An unusual case of Brodie's abscess in the humerus of an adult female.

Brodie's abscess is a manifestation of subacute to chronic osteomyelitis, characterized as intraosseous abscess formation, usually on the metaphysis of the long tubular bones in the lower extremities of male pediatric patients. Clinically, Brodie's abscess presents with atraumatic bone pain of an insidious onset, with absence of systemic findings. Delay in diagnosis is common, as diagnostic imaging, followed by biopsy for culture and histologic examination are generally required to secure a diagnosis of Brodie's abscess. Treatment of Brodie's abscess is non-standardized, and usually consists of surgical debridement and antibacterial therapy. Despite the variability in therapeutic approaches, outcomes of Brodie's abscess treated with surgery and antibiotics are favourable. Herein we report a case of a delayed diagnosis of Brodie's abscess in the upper extremity of an adult female. While she improved with treatment of Brodie's abscess, the case serves to remind clinicians to consider this entity in adult individuals who present with atraumatic bone pain.

Heterogeneous levels of delta-like 4 within a multinucleated niche cell maintains muscle stem cell diversity.

The quiescent muscle stem cell (QSC) pool is heterogeneous and generally characterized by the presence and levels of intrinsic myogenic transcription factors. Whether extrinsic factors maintain the diversity of states across the QSC pool remains unknown. The muscle fiber is a multinucleated syncytium that serves as a niche to QSCs, raising the possibility that the muscle fiber regulates the diversity of states across the QSC pool. Here, we show that the muscle fiber maintains a continuum of quiescent states, through a gradient of Notch ligand, Dll4, produced by the fiber and captured by QSCs. The abundance of Dll4 captured by the QSC correlates with the protein levels of the stem cell (SC) identity marker, Pax7. Niche-specific loss of Dll4 decreases QSC diversity and shifts the continuum to cell states that are biased toward more proliferative and committed fates. We reveal that fiber-derived Mindbomb1 (Mib1), an E3 ubiquitin ligase activates Dll4 and controls the heterogeneous levels of Dll4. In response to injury, with a Dll4-replenished niche, the normal continuum and diversity of the SC pool is restored, demonstrating bidirectionality within the SC continuum. Our data show that a post-translational mechanism controls heterogeneity of Notch ligands in a multinucleated niche cell to maintain a continuum of metastable states within the SC pool during tissue homeostasis.

Deep Convolutional and Recurrent Neural Networks for Cell Motility Discrimination and Prediction.

Cells in culture display diverse motility behaviors that may reflect differences in cell state and function, providing motivation to discriminate between different motility behaviors. Current methods to do so rely upon manual feature engineering. However, the types of features necessary to distinguish between motility behaviors can vary greatly depending on the biological context, and it is not always clear which features may be most predictive in each setting for distinguishing particular cell types or disease states. Convolutional neural networks (CNNs) are machine learning models allowing for relevant features to be learned directly from spatial data. Similarly, recurrent neural networks (RNNs) are a class of models capable of learning long term temporal dependencies. Given that cell motility is inherently spacio-temporal data, we present an approach utilizing both convolutional and long- short-term memory (LSTM) recurrent neural network units to analyze cell motility data. These RNN models provide accurate classification of simulated motility and experimentally measured motility from multiple cell types, comparable to results achieved with hand-engineered features. The variety of cell motility differences we can detect suggests that the algorithm is generally applicable to additional cell types not analyzed here. RNN autoencoders based on the same architecture are capable of learning motility features in an unsupervised manner and capturing variation between myogenic cells in the latent space. Adapting these RNN models to motility prediction, RNNs are capable of predicting muscle stem cell motility from past tracking data with performance superior to standard motion prediction models. This advance in cell motility prediction may be of practical utility in cell tracking applications.

Functionally heterogeneous human satellite cells identified by single cell RNA sequencing.

Although heterogeneity is recognized within the murine satellite cell pool, a comprehensive understanding of distinct subpopulations and their functional relevance in human satellite cells is lacking. We used a combination of single cell RNA sequencing and flow cytometry to identify, distinguish, and physically separate novel subpopulations of human PAX7+ satellite cells (Hu-MuSCs) from normal muscles. We found that, although relatively homogeneous compared to activated satellite cells and committed progenitors, the Hu-MuSC pool contains clusters of transcriptionally distinct cells with consistency across human individuals. New surface marker combinations were enriched in transcriptional subclusters, including a subpopulation of Hu-MuSCs marked by CXCR4/CD29/CD56/CAV1 (CAV1+). In vitro, CAV1+ Hu-MuSCs are morphologically distinct, and characterized by resistance to activation compared to CAV1- Hu-MuSCs. In vivo, CAV1+ Hu-MuSCs demonstrated increased engraftment after transplantation. Our findings provide a comprehensive transcriptional view of normal Hu-MuSCs and describe new heterogeneity, enabling separation of functionally distinct human satellite cell subpopulations.

