Selenium-Stimulated Exosomes Enhance Wound Healing by Modulating Inflammation and Angiogenesis.

Mesenchymal stem cell (MSC)-derived exosomes have emerged as an attractive cell-free tool in tissue engineering and regenerative medicine. The current study aimed to examine the anti-inflammatory, pro-angiogenic, and wound-repair effects of both exosomes and selenium-stimulated exosomes, and check whether the latter had superior wound healing capacity over others. The cellular and molecular network of exosomes, as a paracrine signal, was extensively studied by performing miRNA arrays to explore the key mediators of exosomes in wound healing. Selenium is known to play a critical role in enhancing the proliferation, multi-potency, and anti-inflammatory effects of MSCs. Selenium-stimulated exosomes showed significant effects in inhibiting inflammation and improving pro-angiogenesis in human umbilical vein endothelial cells. Cell growth and the migration of human dermal fibroblasts and wound regeneration were more enhanced in the selenium-stimulated exosome group than in the selenium and exosome groups, thereby further promoting the wound healing in vivo. Taken together, selenium was found to augment the therapeutic effects of adipose MSC-derived exosomes in tissue regeneration. We concluded that selenium may be considered a vital agent for wound healing in stem cell-based cell-free therapies.

Human adipose mesenchymal stem cells modulate inflammation and angiogenesis through exosomes.

Stem cell-derived exosomes are efficient and safe therapeutic tools for transferring endogenous biological cargo or functional biomolecules for regenerative medicine. The regulation of inflammation and angiogenesis plays a pivotal role in wound healing and tissue regeneration. The purpose of this study was to investigate the anti-inflammatory and pro-angiogenic roles of human adipose mesenchymal stem cell-derived exosomes, focusing on the underlying mechanisms. Exosomes inhibited LPS-induced inflammation by activating ROCK1 and PTEN expression. Moreover, microRNAs (miR-132 and miR-146a) released from exosomes upregulated the expression of pro-angiogenic genes and promoted proliferation activity and tube formation in human umbilical vein endothelial cells. Exosomal effects were verified using ROCK1/PTEN inhibitors for anti-inflammation and miR-132/miR-146a inhibitors for pro-angiogenesis. Our findings suggest that exosomes exert anti-inflammatory effects by targeting the ROCK1/PTEN pathway and exhibit pro-angiogenic effects via delivery of miR-132 and miR-146a. Taken together, these results suggest that exosomes may be promising therapeutic candidates for curing diseases involved in inflammation and angiogenesis.

A cancer cell-specific benzoxadiazole-based fluorescent probe for hydrogen sulfide detection in mitochondria.

The present work describes the design and biological applications of a novel colorimetric and fluorescence turn-on probe for hydrosulfide detection. The probe was designed to introduce hemicyanine as the fluorescent skeleton and 7-nitro-1,2,3-benzoxadiazole as the recognition site. The optical properties and responses of the probe towards HS-, anions and some biothiols indicate an impressively high selectivity of the probe towards HS- such that it can be effectively used as an indicator for monitoring the level of HS- in living cells. In biological experiments using the probe, the H2S levels are found to be higher in cancer cells than in normal cells. In addition, the probe is shown to specifically and rapidly detect endogenous H2S, which is produced primarily in the mitochondria of cancer cells, as demonstrated by a co-localization experiment using specific trackers for the detection of cellular organelles in pharmacological inhibition or stimulation studies, without any significant cytotoxic effects. Thus, the results of the chemical and biological experiments described herein demonstrate the potential of this novel probe to specifically, safely, and rapidly detect H2S to distinguish cancer cells from normal cells by targeting it specifically in mitochondria.

Human Adipose Mesenchymal Stem Cell-Derived Exosomes: A Key Player in Wound Healing.

