# Extracellular vesicles

Extracellular vesicles (EVs) is a collective term covering various subtypes of cell-released membranous structures that can be found in various body fluids, including blood plasma, urine, saliva, amniotic fluid, breast milk, and fluid that accumulates in pleural ascites. Released by almost all cell types, they play an important role in cellular communication transporting biological molecules between cells, as they carry bioactive proteins, lipids, and nucleic acids as part of their functional cargo, transporting biological molecules between cells.
EVs acts as one of the SASP (senescence-associated secretory phenotype) factors, which enhance the proliferation of cancer cells, paracrine senescence, and chromosomal instability. Among the hallmarks of senescence, the SASP, especially SASP-related extracellular vesicle (EV) signalling, plays the leading role in aging transmission via paracrine and endocrine mechanisms.

## EV classification and composition

EVs encompass various types of vesicles, including:
* Exosomes (30–100 nm, the smallest extracellular vesicle) formation and release occur through the endosomal pathway and into the extracellular medium after fusion with the plasma membrane. Its content corresponds to that existing in the endosomal compartment.
* Ectosomes (100–350 nm) are vesicles found everywhere in organisms and released from the plasma membrane. Their function is analogous to exosomes.
* Microvesicles (MVs; formerly called microparticles or MPs) have a size from 100 nm–1 µm. They are secreted outside the cell by the process of evagination or sprouting of the plasma membrane, which involves: (a) relocation of phospholipids in the outer membrane so that the phosphatidylserine (PS), generally located on the inner side of the membrane, is exposed on the surface of the vesicle, (b) rearrangement of the cytoskeleton, (c) generation of the curvature of the membrane, and (d) liberation of the vesicle.
* Apoptotic bodies (1–5 µm) are released as vesicles after cellular apoptosis, followed by increased membrane permeability, DNA fragmentation, and changes in mitochondrial membrane potential. Apoptotic bodies also expose PS on their surface and contain cellular organelles and genetic material.
* Exophers are the 3.5–4-μm large type of EV, which contain damaged mitochondria and protein aggregates

## The role of extracellular vesicles in cellular senescence

Extracellular vesicles production is upregulated in senescent cells up to 50-fold, with senescent-induced changes to their cargo (e.g., of proteins, miRNAs, and mRNAs).
It was found that senescent human fibroblast cells can induce a bystander effect, spreading senescence in intact neighboring fibroblasts in vitro, and that small extracellular vesicles from senescent cells are responsible for mediating paracrine senescence to nearby cells. The outcome of their production can be either beneficial or detrimental, depending on the context.

## Diagnostic Roles of EV in Aging-Related Diseases

Urinal EVs have a major role in the prediction of the response to therapy in urogenital system diseases. Indeed, the identification of specific biomarkers, including protein, lipids, and miRNAs, within urinary EVs unveils the prognosis of the prostate, bladder, and renal cancers EVs not only serve as biomarker reservoir but also as messengers to and from kidney tubular cells.

