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Tissue fibrosis is the deposition of excessive extracellular matrix and can occur as part of the body's natural wound healing process upon injury, or as a consequence of diseases such as systemic sclerosis. Skin fibrosis contributes to significant morbidity due to the prevalence of injuries resulting from trauma and burn. Fibroblasts, the principal cells of the dermis, synthesize extracellular matrix to maintain the skin during homeostasis and also play a pivotal role in all stages of wound healing. Although it was previously believed that fibroblasts are homogeneous and mostly quiescent cells, it has become increasingly recognized that numerous fibroblast subtypes with unique functions and morphologies exist. This Review provides an overview of fibroblast heterogeneity in the mammalian dermis. We explain how fibroblast identity relates to their developmental origin, anatomical site and precise location within the skin tissue architecture in both human and mouse dermis. We discuss current evidence for the varied functionality of fibroblasts within the dermis and the relationships between fibroblast subtypes, and explain the current understanding of how fibroblast subpopulations may be controlled through transcriptional regulatory networks and paracrine communications. We consider how fibroblast heterogeneity can influence wound healing and fibrosis, and how insight into fibroblast heterogeneity could lead to novel therapeutic developments and targets for skin fibrosis. Finally, we contemplate how future studies should be shaped to implement knowledge of fibroblast heterogeneity into clinical practice in order to lessen the burden of skin fibrosis.

The regrowth of amputated limbs and the distal tips of digits represent models of tissue regeneration in amphibians, fish and mice. For decades it had been assumed that limb regeneration derived from the blastema, an undifferentiated pluripotent cell population thought to be derived from mature cells via dedifferentiation. Here we show that a wide range of tissue stem/progenitor cells contribute towards the restoration of the mouse distal digit. Genetic fate mapping and clonal analysis of individual cells revealed that these stem cells are lineage restricted, mimicking digit growth during development. Transplantation of cyan-fluorescent-protein-expressing haematopoietic stem cells, and parabiosis between genetically marked mice, confirmed that the stem/progenitor cells are tissue resident, including the cells involved in angiogenesis. These results, combined with those from appendage regeneration in other vertebrate subphyla, collectively demonstrate that tissue stem cells rather than pluripotent blastema cells are an evolutionarily conserved cellular mode for limb regeneration after amputation. Stem cells needed in digit tip regrowth Newborn and adult mice are able to regrow forelimb and hindlimb digit tips after their amputation through the distal interphalangeal joint, but the cellular origin of the regenerated tissue in adult mice has been unclear. Using in vivo genetic fate-mapping techniques, Rinkevich et al . show that a range of tissue-specific stem cells contribute to the restoration of the mouse's distal phalanx. These findings, together with work in chick and salamander limb-regeneration models, suggest that lineage restriction of tissue stem cells — as opposed to dedifferentiation into a multipotent cell state — is evolutionarily conserved.

In the skin, tissue injury results in fibrosis in the form of scars composed of dense extracellular matrix deposited by fibroblasts. The therapeutic goal of regenerative wound healing has remained elusive, in part because principles of fibroblast programming and adaptive response to injury remain incompletely understood. Here, we present a multimodal -omics platform for the comprehensive study of cell populations in complex tissue, which has allowed us to characterize the cells involved in wound healing across both time and space. We employ a stented wound model that recapitulates human tissue repair kinetics and multiple Rainbow transgenic lines to precisely track fibroblast fate during the physiologic response to skin injury. Through integrated analysis of single cell chromatin landscapes and gene expression states, coupled with spatial transcriptomic profiling, we are able to impute fibroblast epigenomes with temporospatial resolution. This has allowed us to reveal potential mechanisms controlling fibroblast fate during migration, proliferation, and differentiation following skin injury, and thereby reexamine the canonical phases of wound healing. These findings have broad implications for the study of tissue repair in complex organ systems.

