Exosomes Derived from Mesenchymal Stem Cells Suppress Angiogenesis by Down-Regulating VEGF Expression in Breast Cancer Cells

Exosomes are small membrane vesicles released by a variety of cell types. Exosomes contain genetic materials, such as mRNAs and microRNAs (miRNAs), implying that they may play a pivotal role in cell-to-cell communication. Mesenchymal stem cells (MSCs), which potentially differentiate into multiple cell types, can migrate to the tumor sites and have been reported to exert complex effects on tumor progression. To elucidate the role of MSCs within the tumor microenvironment, previous studies have suggested various mechanisms such as immune modulation and secreted factors of MSCs. However, the paracrine effects of MSC-derived exosomes on the tumor microenvironment remain to be explored. The hypothesis of this study was that MSC-derived exosomes might reprogram tumor behavior by transferring their molecular contents. To test this hypothesis, exosomes from MSCs were isolated and characterized. MSC-derived exosomes exhibited different protein and RNA profiles compared with their donor cells and these vesicles could be internalized by breast cancer cells. The results demonstrated that MSC-derived exosomes significantly down-regulated the expression of vascular endothelial growth factor (VEGF) in tumor cells, which lead to inhibition of angiogenesis in vitro and in vivo. Additionally, miR-16, a miRNA known to target VEGF, was enriched in MSC-derived exosomes and it was partially responsible for the anti-angiogenic effect of MSC-derived exosomes. The collective results suggest that MSC-derived exosomes may serve as a significant mediator of cell-to-cell communication within the tumor microenvironment and suppress angiogenesis by transferring anti-angiogenic molecules.     

Mesenchymal stem cells protect against ferroptosis via exosome-mediated stabilization of SLC7A11 in acute liver injury

Mesenchymal stem cells (MSCs) have attracted interest for their potential to alleviate liver injury. Here, the protective effect of MSCs on carbon tetrachloride (CCl₄)-induced acute liver injury (ALI) was investigated. In this study, we illustrated a novel mechanism that ferroptosis, a newly recognized form of regulated cell death, contributed to CCl₄-induced ALI. Subsequently, based on the in vitro and in vivo evidence that MSCs and MSC-derived exosomes (MSC-Exo) treatment achieved pathological remission and inhibited the production of lipid peroxidation, we proposed an MSC-based therapy for CCl₄-induced ALI. More intriguingly, treatment with MSCs and MSC-Exo downregulated the mRNA level of prostaglandin-endoperoxide synthase 2 (Ptgs2) and lipoxygenases (LOXs) while it restored the protein level of SLC7A11 in primary hepatocytes and mouse liver, indicating that the inhibition of ferroptosis partly accounted for the protective effect of MSCs and MSC-Exo on ALI. We further revealed that MSC-Exo-induced expression of SLC7A11 protein was accompanied by increasing of CD44 and OTUB1. The aberrant expression of ubiquitinated SLC7A11 triggered by CCl₄ could be rescued with OTUB1-mediated deubiquitination, thus strengthening SLC7A11 stability and thereby leading to the activation of system X_C⁻ to prevent CCl₄-induced hepatocyte ferroptosis. In conclusion, we showed that MSC-Exo had a protective role against ferroptosis by maintaining SLC7A11 function, thus proposing a novel therapeutic strategy for ferroptosis-induced ALI.Subject terms: Hepatotoxicity, Experimental models of disease

Schematic diagram of the protective effect of MSC-derived exosomes on maintaining SLC7A11 function during ferroptosis involved in CCl₄-induced ALI.     

