Platelet factors attenuate inflammation and rescue cognition in ageing.

Identifying therapeutics to delay, and potentially reverse, age-related cognitive decline is critical in light of the increased incidence of dementia-related disorders forecasted in the growing older population1. Here we show that platelet factors transfer the benefits of young blood to the ageing brain. Systemic exposure of aged male mice to a fraction of blood plasma from young mice containing platelets decreased neuroinflammation in the hippocampus at the transcriptional and cellular level and ameliorated hippocampal-dependent cognitive impairments. Circulating levels of the platelet-derived chemokine platelet factor 4 (PF4) (also known as CXCL4) were elevated in blood plasma preparations of young mice and humans relative to older individuals. Systemic administration of exogenous PF4 attenuated age-related hippocampal neuroinflammation, elicited synaptic-plasticity-related molecular changes and improved cognition in aged mice. We implicate decreased levels of circulating pro-ageing immune factors and restoration of the ageing peripheral immune system in the beneficial effects of systemic PF4 on the aged brain. Mechanistically, we identified CXCR3 as a chemokine receptor that, in part, mediates the cellular, molecular and cognitive benefits of systemic PF4 on the aged brain. Together, our data identify platelet-derived factors as potential therapeutic targets to abate inflammation and rescue cognition in old age.

Tissue-Resident Group 2 Innate Lymphoid Cells Differentiate by Layered Ontogeny and In Situ Perinatal Priming.

The perinatal period is a critical window for distribution of innate tissue-resident immune cells within developing organs. Despite epidemiologic evidence implicating the early-life environment in the risk for allergy, temporally controlled lineage tracing of group 2 innate lymphoid cells (ILC2s) during this period remains unstudied. Using complementary fate-mapping approaches and reporters for ILC2 activation, we show that ILC2s appeared in multiple organs during late gestation like tissue macrophages, but, unlike the latter, a majority of peripheral ILC2 pools were generated de novo during the postnatal window. This period was accompanied by systemic ILC2 priming and acquisition of tissue-specific transcriptomes. Although perinatal ILC2s were variably replaced across tissues with age, the dramatic increases in tissue ILC2s following helminth infection were mediated through local expansion independent of de novo generation by bone marrow hematopoiesis. We provide comprehensive temporally controlled fate mapping of an innate lymphocyte subset with notable nuances as compared to tissue macrophage ontogeny.

Collagen architecture and biomechanics of gracilis and adductor longus muscles from children with cerebral palsy.

Cerebral palsy (CP) describes some upper motoneuron disorders due to non-progressive disturbances occurring in the developing brain that cause progressive changes to muscle. While longer sarcomeres increase muscle stiffness in patients with CP compared to typically developing (TD) patients, changes in extracellular matrix (ECM) architecture can increase stiffness. Our goal was to investigate how changes in muscle and ECM architecture impact muscle stiffness, gait and joint function in CP. Gracilis and adductor longus biopsies were collected from children with CP undergoing tendon lengthening surgery for hamstring and hip adduction contractures, respectively. Gracilis biopsies were collected from TD patients undergoing anterior cruciate ligament reconstruction surgery with hamstring autograft. Muscle mechanical testing, two-photon imaging and hydroxyproline assay were performed on biopsies. Corresponding data were compared to radiographic hip displacement in CP adductors (CPA), gait kinematics in CP hamstrings (CPH), and joint range of motion in CPA and CPH. We found at matched sarcomere lengths muscle stiffness and collagen architecture were similar between TD and CP hamstrings. However, CPH stiffness (R2 = 0.1973), collagen content (R2 = 0.5099) and cross-linking (R2 = 0.3233) were correlated to decreased knee range of motion. Additionally, we observed collagen fibres within the muscle ECM increase alignment during muscular stretching. These data demonstrate that while ECM architecture is similar between TD and CP hamstrings, collagen fibres biomechanics are sensitive to muscle strain and may be altered at longer in vivo sarcomere lengths in CP muscle. Future studies could evaluate the impact of ECM architecture on TD and CP muscle stiffness across in vivo operating ranges. KEY POINTS: At matched sarcomere lengths, gracilis muscle mechanics and collagen architecture are similar in TD patients and patients with CP. In both TD and CP muscles, collagen fibres dynamically increase their alignment during muscle stretching. Aspects of muscle mechanics and collagen architecture are predictive of in vivo knee joint motion and radiographic hip displacement in patients with CP. Longer sarcomere lengths in CP muscle in vivo may alter collagen architecture and biomechanics to drive deficits in joint mobility and gait function.

