Hypoxic conditioned medium from human amniotic fluid-derived mesenchymal stem cells accelerates skin wound healing through TGF-?/SMAD2 and PI3K/Akt pathways.
ABSTRACT: In a previous study, we isolated human amniotic fluid (AF)-derived mesenchymal stem cells (AF-MSCs) and utilized normoxic conditioned medium (AF-MSC-norCM) which has been shown to accelerate cutaneous wound healing. Because hypoxia enhances the wound healing function of mesenchymal stem cell-conditioned medium (MSC-CM), it is interesting to explore the mechanism responsible for the enhancement of wound healing function. In this work, hypoxia not only increased the proliferation of AF-MSCs but also maintained their constitutive characteristics (surface marker expression and differentiation potentials). Notably, more paracrine factors, VEGF and TGF-?1, were secreted into hypoxic conditioned medium from AF-MSCs (AF-MSC-hypoCM) compared to AF-MSC-norCM. Moreover, AF-MSC-hypoCM enhanced the proliferation and migration of human dermal fibroblasts in vitro, and wound closure in a skin injury model, as compared to AF-MSC-norCM. However, the enhancement of migration of fibroblasts accelerated by AF-MSC-hypoCM was inhibited by SB505124 and LY294002, inhibitors of TGF-?/SMAD2 and PI3K/AKT, suggesting that AF-MSC-hypoCM-enhanced wound healing is mediated by the activation of TGF-?/SMAD2 and PI3K/AKT. Therefore, AF-MSC-hypoCM enhances wound healing through the increase of hypoxia-induced paracrine factors via activation of TGF-?/SMAD2 and PI3K/AKT pathways.
Project description:BACKGROUND:Mesenchymal stem cell-derived conditioned medium (MSC-CM) has emerged as a promising cell-free tool for restoring degenerative diseases and treating traumatic injuries. The present study describes the effect of selenium as a reactive oxygen species (ROS) scavenger and its additive effect with basic fibroblast growth factor (bFGF) on in vitro expansion of amniotic fluid (AF)-MSCs and the paracrine actions of AF-MSC-CM as well as the associated cellular and molecular mechanisms. METHODS:In this study, we obtained CM from human AF-MSCs cultured with selenium. The stemness of selenium-treated AF-MSCs was evaluated by cell growth and differentiation potential. Human fibroblasts were treated with AF-MSC-CM and analyzed for cell signaling changes. For in vivo wound healing assay, ICR mice with a full-thickness skin wound were used. RESULTS:Selenium played a critical role in in vitro expansion of AF-MSCs through activation of the AKT-ERK1/2, Smad2, and Stat3 signaling pathways along with inactivation of GSK3?. When administered together with bFGF, it showed remarkable effect in inhibiting ROS accumulation and preserving their multipotency. Proliferation and migration of human dermal fibroblasts and in vivo wound healing were improved in the CMs derived from AF-MSCs exposed to selenium and bFGF, which was caused by the Smad2, AKT-MEK1/2-ERK, and NF?B signaling triggered by the paracrine factors of AF-MSCs, such as TGF-?, VEGF, and IL-6. Our results suggest the following: (a) supplementation of selenium in AF-MSC culture contributes to in vitro expansion and preservation of multipotency, (b) ROS accumulation causes progressive losses in proliferative and differentiation potential, (c) the separate activities of bFGF and selenium in MSCs exert an additive effect when used together, and (d) the additive combination improves the therapeutic effects of AF-MSC-derived CMs on tissue repair and regeneration. CONCLUSION:Antioxidants, such as selenium, should be considered as an essential supplement for eliciting the paracrine effects of MSC-CMs.
