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Wound healing is a complex process involving cell proliferation, migration, and extracellular matrix (ECM) remodeling. Extracellular vesicles (EVs) or exosomes derived from adipose tissue-derived stem cells (ASCs) are emerging as promising alternatives to cell therapy for advanced wound healing. Hyaluronic acid (HA), a major component of the skin ECM, is widely utilized in wound dressings and dermal fillers. This study aimed to investigate the effects of ASC-derived exosomes (ASC-EXOs) on human dermal fibroblasts (HDFs) and their potential combination with HA in in vivo wound healing and dermal filler models. In HDFs, ASC-EXOs increased cell proliferation and migration. ASC-EXOs also upregulated the expression of genes involved in cell proliferation and wound healing while stimulating collagen production in HDFs. In a porcine wound healing model, topical treatment with a combination of HA and ASC-EXOs led to higher wound closure rates compared to HA alone. Histological examination showed increased re-epithelialization and collagen type III deposition in wounds treated with the combination of HA and ASC-EXOs. In a mouse dermal filler model, tissues injected with the combination of HA and ASC-EXOs exhibited thicker tissue layers, increased vascularization, enhanced infiltration of myofibroblasts, and higher levels of collagen III and collagen fiber content compared to HA alone. These findings suggest that ASC-EXOs have beneficial effects on cell proliferation, migration, and gene expression related to wound healing, and they may accelerate wound closure and promote tissue regeneration. Furthermore, the combination of HA and ASC-EXOs may enhance wound healing and tissue remodeling, indicating its potential for both clinical and regenerative aesthetic applications in skin repair and regeneration.

Obesity and its associated metabolic comorbidities are a rising global health and social issue, with novel therapeutic approaches urgently needed. Adipose tissue plays a key role in the regulation of energy balance and adipose tissue-derived mesenchymal stem cells (AT-MSCs) have gained great interest in cell therapy. Carnitine palmitoyltransferase 1A (CPT1A) is the gatekeeper enzyme for mitochondrial fatty acid oxidation. Here, we aimed to generate adipocytes expressing a constitutively active CPT1A form (CPT1AM) that can improve the obese phenotype in mice after their implantation. AT-MSCs were differentiated into mature adipocytes, subjected to lentivirus-mediated expression of CPT1AM or the GFP control, and subcutaneously implanted into mice fed a high-fat diet (HFD). CPT1AM-implanted mice showed lower body weight, hepatic steatosis and serum insulin and cholesterol levels alongside improved glucose tolerance. HFD-induced increases in adipose tissue hypertrophy, fibrosis, inflammation, endoplasmic reticulum stress and apoptosis were reduced in CPT1AM-implanted mice. In addition, the expression of mitochondrial respiratory chain complexes was enhanced in the adipose tissue of CPT1AM-implanted mice. Our results demonstrate that implantation of CPT1AM-expressing AT-MSC-derived adipocytes into HFD-fed mice improves the obese metabolic phenotype, supporting the future clinical use of this ex vivo gene therapy approach. info:eu-repo/semantics/publishedVersion

The aim of this in vitro study was to compare three disinfection protocols of biofilm-coated machined (MAC) and acid etched (SLA) commercial pure Grade 4 Titanium disks. Samples were infected with a vial of polymicrobial biofilm to simulate peri-implantitis in vitro. Seventeen MAC and twenty SLA titanium disks were randomly assigned to: (1) glycine powder air-flow (GYPAP) for 1 min; (2) a local delivered triple paste antibiotic composed by a gel mixture with ciprofloxacin, metronidazole, and clarithromycin (3MIX) for 1 h; and (3) a combination of both (GYPAP + 3MIX). Biocompatibility of the titanium disks after each treatment protocol was assessed by measurement of adhesion and growth of adipose-derived mesenchymal stem cells (ASCs) after 24 and 72 h. A confocal laser scanning microscope (CLSM) assessed the antibacterial effect of each treatment. Data of the antibacterial efficacy and cell viability were presented as mean with standard deviation and calculated by one-way ANOVA with multiple comparisons via Bonferroni tests. Results were considered significant with p < 0.05. The higher cell viability was achieved by the 3MIX and GYPAP combination on the SLA surfaces after 72 h. CLSM analysis showed a mean ratio of dead bacteria statistically higher in the 3MIX + GYPAP group compared with the GYPAP and 3MIX subgroups (p < 0.05). In conclusion, data showed that the combination of GYPAP and 3MIX could be preferred to the other protocols, especially in presence of SLA titanium surface.

Background and objectives Adipose tissue-derived mesenchymal stem cells (ASCs) are recognized as an advantaged source for the prevention and treatment of diverse diseases including type 2 diabetes mellitus (T2DM). However, alterations in characteristics of ASCs from the aforementioned T2DM patients are still obscure, which also hinder the rigorous and systematic illumination of progression and pathogenesis. Methods and results In this study, we originally isolated peripancreatic adipose tissue-derived mesenchymal stem cells from both human type 2 diabetic and non-diabetic donors (T2DM-ASCs, ND-ASCs) with the parental consent, respectively. We noticed that T2DM-ASCs exhibited indistinguishable immunophenotype, cell vitality, chondrogenic differentiation and stemness as ND-ASCs. Simultaneously, there's merely alterations in migration and immunoregulatory capacities in T2DM-ASCs. However, differing from ND-ASCs, T2DM-ASCs exhibited deficiency in adipogenic and osteogenic differentiation, and in particular, the delayed cell cycle and different cytokine expression spectrum. Conclusions The conservative alterations of T2DM-ASCs in multifaceted characteristics indicated the possibility of autologous application of ASCs for cell-based T2DM treatment in the future.

Purpose This study aims to assess the effectiveness of Microfragmented Autologous Fat Tissue (MFAT) treatment for knee osteoarthritis and to investigate whether patients’ pre-treatment clinical condition, such as synovitis, correlates with clinical outcomes, to identify potential predicting factors for the success or failure of the treatment. Methods In this prospective Cohort Study Level II multicentric trial, consecutive patients with a diagnosis of early/mild osteoarthritis and failure of previous conservative measures were enrolled to undergo diagnostic arthroscopy and a single MFAT injection. Patients were assessed with repeated scoring systems at baseline, 6 months, and 12 months after surgery. The demographic features, the arthroscopic findings, the immunophenotype of injected tissue and the histologic examination of synovia of failed patients were analyzed. Results Data from 91 patients showed a significant improvement in Lysholm, WOMAC scores at 1-year follow-up (p Age and synovitis were identified as significant factors influencing the clinical outcome (p Conclusion MFAT is effective in reducing pain when used with a single dose injection in early/mild OA of the knee, without major complications. Age over 60 and synovitis may be predictive for persistent pain at one year and should be considered before indications.

This research was funded by the Andalusian Government, Spain (grant no. P20-01255 to M.D. and FEDER program grant no. A1-CTS-324-UGR18 to M.R.S.) and by the Spanish Ministry for Economy and Competition, Spain (grant no. SAF2017-85602-R and PID2020-119638RB-I00, both to E.G.-R.). A.I.S.-C. was the awardee of a Research Starting Fellowship for master´s students at the University of Granada, Spain. The APC was funded by MDPI., Neurological disorders, including neurodegenerative diseases, are often characterized by neuroinflammation, which is largely driven by microglia, the resident immune cells of the central nervous system (CNS). Under these conditions, microglia are able to secrete neurotoxic substances, provoking neuronal cell death. However, microglia in the healthy brain carry out CNS-supporting functions. This is due to the ability of microglia to acquire different phenotypes that can play a neuroprotective role under physiological conditions or a pro-inflammatory, damaging one during disease. Therefore, therapeutic strategies focus on the downregulation of these neuroinflammatory processes and try to re-activate the neuroprotective features of microglia. Mesenchymal stem cells (MSC) of different origins have been shown to exert such effects, due to their immunomodulatory properties. In recent years, MSC derived from adipose tissue have been made the center of attention because of their easy availability and extraction methods. These cells induce a neuroprotective phenotype in microglia and downregulate neuroinflammation, resulting in an improvement of clinical symptoms in a variety of animal models for neurological pathologies, e.g., Alzheimer’s disease, traumatic brain injury and ischemic stroke. In this review, we will discuss the application of adipose tissue-derived MSC and their conditioned medium, including extracellular vesicles, in neurological disorders, their beneficial effect on microglia and the signaling pathways involved., Andalusian Government, Spain P20-01255, FEDER program grant no. A1-CTS-324-UGR18, Spanish Ministry for Economy and Competition, Spain (grant no. SAF2017-85602-R and PID2020-119638RB-I00), Research Starting Fellowship for master´s students at the University of Granada, Spain, MDPI

