The Potential Use of Mitochondrial Extracellular Vesicles as Biomarkers or Therapeutical Tools.

The mitochondria play a crucial role in cellular metabolism, reactive oxygen species (ROS) production, and apoptosis. Aberrant mitochondria can cause severe damage to the cells, which have established a tight quality control for the mitochondria. This process avoids the accumulation of damaged mitochondria and can lead to the release of mitochondrial constituents to the extracellular milieu through mitochondrial extracellular vesicles (MitoEVs). These MitoEVs carry mtDNA, rRNA, tRNA, and protein complexes of the respiratory chain, and the largest MitoEVs can even transport whole mitochondria. Macrophages ultimately engulf these MitoEVs to undergo outsourced mitophagy. Recently, it has been reported that MitoEVs can also contain healthy mitochondria, whose function seems to be the rescue of stressed cells by restoring the loss of mitochondrial function. This mitochondrial transfer has opened the field of their use as potential disease biomarkers and therapeutic tools. This review describes this new EVs-mediated transfer of the mitochondria and the current application of MitoEVs in the clinical environment.

Small extracellular vesicles from senescent stem cells trigger adaptive mechanisms in young stem cells by increasing antioxidant enzyme expression.

Extracellular vesicles' biogenesis, shedding, and uptake are redox-sensitive. Indeed, oxidative stress conditions influence extracellular vesicles' release and content, which can modulate the redox status of the receiving cells. In this study, we aimed to assess the effect of extracellular vesicles from human dental pulp stem cells cultured under 21% O2 (senescent stem cells) on human dental pulp stem cells cultured under 3% O2 (young stem cells). Extracellular vesicles were isolated by ultracentrifugation from senescent stem cells and prepared for the treatment of young stem cells at a final concentration of 10 µg/mL. Cells were analyzed for antioxidant gene expression, mitochondrial bioenergetic parameters, ROS production, culture kinetics, and apoptosis. The results show that extracellular vesicles from senescent stem cells induce overexpression of antioxidant genes (MnSOD, CAT, and GPx) in young stem cells, which show an increased non-mitochondrial oxygen consumption, accompanied by reduced maximal respiration and spare respiratory capacity without altering mitochondrial membrane potential. This is accompanied by improved cell proliferation, viability, and migration rates and a reduction of apoptosis. In conclusion, extracellular vesicles from senescent stem cells trigger an adaptive response in young stem cells which improves their antioxidant defenses and their proliferation, migration, and survival rates. This suggests that extracellular vesicles can modulate the cells' microenvironment and the balance between proliferation and senescence.

Extracellular Vesicles: The Future of Diagnosis in Solid Organ Transplantation?

Solid organ transplantation (SOT) is a life-saving treatment for end-stage organ failure, but it comes with several challenges, the most important of which is the existing gap between the need for transplants and organ availability. One of the main concerns in this regard is the lack of accurate non-invasive biomarkers to monitor the status of a transplanted organ. Extracellular vesicles (EVs) have recently emerged as a promising source of biomarkers for various diseases. In the context of SOT, EVs have been shown to be involved in the communication between donor and recipient cells and may carry valuable information about the function of an allograft. This has led to an increasing interest in exploring the use of EVs for the preoperative assessment of organs, early postoperative monitoring of graft function, or the diagnosis of rejection, infection, ischemia-reperfusion injury, or drug toxicity. In this review, we summarize recent evidence on the use of EVs as biomarkers for these conditions and discuss their applicability in the clinical setting.

Extracellular Vesicles as Therapeutic Resources in the Clinical Environment.

The native role of extracellular vesicles (EVs) in mediating the transfer of biomolecules between cells has raised the possibility to use them as therapeutic vehicles. The development of therapies based on EVs is now expanding rapidly; here we will describe the current knowledge on different key points regarding the use of EVs in a clinical setting. These points are related to cell sources of EVs, isolation, storage, and delivery methods, as well as modifications to the releasing cells for improved production of EVs. Finally, we will depict the application of EVs therapies in clinical trials, considering the impact of the COVID-19 pandemic on the development of these therapies, pointing out that although it is a promising therapy for human diseases, we are still in the initial phase of its application to patients.