Project description:BACKGROUND:Stem cell therapy is the next generation a well-established technique. Cell therapy with mesenchymal stem cells (MSC) has been demonstrated to enhance wound healing in diabetic mice, at least partly due to improved growth factor production. However, it is unclear whether MSC can biomechanically affect wound closure. Utilizing the well-established cell-populated collagen gel contraction model we investigated the interactions between MSC and the extracellular matrix. METHODS:Murine fetal liver-derived Mesenchymal Stem Cells (MSCs) or fetal Dermal Fibroblasts (DFs) were cultured in cell-populated collagen gels (CPCGs). The effect of cell density, conditioned media, growth factors (TGF-B1, FGF, PDGF-BB), cytoskeletal disruptors (colchicine, cytochalasin-D), and relative hypoxia on gel contraction were evaluated. Finally, we also measured the expression of integrin receptors and some growth factors by MSCs within the contracting gels. RESULTS:Our results show that at different densities, MSCs induced a higher gel contraction compared to DFs. Higher cell density resulted in faster and more complete contraction of CPCGs. Cytoskeletal inhibitors either inhibited or prevented MSC-mediated contraction in a dose dependent fashion. Growth factors, conditioned media from both MSC and DF, and hypoxia all influenced CPCG contraction. DISCUSSION:The results suggest that MSCs are capable of directly contributing to wound closure through matrix contraction, and they are more effective than DF. In addition, this study demonstrates the importance of how other factors such as cell concentration, cytokines, and oxygen tension can provide potential modulation of therapies to correct wound healing impairments.
Project description:The prevalence of nonhealing wounds is predicted to increase due to the growing aging population. Despite the use of novel skin substitutes and wound dressings, poorly vascularized wound niches impair wound repair. Mesenchymal stem cells (MSCs) have been reported to provide paracrine signals to promote wound healing, but the effect of human Wharton's jelly-derived MSCs (WJ-MSCs) has not yet been described in human normal skin.Human WJ-MSCs and normal skin fibroblasts were isolated from donated umbilical cords and normal adult human skin. Fibroblasts were treated with WJ-MSC-conditioned medium (WJ-MSC-CM) or nonconditioned medium.Expression of genes involved in re-epithelialization (transforming growth factor-?2), neovascularization (hypoxia-inducible factor-1?) and fibroproliferation (plasminogen activator inhibitor-1) was upregulated in WJ-MSC-CM-treated fibroblasts (P?0.05). WJ-MSC-CM enhanced normal skin fibroblast proliferation (P?0.001) and migration (P?0.05), and promoted wound healing in an excisional full-thickness skin murine model.Under our experimental conditions, WJ-MSCs enhanced skin wound healing in an in vivo mouse model.
Project description:Transplantation of umbilical cord mesenchymal stem cells (UC-MSCs) is currently considered a novel therapeutic strategy for diabetic nephropathy (DN). However, the mechanisms by which UC-MSCs ameliorate renal fibrosis in DN are not well understood. Herein, we firstly investigated the therapeutic effects of mouse UC-MSC infusion on kidney structural and functional impairment in streptozotocin- (STZ-) induced diabetic mice. We found that the repeated injection with mUC-MSCs alleviates albuminuria, glomerulus injury, and fibrosis in DN mouse models. Next, mesangial cells were exposed to 5.6?mM glucose, 30?mM glucose, or mUC-MSC-conditioned medium, and then we performed western blotting, immunofluorescence, wound healing assay, and cell proliferation assay to measure extracellular matrix (ECM) proteins and matrix metalloproteinases (MMPs), myofibroblast transdifferentiation (MFT), and cell proliferation. We demonstrated that mUC-MSC paracrine decreased the deposition of fibronectin and collagen I by inhibiting TGF-?1-triggered MFT and cell proliferation mediated by PI3K/Akt and MAPK signaling pathways, and elevating the levels of MMP2 and MMP9. Importantly, we provided evidence that the antifibrosis role of mUC-MSC paracrine in DN might be determined by exosomes shed by MSCs. Together, these findings reveal the mechanisms underlying the therapeutic effects of UC-MSCs on renal fibrosis in DN and provide the evidence for DN cell-free therapy based on UC-MSCs in the future.
Project description:BACKGROUND:Cutaneous wound healing represents a morphogenetic response to injury and is designed to restore anatomic and physiological function. Human bone marrow mesenchymal stem cell-derived exosomes (hBM-MSC-Ex) are a promising source for cell-free therapy and skin regeneration. METHODS:In this study, we investigated the cell regeneration effects and its underlying mechanism of hBM-MSC-Ex on cutaneous wound healing in rats. In vitro studies, we evaluated the role of hBM-MSC-Ex in the two types of skin cells: human keratinocytes (HaCaT) and human dermal fibroblasts (HDFs) for the proliferation. For in vivo studies, we used a full-thickness skin wound model to evaluate the effects of hBM-MSC-Ex on cutaneous wound healing in vivo. RESULTS:The results demonstrated that hBM-MSC-Ex promote both two types of skin cells' growth effectively and accelerate the cutaneous wound healing. Interestingly, we found that hBM-MSC-Ex significantly downregulated TGF-?1, Smad2, Smad3, and Smad4 expression, while upregulated TGF-?3 and Smad7 expression in the TGF-?/Smad signaling pathway. CONCLUSIONS:Our findings indicated that hBM-MSC-Ex effectively promote the cutaneous wound healing through inhibiting the TGF-?/Smad signal pathway. The current results provided a new sight for the therapeutic strategy for the treatment of cutaneous wounds.