Mesenchymal stem cells (MSCs) can improve chronic wound healing, however, recent studies suggest that the therapeutic effect of MSCs is mediated mainly through the growth factors and cytokines secreted by these cells, referred to as the MSC secretome. To overcome difficulties related to the translation of cell therapy into clinical use such as efficacy, safety and cost, we propose a hydrogel loaded with a secretome from the recently established human adipose tissue mesenchymal stem cell line (HATMSC2) as a potential treatment for chronic wounds. Biocompatibility and biological activity of hydrogel-released HATMSC2 supernatant were investigated in vitro by assessing the proliferation and metabolic activity of human fibroblast, endothelial cells and keratinocytes. Hydrogel degradation was measured using hydroxyproline assay while protein released from the hydrogel was assessed by interleukin-8 (IL-8) and macrophage chemoattractant protein-1 (MCP-1) ELISAs. Pro-angiogenic activity of the developed treatment was assessed by tube formation assay while the presence of pro-angiogenic miRNAs in the HATMSC2 supernatant was investigated using real-time RT-PCR. The results demonstrated that the therapeutic effect of the HATMSC2-produced factors is maintained following incorporation into collagen hydrogel as confirmed by increased proliferation of skin-origin cells and improved angiogenic properties of endothelial cells. In addition, HATMSC2 supernatant revealed antimicrobial activity, and which therefore, in combination with the hydrogel has a potential to be used as advanced wound-healing dressing.

Background Our previous study shows that Adipose tissue‐derived mesenchymal stem cells (ASCs) are a promising strategy for cell‐based therapy against pulmonary infection with Pseudomonas aeruginosa (P. aeruginosa), but the underlying mechanisms remain unclear. Methods cDNA microarray assay was performed to explore the transcriptome of ASCs primed by P. aeruginosa. Small interfering RNA (siRNA) was constructed to select the receptor candidates for P. aeruginosa recognition and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) production in ASCs. The soluble protein chimeras containing the extracellular domain of human CD69 fused to the Fc region of human immunoglobulin IgG1 were used as a probe to validate the recognition of P. aeruginosa. The association between CD69 and extracellular regulated protein kinases 1/2 (ERK1/2) was explored via co‐immunoprecipitation, siRNA, and inhibitor. The murine models of P. aeruginosa pneumonia treated with WT‐ASCs, GM‐CSF −/−‐ASCs Cd69 −/−‐ASCs or Erk1 −/−‐ASCs were used to determine the role of GM‐CSF, CD69, and ERK1 in ASCs against P. aeruginosa infection. Results We showed that C‐type lectin receptor CD69 mediated the protective effects of ASCs partly through GM‐CSF. CD69 could specifically recognize P. aeruginosa and regulate GM‐CSF secretion of ASCs. CD69 regulated the production of GM‐CSF via ERK1 in ASCs after P. aeruginosa infection. Moreover, the Administration of ASCs with deficiency of CD69 or ERK1 completely blocked its protective effects in a murine model of P. aeruginosa pneumonia. Conclusions CD69 recognizes P. aeruginosa and further facilitates ERK1 activation, which plays a crucial role in ASCs‐based therapy against P. aeruginosa pneumonia. CD69 may be a novel target molecule to improve ASCs‐based therapy against P. aeruginosa infection., 1. CD69 mediates the protective effects of ASCs partly through GM‐CSF. 2. ASCs use CD69 to recognize P. aeruginosa for ERK1 activation and subsequent GM‐CSF production. 3. CD69‐ERK1 pathway plays a key role in ASCs‐based therapy against P. aeruginosa pneumonia.

Objective To assess the effects of acellular xenogeneic nerve scaffold seeded with composite Schwann-like cells for repair of facial nerve defects in rabbits. Methods Adipose tissue-derived mesenchymal stem cells (ADSCs) were isolated from the groin fat pads of Japanese white rabbits using single enzyme digestion and were induced into Schwann cell-like cell lineages using chemical reagents and autologous platelet-rich plasma. The acellular xenogeneic nerve scaffold was prepared by chemical treatment combined with freezing and thawing of the bilateral sciatic nerve grafts from Wistar rats. The Schwann-like cells were labeled with DiOC16(3) fluorescent probe before seeding onto the scaffold. In rabbit models of facial nerve defect (5 mm), the defect was repaired using autologous nerve grafts (group A, n=10), xenogeneic nerve scaffold seeded with the labeled Schwann-like cells (group B, n=10), or xenogeneic nerve scaffold only (group C, n=10). At 8 weeks after the operation, the nerve function was evaluated quantitatively by assessing the electrophysiology of the regenerated nerve, density of myelinated nerve fibers and expression of specific proteins. Results All the rabbit models survived after the operation. In all the rabbits, the regenerated nerve showed a good continuity and integrity without the formation of obvious neuromas. At 8 weeks after the operation, the latency of the action potential of the repaired facial nerve was prolonged in all the groups; the latency was significantly shorter in groups A and B than in group C, and was the shortest in group A (P < 0.05). The density of the regenerated myelinated nerve fibers, as calculated using toluidine blue staining, was significantly higher in groups A and B than in group C (P < 0.05) and similar between groups A and B. The expression levels of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in the regenerated nerve were significantly higher in groups A and B than in group C (P < 0.05); the expression of NGF was the highest in group A, and BDNF expression was the highest in group B. Conclusion The acellular xenogeneic scaffold seeded with Schwann-like cells achieves similar effect to autogenous nerve graft for repairing facial nerve defect in the early stage of injury.

Objectives CD49f is expressed on a variety of stem cells and has certain effects on their cytological functions, such as proliferation and differentiation potential. However, whether CD49f is expressed on the surface of adipose tissue‐derived mesenchymal stem cells (ADSCs) and its effect on ADSCs has not been clarified. Materials and methods The effects of in vitro culture passage and inflammatory factor treatment on CD49f expression and the adhesion ability of ADSCs from mice and rats were investigated. CD49f+ cells were selected from rat ADSCs (rADSCs) by magnetic‐activated cell sorting (MACS), and the cellular functions of CD49f+ ADSCs and unsorted ADSCs, including their clonogenic, proliferation, adipogenic and osteogenic differentiation, migration and anti‐apoptotic capacities, were compared. Results CD49f expression and the adhesion ability of ADSCs decreased with increasing in vitro culture passage number. TNF‐α and IFN‐γ treatment decreased CD49f expression but increased the adhesion ability of ADSCs. After CD49f was blocked with an anti‐CD49f antibody, the adhesion ability of ADSCs was decreased. No significant difference in clonogenic activity was observed between unsorted ADSCs and CD49f+ ADSCs. CD49f+ ADSCs had greater proliferation, adipogenic and osteogenic differentiation, migration and anti‐apoptotic capacities than unsorted ADSCs. Conclusion In the current study, the expression of CD49f on ADSCs was identified for the first time. The expression of CD49f on ADSCs was influenced by in vitro culture passage number and inflammatory factor treatment. Compared with unsorted ADSCs, CD49f + ADSCs exhibited superior cellular functions, thus may have great application value in mesenchymal stem cell (MSC)‐based therapies., Although mesenchymal stem cells (MSCs) is one of the most promising seed cells in tissue engineering, its application faces some problems, such as an insufficient cell source, cell senescence and the loss of ‘stemness’ during in vitro culture, and rapid cell death after in vivo transplantation. To address these issues, we isolated CD49f+ cells from ADSCs by magnetic activated cell sorting and confirmed that CD49f+ ADSCs possessed greater proliferation, multiple differentiation, migration and anti‐apoptotic potential than unsorted ADSCs. After treatment with anti‐CD49f antibody, ADSCs exhibited a significantly reduced adhesion ability. These results indicated that CD49f+ ADSCs have superior cellular functions and considerable application prospects.

