Progression of irradiated mesenchymal stromal cells from early to late senescence: Changes in SASP composition and anti-tumour properties.

Genotoxic injuries converge on senescence-executive program that promotes production of a senescence-specific secretome (SASP). The study of SASP is particularly intriguing, since through it a senescence process, triggered in a few cells, can spread to many other cells and produce either beneficial or negative consequences for health. We analysed the SASP of quiescent mesenchymal stromal cells (MSCs) following stress induced premature senescence (SIPS) by ionizing radiation exposure. We performed a proteome analysis of SASP content obtained from early and late senescent cells. The bioinformatics studies evidenced that early and late SASPs, besides some common ontologies and signalling pathways, contain specific factors. In spite of these differences, we evidenced that SASPs can block in vitro proliferation of cancer cells and promote senescence/apoptosis. It is possible to imagine that SASP always contains core components that have an anti-tumour activity, the progression from early to late senescence enriches the SASP of factors that may promote SASP tumorigenic activity only by interacting and instructing cells of the immune system. Our results on Caco-2 cancer cells incubated with late SASP in presence of peripheral white blood cells strongly support this hypothesis. We evidenced that quiescent MSCs following SIPS produced SASP that, while progressively changed its composition, preserved the capacity to block cancer growth by inducing senescence and/or apoptosis only in an autonomous manner.

Role of glycosphingolipid SSEA-3 and FGF2 in the stemness and lineage commitment of multilineage differentiating stress enduring (MUSE) cells.

OBJECTIVES: Multilineage differentiating Stress Enduring (MUSE) cells are endogenous, stress-resistant stem cells, expressing pluripotency master genes and able to differentiate in cells of the three embryonic sheets. Stage-Specific Embryonic Antigen 3 (SSEA-3), a glycosphingolipid (GSL), is the marker for identifying MUSE cells and is used to isolate this population from mesenchymal stromal cells. GSLs modulate signal transduction by interacting with plasma membrane components. The growth factor FGF2, important for MUSE cells biology, may interact with GSLs. Specific cell surface markers represent an invaluable tool for stem cell isolation. Nonetheless their role, if any, in stem cell biology is poorly investigated. Functions of stem cells, however, depend on niche external cues, which reach cells through surface markers. We addressed the role of SSEA-3 in MUSE cell behaviour, trying to define whether SSEA-3 is just a marker or if it plays a functional role in this cell population by determining if it has any relationship with FGF2 activity. RESULTS: We evidenced how the SSEA-3 and FGF2 cooperation affected the self-renewal and clonogenic capacity of MUSE cells. The block of SSEA-3 significantly reduced the multilineage potential of MUSE cells with production of nullipotent clones. CONCLUSIONS: We contributed to dissecting the mechanisms underlying MUSE cell properties for establishing successful stem-cell-based therapies and the promotion of MUSE cells as a tool for the in vitro disease model.

Cellular Senescence in Physiological and Pathological Processes.

This Special Issue aims to address the impact of cellular senescence on human biology, looking at both physiological and pathological processes [...].

Environmental microplastics (EMPs) exposure alter the differentiation potential of mesenchymal stromal cells.

Humans are exposed to environmental microplastic (MPs) that can be frequent in surrounding environment. The mesenchymal stromal cells are a heterogeneous population, which contain fibroblasts and stromal cells, progenitor cells and stem cells. They are part of the stromal component of most tissue and organs in our organisms. Any injury to their functions may impair tissue renewal and homeostasis. We evaluated the effects of different size MPs that could be present in water bottles on human bone marrow mesenchymal stromal cells (BMMSCs) and adipose mesenchymal stromal cells (AMSCs). MPs of polyethylene terephthalate (MPs-PET) (<1 µm and <2.6 µm) were tested in this study. PET treatments induced a reduction in proliferating cells (around 30%) associated either with the onset of senescence or increase in apoptosis. The AMSCs and BMMSCs exposed to PET showed an alteration of differentiation potential. AMSCs remained in an early stage of adipocyte differentiation as shown by high levels of mRNA for Peroxisome Proliferator Activated Receptor Gamma (PPARG) (7.51 vs 1.00) and reduction in Lipoprotein Lipase (LPL) mRNA levels (0.5 vs 1.0). A loss of differentiation capacity was also observed for the osteocyte phenotype in BMMSCs. In particular, we observed a reduction in Bone Gamma-Carboxy glutamate Protein (BGLAP) (0.4 for PET1 and 0.6 for PET2.6 vs 0.1 CTRL) and reduction in Osteopontin (SPP1) (0.3 for PET 1 and 0.64 for PET 2.6 vs 0.1 CTRL). This pioneering mesenchymal cell response study demonstrated that environmental microplastic could be bioavailable for cell uptake and may further lead to irreversible diseases.