Project description:Mutations in the CD18 gene encoding the common ?-chain of ?2 integrins result in impaired wound healing in humans and mice suffering from leukocyte adhesion deficiency syndrome type 1 (LAD1). Transplantation of adipose tissue-derived mesenchymal stem cells (MSCs) restores normal healing of CD18-/- wounds by restoring the decreased TGF-?1 concentrations. TGF-?1 released from MSCs leads to enhanced myofibroblast differentiation, wound contraction, and vessel formation. We uncover that MSCs are equipped with a sensing mechanism for TGF-?1 concentrations at wound sites. Low TGF-?1 concentrations as occurring in CD18-/- wounds induce TGF-?1 release from MSCs, whereas high TGF-?1 concentrations suppress TGF-?1 production. This regulation depends on TGF-? receptor sensing and is relayed to microRNA-21 (miR-21), which subsequently suppresses the translation of Smad7, the negative regulator of TGF-?1 signaling. Inactivation of TGF-? receptor, or overexpression or silencing of miR-21 or Smad7, abrogates TGF-?1 sensing, and thus prevents the adaptive MSC responses required for tissue repair.
Project description:BACKGROUND:Diabetic polyneuropathy (DPN) is the most common and early developing complication of diabetes mellitus, and the key contributor for foot ulcers development, with no specific therapies available. Different studies have shown that mesenchymal stem cell (MSC) administration is able to ameliorate DPN; however, limited cell survival and safety reasons hinder its transfer from bench to bedside. MSCs secrete a broad range of antioxidant, neuroprotective, angiogenic, and immunomodulatory factors (known as conditioned medium), which are all decreased in the peripheral nerves of diabetic patients. Furthermore, the abundance of these factors can be boosted in vitro by incubating MSCs with a preconditioning stimulus, enhancing their therapeutic efficacy. We hypothesize that systemic administration of conditioned medium derived from preconditioned MSCs could reverse DPN and prevent foot ulcer formation in a mouse model of type II diabetes mellitus. METHODS:Diabetic BKS db/db mice were treated with systemic administration of conditioned medium derived from preconditioned human MSCs; conditioned medium derived from non-preconditioned MSCs or vehicle after behavioral signs of DPN was already present. Conditioned medium or vehicle administration was repeated every 2 weeks for a total of four administrations, and several functional and structural parameters characteristic of DPN were evaluated. Finally, a wound was made in the dorsal surface of both feet, and the kinetics of wound closure, re-epithelialization, angiogenesis, and cell proliferation were evaluated. RESULTS:Our molecular, electrophysiological, and histological analysis demonstrated that the administration of conditioned medium derived from non-preconditioned MSCs or from preconditioned MSCs to diabetic BKS db/db mice strongly reverts the established DPN, improving thermal and mechanical sensitivity, restoring intraepidermal nerve fiber density, reducing neuron and Schwann cell apoptosis, improving angiogenesis, and reducing chronic inflammation of peripheral nerves. Furthermore, DPN reversion induced by conditioned medium administration enhances the wound healing process by accelerating wound closure, improving the re-epithelialization of the injured skin and increasing blood vessels in the wound bed in a skin injury model that mimics a foot ulcer. CONCLUSIONS:Studies conducted indicate that MSC-conditioned medium administration could be a novel cell-free therapeutic approach to reverse the initial stages of DPN, avoiding the risk of lower limb amputation triggered by foot ulcer formation and accelerating the wound healing process in case it occurs.
