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BackgroundPancreatic ductal adenocarcinoma (PDAC) is resistant to many available therapies including immunotherapy because of its highly complex tumor microenvironment (TME). PDAC TME consists of a significant proportion of stromal cells, such as endothelial cells, perivascular cells, and cancer-associated fibroblasts (CAFs). Recent work indicates how CAFs can orchestrate the crosstalk cancer and immune cells, and contribute to many aspects of tumor progression, including angiogenesis, senescence, and inflammation. Recent studies based on scRNA-seq have increased understanding of CAF heterogeneity in PDAC in both human and genetically engineered mouse models (GEMMs) is of high interest. To understand the translatability of GEMMs in the setting of PDAC, we conducted a thorough scRNA-seq meta-analysis on CAFs across GEMMs and PDAC human samples. Hereafter, we characterized CAFs multi-dimensionally based on transcriptional, chromatin accessibility, and spatial profiles. Finally, we suggested certain transcription factors may be regulatory drivers of heterogeneous CAF phenotypes in both human and GEMMs.MethodsWe collected publicly available and internally generated scRNA-seq data of PDAC CAFs from human and mouse. After dataset alignment and label transfer, we conducted differential expression analysis across CAF subsets to characterize myofibroblasts (myCAFs) and other CAF subsets of interest. Bioinformatically, we further interrogated CAF heterogeneity in terms of regulatory potential of transcription factors, gene set enrichment, and functional state transition. Complemented by epigenomic assessment, we investigated chromatin accessibility and transcription factor binding availability on the single-cell level. Finally, to investigate the TME organization and spatial neighborhood of cell-to-cell interaction, we explored potential functional differences across location and transcriptional changes of CAF subsets by spatial transcriptomics.ResultsWe found that myofibroblasts (myCAFs) make up a substantial proportion of the CAF population, in both human and mouse TME. In a combination of transcriptional profiling, chromatin accessibility assessment, and spatial transcriptomics, we elucidated potential functional and phenotypic differences within myCAF population and compared to other CAF subsets in the TME. While myofibroblasts are traditionally described as matrix remodeling related, heterogeneity in myofibroblasts may suggest additional roles played by this specific subset. In addition, CAFs in human and mouse share similarities, in terms of transcriptional profiles and phenotypes. The use of GEMMs facilitates our understanding of CAF heterogenous behavior and phenotypes in the PDAC TME.ConclusionsHere, we presented a comprehensive overview of CAF heterogeneity in mouse PDAC models and human datasets. Our observations highlight molecular differences in CAFs, which facilitates our understanding on PDAC stromal microenvironment and translatability in GEMMs in imitating human TME.

Volumetric muscle loss (VML) overwhelms the innate regenerative capacity of mammalian skeletal muscle (SkM), leading to numerous disabilities and reduced quality of life. Immune cells are critical responders to muscle injury and guide tissue resident stem cell and progenitor mediated myogenic repair. However, how immune cell infiltration and inter-cellular communication networks with muscle stem cells are altered following VML and drive pathological outcomes remains underexplored. Herein, we contrast the cellular and molecular mechanisms of VML injuries that result in fibrotic degeneration or regeneration of SkM. Following degenerative VML injuries, we observe heightened infiltration of natural killer (NK) cells as well as persistence of neutrophils beyond two weeks post injury. Functional validation of NK cells revealed an antagonistic role on neutrophil accumulation in part via inducing apoptosis and CCR1 mediated chemotaxis. The persistent infiltration of neutrophils in degenerative VML injuries was found to contribute to impairments in muscle stem cell regenerative function, which was also attenuated by transforming growth factor beta 1 (TGFβ1). BlockingTGFβsignaling reduced neutrophil accumulation and fibrosis, as well as improved muscle specific force. Collectively, these results enhance our understanding of immune cell-stem cell crosstalk that drives regenerative dysfunction and provide further insight into possible avenues for fibrotic therapy exploration.SINGLE SENTENCE SUMMARYComparison of muscle injuries resulting in regeneration or fibrosis reveals inter-cellular communication between neutrophils and natural killer cells impacts muscle stem cell mediated repair.

Cellular identity and state are determined by a collection of molecular components that are specified during development and stabilized thereafter to maintain and protect tissue functions. Alteration of the molecular elements (gene expression program and chromatin state) as a result of disease or age can induce somatic cells to assume different identities or modulate functions. Therapeutic use of this technique, called 'cellular reprogramming', is very promising for regenerative medicine, but implementation of reprogramming-based strategies in vivo has been precluded by technological and safety limitations. Recent advances in transcriptional control and improved transmembrane delivery strategies now offer exciting potential to more efficiently reprogram cell fates as well as to control the reprogramming timeline and scale of delivery to improve safety.

