Production of reactive oxygen species in mitochondria of HeLa cells under oxidative stress

Mitochondria can be a source of reactive oxygen species (ROS) and a target of oxidative damage during oxidative stress. In this connection, the effect of photodynamic treatment (PDT) with Mitotracker Red (MR) as a mitochondria-targeted photosensitizer has been studied in HeLa cells. It is shown that MR produces both singlet oxygen and superoxide anion upon photoactivation and causes photoinactivation of gramicidin channels in a model system (planar lipid bilayer). Mitochondria-targeted antioxidant (MitoQ) inhibits this effect. In living cells, MR-mediated PDT initiates a delayed ("dark") accumulation of ROS, which is accelerated by inhibitors of the respiratory chain (piericidin, rotenone and myxothiazol) and inhibited by MitoQ and diphenyleneiodonium (an inhibitor of flavin enzymes), indicating that flavin of Complex I is involved in the ROS production. PDT causes necrosis that is prevented by MitoQ. Treatment of the cell with hydrogen peroxide causes accumulation of ROS, and the effects of inhibitors and MitoQ are similar to that described for the PDT model. Apoptosis caused by H2O2 is augmented by the inhibitors of respiration and suppressed by MitoQ. It is concluded that the initial segments of the respiratory chain can be an important source of ROS, which are targeted to mitochondria, determining the fate of the cell subjected to oxidative stress.     

SIRT6 safeguards human mesenchymal stem cells from oxidative stress by coactivating NRF2

SIRT6 belongs to the mammalian homologs of Sir2 histone NAD⁺-dependent deacylase family. In rodents, SIRT6 deficiency leads to aging-associated degeneration of mesodermal tissues. It remains unknown whether human SIRT6 has a direct role in maintaining the homeostasis of mesodermal tissues. To this end, we generated SIRT6 knockout human mesenchymal stem cells (hMSCs) by targeted gene editing. SIRT6-deficient hMSCs exhibited accelerated functional decay, a feature distinct from typical premature cellular senescence. Rather than compromised chromosomal stability, SIRT6-null hMSCs were predominately characterized by dysregulated redox metabolism and increased sensitivity to the oxidative stress. In addition, we found SIRT6 in a protein complex with both nuclear factor erythroid 2-related factor 2 (NRF2) and RNA polymerase II, which was required for the transactivation of NRF2-regulated antioxidant genes, including heme oxygenase 1 (HO-1). Overexpression of HO-1 in SIRT6-null hMSCs rescued premature cellular attrition. Our study uncovers a novel function of SIRT6 in maintaining hMSC homeostasis by serving as a NRF2 coactivator, which represents a new layer of regulation of oxidative stress-associated stem cell decay.     

Different protective mechanisms of human embryonic and endometrium-derived mesenchymal stem cells under oxidative stress

Oxidative stress has been shown to cause either apoptosis or stress-induced premature senescence (SIPS) in different cell types. At present, it is generally accepted that stem cells have high resistance to oxidative stress; however, data reported by various authors are disputed. In this study, we investigated stress responses of human embryonic stem cells (hESC) and human mesenchymal stem cells (hMESC) derived from desquamated endometrium to hydrogen peroxide (H₂O₂). Cell viability was evaluated by MTT assay. LD₅₀ were determined as 300–350, 370–400, and 600–700 μM for hESC, human embryonic fibroblasts, and hMESC, respectively. Thus, of the studied cell lines, hMESC exhibited the greatest resistance to increased H₂O₂ concentration. We found for the first time that a sublethal concentration of H₂O₂ induced premature senescence phenotype in hMESC, like in HEF, that was characterized by increased expression of cyclin-dependent kinase inhibitor p21^Waf1/Cip1, an irreversible cell cycle arrest, the permanent loss of proliferative potential, cell hypertrophy, and the SA-β-Gal staining. Whereas the sublethal H₂O₂ concentration (200 μM) promoted in hMESC only SIPS, higher H₂O₂ concentrations also induced apoptosis in a small part of the cell population. On the contrary, in hESC, H₂O₂, regardless of the tested concentrations (from 50 to 500 μM), triggered apoptosis, which was the only pronounced response of these cells to oxidative damage. The obtained data demonstrate that stem cells of different origins under conditions of oxidative stress use different protective mechanisms: hESC rapidly eliminate damaged cells through apoptosis, whereas hMESC are subjected to premature senescence.     

