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.     

IGF-1 prevents oxidative stress induced-apoptosis in induced pluripotent stem cells which is mediated by microRNA-1

Oxidative stress contributes to tissue injury and cell death during the development of various diseases. The present study aims at investigating whether oxidative stress triggered by the exposure to hydrogen peroxide (H₂O₂) can induce apoptosis of induced pluripotent stem cells (iPS cells) in a mechanism mediated by insulin-like growth factor (IGF-1) and microRNA-1 (miR-1). iPS cells treated with H₂O₂ showed increases in miR-1 expression, mitochondria dysfunction, cytochrome-c release and apoptosis, Addition of IGF-1 into the iPS cell cultures reduced the H₂O₂ cytotoxicity. Prediction algorithms showed that 3′-untranslated regions of IGF-1 gene as a target of miR-1. Moreover, miR-1 mimic, but not miR-1 mimic negative control, diminished the protective effect of IGF-1 on H₂O₂-induced mitochondrial dysfunction, cytochrome-c release and apoptosis in iPS cells. In conclusion, IGF-1 inhibits H₂O₂-induced mitochondrial dysfunction, cytochrome-c release and apoptosis. IGF-1′s effect is, at least partially, regulated by miR-1 in iPS cells.     

Chloride channels involve in hydrogen peroxide-induced apoptosis of PC12 cells

Chloride channel activity is one of the critical factors responsible for cell apoptotic volume decrease (AVD). However, the roles of chloride channels in apoptosis have not been fully understood. In the current study, we assessed the role of chloride channels in hydrogen peroxide (H₂O₂)-induced apoptosis of pheochromocytoma cells (PC12). Extracellular application of H₂O₂ activated a chloride current and induced cell volume decrease in a few minutes. Incubation of cells with H₂O₂ elevated significantly the membrane permeability to the DNA dye Hoechst 33258 in 1 h and induced apoptosis of most PC12 cells tested in 24 h. The chloride channel blocker NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate) prevented appearance of H₂O₂-induced high membrane permeability and cell shrinkage, suppressed H₂O₂-activated chloride currents and protected PC12 cells from apoptosis induced by H₂O₂. The results suggest that chloride channels may contribute to H₂O₂-induced apoptosis by ways of elevation of membrane permeability and AVD in PC12 cells.     

Magnetic stromal layers for enhanced and unbiased recovery of co-cultured hematopoietic cells

Cell co-culture systems have a long history of application in hematology and hold promise for successful hematopoietic stem and progenitor cell expansion. Here we report that various types of stromal cells used in such co-cultures can be rapidly and efficiently labeled with l-lysine-modified Fe₃O₄ magnetic nanoparticles. Hematopoiesis-supporting activity does not seem to be compromised after magnetic labeling of stromal cells, and the loss of the label by stromal layers during extended culturing is negligible. Magnetic labeling allows for simple and efficient removal of stromal component, yielding unbiased hematopoietic cell populations. When Lin^– bone mouse marrow fraction was co-cultured with magnetic stromal layers and resulting cell populations were harvested by trypsinization, the yields of total nucleated cells, colony forming cells, and phenotypically primitive Lin^–Sca-1⁺c-kit⁺ subset were substantially higher as compared with nonadherent cell fractions harvested after conventional stromal co-culture. The advantage offered by the magnetic stroma approach over the traditional one was even more significant after a second round of co-culture and was more dramatic for more primitive hematopoietic cells. We conclude that magnetic stromal layers represent a simple, efficient, and convenient tool for co-culturing and subsequent recovery of sufficiently pure unbiased populations of hematopoietic cells.     

Redox regulation of stem/progenitor cells and bone marrow niche

Bone marrow (BM)-derived stem and progenitor cell functions including self-renewal, differentiation, survival, migration, proliferation, and mobilization are regulated by unique cell-intrinsic and -extrinsic signals provided by their microenvironment, also termed the “niche.” Reactive oxygen species (ROS), especially hydrogen peroxide (H₂O₂), play important roles in regulating stem and progenitor cell functions in various physiologic and pathologic responses. The low level of H₂O₂ in quiescent hematopoietic stem cells (HSCs) contributes to maintaining their “stemness,” whereas a higher level of H₂O₂ within HSCs or their niche promotes differentiation, proliferation, migration, and survival of HSCs or stem/progenitor cells. Major sources of ROS are NADPH oxidase and mitochondria. In response to ischemic injury, ROS derived from NADPH oxidase are increased in the BM microenvironment, which is required for hypoxia and hypoxia-inducible factor-1α expression and expansion throughout the BM. This, in turn, promotes progenitor cell expansion and mobilization from BM, leading to reparative neovascularization and tissue repair. In pathophysiological states such as aging, atherosclerosis, heart failure, hypertension, and diabetes, excess amounts of ROS create an inflammatory and oxidative microenvironment, which induces cell damage and apoptosis of stem and progenitor cells. Understanding the molecular mechanisms of how ROS regulate the functions of stem and progenitor cells and their niche in physiological and pathological conditions will lead to the development of novel therapeutic strategies.     