Specialized pro‐resolving mediators actively limit inflammation and support tissue regeneration, but their role in age‐related muscle dysfunction has not been explored. We profiled the mediator lipidome of aging muscle via liquid chromatography‐tandem mass spectrometry and tested whether treatment with the pro‐resolving mediator resolvin D1 (RvD1) could rejuvenate the regenerative ability of aged muscle. Aged mice displayed chronic muscle inflammation and this was associated with a basal deficiency of pro‐resolving mediators 8‐oxo‐RvD1, resolvin E3, and maresin 1, as well as many anti‐inflammatory cytochrome P450‐derived lipid epoxides. Following muscle injury, young and aged mice produced similar amounts of most pro‐inflammatory eicosanoid metabolites of cyclooxygenase (e.g., prostaglandin E2) and 12‐lipoxygenase (e.g., 12‐hydroxy‐eicosatetraenoic acid), but aged mice produced fewer markers of pro‐resolving mediators including the lipoxins (15‐hydroxy‐eicosatetraenoic acid), D‐resolvins/protectins (17‐hydroxy‐docosahexaenoic acid), E‐resolvins (18‐hydroxy‐eicosapentaenoic acid), and maresins (14‐hydroxy‐docosahexaenoic acid). Similar absences of downstream pro‐resolving mediators including lipoxin A4, resolvin D6, protectin D1/DX, and maresin 1 in aged muscle were associated with greater inflammation, impaired myofiber regeneration, and delayed recovery of strength. Daily intraperitoneal injection of RvD1 had minimal impact on intramuscular leukocyte infiltration and myofiber regeneration but suppressed inflammatory cytokine expression, limited fibrosis, and improved recovery of muscle function. We conclude that aging results in deficient local biosynthesis of specialized pro‐resolving mediators in muscle and that immunoresolvents may be attractive novel therapeutics for the treatment of muscular injuries and associated pain in the elderly, due to positive effects on recovery of muscle function without the negative side effects on tissue regeneration of non‐steroidal anti‐inflammatory drugs., Chronic low‐grade inflammation of aging muscle was associated with a basal deficiency of maresin 1, resolvin E3, 8‐oxo‐resolvin D1, and anti‐inflammatory fatty acid epoxides. Aged mice produced normal amounts of most prostaglandins following muscle injury but were deficient in local biosynthesis of markers of the lipoxins, E‐resolvins, D‐resolvins, and maresins. Systemic treatment with resolvin D1 suppressed inflammatory cytokine expression, limited muscle fibrosis, and improved functional recovery.

Chronic inflammation and deregulated acute immune cell responses to injury contribute to age-associated skeletal muscle dysfunction. Specialized pro-resolving mediators (SPMs) control inflammation and support myofiber regeneration in young mice, but their role in aging muscle remains unknown. Here we examined the effect of age on the mediator lipidome of skeletal muscle via LC-MS based lipidomic profiling and tested whether systemic administration of the SPM resolvin D1 (RvD1) could limit excessive inflammation and improve the regenerative capacity of aged muscle. Aged mice displayed chronic low-grade muscle inflammation prior to injury and this was associated with a basal deficiency of lipoxygenase (LOX) derived SPMs as well as anti-inflammatory cytochrome P450 (CYP) derived lipid epoxides. Following muscle damage, young and aged mice produced similar amounts of pro-inflammatory cyclooxygenase (COX) and 12-LOX metabolites, but aged mice mounted a markedly deficient SPM response. This was associated with heightened leukocyte recruitment, impaired myofiber regeneration, and delayed recovery of strength. Systemic treatment with RvD1 had minimal impact on excessive myeloid cell infiltration and defective myofiber regeneration in aged mice. Nevertheless, RvD1 treatment did suppress inflammatory cytokines, modulated muscle stem cells, limited maladaptive tissue remodeling, and improved recovery of specific muscle force. We conclude that aging results in a marked deficiency of local SPM biosynthesis within muscle and that immunoresolvents may be attractive novel therapeutics for the treatment of muscular injuries and associated pain in the elderly, due to positive effects on recovery of muscle function without the negative side effects on myofiber regeneration of traditional anti-inflammatory treatments.

During aging and neuromuscular diseases, there is a progressive loss of skeletal muscle volume and function in that impacts mobility and quality of life. Muscle loss is often associated with denervation and a loss of resident muscle stem cells (satellite cells or MuSCs), but the relationship between MuSCs and neural control has not been established. Herein, using a combination of single-cell transcriptomic analysis, high-resolution immunofluorescence imaging and transgenic young and aged mice as well as from mice with neuromuscular degeneration (Sod1-/-), a compensatory neuro-responsive function for a subset of MuSCs was identified. Genetic rescue of motor neurons in Sod1-/- mice reduced this subset of MuSCs and restored integrity of the neuromuscular junction (NMJ) in a manner akin to young muscle. Administration of severe neuromuscular trauma induced young MuSCs to specifically engraft in a position proximal to the NMJ but in aging, this behavior was abolished. Contrasting the expression programs of young and aged MuSCs after muscle injury at the single cell level, we observed distinctive gene expression programs between responses to neuro-muscular degeneration and muscle trauma. Collectively, these data reveal MuSCs sense synaptic perturbations during aging and neuro-muscular deterioration, and can exert support for the NMJ, particularly in young muscle.HighlightsTranscriptional landscapes of single satellite cells from different ages before and after injury as well as neurodegenerative models before and after nervous rescueA population of satellite cells reside in close proximity to neuromuscular synapse, which are lost with ageDenervation promotes satellite cell engraftment into post-synaptic regions of young as opposed to aged muscle