Human adipose-derived stromal cells (hASCs) represent a multipotent cell stromal cell type with proven capacity to differentiate along an osteogenic lineage. This suggests that they may be used to heal defects of the craniofacial or appendicular skeleton. We sought to substantiate the use of undifferentiated hASCs in the regeneration of a non-healing mouse skeletal defect. Human ASCs were harvested from female lipoaspirate. Critical-sized (4 mm) calvarial defects were created in the parietal bone of adult male nude mice. Defects were either left empty, treated with an apatite coated PLGA scaffold alone, or a scaffold with human ASCs. MicroCT scans were obtained at stratified time points post-injury. Histology, in situ hybridization, and histomorphometry were performed. Near complete healing was observed among hASC engrafted calvarial defects. This was in comparison to control groups that showed little healing (*P<0.01). Human ASCs once engrafted differentiate down an osteogenic lineage, determined by qRT-PCR and histological co-expression assays using GFP labeled cells. ASCs were shown to persist within a defect site for two weeks (shown by sex chromosome analysis and quantified using Luciferase+ ASCs). Finally, rBMP-2 was observed to increase hASC osteogenesis in vitro and osseous healing in vivo. Human ASCs ossify critical sized mouse calvarial defects without the need for pre-differentiation. Recombinant differentiation factors such as BMP-2 may be used to supplement hASC mediated repair. Interestingly, ASC presence gradually dissipates from the calvarial defect site. This study supports the potential translation for ASC use in the treatment of human skeletal defects.

Cutaneous wound repair is a highly coordinated cascade of cellular responses to injury which restores the epidermal integrity and its barrier functions. Even under optimal healing conditions, normal wound repair of adult human skin is imperfect and delayed healing and scarring are frequent occurrences. Dysregulated wound healing is a major concern for global healthcare, and, given the rise in diabetic and aging populations, this medicoeconomic disease burden will continue to rise. Therapies to reliably improve nonhealing wounds and reduce scarring are currently unavailable. Mesenchymal stromal cells (MSCs) have emerged as a powerful technique to improve skin wound healing. Their differentiation potential, ease of harvest, low immunogenicity, and integral role in native wound healing physiology make MSCs an attractive therapeutic remedy. MSCs promote cell migration, angiogenesis, epithelialization, and granulation tissue formation, which result in accelerated wound closure. MSCs encourage a regenerative, rather than fibrotic, wound healing microenvironment. Recent translational research efforts using modern bioengineering approaches have made progress in creating novel techniques for stromal cell delivery into healing wounds. This paper discusses experimental applications of various stromal cells to promote wound healing and discusses the novel methods used to increase MSC delivery and efficacy.

Tissue repair and healing remain among the most complicated processes that occur during postnatal life. Humans and other large organisms heal by forming fibrotic scar tissue with diminished function, while smaller organisms respond with scarless tissue regeneration and functional restoration. Well-established scaling principles reveal that organism size exponentially correlates with peak tissue forces during movement, and evolutionary responses have compensated by strengthening organ-level mechanical properties. How these adaptations may affect tissue injury has not been previously examined in large animals and humans. Here, we show that blocking mechanotransduction signaling through the focal adhesion kinase pathway in large animals significantly accelerates wound healing and enhances regeneration of skin with secondary structures such as hair follicles. In human cells, we demonstrate that mechanical forces shift fibroblasts toward pro-fibrotic phenotypes driven by ERK-YAP activation, leading to myofibroblast differentiation and excessive collagen production. Disruption of mechanical signaling specifically abrogates these responses and instead promotes regenerative fibroblast clusters characterized by AKT-EGR1. Humans and other large mammals heal wounds by forming fibrotic scar tissue with diminished function. Here, the authors show that disrupting mechanotransduction through the focal adhesion kinase pathway in large animals accelerates healing, prevents fibrosis, and enhances skin regeneration.

Bone-related maladies are a major health burden on modern society. Loss of skeletal integrity and regeneration capacity through aging, obesity, and disease follows from a detrimental shift in bone formation and resorption dynamics. Targeting tissue-resident adult stem cells offers a potentially innovative paradigm in the development of therapeutic strategies against organ dysfunction. While the essential role of skeletal stem cells (SSCs) for development, growth, and maintenance of the skeleton has been generally established, a common consensus on the exact identity and definition of a pure SSC population remains elusive. The controversies stem from conflicting results between different approaches and criteria for isolation, detection, and functional evaluation; along with the interchangeable usage of the terms SSC and \"mesenchymal stromal/stem cell (MSC)\". A great number of prospective bone-forming stem cell populations have been reported with various characteristic markers, often describing overlapping cell populations with widely unexplored heterogeneity, species specificity, and distribution at distinct skeletal sites, bone regions, and microenvironments, thereby creating confusion that may complicate future advances in the field. In this review, we examine the state-of-the-art knowledge of SSC biology and try to establish a common ground for the definition and terminology of specific bone-resident stem cells. We also discuss recent advances in the identification of highly purified SSCs, which will allow detailed interrogation of SSC diversity and regulation at the single-cell level.

