Senescence Phenomena and Metabolic Alteration in Mesenchymal Stromal Cells from a Mouse Model of Rett Syndrome.

Chromatin modifiers play a crucial role in maintaining cell identity through modulation of gene expression patterns. Their deregulation can have profound effects on cell fate and functions. Among epigenetic regulators, the MECP2 protein is particularly attractive. Mutations in the Mecp2 gene are responsible for more than 90% of cases of Rett syndrome (RTT), a progressive neurodevelopmental disorder. As a chromatin modulator, MECP2 can have a key role in the government of stem cell biology. Previously, we showed that deregulated MECP2 expression triggers senescence in mesenchymal stromal cells (MSCs) from (RTT) patients. Over the last few decades, it has emerged that senescent cells show alterations in the metabolic state. Metabolic changes related to stem cell senescence are particularly detrimental, since they contribute to the exhaustion of stem cell compartments, which in turn determine the falling in tissue renewal and functionality. Herein, we dissect the role of impaired MECP2 function in triggering senescence along with other senescence-related aspects, such as metabolism, in MSCs from a mouse model of RTT. We found that MECP2 deficiencies lead to senescence and impaired mitochondrial energy production. Our results support the idea that an alteration in mitochondria metabolic functions could play an important role in the pathogenesis of RTT.

Neural stem cells from a mouse model of Rett syndrome are prone to senescence, show reduced capacity to cope with genotoxic stress, and are impaired in the differentiation process.

Several aspects of stem cell life are governed by epigenetic variations, such as DNA methylation, histone modifications, and chromatin remodeling. Epigenetic events are also connected with the impairment of stem cell functions. For example, during senescence, there are significant changes in chromatin organization that alter transcription. The MECP2 protein can bind methylated cytosines and contribute to regulating gene expression at one of the highest hierarchical levels. Researchers are particularly interested in this protein, as up to 90% of Rett syndrome patients have an MECP2 gene mutation. Nevertheless, the role of MECP2 in this disease remains poorly understood. We used a mouse model of Rett syndrome to evaluate whether residual MECP2 activity in neural stem cells (NSCs) induced the senescence phenomena that could affect stem cell function. Our study clearly demonstrated that the reduced expression of MECP2 is connected with an increase in senescence, an impairment in proliferation capacity, and an accumulation of unrepaired DNA foci. Mecp2 +/- NSCs did not cope with genotoxic stress in the same way as the control cells did. Indeed, after treatment with different DNA-damaging agents, the NSCs from mice with mutated Mecp2 accumulated more DNA damage foci (γ-H2AX+) and were more prone to cell death than the controls. Senescence in Mecp2 +/- NSCs decreased the number of stem cells and progenitors and gave rise to a high percentage of cells that expressed neither stem/progenitor nor differentiation markers. These cells could be senescent and dysfunctional.

Misidentified Human Gene Functions with Mouse Models: The Case of the Retinoblastoma Gene Family in Senescence.

Although mice models rank among the most widely used tools for understanding human genetics, biology, and diseases, differences between orthologous genes among species as close as mammals are possible, particularly in orthologous gene pairs in which one or more paralogous (i.e., duplicated) genes appear in the genomes of the species. Duplicated genes can possess overlapping functions and compensate for each other. The retinoblastoma gene family demonstrates typical composite functionality in its three member genes (i.e., RB1, RB2/P130, and P107), all of which participate in controlling the cell cycle and associated phenomena, including proliferation, quiescence, apoptosis, senescence, and cell differentiation. We analyzed the role of the retinoblastoma gene family in regulating senescence in mice and humans. Silencing experiments with each member of the gene family in mesenchymal stromal cells (MSCs) and fibroblasts from mouse and human tissues demonstrated that RB1 may be indispensable for senescence in mouse cells, but not in human ones, as an example of species specificity. Furthermore, although RB2/P130 seems to be implicated in maintaining human cell senescence, the function of RB1 within any given species might differ by cell type, as an example of cell specificity. For instance, silencing RB1 in mouse fibroblasts induced a reduced senescence not observed in mouse MSCs. Our findings could be useful as a general paradigm of cautions to take when inferring the role of human genes analyzed in animal studies and when examining the role of the retinoblastoma gene family in detail.

Mesenchymal stromal cells having inactivated RB1 survive following low irradiation and accumulate damaged DNA: Hints for side effects following radiotherapy.

