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. 2024 Jun 26;22(1):373.
doi: 10.1186/s12951-024-02650-x.

Activation of the Wnt/β-catenin signalling pathway enhances exosome production by hucMSCs and improves their capability to promote diabetic wound healing

Liming Wang  1 , Jun Chen  1   2   3   4 , Jia Song  1   2   3   4 , Yingyue Xiang  1 , Mengmeng Yang  1 , Longqing Xia  1 , Jingwen Yang  1 , Xinguo Hou  1   2   3   4 , Li Chen  5   6   7   8 , Lingshu Wang  9   10   11   12
Affiliations

Activation of the Wnt/β-catenin signalling pathway enhances exosome production by hucMSCs and improves their capability to promote diabetic wound healing

Liming Wang et al. J Nanobiotechnology. .

Abstract

Background: The use of stem cell-derived exosomes (Exos) as therapeutic vehicles is receiving increasing attention. Exosome administration has several advantages over cell transplantation, thus making exosomes promising candidates for large-scale clinical implementation and commercialization. However, exosome extraction and purification efficiencies are relatively low, and therapeutic heterogeneity is high due to differences in culture conditions and cell viability. Therefore, in this study, we investigated a priming procedure to enhance the production and therapeutic effects of exosomes from human umbilical cord mesenchymal stem cells (hucMSCs). After preconditioning hucMSCs with agonists/inhibitors that target the Wnt/β-catenin pathway, we assessed both the production of exosomes and the therapeutic efficacy of the optimized exosomes in the context of diabetic wound healing, hoping to provide a safer, more stable and more effective option for clinical application.

Results: The Wnt signalling pathway agonist CHIR99021 increased exosome production by 1.5-fold without causing obvious changes in the characteristics of the hucMSCs or the size of the exosome particles. Further studies showed that CHIR99021 promoted the production of exosomes by facilitating exocytosis. This process was partly mediated by SNAP25. To further explore whether CHIR99021 changed the cargo that was loaded into the exosomes and its therapeutic effects, we performed proteomic and transcriptomic analyses of exosomes from primed and control hucMSCs. The results showed that CHIR99021 significantly upregulated the expression of proteins that are associated with cell migration and wound healing. Animal experiments confirmed that, compared to control hucMSC-derived exosomes, CHIR99021-pretreated hucMSC-derived exosomes (CHIR-Exos) significantly accelerated wound healing in diabetic mice, enhanced local collagen deposition, promoted angiogenesis, and reduced chronic inflammation. Subsequent in vitro experiments confirmed that the CHIR-Exos promoted wound healing by facilitating cell migration, inhibiting oxidative stress-induced apoptosis, and preventing cell cycle arrest.

Conclusions: The Wnt agonist CHIR99021 significantly increased exosome secretion by hucMSCs, which was partly mediated by SNAP25. Notably, CHIR99021 treatment also significantly increased the exosomal levels of proteins that are associated with wound healing and cell migration, resulting in enhanced acceleration of wound healing. All of these results suggested that pretreatment of hucMSCs with CHIR99021 not only promoted exosome production but also improved the exosome therapeutic efficacy, thus providing a promising option for large-scale clinical implementation and commercialization.

Keywords: Exocytosis; Exosomes; HucMSCs; Wnt/β-catenin signalling; Wound healing.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
The characteristics of hucMSCs and exosomes remained unchanged after Wnt signalling priming. (A) Alizarin red and oil red O staining after treatment of hucMSCs with Wnt agonists or inhibitors (B) Flow cytometry identification of hucMSCs maker after treatment of hucMSCs with Wnt agonists or inhibitors. (C) Western blot analysis of exosome marker from different pretreated hucMSCs. (D) Exosome transmission electron microscopy. (E) Exosome NTA assay
Fig. 2
Fig. 2
Wnt agonist treatment increased hucMSC exosome production. (A) ELISA detection of AChE in hucMSC supernatants from different treatment groups (B) Exosome particle size distribution. (C) Quantification of different treatment groups of exosomes by NTA. (D-F) Representative TEM images of hucMSCs and the quantitation analysis on the MVBs and ILVs after hucMSCs were treated with Wnt agonists or inhibitors. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01
Fig. 3
Fig. 3
CHIR99021 increased exosome secretion by promoting SNAP25-dependent exocytosis. (A) Volcano plot showing differential gene expression of hucMSCs in the CHIR99021-treated group versus the control group. (B) GO enrichment analysis of hucMSCs in CHIR99021-treated group versus control group. (C) KEGG enrichment analysis of hucMSCs in CHIR99021-treated group versus control group. (D) Heatmap of gene clustering of hucMSCs transcriptomics in synaptic vesicle cycle between CHIR99021-treated and control groups. (E) Western blot analysis of β-catenin, Rab27a, Rab7, SNAP25 in hucMSCs treated with Wnt signaling pathway agonists and inhibitors. (F) Co-location of SNAP25 with VAMP3 was observed using immunofluorescence after treatment of hucMSCs with Wnt signaling pathway agonists and inhibitors. (G) Co-location of LAMP2 with CD63 was observed using immunofluorescence after treatment of hucMSCs with Wnt signaling pathway agonists and inhibitors. (H) Western blot analysis of SNAP25 knockdown efficiency. (I) After knocking down of SNAP25, hucMSCs were treated with Wnt signaling pathway agonists for 48 h, supernatants were collected, and AChE concentration was analysis by ELISA. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01
Fig. 4
Fig. 4
CHIR99021 enhanced the loading of wound healing-related proteins into exosomes. (A) Volcano plot showing differential protein of exosome between CHIR99021-treated and control groups. (B) GO enrichment analysis between CHIR99021 treatment group and control group. (C-D) Heatmap showing differential protein between CHIR99021 treatment group and control group. (E-F) Heatmap of hucMSCs transcriptomics clustering in CHIR99021-treated group vs. control group
Fig. 5
Fig. 5
CHIR-Exos promoted diabetic cutaneous wound healing more strongly than untreated hucMSC exosomes. (A) Flow chart of the animal experiment. (n = 5 in each group) (B) Skin wound healing in 4 groups of mice on days 0, 3, 7, 10 and 14. (C) Pattern diagrams of skin healing in the 4 groups of mice, with coloured areas showing the area of unhealed wounds at different observation times. (D) Statistics on the percentage of wound healing area of mice. (E-F) H&E staining to observe the epidermal thickness of the skin at the wound in each group of mice. (G-K) Masson staining and immunohistochemical analysis of collagen deposition, IL-1β, CD31 expression in the skin of mice in each group. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01
Fig. 6
Fig. 6
CHIR-Exos promoted fibroblast migration and proliferation and suppressed apoptosis in vitro. (A-B). Migration of NIH/3T3 cells at 0, 6, 12, and 24 h in scratch assay. (C) Apoptosis was detected in each group using Annexin5/PI staining, and the histogram showed the percentage of both Annexin5 (+) PI (−) and Annexin5 (+) PI (+) cells. (D) Cell cycle was detected using PI staining. (E) KEGG enrichment analysis of exosome between CHIR99021-treated and control groups. (F) Western blot analysis of AKT, p-AKT, p38, p-p38, caspase3 and cleaved-caspase3 in each group. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01
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