Adipose-derived stem cell exosomes act as delivery vehicles of microRNAs in a dog model of chronic hepatitis.

Exosomes are nanosized extracellular vesicles secreted by all cell types, including canine adipose-derived stem cells (cADSCs). By mediating intercellular communication, exosomes modulate the biology of adjacent and distant cells by transferring their cargo. In the present work after isolation and characterization of exosomes derived from canine adipose tissue, we treated the same canine donors affected by hepatopathies with the previously isolated exosomes. We hypothesize that cADSC-sourced miRNAs are among the factors responsible for a regenerative and anti-inflammatory effect in the treatment of hepatopathies in dogs, providing the clinical veterinary field with an effective and innovative cell-free therapy. Exosomes were isolated and characterized for size, distribution, surface markers, and for their miRNomic cargo by microRNA sequencing. 295 dogs affected with hepatopathies were treated and followed up for 6 months to keep track of their biochemical marker levels. Results confirmed that exosomes derived from cADSCs exhibited an average diameter of 91 nm, and positivity to 8 known exosome markers. The administration of exosomes to dogs affected by liver-associated inflammatory pathologies resulted in the recovery of the animal alongside the normalization of biochemical parameters of kidney function. In conclusion, cADSCs-derived exosomes are a promising therapeutic tool for treating inflammatory disorders in animal companions.

A mitochondrial NADPH-cholesterol axis regulates extracellular vesicle biogenesis to support hematopoietic stem cell fate.

Mitochondrial fatty acid oxidation (FAO) is essential for hematopoietic stem cell (HSC) self-renewal; however, the mechanism by which mitochondrial metabolism controls HSC fate remains unknown. Here, we show that within the hematopoietic lineage, HSCs have the largest mitochondrial NADPH pools, which are required for proper HSC cell fate and homeostasis. Bioinformatic analysis of the HSC transcriptome, biochemical assays, and genetic inactivation of FAO all indicate that FAO-generated NADPH fuels cholesterol synthesis in HSCs. Interference with FAO disturbs the segregation of mitochondrial NADPH toward corresponding daughter cells upon single HSC division. Importantly, we have found that the FAO-NADPH-cholesterol axis drives extracellular vesicle (EV) biogenesis and release in HSCs, while inhibition of EV signaling impairs HSC self-renewal. These data reveal the existence of a mitochondrial NADPH-cholesterol axis for EV biogenesis that is required for hematopoietic homeostasis and highlight the non-stochastic nature of HSC fate determination.

Link between organic nanovescicles from vegetable kingdom and human cell physiology: intracellular calcium signalling.

BACKGROUND: Plant-derived nanovesicles (PDNVs) are a novelty in medical and agrifood environments, with several studies exploring their functions and potential applications. Among fruits, apples (sp. Malus domestica) have great potential as PDNVs source, given their widespread consumption, substantial waste production, and recognized health benefits. Notably, apple-derived nanovesicles (ADNVs) can interact with human cell lines, triggering anti-inflammatory and antioxidant responses. This work is dedicated to the comprehensive biochemical characterization of apple-derived nanovesicles (ADNVs) through proteomic and lipidomic analysis, and small RNAs sequencing. This research also aims to shed light on the underlying mechanism of action (MOA) when ADNVs interface with human cells, through observation of intracellular calcium signalling in human fibroblasts, and to tackles differences in ADNVs content when isolated from fruits derived from integrated and organic production methods cultivars. RESULTS: The ADNVs fraction is mainly composed of exocyst-positive organelles (EXPOs) and MVB-derived exosomes, identified through size and molecular markers (Exo70 and TET-3-like proteins). ADNVs' protein cargo is heterogeneous and exhibits a diverse array of functions, especially in plant's protection (favouring ABA stress-induced signalling, pathogen resistance and Reactive Oxygen Species (ROS) metabolism). Noteworthy plant miRNAs also contribute to phytoprotection. In relation with human cells lines, ADNVs elicit spikes of intracellular Ca2+ levels, utilizing the cation as second messenger, and produce an antioxidant effect. Lastly, organic samples yield a substantial increase in ADNV production and are particularly enriched in bioactive lysophospholipids. CONCLUSIONS: We have conclusively demonstrated that ADNVs confer an antioxidant effect upon human cells, through the initiation of a molecular pathway triggered by Ca2+ signalling. Within ADNVs, a plethora of bioactive proteins, small RNAs, and lipids have been identified, each possessing well-established functions within the realm of plant biology. While ADNVs predominantly function in plants, to safeguard against pathogenic agents and abiotic stressors, it is noteworthy that proteins with antioxidant power might act as antioxidants within human cells.

It takes two to remyelinate: A bioengineered platform to study astrocyte-oligodendrocyte crosstalk and potential therapeutic targets in remyelination.

