Mesenchymal stromal cells from amniotic fluid are less prone to senescence compared to those obtained from bone marrow: An in vitro study

Mesenchymal stromal cells (MSCs) are considered to be an excellent source in regenerative medicine. They contain several cell subtypes, including multipotent stem cells. MSCs are of particular interest as they are currently being tested using cell and gene therapies for a number of human diseases. They represent a rare population in tissues; for this reason, they require, before being transplanted, an in vitro amplification. This process may induce replicative senescence, thus affecting differentiation and proliferative capacities. Increasing evidence suggests that MSCs from fetal tissues are significantly more plastic and grow faster than MSCs from bone marrow. Here, we compare amniotic fluid mesenchymal stromal cells (AF‐MSCs) and bone marrow mesenchymal stromal cells (BM‐MSCs) in terms of cell proliferation, surface markers, multidifferentiation potential, senescence, and DNA repair capacity. Our study shows that AF‐MSCs are less prone to senescence with respect to BM‐MSCs. Moreover, both cell models activate the same repair system after DNA damage, but AF‐MSCs are able to return to the basal condition more efficiently with respect to BM‐MSCs. Indeed, AF‐MSCs are better able to cope with genotoxic stress that may occur either during in vitro cultivation or following transplantation in patients. Our findings suggest that AF‐MSCs may represent a valid alternative to BM‐MSCs in regenerative medicine, and, of great relevance, the investigation of the mechanisms involved in DNA repair capacity of both AF‐MSCs and BM‐MSCs may pave the way to their rational use in the medical field.     

Induction of DNA damage in mammalian cells by hydrogen peroxide generated by glucose oxidase immobilized in agarose slides

A new model system has been developed to study the influence of reactive oxygen species on isolated mammalian cells in conjunction with the comet assay. The glucose-glucose oxidase system was used as a hydrogen peroxide generating source. The level of DNA damage was assessed in the splenocytes and the cells of bone marrow of mouse and in human leukocytes both in untreated cells and in cells treated with hydrogen peroxide generated by glucose oxidase using the alkaline comet assay in vitro. Various options for the location of the enzyme in the slides have been studied: in the layer with the cells, in the layer above the cells, or in solution on the surface of the slides. The option where glucose oxidase was in the upper layer of 0.5% agarose over the layer of the cells was optimal. It provided separation of the enzyme from the cells and avoided obstruction to the hydrogen peroxide exposure. For the whole blood study, the content of endogenous glucose must be taken into account. This approach can be used to study the level of DNA damage induced in vitro and for the detection of DNA repair, thereby expanding the possibilities of the method, while the experiments are conducted under controlled conditions.     

Mitogen stimulation cooperates with telomere shortening to activate DNA damage responses and senescence signaling

Replicative senescence is induced by critical telomere shortening and limits the proliferation of primary cells to a finite number of divisions. To characterize the activity status of the replicative senescence program in the context of cell cycle activity, we analyzed the senescence phenotypes and signaling pathways in quiescent and growth-stimulated primary human fibroblasts in vitro and liver cells in vivo. This study shows that replicative senescence signaling operates at a low level in cells with shortened telomeres but becomes fully activated when cells are stimulated to enter the cell cycle. This study also shows that the dysfunctional telomeres and nontelomeric DNA lesions in senescent cells do not elicit a DNA damage signal unless the cells are induced to enter the cell cycle by mitogen stimulation. The amplification of senescence signaling and DNA damage responses by mitogen stimulation in cells with shortened telomeres is mediated in part through the MEK/mitogen-activated protein kinase pathway. These findings have implications for the further understanding of replicative senescence and analysis of its role in vivo.     

