TNF-α Preconditioning Promotes a Proangiogenic Phenotype in hiPSC-Derived Vascular Smooth Muscle Cells.

Introduction: hiPSC-VSMCs have been suggested as therapeutic agents for wound healing and revascularization through the secretion of proangiogenic factors. However, methods of increasing cell paracrine secretion and survivability have thus far yielded inconsistent results. This study investigates the effect of pre-conditioning of hiPSC-VSMCs with TNF-α and their integration into 3D collagen scaffolds on cellular viability and secretome. Methods: hiPSC-VSMCs were dual-plated in a 2D environment. TNF-α was introduced to one plate. Following incubation, cells from each plate were divided and added to type-I collagen scaffolds. TNF-α was introduced to two sets of scaffolds, one from each 2D plate. Following incubation, scaffolds were harvested for their media, tested for cell survivability, cytotoxicity, and imaged. Intra-media VEGF and bFGF levels were evaluated using ELISA testing. Results: hiPSC-VSMCs exposed to TNF-α during collagen scaffold proliferation and preconditioning showed an increase in cell viability and less cytotoxicity compared to non-exposed cells and solely-preconditioned cells. Significant increases in bFGF expression were found in pre-conditioned cell groups with further increases found in cells subsequently exposed during intra-scaffold conditioning. A significant increase in VEGF expression was found in cell groups exposed during both pre-conditioning and intra-scaffold conditioning. Fibroblasts treated with any conditioned media demonstrated increased migration potential. Conclusions: Conditioning hiPSC-VSMCs embedded in scaffolds with TNF-α improves cellular viability and increases the secretion of paracrine factors necessary for wound healing mechanisms such as migration. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00764-0.

Transcriptional heterogeneity in human diabetic foot wounds.

Wound repair requires the coordination of multiple cell types including immune cells and tissue resident cells to coordinate healing and return of tissue function. Diabetic foot ulceration is a type of chronic wound that impacts over 4 million patients in the US and over 7 million worldwide (Edmonds et al., 2021). Yet, the cellular and molecular mechanisms that go awry in these wounds are not fully understood. Here, by profiling chronic foot ulcers from non-diabetic (NDFUs) and diabetic (DFUs) patients using single-cell RNA sequencing, we find that DFUs display transcription changes that implicate reduced keratinocyte differentiation, altered fibroblast function and lineages, and defects in macrophage metabolism, inflammation, and ECM production compared to NDFUs. Furthermore, analysis of cellular interactions reveals major alterations in several signaling pathways that are altered in DFUs. These data provide a view of the mechanisms by which diabetes alters healing of foot ulcers and may provide therapeutic avenues for DFU treatments.

Multifunctional Elastin-Like Polypeptide Fusion Protein Coacervates Inhibit Receptor-Mediated Proinflammatory Signals and Promote Angiogenesis in Mouse Diabetic Wounds.

Objective: Chronic skin wounds are one of the most devastating complications in diabetic patients due to the formation of advanced glycation end-products (AGEs) resulting from nonenzymatic glycation of proteins and lipids in hyperglycemia. AGEs, upon binding their receptors (RAGEs), trigger proinflammatory signals that impair wound healing in diabetes and contribute to the pathology of chronic skin wounds. Approach: We previously developed a recombinant fusion protein containing the binding domain of RAGE (vRAGE) linked to elastin-like polypeptides (ELPs) that acts as a competitive inhibitor of AGEs, and another ELP fusion protein containing stromal cell-derived factor 1 (SDF1) that promotes revascularization. In this study, we report the effects of protein coacervates incorporating both vRAGE-ELP and SDF1-ELP on wound healing in an in vitro diabetes-mimicking cell culture system, and in in vivo in full-thickness wounds on diabetic mice. Results: The combination of vRAGE-ELP and SDF1-ELP increased cell metabolic activity in AGE-stimulated endothelial cells, promoted in vitro tube formation and accelerated healing in an in vitro cell migration assay. When used in a single topical application on full-thickness excisional skin wounds in diabetic mice, wound closure in the combination groups reached almost 100% on postwounding day 35, compared to 62% and 85% on the same days in animals treated with fibrin gel control and vehicle control consisting of ELP alone. Innovation: To our knowledge, this is the first study that attempts to reverse the AGE-RAGE-mediated signaling as well as to promote cell proliferation and vascularization in one single treatment. Conclusion: The codelivery of vRAGE-ELP and SDF1-ELP has potential for the treatment of diabetic wounds.

