Michael S. Hu

Skin fibrosis. Identification and isolation of a dermal lineage with intrinsic fibrogenic potential

Fibroblasts in fibrosis Excess fibrous connective tissue, similar to scarring, forms during the repair of injuries. Fibroblasts are known to be involved, but their role is poorly characterized. Rinkevich et al. identify two lineages of dermal fibroblasts in the dorsal skin of mice (see the Perspective by Sennett and Rendl). A fibrogenic lineage, defined by embryonic expression of Engrailed-1, plays a central role in dermal development, wound healing, radiation-induced fibrosis, and cancer stroma formation. Targeted inhibition of this lineage results in reduced melanoma growth and scar formation, with no effect on the structural integrity of the healed skin, thus indicating therapeutic approaches for treating fibrotic disease. Science, this issue 10.1126/science.aaa2151; see also p. 284 An embryonic fibroblast lineage deposits connective tissue in wounds. [Also see Perspective by Sennett and Rendl] INTRODUCTION Fibroblasts are the predominant cell type that synthesizes and remodels the extracellular matrix in organs during both embryonic and adult life and are central to the fibrotic response across a range of pathologic states. Morphologically, they are most commonly defined as elongated, spindle-shaped cells that readily adhere to and migrate over tissue culture substrates. However, fibroblasts exhibit a variety of shapes and sizes, depending on the physiologic or pathologic state of the host tissue, and represent a heterogeneous population of cells with diverse features that remain largely undefined. In cutaneous tissues, fibroblasts display considerable functional variation during wound repair, depending on developmental time, and between anatomic sites. For example, wounds in the oral cavity remodel with minimal scar formation, whereas scar tissue deposition within cutaneous wounds is substantial. The mechanisms underlying this diversity of regenerative responses in cutaneous tissues have remained largely underexplored. RATIONALE The effective development of treatments for fibrosis depends on a mechanistic understanding of its pathogenesis. The identification and characterization of distinct lineages of fibroblasts, based on functional role, hold potential value for developing therapeutic approaches to fibrosis. We employed a nonselective depletion-based fluorescence-activated cell sorting strategy to isolate fibroblasts from a murine model that labels a particular lineage of cells based on the gene expression of Engrailed-1 (En1) in its embryonic progenitors. Using this reporter mouse, we reveal the presence of at least two functionally distinct embryonic fibroblast lineages in murine dorsal skin and characterize a single lineage that plays a primary role in connective tissue formation. RESULTS Genetic lineage tracing and transplantation assays demonstrate that a single somitic-derived fibroblast lineage that is defined by embryonic expression of En1 is responsible for the bulk of connective tissue deposition during embryonic development, cutaneous wound healing, radiation fibrosis, and cancer stroma formation. Reciprocal transplantation of distinct fibroblast lineages between the dorsal back and oral cavity induces ectopic dermal architectures that mimic their place of origin rather than their site of transplantation. Lineage-specific cell ablation using transgenic-mediated expression of the simian diphtheria toxin receptor in conjunction with localized administration of diphtheria toxin leads to diminished connective tissue deposition in wounds and significantly reduces melanoma growth in the dorsal skin of mice. Tensile strength testing reveals that, although scar formation is significantly reduced in wounds treated with diphtheria toxin to ablate the En1 lineage, as compared with control wounds, tensile strength in lineage-ablated wounds is not significantly affected. Using flow cytometry and in silico approaches, we identify CD26/dipeptidyl peptidase-4 (DPP4) as a surface marker that allows for the isolation of this fibrogenic, scar-forming lineage. Small molecule–based inhibition of CD26/DPP4 enzymatic activity in the wound bed of wild-type mice during wound healing results in diminished cutaneous scarring after excisional wounding. CONCLUSION We have identified multiple lineages of fibroblasts in the dorsal skin. Among these, we have characterized a single lineage responsible for the fibrotic response to injury in the dorsal skin of mice and demonstrated that targeted inhibition of this lineage results in reduced scar formation with no effect on the structural integrity of the healed skin. Furthermore, these studies demonstrate that intra- and intersite diversity of dermal architectures are set embryonically and are maintained postnatally by distinct lineages of fibroblasts in different anatomic locations. These results hold promise for the development of therapeutic approaches to fibrotic disease, wound healing, and cancer progression in humans. Schematic showing reduced scarring with targeted ablation/inhibition of En1 fibroblasts. Fibroblasts derived from embryonic precursors expressing En1 are responsible for most connective tissue deposition in skin fibrosis. Targeted ablation/inhibition of this lineage leads to a reduction in fibrosis during wound repair and tumor stroma formation. These findings may lead to the elimination of scarring and other types of fibrotic tissue disease. Green cells, En1-positive fibroblasts; red cells, En1-negative fibroblasts. CREDIT: SILHOUETTES FROM PHYLOPIC.ORG Dermal fibroblasts represent a heterogeneous population of cells with diverse features that remain largely undefined. We reveal the presence of at least two fibroblast lineages in murine dorsal skin. Lineage tracing and transplantation assays demonstrate that a single fibroblast lineage is responsible for the bulk of connective tissue deposition during embryonic development, cutaneous wound healing, radiation fibrosis, and cancer stroma formation. Lineage-specific cell ablation leads to diminished connective tissue deposition in wounds and reduces melanoma growth. Using flow cytometry, we identify CD26/DPP4 as a surface marker that allows isolation of this lineage. Small molecule–based inhibition of CD26/DPP4 enzymatic activity during wound healing results in diminished cutaneous scarring. Identification and isolation of these lineages hold promise for translational medicine aimed at in vivo modulation of fibrogenic behavior.