Accumulation of apoptosis‐insensitive human bone marrow‐mesenchymal stromal cells after long‐term expansion

Cells undergo replicative senescence during in vitro expansion, which is induced by the accumulation of cellular damage caused by excessive reactive oxygen species. In this study, we investigated whether long‐term‐cultured human bone marrow mesenchymal stromal cells (MSCs) are insensitive to apoptotic stimulation. To examine this, we established replicative senescent cells from long‐term cultures of human bone marrow MSCs. Senescent cells were identified based on declining population doublings, increased expression of senescence markers p16 and p53 and increased senescence‐associated β‐gal activity. In cell viability assays, replicative senescent MSCs in late passages (i.e. 15–19 passages) resisted damage induced by oxidative stress more than those in early passages did (i.e. 7–10 passages). This resistance occurred via caspase‐9 and caspase‐3 rather than via caspase‐8. The senescent cells are gradually accumulated during long‐term expansion. The oxidative stress‐sensitive proteins ataxia‐telangiectasia mutated and p53 were phosphorylated, and the expression of apoptosis molecules Bax increased, and Bcl‐2 decreased in early passage MSCs; however, the expression of the apoptotic molecules did less change in response to apoptotic stimulation in late‐passage MSCs, suggesting that the intrinsic apoptotic signalling pathway was not induced by oxidative stress in long‐term‐cultured MSCs. Based on these results, we propose that some replicative senescent cells may avoid apoptosis signalling via impairment of signalling molecules and accumulation during long‐term expansion. Copyright © 2016 John Wiley & Sons, Ltd.     

The comet assay in male reproductive toxicology

Due to our lifestyle and the environment we live in, we are constantly confronted with genotoxic or potentially genotoxic compounds. These toxins can cause DNA damage to our cells, leading to an increase in mutations. Sometimes such mutations could give rise to cancer in somatic cells. However, when germ cells are affected, then the damage could also have an effect on the next and successive generations. A rapid, sensitive and reliable method to detect DNA damage and assess the integrity of the genome within single cells is that of the comet or single-cell gel electrophoresis assay. The present communication gives an overview of the use of the comet assay utilising sperm or testicular cells in reproductive toxicology. This includes consideration of damage assessed by protocol modification, cryopreservation vs the use of fresh sperm, viability and statistics. It further focuses on in vivo and in vitro comet assay studies with sperm and a comparison of this assay with other assays measuring germ cell genotoxicity. As most of the de novo structural aberrations occur in sperm and spermatogenesis is functional from puberty to old age, whereas female germ cells are more complicated to obtain, the examination of male germ cells seems to be an easier and logical choice for research and testing in reproductive toxicology. In addition, the importance of such an assay for the paternal impact of genetic damage in offspring is undisputed. As there is a growing interest in the evaluation of genotoxins in male germ cells, the comet assay allows in vitro and in vivo assessments of various environmental and lifestyle genotoxins to be reliably determined.     

Detection of DNA damage and identification of UV-induced photoproducts using the CometAssay kit.

We introduce the first commercially available comet assay for the detection and quantification of DNA damage in individual eukaryotic cells. The major difficulty of the comet assay is the preparation of the slides needed to immobilize the samples throughout the lysis and electrophoretic procedures. The CometAssay kit uses a proprietary technology to precoat glass microscope slides to allow direct application of the agarose embedded sample without any additional slide treatment. In this report, we discuss the detection of DNA damage in individual cells exposed to ultraviolet irradiation using the new CometSlides and their cost compared to traditional slides.     

Defects in telomere maintenance molecules impair osteoblast differentiation and promote osteoporosis

Osteoporosis and the associated risk of fracture are major clinical challenges in the elderly. Telomeres shorten with age in most human tissues, including bone, and because telomere shortening is a cause of cellular replicative senescence or apoptosis in cultured cells, including mesenchymal stem cells (MSCs) and osteoblasts, it is hypothesized that telomere shortening contributes to the aging of bone. Osteoporosis is common in the Werner (Wrn) and dyskeratosis congenita premature aging syndromes, which are characterized by telomere dysfunction. One of the targets of the Wrn helicase is telomeric DNA, but the long telomeres and abundant telomerase in mice minimize the need for Wrn at telomeres, and thus Wrn knockout mice are relatively healthy. In a model of accelerated aging that combines the Wrn mutation with the shortened telomeres of telomerase (Terc) knockout mice, synthetic defects in proliferative tissues result. Here, we demonstrate that deficiencies in Wrn^−/– Terc^−/– mutant mice cause a low bone mass phenotype, and that age-related osteoporosis is the result of impaired osteoblast differentiation in the context of intact osteoclast differentiation. Further, MSCs from single and Wrn^−/– Terc^−/– double mutant mice have a reduced in vitro lifespan and display impaired osteogenic potential concomitant with characteristics of premature senescence. These data provide evidence that replicative aging of osteoblast precursors is an important mechanism of senile osteoporosis.     