Mezenkimal kök hücreler deneysel ve klinik çalışmalarda kullanılan önemli tedavi yöntemleridir. Günümüzde mezenkimal kök hücre tedavisi birçok hastalıkta uygulanmaktadır. Organ nakillerinden sonra kullanılan immünsupresif ilaç tedavisinin yanı sıra hastalarda mezenkimal kök hücre uygulaması da yapılmaktadır. Yapılan bu deneysel çalışmada organ nakillerinde immünsupresif amaçla kullanılan sunitinibin sıçan adipoz doku kaynaklı mezenkimal kök hücreler üzerindeki sitotoksik etkilerinin araştırılması ve antioksidant özelliği olduğu bilinen oksitosinin bu toksisite üzerindeki etkileri gözlemlenmiştir. Adipoz doku kaynaklı mezenkimal kök hücreler hattı ticari olarak temin edilmiştir. Yapılacak çalışmaların gerektirdiği doğrultuda, mezenkimal kök hücreler yeterli sayıya ulaşana kadar özel besiyeri içerisinde 37oC’de inkübe edilmiştir. Etken maddelerin sitotoksik etkileri WST-1 testi ile zaman ve doz bağımlı olarak değerlendirilmiştir. Canlılık ve proliferasyon analizleri ışık mikroskobu kullanılarak ve hücre sayımı yapılarak belirlenmiştir. BD Accuri C6 flow sitometri cihazı kullanılarak apoptoz ve hücre canlılığı değerlendirmeleri yapılmıştır. Biyokimyasal yöntemlerle lipit peroksidasyonu, glutatyon peroksidaz (GPx), katalaz (CAT) aktivitesi ve superoksit dismutaz (SOD) aktivitesi ölçülmüştür. Gruplara uygulanan ajanların sitotoksik dozları WST-1 testi ile değerlendirilmiştir. Anneksin V yöntemi ile nekrotik, erken apoptotik, geç apoptotik, toplam apoptotik ve canlı hücreler hem yüzde olarak hem de hücre/ml olarak hesaplanmıştır. Oksidatif stresle oluşan ve reaktif oksijen metabolitlerinin verdiği zararı belirlemek amacıyla lipit peroksidasyonu, glutatyon peroksidaz (GPx), katalaz (CAT) aktivitesi ve superoksit dismutaz (SOD) aktivitesi ölçülmüştür. Sıçan adipoz doku kaynaklı mezenkimal kök hücreler için Sunitinib IC50 dozu (48. saat) için 44, 57 μM olarak hesaplanmıştır. Oksitosin için ise çalışılan dozlarda sitotoksik etki gözlemlenmemiştir. Bu yüzden en yüksek sabit doz olan 100 μM ile çalışmaya devam edilmiştir. BD Accuri C6 flow sitometri cihazı ile yapılan ölçümler ve yapılan hesaplamalar doğrultusunda kontrol grubundaki hücrelerde ölü hücre yüzdesi 1,03; canlı hücre yüzdesi 98,97 olarak hesaplanmıştır. Oksitosin grubundaki hücrelerde ölü hücre yüzdesi 2,59, canlı hücre yüzdesi 97,41 olarak hesaplanmıştır. Sunitinib grubunda erken apoptoz oranı %7,69; geç apoptoz oranı %4,31; ölü hücre %8,10; canlı hücre %79,90 olarak hesaplanmıştır. Sunitinib+Oksitosin kombine grup sonuçlarında ise erken apoptoz oranı %3,90; geç apoptoz oranı %1,34; ölü hücreler %7,81; canlı hücreler %25,5 olarak ölçülmüştür. Biyokimyasal bulgular ise MDA düzeylerinde Kontrol grubu, GSH-Px düzeyleri açısından Sunitinib grubu, CAT düzeyleri açısından oksitosin grubunda değişiklik olmadığı için güven aralıkları diğer gruplara göre hesaplandı, SOD düzeyleri bakımından ise kontrol ile diğer üç grup arasında farklar anlamlı bulunurken, diğer gruplar arasında hiçbir ikili farklılık anlamlı bulunmadı. Tüm gruplar arası ikili farkların tümü istatistiksel olarak anlamlı olarak saptanmıştır (p, Mesenchymal stem cells are essential treatment methods used in experimental and clinical studies. Nowadays, mesenchymal stem cell therapy is used in many various diseases. In addition to immunosuppressive drug therapy used after organ transplants, a mesenchymal stem cell application can also be utilized in the patient's treatment. In this experimental study, the cytotoxic effects of Sunitinib drug, which is used for immunosuppressive purposes in organ transplants, on mesenchymal stem cells which is derivated from adipose tissue and the toxicity effect on oxytocin (known to have antioxidant properties) were inspected. A mesenchymal stem cell line derived from mouse adipose tissue was commercially available. In line with the work to be done, stromal stem cells were incubated at 37°C in a special medium until they reached a sufficient number. The cytotoxic effects of the active ingredients were evaluated using the WST-1 assay in a time and dose-dependent manner. Viability and prevalence analyze were determined using light microscopy and cell counting. Apoptosis and cell viability were assessed with a BD Accuri C6 flow cytometer analyzer. Lipid peroxidation, glutathione peroxidase (GPx), catalase activity (CAT), and superoxide dismutase (SOD) activity were measured by biochemical methods. The cytotoxic doses of agents applied to the groups were assessed by the WST-1 assay. Necrotic, early apoptotic, late apoptotic, total, and viable cells were calculated in both percentage and cell/ml by the Annexin V method. The effect of lipid peroxidation, glutathione peroxidase (GPx), catalase activity (CAT), and superoxide dismutase activity (SOD) was measured in order to determine the damage due to oxidative stress and reactive oxygen metabolites. The IC50 dose of Sunitinib in mesenchymal stem cells derived from adipose tissue of mice (48 h) was calculated as 44.57 μM. For oxytocin, no cytotoxic effect was observed at the studied doses. Therefore, the study continued with the highest stable dose of 100 μM. According to the measurements and calculations made with the BD Accuri C6 cellular device, the percentage of dead cells in cells in the control group was calculated at 1.03 and the percentage of live cells was 98.97. The percentage of dead cells in the cells in the oxytocin group was 2.59 and the percentage of live cells was 97.41. In the sunitinib group, the rate of early apoptosis was 7.69%, late apoptosis rate 4.31%, dead cells 8.10%, and live-cell 79.90%. In the results of the pooled Sunitinib + oxytocin group, the rate of early apoptosis was 3.90%, the rate of late apoptosis was 1.34%, the percentage of dead cells 7.81, and the percentage of live cells 25.5. As for the biochemical results, confidence intervals were calculated according to the other groups as there was no change in MDA levels in the control group, GSH-Px levels in the Sunitinib group, and CAT levels in the oxytocin group, and there was no significant difference between the groups. All marital differences between all groups were statistically significant (p

Mesenchymal stem cells (MSCs) have been recently introduced in veterinary medicine as a potential therapeutic tool for several pathologies. The large-scale in vitro expansion needed to ensure the preparation of a suitable number of MSCs for clinical application usually requires the use of xenogeneic supplements like the fetal bovine serum (FBS). The substitution of FBS with species-specific supplements would improve the safety of implanted cells, reducing the risk of undesired immune responses following cell therapy. We have evaluated the effectiveness of canine adipose tissue-derived stromal vascular fraction (SVF) and MSCs (ADMSCs) expansion in the presence of canine blood-derived supplements. Cells were cultured on traditional plastic surface and inside a 3D environment derived from the jellification of different blood-derived products, i.e., platelet-poor plasma (PPP), platelet-rich plasma (PRP), or platelet lysate (PL). PPP, PRP, and PL can contribute to canine ADMSCs in vitro expansion. Both allogeneic and autologous PPP and PL can replace FBS for ADMSCs culture on a plastic surface, exhibiting either a similar (PPP) or a more effective (PL) stimulus to cell replication. Furthermore, the 3D environment based on homospecific blood-derived products polymerization provides a strong stimulus to ADMSCs replication, producing a higher number of cells in comparison to the plastic surface environment. Allogeneic or autologous blood products behave similarly. The work suggests that canine ADMSCs can be expanded in the absence of xenogeneic supplements, thus increasing the safety of cellular preparations. Furthermore, the 3D fibrin-based matrices could represent a simple, readily available environments for effective in vitro expansion of ADMSCs using allogeneic or autologous blood-products.

