Small extracellular vesicles from young mice prevent frailty, improve healthspan and decrease epigenetic age in old mice

Aging is associated with an increased risk of frailty, disability, comorbidities, institutionalization, falls, fractures, hospitalization, and mortality. Searching for strategies to delay the degenerative changes associated with aging and frailty is interesting. We treated old animals intravenously with small extracellular vesicles (sEVs) derived from adipose mesenchymal stem cells (ADSCs) of young animals, and we found an improvement of several functional parameters usually altered with aging, such as motor coordination, grip strength, fatigue resistance, fur regeneration, and renal function. Frailty index analysis showed that 40% of old control mice were frail, whereas none of the old ADSCs-sEVs treated mice were. Molecular and structural benefits in muscle and kidney accompanied this functional improvement. ADSCs-sEVs induced pro-regenerative effects and a decrease in oxidative stress, inflammation, and senescence markers. Moreover, predicted epigenetic age was lower in tissues of old mice treated with ADSCs-sEVs and their metabolome changed to a youth-like pattern. Finally, we gained some insight into the miRNAs contained in sEVs that might be, at least in part, responsible for the effects observed. We propose that young sEVs treatment can be beneficial against frailty and therefore can promote healthy aging.


Introduction
To add health to the years gained, as well as to promote the ability to live autonomously is a public health priority in modern societies. The search for strategies to delay the degenerative changes associated with aging and frailty is particularly interesting.
Aging is accompanied by an impairment in the physical condition and an increased risk of frailty 1 . It is characterized by several changes at the cellular and organismal levels that result in a decreased functionality of several tissues. Alterations in intercellular communication have been described as potential drivers of age-related dysfunctions 2 . Cellular function not only depends on cell-autonomous factors, but is also affected by the extracellular environment, and its modification can have a great effect on the performance of several tissues 3,4 . Parabiosis experiments conducted in mice demonstrated that factors present in the blood of a young organism are beneficial for an aged one, improving several parameters affected by aging 5,6,7 . Allogenic transplantation of mesenchymal stem cells in frail people has shown very promising results to treat frailty 9 . Stem cells have intrinsic regenerative effects that are not only mediated by the repopulation of damaged tissue. The releasing of regulatory molecules is also proposed as one of the most important mechanisms in stem cell therapies 10,11 . More specifically, sEVs derived from multiple stem cells have demonstrated their capacity to promote tissue regeneration after several types of damage 12,13 . 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 14,15,16,17 .
It is important to consider that the way of culturing stem cells affects dramatically paracrine signals. We previously showed that oxygen tension in culture is one of the As physical tests depend on the weight of mice, we measured the total body weight of mice during the experiment, and we did not find differences between groups. In addition, we failed to observe any signs of toxicity (excessive weight loss) during the entire experiment (Figure 1b). Physical tests were repeated on days 14 and 30 after ADSCs-sEVs or PBS treatment. On day 14 we could observe an improvement in the strength test on ADSCs-sEVs treated mice. The maximum benefit was observed on day 30: mice treated with ADSCs-sEVs showed an improvement in grip strength, motor coordination, and fatigue resistance when compared to controls (Figure 1c-e).
Interestingly, 60 days after treatment, the positive effect was lost, and we could not find any differences between groups (Supplementary Table 1). Thus, the protective effects of ADSCs-sEVs are, as expected, transitory.
For a quantitative assessment of frailty, we used a score based on the clinical phenotype of frail humans developed by our group 26 . This enabled us to classify each mouse as frail or non-frail at each time point. On day 30, there were no frail mice in the ADSCs-sEVs treated group, while the control group shed 40% of frail mice, which is in accordance with previous results in mice of this age 26 (Figure 1f).
Fur density is reduced and capacity to re-growth hair becomes impaired with aging 27 .
To check the effect of ADSCs-sEVs on the growth of hair, we plucked a square of 1cm x 1cm of the dorsal fur of each mouse on day 1 just before injection. On day 14, we observed that most mice treated with ADSCs-sEVs had regenerated the entire area. In contrast, mice injected with PBS had a much lower capacity for hair re-growth ( Figure   1g-h).
To determine possible changes in the renal function we used plasma values of urea, as it is a well-known marker of kidney function. We obtained plasma samples of each mouse on days 0, 14, 30, and 60 and measured urea levels at each time point. Day 0 levels were used as a baseline. Fourteen days after being injected with ADSCs-sEVs, mice showed a decrease in plasma concentration of urea, whereas the control group showed no modification. On day 30, urea levels raised 50% from baseline in the control group. By contrast, the ADSCs-sEVs group showed a plasmatic urea level like day 0 ( Figure 1i).
