Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Sep 21;9(23):6976-6990.
doi: 10.7150/thno.35305. eCollection 2019.

Embryonic stem cell-derived extracellular vesicles enhance the therapeutic effect of mesenchymal stem cells

Yan Zhang  1   2 , Jia Xu  1   2 , Siying Liu  1   2 , Meikuang Lim  3 , Shuang Zhao  2   4 , Kaige Cui  1   2 , Kaiyue Zhang  1   2 , Lingling Wang  4 , Qian Ji  5 , Zhongchao Han  3 , Deling Kong  2 , Zongjin Li  1   2 , Na Liu  1   2
Affiliations

Embryonic stem cell-derived extracellular vesicles enhance the therapeutic effect of mesenchymal stem cells

Yan Zhang et al. Theranostics. .

Abstract

Background: Embryonic stem cells (ES) have a great potential for cell-based therapies in a regenerative medicine. However, the ethical and safety issues limit its clinical application. ES-derived extracellular vesicles (ES-EVs) have been reported suppress cellular senescence. Mesenchymal stem cells (MSCs) are widely used for clinical cell therapy. In this study, we investigated the beneficial effects of ES-EVs on aging MSCs to further enhancing their therapeutic effects. Methods:In vitro, we explored the rejuvenating effects of ES-EVs on senescent MSCs by senescence-associated β-gal (SA-β-gal) staining, immunostaining, and DNA damage foci analysis. The therapeutic effect of senescent MSC pre-treated with ES-EVs was also evaluated by using mouse cutaneous wound model. Results: We found that ES-EVs significantly rejuvenated the senescent MSCs in vitro and improve the therapeutic effects of MSCs in a mouse cutaneous wound model. In addition, we also identified that the IGF1/PI3K/AKT pathway mediated the antisenescence effects of ES-EVs on MSCs. Conclusions: Our results suggested that ES cells derived-extracellular vesicles possess the antisenescence properties, which significantly rejuvenate the senescent MSCs and enhance the therapeutic effects of MSCs. This strategy might emerge as a novel therapeutic strategy for MSCs clinical application.