MiR-124 protects human hepatic L02 cells from H2O2-induced apoptosis by targeting Rab38 gene

Background

Hepatic ischemia reperfusion injury (IRI) is an inevitable clinical problem for liver surgeons. Because microRNAs (miRNAs) participate in various hepatic pathophysiological processes, this study aimed to explore the role and potential mechanism of miR-124 in hepatic IRI.

Methods

A liver IRI model was established in rats. The differential expression of miRNAs was detected using microarrays, and the expression of miR-124 was measured by qRT-PCR. A hydrogen peroxide (H₂O₂)-induced oxidative stress apoptosis model was also established. Cell apoptosis was detected by flow cytometry, and viability was detected by CCK8. The expression of Rab38 was detected by Western blotting and qRT-PCR, and a luciferase reporter assay was used to verify the expression of the miR-124 target gene.

Results

The miRNA spectrum changes dramatically after hepatic IRI in rats, and miR-124 is significantly down-regulated after liver IRI. MiR-124 decreases the H₂O₂-induced apoptosis of human hepatic L02 cells by up-regulating the activation of the AKT pathway. Rab38 is a target gene of miR-124 and is involved in H₂O₂-induced apoptosis. Interference with the expression of the Rab38 gene can protect hepatic L02 from H₂O₂-induced apoptosis by increasing the phosphorylation of AKT. These protective effects of miR-124 are attenuated by over-expression of Rab38.

Conclusions

Many miRNAs are involved in hepatic IRI in rats, and miR-124 is significantly decreased in this model. MiR-124 significantly decreases the H₂O₂-induced apoptosis of human hepatic L02 cells by targeting the Rab38 gene and activating the AKT pathway.     

Apoptosis of mesenchymal stem cells induced by hydrogen peroxide concerns both endoplasmic reticulum stress and mitochondrial death pathway through regulation of caspases,p38 and JNK

Poor survival of mesenchymal stem cells (MSCs) compromised the efficacy of stem cell therapy for myocardial infarction. The increase of exogenous reactive oxygen species (ROS) in infracted heart is one of the important factors that challenged the survival of donor MSCs. In the study we aimed to evaluate the effect of oxidative stress on the cell death of MSCs and investigate its mechanisms in order to help with the identification of new biological compounds to reduce donor cells damage. Apoptosis of MSCs were evaluated with Hoechst 33342 staining and flow cytometry analysis. The mitochondrial membrane potential of MSCs was analyzed with JC‐1 staining. Signaling pathways involved in H₂O₂ induced apoptosis were analyzed with Western blot. H₂O₂ induced apoptosis of MSCs in a dose‐ and time‐dependent manner. H₂O₂ induced apoptosis of MSCs via both endoplasmic reticulum (ER) and mitochondrial pathways rather than extrinsic apoptosis pathway. H₂O₂ caused transient rather than sustained activation of p38 and JNK with no effect on ERK1/2 pathway. P38 was involved in the regulation of early apoptosis of MSCs while JNK was involved in the late apoptosis. P38 directed both ER stress and mitochondria death pathway in the early apoptosis. In conclusion, exogenous ROS was a major factor to induce apoptosis of MSCs. Both ER stress and mitochondria death pathway were involved in the apoptosis of MSCs. H₂O₂ activated p38 that directed the above two pathways in the regulation of early apoptosis of MSCs while JNK was involved in the late apoptosis of MSCs. J. Cell. Biochem. 111: 967–978, 2010. © 2010 Wiley‐Liss, Inc.     