In addition, as a control to further test the possible effect of age in the quality of sEVs and ADSCs, we performed a pilot study with 3 old mice treated with ADSCs-sEVs isolated from old mice, we did not find any effect of these old-sEVs in physical performance tests, indicating that the age of ADSCs donor is essential in the beneficial effects observed (Supplementary Table 2).
Taken together, we find that only ADSCs-sEVs isolated from young mice induce an improvement in the global healthspan of aged mice.  ADSCs-sEVs reverse age-related structural changes in kidney and muscle of old mice.
As an organism begins to age, its tissues suffer from several structural changes, which are detrimental to the normal function of the tissue, such as a loss of regenerative capacity and fibrosis 2, 28 . We selected kidney and muscle to test the effect of ADSCs-sEVs on tissue structure, as these organs are specially involved in the aging process: kidney function is one of the organs that age faster in normal individuals 29 , and muscle function is essential for the activities of daily life and is directly related to frailty 30 .
Therefore, we obtained kidney and muscle from old mice 30 days after treatment with ADSCs-sEVs/PBS. In the kidney, we performed histological analysis of the renal cortex, looking for the presence of tubular atrophy and interstitial fibrosis, two distinctive changes associated with aging that leads to the loss of renal function 31 . The macroscopic appearance of kidneys was very different between old untreated and treated mice, kidneys isolated from old mice treated with sEVs were more like those isolated from young mice (Figure 2a). Control mice showed widespread tubular atrophy, defined by a reduced tubular density and the presence of dilated tubules. In contrast, tubular density was much higher in mice treated with ADSCs-sEVs, as well as very low tubular dilation. We did not observe differences in glomerular density between groups (Figure 2b-e). For the study of interstitial fibrosis, we used Sirius red staining to measure collagen deposition. We noted a mild decrease in the ADSCs-sEVs treated group, although no statistically significant differences were observed ( Figure   2f).
These observations suggested that ADSCs-sEVs may induce the proliferation of tubular cells as a mean of regeneration, as these cells can repopulate renal tubules after damage 32 . Using Ki67 as a proliferation marker, we looked for the presence of Ki67+ cells in the cortex tubules of our mice. We found very low levels of proliferating cells in the control group, however, in the treated group we identified a clear population of Muscle loss of function and atrophy lead to a global dysfunction of the organism as it ages, as the musculoskeletal system is essential for the activities of daily life. The prevalence of sarcopenia (defined by a loss of muscle mass and function) increases with age 33 . To investigate the effect of ADSCs-sEVs on muscle tissue of old mice, we measured the cross-sectional area (CSA) of muscle fibers, which is a parameter related to muscle atrophy and loss of strength. Interestingly, we found an increase in the mean CSA with ADSCs-sEVs treatment, which was supported by a higher protein concentration in the muscles of ADSCs-sEVs treated mice (Figure 2i-k).
In sum, these results show that ADSCs-sEVs induce pro-regenerative effects in kidney and muscle, partially reverting structural changes associated with aging in these tissues. Telomere attrition and damage to telomeres are two of the most studied factors that lead to genomic instability and loss of proliferative capacity. Telomere shortening is observed in many species during normal aging, and excessively short telomeres are associated with multiple conditions characterized by a loss of regenerative capacity, such as pulmonary fibrosis, dyskeratosis congenita, and aplastic anaemia 34 . We explored the effect of ADSCs-sEVs on telomere length and damage, as possible mediators of the observed effects in mice. For this purpose, we measured mean telomere length and DNA damage at the telomeres quantified as telomere dysfunction induced foci (TIF). We did not find significant differences between both groups ( Figure   3c-d), although we found that the muscle of treated mice showed slightly lower telomere damage than the non-treated ones. Chronic inflammation is a condition closely related to aging and senescence, an increase in pro-inflammatory cytokines in several tissues has been linked to aging, frailty, and age-related diseases 38 . IL-6 is one of the most important factors of the SASP, so we measured levels of this interleukin in the muscle of old mice, observing a decrease in its levels in muscle of ADSCs-sEVs treated mice ( Figure 3e).
Finally, to test the effect of ADSCs-sEVs on a more controlled environment, we developed an in vitro model of senescence in muscle progenitor cells. For this purpose, we used C2C12 mouse myoblasts and induced cellular senescence with Palbociclib (5 μM). We treated the senescent cells with young ADSCs-sEVs (5 μg/mL), and we measured β-galactosidase (SABG) as a senescence marker and Annexin V as an apoptosis marker (Figure 3i). We found that cells that received ADSCs-sEVs showed a decrease in SABG, as well as a decrease in the percentage of apoptotic cells (Figure 3jk).
Overall, these findings prove that treatment with ADSCs-sEVs can alleviate molecular and cellular traits of aging in kidney and muscle of aged mice, as well as a decrease in senescence and apoptosis in an in vitro model. Palbociclib + sEVs n=5. All data are shown as mean±SD.