Following radiotherapy, bone sarcomas account for a significant percentage of recurring tumors. This risk is further increased in patients with hereditary retinoblastoma that undergo radiotherapy. We analyzed the effect of low and medium dose radiation on mesenchymal stromal cells (MSCs) with inactivated RB1 gene to gain insights on the molecular mechanisms that can induce second malignant neoplasm in cancer survivors. MSC cultures contain subpopulations of mesenchymal stem cells and committed progenitors that can differentiate into mesodermal derivatives: adipocytes, chondrocytes, and osteocytes. These stem cells and committed osteoblast precursors are the cell of origin in osteosarcoma, and RB1 gene mutations have a strong role in its pathogenesis. Following 40 and 2000 mGy X-ray exposure, MSCs with inactivated RB1 do not proliferate and accumulate high levels of unrepaired DNA as detected by persistence of gamma-H2AX foci. In samples with inactivated RB1 the radiation treatment did not increase apoptosis, necrosis or senescence versus untreated cells. Following radiation, CFU analysis showed a discrete number of cells with clonogenic capacity in cultures with silenced RB1. We extended our analysis to the other members of retinoblastoma gene family: RB2/P130 and P107. Also in the MSCs with silenced RB2/P130 and P107 we detected the presence of cells with unrepaired DNA following X-ray irradiation. Cells with unrepaired DNA may represent a reservoir of cells that may undergo neoplastic transformation. Our study suggests that, following radiotherapy, cancer patients with mutations of retinoblastoma genes may be under strict controls to evaluate onset of secondary neoplasms following radiotherapy.

Changes in autophagy, proteasome activity and metabolism to determine a specific signature for acute and chronic senescent mesenchymal stromal cells.

A sharp definition of what a senescent cell is still lacking since we do not have in depth understanding of mechanisms that induce cellular senescence. In addition, senescent cells are heterogeneous, in that not all of them express the same genes and present the same phenotype. To further clarify the classification of senescent cells, hints may be derived by the study of cellular metabolism, autophagy and proteasome activity. In this scenario, we decided to study these biological features in senescence of Mesenchymal Stromal Cells (MSC). These cells contain a subpopulation of stem cells that are able to differentiate in mesodermal derivatives (adipocytes, chondrocytes, osteocytes). In addition, they can also contribute to the homeostatic maintenance of many organs, hence, their senescence could be very deleterious for human body functions. We induced MSC senescence by oxidative stress, doxorubicin treatment, X-ray irradiation and replicative exhaustion. The first three are considered inducers of acute senescence while extensive proliferation triggers replicative senescence also named as chronic senescence. In all conditions, but replicative and high IR dose senescence, we detected a reduction of the autophagic flux, while proteasome activity was impaired in peroxide-treated and irradiated cells. Differences were observed also in metabolic status. In general, all senescent cells evidenced metabolic inflexibility and prefer to use glucose as energy fuel. Irradiated cells with low dose of X-ray and replicative senescent cells show a residual capacity to use fatty acids and glutamine as alternative fuels, respectively. Our study may be useful to discriminate among different senescent phenotypes.

Low dose radiation induced senescence of human mesenchymal stromal cells and impaired the autophagy process.

Low doses of radiation may have profound effects on cellular function. Individuals may be exposed to low doses of radiation either intentionally for medical purposes or accidentally, such as those exposed to radiological terrorism or those who live near illegal radioactive waste dumpsites.We studied the effects of low dose radiation on human bone marrow mesenchymal stromal cells (MSC), which contain a subpopulation of stem cells able to differentiate in bone, cartilage, and fat; support hematopoiesis; and contribute to body's homeostasis.The main outcome of low radiation exposure, besides reduction of cell cycling, is the triggering of senescence, while the contribution to apoptosis is minimal. We also showed that low radiation affected the autophagic flux. We hypothesize that the autophagy prevented radiation deteriorative processes, and its decline contributed to senescence.An increase in ATM staining one and six hours post-irradiation and return to basal level at 48 hours, along with persistent gamma-H2AX staining, indicated that MSC properly activated the DNA repair signaling, though some damages remained unrepaired, mainly in non-cycling cells. This suggested that the impaired DNA repair capacity of irradiated MSC seemed mainly related to the reduced activity of a non-homologous end-joining (NHEJ) system rather than HR (homologous recombination).

Sera of overweight people promote in vitro adipocyte differentiation of bone marrow stromal cells.

INTRODUCTION: Overweight status should not be considered merely an aesthetic concern; rather, it can incur health risks since it may trigger a cascade of events that produce further fat tissue through altered levels of circulating signaling molecules. METHODS: We decided to investigate the influence of overweight individuals' sera on in vitro MSC proliferation and differentiation. RESULTS: We observed that in vitro incubation of bone marrow stromal cells with the sera of overweight individuals promotes the adipogenic differentiation of MSCs while partially impairing proper osteogenesis. CONCLUSIONS: These results, which represent a pilot study, might suggest that becoming overweight triggers further weight gains by promoting a bias in the differentiation potential of MSCs toward adipogenesis. The circulating factors involved in this phenomenon remain to be determined, since the great majority of the well known pro-inflammatory cytokines and adipocyte-secreted factors we investigated did not show relevant modifications in overweight serum samples compared with controls.

