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

Abstract

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.

Fig. 1.. Characterization of sEVs from ADSC culture medium, which are delivered to kidney and muscle of old mice.

(A) Nanoparticle tracking analysis (NTA) of sEVs from young ADSCs. (B) Western blot image of staining with TSG101 and bovine serum albumin, a sample of purified sEVs from the cell culture medium of ADSCs, and a sample of FBS present in the same medium are shown. (C) Flow cytometry determination of CD63 presence in sEVs obtained from the cell culture medium of young ADSCs. (D and E) Representative image of ADSC-sEVs as observed by transmission electron microscopy (TEM) and CD63 determination with immuno-gold labeling. (F) Images of kidney and muscle from old mice treated with PKH26-labeled sEVs from young ADSCs and vehicle as a control.

Fig. 2.. ADSC-sEVs improve health span and prevent frailty in old mice.

(A) Schematic representation of the experimental design. (B) Quantification of the change in total body weight; data are shown as the decrease in percentage from the baseline before treatment with sEVs/PBS. PBS: day 14, n = 12; day 30, n = 12; sEVs: day 14, n = 14; day 30, n = 15. (C to E) Quantification of the change in physical condition tests; data are shown as the change in percentage from baseline before treatment with sEVs/PBS. Grip strength: PBS: day 14, n = 11; day 30, n = 11; sEVs: day 14, n = 13; day 30, n = 11. Motor coordination: PBS: day 14, n = 14; day 30, n = 12; sEVs: day 14, n = 16; day 30, n = 18. Fatigue resistance: PBS: day 14, n = 12; day 30, n = 11; sEVs: day 14, n = 14; day 30, n = 15. (F) Quantification of the number of frail mice before and 30 days after treatment with sEVs/PBS, determined by a score based on the clinical phenotype of frailty. PBS: day 0, n = 12; day 30 n = 12; sEVs: day 0, n = 15; day 30, n = 15. (G) Quantification of the hair regrowth capacity of dorsal skin in old mice. PBS, n = 7; sEVs, n = 7. a.u., arbitrary units. (H) Representative image of hair regeneration in both male and female mice before and 14 days after treatment with sEVs/PBS. (I) Quantification of the changes in plasmatic urea; data are shown as the change in percentage from basal levels of urea in plasma before treatment with sEVs/PBS. PBS: day 14, n = 5; day 30, n = 4; sEVs: day 14, n = 5; day 30, n = 6. All data are shown as means ± SD.

Fig. 3.. ADSC-sEVs induce proregenerative effects on kidney and muscle of old mice.

(A) Macroscopic image of kidneys isolated from old mice treated with PBS/sEVs. (B to D) Quantification of histological changes in the kidney (renal cortex) from old mice induced by sEVs. PBS, n = 6; sEVs, n = 6. (E) Hematoxylin and eosin representative images of the effects on the structure of the renal cortex of old mice. (F) Representative image of Sirius red staining of the renal cortex of old mice. (G) Representative immunofluorescence images showing Ki67⁺ cells in the renal tubules of old mice. Morphology marker refers to background fluorescence to better depict tubular morphology. (H) Quantification of extracellular fibrosis by Sirius red staining. PBS, n = 6; sEVs, n = 6. (I) Quantification of Ki67⁺ cells in the renal tubules of old mice. PBS, n = 4; sEVs, n = 4. (J) Hematoxylin and eosin representative images of gastrocnemius of old mice. (K and L) Quantification of the mean cross-sectional area (CSA) of muscle fibers and total protein content in the gastrocnemius of old mice. CSA: PBS, n = 6; sEVs, n = 6. Protein content: PBS, n = 11; sEVs, n = 6. (M and N) Representative immunofluorescence images of MHC2A and LipidTOX staining in gastrocnemius of old mice. (O) Quantification of the percentage of MHC2A⁺ fibers in gastrocnemius of old mice. PBS, n = 6; sEVs, n = 6. (P) Quantification of LipidTOX staining intensity in gastrocnemius of old mice. PBS, n = 4; sEVs, n = 4. All data are shown as means ± SD.

Fig. 4.. sEVs from young ADSCs ameliorate molecular traits of aging in the kidney and muscle and lower senescence in old mice and in an in vitro model.