Predicted epigenetic age is lower in tissues of old mice treated with ADSCs-sEVs.
We used multiple mouse clocks trained on different tissues to estimate the age of our treated and untreated animals. We grouped the clocks into two groups. The general clocks were trained across a wide spectrum of animals. The interventions group of clocks were trained on animals with age affecting interventions. We used mixed effects modelling to discern a trend across multiple clocks. In kidney tissue samples, age  ADSCs-sEVs induce a change in the metabolome of old mice to a youth-like pattern.
We quantified 72 metabolites in blood plasma samples from 7 PBS-treated and 6 ADSCs-sEVs-treated old mice from 4 control young mice. Fourteen of these metabolites show statistically significant differences between ADSCs-sEVs-treated and untreated old animals (Supplementary Table 3 and Figure 5a). Most of the significant metabolites were amino acids including essential (isoleucine, tryptophan, threonine, and valine) and non-essential (aspartate, arginine, tyrosine, glycine, and proline). Some of these amino acids have been previously associated with metabolic health like the branched-chain amino acids isoleucine and valine 40 as well as tryptophan 41 . Lactate is formed by the anaerobic glycolysis in most mammalian tissues. Together with acetate, it is also a product of fermentation of fructans and other prebiotics by gut microbiota 42 .
They are further processed into butyrate and other short-chain fatty acids by some strains of bifidobacteria and lactobacilli, which suggest some impact of ADSCs-sEVs treatment in gut microbiota ecosystems. The heatmap of these metabolites shows a clustering trend among groups and some resemblance between ADSCs-sEVs-treated old mice and young mice (Figure 5b), i.e., red dots (old-sEVs mice) differ from black ones (old-PBS mice) but coincide with blue dots (young mice).
To further explore this resemblance, we  As we injected the young ADSCs-sEVs in the blood of old mice, we reasoned that miRNAs upregulated in the young ADSCs-sEVs and with low levels in plasmatic sEVs from aged mice maybe, at least in part, responsible for the beneficial effects of ADSCs-sEVs. Thereafter, we explored the predicted mRNA targets for the upregulated 25 miRNAs and their role in biological processes and molecular pathways. However, as the number of predicted targets was too high, we outlined 6 miRNAs as plausible biologically relevant, these miRNAs (highlighted in red in Figure 6e) were the ones that shared their nucleotide sequence among several species, including humans, as many features of the aging process are highly conserved across species 48 .
Altogether, these 6 miRNAs were predicted to target 5.379 genes with very high stringency (top 1% in confidence, present in at least five databases as curated by miRDIP). The target gene dataset was then introduced in PANTHER to study over-

Discussion
Aging is the largest risk factor for most diseases that affect people at late stages of their lives. Our understanding of the aging process has grown greatly in the last decades, providing us with several biochemical changes that are considered as drivers of this Given this background, the effect of sEVs from young MSCs on the function of tissues affected by aging in physiologically aged mice remained to be elucidated. In the present work, we have shown that young-derived ADSCs-sEVs have great potential as anti-aging factors, as old mice injected with these sEVs showed an improvement in several functions affected by aging, such as physical condition, fur regeneration, and renal function, as well as a reduction of frailty. Very interestingly, the effect of ADSCs-sEVs seems to be finite, as we observed in a more long-term experiment that the effect on the functionality of old mice was lost two months after treatment with ADSC-sEVs.
Moreover, sEVs induced structural changes in tissues from old mice, exhibiting a potent pro-regenerative effect in these tissues, which is in accordance with previous results that proposed sEVs as a regenerative tool 23 . We observed a reduction of senescence in tissues and in vitro when sEVs were introduced, however, the mechanism of action remains unclear, as we did not find senolytic activity. They may probably act as senomorphics, molecules that suppress the senescent phenotype, as has been described recently 51 . Indeed, we found a reduced level of IL-6, one of the main SASP factors, in the tissues from ADSCs-sEVs treated mice. All these effects found in tissues of ADSCs-sEVs treated mice were accompanied by a decrease in the epigenetic age estimation from epigenetic clocks, which are recognized as a feasible estimation of biological age 52 . Some of the most studied interventions in aging, such as caloric restriction or rapamycin treatment, have demonstrated a strong impact on predicted epigenetic age 53, 54, 55 . To our knowledge, this study is the first to show that intervention with sEVs can decrease epigenetic age in an old organism.
sEVs induced effects not only in individual tissues but also in the whole metabolome of old mice, which turned to a youth-like pattern, indicating that the effects observed are probably part of a pleiotropic effect on the organism. Now as it is time to continue investigating the molecules that are included in sEVs, we have explored miRNAs contained in young ADSCs-sEVs and found that they are involved in several processes and pathways affected by aging, thus proposing miRNAs as possible mediators of the effects shown in mice,miR-214-3p seems to play a key role in senescence 56,57 . More studies are needed to identify factors derived from stem cells that can assist tissue function and regeneration, as they could have an enormous impact on age-related pathologies, such as frailty or renal failure.