Proteomic Analysis of the Extracellular Matrix Produced by Mesenchymal Stromal Cells: Implications for Cell Therapy Mechanism

Mesenchymal stromal cells (MSCs) transiently transfected with notch1 intracellular domain (NICD) are beneficial for neurological disorders as observed in several preclinical studies. Extracellular matrix (ECM) derived from NICD-transfected MSCs has been previously shown to support in vitro neural cell growth and survival better than that of un-transfected MSCs. To understand the underlying mechanism(s) by which NICD-transfected MSC-derived ECM supports neural cell growth and survival, we investigated the differences in NICD-transfected MSC- and MSC-derived ECM protein quantity and composition. To compare the ECM derived from MSCs and NICD-transfected MSCs, the proteins were sequentially solubilized using sodium dodecyl sulfate (SDS) and urea, quantified, and compared across four human donors. We then analyzed ECM proteins using either in-gel digests or in-solution surfactant-assisted trypsin digests (SAISD) coupled with reverse phase nano-liquid chromatography and tandem mass spectrometry (nLC-MS/MS). Analyses using nLC-MS/MS identified key components of ECM from NICD-transfected MSCs and un-transfected MSCs and revealed significant differences in their respective compositions. This work provides a reproducible method for identifying and comparing in vitro cell-derived ECM proteins, which is crucial for exploring the mechanisms underlying cellular therapy.     

Skeletal Muscle Regeneration by the Exosomes of Adipose Tissue-Derived Mesenchymal Stem Cells

Profound skeletal muscle loss can lead to severe disability and cosmetic deformities. Mesenchymal stem cell (MSC)-derived exosomes have shown potential as an effective therapeutic tool for tissue regeneration. This study aimed to determine the regenerative capacity of MSC-derived exosomes for skeletal muscle regeneration. Exosomes were isolated from human adipose tissue-derived MSCs (AD-MSCs). The effects of MSC-derived exosomes on satellite cells were investigated using cell viability, relevant genes, and protein analyses. Moreover, NOD-SCID mice were used and randomly assigned to the healthy control (n = 4), muscle defect (n = 6), and muscle defect + exosome (n = 6) groups. Muscle defects were created using a biopsy punch on the quadriceps of the hind limb. Four weeks after the surgery, the quadriceps muscles were harvested, weighed, and histologically analyzed. MSC-derived exosome treatment increased the proliferation and expression of myocyte-related genes, and immunofluorescence analysis for myogenin revealed a similar trend. Histologically, MSC-derived exosome-treated mice showed relatively preserved shapes and sizes of the muscle bundles. Immunohistochemical staining revealed greater expression of myogenin and myoblast determination protein 1 in the MSC-derived exosome-treated group. These results indicate that exosomes extracted from AD-MSCs have the therapeutic potential for skeletal muscle regeneration.     

Heterotopic bone formation derived from multipotent stromal cells is not inhibited in aged mice

Background aimsDecreased bone formation with age is believed to arise, at least in part, because of the influence of the senescent microenvironment. In this context, it is unclear whether multipotent stromal cell (MSC)-based therapies would be effective for the treatment of bone diseases.MethodsWith the use of a heterotopic bone formation model, we investigated whether MSC-derived osteogenesis is impaired in aged mice compared with young mice.ResultsWe found that bone formation derived from MSCs is not reduced in aged mice. These results are supported by the unexpected finding that conditioned media collected from ionizing radiation–induced senescent MSCs can stimulate mineralization and delay osteoclastogenesis in vitro.ConclusionsOverall, our results suggest that impaired bone formation with age is mainly cell-autonomous and provide a rationale for the use of MSC-based therapies for the treatment of bone diseases in the elderly.     

Myokine Irisin promotes osteogenesis by activating BMP/SMAD signaling via αV integrin and regulates bone mass in mice

