Unraveling the aging-reversal potency of stem cell-derived extracellular vesicles in a rat model of premature cardiac senescence

Figures

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  Figure 1. Donor chronological age vs. CDC biological age effect on different CDC properties (A) Representative images from one chronologically aged donor (70-years old), but biologically young (9.1% of senescent CDCs) vs. one chronologically young donor (7-months old), but biologically aged (14.8% of senescent CDCs) after 72 h of culture. (B) Chronological age is not significantly related to CDC senescence (n = 34 donors), population doubling time (PDT, n = 14), or DNA synthesis capacity (% Ki-67+ CDCs, n = 16) after 72 h of culture. Chronological age significantly affects CDC migration capacity after 24 h of wound generation (n = 16). (C) CDC senescence however relates to PDT (n = 14), DNA synthesis (n = 16), and migration capacity (n= 16) significantly and to a higher extent. Adult samples are represented with a square, while pediatric samples are represented with a diamond. Male samples are shown in black and female samples in purple. White scale bar corresponds to 100 μm, black scale bar to 400 μm. p values obtained with t test. Data are represented as mean ± SEM.

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  Figure 2. Correlation between EDC senescence (n= 17 donors) and CDC senescence (n = 18) vs. derived CDC-EVs anti-senescent potency in one of the CSC target donors (n = 1199 ± 27 cells/donor) Adult samples are represented with a square, while pediatric samples are represented with a diamond. Male samples are shown in black and female samples in purple. p values obtained with t test. Data are represented as mean ± SEM.

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  Figure 3. CDC-EV potency assay results for the different donors (A) Characterization and performance of CDC-EVs from the different donors in the different tests in the potency matrix assay. The values in the table indicate donor position according to results in the potency test, age, gender, and performance in each of the different tests in the potency assay matrix. Reduction in cardiac stroma cell (CSC) senescence (in two target donors) induced by the different CDC-EVs (n = 1199 ± 27 cells/condition in target donor 1 and n = 1520 ± 18 cells/condition in target donor 2). Increment in CSC IL-6 secretion vs. serum-free media (SFM) alone (in two target donors) induced by the different CDC-EVs (n = 3 samples/condition). CDKN1A, CDKN2A, TP53, and TGFB1 relative expression in CSC (from two target donors) under the effect of the different CDC-EVs (n = 3 samples/condition). Relative tube formation in endothelial cells (HUVEC) under the effect of the different CDC-EVs (n = 19 ± 1 images/condition). All data are with respect to untreated CSCs or endothelial cells under SFM. Values in green indicate the donors that showed the most effect in the corresponding test (belonged to the best tercile) and…

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  Figure 4. Performance of the P-EVs (donor 2) and NP-EVs (donor 18) vs. control conditions (SFM) in some potency tests in the assay (A) Representative images from the senescence and the tube formation test under control conditions, P-EVs and NP-EVs. (B) Both P-EVs and NP-EVs reduced senescence of CSCs of target 2, but P-EVs did it to a significantly larger extent (n = 1520 ± 18 cells/condition). Relative tube formation in endothelial cells (HUVEC) under the effect of P-EVs (n = 19 ± 1 images/condition) significantly increased, while it kept similar (slightly reduced, difference not significant) under the effect of NP-EVs. P, potent; NP, non-potent; SFM, serum free media. ∗p < 0.05. Black scale bar corresponds to 100 μm, white scale bar to 400 μm. p values obtained with t test. Data are represented as mean ± SEM.

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  Figure 5. Cardiac effects of D-gal and P- and NP-EV administration (A) P-EVs and NP-EVs rejuvenating and pro-angiogenic potency was tested in a rodent model of cardiac aging. Timeline including group distribution, product administration and tests performed. (B) Effects on cardiac senescence at genetic level (Healthy: n = 6, D-gal: n = 11, D-gal + P-EVs: n = 7, D-gal + NP-EVs: n = 6). (C) Effects on cardiac hypertrophy on the whole population (Healthy: n = 8, D-gal: n = 12, D-gal + P-EVs: n = 7, D-gal + NP-EVs: n = 6). (D and E) (D) Effects on cardiac fibrosis at genetic (Healthy: n = 6, D-gal: n = 11, D-gal + P-EVs: n = 7, D-gal + NP-EVs: n = 6) and (E) tissue level (Healthy: n = 4, D-gal: n = 12, D-gal + P-EVs: n = 7, D-gal + NP-EVs: n = 6). Representative images of interstitial fibrosis in animals in each group. (F) Effects on cardiac perfusion (Healthy: n = 4, D-gal: n = 4, D-gal + P-EVs: n = 6, D-gal + NP-EVs: n = 4). Representative images of single photon emission computer tomography (SPECT) intensity corrected by injected activity, animal weight, and acquisition time in animals in the different groups. F, female; D-gal, D-galactose; EVs, extracellular vesicles; P, potent; NP,…

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  Figure 6. Effect of of D-gal and P- and NP-EV administration in TNF-α and IL-6 levels in peripheral blood (Healthy: n = 4, D-gal: n = 4, D-gal + P-EVs: n = 4, D-gal + NP-EVs: n = 4) D-gal, D-galactose; EVs, extracellular vesicles; P, potent; NP, non-potent. ∗p < 0.05 vs. D-gal, #p < 0.05 vs. Healthy, Ϯ p < 0.05 vs. NP-EVs in Student t test. Data are represented as mean ± SEM.