Aging induces aberrant state transition kinetics in murine muscle stem cells.

Murine muscle stem cells (MuSCs) experience a transition from quiescence to activation that is required for regeneration, but it remains unknown if the trajectory and dynamics of activation change with age. Here, we use time-lapse imaging and single cell RNA-seq to measure activation trajectories and rates in young and aged MuSCs. We find that the activation trajectory is conserved in aged cells, and we develop effective machine-learning classifiers for cell age. Using cell-behavior analysis and RNA velocity, we find that activation kinetics are delayed in aged MuSCs, suggesting that changes in stem cell dynamics may contribute to impaired stem cell function with age. Intriguingly, we also find that stem cell activation appears to be a random walk-like process, with frequent reversals, rather than a continuous linear progression. These results support a view of the aged stem cell phenotype as a combination of differences in the location of stable cell states and differences in transition rates between them.

Wnt4 from the Niche Controls the Mechano-Properties and Quiescent State of Muscle Stem Cells.

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.

Lineage Tracing Reveals a Subset of Reserve Muscle Stem Cells Capable of Clonal Expansion under Stress.

Stem cell heterogeneity is recognized as functionally relevant for tissue homeostasis and repair. The identity, context dependence, and regulation of skeletal muscle satellite cell (SC) subsets remains poorly understood. We identify a minor subset of Pax7+ SCs that is indelibly marked by an inducible Mx1-Cre transgene in vivo, is enriched for Pax3 expression, and has reduced ROS (reactive oxygen species) levels. Mx1+ SCs possess potent stem cell activity upon transplantation but minimally contribute to endogenous muscle repair, due to their relative low abundance. In contrast, a dramatic clonal expansion of Mx1+ SCs allows extensive contribution to muscle repair and niche repopulation upon selective pressure of radiation stress, consistent with reserve stem cell (RSC) properties. Loss of Pax3 in RSCs increased ROS content and diminished survival and stress tolerance. These observations demonstrate that the Pax7+ SC pool contains a discrete population of radiotolerant RSCs that undergo clonal expansion under severe stress.

Thermal stress induces glycolytic beige fat formation via a myogenic state.

Environmental cues profoundly affect cellular plasticity in multicellular organisms. For instance, exercise promotes a glycolytic-to-oxidative fibre-type switch in skeletal muscle, and cold acclimation induces beige adipocyte biogenesis in adipose tissue. However, the molecular mechanisms by which physiological or pathological cues evoke developmental plasticity remain incompletely understood. Here we report a type of beige adipocyte that has a critical role in chronic cold adaptation in the absence of ß-adrenergic receptor signalling. This beige fat is distinct from conventional beige fat with respect to developmental origin and regulation, and displays enhanced glucose oxidation. We therefore refer to it as glycolytic beige fat. Mechanistically, we identify GA-binding protein α as a regulator of glycolytic beige adipocyte differentiation through a myogenic intermediate. Our study reveals a non-canonical adaptive mechanism by which thermal stress induces progenitor cell plasticity and recruits a distinct form of thermogenic cell that is required for energy homeostasis and survival.

Muscle Stem Cells and Aging.

Satellite cells (SCs) are a population of muscle-resident stem cells that are essential for efficient tissue repair. SCs reside in a relatively quiescent state during normal tissue turnover, but are activated in response to injury through the microenvironment and cell-intrinsic signals. During aging, SC dysfunction is a major contributor to the decline in regenerative potential of muscle tissue. Recent studies have demonstrated that both cell-intrinsic and cell-extrinsic factors are deregulated during aging. Interventions that reverse age-associated changes in SCs or the niche have shown to partially rejuvenate the regenerative capacity of aged muscle SCs. In this review, we discuss recent advances in SC biology as it pertains to the deleterious effects of aging. A better understanding of how age-dependent changes in the SC and its environment niche impact muscle regeneration could lead to interventions to ameliorate the effects of aging in humans.

Inferring cell state by quantitative motility analysis reveals a dynamic state system and broken detailed balance.