BACKGROUND: Human adipose-derived mesenchymal stem cells (AMSCs) are an attractive resource for wound healing because their regenerative capacity improves injury repair. Recently, stem cell-derived exosomes have been shown to play a positive role in stem cell-based therapies. However, the effects of exosomes derived from AMSCs (AEXOs) on wound healing are unclear. In this study, we aimed to examine the role of AEXOs in attenuating inflammation and explore their effects in normal wound healing. METHODS: We isolated exosomes from AMSCs and established a cellular model of inflammation by treatment with the inflammatory cytokines, interferon gamma and tumor necrosis factor alpha, to determine whether AEXOs can inhibit inflammation. We examined the wound healing effects of AEXOs in in vitro wound healing models and performed a miRNA array to understand the role of AEXOs in inflammation and wound healing. RESULTS: A significant difference was observed in wound closure and the expression of anti-inflammatory and wound-healing-related factors between control and AEXO-treated cells. CONCLUSION: Our results showed that besides alleviating the inflammation response, AEXOs also promote wound healing. Thus, AEXOs represent a novel, stem-cell-based, therapeutic strategy for wound healing.

Exosome and Melatonin Additively Attenuates Inflammation by Transferring miR-34a, miR-124, and miR-135b.

The positive effects of mesenchymal stem cells (MSCs) are primarily activated through molecular secretions known as paracrine activity, which regulates the function of various cell types including immune cells. Accumulating evidence shows that exosomes of soluble factors released from MSCs are potential alternative agents for stem cell-based therapy, although the exact underlying mechanism has not been elucidated. The purpose of this study was to evaluate the potential effects of exosomes produced by adipose-derived MSCs and to examine the changes in anti-inflammatory genes in concurrence with the polarization of M2 macrophages in cellular models ex vivo. Isolated exosomes were used to investigate the inflammatory modulation in pro-inflammatory cytokine-treated fibroblasts and THP-1 cells. The anti-inflammatory mRNA expression associated with M2 macrophages was significantly upregulated after exosome treatment in an interferon gamma and tumor necrosis factor alpha-treated inflammatory environment. Furthermore, melatonin-stimulated exosomes exerted superior anti-inflammatory modulation via exosomal miRNAs miR-34a, miR-124, and miR-135b, compared with exosomes. Our results indicate that melatonin-stimulated exosomes originating from adipose-derived MSCs are safe and efficient tools for regenerative medicine to treat inflammatory diseases.

Adipose-Derived Mesenchymal Stem Cells Promote M2 Macrophage Phenotype through Exosomes.

Accumulating evidence has shown that the paracrine factors derived from mesenchymal stem cells (MSCs) are capable of regulating the immune system via interaction with various immune cells. In this study, adipose-derived MSCs (AdMSCs) and human peripheral blood monocytes (PBMCs) were isolated and cultured to examine the effects of MSC-induced macrophages (iMΦ) on inflammation and immune modulation. Indirect coculture with MSCs increased the expression of arginase-1 and mannose receptor (CD206), markers of activated M2 macrophages, in the PBMCs demonstrating that MSC-secreted factors promoted M2-MΦ polarization. Additionally, iMΦ exhibited a similar higher inhibitory effect on the growth of activated T cells compared to that in the other groups (AdMSCs only, AdMSCs plus iMΦ), implying that iMΦ can play a sufficient functional role. Interestingly, the population of FoxP3 Treg cells significantly increased when cocultured with iMΦ, suggesting that iMΦ have an immunomodulatory effect on the Treg cells through the modulation of the FoxP3 expression. Notably, iMΦ expressed high levels of immunosuppressive and anti-inflammatory cytokines, namely IL-10 and TSG-6. Furthermore, we confirmed that the AdMSC-derived exosomes modulated macrophage polarization by upregulating the expression of M2 macrophage markers. Conclusively, our results suggest that iMΦ play a significant role in regulating the immunomodulatory- and inflammatory-mediated responses. Thus, iMΦ may be used as a novel stem cell-based cell-free therapy for the treatment of immune-mediated inflammatory disorders.

