Small extracellular vesicles secreted by urine-derived stem cells enhanced wound healing in aged mice by ameliorating cellular senescence

doi:10.1016/j.jmst.2020.03.014

Abstract

Abstract:

Regeneration of chronic skin wounds in tissue is still a key challenge in regenerative medicine because of the accumulation of senescent cells and increasing secretion of "senescence-associated secretory phenotype"-(SASP) in the wound site. Recently, some studies have reported that small extracellular vesicles (sEVs) derived from stem cells can alleviate cellular senescence with very low risk of tumorigenesis and immune responses. As our previous studies have shown that urine-derived stem cells (USCs) can be obtained easily and noninvasively and sEVs derived from USCs (USC-sEVs) have capabilities of regenerating tissue injuries, using USC-sEVs to enhance chronic skin wound healing in aged tissue might be a feasible and efficient strategy. Therefore, in this study, the USC-sEVs were collected and firstly loaded in a human acellular amniotic membrane (HAAM) for controlled releasing and locating the USC-sEVs in the wound site before they were implanted into a chronic skin wound in aged mice. In vivo results showed that the USC-sEVs in HAAM could effectively accelerate the wound healing by ameliorating cellular senescence and reducing the secretion of SASP in the aged skin wounds. To elucidate the mechanism, USC-sEVs were used to in vitro culture human dermal fibroblasts (HDFs) and results showed that USC-sEVs could rejuvenate senescent fibroblasts by reversing the aging phenotypes of senescent HDFs and efficiently reducing the secretion of SASP after they activated the Sirt1 pathway. Therefore, USC-sEVs are efficient for enhancing wound healing in aged mice by ameliorating cellular senescence.

Key words: Stem cells, Extracellular vesicle, Aged tissue, Wound healing

Yongjin Sun, Juntao Zhang, Bi Chen, Yunlong Yang, Haiyan Li, Xin Niu, Qing Li, Weidong Wu, Zongping Xie, Yunfeng Chen, Fuyue Wu, Yang Wang. Small extracellular vesicles secreted by urine-derived stem cells enhanced wound healing in aged mice by ameliorating cellular senescence[J]. J. Mater. Sci. Technol., 2021, 63: 216-227.

Figures/Tables 8

Fig. 1. Characterization of USCs, USC-derived sEVs and HAAM. (a) Flow cytometric analyses of phenotypic markers of USCs. USCs were negative for CD34, CD45, and HLA-DR and were positive for CD29, CD44, and CD73. (b) Western blot analysis showed the presence of sEVs characteristic CD9, CD63, and TSG101 proteins, but the absence of GM130 protein. (c) Morphology of sEVs under TEM. Scale bar, 100 nm. (d) Particle size distribution of UCS-sEVs measured by qNano analysis.

Fig. 2. Characterization of HAAM. (a) H&E staining of HAAM section. Scale bar, 25 μm. (b) SEM images of the surface of HAAM. Scale bar, 1 μm. (c) SEM images of the cross section of HAAM. Scale bar, 1 μm. (d) Pore size distribution of HAAM. (e) Controlled-release of particles of HAAM loaded with USC-sEVs. Note significant difference of 180 min group and 240 min group from 120 min group (from 12 h until day 6; P < 0.05). No difference between 180 min and 240 min group. (f) The fluorescent images of live and dead cells after HDFs cultured on HAAM with or without USC-sEVs. Scale bar, 50 μm. Statistical significance was determined by one-way ANOVA (Tukey’s multiple-comparisons test).

Fig. 3. Wound healing was significantly promoted by HAAM loaded with USC-sEVs in aged mice. (a) Representative images of wounds treated with PBS in young mice (Control), and wounds treated with PBS (Aged), HAAM (Aged-HAAM), USC-sEVs (Aged-sEVs), HAAM loaded with USC-sEVs (Aged-HAAM-sEVs) in aged mice, at 0, 3, 7, 14 and 21days after initial treatment. (b) Percent wound closure. n = 6 per group. (c) H&E staining of wound sections obtaining from control, Aged, Aged-HAAM, Aged-sEVs, Aged-HAAM-sEVs groups at 21 days after initial treatment. The black arrows indicate the edges of the scar. Scale bar, 200 μm. (d) Quantification of scar width. n = 3 per group. (e) Masson’s trichrome staining of wound sections obtaining from control, Aged, Aged-HAAM, Aged-sEVs, Aged-HAAM-sEVs groups at 21 days after initial treatment. Scale bar, 100 μm (100×) or 25 μm (400×). (*, P < 0.05, compared with control; #, P < 0.05, compared with Aged; %, P < 0.05, compared with Aged-HAAM; &, P < 0.05, compared with Aged-sEVs). Statistical significance was determined by one-way ANOVA (Tukey’s multiple-comparisons test).