Irisin is well-known to contribute to bone homeostasis due to its bidirectional regulation on osteogenesis and osteoclastogenesis. However, the mechanisms of irisin involved in mesenchymal stem/stromal cells (MSCs)-derived osteogenesis are still under investigated. Fibronectin type III domain-containing protein 5 (FNDC5) is the precursor protein of irisin, compare with wild type (WT) littermates, FNDC5^-/- mice lost bone mass significantly, collectively evidenced by the decrease of bone mineral density (BMD), impaired bone formation and reduced N-terminal propertied of type I procollagen (P1NP) in sera. Meanwhile, the bone resorbing of FNDC5^-/- mice has enhanced accompanied by increased tartrate phosphatase (TRAP) staining cells morphologically and cross-Linked C-telopeptide of type 1 collagen (CTX) level in sera. In vitro study showed that lack of irisin impeded the MSC-derived osteogenesis of FNDC5^-/- mice. The addition of irisin promote the osteogenesis of WT and irisin-deficient MSCs, by activating αV integrin-induced ERK/STAT pathway, subsequently enhancing bone morphogenetic protein 2 (BMP2) expression and BMP/SMAD signaling activation. Taken together, these findings further indicate that irisin regulates bone homeostasis. Moreover, irisin promotes MSC-derived osteogenesis by binding to αV integrin and activating BMP/SMAD signaling consequently. Thus, irisin may be a promising therapeutic target for osteoporosis and bone defects.     

Exosomal miR-21-5p derived from bone marrow mesenchymal stem cells promote osteosarcoma cell proliferation and invasion by targeting PIK3R1

Mesenchymal stem cells (MSCs) are a class of pluripotent cells that can release a large number of exosomes which act as paracrine mediators in tumour-associated microenvironment. However, the role of MSC-derived exosomes in pathogenesis and progression of cancer cells especially osteosarcoma has not been thoroughly clarified until now. In this study, we established a co-culture model for human bone marrow-derived MSCs with osteosarcoma cells, then extraction of exosomes from induced MSCs and study the role of MSC-derived exosomes in the progression of osteosarcoma cell. The aim of this study was to address potential cell biological effects between MSCs and osteosarcoma cells. The results showed that MSC-derived exosomes can significantly promote osteosarcoma cells’ proliferation and invasion. We also found that miR-21-5p was significantly over-expressed in MSCs and MSC-derived exosomes by quantitative real-time polymerase chain reaction (qRT-PCR), compared with human foetal osteoblastic cells hFOB1.19. MSC-derived exosomes transfected with miR-21-5p could significantly enhance the proliferation and invasion of osteosarcoma cells in vitro and in vivo. Bioinformatics analysis and dual-luciferase reporter gene assays validated the targeted relationship between exosomal miR-21-5p and PIK3R1; we further demonstrated that miR-21-5p-abundant exosomes derived human bone marrow MSCs could activate PI3K/Akt/mTOR pathway by suppressing PIK3R1 expression in osteosarcoma cells. In summary, our study provides new insights into the interaction between human bone marrow MSCs and osteosarcoma cells in tumour-associated microenvironment.     

Weak Functional Coupling of the Melanocortin-1 Receptor Expressed in Human Adipocytes

The melanocortin (MC) receptor type-1 (MC1-R) is the only one of the five MC receptor subtypes expressed in human adipose tissue explants, human mesenchymal stem cells (MSCs), and MSC-derived adipocytes. Following our recent expression studies (Obesity 2007, 15, 40–49), we now investigated the functional role of MC1-R in these tissues and cells to deduce the coupling state of MC1-R to intracellular output signals in human fat cells and tissue. Expression of MC1-R by undifferentiated and differentiated MSCs was quantified by real-time TaqMan PCR. Intracellular output signals (cAMP, lipolysis, secretion of IL-6, IL-10, and TNF-α), as well as effects on the metabolic rate and proliferation of human MSCs were analyzed by standard assays, exposing undifferentiated and differentiated MSCs and, in part, human adipose tissue explants to the potent MC1-R agonist, [Nle⁴, D-Phe⁷]-α -MSH (NDP-MSH). This agonist induced a weak cAMP signal in MSC-derived adipocytes. However, it did not affect lipolysis in these cells or in adipose tissue explants, nor did it modulate cytokine release and mRNA expression of IL-6, IL-8, and TNF-α upon LPS stimulation. In undifferentiated MSCs, NDP-MSH did not alter the metabolic rate, but it showed a significant antiproliferative effect. Therefore, it appears that MC1-R–effector coupling in (differentiated) human adipocytes is too weak to induce a regulatory effect on lipolysis or inflammation; by contrast, MC1-R stimulation in undifferentiated MSCs induces an inhibitory signal on cell proliferation.     

