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Mast cells, immune sentinels that respond to various stimuli in barrier organs, provide defense by expressing pattern recognition receptors, such as Toll-like receptors (TLRs). They may affect inflammatory responses and wound healing. Here, we investigated the effect of TLR2/6-stimulated mast cells on wound healing in keratinocytes. The human mast cell line HMC-1 was treated with the TLR2/6 agonist FSL-1, and the conditioned medium (CM) was collected from untreated cells (HMC-1 CM) and FSL-1-stimulated cells (FSL-1-HMC-1 CM). Cell migration was evaluated in keratinocyte cells (HaCaT) treated with HMC-1 CM and FSL-1-HMC-1 CM via scratch and Transwell assays. Mice were treated with HMC-1 CM, FSL-1-HMC-1 CM, and FSL-1. Wound closure was measured, and tissue regeneration was assessed using hematoxylin and eosin staining. Growth factor expression levels were evaluated to identify the factors affecting wound healing. The tryptase inhibitor APC 366 was treated with HMC-1 CM and FSL-1-HMC-1 CM in a scratch assay. This study revealed that HMC-1 CM affected HaCaT cell migration, which was facilitated by FSL-1-HMC-1 CM. HMC-1 CM promoted tryptase-dependent HaCaT migration. Moreover, FSL-1-HMC-1 CM enhanced wound healing in C57BL/6J mice in vivo. Our findings indicated that TLR2/6-stimulated mast cells contributed to skin homeostasis by promoting tryptase-dependent wound healing.

One of the most affected aspects of the aging process is immunity, with age-related immune system decline beingresponsible for an increase in susceptibility to infectious diseases and cancer risk. On the other hand, the agingprocess is accompanied with low-grade pro-inflammatory status. This condition involves a persistent rise in cytokinelevels that can activate both innate and adaptive immune systems. Finally, despite the fact that immunologicalresponses to antigenic stimulations decrease with age, the incidence and prevalence of many common autoimmunediseases increase in the elderly population. Overall, the co-existence of a prolonged, low-grade inflammatory statusand declining immune activity appears to be a paradoxical phenomenon. This study characterized skin inflammation inmouse dermatitis model of various ages to monitor possible changes of inflammatory responses during aging.

Abstract Tonsil-derived mesenchymal stem cells (TMSCs) showed therapeutic effects on acute and chronic murine colitis models, owing to their immunomodulatory properties; therefore, we evaluated enhanced therapeutic effects of TMSCs on a murine colitis model using three-dimensional (3D) culture method. The expression of angiogenic factors, VEGF, and anti-inflammatory cytokines, IL-10, TSG-6, TGF-β, and IDO-1, was significantly higher in the 3D-TMSC-treated group than in the 2D-TMSC-treated group (P

Abstract Background Co-transplantation of bone marrow cells (BMCs) and mesenchymal stem cells (MSCs) is used as a strategy to improve the outcomes of bone marrow transplantation. Tonsil-derived MSCs (TMSCs) are a promising source of MSCs for co-transplantation. Previous studies have shown that TMSCs or conditioned media from TMSCs (TMSC-CM) enhance BMC engraftment. However, the factors in TMSCs that promote better engraftment have not yet been identified. Methods Mice were subjected to a myeloablative regimen of busulfan and cyclophosphamide, and the mRNA expression in the bone marrow was analyzed using an extracellular matrix (ECM) and adhesion molecule-targeted polymerase chain reaction (PCR) array. Nano-liquid chromatography with tandem mass spectrometry, real-time quantitative PCR, western blots, and enzyme-linked immunosorbent assays were used to compare the expression levels of metalloproteinase 3 (MMP3) in MSCs derived from various tissues, including the tonsils, bone marrow, adipose tissue, and umbilical cord. Recipient mice were conditioned with busulfan and cyclophosphamide, and BMCs, either as a sole population or with control or MMP3-knockdown TMSCs, were co-transplanted into these mice. The effects of TMSC-expressed MMP3 were investigated. Additionally, Enzchek collagenase and Transwell migration assays were used to confirm that the collagenase activity of TMSC-expressed MMP3 enhanced BMC migration. Results Mice subjected to the myeloablative regimen exhibited increased mRNA expression of collagen type IV alpha 1/2 (Col4a1 and Col4a2). Among the various extracellular matrix-modulating proteins secreted by TMSCs, MMP3 was expressed at higher levels in TMSCs than in other MSCs. Mice co-transplanted with BMCs and control TMSCs exhibited a higher survival rate, weight recovery, and bone marrow cellularity compared with mice co-transplanted with BMCs and MMP3-knockdown TMSCs. Control TMSC-CM possessed higher collagenase activity against collagen IV than MMP3-knockdown TMSC-CM. TMSC-CM also accelerated BMC migration by degrading collagen IV in vitro. Conclusions Collectively, these results indicate that TMSCs enhance BMC engraftment by the secretion of MMP3 for the modulation of the bone marrow extracellular matrix.