The Double-Edged Role of Extracellular Vesicles in the Hallmarks of Aging.

The exponential growth in the elderly population and their associated socioeconomic burden have recently brought aging research into the spotlight. To integrate current knowledge and guide potential interventions, nine biochemical pathways are summarized under the term hallmarks of aging. These hallmarks are deeply inter-related and act together to drive the aging process. Altered intercellular communication is particularly relevant since it explains how damage at the cellular level translates into age-related loss of function at the organismal level. As the main effectors of intercellular communication, extracellular vesicles (EVs) might play a key role in the aggravation or mitigation of the hallmarks of aging. This review aims to summarize this role and to provide context for the multiple emerging EV-based gerotherapeutic strategies that are currently under study.

Therapeutic Potential of Extracellular Vesicles in Aging and Age-Related Diseases.

Aging is associated with an alteration of intercellular communication. These changes in the extracellular environment contribute to the aging phenotype and have been linked to different aging-related diseases. Extracellular vesicles (EVs) are factors that mediate the transmission of signaling molecules between cells. In the aging field, these EVs have been shown to regulate important aging processes, such as oxidative stress or senescence, both in vivo and in vitro. EVs from healthy cells, particularly those coming from stem cells (SCs), have been described as potential effectors of the regenerative potential of SCs. Many studies with different animal models have shown promising results in the field of regenerative medicine. EVs are now viewed as a potential cell-free therapy for tissue damage and several diseases. Here we propose EVs as regulators of the aging process, with an important role in tissue regeneration and a raising therapy for age-related diseases.

Small extracellular vesicles from young adipose-derived stem cells prevent frailty, improve health span, and decrease epigenetic age in old mice.

Aging is associated with an increased risk of frailty, disability, and mortality. Strategies to delay the degenerative changes associated with aging and frailty are particularly interesting. We treated old animals with small extracellular vesicles (sEVs) derived from adipose mesenchymal stem cells (ADSCs) of young animals, and we found an improvement in several parameters usually altered with aging, such as motor coordination, grip strength, fatigue resistance, fur regeneration, and renal function, as well as an important decrease in frailty. ADSC-sEVs induced proregenerative effects and a decrease in oxidative stress, inflammation, and senescence markers in muscle and kidney. Moreover, predicted epigenetic age was lower in tissues of old mice treated with ADSC-sEVs and their metabolome changed to a youth-like pattern. Last, we gained some insight into the microRNAs contained in sEVs that might be responsible for the observed effects. We propose that young sEV treatment can promote healthy aging.

Relationship between Diet, Microbiota, and Healthy Aging.

Due to medical advances and lifestyle changes, population life expectancy has increased. For this reason, it is important to achieve healthy aging by reducing the risk factors causing damage and pathologies associated with age. Through nutrition, one of the pillars of health, we are able to modify these factors through modulation of the intestinal microbiota. The Mediterranean and Oriental diets are proof of this, as well as the components present in them, such as fiber and polyphenols. These generate beneficial effects on the body thanks, in part, to their interaction with intestinal bacteria. Likewise, the low consumption of products with high fat content favors the state of the microbiota, contributing to the maintenance of good health.

Extracellular Vesicles from Healthy Cells Improves Cell Function and Stemness in Premature Senescent Stem Cells by miR-302b and HIF-1α Activation.

Aging is accompanied by the accumulation of senescent cells that alter intercellular communication, thereby impairing tissue homeostasis and reducing organ regenerative potential. Recently, the administration of mesenchymal stem cells (MSC)-derived extracellular vesicles has proven to be more effective and less challenging than current stem cell-based therapies. Extracellular vesicles (EVs) contain a cell-specific cargo of proteins, lipids and nucleic acids that are released and taken up by probably all cell types, thereby inducing functional changes via the horizontal transfer of their cargo. Here, we describe the beneficial properties of extracellular vesicles derived from non-senescent MSC, cultured in a low physiological oxygen tension (3%) microenvironment into prematurely senescent MSC, cultured in a hyperoxic ambient (usual oxygen culture conditions, i.e., 21%). We observed that senescent MCS, treated with EVs from non-senescent MCS, showed reduced SA-ß-galactosidase activity levels and pluripotency factor (OCT4, SOX2, KLF4 and cMYC, or OSKM) overexpression and increased glycolysis, as well as reduced oxidative phosphorylation (OXPHOS). Moreover, these EVs' cargo induced the upregulation of miR-302b and HIF-1α levels in the target cells. We propose that miR-302b triggered HIF-1α upregulation, which in turn activated different pathways to delay premature senescence, improve stemness and switch energetic metabolism towards glycolysis. Taken together, we suggest that EVs could be a powerful tool to restore altered intercellular communication and improve stem cell function and stemness, thus delaying stem cell exhaustion in aging.