The loss of the myelin sheath insulating axons is the hallmark of demyelinating diseases. These pathologies often lead to irreversible neurological impairment and patient disability. No effective therapies are currently available to promote remyelination. Several elements contribute to the inadequacy of remyelination, thus understanding the intricacies of the cellular and signaling microenvironment of the remyelination niche might help us to devise better strategies to enhance remyelination. Here, using a new in vitro rapid myelinating artificial axon system based on engineered microfibres, we investigated how reactive astrocytes influence oligodendrocyte (OL) differentiation and myelination ability. This artificial axon culture system enables the effective uncoupling of molecular cues from the biophysical properties of the axons, allowing the detailed study of the astrocyte-OL crosstalk. Oligodendrocyte precursor cells (OPCs) were cultured on poly(trimethylene carbonate-co-ε-caprolactone) copolymer electrospun microfibres that served as surrogate axons. This platform was then combined with a previously established tissue engineered glial scar model of astrocytes embedded in 1 % (w/v) alginate matrices, in which astrocyte reactive phenotype was acquired using meningeal fibroblast conditioned medium. OPCs were shown to adhere to uncoated engineered microfibres and differentiate into myelinating OL. Reactive astrocytes were found to significantly impair OL differentiation ability, after six and eight days in a co-culture system. Differentiation impairment was seen to be correlated with astrocytic miRNA release through exosomes. We found significantly reduction on the expression of pro-myelinating miRNAs (miR-219 and miR-338) and an increase in anti-myelinating miRNA (miR-125a-3p) content between reactive and quiescent astrocytes. Additionally, we show that OPC differentiation inhibition could be reverted by rescuing the activated astrocytic phenotype with ibuprofen, a chemical inhibitor of the small rhoGTPase RhoA. Overall, these findings show that modulating astrocytic function might be an interesting therapeutic avenue for demyelinating diseases. The use of these engineered microfibres as an artificial axon culture system will enable the screening for potential therapeutic agents that promote OL differentiation and myelination while providing valuable insight on the myelination/remyelination processes.

Mitochondrial Metabolism and EV Cargo of Endothelial Cells Is Affected in Presence of EVs Derived from MSCs on Which HIF Is Activated.

Small extracellular vesicles (sEVs) derived from mesenchymal stem cells (MSCs) have attracted growing interest as a possible novel therapeutic agent for the management of different cardiovascular diseases (CVDs). Hypoxia significantly enhances the secretion of angiogenic mediators from MSCs as well as sEVs. The iron-chelating deferoxamine mesylate (DFO) is a stabilizer of hypoxia-inducible factor 1 and consequently used as a substitute for environmental hypoxia. The improved regenerative potential of DFO-treated MSCs has been attributed to the increased release of angiogenic factors, but whether this effect is also mediated by the secreted sEVs has not yet been investigated. In this study, we treated adipose-derived stem cells (ASCs) with a nontoxic dose of DFO to harvest sEVs (DFO-sEVs). Human umbilical vein endothelial cells (HUVECs) treated with DFO-sEVs underwent mRNA sequencing and miRNA profiling of sEV cargo (HUVEC-sEVs). The transcriptomes revealed the upregulation of mitochondrial genes linked to oxidative phosphorylation. Functional enrichment analysis on miRNAs of HUVEC-sEVs showed a connection with the signaling pathways of cell proliferation and angiogenesis. In conclusion, mesenchymal cells treated with DFO release sEVs that induce in the recipient endothelial cells molecular pathways and biological processes strongly linked to proliferation and angiogenesis.

Playing with Biophysics: How a Symphony of Different Electromagnetic Fields Acts to Reduce the Inflammation in Diabetic Derived Cells.

Several factors, such as ischemia, infection and skin injury impair the wound healing process. One common pathway in all these processes is related to the reactive oxygen species (ROS), whose production plays a vital role in wound healing. In this view, several strategies have been developed to stimulate the activation of the antioxidative system, thereby reducing the damage related to oxidative stress and improving wound healing. For this purpose, complex magnetic fields (CMFs) are used in this work on fibroblast and monocyte cultures derived from diabetic patients in order to evaluate their influence on the ROS production and related wound healing properties. Biocompatibility, cytotoxicity, mitochondrial ROS production and gene expression have been evaluated. The results confirm the complete biocompatibility of the treatment and the lack of side effects on cell physiology following the ISO standard indication. Moreover, the results confirm that the CMF treatment induced a reduction in the ROS production, an increase in the macrophage M2 anti-inflammatory phenotype through the activation of miRNA 5591, a reduction in inflammatory cytokines, such as interleukin-1 (IL-1) and IL-6, an increase in anti-inflammatory ones, such as IL-10 and IL-12 and an increase in the markers related to improved wound healing such as collagen type I and integrins. In conclusion, our findings encourage the use of CMFs for the treatment of diabetic foot.