Aging disrupts cell subpopulation dynamics and diminishes the function of mesenchymal stem cells

Advanced age is associated with an increased risk of vascular morbidity, attributable in part to impairments in new blood vessel formation. Mesenchymal stem cells (MSCs) have previously been shown to play an important role in neovascularization and deficiencies in these cells have been described in aged patients. Here we utilize single cell transcriptional analysis to determine the effect of aging on MSC population dynamics. We identify an age-related depletion of a subpopulation of MSCs characterized by a pro-vascular transcriptional profile. Supporting this finding, we demonstrate that aged MSCs are also significantly compromised in their ability to support vascular network formation in vitro and in vivo. Finally, aged MSCs are unable to rescue age-associated impairments in cutaneous wound healing. Taken together, these data suggest that age-related changes in MSC population dynamics result in impaired therapeutic potential of aged progenitor cells. These findings have critical implications for therapeutic cell source decisions (autologous versus allogeneic) and indicate the necessity of strategies to improve functionality of aged MSCs.

Diabetes impairs the angiogenic potential of adipose-derived stem cells by selectively depleting cellular subpopulations

IntroductionPathophysiologic changes associated with diabetes impair new blood vessel formation and wound healing. Mesenchymal stem cells derived from adipose tissue (ASCs) have been used clinically to promote healing, although it remains unclear whether diabetes impairs their functional and therapeutic capacity.MethodsIn this study, we examined the impact of diabetes on the murine ASC niche as well as on the potential of isolated cells to promote neovascularization in vitro and in vivo. A novel single-cell analytical approach was used to interrogate ASC heterogeneity and subpopulation dynamics in this pathologic setting.ResultsOur results demonstrate that diabetes alters the ASC niche in situ and that diabetic ASCs are compromised in their ability to establish a vascular network both in vitro and in vivo. Moreover, these diabetic cells were ineffective in promoting soft tissue neovascularization and wound healing. Single-cell transcriptional analysis identified a subpopulation of cells which was diminished in both type 1 and type 2 models of diabetes. These cells were characterized by the high expression of genes known to be important for new blood vessel growth.ConclusionsPerturbations in specific cellular subpopulations, visible only on a single-cell level, represent a previously unreported mechanism for the dysfunction of diabetic ASCs. These data suggest that the utility of autologous ASCs for cell-based therapies in patients with diabetes may be limited and that interventions to improve cell function before application are warranted.