Polyphenols, the Healthy Brand of Olive Oil: Insights and Perspectives.

Given their beneficial potential on human health, plant food bioactive molecules are important components influencing nutrition. Polyphenols have been widely acknowledged for their potentially protective role against several complex diseases. In particular, the polyphenols of olive oil (OOPs) emerge as the key components of many healthy diets and have been widely studied for their beneficial properties. The qualitative and quantitative profile defining the composition of olive oil phenolic molecules as well as their absorbance and metabolism once ingested are key aspects that need to be considered to fully understand the health potential of these molecules. In this review, we provide an overview of the key aspects influencing these variations by focusing on the factors influencing the biosynthesis of OOPs and the findings about their absorption and metabolism. Despite the encouraging evidence, the health potential of OOPs is still debated due to limitations in current studies. Clinical trials are necessary to fully understand and validate the beneficial effects of olive oil and OOPs on human health. We provide an update of the clinical trials based on olive oil and/or OOPs that aim to understand their beneficial effects. Tailored studies are needed to standardize the polyphenolic distribution and understand the variables associated with phenol-enriched OO. An in-depth knowledge of the steps that occur following polyphenol ingestion may reveal useful insights to be used in clinical settings for the prevention and treatment of many diseases.

Biomolecular Evaluation of Piceatannol's Effects in Counteracting the Senescence of Mesenchymal Stromal Cells: A New Candidate for Senotherapeutics?

Several investigations on senescence and its causative role in aging have underscored the importance of developing senotherapeutics, a field focused on killing senescent cells and/or preventing their accumulation within tissues. Using polyphenols in counteracting senescence may facilitate the development of senotherapeutics given their presence in the human diet, their confirmed tolerability and absence of severe side effects, and their role in preventing senescence and inducing the death of senescent cells. Against that background, we evaluated the effect of piceatannol, a natural polyphenol, on the senescence of mesenchymal stromal cells (MSCs), which play a key role in the body's homeostasis. Among our results, piceatannol reduced the number of senescent cells both after genotoxic stress that induced acute senescence and in senescent replicative cultures. Such senotherapeutics activity, moreover, promoted the recovery of cell proliferation and the stemness properties of MSCs. Altogether, our findings demonstrate piceatannol's effectiveness in counteracting senescence by targeting its associated pathways and detecting and affecting P53-dependent and P53-independent senescence. Our study thus suggests that, given piceatannol's various mechanisms to accomplish its pleiotropic activities, it may be able to counteract any senescent phenotypes.

MUSE Stem Cells Can Be Isolated from Stromal Compartment of Mouse Bone Marrow, Adipose Tissue, and Ear Connective Tissue: A Comparative Study of Their In Vitro Properties.

The cells present in the stromal compartment of many tissues are a heterogeneous population containing stem cells, progenitor cells, fibroblasts, and other stromal cells. A SSEA3(+) cell subpopulation isolated from human stromal compartments showed stem cell properties. These cells, known as multilineage-differentiating stress-enduring (MUSE) cells, are capable of resisting stress and possess an excellent ability to repair DNA damage. We isolated MUSE cells from different mouse stromal compartments, such as those present in bone marrow, subcutaneous white adipose tissue, and ear connective tissue. These cells showed overlapping in vitro biological properties. The mouse MUSE cells were positive for stemness markers such as SOX2, OCT3/4, and NANOG. They also expressed TERT, the catalytic telomerase subunit. The mouse MUSE cells showed spontaneous commitment to differentiation in meso/ecto/endodermal derivatives. The demonstration that multilineage stem cells can be isolated from an animal model, such as the mouse, could offer a valid alternative to the use of other stem cells for disease studies and envisage of cellular therapies.

Micro-RNAs: Crossroads between the Exposure to Environmental Particulate Pollution and the Obstructive Pulmonary Disease.