Human iPSC-Vascular smooth muscle cell spheroids demonstrate size-dependent alterations in cellular viability and secretory function.

Human-induced pluripotent stem cells (hiPSC) and their differentiated vascular cells have been revolutionizing the field of regenerative wound healing. These cells are shown to be rejuvenated with immense potentials in secreting paracrine factors. Recently, hiPSC-derived vascular smooth muscle cells (hiPSC-VSMC) have shown regenerative wound healing ability via their paracrine secretion. The quest to modulate the secretory function of these hiPSC-VSMC is an ongoing effort and involves the use of both biochemical and biophysical stimuli. This study explores the development and optimization of a reproducible, inexpensive protocol to form hiPSC-VSMC derived spheroids to investigate the implications of spheroid size on viability and paracrine secretion. Our data show the successful formation of different sizes of spheroids using various amount of hiPSC-VSMC. The hiPSC-VSMC spheroids formed with 10,000 cells strike an ideal balance between overall cell health and maximal paracrine secretion. The conditioned medium from these spheroids was found to be bioactive in enhancing human dermal fibroblast cell proliferation and migration. This research will inform future studies on the optimal spheroid size for regenerative wound healing applications.

Single cell transcriptomic landscape of diabetic foot ulcers.

Diabetic foot ulceration (DFU) is a devastating complication of diabetes whose pathogenesis remains incompletely understood. Here, we profile 174,962 single cells from the foot, forearm, and peripheral blood mononuclear cells using single-cell RNA sequencing. Our analysis shows enrichment of a unique population of fibroblasts overexpressing MMP1, MMP3, MMP11, HIF1A, CHI3L1, and TNFAIP6 and increased M1 macrophage polarization in the DFU patients with healing wounds. Further, analysis of spatially separated samples from the same patient and spatial transcriptomics reveal preferential localization of these healing associated fibroblasts toward the wound bed as compared to the wound edge or unwounded skin. Spatial transcriptomics also validates our findings of higher abundance of M1 macrophages in healers and M2 macrophages in non-healers. Our analysis provides deep insights into the wound healing microenvironment, identifying cell types that could be critical in promoting DFU healing, and may inform novel therapeutic approaches for DFU treatment.

Unlocking the Potential of Induced Pluripotent Stem Cells for Wound Healing: The Next Frontier of Regenerative Medicine.

Significance: Nonhealing wounds are a significant burden for the health care system all over the world. Existing treatment options are not enough to promote healing, highlighting the urgent need for improved therapies. In addition, the current advancements in tissue-engineered skin constructs and stem cell-based therapies are facing significant hurdles due to the absence of a renewable source of functional cells. Recent Advances: Induced pluripotent stem cell technology (iPSC) is emerging as a novel tool to develop the next generation of personalized medicine for the treatment of chronic wounds. The iPSC provides unlimited access to various skin cells to generate complex personalized three-dimensional skin constructs for disease modeling and autologous grafts. Furthermore, the iPSC-based therapies can target distinct wound healing phases and have shown accelerating wound closure by enhancing angiogenesis, cell migration, tissue regeneration, and modulating inflammation. Critical Issues: Since the last decade, iPSC has been revolutionizing the field of wound healing and skin tissue engineering. Despite the current progress, safety and heterogeneity among iPSC lines are still major hurdles in addition to the lack of large animal studies. These challenges need to be addressed before translating an iPSC-based therapy to the clinic. Future Directions: Future considerations should be given to performing large animal studies to check the safety and efficiency of iPSC-based therapy in a wound healing setup. Furthermore, strategies should be developed to overcome variation between hiPSC lines, develop an efficient manufacturing process for iPSC-derived products, and generate complex skin constructs with vasculature and skin appendages.