The administration of adipose tissue-derived mesenchymal stem cells (ADMSCs) represents a promising therapeutic option after myocardial ischemia or myocardial infarction. However, their potential is reduced due to the high post-transplant cell mortality probably caused by oxidative stress and mitogen-deficient microenvironments. To identify protection strategies for ADMSCs, this study investigated the influence of the non-psychoactive phytocannabinoid cannabidiol (CBD) and the endocannabinoid analogue R(+)-methanandamide (MA) on the induction of heme oxygenase-1 (HO-1) and autophagy under serum-free conditions. At a concentration of 3 &micro, M, CBD induced an upregulation of HO-1 mRNA and protein within 6 h, whereas for MA only a late and comparatively lower increase in the HO-1 protein could be detected after 48 h. In addition, both cannabinoids induced time- and concentration-dependent increases in LC3A/B-II protein, a marker of autophagy, and in metabolic activity. A participation of several cannabinoid-binding receptors in the effect on metabolic activity and HO-1 was excluded. Similarly, knockdown of HO-1 by siRNA or inhibition of HO-1 activity by tin protoporphyrin IX (SnPPIX) had no effect on CBD-induced autophagy and metabolic activity. On the other hand, the inhibition of autophagy by bafilomycin A1 led to a significant decrease in cannabinoid-induced metabolic activity and to an increase in apoptosis. Under these circumstances, a significant induction of HO-1 expression after 24 h could also be demonstrated for MA. Remarkably, inhibition of HO-1 by SnPPIX under conditions of autophagy deficit led to a significant reversal of apoptosis in cannabinoid-treated cells. In conclusion, the investigated cannabinoids increase metabolic viability of ADMSCs under serum-free conditions by inducing HO-1-independent autophagy but contribute to apoptosis under conditions of additional autophagy deficit via an HO-1-dependent pathway.

The therapeutic and differentiation potential of human mesenchymal stems cells (hMSCs) makes these cells a promising candidate for cellular therapies and tissue engineering. On the path of a successful medical application of hMSC, the cultivation of cells in a three-dimensional (3D) environment was a landmark for the transition from simple two-dimensional (2D) testing platforms to complex systems that mimic physiological in vivo conditions and can improve hMSC curative potential as well as survival after implantation. A 3D arrangement of cells can be mediated by scaffold materials where cells get entrapped in pores, or by the fabrication of spheroids, scaffold-free self-organized cell aggregates that express their own extracellular matrix. Independently from the cultivation method, cells expanded in 3D experience an inhomogeneous microenvironment. Many gradients in nutrient supply, oxygen supply, and waste disposal from one hand mimic in vivo microenvironment, but also put every cell in the 3D construct in a different context. Since oxygen concentration in spheroids is compromised in a size-dependent manner, it is crucial to have a closer insight on the thresholds of hypoxic response in such systems. In this work, we want to improve our understanding of oxygen availability and consequensing hypoxia onset in hMSC spheroids. Therefore, we utilized human adipose tissue-derived MSCs (hAD-MSCs) modified with a genetical sensor construct to reveal (I) the influence of spheroid production methods and (II) hMSCs cell number per spheroid to detect the onset of hypoxia in aggregates. We could demonstrate that not only higher cell numbers of MSCs, but also spheroid formation method plays a critical role in onset of hypoxia.

Background: Activated T lymphocytes play an important role in the pathogenesis of rheumatic diseases (RD). Mesenchymal stem cells (MSCs) possess immunoregulatory activities but such functions of MSCs from bone marrow of systemic lupus erythematosus (SLE), systemic sclerosis (SSc), and ankylosing spondylitis (AS) patients are impaired. Adipose tissue–derived MSCs (ASCs) are an optional pool of therapeutically useful MSCs, but biology of these cells in RD is poorly known. This study aimed at investigating the effect of ASCs from RD patients and healthy donors (HD) on the expression of the key T-cell activation markers. Methods: ASCs were isolated from subcutaneous abdominal fat from SLE ( n = 16), SSc ( n = 18), and AS ( n = 16) patients, while five human ASCs lines from HD were used as a control. Untreated and cytokine (tumor necrosis factor α + interferon γ)-treated ASCs were co-cultured with allogenic, mitogen (phytohemagglutinin)-stimulated peripheral blood mononuclear cells (PBMCs) or purified anti-CD3/CD28-activated CD4+ T lymphocytes. Contacting and noncontacting ASCs-PBMCs co-cultures were performed. RD/ASCs were analyzed in co-cultures with both allogeneic and autologous PBMCs. Flow cytometry analysis was used to evaluate expression of CD25, HLA-DR, and CD69 molecules on CD4+ and CD8+ cells. Results: In co-cultures with allogeneic, activated CD4+ T cells and PBMCs, HD/ASCs and RD/ASCs downregulated CD25 and HLA-DR, while upregulated CD69 molecules expression on both CD4+ and CD8+ cells with comparable potency. This modulatory effect was similar in contacting and noncontacting co-cultures. RD/ASCs exerted weaker inhibitory effect on CD25 expression on autologous than allogeneic CD4+ and CD8+ T cells. Conclusion: RD/ASCs retain normal capability to regulate expression of activation markers on allogeneic T cells. Both HD/ASCs and RD/ASCs exert this effect independently of their activation status, mostly through the indirect pathway and soluble factors. However, autologous CD4+ and CD8+ T cells are partially resistant to RD/ASCs inhibition of CD25 expression, suggesting weaker control of T-cell activation in vivo.

BackgroundAlthough it has been preclinically suggested that adipose tissue-derived mesenchymal stem cell (ADSC)-based therapy could effectively treat chronic liver diseases, the hepatic engraftment of ADSCs is still extremely low, which severely limits their long-term efficacy for chronic liver diseases. This study was designed to investigate the impact of antioxidant preconditioning on hepatic engraftment efficiency and therapeutic outcomes of ADSC transplantation in liver fibrotic mice.MethodsLiver fibrosis model was established by using intraperitoneal injection of carbon tetrachloride (CCl4) in the male C57BL/6 mice. Subsequently, the ADSCs with or without antioxidant pretreatment (including melatonin and reduced glutathione (GSH)) were administrated into fibrotic mice via tail vein injection. Afterwards, the ADSC transplantation efficiency was analyzed by ex vivo imaging, and the liver functions were assessed by biochemical analysis and histopathological examination, respectively. Additionally, a typical hydrogen peroxide (H2O2)-induced cell injury model was applied to mimic the cell oxidative injury to further investigate the protective effects of antioxidant preconditioning on cell migration, proliferation, and apoptosis of ADSCs.ResultsOur data showed that antioxidant preconditioning could enhance the therapeutic effects of ADSCs on liver function recovery by reducing the level of AST, ALT, and TBIL, as well as the content of hepatic hydroxyproline and fibrotic area in liver tissues. Particularly, we also found that antioxidant preconditioning could enhance hepatic engraftment efficiency of ADSCs in liver fibrosis model through inhibiting oxidative injury.ConclusionsAntioxidant preconditioning could effectively improve therapeutic effects of ADSC transplantation for liver fibrosis through enhancing intrahepatic engraftment efficiency by reducing oxidative injuries. These findings might provide a practical strategy for enhancing ADSC transplantation and therapeutic efficiency.

Osteoarthritis (OA) is a major joint disease that promotes locomotor deficiency during the middle- to old-age, with the associated disability potentially decreasing quality of life. Recently, surgical strategies to reconstruct both articular cartilage and subchondral bone for OA have been diligently investigated for restoring joint structure and function. Adipose tissue-derived mesenchymal stem cells (AT-MSCs), which maintain pluripotency and self-proliferation ability, have recently received attention as a useful tool to regenerate osteocartilage for OA. In this review, several studies were described related to AT-MSC spheroids, with scaffold and scaffold-free three-dimensional (3D) constructs produced using “mold” or “Kenzan” methods for osteochondral regeneration. First, several examples of articular cartilage regeneration using AT-MSCs were introduced. Second, studies of osteochondral regeneration (not only cartilage but also subchondral bone) using AT-MSCs were described. Third, examples were presented wherein spheroids were produced using AT-MSCs for cartilage regeneration. Fourth, osteochondral regeneration following autologous implantation of AT-MSC scaffold-free 3D constructs, fabricated using the “mold” or “Kenzan” method, was considered. Finally, prospects of osteochondral regeneration by scaffold-free 3D constructs using AT-MSC spheroids were discussed.