The Relationship between Diet and Frailty in Aging.

The increase in lifespan in the 20th century entails an increase in the elderly population. This brings a new challenge for society, causing people to have physical and mental limitations caused by age-related diseases, such as frailty. Frailty is clinically characterized by multisystem pathophysiological processes, such as chronic inflammation, immune activation, dysregulation of the musculoskeletal and endocrine systems, oxidative stress, energy imbalances, mitochondrial dysfunction, and sarcopenia. The elderly should consume energy in amounts close to those in what is currently accepted as a balanced diet. However, an increase in protein intake may be recommended for elderly people as long as there is no kidney damage. This increase could help fight the loss of muscle mass associated with age. Additionally, vitamin and mineral intakes are often insufficient in their diets. Therefore, the diet should be adapted not only to their age, but also to the pathologies associated with aging. Through these measures, we can reduce the prevalence of comorbidity and thereby increase health span. Therefore, both physical and nutritional interventions, including functional foods and nutraceuticals, should be taken into account.

BCL-xL, a Mitochondrial Protein Involved in Successful Aging: From C. elegans to Human Centenarians.

B-Cell Lymphoma-extra-large (BCL-xL) is involved in longevity and successful aging, which indicates a role for BCL-xL in cell survival pathway regulation. Beyond its well described role as an inhibitor of apoptosis by preventing cytochrome c release, BCL-xL has also been related, indirectly, to autophagy and senescence pathways. Although in these latter cases, BCL-xL has dual roles, either activating or inhibiting, depending on the cell type and the specific conditions. Taken together, all these findings suggest a precise mechanism of action for BCL-xL, able to regulate the crosstalk between apoptosis, autophagy, and senescence, thus promoting cell survival or cell death. All three pathways can be both beneficial or detrimental depending on the circumstances. Thus, targeting BCL-xL would in turn be a "double-edge sword" and therefore, additional studies are needed to better comprehend this dual and apparently contradictory role of BCL-XL in longevity.

Extracellular vesicles and redox modulation in aging.

Extracellular vesicles (EVs) are nowadays known to be mediators of cell-to-cell communication involved in physiological and pathological processes. The current expectation is their use as specific biomarkers and therapeutic tools due to their inner characteristics. However, several investigations still need to be done before we can use them in the clinic. First, their categorization is still under debate, although an accurate classification of EVs subtypes should be based on physical characteristics, biochemical composition or condition description of the cell of origin. Second, EVs carry lipids, proteins and nucleic acids that can induce epigenetic modifications on target cells. These cargos, as well as EVs biogenesis, shedding and uptake is both ageing and redox sensitive. More specifically, senescence and oxidative stress increase EVs release, and their altered content can trigger antioxidant but also prooxidant responses in target cells thereby modulating the redox status. Further analysis would help to asses EVs role in the development and progression of oxidative stress-related pathologies. In this review we aimed to summarize the current knowledge on EVs and their involvement in redox modulation on age-related pathologies. We also discuss future directions and prospective that could be performed to improve EVs usage as biomarkers or therapeutic tools.

Relevance of Oxygen Concentration in Stem Cell Culture for Regenerative Medicine.

The key hallmark of stem cells is their ability to self-renew while keeping a differentiation potential. Intrinsic and extrinsic cell factors may contribute to a decline in these stem cell properties, and this is of the most importance when culturing them. One of these factors is oxygen concentration, which has been closely linked to the maintenance of stemness. The widely used environmental 21% O2 concentration represents a hyperoxic non-physiological condition, which can impair stem cell behaviour by many mechanisms. The goal of this review is to understand these mechanisms underlying the oxygen signalling pathways and their negatively-associated consequences. This may provide a rationale for culturing stem cells under physiological oxygen concentration for stem cell therapy success, in the field of tissue engineering and regenerative medicine.