Nanotechnology in bone tissue engineering

UNLABELLED Nanotechnology represents a major frontier with potential to significantly advance the field of bone tissue engineering. Current limitations in regenerative strategies include impaired cellular proliferation and differentiation, insufficient mechanical strength of scaffolds, and inadequate production of extrinsic factors necessary for efficient osteogenesis. Here we review several major areas of research in nanotechnology with potential implications in bone regeneration: 1) nanoparticle-based methods for delivery of bioactive molecules, growth factors, and genetic material, 2) nanoparticle-mediated cell labeling and targeting, and 3) nano-based scaffold construction and modification to enhance physicochemical interactions, biocompatibility, mechanical stability, and cellular attachment/survival. As these technologies continue to evolve, ultimate translation to the clinical environment may allow for improved therapeutic outcomes in patients with large bone deficits and osteodegenerative diseases. FROM THE CLINICAL EDITOR Traditionally, the reconstruction of bony defects has relied on the use of bone grafts. With advances in nanotechnology, there has been significant development of synthetic biomaterials. In this article, the authors provided a comprehensive review on current research in nanoparticle-based therapies for bone tissue engineering, which should be useful reading for clinicians as well as researchers in this field.

Scarless wound healing: chasing the holy grail.

Summary: Over 100 million patients acquire scars in the industrialized world each year, primarily as a result of elective operations. Although undefined, the global incidence of scarring is even larger, extending to significant numbers of burn and other trauma-related wounds. Scars have the potential to exert a profound psychological and physical impact on the individual. Beyond aesthetic considerations and potential disfigurement, scarring can result in restriction of movement and reduced quality of life. The formation of a scar following skin injury is a consequence of wound healing occurring through reparative rather than regenerative mechanisms. In this article, the authors review the basic stages of wound healing; differences between adult and fetal wound healing; various mechanical, genetic, and pharmacologic strategies to reduce scarring; and the biology of skin stem/progenitor cells that may hold the key to scarless regeneration.

Tissue Engineering and Regenerative Repair in Wound Healing

Wound healing is a highly evolved defense mechanism against infection and further injury. It is a complex process involving multiple cell types and biological pathways. Mammalian adult cutaneous wound healing is mediated by a fibroproliferative response leading to scar formation. In contrast, early to mid-gestational fetal cutaneous wound healing is more akin to regeneration and occurs without scar formation. This early observation has led to extensive research seeking to unlock the mechanism underlying fetal scarless regenerative repair. Building upon recent advances in biomaterials and stem cell applications, tissue engineering approaches are working towards a recapitulation of this phenomenon. In this review, we describe the elements that distinguish fetal scarless and adult scarring wound healing, and discuss current trends in tissue engineering aimed at achieving scarless tissue regeneration.

Studies in fat grafting: Part I. Effects of injection technique on in vitro fat viability and in vivo volume retention.

Background: Fat grafting has become increasingly popular for the correction of soft-tissue deficits at many sites throughout the body. Long-term outcomes, however, depend on delivery of fat in the least traumatic fashion to optimize viability of the transplanted tissue. In this study, the authors compare the biological properties of fat following injection using two methods. Methods: Lipoaspiration samples were obtained from five female donors, and cellular viability, proliferation, and lipolysis were evaluated following injection using either a modified Coleman technique or an automated, low-shear device. Comparisons were made to minimally processed, uninjected fat. Volume retention was also measured over 12 weeks after injection of fat under the scalp of immunodeficient mice using either the modified Coleman technique or the Adipose Tissue Injector. Finally, fat grafts were analyzed histologically. Results: Fat viability and cellular proliferation were both significantly greater with the Adipose Tissue Injector relative to injection with the modified Coleman technique. In contrast, significantly less lipolysis was noted using the automated device. In vivo fat volume retention was significantly greater than with the modified Coleman technique at the 4-, 6-, 8-, and 12-week time points. This corresponded to significantly greater histologic scores for healthy fat and lower scores for injury following injection with the device. Conclusion: Biological properties of injected tissues reflect how disruptive and harmful techniques for placement of fat may be, and the authors’ in vitro and in vivo data both support the use of the automated, low-shear devices compared with the modified Coleman technique.

Peripheral Blood-Derived Mesenchymal Stem Cells: Candidate Cells Responsible for Healing Critical-Sized Calvarial Bone Defects

