Umbilical Cord-Derived Mesenchymal Stem Cell-Derived Exosomal MicroRNAs Suppress Myofibroblast Differentiation by Inhibiting the Transforming Growth Factor-β/SMAD2 Pathway During Wound Healing

Abstract

: Excessive scar formation caused by myofibroblast aggregations is of great clinical importance during skin wound healing. Studies have shown that mesenchymal stem cells (MSCs) can promote skin regeneration, but whether MSCs contribute to scar formation remains undefined. We found that umbilical cord-derived MSCs (uMSCs) reduced scar formation and myofibroblast accumulation in a skin-defect mouse model. We found that these functions were mainly dependent on uMSC-derived exosomes (uMSC-Exos) and especially exosomal microRNAs. Through high-throughput RNA sequencing and functional analysis, we demonstrated that a group of uMSC-Exos enriched in specific microRNAs (miR-21, -23a, -125b, and -145) played key roles in suppressing myofibroblast formation by inhibiting the transforming growth factor-β2/SMAD2 pathway. Finally, using the strategy we established to block miRNAs inside the exosomes, we showed that these specific exosomal miRNAs were essential for the myofibroblast-suppressing and anti-scarring functions of uMSCs both in vitro and in vivo. Our study revealed a novel role of exosomal miRNAs in uMSC-mediated therapy, suggesting that the clinical application of uMSC-derived exosomes might represent a strategy to prevent scar formation during wound healing.

Significance: Exosomes have been identified as a new type of major paracrine factor released by umbilical cord-derived mesenchymal stem cells (uMSCs). They have been reported to be an important mediator of cell-to-cell communication. However, it is still unclear precisely which molecule or group of molecules carried within MSC-derived exosomes can mediate myofibroblast functions, especially in the process of wound repair. The present study explored the functional roles of uMSC-exosomal microRNAs in the process of myofibroblast formation, which can cause excessive scarring. This is an unreported function of uMSC exosomes. Also, for the first time, the uMSC-exosomal microRNAs were examined by high-throughput sequencing, with a group of specific microRNAs (miR-21, miR-23a, miR-125b, and miR-145) found to play key roles in suppressing myofibroblast formation by inhibiting excess α-smooth muscle actin and collagen deposition associated with activity of the transforming growth factor-β/SMAD2 signaling pathway.

Keywords: Exosome; Mesenchymal stem cells; MicroRNA; Myofibroblast; Transforming growth factor-β.

Figure 1.

uMSC-Exos suppress myofibroblast aggregation and scar formation in a full-thickness skin defect mouse model. (A): Upper: Images of indicated cell-transplanted wound at 14th day after initial treatment. Lower: Scar formation of mice in different treatment groups at 25 days after transplantation. (B): Quantification of wound diameter (upper) and the scar length (lower) of different treatment groups indicated in (A). ∗∗, p < .01. (C): Representative images of immunohistochemistry showing α-SMA expression in the indicated treatment groups. Scale bar = 500 μm. (D): Representative image of purified exosome particles (left) and the particle size distribution in purified uMSC-Exos (right) as determined by NanoSight. The red arrow indicates exoxomes. Scale bar = 1 μm. (E): Precise particle size distribution of purified uMSC-Exos determined by laser light scattering assessment. The dashed dot line indicates the peak particle size of purified uMSC-Exos. (F): Western blot analysis identifying purified uMSC-Exos using CD81 and CD63 antibody. (G): Representative immunohistochemistry showing α-SMA expression in the indicated exosome-treated groups. Phosphate-buffered saline used as control. Scale bar = 500 μm. Abbreviations: Medium, umbilical cord-derived mesenchymal stem cell culture medium; Mock, phosphate-buffered saline group; N, normal region; SMA, α-smooth muscle actin; UEFS, umbilical cord-derived mesenchymal stem cell exosome-free supernatant; uMSC-Exos, umbilical cord-derived mesenchymal stem cell-derived exosomes; W, wound region.

Figure 2.

uMSC-Exos suppress TGF-β-induced myofibroblast formation in vitro. (A): α-SMA expression in different TGF-β dosage-stimulated fibroblasts. Immunohistochemistry images of stimulated fibroblasts (left) and RNA level of SMA and collagen I (right). Scale bar = 20 μm. (B): Exosomes were added to fibroblasts labeled with PKH67. Nuclei were counterstained with Hoechst 33342. The cells were subject to fluorescence microscopy after 12 hours. Scale bar = 20 μm. (C): Fluorescent microscopy images illustrate the expression of SMA (green) followed by indicated treatment. Scale bar = 20 μm. (D): Flow cytometry comparing the percentage of SMA-negative cells of differently treated fibroblasts. Percentage of SMA-negative cells shown in upper left corner as standardized using the isotype control antibody-incubated cells (NC). (E): Expression levels of α-SMA and collagen I in different treatments using quantitative reverse transcription-polymerase chain reaction. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. ∗∗, p < .01. (F): Representative photographs of collagen gel contraction assay in the indicated treatment groups (left). The contracted gel diameter was measured 24 hours after treatment and is presented as the fold change of diameter compared with contraction inhibitor (1 M BDM) (right). (G): Cell cycle assay of differently treated fibroblasts showing representative images (left) and percentage of G₂ population (right). ∗∗, p < .01. (H): Scratch wound assay of differently treated fibroblasts showing representative images 48 hours after treatment (left) and the interval distance (right). Data are presented as mean ± SD; n = 3; ∗∗, p < .01 compared with negative controls. Scale bar = 200 μm. Abbreviations: BDM, 2,3-butanedione monoxime; h, hours; NC, negative control; SMA, smooth muscle actin; TGF-β, transforming growth factor-β; UEFS, umbilical cord-derived mesenchymal stem cell exosome-free supernatant; uMSC-Exos, umbilical cord-derived mesenchymal stem cell-derived exosomes.

