Exosomes derived from pro‐inflammatory bone marrow‐derived mesenchymal stem cells reduce inflammation and myocardial injury via mediating macrophage polarization

Exosomes are served as substitutes for stem cell therapy, playing important roles in mediating heart repair during myocardial infarction injury. Evidence have indicated that lipopolysaccharide (LPS) pre‐conditioning bone marrow‐derived mesenchymal stem cells (BMSCs) and their secreted exosomes promote macrophage polarization and tissue repair in several inflammation diseases; however, it has not been fully elucidated in myocardial infarction (MI). This study aimed to investigate whether LPS‐primed BMSC‐derived exosomes could mediate inflammation and myocardial injury via macrophage polarization after MI. Here, we found that exosomes derived from BMSCs, in both Exo and L‐Exo groups, increased M2 macrophage polarization and decreased M1 macrophage polarization under LPS stimulation, which strongly depressed LPS‐dependent NF‐κB signalling pathway and partly activated the AKT1/AKT2 signalling pathway. Compared with Exo, L‐Exo had superior therapeutic effects on polarizing M2 macrophage in vitro and attenuated the post‐infarction inflammation and cardiomyocyte apoptosis by mediating macrophage polarization in mice MI model. Consequently, we have confidence in the perspective that low concentration of LPS pre‐conditioning BMSC‐derived exosomes may develop into a promising cell‐free treatment strategy for clinical treatment of MI.     

Exosomes derived from miR‐181‐5p‐modified adipose‐derived mesenchymal stem cells prevent liver fibrosis via autophagy activation

Proliferating hepatic stellate cells (HSCs) respond to liver damage by secreting collagens that form fibrous scar tissue, which can lead to cirrhosis if in appropriately regulated. Advancement of microRNA (miRNA) hepatic therapies has been hampered by difficulties in delivering miRNA to damaged tissue. However, exosomes secreted by adipose‐derived mesenchymal stem cells (ADSCs) can be exploited to deliver miRNAs to HSCs. ADSCs were engineered to overexpress miRNA‐181‐5p (miR‐181‐5p‐ADSCs) to selectively home exosomes to mouse hepatic stellate (HST‐T6) cells or a CCl4‐induced liver fibrosis murine model and compared with non‐targeting control Caenorhabditis elegans miR‐67 (cel‐miR‐67)‐ADSCs. In vitro analysis confirmed that the transfer of miR‐181‐5p from miR‐181‐5p‐ADSCs occurred via secreted exosomal uptake. Exosomes were visualized in HST‐T6 cells using cyc3‐labelled pre‐miRNA‐transfected ADSCs with/without the exosomal inhibitor, GW4869. The effects of miRNA‐181‐5p overexpression on the fibrosis associated STAT3/Bcl‐2/Beclin 1 pathway and components of the extracellular matrix were assessed. Exosomes from miR181‐5p‐ADSCs down‐regulated Stat3 and Bcl‐2 and activated autophagy in the HST‐T6 cells. Furthermore, the up‐regulated expression of fibrotic genes in HST‐T6 cells induced by TGF‐β1 was repressed following the addition of isolated miR181‐5p‐ADSC exosomes compared with miR‐67‐ADSCexosomes. Exosome therapy attenuated liver injury and significantly down‐regulated collagen I, vimentin, α‐SMA and fibronectin in liver, compared with controls. Taken together, the effective anti‐fibrotic function of engineered ADSCs is able to selectively transfer miR‐181‐5p to damaged liver cells and will pave the way for the use of exosome‐ADSCs for therapeutic delivery of miRNA targeting liver disease.     

In vitro screening system for hepatotoxicity: Comparison of bone‐marrow‐derived mesenchymal stem cells and Placenta‐derived stem cells