Skeletal muscle possesses a remarkable capacity to regenerate when injured, but when confronted with major traumatic injury resulting in volumetric muscle loss (VML), the regenerative process consistently fails. The loss of muscle tissue and function from VML injury has prompted development of a suite of therapeutic approaches but these strategies have proceeded without a comprehensive understanding of the molecular landscape that drives the injury response. Herein, we administered a VML injury in an established rodent model and monitored the evolution of the healing phenomenology over multiple time points using muscle function testing, histology, and expression profiling by RNA sequencing. The injury response was then compared to a regenerative medicine treatment using orthotopic transplantation of autologous minced muscle grafts (~1 mm3 tissue fragments). A chronic inflammatory and fibrotic response was observed at all time points following VML. These results suggest that the pathological response to VML injury during the acute stage of the healing response overwhelms endogenous and therapeutic regenerative processes. Overall, the data presented delineate key molecular characteristics of the pathobiological response to VML injury that are critical effectors of effective regenerative treatment paradigms.

Fibronectin (Fn) is an extracellular matrix protein that orchestrates complex cell adhesion and signaling through cell surface integrin receptors during tissue development, remodeling, and disease, such as fibrosis. Fn is sensitive to mechanical forces in its tandem type III repeats, resulting in extensive molecular enlongation. As such, it has long been hypothesized that cell- and tissue-derived forces may activate an "integrin switch" within the critical integrin-binding ninth and 10th type III repeats-conferring differential integrin-binding specificity, leading to differential cell responses. Yet, no direct evidence exists to prove the hypothesis nor demonstrate the physiological existence of the switch. We report direct experimental evidence for the Fn integrin switch both in vitro and ex vivo using a scFv engineered to detect the transient, force-induced conformational change, representing an opportunity for detection and targeting of early molecular signatures of cell contractile forces in tissue repair and disease.

The Biointerfaces Interlaboratory Committees, the student organization for the Biointerfaces Institute at the University of Michigan, organized and executed an 8 hour “BioHackathon” on the broadly defined clinical topic of ‘Aging.’ The event began with experts in the field (a clinician and an engineer) highlighting the areas of greatest need in which engineers could improve the lives of the aging population. Attendees separated into teams based on shared interests in pursuing specific needs, and rapidly developed need statements and solution models to help the elderly. Solutions ranged from a smart toothbrush to help detect pneumonia in early stages, to small clothing pads to help reduce the impact of falls on hip fractures. Based on a follow-up anonymous internet survey, the attendees indicated they enjoyed the event, and would likely attend a similar event in the future. We conclude by reflecting upon the event, and suggest ways in which we could improve the style of the event for future hackathons.

During aging, there is a progressive loss of volume and function in skeletal muscle that impacts mobility and quality of life. The repair of skeletal muscle is regulated by tissue resident stem cells called satellite cells (MuSCs), but in aging, MuSCs decrease in numbers and regenerative capacity. The transcriptional networks and epigenetic changes that confer diminished regenerative function in MuSCs as a result of natural aging are partially understood. Herein, an integrative genomics approach was utilized to profile MuSCs from young and aged animals before and after injury. Integration of these datasets revealed aging impacts multiple regulatory changes through significant differences in gene expression, metabolic flux, chromatin accessibility and patterns of transcription factor (TF) binding activities. Collectively, these datasets facilitate a deeper understanding of the regulation tissue resident stem cells utilize during aging and healing.

Correction to:Cell Death and Discovery (2018) 4, 33; https://doi.org/10.1038/s41420-018-0027-8; published online 20 February 2018.

Significance: The immune system plays a central role in orchestrating the tissue healing process. Hence, controlling the immune system to promote tissue repair and regeneration is an attractive approach when designing regenerative strategies. This review discusses the pathophysiology of both acute and chronic wounds and possible strategies to control the immune system to accelerate chronic wound closure and promote skin regeneration (scar-less healing) of acute wounds. Recent Advances: Recent studies have revealed the key roles of various immune cells and immune mediators in skin repair. Thus, immune components have been targeted to promote chronic wound repair or skin regeneration and several growth factors, cytokines, and biomaterials have shown promising results in animal models. However, these novel strategies are often struggling to meet efficacy standards in clinical trials, partly due to inadequate drug delivery systems and safety concerns. Critical Issues: Excess inflammation is a major culprit in...