Obesity, which has become a major global health problem, involves a constitutive increase in adipocyte differentiation signaling. Previous studies show that mesenchymal stem cells (MSCs) induce weight loss and glycemic control. However, the mechanisms by which MSCs regulate adipocyte differentiation are not yet known. In this study, we investigated the effects of conditioned medium obtained from human tonsil-derived MSCs (T-MSC CM) on adipocyte differentiation. We found that T-MSC CM attenuated adipocyte differentiation from early stages via inhibiting glucocorticoid signaling. T-MSC CM also increased the phosphorylation of p38 mitogen-activated protein kinase and glucocorticoid receptors and decreased the subsequent nucleus translocation of glucocorticoid receptors. Chronic treatment of mice with synthetic glucocorticoids induced visceral and bone marrow adipose tissue expansion, but these effects were not observed in mice injected with T-MSC CM. Furthermore, T-MSC CM injection protected against reductions in blood platelet counts induced by chronic glucocorticoid treatment, and enhanced megakaryocyte differentiation was also observed. Collectively, these results demonstrate that T-MSC CM exerts inhibitory effects on adipocyte differentiation by regulating glucocorticoid signal transduction. These findings suggest that the therapeutic application of T-MSC CM could reduce obesity by preventing adipose tissue expansion.

Sphingosine-1-phosphate (S1P) is a signalling sphingolipid metabolite that regulates important cell processes, including cell proliferation and apoptosis. Circulating S1P levels have been reported to be increased in patients with psoriasis relative to healthy patients. The aim of this study was to examine the potency of S1P inhibition using an imiquimod-induced psoriasis mouse model. Both topical ceramidase and sphingosine kinase 1/2 inhibition, which blocks S1P generation, alleviated imiquimod-induced skin lesions and reduced the serum interleukin 17-A levels induced by application of imiquimod. These treatments also normalized skin mRNA levels of genes associated with inflammation and keratinocyte differentiation. Inhibition of sphingosine kinase 2, but not sphingosine kinase 1, diminished levels of suppressor of cytokine signalling 1 and blocked T helper type 17 differentiation of naïve CD4+ T cells; imiquimod-induced psoriasis-like skin symptoms were also ameliorated. These results indicate the distinct effects of sphingosine kinase 1 and sphingosine kinase 2 inhibition on T helper type 17 generation and suggest molecules that inhibit S1P formation, including ceramidase and sphingosine kinase 2 inhibitors, as novel therapeutic candidates for psoriasis.

Reactive oxygen species (ROS) generated by neutrophils provide a frontline defence against invading pathogens. We investigated the supportive effect of tonsil-derived mesenchymal stem cells (TMSCs) on ROS generation from neutrophils using promyelocytic HL-60 cells. Methods: Differentiated HL-60 (dHL-60) cells were cocultured with TMSCs isolated from 25 independent donors, and ROS generation in dHL-60 cells was measured using luminescence. RNA sequencing and real-time PCR were performed to identify the candidate genes of TMSCs involved in augmenting the oxidative burst of dHL-60 cells. Transcriptome analysis of TMSCs derived from 25 independent donors revealed high levels of procollagen C-endopeptidase enhancer 2 (PCOLCE2) in TMSCs, which were highly effective in potentiating ROS generation in dHL-60 cells. In addition, PCOLCE2 knockdown in TMSCs abrogated TMSC-induced enhancement of ROS production in dHL-60 cells, indicating that TMSCs increased the oxidative burst in dHL-60 cells via PCOLCE2. Furthermore, the direct addition of recombinant PCOLCE2 protein increased ROS production in dHL-60 cells. These results suggest that PCOLCE2 secreted by TMSCs may be used as a therapeutic candidate to enhance host defences by increasing neutrophil oxidative bursts. PCOLCE2 levels in TMSCs could be used as a marker to select TMSCs exhibiting high efficacy for enhancing neutrophil oxidative bursts.