Several studies have investigated strategies to improve the clinical efficacy of radiotherapy (RT) against hepatocellular carcinoma (HCC), yet the prognosis remains poor. Human adipose tissue-derived mesenchymal stem cells (AT-MSCs), easily accessible and abundant in quantity, have represented as an attractive therapeutic tool for the stem cell-based treatment for cancer diseases. Through direct co-culture and indirect separate culture experiments, we showed that AT-MSCs could enhance inhibitory effect of RT on reducing HCC cell growth, migration and invasion in both in vitro and in vivo experiments. RNA-sequencing analysis revealed a noticeable interferon-induced transmembrane 1 (IFITM1)-induced tumor gene signature. Gain and loss of mechanistic studies indicated that mechanism was attributed to downregulated expression of signal transducer and activator of transcription 3 (STAT3) and matrix metallopeptidases (MMPs) and upregulated expression of P53 and caspases. Collectively, our findings suggest that AT-MSCs might enhance the therapeutic effects of RT on HCC, providing a rationale for AT-MSCs and RT combination therapy as a new remedy for HCC.

Exosomes are nano-sized vesicles (30–200 nm) constantly released by almost all cells. The ability of exosomes to travel between cells and deliver their cargo, which includes lipids, proteins, and nucleic acids, makes them an appealing cell-free therapy option to treat multiple diseases. Here, we investigated for the first time whether human adipose tissue-derived mesenchymal stem cell-derived exosomes (ASC-exosomes) can ameliorate atopic dermatitis (AD) in an in vivo mouse model. When injected either intravenously (IV) or subcutaneously (SC) into NC/Nga mice treated with house dust mite antigens, ASC-exosomes were found to reduce pathological symptoms such as clinical score, the levels of serum IgE, the number of eosinophils in blood, and the infiltration of mast cells, CD86+, and CD206+ cells in skin lesions. ASC-exosomes also significantly reduced mRNA expression of various inflammatory cytokines such as interleukin (IL)-4, IL-23, IL-31, and tumor necrosis factor-α (TNF-α) in AD skin lesions of Nc/Nga mice. Taken together, these results suggest that ASC-exosomes can be a novel promising cell-free therapeutic modality for AD treatment. Electronic supplementary material The online version of this article (10.1186/s13287-018-0939-5) contains supplementary material, which is available to authorized users.

Pancreatic and duodenal homeobox 1 (Pdx1) and Sonic hedgehog (Shh) are the key regulators of beta‐cell function. In vitro experiments have shown that there is significant cooperation between Pdx1 and Shh with regard to the production and maintenance of insulin‐producing cells (IPCs). In this study, the combined effect of Pdx1 overexpression and Shh manipulation on the function of adipose tissue‐derived IPCs was determined. A eukaryotic expression vector (Pdx1‐ pCDNA3.1(+)) was constructed and transfected into a Chinese hamster ovary (CHO) cell line. Adipose tissue‐derived mesenchymal stem cells (ADMSCs) obtained from rats were assigned to two groups [control (C) and manipulated (M)] and differentiated into IPCs. Manipulated cells were treated with a mixture of FGF‐β and cyclopamine and recombinant Shh protein at days 3 and 11, respectively, and transfected with Pdx1‐ pCDNA3.1(+) at day 10. The expression of multiple genes related to function of beta cells was analyzed using real‐time PCR. The functionality of IPCs in vitro was analyzed through dithizone (DTZ) staining and ELISA. IPCs were injected into the tail vein of diabetic rats, and blood glucose and insulin concentrations were measured. CHO cells transfected with Pdx1‐ pCDNA3.1(+) showed a significantly higher expression of Pdx1 compared with nontransfected cells. Manipulated IPCs exhibited a significantly higher expression of MafA, Nkx2.2, Nkx6.1, Ngn3, insulin, and Isl1 and a higher insulin secretion in response to glucose challenge in relation to control cells. Rats that received manipulated IPCs exhibited a higher ability to normalize blood glucose and insulin secretion when compared to controls. Our protocol might be used for more efficient cell therapy of patients with diabetes in the future.

Mesenchymal stem cell (MSC) therapy has emerged as a potential novel method of treating liver fibrosis. To date, bone marrow-derived MSCs (BM-MSCs) and adipose tissue-derived MSCs (AD-MSCs) have not been analyzed with respect to their ability to combat liver fibrosis. The present study aimed to compare the capabilities of BM-MSCs and AD-MSCs in the treatment of liver fibrosis. BM-MSCs and AD-MSCs were taken from male Sprague-Dawley rats and cultured. Hepatic stellate cells (HSCs) were co-cultured with either BM-MSCs or AD-MSCs, and the effects of BM-MSCs or AD-MSCs on the proliferation, activation and apoptosis of HSCs were determined. The secretion of a selected group of cytokines by BM-MSCs and AD-MSCs was measured using enzyme-linked immunosorbent assays. Using a CCl4-induced liver fibrosis animal model, the anti-inflammatory and anti-fibrotic effects of BM-MSCs or AD-MSCs against liver fibrosis in vivo were evaluated. The morphological examination and analysis of specific surface markers confirmed the successful preparation of BM-MSCs and AD-MSCs. Furthermore, the proliferation, activation and apoptosis of HSCs were significantly inhibited by BM-MSCs and AD-MSCs, with statistically greater reductions achieved by AD-MSCs compared with BM-MSCs. Direct comparison of the secretion of selected cytokines by BM-MSCs and AD-MSCs revealed that significantly higher levels of nerve growth factor and transforming growth factor-β1 were secreted in the AD-MSC culture medium, whereas levels of vascular endothelial growth factor and interleukin-10 did not differ significantly between AD-MSCs and BM-MSCs. In vivo studies using a CCl4-induced liver fibrosis model demonstrated that inflammatory activity and fibrosis staging scores were significantly lower in the MSC-treated groups compared with controls. Although AD-MSCs improved anti-inflammatory and anti-fibrotic effects compared with BM-MSCs, these differences were not significant. Thus, the current study demonstrated that BM-MSCs and AD-MSCs are similarly effective at attenuating liver fibrosis by inhibiting the activation and proliferation of HSCs, as well as promoting the apoptosis of HSCs.

Background To analyze protective/regenerative effects of adipose tissue-derived mesenchymal stem cells (ADMSC) on 131I-Radioiodine (RAI)-induced salivary gland damage in rats. Materials and Methods Study population consisted of controls (n:6) and study groups (n:54): RAI (Group 1), ADMSC (Group 2), amifostine (Group 3), RAI+amifostine (Group 4), concomitant RAI+ADMSC (Group 5) and RAI+ADMSC after 48 h (Group 6). We used light microscopy (LM), transmission electron microscopy (TEM), and salivary gland scintigraphy (SGS), and analyzed data statistically. Results We observed the homing of ADMSC in salivary glands at 1st month on LM. RAI exposure affected necrosis, periductal fibrosis, periductal sclerosis, vascular sclerosis and the total sum score were in a statistically significant manner (P < 0.05). Intragroup comparisons with LM at 1st and 6th months revealed statistically significant improvements in Group 6 (P < 0.05) but not in Groups 4 and 5. Intergroup comparisons of the total score showed that Groups 4 and 5 in 1st month and Group 6 in 6th month had the lowest values. TEM showed vacuolization, edema, and fibrosis at 1st month, and an improvement in damage in 6th month in Groups 5 and 6. SGSs revealed significant differences for the maximum secretion ratio (Smax) (P = 0.01) and the gland-to-background ratio at a maximum count (G/BGmax) (P = 0. 01) at 1st month, for G/BGmax (P = 0.01), Smax (P = 0.01) and the time to reach the maximum count ratio over the time to reach the minimum count (Tmax/Tmin) (P = 0.03) at 6th month. 1st and 6th month scans showed differences for Smax and G/BGmax (P = 0.04), but not for Tmax/Tmin (p > 0.05). We observed a significant deterioration in gland function in group 1, whereas, mild to moderate deteriorations were seen in protective treatment groups. Conclusions Our results indicated that ADMSC might play a promising role as a protective/regenerative agent against RAI-induced salivary gland dysfunction.