Regulation of plasticity and biological features of endothelial progenitor cells by MSC-derived SDF-1

Bone marrow (BM) is a source of mesenchymal stromal cells (MSCs) and endothelial progenitor cells (EPCs). MSCs provide a specific niche in the BM and biological features of EPCs may be changed with this niche. Stromal cell-derived factor 1 (SDF-1) secreted from primary BM-MSCs and biological features of this niche on EPC development are still yet to be understood. The aim of this study was to evaluate the role of SDF-1 produced by MSCs on EPC development. We applied the CRISPR/Cas9 system for the knock-out of the SDF-1 gene in BM-derived MSCs. BM-derived EPCs were then cocultured with MSCs^SDF-1−/− or MSCs^SDF-1+/+ to identify the role of MSC-derived SDF-1α on proliferation, migration and angiogenic activity of EPCs. Next, pre-expanded EPCs were harvested and co-transplanted with MSCs^SDF-1−/− or MSCs^SDF-1+/+ into sublethally irradiated mice to analyze the potency of these cells for marrow reconstitution. Our results revealed that proliferation, colony formation, migration and angiogenic activity of EPCs was significantly increased after coculture with MSCs^SDF-1+/+. We also found that co-transplantation of EPCs with MSCs^SDF-1+/+, in contrast to MSCs^SDF-1−/−, into irradiated mice resulted in marrow repopulation and hematologic recovery, leading to improved survival of transplanted mice. In conclusions, MSC-derived SDF-1 niche plays an important role in the development of EPCs and this niche is essential for bone marrow repopulation by these cells and can enhance the efficiency of EPC therapy for ischemic diseases.     

Bone marrow-derived mesenchymal stromal cells from patients with end-stage renal disease are suitable for autologous therapy

Background aimsMesenchymal stromal cells (MSCs) are pluripotent cells that have immunosuppressive and reparative properties in vitro and in vivo. Although autologous bone marrow (BM)-derived MSCs are already clinically tested in transplant recipients, it is unclear whether these BM cells are affected by renal disease. We assessed whether renal failure affected the function and therapeutic potential of BM-MSCs.MethodsMSCs from 10 adults with end-stage renal disease (ESRD) and 10 age-matched healthy controls were expanded from BM aspirates and tested for phenotype and functionality in vitro.ResultsMSCs from ESRD patients were >90% positive for CD73, CD90 and CD105 and negative for CD34 and CD45 and showed a similar morphology and differentiation capacity as MSCs from healthy controls. Of importance for their clinical utility, growth characteristics were similar in both groups, and sufficient numbers of MSCs were obtained within 4 weeks. Messenger RNA expression levels of self-renewal genes and factors involved in repair and inflammation were also comparable between both groups. Likewise, microRNA expression profiling showed a broad overlap between ESRD and healthy donor MSCs. ESRD MSCs displayed the same immunosuppressive capacities as healthy control MSCs, demonstrated by a similar dose-dependent inhibition of peripheral blood mononuclear cell proliferation, similar inhibition of proinflammatory cytokines tumor necrosis factor-α and interferon-γ production and a concomitant increase in the production of interleukin-10.ConclusionsExpanded BM-MSCs procured from ESRD patients and healthy controls are both phenotypically and functionally similar. These findings are important for the potential autologous clinical application of BM-MSCs in transplant recipients.     