Biological effects of melatonin on human adipose­derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are capable of differentiating into other cell types and exhibit immunomodulatory effects. MSCs are affected by several intrinsic and extrinsic signaling modulators, including growth factors, cytokines, extracellular matrix and hormones. Melatonin, produced by the pineal gland, is a hormone that regulates sleep cycles. Recent studies have shown that melatonin improves the therapeutic effects of stem cells. The present study aimed to investigate whether melatonin enhances the biological activities of human adipose­derived MSCs. The results demonstrated that treatment with melatonin promoted cell proliferation by inducing SRY­box transcription factor 2 gene expression and preventing replicative senescence. In addition, melatonin exerted anti­adipogenic effects on MSCs. PCR analysis revealed that the expression of the CCAAT enhancer binding protein a gene, a key transcription factor in adipogenesis, was decreased following melatonin treatment, resulting in reduced adipogenic differentiation in an in vitro assay. The present study also examined the effect of melatonin on the immunomodulatory response using a co­culture system of human peripheral blood mononuclear cells and MSCs. Activated T cells were strongly inhibited following melatonin exposure compared with those in the control group. Finally, the favorable effects of melatonin on MSCs were confirmed using luzindole, a selective melatonin receptor antagonist. The proliferation­promoting, anti­inflammatory effects of melatonin suggested that melatonin­treated MSCs may be used for effective cell therapy.

Environmental Benzopyrene Attenuates Stemness of Placenta-Derived Mesenchymal Stem Cells via Aryl Hydrocarbon Receptor.

The toxic effects of particulate matter have been linked to polycyclic aromatic hydrocarbons (PAHs) such as benzopyrene. PAHs are potent inducers of the aryl hydrocarbon receptor (AhR), which is an expressed nuclear receptor that senses environmental stimuli and modulates gene expression. Even though several studies have shown that the benzopyrene (BP) of chemical pollutants significantly impaired stem cell activity, the exact molecular mechanisms were not clearly elucidated. In the present study, we aimed to investigate the effects of BP on placenta-derived mesenchymal stem cells (PD-MSCs) in vitro. We found that the AhR in PD-MSCs was expressed under the treatment of BP, and its activation markedly disrupted osteogenic differentiation through the alteration of stemness activity of PD-MSCs. Moreover, BP treatment significantly reduced the proliferation activity of PD-MSCs and expression of pluripotent markers through the induction of AhR. Treatment with StemRegenin 1 (SR1), a purine derivative that antagonizes the AhR, effectively prevented BP-induced reduction of the proliferation and differentiation activity of PD-MSCs. In this study, we found that BP treatment in PD-MSCs markedly obstructs PD-MSC stemness through AhR signaling. Noteworthy, SR1-mediated MSC application will contribute to new perspectives on MSC-based therapies for air pollution-related bone diseases.

The co-existence of elevated high sensitivity C-reactive protein and homocysteine levels is associated with increased risk of metabolic syndrome: A 6-year follow-up study.

Accumulating evidence has revealed that both high sensitivity C-reactive protein (hsCRP) and homocysteine (HCY) are associated with increased risk of metabolic syndrome (MetS) and cardiovascular disease. However, it is unclear whether the coexistence of these conditions accelerates the risk of metabolic syndrome (MetS). We hypothesized that the combination of high sensitivity C-reactive protein (hsCRP) and homocysteine (HCY) levels could exacerbate the development of MetS in a large prospective cohort study. We selected data from 3,170 individuals (1,614 men and 1,556 women) who participated in the Korean Genome and Epidemiology Study. Participants with high hsCRP and HCY levels were categorized into quartiles. MetS was defined based on the criteria of the modified National Cholesterol Education Program, Adult Treatment Panel III. The prevalence of MetS was higher in participants with concurrent high hsCRP and HCY compared to those with low hsCRP and HCY levels. The incidence of MetS at the 6-year follow-up was the highest in participants with concomitant high hsCRP and HCY levels, regardless of obesity. Even after adjusting for potential confounding factors including body mass index in a multivariate logistic regression model, subjects with elevated hsCRP and HCY levels had a 2.50-fold increased risk of developing MetS at the six-year follow-up compared to those who did not have high hsCRP and HCY level. MetS is more prevalent in the concurrent presence of elevated hsCRP and HCY levels. The combination of the two conditions may contribute to an increased risk of MetS, but these factors may not be synergistic.

Protein-Engineered Large Area Adipose-derived Stem Cell Sheets for Wound Healing.