MHC class I H2-Kb negatively regulates neural progenitor cell proliferation by inhibiting FGFR signaling.

Proteins of the major histocompatibility complex class I (MHC I), predominantly known for antigen presentation in the immune system, have recently been shown to be necessary for developmental neural refinement and adult synaptic plasticity. However, their roles in nonneuronal cell populations in the brain remain largely unexplored. Here, we identify classical MHC I molecule H2-Kb as a negative regulator of proliferation in neural stem and progenitor cells (NSPCs). Using genetic knockout mouse models and in vivo viral-mediated RNA interference (RNAi) and overexpression, we delineate a role for H2-Kb in negatively regulating NSPC proliferation and adult hippocampal neurogenesis. Transcriptomic analysis of H2-Kb knockout NSPCs, in combination with in vitro RNAi, overexpression, and pharmacological approaches, further revealed that H2-Kb inhibits cell proliferation by dampening signaling pathways downstream of fibroblast growth factor receptor 1 (Fgfr1). These findings identify H2-Kb as a critical regulator of cell proliferation through the modulation of growth factor signaling.

Muscle satellite cells and fibro-adipogenic progenitors from muscle contractures of children with cerebral palsy have impaired regenerative capacity.

AIM: To evaluate the mechanosensitivity of muscle satellite cells (MuSCs) and fibro-adipogenic progenitors (FAPs) in cerebral palsy (CP) and the efficacy of the drug verteporfin in restoring cells' regenerative capacity. METHOD: Muscle biopsies were collected from six children with CP and six typically developing children. MuSCs and FAPs were isolated and plated on collagen-coated polyacrylamide gels at stiffnesses of 0.2 kPa, 8 kPa, and 25 kPa. Cells were treated with verteporfin to block mechanosensing or with dimethyl sulfoxide as a negative control. MuSC differentiation and FAP activation into myofibroblasts were measured using immunofluorescence staining. RESULTS: Surprisingly, MuSC differentiation was not affected by stiffness; however, stiff substrates resulted in large myonuclear clustering. Across all stiffnesses, MuSCs from children with CP had less differentiation than those of their typically developing counterparts. FAP activation into myofibroblasts was significantly higher in children with CP than their typically developing peers, but was not affected by stiffness. Verteporfin did not affect differentiation or activation in either cell population, but slightly decreased myonuclear clustering on stiff substrates. INTERPRETATION: Cells from children with CP were less regenerative and more fibrotic compared to those of their typically developing counterparts, with MuSCs being sensitive to increases in stiffness. Therefore, the mechanosensitivity of MuSCs and FAPs may represent a new target to improve differentiation and activation in CP muscle.

Thrown for a loop: fibro-adipogenic progenitors in skeletal muscle fibrosis.

Fibro-adipogenic progenitors (FAPs) are key regulators of skeletal muscle regeneration and homeostasis. However, dysregulation of these cells leads to fibro-fatty infiltration across various muscle diseases. FAPs are the key source of extracellular matrix (ECM) deposition in muscle, and disruption to this process leads to a pathological accumulation of ECM, known as fibrosis. The replacement of contractile tissue with fibrotic ECM functionally impairs the muscle and increases muscle stiffness. FAPs and fibrotic muscle form a progressively degenerative feedback loop where, as a muscle becomes fibrotic, it induces a fibrotic FAP phenotype leading to further development of fibrosis. In this review, we summarize FAPs' role in fibrosis in terms of their activation, heterogeneity, contributions to fibrotic degeneration, and role across musculoskeletal diseases. We also discuss current research on potential therapeutic avenues to attenuate fibrosis by targeting FAPs.