Role of mesenchymal stem cells in neurogenesis and nervous system repair

Bone marrow-derived mesenchymal stem cells (MSCs) are attractive candidates for use in regenerative medicine since they are easily accessible and can be readily expanded in vivo, and possess unique immunogenic properties. Moreover, these multipotent cells display intriguing environmental adaptability and secretory capacity. The ability of MSCs to migrate and engraft in a range of tissues has received significant attention. Evidence indicating that MSC transplantation results in functional improvement in animal models of neurological disorders has highlighted exciting potential for their use in neurological cell-based therapies. The manner in which MSCs elicit positive effects in the damaged nervous system remains unclear. Cell fusion and/or ‘transdifferentiation’ phenomena, by which MSCs have been proposed to adopt neural cell phenotypes, occur at very low frequency and are unlikely to fully account for observed neurological improvement. Alternatively, MSC-mediated neural recovery may result from the release of soluble molecules, with MSC-derived growth factors and extracellular matrix components influencing the activity of endogenous neural cells. This review discusses the potential of MSCs as candidates for use in therapies to treat neurological disorders and the molecular and cellular mechanisms by which they are understood to act.     

Extracellular signal-regulated kinase 2 (ERK2) phosphorylation sites and docking domain on the nuclear pore complex protein Tpr cooperatively regulate ERK2-Tpr interaction

Identifying direct substrates of mitogen-activated protein kinases (MAPKs) and understanding how those substrates are selected is central to understanding how these ubiquitously activated enzymes generate diverse biological responses. In previous work, we identified several new candidate substrates for the MAPK ERK2 (extracellular signal-regulated kinase 2), including the nuclear pore complex protein Tpr (translocated promoter region). In this report, we identify sites on Tpr for ERK2 phosphorylation and binding and demonstrate their functional interaction. ERK2 phosphorylation and dimerization are necessary for ERK2-Tpr binding, and this occurs through a DEF (docking site for ERK2, FXF) domain on Tpr. Surprisingly, the DEF domain and the phosphorylation sites displayed positive cooperativity to promote ERK2 binding to Tpr, in contrast to substrates where phosphorylation reduces binding. Ectopic expression or depletion of Tpr resulted in decreased movement of activated ERK2 from the cytoplasm to the nucleus, implying a role for Tpr in ERK2 translocation. Collectively, the data provide direct evidence that a component of the nuclear pore complex is a bona fide substrate of ERK2 in vivo and that activated ERK2 stably associates with this substrate after phosphorylation, where it could play a continuing role in nuclear pore function. We propose that Tpr is both a substrate and a scaffold for activated ERKs.     

Mesenchymal stem cells in regenerative medicine applied to rheumatic diseases: Role of secretome and exosomes

Over the last decades, mesenchymal stem cells (MSCs) have been extensively studied with regard to their potential applications in regenerative medicine. In rheumatic diseases, MSC-based therapy is the subject of great expectations for patients who are refractory to proposed treatments such as rheumatoid arthritis (RA), or display degenerative injuries without possible curative treatment, such as osteoarthritis (OA). The therapeutic potential of MSCs has been demonstrated in several pre-clinical models of OA or RA and both the safety and efficacy of MSC-based therapy is being evaluated in humans. The predominant mechanism by which MSCs participate to tissue repair is through a paracrine activity. Via the production of a multitude of trophic factors with various properties, MSCs can reduce tissue injury, protect tissue from further degradation and/or enhance tissue repair. However, a thorough in vivo examination of MSC-derived secretome and strategies to modulate it are still lacking. The present review discusses the current understanding of the MSC secretome as a therapeutic for treatment of inflammatory or degenerative pathologies focusing on rheumatic diseases. We provide insights on and perspectives for future development of the MSC secretome with respect to the release of extracellular vesicles that would have certain advantages over injection of living MSCs or administration of a single therapeutic factor or a combination of factors.     

Cyclooxygenase-2 dependent metabolism of 20-HETE increases adiposity and adipocyte enlargement in mesenchymal stem cell-derived adipocytes