Environmental pollution has reached a global echo and represents a serious problem for human health. Air pollution encompasses a set of hazardous substances, such as particulate matter and heavy metals (e.g., cadmium, lead, and arsenic), and has a strong impact on the environment by affecting groundwater, soil, and air. An adaptive response to environmental cues is essential for human survival, which is associated with the induction of adaptive phenotypes. The epigenetic mechanisms regulating the expression patterns of several genes are promising candidates to provide mechanistic and prognostic insights into this. Micro-RNAs (miRNAs) fulfil these features given their ability to respond to environmental factors and their critical role in determining phenotypes. These molecules are present in extracellular fluids, and their expression patterns are organ-, tissue-, or cell-specific. Moreover, the experimental settings for their quantitative and qualitative analysis are robust, standardized, and inexpensive. In this review, we provide an update on the role of miRNAs as suitable tools for understanding the mechanisms behind the physiopathological response to toxicants and the prognostic value of their expression pattern associable with specific exposures. We look at the mechanistic evidence associable to the role of miRNAs in the processes leading to environmental-induced pulmonary disease (i.e., chronic obstructive pulmonary disease).

Obesity is associated with senescence of mesenchymal stromal cells derived from bone marrow, subcutaneous and visceral fat of young mice.

White adipose tissue (WAT) is distributed in several depots with distinct metabolic and inflammatory functions. In our body there are subcutaneous (sWAT), visceral (vWAT) and bone marrow (bWAT) fat depots. Obesity affects the size, function and inflammatory state of WATs. In particular, obesity may affect the activity of mesenchymal stromal cells (MSCs) present in WAT. MSCs are a heterogeneous population containing stromal cells, progenitor cells, fibroblasts and stem cells that are able to differentiate among adipocytes, chondrocytes, osteocytes and other mesodermal derivatives.In the first study of this kind, we performed a comparison of the effects of obesity on MSCs obtained from sWAT, vWAT and bWAT. Our study showed that obesity affects mainly the biological functions of MSCs obtained from bone marrow and vWAT by decreasing the proliferation rate, reducing the percentage of cells in S phase and triggering senescence. The onset of senescence was confirmed by expression of genes belonging to RB and P53 pathways.Our study revealed that the negative consequences of obesity on body physiology may also be related to impairment in the functions of the stromal compartment present in the several adipose tissues. This finding provides new insights as to the targets that should be considered for an effective treatment of obesity-related diseases.

Long non-coding RNAs in regulation of adipogenesis and adipose tissue function.

Complex interaction between genetics, epigenetics, environment, and nutrition affect the physiological activities of adipose tissues and their dysfunctions, which lead to several metabolic diseases including obesity or type 2 diabetes. Here, adipogenesis appears to be a process characterized by an intricate network that involves many transcription factors and long noncoding RNAs (lncRNAs) that regulate gene expression. LncRNAs are being investigated to determine their contribution to adipose tissue development and function. LncRNAs possess multiple cellular functions, and they regulate chromatin remodeling, along with transcriptional and post-transcriptional events; in this way, they affect gene expression. New investigations have demonstrated the pivotal role of these molecules in modulating white and brown/beige adipogenic tissue development and activity. This review aims to provide an update on the role of lncRNAs in adipogenesis and adipose tissue function to promote identification of new drug targets for treating obesity and related metabolic diseases.

Increase of circulating IGFBP-4 following genotoxic stress and its implication for senescence.

Senescent cells secrete several molecules, collectively named senescence-associated secretory phenotype (SASP). In the SASP of cells that became senescent following several in vitro chemical and physical stress, we identified the IGFBP-4 protein that can be considered a general stress mediator. This factor appeared to play a key role in senescence-paracrine signaling. We provided evidences showing that genotoxic injury, such as low dose irradiation, may promote an IGFBP-4 release in bloodstream both in mice irradiated with 100 mGy X-ray and in human subjects that received Computer Tomography. Increased level of circulating IGFBP-4 may be responsible of pro-aging effect. We found a significant increase of senescent cells in the lungs, heart, and kidneys of mice that were intraperitoneally injected with IGFBP-4 twice a week for two months. We then analyzed how genotoxic stressors may promote the release of IGFBP-4 and the molecular pathways associated with the induction of senescence by this protein.

The senescence-associated secretory phenotype (SASP) from mesenchymal stromal cells impairs growth of immortalized prostate cells but has no effect on metastatic prostatic cancer cells.

