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. 2021 Aug 3;12(1):434.
doi: 10.1186/s13287-021-02517-0.

Exosomes derived from human umbilical cord blood mesenchymal stem cells stimulate regenerative wound healing via transforming growth factor-β receptor inhibition

Yan Zhang  1   2 , Yingjin Pan  3 , Yanhong Liu  4 , Xiheng Li  1   2 , Liang Tang  1   2 , Mengna Duan  5 , Jiang Li  6   7 , Guokun Zhang  8   9
Affiliations

Exosomes derived from human umbilical cord blood mesenchymal stem cells stimulate regenerative wound healing via transforming growth factor-β receptor inhibition

Yan Zhang et al. Stem Cell Res Ther. .

Abstract

Background: Scar formation is a common consequence of skin wound healing, and no effective treatment exists. Umbilical cord blood mesenchymal stem cells (UCB-MSCs) can improve wound healing; however, the role of UCB-MSCs remains unclear and whether they can ameliorate scar formation has not been fully elucidated.

Methods: To determine the function of UCB-MSCs, we examined and compared the therapeutic effects of UCB-MSCs and UCB-MSC-derived exosomes (UCB-MSC-exo) on skin healing in rats. Moreover, UCB-MSC-exo-specific miRNAs were identified and their effects in inhibiting the human dermal fibroblast (HDF) differentiation into myofibroblasts were investigated.

Results: Both UCB-MSCs and UCB-MSC-exo accelerated wound closure; reduced scar formation; improved the regeneration of skin appendages, nerves, and vessels; and regulated the natural distribution of collagen fibers in wound healing. Additionally, UCB-MSC-exo suppressed the excessive formation of myofibroblasts and collagen I and increased the proliferation and migration of skin cells in vivo and in vitro. Functional analysis showed that UCB-MSC-derived miRNAs were closely related to the transforming growth factor-β (TGF-β) signaling pathway, which could induce myofibroblast differentiation. We identified abundant miRNAs that were highly expressed in UCB-MSC-exo. miR-21-5p and miR-125b-5p were predicted to contribute to TGF-β receptor type II (TGFBR2) and TGF-β receptor type I (TGFBR1) inhibition, respectively. Using miRNA mimics, we found that miR-21-5p and miR-125b-5p were critical for anti-myofibroblast differentiation in the TGF-β1-induced HDF.

Conclusion: The effect of UCB-MSCs in stimulating regenerative wound healing might be achieved through exosomes, which can be, in part, through miR-21-5p- and miR-125b-5p-mediated TGF-β receptor inhibition, suggesting that UCB-MSC-exo might represent a novel strategy to prevent scar formation during wound healing.

Keywords: Cord blood stem cell transplantation; Exosomes; MicroRNAs; Myofibroblasts; Regeneration; Transforming growth factor beta; Wound healing.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
UCB-MSC-exo accelerate wound closure and suppress scar formation in full-thickness skin-wounded rats. A Schematic representation of UCB-MSC-exo preparation. B UCB-MSC-exo particle size was measured using NanoSight. C Morphological characteristics of UCB-MSC-exo were measured using transmission electron microscopy (scale bar = 100 nm). D Exosomal markers of UCB-MSC-exo were measured via western blot. E Schematic representation of animal experiments. The full-thickness excisional wound (12 mm diameter) was created on the dorsal region. The rats were then administered by tail vein injection every week: PBS (control group), UCM-MSCs (2 × 106 cells), or UCB-MSC-exo (20 μg). F Morphological changes during wound healing; scale bar = 5 mm. G Changes in the wound area during wound healing. *p < 0.05; **p < 0.01; means ± SD; n = 5
Fig. 2
Fig. 2
UCB-MSC-exo improve the regenerative wound healing in rats. A H&E and Masson staining of the healing skin; scale bar = 200 μm. B Number of skin appendages (hair follicles and sebaceous glands) in the healing skin. C–E Levels of collagen fibers (blue), myofibers (red) and the ratio of collagen fibers to myofibers in the healing skin, per Masson staining. F IF staining of Nestin (nerves) and CD31 (vessels) in the healing skin; scale bar = 300 μm. *p < 0.05; **p < 0.01; ***p < 0.001; means ± SD; n = 3
Fig. 3
Fig. 3
UCB-MSC-exo promote the proliferation and migration of skin cells. A IF staining of Ki67 in the healing skin; scale bar = 300 μm. B, C Effects of UCB-MSC-exo (25 ng/ml) on proliferation and migration of EPSCs and HDFs using the CCK8 and cell scratch assays in vitro; scale bar = 100 μm; *p < 0.05; **p < 0.01; means ± SD; n = 3
Fig. 4
Fig. 4
UCB-MSC-exo suppress myofibroblast formation. A IF staining of α-SMA and collagen I in the wound healing skin; scale bar = 300 μm. B, C mRNA levels of α-SMA and Col1a2 in the wound healing skin. HDFs were cultured for 48 h with TGF-β1 (25 ng/ml) to induce myofibroblast differentiation. Some cells were also treated with UCB-MSC-exo (25 ng/ml). D–F IF staining and qRT-PCR analysis of α-SMA and collagen I in HDFs; scale bar = 100 μm. *p < 0.05; **p < 0.01; ***p < 0.001; means ± SD; n = 3
Fig. 5
Fig. 5
Functional analysis of UCB-MSC-derived miRNAs and identification of UCB-MSC-exo-derived miRNAs. A UCB-MSC-miRNA abundance analysis according to Meng X et al. The top 10 abundant miRNAs in UCB-MSC are color-labeled. B Target mRNA prediction of UCB-MSC-miRNAs via Gene Ontology analysis; the TGF-β signaling pathway was highly related to myofibroblast formation. C UCB-MSC-exo-miRNAs, enriched in UCB-MSCs, measured by qRT-PCR analysis
Fig. 6
Fig. 6
UCB-MSC-exo-derived specific miRNAs target TGF-β receptors to inhibit myofibroblast differentiation. A List of predicted binding sites for UCB-MSC-exo-miRNAs and their targets. B–D Expression levels of TGFBR2 and α-SMA in HDFs treated with miR-21-5p-mimics. E–G Expression levels of TGF-βR1 and α-SMA in HDFs treated with miR-125b-5p-mimics; scale bar = 100 μm; *p < 0.05; **p < 0.01; ***p < 0.001; means ± SD; n = 3
Fig. 7
Fig. 7
UCB-MSC-exo inhibit TGFBR2 and TGFBR1 expression in wound healing in rats. A IF staining of TGFBR2 and TGFBR1 in the wound healing skin. B, C mRNA levels of TGFBR2 and TGFBR1 in the healing skin; scale bar = 300 μm; *p < 0.05; **p < 0.01; ***p < 0.001; means ± SD; n = 3
Fig. 8
Fig. 8
UCB-MSC-exo improve regenerative wound healing and suppress scar formation by inhibiting the expression of TGF-β receptors. When UCB-MSCs are transplanted into the skin wound, multiple vesicles bud from the plasma membrane, releasing the exosomes into the extracellular space where they then fuse with the plasma membrane of receptor cells. UCB-MSC-exo, carrying cargos of proteins, and RNAs, can travel to and be internalized by recipient cells (HDFs). Then, UCB-MSC-exo-derived specific miRNAs (such as miR-21-5p and miR-125b-5p) can regulate gene expression in HDFs. miR-21-5p targets TGFBR2, whereas miR-125b-5p targets TGFBR1, thereby suppressing myofibroblast differentiation, and ultimately reducing scar formation and promoting regenerative wound healing
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