Stem cells have unique properties such as self‐renewal, plasticity to generate various cell types, and availability of cells of human origin. The characteristics are attentive in the toxicity screening against chemical toxicants. Placenta‐derived stem cells (PDSCs) have been spotlighted as a new cell source in stem cell research recently because they are characterized by their capacity to differentiate into multilineages. However, the use of PDSCs as an in vitro screening model for potential drug candidates has not yet been studied. Here, we analyzed the potentials for bone‐marrow‐derived mesenchymal stem (BM‐MSCs), which is a representative adult stem cells and PDSCs as an in vitro hepatotoxicity screening system, using well‐known hepatotoxicants. BM‐MSCs and PDSCs were analyzed to the potential for hepatogenic differentiation and were cultured with different concentrations of hepatotoxicants for time courses. The viability and ATP‐binding cassette (ABC) transporters were measured by the MTT assay and RT‐PCR, respectively. The sensitivities of PDSCs to hepatotoxicants are more sensitive than those of BM‐MSCs. The viability (IC₅₀) to in PDSCs was less than that of BM‐MSCs after 48 and 72 h (P < 0.05) of CCl₄ exposure. The toxicities of CCl₄ were decreased by fourfold in hepatogenic differentiation inducing PDSCs compared to the undifferentiated cells. The alteration of ABCGs was observed in PDSCs during differentiation. These findings suggest that the naïve PDSCs expressing ABCGs can be used as a source for in vitro screening system as well as the expression patterns of ABCG1 and ABCG2 might be involved in the sensitivity of PDSCs to hepatotoxicants. J. Cell. Biochem. 112: 49–58, 2011. © 2010 Wiley‐Liss, Inc.     

Bone marrow‐derived cells can acquire cardiac stem cells properties in damaged heart

Experimental data suggest that cell‐based therapies may be useful for cardiac regeneration following ischaemic heart disease. Bone marrow (BM) cells have been reported to contribute to tissue repair after myocardial infarction (MI) by a variety of humoural and cellular mechanisms. However, there is no direct evidence, so far, that BM cells can generate cardiac stem cells (CSCs). To investigate whether BM cells contribute to repopulate the Kit⁺ CSCs pool, we transplanted BM cells from transgenic mice, expressing green fluorescent protein under the control of Kit regulatory elements, into wild‐type irradiated recipients. Following haematological reconstitution and MI, CSCs were cultured from cardiac explants to generate ‘cardiospheres’, a microtissue normally originating in vitro from CSCs. These were all green fluorescent (i.e. BM derived) and contained cells capable of initiating differentiation into cells expressing the cardiac marker Nkx2.5. These findings indicate that, at least in conditions of local acute cardiac damage, BM cells can home into the heart and give rise to cells that share properties of resident Kit⁺ CSCs.     

Effects of over‐expression of HIF‐1alpha in bone marrow‐derived mesenchymal stem cells on traumatic brain injury

Mesenchymal stem cells (MSCs) have been shown to play therapeutic effect in traumatic brain injury (TBI). To augment the therapeutic effect, MSCs could be engineered to over‐express genes that are beneficial for treatment. In the present study, we over‐expressed hypoxia inducible factor (HIF)‐1alpha in bone marrow derived MSCs (BM‐MSCs) and sought to investigate whether HIF‐1alpha could enhance the therapeutic effect of MSCs in a mouse model of TBI. Balb/c mice were subjected to controlled cortical impact injury and MSCs were transplanted intravenously at 6 h after injury. The lesion volume and brain water content were measured and the neurological function was assessed by modified neurologic severity score tests. Double‐labeled immunofluorescence for BrdU and NeuU was performed to determine angiogenesis and neurogenesis. The expression of erythropoietin (EPO) and vascular endothelial growth factor (VEGF) was measured by quantitative RT‐PCR and western blotting. After TBI, mice received BM‐MSCs over‐expressing HIF‐1alpha showed significantly more functional recovery, reduced brain damage, increased angiogenesis and neurogenesis and increased expression of VEGF and EPO, compared with control mice or mice treated with non‐transduced BM‐MSCs. Over‐expression of HIF‐1alpha enhanced BM‐MSCs induced improvement of neurological recovery after TBI, by stimulating angiogenesis and neurogenesis.     

Hypoxia changes chemotaxis behaviour of mesenchymal stem cells via HIF‐1α signalling

Mesenchymal stem cells (MSCs) have drawn great attention because of their therapeutic potential. It has been suggested that intra‐venous infused MSCs could migrate the site of injury to help repair the damaged tissue. However, the mechanism for MSC migration is still not clear so far. In this study, we reported that hypoxia increased chemotaxis migration of MSCs. At 4 and 6 hours after culturing in hypoxic (1% oxygen) conditions, the number of migrated MSCs was significantly increased. Meanwhile, hypoxia also increased the expression of HIF‐1α and SDF‐1. Using small interference RNA, we knocked down the expression of HIF‐1α in MSCs to study the role of HIF‐1α in hypoxia induced migration. Our data indicated that knocking down the expression of HIF‐1α not only abolished the migration of MSCs, but also reduced the expression of SDF‐1. Combining the results of migration assay and expression at RNA and protein level, we demonstrated a novel mechanism that controls the increase of MSCs migration. This mechanism involved HIF‐1α mediated SDF‐1 expression. These findings provide new insight into the role of HIF‐1α in the hypoxia induced MSC migration and can be a benefit for the development of MSC‐based therapeutics for wound healing.     