Sublethal heat shock induces premature senescence rather than apoptosis in human mesenchymal stem cells

Stem cells in adult organism are responsible for cell turnover and tissue regeneration. The study of stem cell stress response contributes to our knowledge on the mechanisms of damaged tissue repair. Previously, we demonstrated that sublethal heat shock (HS) induced apoptosis in human embryonic stem cells. This study aimed to investigate HS response of human adult stem cells. Human mesenchymal stem cells (MSCs) cultivated in vitro were challenged with sublethal HS. It was found that sublethal HS did not affect the cell viability assessed by annexin V/propidium staining. However, MSCs subjected to severe HS exhibited features of stress-induced premature senescence (SIPS): irreversible cell cycle arrest, altered morphology, increased expression of senescence-associated β-galactosidase (SA-β-gal) activity, and induction of cyclin-dependent kinase inhibitor p21 protein. High level of Hsp70 accumulation induced by sublethal HS did not return to the basal level, at least, after 72 h of the cell recovery when most cells exhibited SIPS hallmarks. MSCs survived sublethal HS, and resumed proliferation sustained the properties of parental MSCs: diploid karyotype, replicative senescence, expression of the cell surface markers, and capacity for multilineage differentiation. Our results showed for the first time that in human MSCs, sublethal HS induced premature senescence rather than apoptosis or necrosis. MSC progeny that survived sublethal HS manifested stem cell properties of the parental cells: limited replicative life span and multilineage capacity.     

Melatonin suppresses senescence‐derived mitochondrial dysfunction in mesenchymal stem cells via the HSPA1L–mitophagy pathway

Mesenchymal stem cells (MSCs) are a popular cell source for stem cell‐based therapy. However, continuous ex vivo expansion to acquire large amounts of MSCs for clinical study induces replicative senescence, causing decreased therapeutic efficacy in MSCs. To address this issue, we investigated the effect of melatonin on replicative senescence in MSCs. In senescent MSCs (late passage), replicative senescence decreased mitophagy by inhibiting mitofission, resulting in the augmentation of mitochondrial dysfunction. Treatment with melatonin rescued replicative senescence by enhancing mitophagy and mitochondrial function through upregulation of heat shock 70 kDa protein 1L (HSPA1L). More specifically, we found that melatonin‐induced HSPA1L binds to cellular prion protein (PrP^C), resulting in the recruitment of PrP^C into the mitochondria. The HSPA1L‐PrP^C complex then binds to COX4IA, which is a mitochondrial complex IV protein, leading to an increase in mitochondrial membrane potential and anti‐oxidant enzyme activity. These protective effects were blocked by knockdown of HSPA1L. In a murine hindlimb ischemia model, melatonin‐treated senescent MSCs enhanced functional recovery by increasing blood flow perfusion, limb salvage, and neovascularization. This study, for the first time, suggests that melatonin protects MSCs against replicative senescence during ex vivo expansion for clinical application via mitochondrial quality control.     

In vivo DNA damage in gastric epithelial cells

A number of risk factors have been linked epidemiologically with gastric cancer, but studies of DNA damage in gastric epithelial cells are limited. The comet assay is a simple technique for determining levels of DNA damage in individual cells. In this study, we have validated the comet assay for use in epithelial cells derived directly from human gastric biopsies, determined optimal conditions for biopsy digestion and investigated the effects of oxidative stress and digestion time on DNA damage. Biopsies taken at endoscopy were digested using combinations of pronase and collagenase, ethylenediaminetetra-acetic acid (EDTA) and vigorous shaking. The resultant cell suspension was assessed for cell concentration and epithelial cell and leukocyte content. A score for DNA damage, the comet %, was derived from the cell suspension, and the effect of various digestion conditions was studied. Cells were incubated with H(2)O(2) and DNA damage was assessed. Pronase and collagenase provided optimum digestion conditions, releasing 1. 12x10(5) cells per biopsy, predominantly epithelial. Of the 23 suspensions examined, all but three had leukocyte concentrations of less than 20%. The comet assay had high inter-observer (6.1%) and inter-assay (4.5%) reproducibility. Overnight storage of the biopsy at 4 degrees C had no significant effect on DNA migration. Comet % increased from a median of 46% in untreated cells to 88% in cells incubated for 45 min in H(2)O(2) (p=0.005). Serial 25-min digestions were performed on biopsies from 13 patients to release cells from successively deeper levels in the crypt. Levels of DNA migration were significantly lower with each digestion (r=-0.94, p<0.001), suggesting that DNA damage is lower in younger cells released from low in the gastric crypt. The comet assay is a reproducible measure of DNA damage in gastric epithelial cells. Damage accumulates in older, more superficial cells, and can be induced by oxidative stress.     

