Fig. 8.

Rejuvenation of aged mice by ESC-EVs. (A) Schematic illustrating the procedures of the in vivo experiment. (B) A scatter plot illustrating differentially regulated gene expression from RNA-Seq analysis between the control and ESC-EVs-treated aged mice. Up-regulated and down-regulated genes are shown in red and blue, respectively. (p < 0.05 and FC ≥ 2 or FC ≤ 0.5). (C) PCA of RNA-Seq of PBMCs from young mice, aged mice, and ESC-EVs-treated aged mice. (D) Heat map of differentially regulated genes (p < 0.05 and FC ≥ 1.5 or FC ≤ 0.67) expression from RNA-Seq analysis between the aged mice and ESC-EVs treated aged mice. (E) GSEA analysis identifies enrichment scores for modules of aging, p53 pathway and PI3K-AKT pathway. (F) Representative tissue morphology and SA-β -Gal staining of liver, kidney and spleen after ESC-EVs treatment, and quantification of the percentage of SA-β -Gal positive area. Scale bar represents 100 μm. (G) RT-qPCR analysis of senescence-related genes in liver and kidney after ESC-EVs treatment. (H) Immunofluorescence analysis of p21 expression in the liver after ESC-EVs treatment. Scale bar represents 100 μm. (I) HE staining was used for histological analysis of the liver and kidney after 8 weeks of ESC-EVs treatment. In HE staining of the liver, the arrows indicate the pathological features of hepatocyte enlargement and hepatic cord disruption. In the HE staining of the kidney, the arrows at the top indicate the enlargement of the renal cystic cavity and the glomerular pyknosis, while the arrows at the bottom indicate thinning of the renal tubular wall and enlargement of the lumen. Scale bar represents 100 μm. Data are presented as mean ± SEM. n = 3. Student's t-test: ns, not significance; *p < 0.05 and **p < 0.01.