Project description:WRN mutation causes a premature aging disease called Werner syndrome (WS). However, the mechanism by which WRN loss leads to progeroid features evident with impaired tissue repair and regeneration remains unclear. To determine this mechanism, we performed gene editing in reprogrammed induced pluripotent stem cells (iPSCs) derived from WS fibroblasts. Gene correction restored the expression of WRN. WRN+/+ mesenchymal stem cells (MSCs) exhibited improved pro-angiogenesis. An analysis of paracrine factors revealed that hepatocyte growth factor (HGF) was downregulated in WRN-/- MSCs. HGF insufficiency resulted in poor angiogenesis and cutaneous wound healing. Furthermore, HGF was partially regulated by PI3K/AKT signaling, which was desensitized in WRN-/- MSCs. Consistently, the inhibition of the PI3K/AKT pathway in WRN+/+ MSC resulted in reduced angiogenesis and poor wound healing. Our findings indicate that the impairment in the pro-angiogenic function of WS-MSCs is due to HGF insufficiency and PI3K/AKT dysregulation, suggesting trophic disruption between stromal and epithelial cells as a mechanism for WS pathogenesis.
Project description:BACKGROUND:Hypertrophic scars (HSs) are formed via an aberrant response to the wound healing process. HSs can be cosmetic or can result in functional problems. Prolonged proliferation and remodeling phases disrupt wound healing, leading to excessive collagen production and HS formation. However, there are currently no satisfactory drugs to prevent HS formation. Mesenchymal stem cell (MSC) conditioned medium (CM) has therapeutic effects on wound healing and preventing HS formation. Bone marrow concentrate (BMC) contains various growth factors and cytokines that are crucial for regeneration and has been applied in the clinical setting. In this study, we evaluated the effects of BMC-induced MSC CM on HS formation in a rabbit ear model. METHODS:We established a rabbit ear wound model by generating full-thickness wounds in the ears of rabbits (n = 12) and treated wounds with MSC CM, BMC CM, or BMC-induced MSC CM. Dermal fibroblasts from human hypertrophic scar were stimulated with transforming growth factor beta 1 (TGF-β1) for 24 h and cultured in each culture medium for 72 h. We measured the hypertrophic scar (HS) formation during the skin regeneration by measuring the expression of several remodeling molecules and the effect of these conditioned media on active human HS fibroblasts. RESULTS:Our results showed that BMC-induced MSC CM had greater antifibrotic effects than MSC CM and BMC CM significantly attenuated HS formation in rabbits. BMC-induced MSC CM accelerated wound re-epithelization by increasing cell proliferation. Additionally, BMC-induced MSC CM also inhibited fibrosis by decreasing profibrotic gene and protein expression, promoting extracellular matrix turnover, inhibiting fibroblast contraction, and reversing myofibroblast activation. CONCLUSIONS:BMC-induced MSC CM modulated the proliferation and remodeling phases of wound healing, representing a potential wound healing agent and approach for preventing HS formation.
Project description:The wound healing process initiates after injury to a tissue and involves a series of orchestrated events to minimize the invasion of foreign matters such as bacteria and efficiently regenerate the damaged tissue. A variety of cells must be recruited to the tissue during wound healing. However, this process is severely disrupted in patients suffering from chronic illness, including diabetes, leading to impaired healing or non-healing wounds. Current avenues of treatment include negative-pressure therapy, wound debridement, growth factor replacement, and cell-based therapies. Among these therapies, mesenchymal stem cells (MSCs) delivery to the wound holds a very high promise due to the innate abilities of MSCs that include immunogenicity, plasticity, and self-renewal. Bone marrow derived MSCs have been shown to promote more rapid wound healing by increased cytokine production in diabetic mice. However, the lack of understanding of the mechanical and chemical interaction of the transplanted MSCs with the factors present in the regenerative niches limits their efficacy in the wound bed. In this study, we sought to understand how the changes in MSC biochemical and biophysical properties can affect their function <i>in vitro</i> and <i>in vivo</i>. We demonstrate that pretreatment of MSCs with the mechano-stimulatory soluble factor transforming growth factor (TGF-?1), which is highly expressed in injury sites, improves wound closure in a syngeneic murine wound model. This improved wound closure correlated with increased invasion into the wound bed. <i>In vitro</i> studies demonstrated that TGF-?1 pretreatment expedited wound closure by increasing adhesion, traction force, and migration even after removal of the stimulus. Furthermore, this response was mediated by the cytoskeletal protein focal adhesion kinase. Taken together, this study suggests that defined chemical stimuli can benefit site specific adaptability of MSCs to improve their function and therapeutic usefulness.