Exosomes are a group of small membranous vesicles that are shed into the extracellular environment by tumoral or non-tumoral cells and contribute to cellular communication by delivering micro RNAs (miRNAs). In this study, we aimed to evaluate the role of exosomal miRNAs from colorectal cancer cell lines in tumorigenesis, by affecting cancer-associated fibroblasts (CAFs), which are vital constituents of the tumor microenvironment. To analyze the effect of exosomal miRNA on the tumor microenvironment, migration of the monocytic cell line THP-1 was evaluated via Transwell migration assay using CAFs isolated from colon cancer patients. The migration assay was performed with CAFs ± CCL7-blocking antibody and CAFs that were treated with exosomes isolated from colon cancer cell lines. To identify the associated exosomal miRNAs, miRNA sequencing and quantitative reverse transcription polymerase chain reaction were performed. The migration assay revealed that THP-1 migration was decreased in CCL7-blocking antibody-expressing and exosome-treated CAFs. Colon cancer cell lines contained miRNA let-7d in secreted exosomes targeting the chemokine CCL7. Exosomes from colorectal cancer cell lines affected CCL7 secretion from CAFs, possibly via the miRNA let-7d, and interfered with the migration of CCR2+ monocytic THP-1 cells in vitro.

Skin forms a physical barrier that protects the body against outside agents. The deepest layer of the skin, the stratum basale, contains two cell types: agent-sensing keratinocytes, and melanin-producing melanocytes. Keratinocytes can sense both harmless commensal organisms and harmful pathogens via Toll-like receptors (TLRs), and keratinocytes subsequently drive immune responses. Activation of TLR3 is required for barrier repair because it stimulates essential genes, including tight junction genes, and inflammatory cytokines. Within the basal layer of the skin, resident melanocytes use their dendritic processes to connect with approximately 30-40 neighboring keratinocytes. Most studies have focused on the transfer of melanin-synthesizing melanosomes from melanocytes to keratinocytes, but the potential regulation of melanogenesis by soluble factor(s) produced by keratinocytes remains to be explored. Studying such regulation in vivo is challenging because of the keratinocyte:melanocyte ratio in the epidermis and the location of the cells within the skin. Therefore, in this study, we investigated whether keratinocytes affected melanocyte melanogenesis in vitro under normal or inflammatory conditions. We found that polyinosinic-polycytidylic acid [poly(I:C)] stimulation induced PD-L1 secretion from HaCaT cells and that poly(I:C)-induced PD-L1 inhibited melanin production by B16F10 cells. These data provide key evidence that keratinocytes can alter melanocyte melanogenesis via the production of soluble factors under inflammatory conditions.

Skeletal muscle mass is decreased under a wide range of pathologic conditions. In particular, chemotherapy is well known for inducing muscle loss and atrophy. Previous studies using tonsil-derived mesenchymal stem cells (T-MSCs) or a T-MSC-conditioned medium showed effective recovery of total body weight in the chemotherapy-preconditioned bone marrow transplantation mouse model. This study investigated whether extracellular vesicles of T-MSCs, such as exosomes, are a key player in the recovery of body weight and skeletal muscle mass in chemotherapy-treated mice. T-MSC exosomes transplantation significantly decreased loss of total body weight and muscle mass in the busulfan-cyclophosphamide conditioning regimen in BALB/c recipient mice containing elevated serum activin A. Additionally, T-MSC exosomes rescued impaired C2C12 cell differentiation in the presence of activin A in vitro. We found that T-MSC exosomes possess abundant miR-145-5p, which targets activin A receptors, ACVR2A, and ACVR1B. Indeed, T-MSC exosomes rescue muscle atrophy both in vivo and in vitro via miR-145-5p dependent manner. These results suggest that T-MSC exosomes have therapeutic potential to maintain or improve skeletal muscle mass in various activin A elevated pathologic conditions.