Generation and Encapsulation of Human iPSC-Derived Vascular Smooth Muscle Cells for Proangiogenic Therapy.

iPSC technology is revolutionizing the field of regenerative medicine. The generation of patient-specific cells has huge potential for disease modeling as well as for clinical applications. iPSCs have been used as a renewable source of vascular cells, and in particular vascular smooth muscle cells. The use of these human iPSC-derived vascular smooth muscle cells is attractive for vascular tissue engineering. The cells are used in developing vascular grafts as well as in engineering disease models. Recent studies have shown the proangiogenic potentials of human iPSC-derived vascular smooth muscle cells in treating wounds. Here, we describe the VSMC differentiation protocol from human iPSCs and encapsulation methods in collagen scaffolds to promote proangiogenic potentials.

Human iPSC-Derived Vascular Smooth Muscle Cells in a Fibronectin Functionalized Collagen Hydrogel Augment Endothelial Cell Morphogenesis.

Tissue-engineered constructs have immense potential as autologous grafts for wound healing. Despite the rapid advancement in fabrication technology, the major limitation is controlling angiogenesis within these constructs to form a vascular network. Here, we aimed to develop a 3D hydrogel that can regulate angiogenesis. We tested the effect of fibronectin and vascular smooth muscle cells derived from human induced pluripotent stem cells (hiPSC-VSMC) on the morphogenesis of endothelial cells. The results demonstrate that fibronectin increases the number of EC networks. However, hiPSC-VSMC in the hydrogel further substantiated the number and size of EC networks by vascular endothelial growth factor and basic fibroblast growth factor secretion. A mechanistic study shows that blocking αvß3 integrin signaling between hiPSC-VSMC and fibronectin impacts the EC network formation via reduced cell viability and proangiogenic growth factor secretion. Collectively, this study set forth initial design criteria in developing an improved pre-vascularized construct.

Integrin ß3 targeting biomaterial preferentially promotes secretion of bFGF and viability of iPSC-derived vascular smooth muscle cells.

Human-induced pluripotent stem cell-derived-vascular smooth muscle cells (hiPSC-VSMC) and their secretome have been shown to promote angiogenesis and wound healing. However, there is a paucity of research on how the extracellular matrix (ECM) microenvironment may impact the hiPSC-VSMC's functions. In this study, we investigated the effect of specific ECM ligand-integrin interaction on hiPSC-VSMC's paracrine secretion, cell viability, and morphology. Here, we show precise modulation of hiPSC-VSMC in a fibronectin functionalized fibrillar collagen scaffold by targeting their integrin ß3. The secretion of proangiogenic growth factor, basic fibroblast growth factor (bFGF) was found to be fibronectin-dependent via αvß3 integrin interactions. In addition, our data show the possible role of a positive feedback loop between integrin ß3, bFGF, and matrix metalloproteinase-2 in regulating hiPSC-VSMC's morphology and cell viability. Finally, the secretome with enhanced bFGF shows potential for future wound healing applications.

Self-assembled elastin-like polypeptide fusion protein coacervates as competitive inhibitors of advanced glycation end-products enhance diabetic wound healing.

Chronic and non-healing skin wounds are some of the most significant complications in patients with advanced diabetes. A contributing mechanism to this pathology is the non-enzymatic glycation of proteins due to hyperglycemia, leading to the formation of advanced glycation end products (AGEs). AGEs bind to the receptor for AGEs (RAGE), which triggers pro-inflammatory signals that may inhibit the proliferative phase of wound healing. Soluble forms of RAGE (sRAGE) may be used as a competitive inhibitor of AGE-mediated signaling; however, sRAGE is short-lived in the highly proteolytic wound environment. We developed a recombinant fusion protein containing the binding domain of RAGE (vRAGE) linked to elastin-like polypeptides (ELPs) that self-assembles into coacervates at around 30-31 °C. The coacervate size was concentration and temperature-dependent, ranging between 500 and 1600 nm. vRAGE-ELP reversed several AGE-mediated changes in cultured human umbilical vein endothelial cells, including a decrease in viable cell number, an increase in levels of reactive oxygen species (ROS), and an increased expression of the pro-inflammatory marker, intercellular adhesion molecule-1 (ICAM-1). vRAGE-ELP was stable in elastase in vitro for 7 days. When used in a single topical application on full-thickness excisional skin wounds in diabetic mice, wound closure was accelerated, with 90% and 100% wound closure on post-wounding days 28 and 35, respectively, compared to 62% and 85% on the same days in animals treated with vehicle control, consisting of ELP alone. This coacervate system topically delivering a competitive inhibitor of AGEs has potential for the treatment of diabetic wounds.