Alignment, cross linking, and beyond: a collagen architect's guide to the skeletal muscle extracellular matrix.

The muscle extracellular matrix (ECM) forms a complex network of collagens, proteoglycans, and other proteins that produce a favorable environment for muscle regeneration, protect the sarcolemma from contraction-induced damage, and provide a pathway for the lateral transmission of contractile force. In each of these functions, the structure and organization of the muscle ECM play an important role. Many aspects of collagen architecture, including collagen alignment, cross linking, and packing density affect the regenerative capacity, passive mechanical properties, and contractile force transmission pathways of skeletal muscle. The balance between fortifying the muscle ECM and maintaining ECM turnover and compliance is highly dependent on the integrated organization, or architecture, of the muscle matrix, especially related to collagen. While muscle ECM remodeling patterns in response to exercise and disease are similar, in that collagen synthesis can increase in both cases, one outcome leads to a stronger muscle and the other leads to fibrosis. In this review, we provide a comprehensive analysis of the architectural features of each layer of muscle ECM: epimysium, perimysium, and endomysium. Further, we detail the importance of muscle ECM architecture to biomechanical function in the context of exercise or fibrosis, including disease, injury, and aging. We describe how collagen architecture is linked to active and passive muscle biomechanics and which architectural features are acutely dynamic and adapt over time. Future studies should investigate the significance of collagen architecture in muscle stiffness, ECM turnover, and lateral force transmission in the context of health and fibrosis.

Chronic Kidney Disease is Associated With Attenuated Plasma Metabolome Response to Oral Glucose Tolerance Testing.

OBJECTIVE: Chronic kidney disease (CKD) is associated with decreased anabolic response to insulin contributing to protein-energy wasting. Targeted metabolic profiling of oral glucose tolerance testing (OGTT) may help identify metabolic pathways contributing to disruptions to insulin response in CKD. METHODS: Using targeted metabolic profiling, we studied the plasma metabolome response in 41 moderate-to-severe nondiabetic CKD patients and 20 healthy controls at fasting and 2 hours after an oral glucose load. We used linear mixed modeling with random intercepts, adjusting for age, gender, race/ethnicity, body weight, and batch to assess heterogeneity in response to OGTT by CKD status. RESULTS: Mean estimated glomerular filtration rate among CKD participants was 38.9 ± 12.7 mL/min per 1.73 m2 compared to 87.2 ± 17.7 mL/min per 1.73 m2 among controls. Glucose ingestion induced an anabolic response resulting in increased glycolysis products and a reduction in a wide range of metabolites including amino acids, tricarboxylic acid cycle intermediates, and purine nucleotides compared to fasting. Participants with CKD demonstrated a blunted anabolic response to OGTT evidenced by significant changes in 13 metabolites compared to controls. The attenuated metabolome response predominant involved mitochondrial energy metabolism, vitamin B family, and purine nucleotides. Compared to controls, CKD participants had elevated lactate:pyruvate (L:P) ratio and decreased guanosine diphosphate:guanosine triphosphate ratio during OGTT. CONCLUSION: Metabolic profiling of OGTT response suggests a broad disruption of mitochondrial energy metabolism in CKD patients. These findings motivate further investigation into the impact of insulin sensitizers and mitochondrial targeted therapeutics on energy metabolism in patients with nondiabetic CKD.

A mutation in desmin makes skeletal muscle less vulnerable to acute muscle damage after eccentric loading in rats.