Human adipose-derived stem cells (hADSCs) formed robust cell sheets by engineering the cells with soluble cell adhesive molecules (CAMs), which enabled unique approaches to harvest large area hADSC sheets. As a soluble CAM, fibronectin (FN) (100 pg/ml) enhanced the cell proliferation rate and control both cell-to-cell and cell-to-substrate interactions. Through this engineering of FN, a transferrable hADSC sheet was obtained as a free-stranding sheet (122.6 mm2) by a photothermal method. During the harvesting of hADSC sheets by the photothermal method, a collagen layer in-between cells and conductive polymer film (CP) was dissociated, to protect cells from direct exposure to a near infrared (NIR) source. The hADSC sheets were applied to chronic wound of genetically diabetic db/db mice in vivo, to accelerate 30% faster wound closure with a high closure effect (εwc) than that of control groups. These results indicated that the engineering of CAM and collagens allow hADSC sheet harvesting, which could be extended to engineer various stem cell sheets for efficient therapies.

Peptide ligand-mediated endocytosis of nanoparticles to cancer cells: Cell receptor-binding- versus cell membrane-penetrating peptides.

The endocytosis-mediating performances of two types of peptide ligands, cell receptor binding peptide (CRBP) and cell membrane penetrating peptide (CMPP), were analyzed and compared using a common carrier of peptide ligands-human ferritin heavy chain (hFTH) nanoparticle. Twenty-four copies of a CMPP(human immunodeficiency virus-derived TAT peptide) and/or a CRBP (peptide ligand with strong and specific affinity for either human integrin(αv ß3 ) or epidermal growth factor receptor I (EGFR) that is overexpressed on various cancer cells) were genetically presented on the surface of each hFTH nanopariticle. The quantitative level of endocytosis and intracellular localization of fluorescence dye-labeled CRBP- and CMPP-presenting nanoparticles were estimated in the in vitro cultures of integrin- and EGFR-overexpressing cancer and human dermal fibroblast cells(control). From the cancer cell cultures treated with the CMPP- and CRBP-presenting nanoparticles, it was notable that CRBPs resulted in quantitatively higher level of endocytosis than CMPP (TAT) and successfully transported the nanoparticles to the cytosol of cancer cells depending on concentration and treatment period of time, whereas TAT-mediated endocytosis localized most of the nanoparticles within endosomal vesicles under the same conditions. These novel findings provide highly useful informations to many researchers both in academia and in industry who are interested in developing anticancer drug delivery systems/carriers.

Distal-less homeobox 5 is a master regulator of the osteogenesis of human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) differentiate into multiple lineages and are a promising source of cells for clinical use. Previously, we found that the gene distal­less homeobox 5 (DLX5) is specifically expressed in MSCs with osteogenic potential. Understanding the mechanism of osteogenesis is necessary for successful bone regeneration using MSCs. The aim of this study was to examine the function of the DLX5 gene in MSCs during osteogenesis (bone development). We analyzed the possible association between DLX5 expression and osteogenesis-, chondrogenesis- and adipogenesis-related gene expression in different cells isolated from bone marrow and cord blood. Differentiation capacity was assessed by observing morphological changes, monitoring gene expression patterns, and staining with Von Kossa, safranin O, and Oil Red O. Suppression of DLX5 expression by means of a small interfering RNA (siRNA) downregulated osteogenic markers and reduced the signs of calcium mineralization. Tanshinone IIA is a known small molecule activator of bone morphogenetic protein (BMP) signaling. Here, we report that induction of DLX5 by tanshinone IIA in MSCs enhanced osteogenic differentiation. In addition, we showed that tanshinone IIA (as a mediator of BMP2 signaling) activates runt-related transcription factor 2 (RUNX2) in MSCs and initiates calcium mineralization during osteogenesis. Taken together, these findings indicate that, in MSCs, DLX5 is a master regulator of osteogenesis. Furthermore, tanshinone IIA may be valuable for stem cell-based therapies of certain bone diseases.

A bioorthogonal 'turn-on' fluorescent probe for tracking mitochondrial nitroxyl formation.

A bioreductant-resistant 'turn-on' chemodosimetric fluorescent probe Mito-1 has been developed for the detection of mitochondrial HNO in live cells. Mito-1 enables the detection of HNO as low as ∼18 nM. It has the capability to detect both exogenous and endogenous mitochondrial HNO formations in cellular milieus by providing fluorescence images. Its two-photon imaging ability fosters its use as a noninvasive imaging tool for the detection of mitochondrial nitroxyl.