## Therapeutic Roles of EV
### Mesenchymal stem cell‐derived extracellular vesicles

Mesenchymal stem/stromal cells (MSCs) are under clinical development for the treatment of numerous disease indications. There is growing interest surrounding the therapeutic application of purified and concentrated regenerative factors secreted by MSCs. Mesenchymal stem cells (MSCs)-derived EVs have been observed to implement the same therapeutic effects as MSCs with minimal adverse effects and could be used as an alternative treatment method to MSC-based therapy. Donor variance in age, sex, and other genetic differences creates significant variability in the therapeutic potency of MSCs and their secreted EVs.
In contrast to stem cells, EVs like exosomes cannot self-replicate, eliminating concerns about potential tumour formation after stem cell transplantation. Exosomes are also stable enough for long-term frozen storage and storage at room temperature after lyophilization. Their small size further allows sterilization by filtration. In addition, exosomes can be administered by several routes; for example, nebulized or lyophilized lung stem cell-derived exosomes can be administrated by inhalation to treat lung diseases.
Parabiosis experiments in mice demonstrated that a young environment could partially rejuvenate multiple tissues of old organisms and it was applied to demonstrate a lifespan-enhancing effect of young blood. EVs isolated from young (4-to-12-month-old) mouse plasma injected into 26-month-old female mice once a week until sacrifice led to increase of 10.2% and of 15.8% in median and maximal lifespan, respectively, in mice receiving the treatment vs. vehicle-treated mice of the same age. So, it was suggested that the beneficial effects of young blood may be recapitulated by EVs transfusion.
Сonditioned media (CM) from young bone marrow-derived mesenchymal stem cells (BM-MSCs) culture rescued the function of aged, senescent stem cells and senescent murine embryonic fibroblasts (MEFs) in culture, whereas CM from young BM-MSCs depleted of extracellular vesicles was unable to reduce the percentage of senescent aged BM-MSCs. Moreover, the senotherapeutic activity of CM co-purified with extracellular vesicles that were released by young, but not old MSCs and muscle-derived stem/progenitor cells (MDSPC)s. Treatment with EVs isolated from human embryonic stem cell-derived MSCs (hESC-MSC) was capable of significantly reducing the expression of markers of senescence in cultured senescent fibroblasts as well as naturally aged wild-type mice, and improving measures of healthspan in vivo. These results identified EVs as key factors released by young, functional stem cells that can rescue cellular senescence and stem cell dysfunction in culture and reduce senescent cell burden in vivo. Small EVs (sEVs) derived from multiple stem cells, such as exosomes, have demonstrated their capacity to promote tissue regeneration after several types of damage. Compared to stem cells, sEVs are more stable, have no risk of aneuploidy, have a lower chance of immune rejection, and can provide an alternative therapy for various diseases. It has been shown that sEVs can exert proregenerative effects in tissues of old mice and decrease senescence-related damage.
Old animals treated with small extracellular vesicles derived from adipose mesenchymal stem cells (ADSCs) of young animals, revealed 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. Analysis of miRNAs in sEVs from young ADSC cultures showed several differentially expressed miRNAs when compared to sEVs from old ADSC cultures (9 up-regulated and 1 down-regulated) and from plasma of aged mice (25 up-regulated and 4 down-regulated. Six miRNAs were outlined as plausible biologically relevant, i.e., that can be considered relevant because these miRNAs are the ones that share their nucleotide sequence among several species, including humans, as many features of the aging process are highly conserved across species.
sEVs from young cells ameliorate senescence in a variety of tissues in old mice. It was identified that sEVs have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro.
In experiments with exosomes, modified with miRNAs it was found that elevated miR-26a enhanced axonal growth in hippocampal neurons and axonal regeneration in the peripheral nervous system, and that exosomes with overexpressed miR-26a could activate the mammalian target of rapamycin (mTOR) pathway to enhance axonal growth and renewal in the nervous system, thus promoting neurogenesis.

#### Extruded cell nanovesicles

The clinical translation of EVs is constrained by the poor yield of EVs. Extrusion has recently become an effective technique for producing a large scale of nanovesicles (NVs). Proteomics and RNA sequencing data revealed that NVs resemble MSCs more closely than EVs. Intravenous delivery of MSC NVs improved heart repair and cardiac function in a mouse model of myocardial infarction.

### Undifferentiated iPSCs as a source for therapeutic EV production

Production of MSC EVs is currently hampered by donor-specific characteristics and limited ex vivo expansion capabilities before decreased potency, thus restricting their potential as a scalable and reproducible therapeutic. Induced pluripotent stem cells (iPSCs) represent a self-renewing source for obtaining differentiated iPSC-derived MSCs (iMSCs), circumventing both scalability and donor variability concerns for therapeutic EV production. Compared with tissue-derived MSC, iMSC closely resemble their primary counterparts in morphology, immunophenotype, and three-lineage differentiation capacity while showing stronger regeneration ability in animal disease models. Moreover, iPSC can be passed down indefinitely so that the sources of iMSC can be endless and iMSC induced from a single iPSC cell or clone are theoretically more homogeneous.
In experiments utilizing undifferentiated iPSC EVs as a control, it was found that their vascularization bioactivity was similar and their anti-inflammatory bioactivity was superior to donor-matched iMSC EVs in cell-based assays. Combined with the lack of additional differentiation steps required for iMSC generation, these results support the use of undifferentiated certain proven iPSC lines as a source for therapeutic EV production with respect to both scalability and efficacy.