Mesenchymal stem cells attenuate hydrogen peroxide-induced oxidative stress and enhance neuroprotective effects in retinal ganglion cells

The apoptosis of retinal ganglion cells leads to visual impairment and blindness in ocular neurodegenerative diseases, especially in diabetic retinopathy (DR). Mounting evidence suggests that oxidative stress contributes to the pathogenesis of DR. In the present study, we investigated whether bone mesenchymal stem cells (BMSCs) have protective ability to relieve hydrogen peroxide (H₂O₂)-induced injury on retinal ganglion cells in vitro. An immortalized retinal ganglion cells, RGC-5 cells, were exposed to an indicated concentration of H₂O₂ for 24 h. Cell viability was analyzed by CCK-8 assay to find out a certain concentration to build H₂O₂ oxidative damage model. Morphological changes in RGC-5 cells were observed under optical microscope, and cell apoptosis was detected with Hoechst fluorescence staining. Then, BMSCs were co-cultured with RGC-5 cells in a transwell culture system for 24 h and 48 h. Flow cytometry was performed to qualify the apoptosis rate of RGC-5 cells. Conditioned medium was collected for evaluation the inflammatory cytokines by ELISA. The content of intracellular malondialdehyde (MDA) and superoxide dismutase (SOD) was assayed by thiobarbituric acid and xanthine oxidase method, respectively. qRT-PCR and ELISA were conducted for analysis of the expression changes in brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF), respectively. After H₂O₂ exposure, the morphological varieties were observed as cytoplasm shrinking and paramorphia together with nuclear gathering. Meanwhile, the apoptotic cells had hyperfluorescence with Hoechst 33258 staining. Co-culture with BMSCs significantly inhibited retinal cell death. It was found that BMSCs reduced H₂O₂-induced inflammatory factors IL-1β and TNF-α, down-regulated intracellular oxidant factor MDA, up-regulated intracellular antioxidant factor SOD, and increased neurotrophins BDNF and CNTF expression. BMSCs may enhance protective effect of RGC-5 cells in H₂O₂-induced damage through improving antioxidant capacity, inhibiting pro-inflammatory cytokine secretion, and promoting neurotrophin expression.     

Oxidative stress response of human fibroblasts and endometrial mesenchymal stem cells

Human mesenchymal stem cells are a promising cell source for tissue engineering. During transplantation, they may be subjected to oxidative stress due to unfavorable cellular microenvironment characterized by an increased level of reactive oxygen species. Recently, we have demonstrated that oxidative stress response of human mesenchymal stem cells derived from endometrium (hMESCs) depends on the oxidizer concentration. The duration of cell treatment with an oxidizer also may play an important role. In this study, we investigated the dependence of the cell response on H₂O₂ treatment duration. The effects of high H₂O₂ doses on hMESCs and human lung embryonic fibroblasts were compared. In both cell types, H₂O₂ treatment for 60 min caused multiphase cell cycle arrest, with dose-dependent cell death occurring equally in all phases of the cell cycle. However, the cell death dynamics in hMESCs and fibroblasts were different. Interestingly, in both cell types, shortening of H₂O₂ treatment from 60 to 10 min induced growth retardation, G1-phase cell accumulation, and cell size increase. Collectively, these findings suggest that there is induction of premature senescence. Thus, shortening of oxidative stress induced in human endometrial stem cells and embryonic fibroblasts by high H₂O₂ doses enables one to modulate cellular response as both cell death and premature senescence.     

Dominance of parental genomes in embryonic stem cell/fibroblast hybrid cells depends on the ploidy of the somatic partner

Two dozen hybrid clones were produced by fusion of diploid embryonic stem (ES) cells positive for green fluorescent protein (GFP) with tetraploid fibroblasts derived from DD/c and C57BL-I(I)1RK mice. Cytogenetic analysis demonstrated that most cells from these hybrid clones contained near-hexaploid chromosome sets. Additionally, the presence of chromosomes derived from both parental cells was confirmed by polymerase chain reaction (PCR) analysis of polymorphic microsatellites. All hybrid cells were positive for GFP and demonstrated growth characteristics and fibroblast-like morphology. In addition, most hybrid cells were positive for collagen type I, fibronectin, and lamin A/C but were negative for Oct4 and Nanog proteins. Methylation status of the Oct4 and Nanog gene promoters was evaluated by bisulfite genomic sequencing analysis. The methylation sites (CpG-sites) of the Oct4 and Nanog gene promoters were highly methylated in hybrid cells, whereas the CpG-sites were unmethylated in the parental ES cells. Thus, the fibroblast genome dominated the ES genome in the diploid ES cell/tetraploid fibroblast hybrid cells. Immunofluorescent analysis of the pluripotent and fibroblast markers demonstrated that establishment of the fibroblast phenotype occurred shortly after fusion and that the fibroblast phenotype was further maintained in the hybrid cells. Fusion of karyoplasts and cytoplast derived from tetraploid fibroblasts with whole ES cells demonstrated that karyoplasts were able to establish the fibroblast phenotype of the reconstructed cells but not fibroblast cytoplasts. Thus, these data suggest that the dominance of parental genomes in hybrid cells of ES cell/somatic cell type depends on the ploidy of the somatic partner.     