Mesenchymal stem cell (MSC) transplantation alone may be insufficient for treatment of liver fibrosis because of complicated histopathological changes in the liver. Given that miR‐122 plays an essential role in liver fibrosis by negatively regulating the proliferation and transactivation of hepatic stellate cells (HSCs), this study investigated whether miR‐122 modification can improve the therapeutic efficacy of adipose tissue‐derived MSCs in treating liver fibrosis. MiR‐122‐modified AMSCs (AMSC‐122) were constructed through lentivirus‐mediated transfer of pre‐miR‐122. MiR‐122‐modified AMSCs expressed high level of miR‐122, while they retained their phenotype and differentiation potential as naïve AMSCs. AMSC‐122 more effectively suppressed the proliferation of and collagen maturation in HSCs than scramble miRNA‐modified AMSCs. In addition, AMSC‐derived exosomes mediated the miR‐122 communication between AMSCs and HSCs, further affecting the expression levels of miR‐122 target genes, such as insulin‐like growth factor receptor 1 (IGF1R), Cyclin G(1) (CCNG1) and prolyl‐4‐hydroxylase α1 (P4HA1), which are involved in proliferation of and collagen maturation in HSCs. Moreover, miR‐122 modification enhanced the therapeutic efficacy of AMSCs in the treatment of carbon tetrachloride (CCl4)‐induced liver fibrosis by suppressing the activation of HSCs and alleviating collagen deposition. Results demonstrate that miR‐122 modification improves the therapeutic efficacy of AMSCs through exosome‐mediated miR‐122 communication; thus, miR‐122 modification is a new potential strategy for treatment of liver fibrosis.

Background Adipose tissue-derived mesenchymal stem cells (ADSCs) are promising candidates for regenerative medicine. However, long-term in vitro passaging leads to stemness loss and cell senescence of ADSCs, resulting in failure of ADSC-based therapy. Methods In this study, ADSCs were treated with low dose of antioxidants (reduced glutathione and melatonin) with anti-aging and stem cell protection properties in the in vitro passaging, and the cell functions including stem cell senescence, cell migration, cell multidirectional differentiation potential, and ROS content were carefully analyzed. Results We found that GSH and melatonin could maintain ADSC cell functions through reducing cell senescence and promoting cell migration, as well as by preserving stemness and multidirectional differentiation potential, through inhibiting ROS generation during long-term expansion of ADSCs. Conclusions Our results suggested that antioxidant treatment could efficiently prevent the dysfunction and preserve cell functions of ADSCs after long-term passaging, providing a practical strategy to facilitate ADSC-based therapy.

Background Skeletal development and its cellular function are regulated by various transcription factors. The T-box (Tbx) family of transcription factors have critical roles in cellular differentiation as well as heart and limbs organogenesis. These factors possess activator and/or repressor domains to modify the expression of target genes. Despite the obvious effects of Tbx20 on heart development, its impact on bone development is still unknown. Methods To investigate the consequence by forced Tbx20 expression in the osteogenic differentiation of human mesenchymal stem cells derived from adipose tissue (Ad-MSCs), these cells were transduced with a bicistronic lentiviral vector encoding Tbx20 and an enhanced green fluorescent protein. Results Tbx20 gene delivery system suppressed the osteogenic differentiation of Ad-MSCs, as indicated by reduction in alkaline phosphatase activity and Alizarin Red S staining. Consistently, reverse transcription-polymerase chain reaction analyses showed that Tbx20 gain-of-function reduced the expression levels of osteoblast marker genes in osteo-inductive Ad-MSCs cultures. Accordingly, Tbx20 negatively affected osteogenesis through modulating expression of key factors involved in this process. Conclusion The present study suggests that Tbx20 could inhibit osteogenic differentiation in adipose-derived human mesenchymal stem cells.

Nowadays, cardiac regenerative medicine is facing many limitations because of the complexity to find the most suitable stem cell source and to understand the regenerative mechanisms involved. Mesenchymal stem cells (MSCs) have shown great regenerative potential due to their intrinsic properties and ability to restore cardiac functionality, directly by transdifferentiation and indirectly by paracrine effects. Yet, how MSCs could respond to definite cardiac-committing microenvironments, such as that created by resident cardiac progenitor cells in the form of cardiospheres (CSs), has never been addressed. Recently, a putative MSC pool has been described in the mediastinal fat (hmADMSCs), but both its biology and function remain hitherto unexplored. Accordingly, we investigated the potential of hmADMSCs to be committed toward a cardiovascular lineage after preconditioning with CS-conditioned media (CCM). Results indicated that CCM affects cell proliferation. Gene expression levels of multiple cardiovascular and stemness markers (MHC, KDR, Nkx2.5, Thy-1, c-kit, SMA) are significantly modulated, and the percentage of hmADMSCs preconditioned with CCM and positive for Nkx2.5, MHC, and KDR is significantly higher relative to FBS and explant-derived cell conditioned media (EDCM, the unselected stage before CS formation). Growth factor-specific and survival signaling pathways (i.e., Erk1/2, Akt, p38, mTOR, p53) present in CCM are all equally regulated. Nonetheless, earlier BAD phosphorylation (Ser112) occurs associated with the CS microenvironment (and to a lesser extent to EDCM), whereas faster phosphorylation of PRAS40 in FBS, and of Akt (Ser473) in EDCM and 5-azacytidine occurs compared to CCM. For the first time, we demonstrated that the MSC pool held in the mediastinal fat is adequately plastic to partially differentiate in vitro toward a cardiac-like lineage. Besides, we have provided novel evidence of the potent inductive niche-like microenvironment that the CS structure can reproduce in vitro. hmADMSCs can represent an interesting tool in order to exploit their possible role in cardiovascular diseases and treatment.

Background/Aims: Chronic inflammation contributes to cartilage degeneration during the progression of osteoarthritis (OA). Adipose tissue-derived mesenchymal stem cells (ADMSC) show great potential to treat inflammatory and degradative processes in OA and have demonstrated paracrine effects in chondrocytes. In the present work, we have isolated and characterized the extracellular vesicles from human AD-MSC to investigate their role in the chondroprotective actions of these cells. Methods: AD-MSC were isolated by collagenase treatment from adipose tissue from healthy individuals subjected to abdominal lipectomy surgery. Microvesicles and exosomes were obtained from conditioned medium by filtration and differential centrifugation. Chondrocytes from OA patients were used in primary culture and stimulated with 10 ng/ml interleukin(IL)-1 beta in the presence or absence of AD-MSC microvesicles, exosomes or conditioned medium. Protein expression was investigated by ELISA and immunofluorescence, transcription factor-DNA binding by ELISA, gene expression by real-time PCR, prostaglandin E-2 (PGE(2)) by radioimmunoassay, and matrix metalloproteinase (MMP) activity and nitric oxide (NO) production by fluorometry. Results: In OA chondrocytes stimulated with IL-1 beta, microvesicles and exosomes reduced the production of inflammatory mediators tumor necrosis factor-alpha, IL-6, PGE(2) and NO. The downregulation of cyclooxygenase-2 and microsomal prostaglandin E synthase-1 would lead to the decreased PGE(2) production while the effect on NO could depend on the reduction of inducible nitric oxide synthase expression. Treatment of OA chondrocytes with extracellular vesicles also decreased the release of MMP activity and MMP-13 expression whereas the production of the anti-inflammatory cytokine IL-10 and the expression of collagen II were significantly enhanced. The reduction of inflammatory and catabolic mediators could be the consequence of a lower activation of nuclear factor-kappa B and activator protein-1. The upregulation of annexin A1 specially in MV may contribute to the anti-inflammatory and chondroprotective effects of AD-MSC. Conclusions: Our data support the interest of AD-MSC extracellular vesicles to develop new therapeutic approaches in joint conditions. (C) 2018 The Author(s) Published by S. Karger AG, Basel