Alzheimer's disease: Only prevention makes sense.

Alzheimer's disease therapeutics is one of the most important endeavours in today's clinical investigation. Over more than 30 years of research, no disease-modifying treatment has been approved by either the FDA or the EMA to treat Alzheimer's disease. Recently, the evidence of pathological alterations in the brain tissue has been gathered showing that the signs of brain damage appear more than 20 years before the onset of Alzheimer's dementia. The major aim of this review is to underpin the idea that in Alzheimer's therapeutics, only prevention makes sense. It is difficult to visualise that would-be patients may be treated with endovenous administration of antibodies for several years to delay the onset of dementia. Rather, changes in lifestyle that should be specific, stratified and personalised are a likely alternative to delay the transition from asymptomatic Alzheimer's to minimal cognitive impairment and from there to dementia. These efforts are of the utmost importance. If we could delay the onset of full-blown dementia by 5 years, the number of demented patients would be almost halved. Thus, emphasis on preventive measures that can be implemented for decades must be supported. This approach, where even mild changes in cognition are of the greatest importance, cannot be underestimated in terms of both the individual and society's viewpoints.

Influence of Partial O2 Pressure on the Adhesion, Proliferation, and Osteogenic Differentiation of Human Dental Pulp Stem Cells on ß-Tricalcium Phosphate Scaffold.

PURPOSE: To analyze, in vitro, the influence of O2 pressure on the adhesion, proliferation, and osteogenic differentiation of human dental pulp stem cells (DPSC) on ß-tricalcium phosphate (ß-TCP) scaffold. MATERIALS AND METHODS: DPSC, positive for the molecular markers CD133, Oct4, Nestin, Stro-1, and CD34, and negative for CD45, were isolated from extracted third molars. Experiments were started by seeding 200,000 cells on ß-TCP cultured under 3% or 21% O2 pressure. No osteogenic medium was used. Eight different cultures were performed at each time point under each O2 pressure condition. Cell adhesion, proliferation, and differentiation over the biomaterial were evaluated at 7, 13, 18, and 23 days of culture. Cell adhesion was determined by light microscopy, proliferation by DNA quantification, and osteogenic differentiation by alkaline phosphatase (ALP) activity analysis. RESULTS: DPSC adhered to ß-TCP with both O2 conditions. Cell proliferation was found from day 7 of culture. Higher values were recorded at 3% O2 in each time point. Statistically significant differences were recorded at 23 days of culture (P = .033). ALP activity was not detectable at 7 days. There was, however, an increase in ALP activity over time in both groups. At 13, 18, and 23 days of culture, higher ALP activity was recorded under 3% O2 pressure. Statistical differences were found at day 23 (P = .014). CONCLUSION: DPSC display capacity of adhering to ß-TCP under 3% or 21% O2 pressure conditions. Cell proliferation on ß-TCP phosphate is significantly higher at 3% than at 21% O2 pressure, the most frequently used O2 tension. ß-TCP can itself promote osteogenic differentiation of DPSC and is enhanced under 3% O2 compared with 21%.

Role of p16INK4a and BMI-1 in oxidative stress-induced premature senescence in human dental pulp stem cells.

Human dental pulp stem cells (hDPSCs) are a source for cell therapy. Before implantation, an in vitro expansion step is necessary, with the inconvenience that hDPSCs undergo senescence following a certain number of passages, loosing their stemness properties. Long-term in vitro culture of hDPSCs at 21% (ambient oxygen tension) compared with 3-6% oxygen tension (physiological oxygen tension) caused an oxidative stress-related premature senescence, as evidenced by increased ß-galactosidase activity and increased lysil oxidase expression, which is mediated by p16INK4a pathway. Furthermore, hDPSCs cultured at 21% oxygen tension underwent a downregulation of OCT4, SOX2, KLF4 and c-MYC factors, which was recued by BMI-1 silencing. Thus, p16INK4a and BMI-1 might play a role in the oxidative stress-associated premature senescence. We show that it is important for clinical applications to culture cells at physiological pO2 to retain their stemness characteristics and to delay senescence.