Senescent cells secrete inflammatory cytokines, proteases, and other factors, which are indicated as senescence-associated secretory phenotype (SASP). There are contrasting studies on the role of the SASP in cancer. Studies suggested that cancer cells may misuse the senescent secretome for their growth. Other investigations evidenced that the SASP may induce cancer growth arrest, senescence, or apoptosis. These conflicting data can be reconciled considering that cancer cells can coax senescent cells to secrete factors for their survival, thus abrogating the SASP's anti-cancer effect. Cancer stage may also have an impact on the capacity of the SASP to block tumor proliferation and promote senescence. Indeed, senescence is associated with a permanent cell cycle arrest, which needs functional cell cycle checkpoints. We evaluated the SASP effect on the in vitro biological properties of PNT2 and PC3 cells, which are immortalized prostate cells and metastatic prostatic cancer cells, respectively. We evidenced that SASPs, coming either from mesenchymal stromal cells treated with H202 or with low X-ray doses, induced senescence of immortalized cells but not of cancer cells. Hence, the SASP released by acute senescent cells should be considered as an effective weapon against pre-tumorigenesis events rather than an anti-cancer mechanism acting on malignant cells.

The Autophagy Signaling Pathway: A Potential Multifunctional Therapeutic Target of Curcumin in Neurological and Neuromuscular Diseases.

Autophagy is the major intracellular machinery for degrading proteins, lipids, polysaccharides, and organelles. This cellular process is essential for the maintenance of the correct cellular balance in both physiological and stress conditions. Because of its role in maintaining cellular homeostasis, dysregulation of autophagy leads to various disease manifestations, such as inflammation, metabolic alterations, aging, and neurodegeneration. A common feature of many neurologic and neuromuscular diseases is the alteration of the autophagy-lysosomal pathways. For this reason, autophagy is considered a target for the prevention and/or cure of these diseases. Dietary intake of polyphenols has been demonstrated to prevent/ameliorate several of these diseases. Thus, natural products that can modulate the autophagy machinery are considered a promising therapeutic strategy. In particular, curcumin, a phenolic compound widely used as a dietary supplement, exerts an important effect in modulating autophagy. Herein, we report on the current knowledge concerning the role of curcumin in modulating the autophagy machinery in various neurological and neuromuscular diseases as well as its role in restoring the autophagy molecular mechanism in several cell types that have different effects on the progression of neurological and neuromuscular disorders.

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.

A rapid, safe, and quantitative in vitro assay for measurement of uracil-DNA glycosylase activity.

Base excision repair (BER) is a frontline repair mechanism that operates through the G1 phase of the cell cycle, which ensures the genome integrity by repairing thousands of DNA lesions due to endogenous and exogenous agents. Its correct functioning is fundamental for cell viability and the health of the organism. Uracil is one of the most prevalent lesions that appears in DNA arising by spontaneous or enzymatic deamination of cytosine or misincorporation of the deoxyuridine 5'-triphosphate nucleotide (dUTP) in place of deoxythymidine 5'-triphosphate (dTTP) during DNA replication. In the first pathway, the uracil will preferentially pair with adenine, leading to C:G → T:A transition. When uracil in DNA arises from misincorporation of dUTP instead of dTTP, this process will result in A:U pairs. Organisms counteract the mutagenic effects of uracil in DNA using the BER repair system, which is mediated by a member of the uracil-DNA glycosylase (UDG) superfamily. Several assays evaluating the in vitro BER enzyme activity have been described so far. Some of these measure the BER activity by an oligonucleotide incision assay using radiolabeled duplex oligo. Others use circular double-stranded DNA substrates containing a defined lesion. The novelty of our method resides in its rapidity and safety (radioactive free detection) as well as in the possibility of having a reliable quantitative determination of UDG activity in both cell and tissue extracts. We also demonstrated the effectiveness of our method in assessing UDG activity in cell lines with a reduced DNA repair capacity and in different kinds of tissues. KEY MESSAGES: • Base excision repair is a fundamental repair mechanism ensuring the genome integrity. • Uracil is one of the most prevalent lesions that appears in DNA. • The mutagenic effects of uracil in DNA are mitigated by the uracil-DNA glycosylase. • Several assays evaluating the in vitro BER activity have been described so far. • A safe and quantitative assay evaluating the in vitro UDG activity is required.

Circulating factors present in the sera of naturally skinny people may influence cell commitment and adipocyte differentiation of mesenchymal stromal cells.