Regulation of reactive oxygen species in stem cells and cancer stem cells

Stem cells are defined by their ability to self-renew and their multi-potent differentiation capacity. As such, stem cells maintain tissue homeostasis throughout the life of a multicellular organism. Aerobic metabolism, while enabling efficient energy production, also generates reactive oxygen species (ROS), which damage cellular components. Until recently, the focus in stem cell biology has been on the adverse effects of ROS, particularly the damaging effects of ROS accumulation on tissue aging and the development of cancer, and various anti-oxidative and anti-stress mechanisms of stem cells have been characterized. However, it has become increasingly clear that, in some cases, redox status plays an important role in stem cell maintenance, i.e., regulation of the cell cycle. An active area of current research is redox regulation in various cancer stem cells, the malignant counterparts of normal stem cells that are viewed as good targets of cancer therapy. In contrast to cancer cells, in which ROS levels are increased, some cancer stem cells maintain low ROS levels, exhibiting redox patterns that are similar to the corresponding normal stem cell. To fully elucidate the mechanisms involved in stem cell maintenance and to effectively target cancer stem cells, it is essential to understand ROS regulatory mechanisms in these different cell types. Here, the mechanisms of redox regulation in normal stem cells, cancer cells, and cancer stem cells are reviewed.     

Mitochondria-targeted antioxidant mito-TEMPO alleviate oxidative stress induced by antimycin A in human mesenchymal stem cells

The cell therapy of damaged tissue, which is linked to hypoxia condition might fail, in large part due to the emergence of oxidative stress (OS) and/or mitochondrial dysfunctions. Thus, the invigoration of stem cells against oxidative stress could be a reliable strategy to improve the cell therapy outcome. Of various antioxidants, mito-Tempo (mito-T) is one of the potent antioxidants that could target and neutralize the mitochondrial oxidative stress. In this study, for the induction of hypoxia and oxidative stress in mitochondria of the mesenchymal stem cells (MSCs) isolated from human adipose tissue, antimycin A (AMA) was used and then several parameters were analyzed, including cell viability and cell cycle arrest of MSCs exposed to AMA, mito-T, antioxidant potential, redox homeostasis, and signaling pathways in MSCs under oxidative stress. Based on our findings, the treated MSCs were found to impose a high resistance to the OS-induced apoptosis, which correlated with the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway required to manage OS. Upon exposure of the MSCs to high oxidative stress conditions using AMA, the cells failed to scavenge. The use of mito-T was found to alleviate the damage induced by oxidative stress through both direct functions of the free radical scavenging and the interplay in terms of cell signaling pathways including the upregulation of the Nrf2 pathway. These findings may pave the way in the stem cell therapy for the hypoxia-mediated tissue damage.     

Dissimilar characteristics of umbilical cord mesenchymal stem cells from donors of different ages

Increasing attention is being placed on the use of adult stem cells due to their potential therapeutic applications for unmet medical needs. Among all adult stem cell types, umbilical cord mesenchymal stem cells (UCMSCs) can be collected easily and does not harm newborns and the mothers. The excellent pluripotency and proliferation potential of UCMSCs has been previously demonstrated by many groups. However, little is known about the different characteristics of these cells due to diversity of donors (e.g., the mothers’ age). Regarding this, we examined the effect of mothers’ age-related variation on the basic properties (proliferation, colony forming and differentiation potential) of UCMSCs in vitro. The data evidenced that elder mother results in reduction of both proliferative and colony forming capacity of UCMSCs, and impacts proliferative potential more significantly. Data also evidenced that a decline in osteogenic potential, but an increase in adipogenic potential of UCMSCs from the elder donors. Additionally, no phenotypic differences were observed by flow cytometry analysis using a panel of 6 main surface antigen markers. We observed no differences in karyotyping and both UCMSCs populations exhibited diploid cells without chromosomal aberrations. The results of our study are the basis for banking UCMSCs, which may be labeled with donors’ information for different experiments, and the mother age-related differences in characteristics of UCMSCs should be taken into account when these cells are considered for further practical applications.     