Cytogenetic analysis of human cells reveals specific patterns of DNA damage in replicative and oncogene‐induced senescence

Senescence is thought to be triggered by DNA damage, usually indirectly assessed as activation of the DNA damage response (DDR), but direct surveys of genetic damage are lacking. Here, we mitotically reactivate senescent human fibroblasts to evaluate their cytogenetic damage. We show that replicative senescence is generally characterized by telomeric fusions. However, both telomeric and extratelomeric aberrations are prevented by hTERT, indicating that even non‐telomeric damage descends from the lack of telomerase. Compared with replicative senescent cells, oncogene‐induced senescent fibroblasts display significantly higher levels of DNA damage, depicting how oncogene activation can catalyze the generation of further, potentially tumorigenic, genetic damage.     

Comparative in vitro and in vivo assessment of genotoxic effects of etoposide and chlorothalonil by the comet assay.

The alkaline single cell gel electrophoresis (comet) assay was used to assess in vitro and in vivo genotoxicity of etoposide, a topoisomerase II inhibitor known to induce DNA strand breaks, and chlorothalonil, a fungicide widely used in agriculture. For in vivo studies, rats were sacrificed at various times after treatment and the induction of DNA strand breaks was assessed in whole blood, bone marrow, thymus, liver, kidney cortex and in the distal part of the intestine. One hour after injection, etoposide induced DNA damage in all organs studied except kidney, especially in bone marrow, thymus (presence of HDC) and whole blood. As observed during in vitro comet assay on Chinese hamster ovary (CHO) cells, dose- and time-dependent DNA effects occurred in vivo with a complete disappearance of damage 24 h after administration. Even though apoptotic cells were detected in vitro 48 h after cell exposure to etoposide, such a result was not found in vivo. After chlorothalonil treatment, no DNA strand breaks were observed in rat organs whereas a clear dose-related DNA damage was observed in vitro. The discrepancy between in vivo and in vitro models could be explained by metabolic and mechanistic reasons. Our results show that the in vivo comet assay is able to detect the target organs of etoposide and suggest that chlorothalonil is devoid of appreciable in vivo genotoxic activity under the protocol used.     

Death mechanism of chronologically aged yeast

Budding yeast Saccharomyces cerevisiae has two distinct lifespans. The replicative lifespan is defined as the number of progeny cells that a mother cell can have prior to senescence while the chronological lifespan is a measure of the time nondividing cells remain viable. Mechanisms for cells to choose the type of lifespans appropriate to environmental conditions to minimize DNA damage should be critical for maintenance of viability, an interesting question worthy of further investigation. To this end, we hypothesize that chronologically aged cells are defective in the lifespan choosing mechanism so that DNA replication, characteristic of the replicative lifespan, initiates near end of the chronological lifespan. Replication may frequently stall due to the limited resources and oxidative stress, leading to replication fork stall and fatal DNA damage. We will use the 2D DNA gel electrophoresis to examine replication initiation and stall at rDNA in the chronologically aged cells.     