Acknowledgments
We

Declaration of interests
The authors declare no competing interests

Animal model
This study was performed in strict accordance with all applicable federal and institutional policies. The protocol was approved by the University of Valencia Animal Ethics Committee. All the mice used in this study were of a C57BL/6J background.
Aged mice used were treated at 22-24 months of age. Both sexes were used throughout the study. Where feasible, littermates of the same sex were used. These were randomly assigned to experimental groups. All experiments were addressed blindly. After mice were sacrificed, plasma and organs were obtained and stored at -80ºC. For all the experiments conducted in kidney and gastrocnemius, we used organs obtained from mice 30 days after treatment with sEVs/PBS.

Stem cell culture
Mice at 3-6 months of age were used to obtain mesenchymal stem cells (MSCs) from both inguinal fat pads with the previously described protocol 58 . All cells were used at

ADSCs derived sEVs isolation and characterization
ADSCs at passage 2-3 were expanded until they reached 80% confluency, at that moment, media was changed to DMEM high glucose with 2% Exosome-depleted FBS (Gibco) and 1% P/S. After 48h, conditioned media was collected, and sEVs were isolated by differential ultracentrifugation. Media was centrifuged at 2,000g for 10 minutes and then at 20,000g for 30 minutes to remove whole cells, cell debris, and bigger EVs. The supernatant was then ultracentrifuged at 100,000g for 70 minutes.

Physical condition tests
The Grip Strength Meter (Panlab, Harvard Apparatus) was employed in assessing neuromuscular function by sensing the peak amount of force that the mice applied in grasping specially designed pull bar assemblies. Peak force was automatically registered in grams-force by the apparatus. Data were recorded, and 2 additional trials were immediately given. Maximum force was normalized to the weight of the mouse.
Animals were submitted to a graded intensity treadmill test (Treadmill Control LE 8710 Panlab, Harvard Apparatus) to determine their endurance (running time) and running speed along with the study. After a warmup period, the treadmill band velocity was increased until the animals were unable to run further. The initial bout of 4 minutes at 10 cm/s was followed by consecutive 4 cm/s increments every 2 minutes.
Exhaustion was reached when a mouse remained on the shock grid for 5 seconds rather than running. Motor coordination was assessed with a Rota Rod (Panlab, Harvard Apparatus #76-0772), consisting of a 3cm wide wheel that rotates with an increasing speed. Time to fall was recorded for each mouse, with a total of 3 trials. To assess frailty quantitatively, we used a score based on the clinical phenotype of frailty developed in our group 26 , mice with 3 or more criteria were classified as frail. Criteria used: Loss of 5% of body weight, time to fall in Rota Rod under percentile 20 (p20), time to exhaustion in a treadmill under p20, maximum speed reached in a treadmill under p20, and normalized grip strength under p20.

Hair re-growth assay
On day 1, dorsal hair was removed by plucking from a square of approximately 1 cm x 1 cm. Hair re-growth was scored two weeks later, based on digital photographs and a semi-quantitative assessment, using an arbitrary scale from one to four (where four represents complete hair regeneration). Scoring was performed blindly by two independent investigators.

Plasmatic urea values measurement
100 µL of whole blood was obtained from the saphenous vein of each mouse just before treatment, after that, whole blood was obtained on days 14, 30, and 60. Whole blood samples were collected in a microvette with Ethylenediaminetetraacetic acid (EDTA) for plasma separation and spun for 15 minutes at 1,500g. The clear supernatant was transferred into regular 1.5ml tubes, snap-frozen in liquid N2, and stored at -80ºC.
[Urea] was measured using a QuantiChrom Urea Assay Kit (Gentaur). The samples were incubated in a 200µl reaction mix for 20 min at room temperature before absorbance was measured at 520nm.