The application of mesenchymal stem cells (MSCs) for treating bone-related diseases shows promising outcomes in preclinical studies. However, cells that are isolated and defined as MSCs comprise a heterogeneous population of progenitors. This heterogeneity can produce variations in the performance of MSCs, especially in applications that require differentiation potential in vivo, such as the treatment of osteoporosis. Here, we aimed to identify genetic markers in tonsil-derived MSCs (T-MSCs) that can predict osteogenic potential. Using a single-cell cloning method, we isolated and established several lines of nondifferentiating (ND) or osteoblast-prone (OP) clones. Next, we performed transcriptome sequencing of three ND and three OP clones that maintained the characteristics of MSCs and determined the top six genes that were upregulated in OP clones. Upregulation of WNT16 and DCLK1 expression was confirmed by real-time quantitative PCR, but only WNT16 expression was correlated with the osteogenic differentiation of T-MSCs from 10 different donors. Collectively, our findings suggest that WNT16 is a putative genetic marker that predicts the osteogenic potential of T-MSCs. Thus, examination of WNT16 expression as a selection criterion prior to the clinical application of MSCs may enhance the therapeutic efficacy of stem cell therapy for bone-related complications, including osteoporosis.

Cotransplantation of mesenchymal stem cells (MSCs) with hematopoietic stem cells (HSCs) has been widely reported to promote HSC engraftment and enhance marrow stromal regeneration. The present study aimed to define whether MSC conditioned medium could recapitulate the effects of MSC cotransplantation. Mouse bone marrow (BM) was partially ablated by the administration of a busulfan and cyclophosphamide (Bu−Cy)-conditioning regimen in BALB/c recipient mice. BM cells (BMCs) isolated from C57BL/6 mice were transplanted via tail vein with or without tonsil-derived MSC conditioned medium (T-MSC CM). Histological analysis of femurs showed increased BM cellularity when T-MSC CM or recombinant human pleiotrophin (rhPTN), a cytokine readily secreted from T-MSCs with a function in hematopoiesis, was injected with BMCs. Microstructural impairment in mesenteric and BM arteriole endothelial cells (ECs) were observed after treatment with Bu−Cy-conditioning regimen; however, T-MSC CM or rhPTN treatment restored the defects. These effects by T-MSC CM were disrupted in the presence of an anti-PTN antibody, indicating that PTN is a key mediator of EC restoration and enhanced BM engraftment. In conclusion, T-MSC CM administration enhances BM engraftment, in part by restoring vasculature via PTN production. These findings highlight the potential therapeutic relevance of T-MSC CM for increasing HSC transplantation efficacy.

Type 2 diabetes mellitus (T2DM) is a prevalent chronic metabolic disorder accompanied by high blood glucose, insulin resistance, and relative insulin deficiency. Endoplasmic reticulum (ER) stress induced by high glucose and free fatty acids has been suggested as one of the main causes of β-cell dysfunction and death in T2DM. Stem cell-derived insulin-secreting cells were recently suggested as a novel therapy for diabetes. In the present study, we demonstrate the therapeutic potential of tonsil-derived mesenchymal stem cells (TMSCs) to treat high-fat diet (HFD)-induced T2DM. To explore whether TMSC administration can alleviate T2DM, TMSCs were intraperitoneally injected in HFD-induced T2DM mice once every 2 weeks. TMSC injection markedly improved glucose tolerance and glucose-stimulated insulin secretion and prevented HFD-induced pancreatic β-cell hypertrophy and cell death. In addition, TMSC injection relieved the ER-stress response and preserved gene expression related to glucose sensing and insulin secretion. Moreover, administration of TMSC-derived conditioned medium induced similar therapeutic outcomes, suggesting paracrine effects. Finally, proteomic analysis revealed high secretion of insulin-like growth factor-binding protein 5 by TMSCs, and its expression was critical for the protective effects of TMSCs against HFD-induced glucose intolerance and ER-stress response in pancreatic islets. TMSC administration can alleviate HFD-induced-T2DM via preserving pancreatic islets and their function. These results provide novel evidence of TMSCs as an ER-stress modulator that may be a novel, alternative cell therapy for T2DM.