20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE), a product of the cytochrome P450 (CYP)-catalyzed ω-hydroxylation of arachidonic acid, induces oxidative stress and, in clinical studies, is associated with increased body mass index (BMI) and the metabolic syndrome. This study was designed to examine the effects of exogenous 20-HETE on mesenchymal stem cell (MSC)-derived adipocytes. The expression levels of CYP4A11 and CYP4F2 (major 20-HETE synthases in humans) in MSCs decreased during adipocyte differentiation; however, exogenous administration of 20-HETE (0.1–1 μM) increased adipogenesis in a dose-dependent manner in these cells (P < 0.05). The inability of a 20-HETE analog to reproduce these effects suggested the involvement of a metabolic product of 20-HETE in mediating its pro-adipogenic effects. A cyclooxygenase (COX)-1 selective inhibitor enhanced, whereas a COX-2 selective or a dual COX-1/2 inhibitor attenuated adipogenesis induced by 20-HETE. The COX-derived metabolite of 20-HETE, 20-OH-PGE₂, enhanced adipogenesis and lipid accumulation in MSCs. The pro-adipogenic effects of 20-HETE and 20-OH-PGE₂ resulted in the increased expression of the adipogenic regulators PPARγ and β-catenin in MSC-derived adipocytes. Taken together we show for the first time that 20-HETE-derived COX-2-dependent 20-OH-PGE₂ enhances mature inflamed adipocyte hypertrophy in MSC undergoing adipogenic differentiation.     

Partial reconstruction of flavonoid and isoflavonoid biosynthesis in yeast using soybean type I and type II chalcone isomerases

Flavonoids and isoflavonoids are major plant secondary metabolites that mediate diverse biological functions and exert significant ecological impacts. These compounds play important roles in many essential physiological processes. In addition, flavonoids and isoflavonoids have direct but complex effects on human health, ranging from reducing cholesterol levels and preventing certain cancers to improving women's health. In this study, we cloned and functionally characterized five soybean (Glycine max) chalcone isomerases (CHIs), key enzymes in the phenylpropanoid pathway that produces flavonoids and isoflavonoids. Gene expression and kinetics analysis suggest that the soybean type I CHI, which uses naringenin chalcone as substrate, is coordinately regulated with other flavonoid-specific genes, while the type II CHIs, which use a variety of chalcone substrates, are coordinately regulated with an isoflavonoid-specific gene and specifically activated by nodulation signals. Furthermore, we found that some of the newly identified soybean CHIs do not require the 4′-hydroxy moiety on the substrate for high enzyme activity. We then engineered yeast (Saccharomyces cerevisiae) to produce flavonoid and isoflavonoid compounds. When one of the type II CHIs was coexpressed with an isoflavone synthase, the enzyme catalyzing the first committed step of isoflavonoid biosynthesis, various chalcone substrates added to the culture media were converted to an assortment of isoflavanones and isoflavones. We also reconstructed the flavonoid pathway by coexpressing CHI with either flavanone 3β-hydroxylase or flavone synthase II. The in vivo reconstruction of the flavonoid and isoflavonoid pathways in yeast provides a unique platform to study enzyme interactions and metabolic flux.     

Mechanosensitivity of the 2nd Kind: TGF-β Mechanism of Cell Sensing the Substrate Stiffness

Cells can sense forces applied to them, but also the stiffness of their environment. These are two different phenomena, and here we investigate the mechanosensitivity of the 2^nd kind: how the cell can measure an elastic modulus at a single point of adhesion—and how the cell can receive and interpret the chemical signal released from the sensor. Our model uses the example of large latent complex of TGF-β as a sensor. Stochastic theory gives the rate of breaking of latent complex, which initiates the signaling feedback loop after the active TGF-β release and leads to a change of cell phenotype driven by the α-smooth muscle actin. We investigate the dynamic and steady-state behaviors of the model, comparing them with experiments. In particular, we analyse the timescale of approach to the steady state, the stability of the non-linear dynamical system, and how the steady-state concentrations of the key markers vary depending on the elasticity of the substrate. We discover a crossover region for values of substrate elasticity closely corresponding to that of the fibroblast to myofibroblast transition. We suggest that the cell could actively vary the parameters of its dynamic feedback loop to ‘choose’ the position of the transition region and the range of substrate elasticity that it can detect. In this way, the theory offers the unifying mechanism for a variety of phenomena, such as the myofibroblast conversion in fibrosis of wounds and lungs and smooth muscle cell dysfunction in cardiac disease.