Histological analysis
Kidneys and gastrocnemius were fixed in 4% PFA for 48h and then cryoprotected with 30% sucrose in PBS, 10µm slices were obtained in a cryostat for histological staining, and immunofluorescence. Slices were stained with Haematoxylin (Sigma, MHS32) and Eosin (Sigma, E4009), or Sirius Red (Sigma, 365548), mounted, and sealed for further morphometric analysis. Images were obtained using an optical microscope (Leica), three images from different areas of each slice were obtained. All levels were adjusted equally, and the ratios were not altered, morphometric analysis of kidney and muscle sections was performed with ImageJ. Confocal images were acquired as stacks using a Leica SP5-MP confocal microscope and maximum projections were done with the LAS-AF Software. Telomere signal intensity was quantified using Definiens Software and sites of colocalization of telomeric PNA-Cy3 probe and 53BP1 fluorescent signals were counted per cell. Image analysis was performed blindly.

Epigenetic age estimation using epigenetic clocks
DNA from different tissues was obtained using Qiagen DNeasy Blood & Tissue Kit, following the manufacturer's instructions.
Raw files were processed using R package sesame version 1.3.0. The beta values were obtained using the default sesame procedure with nondetection.mask and quality.mask set to FALSE. Differentially methylated cytosines were detected using robust linear regression implemented in R package limma separately for each tissue.
The treatment effect was estimated for each CpG by adjusting for potential confounding from age, sex, and sentrix array row. Obtained p values were corrected for multiple testing using the FDR procedure and those with q < 0.05 were deemed significant. Pathway enrichment analysis was performed by annotating the loci to the nearest gene and ranking them by signed log p-value. The ranked list was passed to g:profiler for enrichment analysis using R package gprofiler2 39 .
Multiple mouse age predictors (epigenetic clocks) were used to estimate the age of the animals. The mouse clocks were trained/developed in a separate data set 60 . The age predictors were grouped into general and intervention clocks. In the general group, the clocks were trained on the following mouse tissues across a wide spectrum of animals: blood, liver, brain, cerebellum, cortex, fibroblast, heart, kidney, muscle, skin, striatum, tail, and a pan tissue clock spanning all different tissues. In the interventions group, the clocks were trained across the same tissues and animals, but the loci used for training were those known to change methylation levels upon known age affecting

Plasma metabolites by Proton Nuclear Magnetic Resonance (1H-NMR) Spectroscopy
For each sample, a mixture of 20 uL of blood plasma and 2 uL of phosphate buffer with trimethylsilyl propanoic acid (TSP) and deuterated water was transferred into a 1 mm high-quality NMR individual tube. Proton NMR spectra for all samples were recorded in a Bruker Avance DRX 600 spectrometer, equipped with a triple resonance

miRNA pathway and biological processes analysis
From the upregulated miRNAs comparing sEVs from young ADSCs to old mice's plasmatic sEVs, the 6 miRNAs conserved across species were analyzed using mirDIP to determine miRNA target genes with very high confidence (top 1% in confidence). A total of 5379 unique genes were further analyzed by PANTHER to identify enriched biological pathways and biological processes with an overrepresentation test, in PANTHER, Fisher's exact test was used with False discovery rate correction. P-value<0,01 was considered statistically significant. For the biological process analysis, we considered only processes with 2-fold enrichment or more.

In vitro model of senescence in muscle progenitor cells
For the in vitro experiments, we used the cell line C2C12 (ATCC CRL-1772), a mouse myoblast established line. The culture media used for the expansion and maintenance of the cells was DMEM high glucose with 10% of foetal bovine serum (FBS) and 1% of Penicillin/Streptomycin (P/S). Cells were maintained at 37°C, 5% CO2. Cells were seeded at a 10.000 cells/cm 2 density, and 24 h after seeding, they were treated with 5µM Palbociclib for 96h. 72h after seeding, myoblasts were treated for 48 hours in EVsdepleted culture medium with 5µg/ml of ADSC-sEVs in PBS or PBS alone as a control.
After that, cells were collected for further analysis by flow cytometry.

Flow cytometry analysis of C2C12 cells
To determine cellular levels of SABG we used FluoReporter lacZ Flow Cytometry Kit until 10000 events were recorded.

Statistical analysis
Ratios comparing physical test and plasma values after treatment compared to baseline were determined and plotted as % over baseline. The baseline was defined as 0%. All groups were tested for the presence of outliers with the ROUT method (Q=2%).