Graham G. Walmsley

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.

Transcriptional Program Induced by Wnt Protein in Human Fibroblasts Suggests Mechanisms for Cell Cooperativity in Defining Tissue Microenvironments

Background The Wnt signaling system plays key roles in development, regulation of stem cell self-renewal and differentiation, cell polarity, morphogenesis and cancer. Given the multifaceted roles of Wnt signaling in these processes, its transcriptional effects on the stromal cells that make up the scaffold and infrastructure of epithelial tissues are of great interest. Methods and Results To begin to investigate these effects, we used DNA microarrays to identify transcriptional targets of the Wnt pathway in human lung fibroblasts. Cells were treated with active Wnt3a protein in culture, and RNA was harvested at 4 hours and 24 hours. Nuclear accumulation of ß-Catenin, as shown by immunofluorescence, and induction of AXIN2 demonstrate that fibroblasts are programmed to respond to extracellular Wnt signals. In addition to several known Wnt targets, we found many new Wnt induced genes, including many transcripts encoding regulatory proteins. Transcription factors with important developmental roles, including HOX genes, dominated the early transcriptional response. Furthermore, we found differential expression of several genes that play direct roles in the Wnt signaling pathway, as well as genes involved in other cell signaling pathways including fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) signaling. The gene most highly induced by Wnt3a was GREMLIN2, which encodes a secreted BMP antagonist. Conclusions Elevated expression of GREMLIN2 suggests a new role for Wnt signals in the maintenance of stem cell niches, whereby Wnt signals induce nearby fibroblasts to produce a BMP antagonist, inhibiting differentiation and promoting expansion of stem cells in their microenvironment. We suggest that Wnt-induced changes in the gene expression program of local stromal cells may play an important role in the establishment of specialized niches hospitable to the self-renewal of normal or malignant epithelial stem cells in vivo.

Clonal precursor of bone, cartilage, and hematopoietic niche stromal cells

Organs are composites of tissue types with diverse developmental origins, and they rely on distinct stem and progenitor cells to meet physiological demands for cellular production and homeostasis. How diverse stem cell activity is coordinated within organs is not well understood. Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells.

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.

Biomaterials for Craniofacial Bone Engineering

Conditions such as congenital anomalies, cancers, and trauma can all result in devastating deficits of bone in the craniofacial skeleton. This can lead to significant alteration in function and appearance that may have significant implications for patients. In addition, large bone defects in this area can pose serious clinical dilemmas, which prove difficult to remedy, even with current gold standard surgical treatments. The craniofacial skeleton is complex and serves important functional demands. The necessity to develop new approaches for craniofacial reconstruction arises from the fact that traditional therapeutic modalities, such as autologous bone grafting, present myriad limitations and carry with them the potential for significant complications. While the optimal bone construct for tissue regeneration remains to be elucidated, much progress has been made in the past decade. Advances in tissue engineering have led to innovative scaffold design, complemented by progress in the understanding of stem cell–based therapy and growth factor enhancement of the healing cascade. This review focuses on the role of biomaterials for craniofacial bone engineering, highlighting key advances in scaffold design and development.

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.

Isolation of Human Adipose-Derived Stromal Cells Using Laser-Assisted Liposuction and Their Therapeutic Potential in Regenerative Medicine

Harvesting adipose‐derived stromal cells (ASCs) for tissue engineering is frequently done through liposuction. However, several different techniques exist. Although third‐generation ultrasound‐assisted liposuction has been shown to not have a negative effect on ASCs, the impact of laser‐assisted liposuction on the quality and differentiation potential of ASCs has not been studied. Therefore, ASCs were harvested from laser‐assisted lipoaspirate and suction‐assisted lipoaspirate. Next, in vitro parameters of cell yield, cell viability and proliferation, surface marker phenotype, osteogenic differentiation, and adipogenic differentiation were performed. Finally, in vivo bone formation was assessed using a critical‐sized cranial defect in athymic nude mice. Although ASCs isolated from suction‐assisted lipoaspirate and laser‐assisted lipoaspirate both successfully underwent osteogenic and adipogenic differentiation, the cell yield, viability, proliferation, and frequency of ASCs (CD34+CD31−CD45−) in the stromal vascular fraction were all significantly less with laser‐assisted liposuction in vitro (p < .05). In vivo, quantification of osseous healing by micro‐computed tomography revealed significantly more healing with ASCs isolated from suction‐assisted lipoaspirate relative to laser‐assisted lipoaspirate at the 4‐, 6‐, and 8‐week time points (p < .05). Therefore, as laser‐assisted liposuction appears to negatively impact the biology of ASCs, cell harvest using suction‐assisted liposuction is preferable for tissue‐engineering purposes.

Transdermal deferoxamine prevents pressure-induced diabetic ulcers

Significance Diabetes is the leading cause of nontraumatic amputations. There are no effective therapies to prevent diabetic ulcer formation and only modestly effective technologies to help with their healing. To enhance diabetic wound healing we designed a transdermal delivery system containing the FDA-approved small molecule deferoxamine, an iron chelator that increases defective hypoxia inducible factor-1 alpha transactivation in diabetes by preventing iron-catalyzed reactive oxygen stress. This system overcomes the challenge of delivering hydrophilic molecules through the normally impermeable stratum corneum and both prevents diabetic ulcer formation and improves the healing of existing diabetic wounds. This represents a prophylactic pharmacological agent to prevent ulcer formation that is rapidly translatable into the clinic and has the potential to ultimately transform the care and prevention of diabetic complications. There is a high mortality in patients with diabetes and severe pressure ulcers. For example, chronic pressure sores of the heels often lead to limb loss in diabetic patients. A major factor underlying this is reduced neovascularization caused by impaired activity of the transcription factor hypoxia inducible factor-1 alpha (HIF-1α). In diabetes, HIF-1α function is compromised by a high glucose-induced and reactive oxygen species-mediated modification of its coactivator p300, leading to impaired HIF-1α transactivation. We examined whether local enhancement of HIF-1α activity would improve diabetic wound healing and minimize the severity of diabetic ulcers. To improve HIF-1α activity we designed a transdermal drug delivery system (TDDS) containing the FDA-approved small molecule deferoxamine (DFO), an iron chelator that increases HIF-1α transactivation in diabetes by preventing iron-catalyzed reactive oxygen stress. Applying this TDDS to a pressure-induced ulcer model in diabetic mice, we found that transdermal delivery of DFO significantly improved wound healing. Unexpectedly, prophylactic application of this transdermal delivery system also prevented diabetic ulcer formation. DFO-treated wounds demonstrated increased collagen density, improved neovascularization, and reduction of free radical formation, leading to decreased cell death. These findings suggest that transdermal delivery of DFO provides a targeted means to both prevent ulcer formation and accelerate diabetic wound healing with the potential for rapid clinical translation.