[EN] Background/Aims: Chronic inflammation contributes to cartilage degeneration during the progression of osteoarthritis (OA). Adipose tissue-derived mesenchymal stem cells (ADMSC) show great potential to treat inflammatory and degradative processes in OA and have demonstrated paracrine effects in chondrocytes. In the present work, we have isolated and characterized the extracellular vesicles from human AD-MSC to investigate their role in the chondroprotective actions of these cells. Methods: AD-MSC were isolated by collagenase treatment from adipose tissue from healthy individuals subjected to abdominal lipectomy surgery. Microvesicles and exosomes were obtained from conditioned medium by filtration and differential centrifugation. Chondrocytes from OA patients were used in primary culture and stimulated with 10 ng/ml interleukin(IL)-1 beta in the presence or absence of AD-MSC microvesicles, exosomes or conditioned medium. Protein expression was investigated by ELISA and immunofluorescence, transcription factor-DNA binding by ELISA, gene expression by real-time PCR, prostaglandin E-2 (PGE(2)) by radioimmunoassay, and matrix metalloproteinase (MMP) activity and nitric oxide (NO) production by fluorometry. Results: In OA chondrocytes stimulated with IL-1 beta, microvesicles and exosomes reduced the production of inflammatory mediators tumor necrosis factor-alpha, IL-6, PGE(2) and NO. The downregulation of cyclooxygenase-2 and microsomal prostaglandin E synthase-1 would lead to the decreased PGE(2) production while the effect on NO could depend on the reduction of inducible nitric oxide synthase expression. Treatment of OA chondrocytes with extracellular vesicles also decreased the release of MMP activity and MMP-13 expression whereas the production of the anti-inflammatory cytokine IL-10 and the expression of collagen II were significantly enhanced. The reduction of inflammatory and catabolic mediators could be the consequence of a lower activation of nuclear factor-kappa B and activator protein-1. The upregulation of annexin A1 specially in MV may contribute to the anti-inflammatory and chondroprotective effects of AD-MSC. Conclusions: Our data support the interest of AD-MSC extracellular vesicles to develop new therapeutic approaches in joint conditions. (C) 2018 The Author(s) Published by S. Karger AG, Basel, This work was supported by grants SAF2013-4874R (MINECO, FEDER) and PROMETEOII/2014/071 (Generalitat Valenciana), Spain.

Adipose-tissue derived mesenchymal stem cell (ADSC)-based therapy is a promising option for patients with atherosclerotic conditions, including coronary artery disease. However, the potential differences in the metabolic characteristics between bone marrow-derived mesenchymal stem cells (BMSCs) and ADSCs have remained to be fully elucidated. The present study aimed to compare the metabolic profiles of BMSCs and ADSCs via liquid chromatography quadrupole time-of-flight mass spectrometry. BMSCs and ADSCs obtained from elderly coronary heart disease patients were cultured, and after three passages, supernatants of each cell type were collected and systematically analysed. Substantial differences were detected between the metabolite signatures of ADSCs and BMSCs. In addition, further analysis using partial least-squares discriminant analysis score plots indicated significant differences between the supernatants of the two cell types. The following metabolites were deemed to be responsible for the potential differences in the metabolic characteristics of BMSCs and ADSCs: D-lactic acid, hydroxyindoleacetaldehyde, α-D-glucose, bovinic acid, 9,10-epoxyoctadecenoic acid, glyceraldehyde, phenylpyruvic acid, L-octanoylcarnitine, retinyl ester, α-ketoisovaleric acid, guanidoacetic acid, N-acetylneuraminic acid, imidazoleacetic acid riboside, sphingosine and pseudouridine 5′-phosphate. Based on these findings, there may be significant differences in the following metabolic pathways: The linoleic acid metabolic pathway, galactose metabolism, argentines and proline metabolism, retinol metabolism, glycine and serine metabolism, galactose metabolism, and amino sugar and nucleotide sugar metabolism. In conclusion, substantial differences in metabolic characteristics were detected between BMSCs and ADSCs, which may be associated with the different efficacies of atherosclerosis therapies employing these cell types.

BackgroundElectrical stimulation (ES) has been successfully used to treat bone defects clinically. Recently, both cellular and molecular approaches have demonstrated that ES can change cell behavior such as migration, proliferation and differentiation.MethodsIn the present study we exposed rat bone marrow- (BM-) and adipose tissue- (AT-) derived mesenchymal stem cells (MSCs) to direct current electrical stimulation (DC ES) and assessed temporal changes in osteogenic differentiation. We applied 100 mV/mm of DC ES for 1 h per day for three, seven and 14 days to cells cultivated in osteogenic differentiation medium and assessed viability and calcium deposition at the different time points. In addition, expression of osteogenic genes, Runx2, Osteopontin, and Col1A2 was assessed in BM- and AT-derived MSCs at the different time points.ResultsResults showed that ES changed osteogenic gene expression patterns in both BM- and AT-MSCs, and these changes differed between the two groups. In BM-MSCs, ES caused a significant increase in mRNA levels of Runx2, Osteopontin and Col1A2 at day 7, while in AT-MSCs, the increase in Runx2 and Osteopontin expression were observed after 14 days of ES.DiscussionThis study shows that rat bone marrow- and adipose tissue-derived stem cells react differently to electrical stimuli, an observation that could be important for application of electrical stimulation in tissue engineering.

Background Autologous adipose tissue–derived mesenchymal stem cells ( ATMSC s) therapy is a promising strategy to improve post–myocardial infarction outcomes. In a porcine model of acute myocardial infarction, we studied the long‐term effects and the mechanisms involved in allogeneic ATMSC s administration on myocardial performance. Methods and Results Thirty‐eight pigs underwent 50 minutes of coronary occlusion; the study was completed in 33 pigs. After reperfusion, allogeneic ATMSC s or culture medium (vehicle) were intracoronarily administered. Follow‐ups were performed at short (2 days after acute myocardial infarction vehicle‐treated, n=10; ATMSC s‐treated, n=9) or long term (60 days after acute myocardial infarction vehicle‐treated, n=7; ATMSC s‐treated, n=7). At short term, infarcted myocardium analysis showed reduced apoptosis in the ATMSC s‐treated animals (48.6±6% versus 55.9±5.7% in vehicle; P =0.017); enhancement of the reparative process with up‐regulated vascular endothelial growth factor, granulocyte macrophage colony‐stimulating factor, and stromal‐derived factor‐1α gene expression; and increased M2 macrophages (67.2±10% versus 54.7±10.2% in vehicle; P =0.016). In long‐term groups, increase in myocardial perfusion at the anterior infarct border was observed both on day‐7 and day‐60 cardiac magnetic resonance studies in ATMSC s‐treated animals, compared to vehicle (87.9±28.7 versus 57.4±17.7 mL/min per gram at 7 days; P =0.034 and 99±22.6 versus 43.3±14.7 22.6 mL/min per gram at 60 days; P =0.0001, respectively). At day 60, higher vascular density was detected at the border zone in the ATMSC s‐treated animals (118±18 versus 92.4±24.3 vessels/mm 2 in vehicle; P =0.045). Cardiac magnetic resonance–measured left ventricular ejection fraction of left ventricular volumes was not different between groups at any time point. Conclusions In this porcine acute myocardial infarction model, allogeneic ATMSC s‐based therapy was associated with increased cardioprotective and reparative mechanisms and with better cardiac magnetic resonance–measured perfusion. No effect on left ventricular volumes or ejection fraction was observed.

Glaucoma remains one of the major causes of blindness in today's world. The progressive field of stem cell proposes an exciting potential for discovering novel therapies. Here, we report the development of an easy and high throughput method for differentiation of retinal ganglion cells (RGC) and bipolar cells from human adipose tissue-derived mesenchymal stem cells (hADSCs) using PAX6 (+5a) gene expression, a master gene in development of the vertebrate visual system. HADSCs was isolated from fat tissues and confirmed by their surface markers and differentiation potential into adipocytes and osteocytes lineages. Then, the coding region of human PAX6 (+5a) gene was cloned and lentiviral particles were produced. HADSCs differentiation was characterized by morphological characteristics, qRT-PCR and immunocytochemistry (ICC). The hADSCs were isolated successfully with high yield and purity (99%). After 30 hours post transduction by pLEX-pax6- pur lentiviral vectors in fibronectin supplemented medium, cells gradually showed the characteristic morphology of neuronal cells. QRT- PCR and ICC confirmed deriving of mainly RGC and marginally bipolar cells. The current investigation demonstrates the feasibility of differentiation of RGCs and bipolar cells from hADSCs using expression of PAX6 (+5a) in the medium supplemented by fibronectin.