Desminopathy is the most common intermediate filament disease in humans. The most frequent mutation causing desminopathy in patients is a R350P DES missense mutation. We have developed a rat model with an analogous mutation in R349P Des. To investigate the role of R349P Des in mechanical loading, we stimulated the sciatic nerve of wild-type littermates (WT) (n = 6) and animals carrying the mutation (MUT) (n = 6) causing a lengthening contraction of the dorsi flexor muscles. MUT animals showed signs of ongoing regeneration at baseline as indicated by a higher number of central nuclei (genotype: P < .0001). While stimulation did not impact central nuclei, we found an increased number of IgG positive fibers (membrane damage indicator) after eccentric contractions with both genotypes (stimulation: P < .01). Interestingly, WT animals displayed a more pronounced increase in IgG positive fibers with stimulation compared to MUT (interaction: P < .05). In addition to altered histology, molecular signaling on the protein level differed between WT and MUT. The membrane repair protein dysferlin decreased with eccentric loading in WT but increased in MUT (interaction: P < .05). The autophagic substrate p62 was increased in both genotypes with loading (stimulation: P < .05) but tended to be more elevated in WT (interaction: P = .05). Caspase 3 levels, a central regulator of apoptotic cell death, was increased with stimulation in both genotypes (stimulation: P < .01) but more so in WT animals (interaction: P < .0001). Overall, our data indicate that R349P Des rats have a lower susceptibility to structural muscle damage of the cytoskeleton and sarcolemma with acute eccentric loading.

Rapid quantification of microRNA-375 through one-pot primer-generating rolling circle amplification.

A recent surge of interest in microRNA has been driven by its discovery as a circulating biomarker of disease, with many diagnostic test platforms currently under development. Alternatives to widely used microRNA quantification methods such as quantitative reverse transcriptase PCR (qRT-PCR) are needed for use in portable and point-of-care devices which are incompatible with complex sample processing workflows and thermal cycling. Rolling circle amplification (RCA) is a one-pot assay technique which directly amplifies nucleic acids using sequence-specific microRNA priming to initiate a single-step isothermal reaction that is compatible with simple devices. Sensitivity remains a limitation of RCA methods, however, and detection limits do not typically reach the femtomolar level in which microRNA targets are present in blood. RCA assays have previously been improved by digestion of the amplification products using a nicking endonuclease to exponentially generate new reaction primers. Here we describe how a ligation-free version of this technique performed in a single tube can be used to improve the limit of detection for microRNA-375, an important blood biomarker for prostate cancer. Endonuclease addition changes a linear process into an exponential amplification reaction which results in a 61-fold improvement of the limit of detection (5.9 fM), a dynamic range wider than 5-log(10), and a shorter reaction time. By eliminating the need for microRNA reverse transcription and thermal cycling, this single-step one-pot method provides a more rapid and simplified alternative to qRT-PCR for ultrasensitive microRNA quantification in blood extracts.

Digital-resolution detection of microRNA with single-base selectivity by photonic resonator absorption microscopy.

Circulating exosomal microRNA (miR) represents a new class of blood-based biomarkers for cancer liquid biopsy. The detection of miR at a very low concentration and with single-base discrimination without the need for sophisticated equipment, large volumes, or elaborate sample processing is a challenge. To address this, we present an approach that is highly specific for a target miR sequence and has the ability to provide "digital" resolution of individual target molecules with high signal-to-noise ratio. Gold nanoparticle tags are prepared with thermodynamically optimized nucleic acid toehold probes that, when binding to a target miR sequence, displace a probe-protecting oligonucleotide and reveal a capture sequence that is used to selectively pull down the target-probe-nanoparticle complex to a photonic crystal (PC) biosensor surface. By matching the surface plasmon-resonant wavelength of the nanoparticle tag to the resonant wavelength of the PC nanostructure, the reflected light intensity from the PC is dramatically and locally quenched by the presence of each individual nanoparticle, enabling a form of biosensor microscopy that we call Photonic Resonator Absorption Microscopy (PRAM). Dynamic PRAM imaging of nanoparticle tag capture enables direct 100-aM limit of detection and single-base mismatch selectivity in a 2-h kinetic discrimination assay. The PRAM assay demonstrates that ultrasensitivity (<1 pM) and high selectivity can be achieved on a direct readout diagnostic.

Myofibrillar Lattice Remodeling Is a Structural Cytoskeletal Predictor of Diaphragm Muscle Weakness in a Fibrotic mdx (mdx Cmah-/-) Model.