The role of oxygen in regulating neural stem cells in development and disease

Oxygen (O₂) is a substrate for energy production in the cell and is a rapid regulator of cellular metabolism. Recent studies have also implicated O₂ and its signal transduction pathways in controlling cell proliferation, fate, and morphogenesis during the development of many tissues, including the nervous system. O₂ tensions in the intact brain are much lower than in room air, and there is evidence that dynamic control of O₂ availability may be a component of the in vivo neural stem cell (NSC) niche. At lower O₂ tensions, hypoxia‐inducible factor 1α (HIF1α) facilitates signal transduction pathways that promote self‐renewal (e.g., Notch) and inhibits pathways that promote NSC differentiation or apoptosis (e.g., bone morphogenetic proteins). Increasing O₂ tension degrades HIF1α, thus promoting differentiation or apoptosis of NSCs and progenitors. These dynamic changes in O₂ tension can be mimicked to optimize ex vivo production methods for cell replacement therapies. Conversely, disrupted O₂ availability may play a critical role in disease states such as stroke or brain tumor progression. Hypoxia during stroke activates precursor proliferation in vivo, while glioblastoma stem cells proliferate maximally in a more hypoxic environment than normal stem cells, which may make them resistant to certain anti‐neoplastic therapies. These findings suggest that O₂ response is central to the normal architecture and dynamics of NSC regulation and in the etiology and treatment of brain diseases. J. Cell. Physiol. 220: 562–568, 2009. © 2009 Wiley‐Liss, Inc.     

In vitro differentiation of reprogrammed murine somatic cells into hepatic precursor cells

Recently, a new approach to reprogram somatic cells into pluripotent stem cells was shown by fusion of somatic cells with embryonic stem (ES) cells, which results in a tetraploid karyotype. Normal hepatocytes are often polyploid, so we decided to investigate the differentiation potential of fusion hybrids into hepatic cells. We chose toxic milk mice (a model of Wilson's disease) and performed initial transplantation experiments using this potential cell therapy approach. Mononuclear bone marrow cells from Rosa26 mice were fused with OG2 (Oct4-GFP transgenic) ES cells. Unfused ES cells were eliminated by selection with G418 for OG2-Rosa26 hybrids and fusion-derived colonies could be subcloned. Using an endodermal differentiation protocol, hepatic precursor cells could be generated. After FACS depletion of contaminating Oct4-GFP-positive cells, the hepatic precursor cells were transplanted into immunosuppressed toxic milk mice by intrasplenic injection. However, five out of eight mice showed teratoma formation within 3-6 weeks after transplantation in the spleen and liver. In conclusion, a hepatic precursor cell type was achieved from mononuclear bone marrow cell-ES cell hybrids and preliminary transplantation experiments confirmed engraftment, but also showed teratoma formation, which needs to be excluded by using more stringent purification strategies.     

Estradiol protects adipose tissue-derived stem cells against H(2) O(2) -induced toxicity

Oxidative stress is associated with various pathophysiological processes, including cell survival, adhesion, apoptosis, and cancer. In the present study, we aimed to evaluate the effects of H₂O₂‐induced toxicity on adipose tissue–derived stem cells (ADSCs) and whether 17β‐estradiol (E₂) has protective effects on these cells. ADSCs derived from adult Sprague–Dawley rats were pretreated with different doses of E₂ for 24 h and then exposed to 200 µM H₂O₂ for 4 h. Incubation of ADSCs with H₂O₂‐decreased cell viability in a concentration‐dependent fashion (p < 0.0001), whereas pretreatment of these cells with E₂ significantly reversed toxicity (p < 0.05), inhibited apoptotic changes, and decreased lipid peroxidation (p < 0.0005). Our findings suggest that E₂ protects ADSCs from oxidative‐induced cell death, and therefore, it may be used to improve the survival rate and regenerative capacity of stem cells. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:301–307, 2012; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21421     

The Role of N-Acetyltransferase 8 in Mesenchymal Stem Cell-Based Therapy for Liver Ischemia/Reperfusion Injury in Rats

Objective

To evaluate the impact of mesenchymal stem cells (MSCs) against hepatic I/R injury and explore the role of N-acetyltransferase 8 (NAT8) in the process.