A major issue in the clinic is excessive, or hypertrophic, scarring of the skin after injury. Geoffrey Gurtner and his colleagues have now shown that mechanical forces during such injury upregulates focal adhesion kinase (FAK), which in turn leads to the release of a cytokine that promotes inflammation and fibrosis. They also show that genetic deletion of FAK or its pharmacological inhibition results in minimal scarring in a mouse model. Exuberant fibroproliferation is a common complication after injury for reasons that are not well understood 1 . One key component of wound repair that is often overlooked is mechanical force, which regulates cell-matrix interactions through intracellular focal adhesion components, including focal adhesion kinase (FAK) 1 , 2 . Here we report that FAK is activated after cutaneous injury and that this process is potentiated by mechanical loading. Fibroblast-specific FAK knockout mice have substantially less inflammation and fibrosis than control mice in a model of hypertrophic scar formation. We show that FAK acts through extracellular-related kinase (ERK) to mechanically trigger the secretion of monocyte chemoattractant protein-1 (MCP-1, also known as CCL2), a potent chemokine that is linked to human fibrotic disorders 3 , 4 , 5 . Similarly, MCP-1 knockout mice form minimal scars, indicating that inflammatory chemokine pathways are a major mechanism by which FAK mechanotransduction induces fibrosis. Small-molecule inhibition of FAK blocks these effects in human cells and reduces scar formation in vivo through attenuated MCP-1 signaling and inflammatory cell recruitment. These findings collectively indicate that physical force regulates fibrosis through inflammatory FAK–ERK–MCP-1 pathways and that molecular strategies targeting FAK can effectively uncouple mechanical force from pathologic scar formation.

Adhesions are fibrotic scars that form between abdominal organs following surgery or infection, and may cause bowel obstruction, chronic pain, or infertility. Our understanding of adhesion biology is limited, which explains the paucity of anti-adhesion treatments. Here we present a systematic analysis of mouse and human adhesion tissues. First, we show that adhesions derive primarily from the visceral peritoneum, consistent with our clinical experience that adhesions form primarily following laparotomy rather than laparoscopy. Second, adhesions are formed by poly-clonal proliferating tissue-resident fibroblasts. Third, using single cell RNA-sequencing, we identify heterogeneity among adhesion fibroblasts, which is more pronounced at early timepoints. Fourth, JUN promotes adhesion formation and results in upregulation of PDGFRA expression. With JUN suppression, adhesion formation is diminished. Our findings support JUN as a therapeutic target to prevent adhesions. An anti- JUN therapy that could be applied intra-operatively to prevent adhesion formation could dramatically improve the lives of surgical patients. Abdominal adhesions are a common cause of bowel obstruction, but knowledge regarding adhesion biology and anti-adhesion therapies remains limited. Here the authors report a systematic analysis of mouse and human adhesion tissues demonstrating that visceral fibroblast JUN and associated PDGFRA expression promote adhesions, and JUN suppression can prevent adhesion formation.

Cranial sutures are major growth centers for the calvarial vault, and their premature fusion leads to a pathologic condition called craniosynostosis. This study investigates whether skeletal stem/progenitor cells are resident in the cranial sutures. Prospective isolation by FACS identifies this population with a significant difference in spatio-temporal representation between fusing versus patent sutures. Transcriptomic analysis highlights a distinct signature in cells derived from the physiological closing PF suture, and scRNA sequencing identifies transcriptional heterogeneity among sutures. Wnt-signaling activation increases skeletal stem/progenitor cells in sutures, whereas its inhibition decreases. Crossing Axin2 LacZ/+ mouse, endowing enhanced Wnt activation, to a Twist1 +/− mouse model of coronal craniosynostosis enriches skeletal stem/progenitor cells in sutures restoring patency. Co-transplantation of these cells with Wnt3a prevents resynostosis following suturectomy in Twist1 +/− mice. Our study reveals that decrease and/or imbalance of skeletal stem/progenitor cells representation within sutures may underlie craniosynostosis. These findings have translational implications toward therapeutic approaches for craniosynostosis. Cranial sutures are major growth centers for the skull vault and premature fusion leads to pathological fusion, craniosynostosis. Here the authors isolate Wnt responsive skeletal stem and progenitor cells from sutures, that can be transplanted together with Wnt3a protein to repair craniosynostosis in a mouse model.