Telomere dynamics in human mesenchymal stem cells after exposure to acute oxidative stress

A gradual shortening of telomeres due to replication can be measured using the standard telomere restriction fragments (TRF) assay and other methods by measuring the mean length of all the telomeres in a cell. In contrast, stress-induced telomere shortening, which is believed to be just as important for causing cellular senescence, cannot be measured properly using these methods. Stress-induced telomere shortening caused by, e.g. oxidative damage happens in a stochastic manner leaving just a few single telomeres critically short. It is now possible to visualize these few ultra-short telomeres due to the advantages of the newly developed Universal single telomere length assay (STELA), and we therefore believe that this method should be considered the method of choice when measuring the length of telomeres after exposure to oxidative stress. In order to test our hypothesis, cultured human mesenchymal stem cells, either primary or hTERT immortalized, were exposed to sub-lethal doses of hydrogen peroxide, and the short term effect on telomere dynamics was monitored by Universal STELA and TRF measurements. Both telomere measures were then correlated with the percentage of senescent cells estimated by senescence-associated β-galactosidase staining. The exposure to acute oxidative stress resulted in an increased number of ultra-short telomeres, which correlated strongly with the percentage of senescent cells, whereas a correlation between mean telomere length and the percentage of senescent cells was absent. Based on the findings in the present study, it seems reasonable to conclude that Universal STELA is superior to TRF in detecting telomere damage caused by exposure to oxidative stress. The choice of method should therefore be considered carefully in studies examining stress-related telomere shortening as well as in the emerging field of lifestyle studies involving telomere length measurements.     

Fas-stimulated generation of reactive oxygen species or exogenous oxidative stress sensitize cells to Fas-mediated apoptosis

Inhibition of Fas-mediated apoptosis in B cell lymphomas by thiol antioxidants (glutathione and N-acetylcysteine) supported previous studies, suggesting that Fas-stimulated ROS generation may play a role in Fas-mediated apoptosis. Thus, the goal of the current study was to determine if Fas stimulation could induce ROS generation and what role, if any, it played in apoptosis. Fas crosslinking induced rapid generation of ROS (within 15 min) well before the appearance of characteristic apoptotic changes. Overexpression of catalase or superoxide dismutase suggested that Fas induced production of both superoxide anion and hydrogen peroxide. ROS generation was only observed, however, in cells that were sensitive to apoptosis and not in B cells inherently resistant to anti-Fas or in those in which resistance was induced by B cell receptor crosslinking. The exogenous addition of 250 microM hydrogen peroxide could reverse the resistant phenotype and sensitize cells to Fas-induced apoptosis. In Fas-sensitive cells, depletion of endogenous antioxidant defenses with buthionine sulfoximine increased the sensitivity to Fas-induced apoptosis, while overexpression of antioxidant enzymes and antiapoptotic proteins suggested a role for Fas-induced production of hydrogen peroxide in apoptosis. Further analysis suggested a redox-sensitive step early in Fas signaling at the level of initiator caspase (caspase-8) activation. Thus, the data suggest that the level of oxidative stress, either from exogenous sources or generated endogenously upon receptor stimulation, regulates the sensitivity to Fas-mediated apoptosis.     

Proteomic profiling of human bone marrow mesenchymal stem cells under shear stress

Diets containing 8% salt or 4% fructose (FR) cause insulin resistance and increase tissue methylglyoxal and advanced glycation end products (AGEs), platelet cytosolic-free calcium, and systolic blood pressure (SBP) in rats. In WKY rats, we have shown that moderately high salt, 4% NaCl (MHS) alone in diet does not cause hypertension, and when given along with 4% FR it does not have an additive effect. N-acetylcysteine (NAC) or l-arginine (ARG), treatment alone does not prevent hypertension in this model. The objectives of this study were to investigate the effect of NAC plus ARG in diet on SBP, platelet cytosolic-free calcium in a MHS + FR model, and to measure the plasma levels of methylglyoxal and the AGE, methylglyoxal-derived hydroimidazolone (MGH). At 7 weeks of age, WKY rats were divided into three groups: control group was given regular rat chow (0.7% NaCl) and water; MHS + FR group, diet containing 4% NaCl and 4% FR in drinking water; and MHS + FR + NAC + ARG group, MHS diet supplemented with 1.5% N-acetylcysteine (NAC) and 1.5% l-arginine (ARG), and 4% FR in drinking water, and followed for 6 weeks. NAC + ARG prevented the increase in platelet cytosolic-free calcium and SBP in MHS + FR treated rats. There was no difference in mean values of plasma methylglyoxal and MGH among the groups. In conclusion, NAC + ARG treatment is effective in preventing hypertension in a moderately high salt + FR-induced animal model. Plasma methylglyoxal and MGH may not represent tissue modification or, alternatively, other tissue AGEs, derived from methylglyoxal or other aldehydes, may be involved in hypertension in this model.     