The Flavonoid Glabridin Induces OCT4 to Enhance Osteogenetic Potential in Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are a promising tool for studying intractable diseases. Unfortunately, MSCs can easily undergo cellular senescence during in vitro expansion by losing stemness. The aim of this study was to improve the stemness and differentiation of MSCs by using glabridin, a natural flavonoid. Assessments of cell viability, cell proliferation, ß-galactosidase activity, differentiation, and gene expression by reverse transcription PCR were subsequently performed in the absence or presence of glabridin. Glabridin enhanced the self-renewal capacity of MSCs, as indicated by the upregulation of the OCT4 gene. In addition, it resulted in an increase in the osteogenic differentiation potential by inducing the expression of osteogenesis-related genes such as DLX5 and RUNX2. We confirmed that glabridin improved the osteogenesis of MSCs with a significant elevation in the expression of OSTEOCALCIN and OSTEOPONTIN genes. Taken together, these results suggest that glabridin enhances osteogenic differentiation of MSCs with induction of the OCT4 gene; thus, glabridin could be useful for stem cell-based therapies.

Erratum to: Direct photo-patterning on anthracene containing polymer for guiding stem cell adhesion.

[This corrects the article DOI: 10.1186/s40824-016-0072-4.].

Direct photo-patterning on anthracene containing polymer for guiding stem cell adhesion.

BACKGROUND: Various micropatterned surfaces capable of guiding the selective adhesion of biomolecules such as proteins and cells are of great interests in biosensor, diagnostics, drug screening, and tissue engineering. In this study, we described a simple photo-patterning method to prepare micro-patterned films for stem cell patterning using anthracene containing polymers (PMAn). This micro patterned polymer film was prepared by the facile photo-reaction of anthracene units in polymer backbone structure. RESULTS: The UV irradiation of PMAn through a photomask resulted in the quenching of fluorescent intensity as well as the changes in surface wettability from hydrophobic to hydrophilic surface. As a result, UV exposed regions of PMAn film show lower fluorescent intensity as well as higher proliferation rate of mesenchymal stem cells (MSCs) than unexposed region of PMAn film. Furthermore, the selective MSC attachment was clearly observed in the UV exposed regions of PMAn film. CONCLUSION: We developed a simple cell patterning method with a fluorescent, biocompatible, and patternable polymer film containing anthracene units. This method provides a facile stem cell patterning method and could be extended to various patterning of biomaterials without labor-intensive preparation and no pre-treatment for complex interactions of cell-microenvironment.

Poly-L-lysine Prevents Senescence and Augments Growth in Culturing Mesenchymal Stem Cells Ex Vivo.

Mesenchymal stem cells (MSCs) possess great therapeutic potential. Efficient in vitro expansion of MSCs is however necessary for their clinical application. The extracellular matrix (ECM) provides structural and biochemical support to the surrounding cells, and it has been used as a coating substrate for cell culture. In this study, we have aimed to improve the functionality and stemness of MSCs during culture using poly-L-lysine (PLL). Functionality of MSCs was analysed by cell cycle analysis, differentiation assay, ß-galactosidase staining, and RT-PCR. Furthermore, we assessed the global gene expression profile of MSCs on uncoated and PLL-coated plates. MSCs on PLL-coated plates exhibited a faster growth rate with increased S-phase and upregulated expression of the stemness markers. In addition, their osteogenic differentiation potential was increased, and genes involved in cell adhesion, FGF-2 signalling, cell cycle, stemness, cell differentiation, and cell proliferation were upregulated, compared to that of the MSCs cultured on uncoated plates. We also confirmed that MSCs on uncoated plates expressed higher ß-galactosidase than the MSCs on PLL-coated plates. We demonstrate that PLL provides favourable microenvironment for MSC culture by reversing the replicative senescence. This method will significantly contribute to effective preparation of MSCs for cellular therapy.

Comparison of molecular profiles of human mesenchymal stem cells derived from bone marrow, umbilical cord blood, placenta and adipose tissue.