Fig. 4. IHC analysis of P16 protein and SASP expression in wound sites of control, Aged, Aged-HAAM, Aged-sEVs, Aged-HAAM-sEVs groups at 7 days after initial treatment. (a) IHC staining for P16. Scale bar, 25 μm. (b) IHC staining for SASP (IL-1β, IL-6, MMP-13). Scale bar, 25 μm. (c) Quantification of IHC staining of P16. (d) Quantification of IHC staining of SASP (IL-1β, IL-6, MMP-13). (*, P < 0.05, compared with control; #, P < 0.05, compared with Aged; %, P < 0.05, compared with Aged-HAAM; &, P < 0.05, compared with Aged-sEVs). Statistical significance was determined by one-way ANOVA (Tukey’s multiple-comparisons test).

Fig. 5. USC-sEVs protected HDFs from senescence induced by d-gal in vitro experiments. HDFs were treated with 10 g/L d-gal to induce senescence, then the senescent HDFs were treated with 1 × 1010 particles/mL USC-sEVs (Agd-sEVs) or PBS (Aged), while HDFs treated without d-gal (Young) were used as control. (a) Representative micrographs of HDFs stained with SA-β-gal. SA-β-gal-positive cells are shown in blue. Scale bar, 150 μm. (b) Percentage of SA-β‐Gal positive cells. n = 3 per group. (c) IF staining for P16 (red). DAPI was used to stain the nuclei. Scale bar, 50 μm. (d) Percentage of P16 positive cells. n = 3 per group. (e) IF staining for Ki-67 (red). DAPI was used to stain the nuclei. Scale bar, 50 μm. (f) Percentage of Ki-67 positive cells. n = 3 per group. (g) The expression of p16 and p21 were assessed by Western blotting. (h) ELISA-based assay for detecting the concentrations of collagen Ⅰ. (*, P < 0.05, compared with young; #, P < 0.05, compared with Aged). Statistical significance was determined by one-way ANOVA (Tukey’s multiple-comparisons test).

Fig. 6. USC-sEVs decreased the expression of SASP in senescent HDFs in vitro experiments. HDFs were treated with d-gal to induce senescence, then the senescent HDFs were treated with USC-sEVs (Agd-sEVs) or PBS (Aged), while HDFs treated without d-gal (Young) were used as control. (a) ELISA-based assay for detecting the concentrations of SASP (IL-1β, IL-6, MMP-13). n = 3 per group. (b) Quantification of mRNA expression for SASP (IL-1β, IL-6, MMP-13). n = 3 per group. (*, P < 0.05, compared with young; #, P < 0.05, compared with Aged). Statistical significance was determined by one-way ANOVA (Tukey’s multiple-comparisons test).

Fig. 7. USC-sEVs ameliorate aging phenotypes of senescent HDFs through activating Sirt1 in vitro experiments. HDFs were treated with d-gal to induce senescence, then the senescent HDFs were treated with USC-sEVs (Agd-sEVs) or co-treated with USC-sEVs and Sirt1 inhibitor (Aged-sEVs-Nicotinamide), while HDFs treated without d-gal (Young) and aged HDFs without treatment (Aged) were set as controls. (a) The expression of Sirt1 were assessed by Western blotting. (b) Representative micrographs of HDFs stained with SA-β-gal. SA-β-gal-positive cells are shown in blue. Scale bar, 150 μm. (c) Percentage of SA‐β‐Gal positive cells. n = 3 per group. (d) The expression of p16 and p21 were assessed by Western blotting. (e) IF staining for P16 (red). DAPI was used to stain the nuclei. Scale bar, 50 μm. (f) Percentage of P16 positive cells. n = 3 per group. (g) IF staining for Ki-67 (red). DAPI was used to stain the nuclei. Scale bar, 50 μm. (h) Percentage of Ki-67 positive cells. n = 3 per group. i) ELISA-based assay for detecting the concentrations of collagen Ⅰ. (*, P < 0.05, compared with Aged; #, P < 0.05, compared with Aged-sEVs). Statistical significance was determined by one-way ANOVA (Tukey’s multiple-comparisons test).

Fig. 8. USC-sEVs decreased the expression of SASP in senescent HDFs through activating Sirt1 in vitro experiments. HDFs were treated with d-gal to induce senescence, then the senescent HDFs were treated with USC-sEVs (Agd-sEVs) or co-treated with USC-sEVs and Sirt1 inhibitor (Aged-sEVs-Nicotinamide), while aged HDFs without treatment (Aged) were set as control. (a) ELISA-based assay for detecting the concentrations of SASP (IL-1β, IL-6, MMP-13). n = 3 per group. (b) Quantification of mRNA expression for SASP (IL-1β, IL-6, MMP-13). n = 3 per group. (*, P < 0.05, compared with Aged; #, P < 0.05, compared with Aged-sEVs). Statistical significance was determined by one-way ANOVA (Tukey’s multiple-comparisons test).

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