IL-6 Secreted from Senescent Mesenchymal Stem Cells Promotes Proliferation and Migration of Breast Cancer Cells

Human mesenchymal stem cells (hMSCs) are currently investigated for a variety of therapeutic applications. However, MSCs isolated from primary tissue cannot meet clinical grade needs and should be expanded in vitro for several passages. Although hMSCs show low possibility for undergoing oncogenic transformation, they do, similar to other somatic cells, undergo cellular senescence and their therapeutic potential is diminished when cultured in vitro. However, the role of senescent MSCs in tumor progression remains largely elusive. In the current study, by establishing senescent human umbilical cord mesenchymal stem cells (s-UCMSCs) through the replicative senescence model and genotoxic stress induced premature senescence model, we show that s-UCMSCs significantly stimulate proliferation and migration of breast cancer cells in vitro and tumor progression in a co-transplant xenograft mouse model compared with ‘young’ counterparts (defined as MSCs at passage 5, in contrast to senescent MSCs at passage 45). In addition, we identified IL-6, a known pleiotropic cytokine, as a principal mediator for the tumor-promoting activity of s-UCMSCs by induction of STAT3 phosphorylation. Depletion of IL-6 from s-UCMSCs conditioned medium partially abrogated the stimulatory effect of s-UCMSCs on the proliferation and migration of breast tumor cells.     

Time-dependent effects of sodium arsenite on DNA breakage and apoptosis observed in the comet assay

To assess genotoxic effects of sodium arsenite (NaAsO2) the single-cell gel electrophoresis (comet assay) had been conducted in various studies indicating genotoxicity. However, DNA fragmentation due to NaAsO2-induced apoptosis may constitute a bias in the interpretation of the results. Apoptotic cells can show typically large and diffuse comets, which are usually excluded during genotoxicity analysis. It is controversial whether there is a time-window in which the apoptotic process generates comets that would falsely be interpreted to be the result of genotoxic DNA damage. Therefore, we evaluated frequency histograms for single-cell measures of tail DNA (% DNA in comet tail) in 30-min intervals after incubation of mouse lymphoma L5178Y cells with sodium arsenite (NaAsO2). In parallel, we evaluated apoptosis by measuring annexin V-positive cells with flow cytometry, and visualized apoptotic cells on slides by Hoechst bisbenzimide 33258 staining. The first observed effect at 30 min after treatment was an increase in annexin V-positive cells. At about 60 min the number of cells with moderate DNA migration increased in the comet-assay analysis. After 90 min, an increase in the number of cells with high levels of DNA migration was observed, which resulted in a bimodal distribution of cells with moderate and high levels of DNA migration. Hoechst-stained apoptotic cells could only be observed at later times (> or = 120 min). This means that the treatment would have been considered to be genotoxic if analysed at 120 min even if the cells with high levels of DNA migration would have been excluded. The occurrence of annexin V-positive cells preceded the appearance of cells with moderate levels of DNA migration. We hypothesize that these cells were early apoptotic cells and not indicative of genotoxic damage. We conclude that DNA-damaging effects of NaAsO2 cannot adequately be interpreted if the comet assay is not accompanied by separate analysis of early endpoints for induction of apoptosis.     

Telomeres as biomarkers for ageing and age-related diseases

Telomeres in telomerase-negative cells shorten during DNA replication in vitro due to numerous causes including the inability of DNA polymerases to fully copy the lagging strand, DNA end processing and random damage, often caused by oxidative stress. Short telomeres activate replicative senescence, an irreversible cell cycle arrest. Thus, telomere length is an indicator of replicative history, of the probability of cell senescence, and of the cumulative history of oxidative stress. Telomeres in most human cells shorten during ageing in vivo as well, suggesting that telomere length could be a biomarker of ageing and age-related morbidity. There are two distinct possibilities: First, in a tissue-specific fashion, short telomeres might indicate senescence of (stem) cells, and this might contribute to age-related functional attenuation in this tissue. Second, short telomeres in one tissue might cause systemic effects or might simply indicate a history of high stress and damage in the individual and could thus act as risk markers for age-related disease residing in a completely different tissue. In recent years, data have been published to support both approaches, and we will review these. While they together paint a fairly promising picture, it needs to be pointed out that until now most of the evidence is correlative, that much of it comes from underpowered studies, and that causal evidence for essential pathways, for instance for the impact of cell senescence on tissue ageing in vivo, is still very weak.     

