Figure 7.

ESC-sEVs Reverse Cognitive Aging and Rejuvenate H-NSC Senescence in C57BL/6 Mice

(A) Spatial learning and memory abilities were tested by an MWM test in 20 month-PBS and 20 month-sEV mice. n = 9 per group. ∗∗∗p < 0.001. (B) Western blot analysis and quantification of Syp, Gap-43, PSD-95, and Syn-IIa in hippocampus of 20 month-PBS and 20 month-sEV mice. n = 6 per group. ∗∗∗p < 0.001. (C) Western blot analysis of p-SMAD5, SMAD5, SMAD4, MYT1, Egln3, HIF-2α, NAMPT, and Sirt1 in isolated neurospheres of 20 month-PBS and 20 month-sEV mice. n = 3 per group. (D) SA-β-gal staining of hippocampus and quantification of hippocampal SA-β-gal activity in 20 month-PBS and 20 month-sEV mice. Scale bars, 250 μm. n = 6 per group. ∗∗∗p < 0.001. (E) IF images for senescent H-NSCs (p16^INK4a+, red; Sox2⁺, green) and the percentage of Sox2⁺/p16^INK4a+ double-stained cells in whole Sox2⁺ cells in the SGZ of 20 month-PBS and 20 month-sEV mice. Scale bars, 50 μm. n = 6 per group. ∗∗∗p < 0.001. (F) SA-β-gal staining of isolated H-NSCs and the percentage of SA-β-gal⁺ cells in 20 month-PBS and 20 month-sEV mice. Scale bars, 100 μm. n = 3 per group. ∗∗∗p < 0.001. (G) Western blot analysis of p16^INK4a, γ-H2AX, p21, and p53 in isolated neurospheres of 20 month-PBS and 20 month-sEV mice. n = 3 per group. (H) IF images for hippocampal Sox2⁺ (green) and GFAP⁺ (red) cells and the number of Sox2⁺/GFAP⁺ double-stained cells in the SGZ of 20 month-PBS and 20 month-sEV mice. Scale bars, 50 μm. n = 6 per group. ∗∗∗p < 0.001. (I) IF images for proliferating H-NSCs (Sox2⁺, green; EdU⁺, red) and the number of Sox2⁺/EdU⁺ double-stained cells in the SGZ of 20 month-PBS and 20 month-sEV mice. Scale bars, 50 μm. n = 6 per group. ∗∗∗p < 0.001.