Self-Assembled Nanomaterials for Chronic Skin Wound Healing.

Significance: Chronic wounds are one of the major burdens of the U.S. health care system with an annual cost of $31.7 billion and affecting an estimated 2.4-4.5 million people. Several underlying molecular and cellular pathophysiological mechanisms, including poor vascularization, excessive extracellular matrix (ECM) degradation by proteases, decreased growth factor activity, and bacterial infection can lead to chronic wounds. More effective wound therapies need to address one or more of these mechanisms to significantly advance wound care. Recent Advances: Self-assembled nanomaterials may provide new therapeutic options for chronic wound healing applications as those materials generally exhibit excellent biocompatibility and can bear multiple functionalities, such as ECM-mimicking properties, drug delivery capabilities, and tunable mechanics. Furthermore, self-assembled nanomaterials can be produced at low cost, and owing to their ability to self-organize, generate complex multifunctional structures that can be tailored to the varying sizes and shapes of chronic wounds. Self-assembled nanomaterials have been engineered to serve as wound dressings, growth factor delivery systems, and antimicrobials. Critical Issues: As there are many different types of self-assembled nanomaterials, which in turn have different mechanisms of self-assembly and physiochemical properties, one type of self-assembled nanomaterials may not be sufficient to address all underlying mechanisms of chronic wounds. However, self-assembled nanomaterials can be easily tailored, and developing multifunctional self-assembled nanomaterials that can address various targets in chronic wounds will be needed. Future Directions: Future studies should investigate combinations of various self-assembled nanomaterials to take full advantage of their multifunctional properties.

An in situ collagen-HA hydrogel system promotes survival and preserves the proangiogenic secretion of hiPSC-derived vascular smooth muscle cells.

Human-induced pluripotent stem cell-derived vascular smooth muscle cells (hiPSC-VSMCs) with proangiogenic properties have huge therapeutic potential. While hiPSC-VSMCs have already been utilized for wound healing using a biomimetic collagen scaffold, an in situ forming hydrogel mimicking the native environment of skin offers the promise of hiPSC-VSMC mediated repair and regeneration. Herein, the impact of a collagen type-I-hyaluronic acid (HA) in situ hydrogel cross-linked using a polyethylene glycol-based cross-linker on hiPSC-VSMCs viability and proangiogenic paracrine secretion was investigated. Our study demonstrated increases in cell viability, maintenance of phenotype and proangiogenic growth factor secretion, and proangiogenic activity in response to the conditioned medium. The optimally cross-linked and functionalized collagen type-I/HA hydrogel system developed in this study shows promise as an in situ hiPSC-VSMC carrier system for wound regeneration.

Induced pluripotent stem cell-derived smooth muscle cells increase angiogenesis and accelerate diabetic wound healing.

Aim: To assess the potential of human induced pluripotent stem cell-derived smooth muscle cells (hiPSC-SMC) to accelerate diabetic wound healing. Methods: hiPSC-SMC were embedded in 3D collagen scaffolds and cultured in vitro for 72 h; scaffolds were then applied to diabetic, nude mouse, splinted back wounds to assess in vivo healing. Cultured medium after scaffold incubation was collected and analyzed for expression of pro-angiogenic cytokines. Results: hiPSC-SMC secrete increased concentration of pro-angiogenic cytokines, compared with murine adipose derived stem cells. Delivery of hiPSC-SMC-containing collagen scaffolds accelerates diabetic wound healing and is associated with an increased number of total and M2 type macrophages. Conclusion: hiPSC-SMC promote angiogenesis and accelerate diabetic wound healing, making them a promising new candidate for treatment of diabetic wounds.