Duchenne muscular dystrophy (DMD) is a degenerative genetic myopathy characterized by complete absence of dystrophin. Although the mdx mouse lacks dystrophin, its phenotype is milder compared to DMD patients. The incorporation of a null mutation in the Cmah gene led to a more DMD-like phenotype (i.e., more fibrosis). Although fibrosis is thought to be the major determinant of 'structural weakness', intracellular remodeling of myofibrillar geometry was shown to be a major cellular determinant thereof. To dissect the respective contribution to muscle weakness, we assessed biomechanics and extra- and intracellular architecture of whole muscle and single fibers from extensor digitorum longus (EDL) and diaphragm. Despite increased collagen contents in both muscles, passive stiffness in mdx Cmah-/- diaphragm was similar to wt mice (EDL muscles were twice as stiff). Isometric twitch and tetanic stresses were 50% reduced in mdx Cmah-/- diaphragm (15% in EDL). Myofibrillar architecture was severely compromised in mdx Cmah-/- single fibers of both muscle types, but more pronounced in diaphragm. Our results show that the mdx Cmah-/- genotype reproduces DMD-like fibrosis but is not associated with changes in passive visco-elastic muscle stiffness. Furthermore, detriments in active isometric force are compatible with the pronounced myofibrillar disarray of the dystrophic background.

Skeletal muscle progenitors are sensitive to collagen architectural features of fibril size and cross linking.

Muscle stem cells (MuSCs) are essential for the robust regenerative capacity of skeletal muscle. However, in fibrotic environments marked by abundant collagen and altered collagen organization, the regenerative capability of MuSCs is diminished. MuSCs are sensitive to their extracellular matrix environment but their response to collagen architecture is largely unknown. The present study aimed to systematically test the effect of underlying collagen structures on MuSC functions. Collagen hydrogels were engineered with varied architectures: collagen concentration, cross linking, fibril size, and fibril alignment, and the changes were validated with second harmonic generation imaging and rheology. Proliferation and differentiation responses of primary mouse MuSCs and immortal myoblasts (C2C12s) were assessed using EdU assays and immunolabeling skeletal muscle myosin expression, respectively. Changing collagen concentration and the corresponding hydrogel stiffness did not have a significant influence on MuSC proliferation or differentiation. However, MuSC differentiation on atelocollagen gels, which do not form mature pyridinoline cross links, was increased compared with the cross-linked control. In addition, MuSCs and C2C12 myoblasts showed greater differentiation on gels with smaller collagen fibrils. Proliferation rates of C2C12 myoblasts were also higher on gels with smaller collagen fibrils, whereas MuSCs did not show a significant difference. Surprisingly, collagen alignment did not have significant effects on muscle progenitor function. This study demonstrates that MuSCs are capable of sensing their underlying extracellular matrix (ECM) structures and enhancing differentiation on substrates with less collagen cross linking or smaller collagen fibrils. Thus, in fibrotic muscle, targeting cross linking and fibril size rather than collagen expression may more effectively support MuSC-based regeneration.

Passive stiffness of fibrotic skeletal muscle in mdx mice relates to collagen architecture.

KEY POINTS: The amount of fibrotic material in dystrophic mouse muscles relates to contractile function, but not passive function. Collagen fibres in skeletal muscle are associated with increased passive muscle stiffness in fibrotic muscles. The alignment of collagen is independently associated with passive stiffness in dystrophic skeletal muscles. These outcomes demonstrate that collagen architecture rather than collagen content should be a target of anti-fibrotic therapies to treat muscle stiffness. ABSTRACT: Fibrosis is prominent in many skeletal muscle pathologies including dystrophies, neurological disorders, cachexia, chronic kidney disease, sarcopenia and metabolic disorders. Fibrosis in muscle is associated with decreased contractile forces and increased passive stiffness that limits joint mobility leading to contractures. However, the assumption that more fibrotic material is directly related to decreased function has not held true. Here we utilize novel measurement of extracellular matrix (ECM) and collagen architecture to relate ECM form to muscle function. We used mdx mice, a model for Duchenne muscular dystrophy that becomes fibrotic, and wildtype mice. In this model, extensor digitorum longus (EDL) muscle was significantly stiffer, but with similar total collagen, while the soleus muscle did not change stiffness, but increased collagen. The stiffness of the EDL was associated with increased collagen crosslinking as determined by collagen solubility. Measurement of ECM alignment using polarized light microscopy showed a robust relationship between stiffness and alignment for wildtype muscle that broke down in mdx muscles. Direct visualization of large collagen fibres with second harmonic generation imaging revealed their relative abundance in stiff muscles. Collagen fibre alignment was linked to stiffness across all muscles investigated and the most significant factor in a multiple linear regression-based model of muscle stiffness from ECM parameters. This work establishes novel characteristics of skeletal muscle ECM architecture and provides evidence for a mechanical function of collagen fibres in muscle. This finding suggests that anti-fibrotic strategies to enhance muscle function and excessive stiffness should target large collagen fibres and their alignment rather than total collagen.