Lipocalin 2 decreases senescence of bone marrow-derived mesenchymal stem cells under sub-lethal doses of oxidative stress

The regenerative potential of mesenchymal stem cells (MSCs) is impaired by cellular senescence, a multi factorial process that has various functions. However, pathways and molecules involved in senescence have not been fully identified. Lipocalin 2 (Lcn2) has been the subject of intensive research, due to its contribution to many physiological and pathophysiological conditions. The implication of Lcn2 has been reported in many conditions where senescence also occurs. In the present study, we evaluated the role of Lcn2 in the occurrence of senescence in human bone marrow-derived mesenchymal stem cells (hB-MSCs) under oxidative conditions. When hB-MSCs were genetically engineered to over-express Lcn2 (MSC-Lcn2) and exposed to H₂O₂, the proliferation rate of the cells increased. However, the number of colonies and the number of cells that made up each colony in both MSC-V and MSC-Lcn2 cells decreased compared to those cultivated under normal conditions. Our results revealed that over-expression of recombinant Lcn2 in hB-MSCs decreases senescence induced by H₂O₂ treatment. Senescent cells were observed in aged hB-MSCs; however, no alteration in the expression level of Lcn2 was detected compared to earlier passages. Finally, a higher amount of Lcn2 protein was detected in the plasma of the elderly than in young people. Our findings suggest that Lcn2 might restore the health and regeneration potential of MSCs by decreasing senescence.     

Trichostatin a modulates intracellular reactive oxygen species through SOD2 and FOXO1 in human bone marrow‐mesenchymal stem cells

Engraft cells are often exposed to oxidative stress and inflammation; therefore, any factor that can provide the stem cells resistance to these stresses may yield better efficacy in stem cell therapy. Studies indicate that histone deacetylase (HDACs) inhibitors alleviate damage induced by oxidative stress. In this study, we investigated whether regulation of reactive oxygen species (ROS) occurs through the HDAC inhibitor trichostatin A (TSA) in human bone marrow‐mesenchymal stem cells (hBM‐MSCs). Intracellular ROS levels increased following exposure to hydrogen peroxide (H₂O₂), and were suppressed by TSA treatment. Levels of the antioxidant enzyme superoxide dismutase 2 (SOD2) increased following treatment with 200 nM TSA and to a lesser level at 1–5 μM TSA. Cell protective effects against oxidative stress were significantly increased in TSA‐MSCs after treatment with low doses of TSA (50–500 nM) and decreased with high doses of TSA (5–10 μM). Consistent results were obtained with immunoblot analysis for caspase3. Investigation of Forkhead box O1 (FOXO1), superoxide dismutase 2 (SOD2), and p53 levels to determine intracellular signaling by TSA in oxidative stress‐induced MSCs demonstrated that expression of phosphorylated‐FOXO1 and phosphorylated‐SOD2 decreased in H₂O₂‐treated MSCs while levels of p53 increased. These effects were reversed by the treatment of 200 nM TSA. These results suggest that the main function of ROS modulation by TSA is activated through SOD2 and FOXO1. Thus, optimal treatment with TSA may protect hBM‐MSCs against oxidative stress. Copyright © 2014 John Wiley & Sons, Ltd.     

Oxidative stress induces senescence in human mesenchymal stem cells

Mesenchymal stem cells (MSCs) contribute to tissue repair in vivo and form an attractive cell source for tissue engineering. Their regenerative potential is impaired by cellular senescence. The effects of oxidative stress on MSCs are still unknown. Our studies were to investigate into the proliferation potential, cytological features and the telomere linked stress response system of MSCs, subject to acute or prolonged oxidant challenge with hydrogen peroxide. Telomere length was measured using the telomere restriction fragment assay, gene expression was determined by rtPCR. Sub-lethal doses of oxidative stress reduced proliferation rates and induced senescent-morphological features and senescence-associated β-galactosidase positivity. Prolonged low dose treatment with hydrogen peroxide had no effects on cell proliferation or morphology. Sub-lethal and prolonged low doses of oxidative stress considerably accelerated telomere attrition. Following acute oxidant insult p21 was up-regulated prior to returning to initial levels. TRF1 was significantly reduced, TRF2 showed a slight up-regulation. SIRT1 and XRCC5 were up-regulated after oxidant insult and expression levels increased in aging cells. Compared to fibroblasts and chondrocytes, MSCs showed an increased tolerance to oxidative stress regarding proliferation, telomere biology and gene expression with an impaired stress tolerance in aged cells.     