Apple Derived Exosomes Improve Collagen Type I Production and Decrease MMPs during Aging of the Skin through Downregulation of the NF-κB Pathway as Mode of Action.

Skin ageing is strictly related to chronic inflammation of the derma and the decay of structural proteins of the extracellular matrix. Indeed, it has become common practice to refer to this phenomenon as inflammageing. Biotech innovation is always in search of new active principles that induce a youthful appearance. In this paper, apple-derived nanovesicles (ADNVs) were investigated as novel anti-inflammatory compounds, which are able to alter the extracellular matrix production of dermal fibroblasts. Total RNA sequencing analysis revealed that ADNVs negatively influence the activity of Toll-like Receptor 4 (TLR4), and, thus, downregulate the NF-κB pro-inflammatory pathway. ADNVs also reduce extracellular matrix degradation by increasing collagen synthesis (COL3A1, COL1A2, COL8A1 and COL6A1) and downregulating metalloproteinase production (MMP1, MMP8 and MMP9). Topical applications for skin regeneration were evaluated by the association of ADNVs with hyaluronic-acid-based hydrogel and patches.

In Vitro Model for the Evaluation of Innovative Transcatheter Debridement Device (TDD): Pericardium-Based Scaffold and Stem Cells to Reproduce Calcificated Valves.

Aortic valve stenosis has become the most common valvular disease in elderly patients. Several treatments are available such as surgical aortic valve replacement and transcatheter aortic valve implantation. To date, however, there is a need to discover alternative treatments that can delay the disease progression and, therefore, the implant of a prosthetic valve. In this regard, a decalcification procedure based on the use of ultrasonic waves could represent an innovative solution in transcatheter cardiovascular therapies. In this article, we describe an innovative transcatheter debridement device (TDD) that uses low-intensity ultrasound shock waves for calcium ablation from the native aortic valve and bioprosthetic valve. Mesenchymal stem cells were seeded onto pericardium-based scaffolds and committed into an osteogenic phenotype. After treatment with TDD, cell proliferation was analyzed, as well as lactate dehydrogenase release and cell morphology. The release of calcium and inflammation events were detected. The results confirmed that the TDD was able to induce a safe decalcification without any adverse inflammatory events.

Biomaterials for Regenerative Medicine in Italy: Brief State of the Art of the Principal Research Centers.

Regenerative medicine is the branch of medicine that effectively uses stem cell therapy and tissue engineering strategies to guide the healing or replacement of damaged tissues or organs. A crucial element is undoubtedly the biomaterial that guides biological events to restore tissue continuity. The polymers, natural or synthetic, find wide application thanks to their great adaptability. In fact, they can be used as principal components, coatings or vehicles to functionalize several biomaterials. There are many leading centers for the research and development of biomaterials in Italy. The aim of this review is to provide an overview of the current state of the art on polymer research for regenerative medicine purposes. The last five years of scientific production of the main Italian research centers has been screened to analyze the current advancement in tissue engineering in order to highlight inputs for the development of novel biomaterials and strategies.

Exosomes Derived from Dental Pulp Stem Cells Show Different Angiogenic and Osteogenic Properties in Relation to the Age of the Donor.

Craniofacial tissue reconstruction still represents a challenge in regenerative medicine. Mesenchymal stem cell (MSC)-based tissue engineering strategies have been introduced to enhance bone tissue repair. However, the risk of related complications is limiting their usage. To overcome these drawbacks, exosomes (EXOs) derived from MSCs have been recently proposed as a cell-free alternative to MSCs to direct tissue regeneration. It was hypothesized that there is a correlation between the biological properties of exosomes derived from the dental pulp and the age of the donor. The aim of the study was to investigate the effect of EXOs derived from dental pulp stem cells of permanent teeth (old donor group) or exfoliated deciduous teeth (young donor group) on MSCs cultured in vitro. Proliferation potential was evaluated by doubling time, and commitment ability by gene expression and biochemical quantification for tissue-specific factors. Results showed a well-defined proliferative influence for the younger donor aged group. Similarly, a higher commitment ability was detected in the young group. In conclusion, EXOs could be employed to promote bone regeneration, likely playing an important role in neo-angiogenesis in early healing phases.

Exosomes as Neurological Nanosized Machines.

In the past few decades, nanomedicine research has advanced dramatically. In spite of this, traditional nanomedicine faces major obstacles, such as blood-brain barriers, low concentrations at target sites, and rapid removal from the body. Exosomes as natural extracellular vesicles contain special bioactive molecules for cell-to-cell communications and nervous tissue function, which could overcome the challenges of nanoparticles. Most recently, microRNAs, long noncoding RNA, and circulating RNA of exosomes have been appealing because of their critical effect on the molecular pathway of target cells. In this review, we have summarized the important role of exosomes of noncoding RNAs in the occurrence of brain diseases.