Contribution of extracellular matrix components to the stiffness of skeletal muscle contractures in patients with cerebral palsy.

Purpose: Joint contractures in children with cerebral palsy contain muscle tissue that is mechanically stiffer with higher collagen content than typically developing children. Interestingly, the correlation between collagen content and stiffness is weak. To date, no data are available on collagen types or other extracellular matrix proteins in these muscles, nor any information regarding their function. Thus, our purpose was to measure specific extracellular protein composition in cerebral palsy and typically developing human muscles along with structural aspects of extracellular matrix architecture to determine the extent to which these explain mechanical properties. Materials and Methods: Biopsies were collected from children with cerebral palsy undergoing muscle lengthening procedures and typically developing children undergoing anterior cruciate ligament reconstruction. Tissue was prepared for the determination of collagen types I, III, IV, and VI, proteoglycan, biglycan, decorin, hyaluronic acid/uronic acid and collagen crosslinking. Results: All collagen types increased in cerebral palsy along with pyridinoline crosslinks, total proteoglycan, and uronic acid. In all cases, type I or total collagen and total proteoglycan were positive predictors, while biglycan was a negative predictor of stiffness. Together these parameters accounted for a greater degree of variance within groups than across groups, demonstrating an altered relationship between extracellular matrix and stiffness with cerebral palsy. Further, stereological analysis revealed a significant increase in collagen fibrils organized in cables and an increased volume fraction of fibroblasts in CP muscle. Conclusions: These data demonstrate a novel adaptation of muscle extracellular matrix in children with cerebral palsy that includes alterations in extracellular matrix protein composition and structure related to mechanical function.

Development of a Linker-Mediated Immunoassay Using Chemically Transitioned Nanosensors.

Sensitive and specific quantification of protein biomarkers is important in medical diagnostics, academic research, and pharmaceutical development. However, multiple binding steps in conventional sandwich immunoassay protocols result in high assay hands-on-time and delayed results. This is particularly relevant for medical diagnostics, where assay turn-around-time can have an immense impact on patient outcomes. To address this limitation, we report the assembly of nanosensors prepared using DNA-antibody conjugates, which combine capture and detection antibody binding steps by facilitating rapid antigen capture. Following antigen binding, detection antibodies are released using chemically induced complex rearrangement. A panel of 12 chemical additives are characterized to identify melting point depressants capable of rapidly denaturing double stranded DNA (dsDNA) linkers, and 8 compounds are demonstrated to be capable of disrupting dsDNA while maintaining the integrity of protein binding. This technique is then validated for the measurement of the heart attack indicator cardiac troponin I and is shown to successfully combine antigen binding steps while also increasing detection sensitivity 42×. Linker-mediated immunoassays are also demonstrated to provide robust quantification in human serum and are shown to be compatible with each of the most commonly used immunoassay detection modalities.

Matrix Metalloproteinase 13 from Satellite Cells is Required for Efficient Muscle Growth and Regeneration.