Mesenchymal stem cells (MSCs) are clinically useful due to their capacity for self-renewal, their immunomodulatory properties and tissue regenerative potential. These cells can be isolated from various tissues and exhibit different potential for clinical applications according to their origin, and thus comparative studies on MSCs from different tissues are essential. In this study, we investigated the immunophenotype, proliferative potential, multilineage differentiation and immunomodulatory capacity of MSCs derived from different tissue sources, namely bone marrow, adipose tissue, the placenta and umbilical cord blood. The gene expression profiles of stemness-related genes [octamer-binding transcription factor 4 (OCT4), sex determining region Y-box (SOX)2, MYC, Krüppel-like factor 4 (KLF4), NANOG, LIN28 and REX1] and lineage­related and differentiation stage-related genes [B4GALNT1 (GM2/GS2 synthase), inhibin, beta A (INHBA), distal-less homeobox 5 (DLX5), runt-related transcription factor 2 (RUNX2), proliferator­activated receptor gamma (PPARG), CCAAT/enhancer-binding protein alpha (C/EBPA), bone morphogenetic protein 7 (BMP7) and SOX9] were compared using RT-PCR. No significant differences in growth rate, colony-forming efficiency and immunophenotype were observed. Our results demonstrated that MSCs derived from bone marrow and adipose tissue shared not only in vitro tri-lineage differentiation potential, but also gene expression profiles. While there was considerable inter-donor variation in DLX5 expression between MSCs derived from different tissues, its expression appears to be associated with the osteogenic potential of MSCs. Bone marrow-derived MSCs (BM-MSCs) significantly inhibited allogeneic T cell proliferation possibly via the high levels of the immunosuppressive cytokines, IL10 and TGFB1. Although MSCs derived from different tissues and fibroblasts share many characteristics, some of the marker genes, such as B4GALNT1 and DLX5 may be useful for the characterization of MSCs derived from different tissue sources. Collectively, our results suggest that, based on their tri-lineage differentiation potential and immunomodulatory effects, BM-MSCs and adipose tissue-derived MSCs (A-MSCs) represent the optimal stem cell source for tissue engineering and regenerative medicine.

Photothermally induced local dissociation of collagens for harvesting of cell sheets.

The local heating of poly(3,4-ethylenedioxythiophene) (PEDOT) by a photothermal effect directed by near-infrared (NIR) light induces unfolding of absorbed collagen triple helices, yielding soluble collagen single-helical structures. This dissociation of collagens allowed the harvesting of a living idiomorphic cell sheet, achieved upon irradiation with NIR light (λ=808 nm). The PEDOT layer was patterned and cells were successfully cultured on the patterned substrate. Cell sheets of various shapes mirroring the PEDOT pattern could be detached after a few minutes of irradiation with NIR light. The PEDOT patterns guided not only the entire shape of the cell sheets but also the spreading direction of the cells in the sheets. This photothermally induced dissociation of collagen provided a fast non-invasive harvesting method and tailor-made cell-sheet patterns.

Highly fluorescent conjugated polyelectrolyte for protein sensing and cell-compatible chemosensing applications.

Using a highly fluorescent, water-soluble polymer derived from a triazine-bridged copolymer (DTMSPV), we explored the tunable fluorescence properties of the water-soluble DTMSPV by solvent polarity to function as a fluorescence sensory probe for protein sensing. The green-blue fluorescence from DTMSPV was significantly enhanced in the presence of bovine serum albumin through hydrophobic interactions. Meanwhile, complete quenching of the fluorescence from DTMSPV occurred in the presence of hemoglobin through iron complexation with the polyelectrolyte. In addition, the DTMSPVs were highly fluorescent and permeated into living mesenchymal stem cells (MSCs), enabling effective imaging of the MSCs. This permeation into stem cells is crucial to the detection of Al(3+) in living MSCs. The interaction between the triazine units in DTMSPV with the Al(3+) ions allows for the detection of Al(3+) in living cells. Thus, a strong fluorescence from living MSCs pretreated with DTMSPV was quenched as a function of the Al(3+) concentration, confirming that DTMSPV is a cell-permeable fluorescent polymer that can function as a versatile probe to detect Al(3+) in living cells.