The effect of inhibited replication on DNA migration in the comet assay in relation to cytotoxicity and clastogenicity

The DNA-replication inhibitors aphidicolin (APC) and hydroxyurea (HU) were tested for their ability to induce effects on DNA in the in vitro alkaline comet assay with V79 cells. APC concentrations up to 15 microM and HU concentrations up to 500 microM did not significantly increase the extent of DNA migration after treatment during 4h. Treatment for 18 h, however, led to inconsistently significant increase in DNA migration. These increases in DNA migration were accompanied by severe cell-cycle disturbances, cytotoxic effects (reduced population doubling and reduced mitotic index) and increased frequencies of cells with chromosome aberrations. The results indicate that substances with such secondary effects on DNA (in contrast to agents that directly damage DNA) only induce effects in the comet assay after prolonged exposure, together with cytotoxic effects. We conclude that slight inhibition of DNA replication and cell-cycle delay per se do not cause significant effects in the in vitro comet assay under standard test conditions. Furthermore, the in vitro comet assay seems to be less sensitive towards this type of secondary DNA effects than the in vitro chromosome aberration test.     

A comparison of DNA damage induced by aflatoxin B1 in hepatocyte-like cells, their progenitor mesenchymal stem cells and CD34(+) cells isolated from umbilical cord blood

This study compared the sensitivity of differentiated hepatocyte-like cells, their progenitor mesenchymal stem cells (MSCs) and CD34(+) stem cells to DNA damage and toxicity induced by aflatoxin B1 (AFB1). The hepatocyte-like cells and their progenitor cells (isolated from umbilical cord blood (UCB)) were each treated with AFB1 on day 15 of differentiation. Cell toxicity and genotoxicity effects were assessed using MTT and alkaline comet assays. AFB1 treatment resulted in a dose- and time-dependent inhibition of cell growth. The IC(50) values of AFB1 for hepatocytes differentiated from CD34(+) and MSCs were within the same range (44.7-46.8μM). The IC(50) calculated for non-differentiated MSCs and CD34(+) cells was slightly lower (42.0-43.4μM) than that calculated for their differentiated counterparts. However, the extent of DNA damage was different in differentiated and non-differentiated cells. The percentages of DNA (% DNA) in comet tails measured in hepatocytes differentiated from MSCs exposed to AFB1 (0, 2.5, 10 and 20μM) for 24h were ～15, 55, 65 and 70%, respectively. In comparison, hepatocytes from CD34(+) cells were more resistant to AFB1-induced DNA damage. Hepatocyte-MSCs were most sensitive to DNA damage, followed by UCB-CD34(+) cells, then UCB-MSCs and finally hepatocyte-CD34(+) cells. These results clearly showed that stem cells from different sources have different sensitivities to DNA damaging agents. These differences can be assigned to the expression levels of cytochrome P450 (CYP) particularly CYP3A4 in non-differentiated and differentiated cells. These data are useful in better understanding the susceptibility/resistance of stem cells in the process of differentiation to environmental toxicants.     

Comet assay using mullet (Mugil sp.) and sea catfish (Netuma sp.) erythrocytes for the detection of genotoxic pollutants in aquatic environment

The development of comet assay for aquatic organisms is of particular relevance in light of the importance of coastal fisheries to several countries around the world. Two of the most common fish species native to southern Brazil are the gray mullet (Mugil sp.) and sea catfish (Netuma sp.) for which we have produced a standardized comet assay using whole erythrocytes taken from samples of these fish. We investigated the potential of the comet assay for monitoring genotoxicity in mullet and sea catfish and made a preliminary investigation of the baseline levels of DNA damage in the erythrocytes of samples of these fish from non-polluted areas as well as assessing the in vitro sensitivity of erythrocyte exposed to 2, 4 and 8 x 10(-5) M of methyl methanesulfonate (MMS) for 1, 2, 6 and 24h at 25 and 37 degrees C. Our results show that there was an increase in baseline DNA damage at higher temperatures and that the amount of MMS-induced DNA damage also increased at higher temperatures and that there was a clear dose/time response to treatment with MMS. To assess the possibility of using fish for environmental biomonitoring we also used the comet assay to investigate the in vitro genotoxic effect of MMS on whole blood cells from human donors and found a clear concentration-related effect at all exposure times, findings which agree with those of other workers. This study demonstrates the potential application of the comet assay to erythrocytes of mullets and sea catfish. However, these findings also suggest that temperature could alter both baseline DNA damage in untreated animals and in vitro cell sensitivity towards genotoxic pollutants.