### Exosomes as the delivery agents for reprogramming

Commonly, viruses are used as delivery agents of DNA/RNA molecules for reprogramming. However, they are problematic in that certain viruses (retrovirus, lentivirus) are integrating and for others (AAVs) there exists a significant number of individuals with immunity against them. In contrast to viruses, exosomes are not recognized by the host immune system and they are naturally adapted to delivering multiple molecules. Moreover, they may display selective transmission of the genetic information to the tissue/cell, probably on account of their high expression levels of adhesion molecules, such as integrins and tetraspanins, with their potential capability to select target. For instance, C166-derived exosomes are an effective delivery agent in that they appear to be selective for cardiac fibroblasts in vivo. C166-derived exosomes are readily taken up by cardiac fibroblasts while rarely internalized by cardiomyocytes or endothelial cells. This made it possible to successfully reprogram in situ adult cardiac fibroblasts into neonatal fibroblasts which tend to produce more collagen, nestin and smooth muscle actin than their adult counterparts and being associated with improved healing outcomes due to their regenerative capacity.

## ‎Further reading

* Xu, D., & Tahara, H. (2013). The role of exosomes and microRNAs in senescence and aging. Advanced drug delivery reviews, 65(3), 368-375. PMID: 22820533 DOI: link</ref>
* Takasugi, M. (2018). Emerging roles of extracellular vesicles in cellular senescence and aging. Aging cell, 17(2), e12734. ̣̻DOI: link
* Urbanelli, L., Buratta, S., Sagini, K., Tancini, B., & Emiliani, C. (2016). Extracellular vesicles as new players in cellular senescence. International Journal of Molecular Sciences, 17(9), 1408. PMID: 27571072 PMC:link DOI: link
* Oh, C., Koh, D., Jeon, H. B., & Kim, K. M. (2022). The Role of Extracellular Vesicles in Senescence. Molecules and Cells, 45(9), 603-609. PMID: 36058888 PMC:link DOI: 10.14348/molcells.2022.0056
* Kostyushev, D., Kostyusheva, A., Brezgin, S., Smirnov, V., Volchkova, E., Lukashev, A., & Chulanov, V. (2020). Gene Editing by Extracellular Vesicles. International Journal of Molecular Sciences, 21(19), 7362. PMID 33028045 DOI: link
* D’Anca, M., Fenoglio, C., Serpente, M., Arosio, B., Cesari, M., Scarpini, E. A., & Galimberti, D. (2019). Exosome determinants of physiological aging and age-related neurodegenerative diseases. Frontiers in aging neuroscience, 11, 232. PMID: 31555123 PMC:link DOI: link
* Guix, F. X. (2020). The interplay between aging‐associated loss of protein homeostasis and extracellular vesicles in neurodegeneration. Journal of neuroscience research, 98(2), 262-283. PMID: 31549445 DOI: link
* Misawa, T., Tanaka, Y., Okada, R., & Takahashi, A. (2020). Biology of extracellular vesicles secreted from senescent cells as senescence‐associated secretory phenotype factors. Geriatrics & Gerontology International, 20(6), 539-546. PMID: 32358923 DOI: link
* Misawa, T., Hitomi, K., Miyata, K., Tanaka, Y., Fujii, R., Chiba, M., ... & Takahashi, A. (2023). Identification of Novel Senescent Markers in Small Extracellular Vesicles. International journal of molecular sciences, 24(3), 2421. PMID: 36768745 PMC:link DOI: link