Polyphenols suppress hydrogen peroxide‐induced oxidative stress in human bone‐marrow derived mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are considered a highly promising candidate cell type for cell‐based tissue engineering and regeneration because of their self‐renewal and multi‐lineage differentiation characteristics. Increased levels of reactive oxygen/nitrogen species (ROS/RNS) are associated with tissue injury and inflammation, impact a number of cellular processes, including cell adhesion, migration, and proliferation, and have been linked to cellular senescence in MSCs, potentially compromising their activities. Naturally occurring polyphenolic compounds (polyphenols), epigallocatechin‐3‐gallate (EGCG), and curcumin, block ROS/RNS and are potent inflammation‐modulating agents. However, their potential protective effects against oxidative stress in hMSCs have not been examined. In this study, we carried out a systematic analysis of the effects of polyphenols on hMSCs in their response to oxidative stress in the form of treatment with H₂O₂ and S‐nitroso‐N‐acetylpenicillamine (SNAP), respectively. Parameters measured included colony forming activity, apoptosis, and the levels of antioxidant enzymes and free reactive species. We found that polyphenols reversed H₂O₂‐induced loss of colony forming activity in hMSCs. In a dose‐dependent manner, polyphenols inhibited increased levels of ROS and NO, produced by H₂O₂ or SNAP, respectively, in MSCs. Notably, polyphenols rapidly and almost completely blocked H₂O₂‐induced ROS in the absence of significant direct effect on H₂O₂ itself. Polyphenols also protected the antioxidant enzymes and reduced apoptotic cell death caused by H₂O₂ exposure. Taken together, these findings demonstrate that EGCG and curcumin are capable of suppressing inducible oxidative stress in hMSCs, and suggest a possible new approach to maintain MSC viability and potency for clinical application. J. Cell. Biochem. 114: 1163–1173, 2013. © 2012 Wiley Periodicals, Inc.     

Hypoxia induces osteogenic/angiogenic responses of bone marrow‐derived mesenchymal stromal cells seeded on bone‐derived scaffolds via ERK1/2 and p38 pathways

Osteogenesis and angiogenesis are tightly coupled processes during bone development and formation. It is thus well known that the enhancement of vascularization is of great importance in bone tissue engineering. As a potential approach for repairing bone defects, bone tissue constructs should therefore replicate the essential components in vivo microenvironments to promote cell osteogenic differentiation while at same time induce angiogenic response. In light of standpoint above, a combination of human bone‐derived scaffolds and BMSCs that subjected to hypoxia was used to mimic in vivo conditions. Also the underlying cellular/molecular regulation was fully investigated. The results showed that hypoxia (5–10% O₂) greatly enhanced the proliferation of BMSCs seeded in scaffolds, although the hypoxia (5% O₂)‐induced proliferative effect on BMSC cellular scaffolds was not apparent to those cultured in plates. However, such a kind of model was able to significantly induce the osteogenic/angiogenic responses of BMSCs as reflected by osteogenesis or angiogenesis‐related highly expressed genes or proteins, such as alkaline phosphatase, osteocalcin, hypoxia‐inducible factor‐1α and vascular endothelial growth factor. Moreover, ERK1/2 and/or p38 pathways were demonstrated to play essential roles in hypoxia‐induced osteogenic/angiogenic responses. Our results indicated that the combination of bone‐derived scaffolds, a material that has a three dimensional network structure, and hypoxia, an environment that replicates in vivo BMSCs hypoxic living conditions, may be a potential approach for creating functional tissue‐engineered bone. Biotechnol. Bioeng. 2013; 110: 1794–1804. © 2013 Wiley Periodicals, Inc.     