Postnatal tissue‐specific stem/progenitor cells hold great promise to enhance repair of damaged tissues. Many of these cells are retrieved from bone marrow or adipose tissue via invasive procedures. Peripheral blood is an ideal alternative source for the stem/progenitor cells because of its ease of retrieval. We present a coculture system that routinely produces a group of cells from adult peripheral blood. Treatment with these cells enhanced healing of critical‐size bone defects in the mouse calvarium, a proof of principle that peripheral blood‐derived cells can be used to heal bone defects. From these cells, we isolated a subset of CD45− cells with a fibroblastic morphology. The CD45− cells were responsible for most of the differentiation‐induced calcification activity and were most likely responsible for the enhanced healing process. These CD45− fibroblastic cells are plastic‐adherent and exhibit a surface marker profile negative for CD34, CD19, CD11b, lineage, and c‐kit and positive for stem cell antigen 1, CD73, CD44, CD90.1, CD29, CD105, CD106, and CD140α. Furthermore, these cells exhibited osteogenesis, chondrogenesis, and adipogenesis capabilities. The CD45− fibroblastic cells are the first peripheral blood‐derived cells that fulfill the criteria of mesenchymal stem cells as defined by the International Society for Cellular Therapy. We have named these cells “blood‐derived mesenchymal stem cells.”

Cell‐Assisted Lipotransfer Improves Volume Retention in Irradiated Recipient Sites and Rescues Radiation‐Induced Skin Changes

Radiation therapy is not only a mainstay in the treatment of many malignancies but also results in collateral obliteration of microvasculature and dermal/subcutaneous fibrosis. Soft tissue reconstruction of hypovascular, irradiated recipient sites through fat grafting remains challenging; however, a coincident improvement in surrounding skin quality has been noted. Cell‐assisted lipotransfer (CAL), the enrichment of fat with additional adipose‐derived stem cells (ASCs) from the stromal vascular fraction, has been shown to improve fat volume retention, and enhanced outcomes may also be achieved with CAL at irradiated sites. Supplementing fat grafts with additional ASCs may also augment the regenerative effect on radiation‐damaged skin. In this study, we demonstrate the ability for CAL to enhance fat graft volume retention when placed beneath the irradiated scalps of immunocompromised mice. Histologic metrics of fat graft survival were also appreciated, with improved structural qualities and vascularity. Finally, rehabilitation of radiation‐induced soft tissue changes were also noted, as enhanced amelioration of dermal thickness, collagen content, skin vascularity, and biomechanical measures were all observed with CAL compared to unsupplemented fat grafts. Supplementation of fat grafts with ASCs therefore shows promise for reconstruction of complex soft tissue defects following adjuvant radiotherapy. Stem Cells 2016;34:668–673

Epidermal or dermal specific knockout of PHD-2 enhances wound healing and minimizes ischemic injury

Introduction Hypoxia-inducible factor (HIF)-1α, part of the heterodimeric transcription factor that mediates the cellular response to hypoxia, is critical for the expression of multiple angiogenic growth factors, cell motility, and the recruitment of endothelial progenitor cells. Inhibition of the oxygen-dependent negative regulator of HIF-1α, prolyl hydroxylase domain-2 (PHD-2), leads to increased HIF-1α and mimics various cellular and physiological responses to hypoxia. The roles of PHD-2 in the epidermis and dermis have not been clearly defined in wound healing. Methods Epidermal and dermal specific PHD-2 knockout (KO) mice were developed in a C57BL/6J (wild type) background by crossing homozygous floxed PHD-2 mice with heterozygous K14-Cre mice and heterozygous Col1A2-Cre-ER mice to get homozygous floxed PHD-2/heterozygous K14-Cre and homozygous floxed PHD-2/heterozygous floxed Col1A2-Cre-ER mice, respectively. Ten to twelve-week-old PHD-2 KO and wild type (WT) mice were subjected to wounding and ischemic pedicle flap model. The amount of healing was grossly quantified with ImageJ software. Western blot and qRT-PCR was run on protein and RNA from primary cells cultured in vitro. Results qRT-PCR demonstrated a significant decrease of PHD-2 in keratinocytes and fibroblasts derived from tissue specific KO mice relative to control mice (*p<0.05). Western blot analysis showed a significant increase in HIF-1α and VEGF protein levels in PHD-2 KO mice relative to control mice (*p<0.05). PHD-2 KO mice showed significantly accelerated wound closure relative to WT (*p<0.05). When ischemia was analyzed at day nine post-surgery in a flap model, the PHD-2 tissue specific knockout mice showed significantly more viable flaps than WT (*p<0.05). Conclusions PHD-2 plays a significant role in the rates of wound healing and response to ischemic insult in mice. Further exploration shows PHD-2 KO increases cellular levels of HIF-1α and this increase leads to the transcription of downstream angiogenic factors such as VEGF.