BACKGROUND/AIMS: Cell migration and extracellular matrix remodeling underlie normal mammalian development and growth as well as pathologic tumor invasion. Skeletal muscle is no exception, where satellite cell migration replenishes nuclear content in damaged tissue and extracellular matrix reforms during regeneration. A key set of enzymes that regulate these processes are matrix metalloproteinases (MMP)s. The collagenase MMP-13 is transiently upregulated during muscle regeneration, but its contribution to damage resolution is unknown. The purpose of this work was to examine the importance of MMP-13 in muscle regeneration and growth in vivo and to delineate a satellite cell specific role for this collagenase. METHODS: Mice with total and satellite cell specific Mmp13 deletion were utilized to determine the importance of MMP-13 for postnatal growth, regeneration after acute injury, and in chronic injury from a genetic cross with dystrophic (mdx) mice. We also evaluated insulin-like growth factor 1 (IGF-1) mediated hypertrophy in the presence and absence of MMP-13. We employed live-cell imaging and 3D migration measurements on primary myoblasts obtained from these animals. Outcome measures included muscle morphology and function. RESULTS: Under basal conditions, Mmp13-/- mice did not exhibit histological or functional deficits in muscle. However, following acute injury, regeneration was impaired at 11 and 14 days post injury. Muscle hypertrophy caused by increased IGF-1 was blunted with minimal satellite cell incorporation in the absence of MMP-13. Mmp13-/- primary myoblasts displayed reduced migratory capacity in 2D and 3D, while maintaining normal proliferation and differentiation. Satellite cell specific deletion of MMP-13 recapitulated the effects of global MMP-13 ablation on muscle regeneration, growth and myoblast movement. CONCLUSION: These results show that satellite cells provide an essential autocrine source of MMP-13, which not only regulates their migration, but also supports postnatal growth and resolution of acute damage.

Skeletal muscle explants: ex-vivo models to study cellular behavior in a complex tissue environment.

Purpose/Aim: Skeletal muscle tissue explants have been cultured and studied for nearly 100 years. These cultures, which retain complex tissue structure in an environment suited to precision manipulation and measurement, have led to seminal discoveries of the extrinsic and intrinsic mechanisms regulating contractility, metabolism and regeneration. This review discusses the two primary models of muscle explant: isolated myofiber and intact muscle.Materials and Methods: Relevant literature was reviewed and synthesized with a focus on the unique challenges and capabilities of each explant model.Results: Impactful past, current and future novel applications are discussed.Conclusions: Experiments using skeletal muscle explants have been integral to our understanding of the fundamentals of muscle physiology. As they are refined and adapted, they are poised to continue to inform the field for years to come.

Mechanosensing of matrix by stem cells: From matrix heterogeneity, contractility, and the nucleus in pore-migration to cardiogenesis and muscle stem cells in vivo.

Stem cells are particularly 'plastic' cell types that are induced by various cues to become specialized, tissue-functional lineages by switching on the expression of specific gene programs. Matrix stiffness is among the cues that multiple stem cell types can sense and respond to. This seminar-style review focuses on mechanosensing of matrix elasticity in the differentiation or early maturation of a few illustrative stem cell types, with an intended audience of biologists and physical scientists. Contractile forces applied by a cell's acto-myosin cytoskeleton are often resisted by the extracellular matrix and transduced through adhesions and the cytoskeleton ultimately into the nucleus to modulate gene expression. Complexity is added by matrix heterogeneity, and careful scrutiny of the evident stiffness heterogeneity in some model systems resolves some controversies concerning matrix mechanosensing. Importantly, local stiffness tends to dominate, and 'durotaxis' of stem cells toward stiff matrix reveals a dependence of persistent migration on myosin-II force generation and also rigid microtubules that confer directionality. Stem and progenitor cell migration in 3D can be further affected by matrix porosity as well as stiffness, with nuclear size and rigidity influencing niche retention and fate choices. Cell squeezing through rigid pores can even cause DNA damage and genomic changes that contribute to de-differentiation toward stem cell-like states. Contraction of acto-myosin is the essential function of striated muscle, which also exhibit mechanosensitive differentiation and maturation as illustrated in vivo by beating heart cells and by the regenerative mobilization of skeletal muscle stem cells.

Stem Cell Differentiation is Regulated by Extracellular Matrix Mechanics.

Stem cells mechanosense the stiffness of their microenvironment, which impacts differentiation. Although tissue hydration anti-correlates with stiffness, extracellular matrix (ECM) stiffness is clearly transduced into gene expression via adhesion and cytoskeleton proteins that tune fates. Cytoskeletal reorganization of ECM can create heterogeneity and influence fates, with fibrosis being one extreme.