BACKGROUND: Research on physiopathology of obesity may receive new hints from studies on skinny people (SP). These are individuals who show a poor or null gaining of body weight, in spite of high-calorie intake, by far exceeding the body requirements. AIM: To evaluate how circulating factors present in the SP sera may affect adipogenesis of mesenchymal stromal cells (MSCs). METHODS: We isolated MSCs from bone marrow of healthy donors with both normal body mass index (BMI) and caloric consumption. MSC cultures were primed with sera collected from SP or normal people (NP). Then biomolecular assays were performed to evaluate effect on proliferation, apoptosis, senescence, cell commitment, and differentiation. RESULTS: SP priming affected adipocyte cell commitment and reduced spontaneous adipogenesis. Moreover, an in-depth analysis of exogenous-induced adipocyte differentiation showed striking differences between differentiation in SP-primed samples compared with NP ones. In adipocytes from SP cultures we observed a reduced size of lipid droplets, an increased expression of adipose triglyceride lipase, along with high mitochondria content and ability to produce ATP in starvation condition. These data and the expression of UCP1 protein, indicated that SP pretreatment produced a bias toward brown adipocyte differentiation. CONCLUSION: Our data suggest that sera from SP may promote brown adipogenesis rather that white adipocyte differentiation. This finding could explain why SP present normal body composition in spite of an excess of caloric intake. We hypothesize that some circulating components present in the blood of these individuals may favor brown adipogenesis at expense of white adipocyte production.

Metabolic syndrome, Mediterranean diet, and polyphenols: Evidence and perspectives.

Metabolic syndrome (MetS) is defined as the co-occurrence of metabolic risk factors that includes insulin resistance, hyperinsulinemia, impaired glucose tolerance, type 2 diabetes mellitus, dyslipidemia, and visceral obesity. The clinical significance of MetS consists of identifying a subgroup of patients sharing a common physiopathological state predisposing to chronic diseases. Clinical and scientific studies pinpoint lifestyle modification as an effective strategy aiming to reduce several features accountable for the risk of MetS onset. Among the healthy dietary patterns, the Mediterranean diet (MedDiet) emerges in terms of beneficial properties associated with longevity. Current evidence highlights the protective effect exerted by MedDiet on the different components of MetS. Interestingly, the effect exerted by polyphenols contained within the representative MedDiet components (i.e., olive oil, red wine, and nuts) seems to be accountable for the beneficial properties associated to this dietary pattern. In this review, we aim to summarize the principal evidence regarding the effectiveness of MedDiet-polyphenols in preventing or delaying the physiopathological components accountable for MetS onset. These findings may provide useful insights concerning the health properties of MedDiet-polyphenols as well as the novel targets destined to a tailored approach to MetS.

Low-Level Radiofrequency Exposure Does Not Induce Changes in MSC Biology: An in vitro Study for the Prevention of NIR-Related Damage.

BACKGROUND: The ubiquitous diffusion of radiofrequency (RF) radiation across human living environments has attracted the attention of scientists. Though the adverse health effects of RF exposure remain debatable, it has been reported that the interaction of such radiation with biological macromolecular structures can be deleterious for stem cells, inducing impairment of their main functions involving self-renewal and differentiation. PURPOSE: The purpose of this study was to determine whether exposure to RF of 169 megahertz (MHz) that is part of very high radiofrequency (VHF) range 30-300 MHz, could cause damage to stem cells by inducing senescence and loss of regenerative and DNA repair capacity. METHODS: The study was conducted on mesenchymal stromal cells (MSCs) containing a subpopulation of stem cells. The MSCs were exposed to RFs of 169 MHz administered via an open meter 2G "Smart Meter" for different durations of time. RESULT: We did not observe modifications in MSC biology as a result of the RF exposure conducted in our experiments. CONCLUSION: We concluded that MSCs are insensitive to RF radiation exposure at 169 MHz for various time intervals, including longer durations.

Stem Cells and DNA Repair Capacity: Muse Stem Cells Are Among the Best Performers.

Stem cells persist for long periods in the body and experience many intrinsic and extrinsic stresses. For this reason, they present a powerful and effective DNA repair system in order to properly fix DNA damage and avoid the onset of a degenerative process, such as neoplastic transformation or aging. In this chapter, we compare the DNA repair ability of pluripotent stem cells (ESCs, iPSCs, and Muse cells) and other adult stem cells. We also describe personal investigations showing a robust and effective capacity of Muse cells in sensing and repairing DNA following chemical and physical stress. Muse cells can repair DNA through base and nucleotide excision repair mechanisms, BER and NER, respectively. Furthermore, they present a pronounced capacity in repairing double-strand breaks by the nonhomologous end joining (NHEJ) process. The studies addressing the role of DNA damage repair in the biology of stem cells are of paramount importance for comprehension of their functions and, also, for setting up effective and safe stem cell-based therapy.