Methods

We investigated the potential of injected MSCs systemically via the tail vein in healing injuried liver of the SD rat model of 70% hepatic I/R injury by measuring the biochemical and pathologic alterations. Subsequently, we evaluated the expression levels of NAT8 by western blotting in vivo. Concurrently, hydrogen peroxide (H₂O₂)-induced apoptosis in the human normal liver cell line L02 was performed in vitro to evaluate the protective effects of MSC conditioned medium (MSC-CM) on L02 cells. In addition, we downregulated and upregulated NAT8 expression in L02 cells and induced apoptosis by using H₂O₂ to study the protective role of NAT8.

Results

MSCs implantation led to a significant reduced liver enzyme levels, an advanced protection in the histopathological findings of the acutely injured liver and a significantly lower percentage of TUNEL-positive cells, which were increased after I/R injury. In vitro assays, MSC-CM inhibited hepatocyte apoptosis induced by H₂O_2. Moreover, overexpression or downregulation of NAT8 prevented or aggravated hepatocyte apoptosis induced by H₂O₂, respectively.

Conclusions

MSC transplantation provides support to the I/R-injured liver by inhibiting hepatocellular apoptosis and stimulating NAT8 regeneration.     

EphA2 overexpression reduces H2O2-induced damage of lens epithelial cells

Age-related cataract (ARC) is a progressive lens opacification that occurs from middle to old age. Eph-receptor tyrosinekinase-type A2 (EphA2) has been reported to be associated with ARC. This work aims to investigate the molecular mechanism of EphA2 in ARC. We treated human lens epithelial cells (SRA01/04) with different concentration of H₂O₂ to induce lens epithelial cell damage. Then, we found that H₂O₂ treatment significantly suppressed cell viability and enhanced the expression of EphA2 in the SRA01/04 cells. H₂O₂ treatment repressed cell viability and enhanced the levels of reactive oxygen species (ROS) in SRA01/04 cells, which was partly abolished by EphA2 up-regulation. Moreover, EphA2 overexpression reduced H₂O₂-induced apoptosis of SRA01/04 cells. EphA2 up-regulation caused an up-regulation of Bcl-2, and repressed the expression of Bax and Cleaved-caspase-3 in the SRA01/04 cells following H₂O₂ treatment. In conclusion, our data confirm that EphA2 overexpression enhances cell viability and inhibits apoptosis in the H₂O₂-treated SRA01/04 cells, thereby reducing H₂O₂-induced damage of lens epithelial cells. Thus, this work provides new insights into the mechanism of EphA2 in ARC.     

Activation of mitogen-activated protein kinases is essential for hydrogen peroxide -induced apoptosis in retinal pigment epithelial cells

Retinal pigment epithelial (RPE) cells are constantly exposed to oxidative injury while clearing byproducts of photoreceptor turnover, a circumstance thought to be responsible for degenerative retinal diseases. The mechanisms of hydrogen peroxide (H₂O₂)-induced apoptosis in RPE cells are not fully understood. We studied signal transduction mechanisms of H₂O₂-induced apoptosis in the human RPE cell line ARPE-19. Activation of two stress kinases (JNK and p38) occurs during H₂O₂ stimulation, and H₂O₂-mediated cell death was significantly reduced by their specific inhibition. Exposure to a lethal dose of H₂O₂ elicited Bax translocation to the mitochondria and release of apoptosis-inducing factor (AIF) from the mitochondria, both of which were abolished by either JNK- or p38-specific inhibitors. Both H₂O₂-induced cell death and JNK/p38 phosphorylation were partially inhibited by C. difficile toxin B, inhibitor of Rho, Rac, and cdc42. Use of pull-down assays revealed that the small GTPase activated by H₂O₂ is Rac1. This study is the first to demonstrate that H₂O₂ induces a Rac1/JNK1/p38 signaling cascade, and that JNK and p38 activation is important for H₂O₂-induced apoptosis as well as AIF/Bax translocation of RPE cells. Y.-C. Yang and T.-C. Ho contributed equally to the work described herein.