism in mice fed either regular chow or a high-fat diet, demonstrating that these metabolic effects are the result of enhanced muscling. We observed no effect, however, on either bone density or muscle regeneration in mice in which signaling was targeted in myofibers. The latter finding implies that MSTN likely signals to other cells, such as satellite cells, in addition to myofibers to regulate muscle homeostasis.

Intronic polyadenylation of PDGFRα in resident stem cells attenuates muscle fibrosis.

Platelet-derived growth factor receptor α (PDGFRα) exhibits divergent effects in skeletal muscle. At physiological levels, signalling through this receptor promotes muscle development in growing embryos and angiogenesis in regenerating adult muscle. However, both increased PDGF ligand abundance and enhanced PDGFRα pathway activity cause pathological fibrosis. This excessive collagen deposition, which is seen in aged and diseased muscle, interferes with muscle function and limits the effectiveness of gene- and cell-based therapies for muscle disorders. Although compelling evidence exists for the role of PDGFRα in fibrosis, little is known about the cells through which this pathway acts. Here we show in mice that PDGFRα signalling regulates a population of muscle-resident fibro/adipogenic progenitors (FAPs) that play a supportive role in muscle regeneration but may also cause fibrosis when aberrantly regulated. We found that FAPs produce multiple transcriptional variants of Pdgfra with different polyadenylation sites, including an intronic variant that codes for a protein isoform containing a truncated kinase domain. This variant, upregulated during regeneration, acts as a decoy to inhibit PDGF signalling and to prevent FAP over-activation. Moreover, increasing the expression of this isoform limits fibrosis in vivo in mice, suggesting both biological relevance and therapeutic potential of modulating polyadenylation patterns in stem-cell populations.

Lineage of origin in rhabdomyosarcoma informs pharmacological response.

Lineage or cell of origin of cancers is often unknown and thus is not a consideration in therapeutic approaches. Alveolar rhabdomyosarcoma (aRMS) is an aggressive childhood cancer for which the cell of origin remains debated. We used conditional genetic mouse models of aRMS to activate the pathognomonic Pax3:Foxo1 fusion oncogene and inactivate p53 in several stages of prenatal and postnatal muscle development. We reveal that lineage of origin significantly influences tumor histomorphology and sensitivity to targeted therapeutics. Furthermore, we uncovered differential transcriptional regulation of the Pax3:Foxo1 locus by tumor lineage of origin, which led us to identify the histone deacetylase inhibitor entinostat as a pharmacological agent for the potential conversion of Pax3:Foxo1-positive aRMS to a state akin to fusion-negative RMS through direct transcriptional suppression of Pax3:Foxo1.

Computational modeling of malignant ascites reveals CCL5-SDC4 interaction in the immune microenvironment of ovarian cancer.

Fluid accumulation in the abdominal cavity is commonly found in advanced-stage ovarian cancer patients, which creates a specialized tumor microenvironment for cancer progression. Using single-cell RNA sequencing (scRNA-seq) of ascites cells from five patients with ovarian cancer, we identified seven cell types, including heterogeneous macrophages and ovarian cancer cells. We resolved a distinct polarization state of macrophages by MacSpectrum analysis and observed subtype-specific enrichment of pathways associated with their functions. The communication between immune and cancer cells was predicted through a putative ligand-receptor pair analysis using NicheNet. We found that CCL5, a chemotactic ligand, is enriched in immune cells (T cells and NK cells) and mediates ovarian cancer cell survival in the ascites, possibly through SDC4. Moreover, SDC4 expression correlated with poor overall survival in ovarian cancer patients. Our study highlights the potential role of T cells and NK cells in long-term survival patients with ovarian cancer, indicating SDC4 as a potential prognostic marker in ovarian cancer patients.

Monitoring disease activity noninvasively in the mdx model of Duchenne muscular dystrophy.

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.

Angiotensin receptor blockade mimics the effect of exercise on recovery after orthopaedic trauma by decreasing pain and improving muscle regeneration.

KEY POINTS: Our tibial fracture orthopaedic injury model in mice recapitulates the major manifestations of complex trauma, including nociceptive sensitization, bone fracture, muscle fibrosis and muscle fibre loss. Delayed exercise after complex orthopaedic trauma results in decreased muscle fibrosis and improved pain Losartan, an angiotensin-receptor blocker with anti-fibrotic abilities, recapitulates the effect of exercise on post-injury recovery and may provide an enhanced recovery option for those who are unable to exercise after injury ABSTRACT: Chronic pain and disability after limb injury are major public health problems. Early mobilization after injury improves functional outcomes for patients, although when and how to implement rehabilitation strategies remains a clinical challenge. Additionally, whether the beneficial effects of exercise can be reproduced using pharmacological tools remains unknown and may benefit patients who are unable to exercise as a result of immobilization. We developed a murine model of orthopaedic trauma combining tibia fracture and pin fixation with muscle damage. Behavioural measures included mechanical nociceptive thresholds and distances run on exercise wheels. Bone healing was quantified using microcomputed tomagraphic scanning, and muscle fibre size distribution and fibrosis were followed using immunohistochemistry. We found that the model provided robust mechanical allodynia, fibrosis and a shift to smaller average muscle fibre size lasting up to 5 weeks from injury. We also observed that allowing 'late' (weeks 1-2) rather than 'early' (weeks 0-1) exercise after injury resulted in greater overall running activity and greater reversal of allodynia. In parallel, the late running paradigm was associated with reduced muscle fibrosis, earlier increase in muscle fibre diameter and a short-term benefit in reducing callus volume. Providing the anti-fibrotic angiotensin receptor blocker losartan to mice in drinking water reduced both allodynia and muscle fibrosis. Combining losartan and late exercise provided no additional benefit. We conclude that early healing after orthopaedic trauma must be allowed prior to the initiation of exercise to achieve optimal pain, functional and physiological outcomes and that losartan is a viable candidate for translational studies.