The aim of this review was to describe the current state-of-the-art regarding isolation, characterization and aging of adipose tissue-derived mesenchymal stem cells (ADSCs). Mesenchymal stem cells (MSCs) have recently received widespread attention because of their potential use in tissue-engineering applications. Various studies have indicated that MSCs with a fibroblast-like morphology migrate to the sites of injury and help to regenerate damaged tissue. Over the past few years, it has been recognized that fat is not only an energy supply, but also a rich source of multipotent stem cells that can be easily harvested, isolated and selected as compared with other tissues. ADSCs are particularly interesting because of their rapid proliferation and multidirectional differentiation potential.

Recent studies have suggested that adipose tissue-derived mesenchymal stem cell (ADSC) therapy and OX40 costimulation blockade are two immunomodulatory strategies used to suppress the immune response to alloantigens. However, relatively little has been reported regarding the immunomodulatory potential of the abilityof these two strategies to synergize. Thus, in the present study, we aimed to investigate OX40-Ig fusion protein (OX40Ig) expression in ADSCs and to validate their more potent immunosuppressive activity in preventing renal allograft rejection. For this purpose, ADSCs from Lewis rats were transfected with the recombinant plasmid, pcDNA3.1(-)OX40Ig, by nucleofection. The ADSCs transduced with the plasmid (termed ADSCsOX40Ig) or untransduced ADSCs (termed ADSCsnative) were added to allostimulated mixed lymphocyte reaction (MLR) in vitro. In vivo, ADSCsOX40Ig, ADSCsnative, or PBS were administered to an allogeneic renal transplantation model, and the therapeutic effects, as well as the underlying mechanisms were examined. The results revealed that both the ADSCsnative and ADSCsOX40Ig significantly suppressed T cell proliferation and increased the percentage of CD4+CD25+ regulatory T cells in allogeneic MLR assays, with the ADSCsOX40Ig being more effective. Furthermore, the results from our in vivo experiments revealed that compared with the ADSCsnative or PBS group, the administration of autologous ADSCsOX40Ig markedly prolonged the mean survival time of renal grafts, reduced allograft rejection, and significantly downregulated the mRNA expression of intragraft interferon-γ (IFN-γ) , and upregulated the mRNA expression of interleukin (IL)‑10, transforming growth factor-β (TGF-β) and forkhead box protein 3 (Foxp3). The findings of our study indicate that the use of ADSCsOX40Ig is a promising strategy for preventing renal allograft rejection. This strategy provides the synergistic benefits of ADSC immune modulation and OX40-OX40L pathway blockade, and may therefore have therapeutic potential in clinical renal transplantation.

Adipose tissue‑derived mesenchymal stem cells (ADSCs) have been considered to be attractive and readily available adult mesenchymal stem cells, and they are becoming increasingly popular for use in regenerative cell therapy, as they are readily accessible through minimally invasive techniques. The present study investigated whether autologous ADSC transplantation promoted liver regeneration following a repeat partial hepatectomy in rats. The rats were divided into three groups as follows: 70% partial hepatectomy (PH) group; repeat PH (R‑PH) group and R‑PH/ADSC group, subjected to R‑PH and treated with autologous ADSCs via portal vein injection. In each group, the rats were sacrificed at different time points postoperatively in order to evaluate the changes in liver function and to estimate the liver regenerative response. The expression of proliferating cell nuclear antigen (PCNA) labeling index in the liver was measured using immunohistochemistry. The expression levels of hepatocyte growth factor (HGF) mRNA were measured using reverse transcription polymerase chain reaction. The results showed that regeneration of the remaining liver following R‑PH was significantly promoted by ADSC transplantation, as shown by a significant increase in liver to body weight ratio and the PCNA labeling index at 24 h post‑hepatectomy. Additionally, ADSC transplantation markedly inhibited the elevation of serum levels of alanine aminotransferase, aspartate aminotransferase and total bilirubin, increased HGF content and also attenuated hepatic vacuolar degeneration 24 h postoperatively. Furthermore, the liver was found to almost fully recover from hepatocellular damage due to hepatectomy among the three groups at 168 h postoperatively. These results indicated that autologous ADSC transplantation enhanced the regenerative capacity of the remnant liver tissues in the early phase following R‑PH.

Mesenchymal stem cells (MSCs) isolated from various tissues have been well characterized for therapeutic application to clinical diseases. However, in contrast to MSCs from other animal species, the characteristics of feline MSCs have not been fully documented. In this study, we conducted extensive characterization of feline adipose tissue-derived MSCs (fAD-MSCs). Study fAD-MSCs were individually isolated from the intra-abdominal adipose tissues of six felines. The expression levels of cell surface markers and pluripotent markers were evaluated. Next, proliferation capacity was analyzed by performing cumulative population doubling level (CPDL) and doubling time (DT) calculation assays. Differentiation potentials of fAD-MSCs into mesodermal cell lineages were analyzed by examining specific staining and molecular markers. All fAD-MSCs positively expressed cell surface markers such as CD29, CD44, CD90, CD105, CD166, and MHC-I, while CD14, CD34, CD45, and CD73 were negatively expressed. The CPDL of the fAD-MSCs was maintained until passage 5 to 6 (P5 to P6), whereas DT increased after P3 to P4. Also, stem cell-specific pluripotent markers (Oct3/4, Nanog, and SSEA-4) were detected. Importantly, all fAD-MSCs demonstrated mesodermal differentiation capacity. These results suggest that fully characterized fAD-MSCs could be beneficial when considering the use of these cells in feline disease research.

Contains fulltext : 70683.pdf (Publisher’s version ) (Open Access) For bone tissue engineering, it is important that mesenchymal stem cells (MSCs) differentiate into osteoblasts. To develop a method for differentiation of adipose tissue-derived mesenchymal stem cells (AT-MSCs) along the osteogenic lineage, we studied the effect of polyamines, which are organic cations implicated in bone growth and development, on differentiation of AT-MSCs. Treatment of goat-derived AT-MSCs with 1,25-dihydroxyvitamin-D3 (1,25(OH)(2)D(3)), which stimulates osteogenic differentiation, for 7 days induced gene expression of the polyamine-modulated transcription factor-1 (PMF-1) and spermidine/spermine N (1)-acetyltransferase (SSAT), which are both involved in polyamine metabolism, suggesting that polyamines are involved in osteogenic differentiation of AT-MSCs. Furthermore, treatment of AT-MSCs with the polyamine spermine-regulated gene expression of runx-2, a transcription factor involved in early stages of osteogenic differentiation, and that of osteopontin, a bone matrix protein expressed in later stages of osteogenic differentiation. Runx-2 gene expression was increased 4 and 14 days after a short 30 min. treatment with spermine, while osteopontin gene expression was only increased 4 days after spermine treatment. Finally, alkaline phosphatase activity, which is intimately involved in the formation of extracellular matrix of bone, was increased 4 weeks after the 30 min.-spermine treatment of AT-MSCs. In conclusion, this study shows for the first time that the polyamine spermine regulates differentiation of AT-MSCs along the osteogenic lineage, which can be used as a new method for differentiation of AT-MSCs along the osteogenic lineage. Therefore, polyamines may constitute a promising tool for bone tissue engineering approaches using AT-MSCs, such as a one-step surgical procedure for spinal interbody fusion.

Composite biological and synthetic grafts with progenitor cells offer an alternative approach to auto- or allografts for fracture repair. This study was conducted to evaluate osteogenesis of autologous serum-derived albumin (ASA) scaffolds seeded with canine adipose tissue-derived mesenchymal stem cells (Ad-MSCs) in a canine segmental bone defect model. ASA scaffold was prepared with canine serum using cross-linking and freeze-drying procedures. Beta-tricalcium phosphate (β-TCP) was mixed at the cross-linking stage. Ad-MSCs were seeded into the scaffold and incubated for one day before implantation. After 16 weeks, the grafts were harvested for histological analysis. The dogs were divided into five groups: control, ASA scaffolds with and without Ad-MSCs, and ASA scaffolds including β-TCP with and without Ad-MSCs. ASA scaffolds with Ad-MSCs had a significantly larger area of increased opacity at the proximal and distal host cortex-implant interfaces in radiographs 16 weeks after implantation compared to the groups with β-TCP (p < 0.05). Histomorphometric analysis showed that ASA scaffolds with Ad-MSCs had significantly greater new bone formation than other groups (p < 0.05). These results suggest that Ad-MSCs seeded into ASA scaffolds enhanced osteogenesis in the bone defect model, but that β-TCP in the ASA scaffold might prevent penetration of the cells required for bone healing.