Cell populations display heterogeneous and dynamic phenotypic states at multiple scales. Similar to molecular features commonly used to explore cell heterogeneity, cell behavior is a rich phenotypic space that may allow for identification of relevant cell states. Inference of cell state from cell behavior across a time course may enable the investigation of dynamics of transitions between heterogeneous cell states, a task difficult to perform with destructive molecular observations. Cell motility is one such easily observed cell behavior with known biomedical relevance. To investigate heterogenous cell states and their dynamics through the lens of cell behavior, we developed Heteromotility, a software tool to extract quantitative motility features from timelapse cell images. In mouse embryonic fibroblasts (MEFs), myoblasts, and muscle stem cells (MuSCs), Heteromotility analysis identifies multiple motility phenotypes within the population. In all three systems, the motility state identity of individual cells is dynamic. Quantification of state transitions reveals that MuSCs undergoing activation transition through progressive motility states toward the myoblast phenotype. Transition rates during MuSC activation suggest non-linear kinetics. By probability flux analysis, we find that this MuSC motility state system breaks detailed balance, while the MEF and myoblast systems do not. Balanced behavior state transitions can be captured by equilibrium formalisms, while unbalanced switching between states violates equilibrium conditions and would require an external driving force. Our data indicate that the system regulating cell behavior can be decomposed into a set of attractor states which depend on the identity of the cell, together with a set of transitions between states. These results support a conceptual view of cell populations as dynamical systems, responding to inputs from signaling pathways and generating outputs in the form of state transitions and observable motile behaviors.

Use of the Mean Abnormal Result Rate (MARR) to Gauge Changes in Family Physicians' Selectivity of Laboratory Test Ordering, 2010-2015.

OBJECTIVES: The mean abnormal result rate (MARR) has recently been advanced as a metric of laboratory test appropriateness. We used the MARR metric to examine patterns of change in family physician test requisitions over time. METHODS: We accessed the Laboratory Information System of Calgary Laboratory Services for family physician-ordered testing on outpatients to gather aggregate test and abnormal result counts from 2010 to 2015. RESULTS: Over the 6 years, there was an annual average of 3,401,553 tests for 411,295 distinct patients on their first test requisition for the year. The MARR increased from 8.1% to 9.0% through this period. CONCLUSIONS: The MARR for Calgary and surrounding area gives tentative evidence of a gradual increase in physician test selectivity in recent years. Further data from other catchment areas are needed before making assertions about broader trends in physician awareness of laboratory resource use.

Is Growth Differentiation Factor 11 a Realistic Therapeutic for Aging-Dependent Muscle Defects?

This "Controversies in Cardiovascular Research" article evaluates the evidence for and against the hypothesis that the circulating blood level of growth differentiation factor 11 (GDF11) decreases in old age and that restoring normal GDF11 levels in old animals rejuvenates their skeletal muscle and reverses pathological cardiac hypertrophy and cardiac dysfunction. Studies supporting the original GDF11 hypothesis in skeletal and cardiac muscle have not been validated by several independent groups. These new studies have either found no effects of restoring normal GDF11 levels on cardiac structure and function or have shown that increasing GDF11 or its closely related family member growth differentiation factor 8 actually impairs skeletal muscle repair in old animals. One possible explanation for what seems to be mutually exclusive findings is that the original reagent used to measure GDF11 levels also detected many other molecules so that age-dependent changes in GDF11 are still not well known. The more important issue is whether increasing blood [GDF11] repairs old skeletal muscle and reverses age-related cardiac pathologies. There are substantial new and existing data showing that GDF8/11 can exacerbate rather than rejuvenate skeletal muscle injury in old animals. There is also new evidence disputing the idea that there is pathological hypertrophy in old C57bl6 mice and that GDF11 therapy can reverse cardiac pathologies. Finally, high [GDF11] causes reductions in body and heart weight in both young and old animals, suggestive of a cachexia effect. Our conclusion is that elevating blood levels of GDF11 in the aged might cause more harm than good.

The ins and outs of muscle stem cell aging.

Skeletal muscle has a remarkable capacity to regenerate by virtue of its resident stem cells (satellite cells). This capacity declines with aging, although whether this is due to extrinsic changes in the environment and/or to cell-intrinsic mechanisms associated to aging has been a matter of intense debate. Furthermore, while some groups support that satellite cell aging is reversible by a youthful environment, others support cell-autonomous irreversible changes, even in the presence of youthful factors. Indeed, whereas the parabiosis paradigm has unveiled the environment as responsible for the satellite cell functional decline, satellite cell transplantation studies support cell-intrinsic deficits with aging. In this review, we try to shed light on the potential causes underlying these discrepancies. We propose that the experimental paradigm used to interrogate intrinsic and extrinsic regulation of stem cell function may be a part of the problem. The assays deployed are not equivalent and may overburden specific cellular regulatory processes and thus probe different aspects of satellite cell properties. Finally, distinct subsets of satellite cells may be under different modes of molecular control and mobilized preferentially in one paradigm than in the other. A better understanding of how satellite cells molecularly adapt during aging and their context-dependent deployment during injury and transplantation will lead to the development of efficacious compensating strategies that maintain stem cell fitness and tissue homeostasis throughout life.