A Dense Fibrillar Collagen Scaffold Differentially Modulates Secretory Function of iPSC-Derived Vascular Smooth Muscle Cells to Promote Wound Healing.

The application of human-induced pluripotent stem cells (hiPSCs) to generate vascular smooth muscle cells (hiPSC-VSMCs) in abundance is a promising strategy for vascular regeneration. While hiPSC-VSMCs have already been utilized for tissue-engineered vascular grafts and disease modeling, there is a lack of investigations exploring their therapeutic secretory factors. The objective of this manuscript was to understand how the biophysical property of a collagen-based scaffold dictates changes in the secretory function of hiPSC-VSMCs while developing hiPSC-VSMC-based therapy for durable regenerative wound healing. We investigated the effect of collagen fibrillar density (CFD) on hiPSC-VSMC's paracrine secretion and cytokines via the construction of varying density of collagen scaffolds. Our study demonstrated that CFD is a key scaffold property that modulates the secretory function of hiPSC-VSMCs. This study lays the foundation for developing collagen-based scaffold materials for the delivery of hiPSC-VSMCs to promote regenerative healing through guiding paracrine signaling pathways.

Targeting Fibrotic Signaling: A Review of Current Literature and Identification of Future Therapeutic Targets to Improve Wound Healing.

Fibrosis is a consequence of aberrant wound healing processes that can be debilitating for patients and often are associated with highly morbid disease processes. Myofibroblasts play an important role in determining an appropriate physiologic response to tissue injury or an excessive response leading to fibrosis. Specifically, "supermature" focal adhesions, α-smooth muscle actin, and the myocardin-related transcription factor/serum response factor pathway likely play a significant role in the differentiation and survival of myofibroblasts in fibrotic lesions. Thus, targeting each of these and disrupting their functioning could lead to the development of therapeutic options for patients suffering from fibrosis and other sequelae of dysregulated wound healing. In this paper, we review the current literature concerning the roles of these three constituents of fibrotic signaling pathways, work already done in attempting to regulate these processes, and discuss the potential of these biomolecular constituents as therapeutic targets in future translational research.

The potential and limitations of induced pluripotent stem cells to achieve wound healing.

Wound healing is the physiologic response to a disruption in normal skin architecture and requires both spatial and temporal coordination of multiple cell types and cytokines. This complex process is prone to dysregulation secondary to local and systemic factors such as ischemia and diabetes that frequently lead to chronic wounds. Chronic wounds such as diabetic foot ulcers are epidemic with great cost to the healthcare system as they heal poorly and recur frequently, creating an urgent need for new and advanced therapies. Stem cell therapy is emerging as a potential treatment for chronic wounds, and adult-derived stem cells are currently employed in several commercially available products; however, stem cell therapy is limited by the need for invasive harvesting techniques, immunogenicity, and limited cell survival in vivo. Induced pluripotent stem cells (iPSC) are an exciting cell type with enhanced therapeutic and translational potential. iPSC are derived from adult cells by in vitro induction of pluripotency, obviating the ethical dilemmas surrounding the use of embryonic stem cells; they are harvested non-invasively and can be transplanted autologously, reducing immune rejection; and iPSC are the only cell type capable of being differentiated into all of the cell types in healthy skin. This review focuses on the use of iPSC in animal models of wound healing including limb ischemia, as well as their limitations and methods aimed at improving iPSC safety profile in an effort to hasten translation to human studies.

Incisional Negative Pressure Wound Therapy Augments Perfusion and Improves Wound Healing in a Swine Model Pilot Study.