Silencing of RB1 and RB2/P130 during adipogenesis of bone marrow stromal cells results in dysregulated differentiation.

Bone marrow adipose tissue (BMAT) is different from fat found elsewhere in the body, and only recently have some of its functions been investigated. BMAT may regulate bone marrow stem cell niche and plays a role in energy storage and thermogenesis. BMAT may be involved also in obesity and osteoporosis onset. Given the paramount functions of BMAT, we decided to better clarify the human bone marrow adipogenesis by analyzing the role of the retinoblastoma gene family, which are key players in cell cycle regulation. Our data provide evidence that the inactivation of RB1 or RB2/P130 in uncommitted bone marrow stromal cells (BMSC) facilitates the first steps of adipogenesis. In cultures with silenced RB1 or RB2/P130, we observed an increase of clones with adipogenic potential and a higher percentage of cells accumulating lipid droplets. Nevertheless, the absence of RB1 or RB2/P130 impaired the terminal adipocyte differentiation and gave rise to dysregulated adipose cells, with alteration in lipid uptake and release. For the first time, we evidenced that RB2/P130 plays a role in bone marrow adipogenesis. Our data suggest that while the inactivation of retinoblastoma proteins may delay the onset of last cell division and allow more BMSC to be committed to adipocyte, it did not allow a permanent cell cycle exit, which is a prerequisite for adipocyte terminal maturation.

Low concentrations of isothiocyanates protect mesenchymal stem cells from oxidative injuries, while high concentrations exacerbate DNA damage.

Isothiocyanates (ITCs) are molecules naturally present in many cruciferous vegetables (broccoli, black radish, daikon radish, and cauliflowers). Several studies suggest that cruciferous vegetable consumption may reduce cancer risk and slow the aging process. To investigate the effect of ITCs on cellular DNA damage, we evaluated the effects of two different ITCs [sulforaphane (SFN) and raphasatin (RPS)] on the biology of human mesenchymal stem cells (MSCs), which, in addition to their ability to differentiate into mesenchymal tissues, contribute to the homeostatic maintenance of many organs. The choice of SFN and RPS relies on two considerations: they are among the most popular cruciferous vegetables in the diet of western and eastern countries, respectively, and their bioactive properties may differ since they possess specific molecular moiety. Our investigation evidenced that MSCs incubated with low doses of SFN and RPS show reduced in vitro oxidative stress. Moreover, these cells are protected from oxidative damages induced by hydrogen peroxide, while no protection was evident following treatment with the UV ray of a double strand DNA damaging drug, such as doxorubicin. High concentrations of both ITCs induced cytotoxic effects in MSC cultures and further increased DNA damage induced by peroxides. In summary, our study suggests that ITCs, at low doses, may contribute to slowing the aging process related to oxidative DNA damage. Moreover, in cancer treatment, low doses of ITCs may be used as an adjuvant to reduce chemotherapy-induced oxidative stress, while high doses may synergize with anticancer drugs to promote cell DNA damage.

Dose-dependent effects of R-sulforaphane isothiocyanate on the biology of human mesenchymal stem cells, at dietary amounts, it promotes cell proliferation and reduces senescence and apoptosis, while at anti-cancer drug doses, it has a cytotoxic effect.

Brassica vegetables are attracting a great deal of attention as healthy foods because of the fact that they contain substantial amounts of secondary metabolite glucosinolates that are converted into isothiocyanates, such as sulforaphane [(-)1-isothiocyanato-4R-(methylsulfinyl)-butane] (R-SFN), through the actions of chopping or chewing the vegetables. Several studies have analyzed the biological and molecular mechanisms of the anti-cancer activity of synthetic R,S-sulforaphane, which is thought to be a result of its antioxidant properties and its ability to inhibit histone deacetylase enzymes (HDAC). Few studies have addressed the possible antioxidant effects of R-SFN, which could protect cells from the free radical damage that strongly contribute to aging. Moreover, little is known about the effect of R-SFN on stem cells whose longevity is implicated in human aging. We evaluated the effects of R-SFN on the biology on human mesenchymal stem cells (MSCs), which, in addition to their ability to differentiate into mesenchymal tissues, support hematopoiesis, and contribute to the homeostatic maintenance of many organs and tissues. Our investigation found evidence that low doses of R-SFN promote MSCs proliferation and protect them from apoptosis and senescence, while higher doses have a cytotoxic effect, leading to the induction of cell cycle arrest, programmed cell death and senescence. The beneficial effects of R-SFN may be ascribed to its antioxidant properties, which were observed when MSC cultures were incubated with low doses of R-SFN. Its cytotoxic effects, which were observed after treating MSCs with high doses of R-SFN, could be attributed to its HDAC inhibitory activity. In summary, we found that R-SFN, like many other dietary supplements, exhibits a hormetic behavior; it is able to induce biologically opposite effects at different doses.