Next-generation sequencing is commonly used to screen for pathogenic mutations in families with Mendelian disorders, but due to the pace of discoveries, gaps have widened for some diseases between genetic and pathophysiological knowledge. We recruited and analyzed 16 families with limb-girdle muscular dystrophy (LGMD) of Arab descent from Saudi Arabia and Sudan who did not have confirmed genetic diagnoses. The analysis included both traditional and next-generation sequencing approaches. Cellular and metabolic studies were performed on Pyroxd1 siRNA C2C12 myoblasts and controls. Pathogenic mutations were identified in eight of the 16 families. One Sudanese family of Arab descent residing in Saudi Arabia harbored a homozygous c.464A>G, p.Asn155Ser mutation in PYROXD1, a gene recently reported in association with myofibrillar myopathy and whose protein product reduces thiol residues. Pyroxd1 deficiency in murine C2C12 myoblasts yielded evidence for impairments of cellular proliferation, migration, and differentiation, while CG10721 (Pyroxd1 fly homolog) knockdown in Drosophila yielded a lethal phenotype. Further investigations indicated that Pyroxd1 does not localize to mitochondria, yet Pyroxd1 deficiency is associated with decreased cellular respiration. This study identified pathogenic mutations in half of the LGMD families from the cohort, including one in PYROXD1. Developmental impairments were demonstrated in vitro for Pyroxd1 deficiency and in vivo for CG10721 deficiency, with reduced metabolic activity in vitro for Pyroxd1 deficiency. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) are often considered to be a good source for the development of regenerative medicine. Previously, we reported that tonsil‑derived MSC conditioned medium (T‑MSC CM) produces visceral fat reducing effects. As reduced visceral adiposity is closely associated with an increase in circulating adiponectin, the present study investigated the effects of T‑MSC CM on adiponectin production. T‑MSC CM was collected from previously isolated and characterized T‑MSCs and injected into senescence‑accelerated mouse prone 6 mice, which exhibit characteristics of aging and obesity. The results demonstrated a reduction in mouse weight and epididymal adipose tissue (eAT) mass following injection of T‑MSC CM. Significant increases in adiponectin expression in the eAT, and total and high molecular weight (HMW) adiponectin in the circulation were observed in the T‑MSC CM‑injected mice compared with control mice using reverse transcription‑quantitative polymerase chain reaction, western blot analysis and ELISA. In 3T3‑L1 adipocytes, T‑MSC CM treatment increased adiponectin secretion and multimerization, as detected using western blotting under non‑reducing and non‑heat‑denaturing conditions. Furthermore, glucose oxidase was used to induce oxidative stress in 3T3‑L1 adipocytes and it was observed that T‑MSC CM reduced reactive oxygen species production and the expression of certain oxidative stress markers. In addition, the results also demonstrated that the production of HMW adiponectin was increased, which indicates that T‑MSC CM may enhance adiponectin multimerization via amelioration of oxidative stress. Further studies are required to elucidate anti‑oxidant molecules secreted from T‑MSCs, and these results highlight the potential therapeutic relevance of T‑MSC CM for the treatment of obesity or obesity‑associated diseases. [ABSTRACT FROM AUTHOR]

Allergic asthma is characterized by infiltration of eosinophils, elevated Th2 cytokine levels, airway hyperresponsiveness, and IgE. In addition to eosinophils, mast cells, and basophils, a variety of cytokines are also involved in the development of allergic asthma. The pivotal role of eosinophils in the progression of the disease has been a subject of controversy. To determine the role of eosinophils in the progression of airway inflammation, we sensitized and challenged BALB/c wild-type (WT) mice and eosinophil-deficient ▵ dblGATA mice with ovalbumin (OVA) and analyzed different aspects of inflammation. We observed increased eosinophil levels and a Th2-dominant response in OVA-challenged WT mice. In contrast, eosinophil-deficient ▵dblGATA mice displayed an increased proportion of mast cells and a Th17-biased response following OVA inhalation. Notably, the levels of IL-33, an important cytokine responsible for Th2 immune deviation, were not different between WT and eosinophil-deficient mice. We also demonstrated that mast cells induced Th17-differentiation via IL-33/ST2 stimulation in vitro. These results indicate that eosinophils are not essential for the development of allergic asthma and that mast cells can skew the immune reaction predominantly toward Th17 responses via IL-33 stimulation. [ABSTRACT FROM AUTHOR]