BACKGROUND: A commonly used treatment for open wounds, negative pressure wound therapy (NPWT) has recently been used to optimize wound healing in the setting of surgically closed wounds; however, the specific mechanisms of action by which NPWT may benefit patients after surgery remain unknown. Using a swine wound healing model, the current study investigates angiogenesis as a candidate mechanism. METHODS: Multiple excisional wounds were created on the dorsa of 10 male Yorkshire pigs and closed by primary suture. The closed wounds underwent treatment with either NPWT dressing or control dressings in the absence of negative pressure. Dressings were maintained for 8 days followed by euthanasia of the animal. Scar evaluation of the wounds by photographic analysis was performed, and wounds were analyzed for angiogenesis markers by enzyme-linked immunosorbent assay and immunohistochemistry. RESULTS: Scar evaluation scores were observed to be significantly higher for the NPWT-treated sites compared with the control sites (P < 0.05). The enzyme-linked immunosorbent assay results demonstrated increases for vascular endothelial growth factor (VEGF) staining at the incision site treated with NPWT compared with other treatment groups (P < 0.05). In addition, an approximately 3-fold elevation in VEGF expression was observed at the NPWT-treated sites (2.8% vs. 1%, respectively; P < 0.0001).). However, there was no significant difference in immunohistochemistry staining. CONCLUSIONS: The use of NPWT improves the appearance of wounds and appears to increase VEGF expression after 8 days in the setting of a closed excisional wound model, suggesting that improved angiogenesis is one mechanism by which NPWT optimizes wound healing when applied to closed surgical wound sites.

Mouse Model of Pressure Ulcers After Spinal Cord Injury.

Pressure ulcers (PUs) are common debilitating complications of traumatic spinal cord injury (SCI) and tend to occur in soft tissues around bony prominences. There is, however, little known about the impact of SCI on skin wound healing in the context of animal models in controlled experimental settings. In this study, a simple, non-invasive, reproducible and clinically relevant mouse model of PUs in the context of complete SCI is presented. Adult male mice (Balb/c, 10 weeks old) were shaved and depilated. Post-depilation (24 h), mice were subjected to laminectomy followed by complete spinal cord transection (T9-T10 vertebrae). Immediately after, a skin fold on the back of the mice was lifted and sandwiched between two magnetic discs held in place for next 12 h, thus creating an ischemic area that developed into a PU over the following days. The wounded areas demonstrated tissue edema and epidermal disappearance by day 3 post-magnet application. PUs spontaneously developed and healed. Healing was, however, slower in the SCI mice compared to control non-SCI mice when the wound was created below the level of SCI. Conversely, no difference in healing was seen between SCI and control non-SCI mice when the wound was created above the level of SCI. This model is a potentially useful tool to study the dynamics of skin PU development and healing after SCI, as well as to test therapeutic approaches that may help heal such wounds.

Myofibroblast proliferation and heterogeneity are supported by macrophages during skin repair.

During tissue repair, myofibroblasts produce extracellular matrix (ECM) molecules for tissue resilience and strength. Altered ECM deposition can lead to tissue dysfunction and disease. Identification of distinct myofibroblast subsets is necessary to develop treatments for these disorders. We analyzed profibrotic cells during mouse skin wound healing, fibrosis, and aging and identified distinct subpopulations of myofibroblasts, including adipocyte precursors (APs). Multiple mouse models and transplantation assays demonstrate that proliferation of APs but not other myofibroblasts is activated by CD301b-expressing macrophages through insulin-like growth factor 1 and platelet-derived growth factor C. With age, wound bed APs and differential gene expression between myofibroblast subsets are reduced. Our findings identify multiple fibrotic cell populations and suggest that the environment dictates functional myofibroblast heterogeneity, which is driven by fibroblast-immune interactions after wounding.

Stem Cells and Engineered Scaffolds for Regenerative Wound Healing.

The normal wound healing process involves a well-organized cascade of biological pathways and any failure in this process leads to wounds becoming chronic. Non-healing wounds are a burden on healthcare systems and set to increase with aging population and growing incidences of obesity and diabetes. Stem cell-based therapies have the potential to heal chronic wounds but have so far seen little success in the clinic. Current research has been focused on using polymeric biomaterial systems that can act as a niche for these stem cells to improve their survival and paracrine activity that would eventually promote wound healing. Furthermore, different modification strategies have been developed to improve stem cell survival and differentiation, ultimately promoting regenerative wound healing. This review focuses on advanced polymeric scaffolds that have been used to deliver stem cells and have been tested for their efficiency in preclinical animal models of wounds.