Keywords: Cellular senescence; Embryonic stem cells; Extracellular vesicles; IGF1/PI3K/AKT pathway; Mesenchymal stem cells.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Effects of ES-CM on the proliferation ability of senescent MSCs. (A) Schematic representation of the experimental strategy for preparation of conditioned medium. (B) Microscopy showed the morphological change of MSCs treated with ES-CM. Scale bar represents 200 μm. (C) Effect ES-CM on the proliferation potential of MSCs was analyzed by MTT. (D) Immunostaining of Ki67 (left) and the percentage of Ki67-positive cells (right). Scale bar represents 200 um. (E) Cell cycle analysis by flow cytometry. (F) Real-time PCR analysis the expression levels of stemness-related genes in late-passaged MSCs treated with F12, MSC-CM, and ES-CM for 48h, respectively. (G) Western blot analysis the protein levels of OCT4, NANOG and SOX2 in late-passaged MSCs treated with F12, MSC-CM, and ES-CM for 48h, respectively (left). Right panel, quantification of protein levels using ImageJ software, normalized to β-actin. Data are presented as the Mean ± SEM. (n = 3; *p <.05, **p < .01).
Figure 2
Figure 2
Antisenescence effects of ES-CM on late-passaged MSCs. (A) Effects of CM on SA-β-gal activity of late-passaged MSCs (left) and the percentages of SA-β-gal positive cells (right). Scale bar represents 100 um. (B) RT-PCR analysis of stress response genes in late-passaged MSCs treated with F12, MSC-CM, and ES-CM for 48h, respectively. (C) Western blot analysis of protein levels of P16 and P53 in late-passaged MSCs treated with F12, MSC-CM, and ES-CM for 48 h. (D) The activities of caspase 3 in late-passaged MSCs treated with F12, MSC-CM, and ES-CM for 48h (left). Right panel, quantification of protein levels using ImageJ software, normalized to β-actin. (E) DNA damage was analyzed by immunofluorescence staining of γ-H2AX. Scale bar represents 10 μm (left). The percentage of γ-H2AX positive cells was also counted (right). Data are presented as the Mean ± SEM. (n = 3; *p <.05, **p < .01, ***p < .001).
Figure 3
Figure 3
Characteristics of extracellular vesicles derived from ES cells. (A) TEM image of ES-EVs. Scale bar, 100 nm. (B) Size distribution of ES-EVs was measured by dynamic light scattering. (C) The expression of CD9 and CD63 in ES-EVs was analyzed using western blotting. (D) Internalization of ES-EVs was analyzed by immunofluorescence detection. Dil-labeled exosomes (red) was detected in the MSCs which expressing green fluorescent protein (GFP, green). Scale bar, 10 μm.
Figure 4
Figure 4
Antisenescence activity of ES-EVs. (A) RT-PCR analysis of the expression levels of senescence-associated genes in late-passaged MSCs treated with F12, MSC-CM, and ES-EVs for 48h respectively. (B) Protein levels of P16 and P53 were detected by western blotting. Right panel, quantification of protein levels normalized to β-actin. (C) The activities of caspase 3 in late-passaged MSCs treated with F12, MSC-CM, and ES-EVs for 48h respectively. (D) Effects of ES-EVs on SA-β-gal activity of MSCs and the percentage of SA-β-gal-positive cells. Scale bar represents 200 μm. (E) DNA damage foci γ-H2AX was detected by immunofluorescence staining. Scale bar represents 10 μm. Data are presented as the Mean ± SEM. (n = 3; *p <.05, **p < .01, ***p < .001).
Figure 5
Figure 5
Enhanced wound-healing process of senescent MSCs by ES-EVs. (A) The fate of MSCs after transplantation was tracked by molecular imaging. Images were from representative animals receiving 5×105 MSCs, which was pretreated with F12, MSC-CM or ES-EVs respectively. (B) Quantitative analysis of BLI signals demonstrate that cell survival was improved by ES-EVs at all time points. (C) Analysis of the wound-healing area at different time poins (left) and the quantitative analysis of wound-healing area (right). (D) Histologic analysis of wound area by HE staining. Scale bar represents 100 μm. (E) Histologic analysis of wound area by Massion trichrome, Scale bar represents 100 μm. Data are presented as the Mean ± SEM. (n = 3; *p <.05, **p < .01).
Figure 6
Figure 6
ES-EVs activate the IGF1/PI3K/AKT pathway in senescent MSCs. (A) Expression levels of PI3K, AKT, and p-AKT in MSCs with different treatment were detected by western blot (left). Histogram showed the quantitative analysis of western blot (right). (B) IGF1 was detected in ES-EVs using western blot analysis. (C) RT-PCR analysis the expression of IGF1R in late-passaged MSCs treated with F12, MSC-CM, and ES-EVs for 48 hours. (D) Expression level of IGF1R in MSCs with ES-EVs treatment for 48 hours was detected by western blot. (E) Protein levels of P16, PI3K, AKT, and p-AKT in late-passaged MSCs treated with picropodophyllin (PPP: IGF1R inhibitor) were analyzed using western blot (left). Histogram showed the quantitative analysis of western blot (right). (F) Effect of PPP on SA-β-gal activity of late-passaged MSCs. Scale bar represents 100 μm. (G) Histogram showed the quantitative analysis of the percentage of SA-β-gal-positive cells. Data are presented as the Mean ± SEM. (n = 3; *p <.05, **p < .01).
Figure 7
Figure 7
Inhibition of IGF1/PI3K/AKT pathway attenuates the in vivo effects of ES-EVs on senescent MSCs. (A) The fate of MSCs after transplantation was tracked by molecular imaging. Images were from representative animals receiving 5×105 MSCs with F12, ES-EVs, or ES-EVs and PPP. (B) Quantitative analysis of BLI signals. (C) Analysis of the wound-healing area at different time points (left). Quantitative analysis of wound-healing area (right). (D) Histologic analysis of wound area by HE staining. Scale bar represents 50um. Data are presented as the Mean ± SEM. (n = 3; *p <.05).
Figure 8
Figure 8
Schematic illustration the role of ES-EVs on MSCs. The ES-EVs transfer the IGF1, a secreted factor derived from ES cells, to senescent MSCs and activate the IGF1R/AKT signaling pathway of MSCs. Then mediating ES-EVs enhances the therapeutic effect of MSCs by improving cellular proliferation, increasing stemness, suppressing the senescence phenotypes, decreasing SA-β-gal activity, and reducing DNA damage.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Kramann R, Goettsch C, Wongboonsin J, Iwata H, Schneider RK, Kuppe C. et al. Adventitial MSC-like Cells Are Progenitors of Vascular Smooth Muscle Cells and Drive Vascular Calcification in Chronic Kidney Disease. Cell Stem Cell. 2016;19:628–42. - PMC - PubMed
    1. Gu W, Hong X, Potter C, Qu A, Xu Q. Mesenchymal stem cells and vascular regeneration. Microcirculation. 2017;24:e12324. - PubMed
    1. Wu Y, Chen L, Scott PG, Tredget EE. Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells. 2007;25:2648–59. - PubMed
    1. Ahmed AS, Sheng MH, Wasnik S, Baylink DJ, Lau KW. Effect of aging on stem cells. World J Exp Med. 2017;7:1–10. - PMC - PubMed
    1. Campisi J. The biology of replicative senescence. Eur J Cancer. 1997;33:703–9. - PubMed

Publication types

MeSH terms

Substances