Lost in Translation: Preserving Satellite Cell Function with Global Translational Control.

Quiescence is highly regulated to preserve stem cell function. In this issue of Cell Stem Cell, Zismanov et al. (2016) show that P-eIF2α, a translational initiation factor, reinforces the quiescent state of muscle stem cells by safeguarding against cell-cycle entry and lineage progression.

GDF11 Increases with Age and Inhibits Skeletal Muscle Regeneration.

Age-related frailty may be due to decreased skeletal muscle regeneration. The role of TGF-ß molecules myostatin and GDF11 in regeneration is unclear. Recent studies showed an age-related decrease in GDF11 and that GDF11 treatment improves muscle regeneration, which were contrary to prior studies. We now show that these recent claims are not reproducible and the reagents previously used to detect GDF11 are not GDF11 specific. We develop a GDF11-specific immunoassay and show a trend toward increased GDF11 levels in sera of aged rats and humans. GDF11 mRNA increases in rat muscle with age. Mechanistically, GDF11 and myostatin both induce SMAD2/3 phosphorylation, inhibit myoblast differentiation, and regulate identical downstream signaling. GDF11 significantly inhibited muscle regeneration and decreased satellite cell expansion in mice. Given early data in humans showing a trend for an age-related increase, GDF11 could be a target for pharmacologic blockade to treat age-related sarcopenia.

The Spindle Assembly Checkpoint Safeguards Genomic Integrity of Skeletal Muscle Satellite Cells.

To ensure accurate genomic segregation, cells evolved the spindle assembly checkpoint (SAC), whose role in adult stem cells remains unknown. Inducible perturbation of a SAC kinase, Mps1, and its downstream effector, Mad2, in skeletal muscle stem cells shows the SAC to be critical for normal muscle growth, repair, and self-renewal of the stem cell pool. SAC-deficient muscle stem cells arrest in G1 phase of the cell cycle with elevated aneuploidy, resisting differentiation even under inductive conditions. p21(CIP1) is responsible for these SAC-deficient phenotypes. Despite aneuploidy's correlation with aging, we find that aged proliferating muscle stem cells display robust SAC activity without elevated aneuploidy. Thus, muscle stem cells have a two-step mechanism to safeguard their genomic integrity. The SAC prevents chromosome missegregation and, if it fails, p21(CIP1)-dependent G1 arrest limits cellular propagation and tissue integration. These mechanisms ensure that muscle stem cells with compromised genomes do not contribute to tissue homeostasis.

Pax7 is back.

Two recent studies have reinvigorated the conversation regarding the role of Pax7 in adult satellite. Studies by Gunther et al (Cell Stem Cell 13:590-601, 2013) and Von Maltzhen et al (Proc Natl Acad Sci U S A 110:16474-16479) show that Pax7 is critical for adult satellite cell function and their contribution to muscle repair. Previously, Lepper et al (Nature 460:627-631, 2009) demonstrated that Pax7 was dispensable for adult muscle repair. In this commentary I have summarized the results from these studies, focusing on the differences in experimental paradigms that led the authors to different conclusions. I also take this opportunity to discuss the potential limitations and hurdles of Cre-lox technology that are responsible for the discrepant results.

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.

Early forming label-retaining muscle stem cells require p27kip1 for maintenance of the primitive state.

Across different niches, subsets of highly functional stem cells are maintained in a relatively dormant rather than proliferative state. Our understanding of proliferative dynamics in tissue-specific stem cells during conditions of increased tissue turnover remains limited. Using a TetO-H2B-GFP reporter of proliferative history, we identify skeletal muscle stem cell, or satellite cells, that retain (LRC) or lose (nonLRC) the H2B-GFP label. We show in mice that LRCs and nonLRCs are formed at birth and persist during postnatal growth and adult muscle repair. Functionally, LRCs and nonLRCs are born equivalent and transition during postnatal maturation into distinct and hierarchically organized subsets. Adult LRCs give rise to LRCs and nonLRCs; the former are able to self-renew, whereas the latter are restricted to differentiation. Expression analysis revealed the CIP/KIP family members p21(cip1) (Cdkn1a) and p27(kip1) (Cdkn1b) to be expressed at higher levels in LRCs. In accordance with a crucial role in LRC fate, loss of p27(kip1) promoted proliferation and differentiation of LRCs in vitro and impaired satellite cell self-renewal after muscle injury. By contrast, loss of p21(cip1) only affected nonLRCs, in which myogenic commitment was inhibited. Our results provide evidence that restriction of self-renewal potential to LRCs is established early in life and is maintained during increased tissue turnover through the cell cycle inhibitor p27(kip1). They also reveal the differential role of CIP/KIP family members at discrete steps within the stem cell hierarchy.