Background: We investigated the reversibility of liver fibrosis induced with a CCl4 injection and the role of stem cells in reversing the hepatic injury. Furthermore, the most effective cell fraction among bone marrow cells (BMCs) in the repair process was analysed. Methods: C57BL/6 mice were divided into four groups after 5 weeks of injection of CCl4: control, sacrificed after 5 weeks, sacrificed at 10 weeks and sacrificed 5 weeks later after GFPdonorBM transplantation. Liver function tests and real-time polymerase chain reaction (PCR) of markers indicating liver fibrosis were compared between the groups. To identify the most effective BMC fraction that repairs liver injury, the mice were divided into three groups after the injection of CCl4 for 2 days: granulocyte colony stimulating factor (G-CSF) only, mononuclear cell (MNC) transplantation and Lin-Sca-11c kit1haematopoietic stem cell (HSC) transplantation. Eight days after transplantation, the mice were harvested and morphometric, immunohistochemical analyses were performed to compare the expression of extracellularmatrix and liver fibrosis-related factors. Results: The liver fibrosis induced by CCl4 was not spontaneously recovered but was persistent until 10 weeks, but the group injected with BMCs had less fibrosis and better liver function. Mobilization with G-CSF increased the recovery of theinjured liver and the best results were seen in those mice administered the MNC fraction and Lin-Sca-11c-kit1HSC fraction, with no difference between the two groups. Conclusion: BMC transplantation and stem cellmobilization with G-CSF effectively treats liver injury in mice. These are promising techniques for autologous transplantation in humans with liver fibrosis. [ABSTRACT FROM AUTHOR]

Background aims. Graft-versus-host disease (GvHD) remains a major complication after allogeneic hematopoietic cell transplantation (HCT). Recent literature demonstrates a potential benefit of human mesenchymal stromal cells (MSC) for the treatment of refractory GvHD; however, the optimal dose remains uncertain. We set out to develop an animal model that can be used to study the effect of MSC on GvHD. Methods. A GvHD mouse model was established by transplanting C3H/he donor bone marrow (BM) cells and spleen cells into lethally irradiated BALB/c recipient mice. MSC were obtained from C3H/he mice and the C3H/10T1/2 murine MSC line. Results. The mRNA expression of Foxp3 in regional lymph nodes (LN) localized with T cells was markedly increased by the addition of C3H10T1/2 cells in a real-time polymerase chain reaction (PCR). Using a mixed lymphocyte reaction, we determined the optimal splenocyte proliferation inhibition dose (MSC:splenocyte ratios 1:2 and 1:1). Three different C3H10T1/2 cell doses (low, 0.5 × 106, intermediate, 1 × 106, and high, 2 × 106) with a consistent splenocyte dose (1 × 106) were evaluated for their therapeutic potential in an in vivo GvHD model. The clinical and histologic GvHD score and Kaplan–Meier survival rate were improved after MSC transplantation, and these results demonstrated a dose-dependent inhibition. Conclusions. We conclude that MSC inhibit GvHD in a dose-dependent manner in this mouse model and this model can be used to study the effects of MSC on GvHD. [ABSTRACT FROM AUTHOR]

The article provides information on a study which analyzed the erythropoietin (EPO)-independent and dependent processes to optimize the timing of EPO addition during in vitro erythropoiesis. The study used umbilical cord blood obtained from the placenta under asceptic condition in vaginal deliver with informed consent of 8 mothers from Weha Women's University in Seoul, South Korea. The research also analyzed the production efficiency of glycophorin A (GPA)-expressing cells according to the timing of EPO addition.

This article presents a study on mesenchymal stem cells (MSCs), which are a subset of stem cells that reside principally in bone marrow and can be isolated from variety of adult and fetal tissues. It has been reported that MSCs exhibit immunoregulatory functions vt suppressing T cells, NK cells and dendritic cell functions. It hypothesized that MSCs mght exert an effect on the differentiation of Th17 cells and might possibly be one of the ummunoregulatory mechanisms exploited by MSCs.