Neural stimulation on human bone marrow‐derived mesenchymal stem cells by extremely low frequency electromagnetic fields

Adult stem cells are considered multipotent. Especially, human bone marrow‐derived mesenchymal stem cells (hBM‐MSCs) have the potential to differentiate into nerve type cells. Electromagnetic fields (EMFs) are widely distributed in the environment, and recently there have been many reports on the biological effects of EMFs. hBM‐MSCs are weak and sensitive pluripotent stem cells, therefore extremely low frequency‐electromagnetic fields (ELF‐EMFs) could be affect the changes of biological functions within the cells. In our experiments, ELF‐EMFs inhibited the growth of hBM‐MSCs in 12 days exposure. Their gene level was changed and expression of the neural stem cell marker like nestin was decreased but the neural cell markers like MAP2, NEUROD1, NF‐L, and Tau were induced. In immunofluorescence study, we confirmed the expression of each protein of neural cells. And also both oligodendrocyte and astrocyte related proteins like O4 and GFAP were expressed by ELF‐EMFs. We suggest that EMFs can induce neural differentiation in BM‐MSCs without any chemicals or differentiation factors. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Bone marrow‐derived mesenchymal stem cells mitigate caspase‐3 and 8‐hydroxy proline induced via β‐adrenergic agonist in pulmonary injured rats

Estimating the ability of bone marrow‐derived mesenchymal stem cells (BM‐MSCs) to alleviate pulmonary injury induced via isoproterenol (ISP). ISP was injected in a dose of (100 mg/kg, subcutaneously twice at an interval of 24 h). One month post BM‐MSCs transplantation by intravenous injection, pulmonary oxidative stress was assessed, and Western blot analyses and histopathological investigations were conducted. Compared with the normal control group, BM‐MSCs transplantation significantly decreased the expression of pulmonary anti‐oxidative stress marker. Western blot analysis revealed that ISP significantly reduced the protein expression of the anti‐oxidative stress marker nuclear related factor‐2 (Nrf2). However, the apoptotic marker (caspase‐3) and collagen content marker (8‐hydroxyproline) were markedly elevated. These biochemical markers were confirmed by histopathological investigations. Finally, it was demonstrated that BM‐MSCs transplantation showed a superior effect in improving pulmonary function through alleviating oxidative stress, apoptosis, and collagen content.     

Bone‐marrow‐derived mesenchymal stem cells inhibit gastric aspiration lung injury and inflammation in rats

Gastric aspiration lung injury is one of the most common clinical events. This study investigated the effects of bone‐marrow‐derived mesenchymal stem cells (BMSCs) on combined acid plus small non‐acidified particle (CASP)‐induced aspiration lung injury. Enhanced green fluorescent protein (EGFP⁺) or EGFP^? BMSCs or 15d‐PGJ₂ were injected via the tail vein into rats immediately after CASP‐induced aspiration lung injury. Pathological changes in lung tissues, blood gas analysis, the wet/dry weight ratio (W/D) of the lung, levels of total proteins and number of total cells and neutrophils in bronchoalveolar lavage fluid (BALF) were determined. The cytokine levels were measured using ELISA. Protein expression was determined by Western blot. Bone‐marrow‐derived mesenchymal stem cells treatment significantly reduced alveolar oedema, exudation and lung inflammation; increased the arterial partial pressure of oxygen; and decreased the W/D of the lung, the levels of total proteins and the number of total cells and neutrophils in BALF in the rats with CASP‐induced lung injury. Bone‐marrow‐derived mesenchymal stem cells treatment decreased the levels of tumour necrosis factor‐α and Cytokine‐induced neutrophil chemoattractant (CINC)‐1 and the expression of p‐p65 and increased the levels of interleukin‐10 and 15d‐PGJ₂ and the expression of peroxisome proliferator‐activated receptor (PPAR)‐γ in the lung tissue in CASP‐induced rats. Tumour necrosis factor‐α stimulated BMSCs to secrete 15d‐PGJ₂. A tracking experiment showed that EGFP⁺ BMSCs were able to migrate to local lung tissues. Treatment with 15d‐PGJ₂ also significantly inhibited CASP‐induced lung inflammation and the production of pro‐inflammatory cytokines. Our results show that BMSCs can protect lung tissues from gastric aspiration injury and inhibit lung inflammation in rats. A beneficial effect might be achieved through BMSC‐derived 15d‐PGJ₂ activation of the PPAR‐γ receptor, reducing the production of proinflammatory cytokines.