Heme oxygenase-1 protects bone marrow mesenchymal stem cells from iron overload through decreasing reactive oxygen species and promoting IL-10 generation

Iron overload (IO) caused by frequent blood transfusion in hematological diseases has become a major concern. In this study, up-regulation of heme oxygenase-1 (HO-1), a protector against oxidative stress, was observed in bone marrow mesenchymal stem cells (BMMSCs) at the early stage of IO and had favorable prognosis in an IO mouse model. Given that the protective role of HO-1 in IO damage of BMMSCs was still unknown, the mechanism was explored in vitro and in vivo. BMMSCs were transfected with HO-1/siHO-1 in vitro, and the mouse model was established to further evaluate the effect of HO-1 on IO in vivo. As a result, HO-1 decreased the apoptotic rate of BMMSCs with IO through reducing intracellular reactive oxygen species (ROS) but increasing IL-10 secretion. In addition, IL-10 was mediated by HO-1 via the ERK pathway. Intracellular iron was down-regulated by hepcidin depending on IL-10. In conclusion, HO-1 protects BMMSCs from ROS by secreting IL-10 upon iron overload.     

Photodynamic therapy regulates fate of cancer stem cells through reactive oxygen species

Photodynamic therapy (PDT) is an effective and promising cancer treatment. PDT directly generates reactive oxygen species (ROS) through photochemical reactions. This oxygen-dependent exogenous ROS has anti-cancer stem cell (CSC) effect. In addition, PDT may also increase ROS production by altering metabolism, endoplasmic reticulum stress, or potential of mitochondrial membrane. It is known that the half-life of ROS in PDT is short, with high reactivity and limited diffusion distance. Therefore, the main targeting position of PDT is often the subcellular localization of photosensitizers, which is helpful for us to explain how PDT affects CSC characteristics, including differentiation, self-renewal, apoptosis, autophagy, and immunogenicity. Broadly speaking, excess ROS will damage the redox system and cause oxidative damage to molecules such as DNA, change mitochondrial permeability, activate unfolded protein response, autophagy, and CSC resting state. Therefore, understanding the molecular mechanism by which ROS affect CSCs is beneficial to improve the efficiency of PDT and prevent tumor recurrence and metastasis. In this article, we review the effects of two types of photochemical reactions on PDT, the metabolic processes, and the biological effects of ROS in different subcellular locations on CSCs.     

Isolation of mesenchymal stem cells from human placenta: comparison with human bone marrow mesenchymal stem cells

The presence within bone marrow of a population of mesenchymal stem cells (MSCs) able to differentiate into a number of different mesenchymal tissues, including bone and cartilage, was first suggested by Friedenstein nearly 40 years ago. Since then MSCs have been demonstrated in a variety of fetal and adult tissues, including bone marrow, fetal blood and liver, cord blood, amniotic fluid and, in some circumstances, in adult peripheral blood. MSCs from all of these sources can be extensively expanded in vitro and when cultured under specific permissive conditions retain their ability to differentiate into multiple lineages including bone, cartilage, fat, muscle, nerve, glial and stromal cells. There has been great interest in these cells both because of their value as a model for studying the molecular basis of differentiation and because of their therapeutic potential for tissue repair and immune modulation. However, MSCs are a rare population in these tissues. Here we tried to identify cells with MSC-like potency in human placenta. We isolated adherent cells from trypsin-digested term placentas and examined these cells for morphology, surface markers, and differentiation potential and found that they expressed several stem cell markers. They also showed endothelial and neurogenic differentiation potentials under appropriate conditions. We suggest that placenta-derived cells have multilineage differentiation potential similar to MSCs in terms of morphology and cell-surface antigen expression. The placenta may prove to be a useful source of MSCs.