(A) Quantification of MDA as a marker of lipid peroxidation in the kidney and muscle from old mice. PBS: kidney, n = 10; muscle, n = 11; sEVs: kidney, n = 6; muscle, n = 6. (B) Quantification of protein carbonylation as a marker of protein oxidation in the kidney and muscle of old mice. PBS: kidney, n = 7; muscle, n = 7; sEVs: kidney, n = 6; muscle n = 6. (C) Mean telomere length measured as arbitrary units of fluorescence (A.U.F.) in the kidney and muscle of old mice. PBS: kidney, n = 6; muscle, n = 6; sEVs: kidney, n = 6; muscle, n = 6. (D) Quantification of telomere dysfunction–induced foci (TIF) measured as number of telomeric probe and DNA damage marker 53BP1 colocalizing foci per cell by Immuno FISH in the kidney and muscle of old mice. PBS: kidney, n = 5; muscle, n = 5; sEVs: kidney, n = 6; muscle, n = 6. (E and F) Quantification of SASP factors IL-6 and IL-1β in the kidney and muscle of old mice. PBS: kidney, n = 5; muscle, n = 5; sEVs: kidney, n = 5; muscle, n = 5. (G to L) Quantification and representative immunofluorescence images of LMNB1 loss and γH2AX⁺ cells in the kidney and gastrocnemius of old mice. PBS: kidney, n = 4; muscle, n = 4; sEVs: kidney, n = 4; muscle, n = 4. (M) Graphical representation of in vitro experiment with senescent murine myoblasts. (N and O) Assessment of senescence-associated β-galactosidase activity and annexin V by flow cytometry in senescent murine myoblasts. Untreated, n = 5; palbociclib, n = 5; palbociclib + sEVs, n = 5. All data are shown as means ± SD. (P) Quantification of Ki67⁺ cells in senescent murine myoblasts. palbociclib, n = 3; palbociclib + sEVs, n = 3. (Q and R) Quantification of SASP factors IL-6 and IL-1β in senescent murine myoblasts. Palbocilib, n = 5; palbociclib + sEVs, n = 5. (S) Assessment of senescence-associated β-galactosidase activity by flow cytometry in senescent murine myoblasts. Palbociclib, n = 6; palbociclib + 3T3-sEVs, n = 6; palbociclib + C2C12-sEVs, n = 6. All data are shown as means ± SD.

Fig. 5.. ADSC-sEVs decrease the epigenetic age of several tissues in old mice.

(A) ADSC-sEV treatment effect on epigenetic age estimation mixed-effects modeling across several tissues. (B to E) Biological process enrichment analysis of differentially methylated cytosines in ADSC-sEV–treated tissues (liver, muscle, kidney, and spleen, respectively). PBS, n = 6; ADSC-sEVs, n = 6. All data are shown as means ± SD. BMP, bone morphogenetic protein; GTPase, guanosine triphosphatase; MAP, mitogen-activated protein; MAPK, MAP kinase.

Fig. 6.. The metabolome of ADSC-sEV–treated mice resembles that of young animals.

(A) Heatmap of metabolite z scores with statistically significant differences between old-PBS (black bar samples) and old-sEV (red bar samples) mice compared with young untreated mice (blue bar samples). (B) Mean and 90% confidence intervals for z scores in SD units for the same metabolites that (A) in old-PBS (black), old-sEVs (red), and young untreated (blue) mice. (C) Score plot for a metabolome partial least-squares discriminant analysis (PLS-DA) model (72 metabolites) for discrimination between young (blue squares) and old-PBS (black circles) mice. Old mice treated with sEVs (red circles) are projected in the score plot. (D) Variable importance in projection (VIP) scores in the metabolome PLS-DA model of (C) for those metabolites significantly associated with treatment. (E) Enrichment ratio (size of the circle) and P values (color of the circle) for the 25 most enriched metabolite sets from a metabolite set enrichment analysis (MSEA) on the Small Molecule Pathway Database (SMPDB) and our 14 significant metabolites. The n used for all metabolomic analyses was young mice, n = 4; old-PBS mice, n = 6; and old-sEV mice, n = 6.

Fig. 7.. Tissue development and regeneration are major targets of miRNAs enriched in young ADSC-sEVs, with an effect on senescence in vitro.

(A) PCA of miRNA profiling in sEVs from young ADSCs, old ADSCs, and plasma of aged mice. Old plasma, n = 4; old ADSC-sEVs, n = 4; and young ADSC-sEVs, n = 4. (B) Summary of differentially expressed miRNAs under the three conditions. (C) Heatmap of differentially expressed miRNAs in sEVs from young and old ADSCs. (D) Heatmap of differentially expressed miRNAs in sEVs from young ADSCs and plasma of aged mice. (E) Volcano plot showing the different miRNA expression between plasma of aged mice and young ADSC-sEVs. NS, not significant. (F and G) Assessment of senescence-associated β-galactosidase activity by flow cytometry in senescent murine myoblasts treated with miRNA mimics. Vehicle (VEH), n = 13; small interfering RNA control, n = 13; miR-125b-5p, n = 9; miR-let7c-5p, n = 17; miR-214-3p, n = 15; miR-145a-5p, n = 17; miR-135a-3p, n = 17; miR-143-3p, n = 9.