Figure 3.

The myofibroblast-suppressing ability of uMSC-Exos mainly depends on its RNA components. (A): Gel electrophoresis (left) showing the RNase digested exosomes were depleted of RNAs compared with proteinase and control treatment. Silver staining (right) showing that after proteinase treatment, exosomes were degraded thoroughly. (B): NanoSight analysis showing that proteinase treatment did not compromise exosome integrity (additional data shown in supplemental online Fig. 3). (C): Quantitative reverse transcription-polymerase chain reaction showing α-SMA and collagen I expression in the indicated enzyme digestion groups. Data are presented as mean ± SD; n = 3; ∗∗, p < .01. (D): Western blot analysis showing that RNase-treated exosomes cannot downregulate the protein level of SMA induced by TGF-β. (E): Collagen gel contraction assay assessing the contraction inhibitory effect of different enzyme-digested exosomes showing representative images (left) and measurement of gel diameter (right). Data are presented as mean ± SD; n = 3; ∗∗, p < .01. (F): Cell cycle analysis of different enzyme-treated exosomes showing representative images (left) and percentage of G₂ population cells measured from three independent experiments (right). Exo-PROse and Exo-RNase groups were compared with Exo group individually. Data are presented as mean ± SD; n = 3; ∗∗, p < .01. Abbreviations: BDM, 2,3-butanedione monoxime; Exo, exosome; Exo-PROse, proteinase-treated uMSC-Exos; Exo-RNase, RNAse-treated uMSC-Exos; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NC, negative control; α-SMA, α-smooth muscle actin; TGF-β, transforming growth factor-β; uMSC-Exos, umbilical cord-derived mesenchymal stem cell-derived exosomes.

Figure 4.

Identification of uMSC-Exo-specific microRNAs by high-throughput sequencing. (A): Exosomal miRNA abundance analysis by high-throughput small RNA sequencing. The top 10 abundant miRNAs in uMSC-derived exosomes are color labeled. (B): Pie chart showing HEK293-derived exosomal miRNA abundance using high-throughput small RNA sequencing. The top 10 abundant miRNAs in (A) are labeled. (C): The miRNA abundance analysis in uMSCs using GSE46989 from the GEO DataSet. The color-labeled miRNAs were the top 10 abundant miRNAs in uMSC-derived exosomes. (D): miRNA abundance analysis in HEK293T cells using GSE56862 from the GEO DataSet. The 10 highly expressed miRNAs in uMSC-derived exosomes are color labeled. (E): Quantitative reverse transcription-polymerase chain reaction analysis of top 10 uMSC-Exo-abundant miRNAs in uMSC-Exo-treated fibroblasts (upper). UEFS-treated fibroblasts served as control. Expression level of pre-miRNAs in the indicated groups (right). Data are presented as mean ± SD; ∗∗, p < .01; ∗∗∗, p < .001. (F): Gene Ontology analysis of the TargetScan-predicted mRNA targets for the 10 most abundantly expressed miRNAs in uMSC-derived exosomes. The red dash-highlighted term, TGF-β receptor pathway, highly correlated with SMA expression and myofibroblasts formation. (G): The miRNA-mRNA interacting network showing the predicted targets for the top 10 abundant miRNAs in uMSC-derived exosomes. Target genes predicted to be targeted by more than 2 of the 10 miRNAs are shown. The yellow dots represents target mRNA, and the red arrow, miRNA. Abbreviations: Exo, exosome; hsa, Homo sapiens; miR, microRNA; miRNA, microRNA; pre-miR, before microRNA; SMA, smooth muscle actin; TGF-β, transforming growth factor-β; UEFS, umbilical cord-derived mesenchymal stem cell exosome-free supernatant; uMSC, umbilical cord-derived mesenchymal stem cell; uMSC-Exo, umbilical cord-derived mesenchymal stem cell-derived exosomes.

Figure 4.

Identification of uMSC-Exo-specific microRNAs by high-throughput sequencing. (A): Exosomal miRNA abundance analysis by high-throughput small RNA sequencing. The top 10 abundant miRNAs in uMSC-derived exosomes are color labeled. (B): Pie chart showing HEK293-derived exosomal miRNA abundance using high-throughput small RNA sequencing. The top 10 abundant miRNAs in (A) are labeled. (C): The miRNA abundance analysis in uMSCs using GSE46989 from the GEO DataSet. The color-labeled miRNAs were the top 10 abundant miRNAs in uMSC-derived exosomes. (D): miRNA abundance analysis in HEK293T cells using GSE56862 from the GEO DataSet. The 10 highly expressed miRNAs in uMSC-derived exosomes are color labeled. (E): Quantitative reverse transcription-polymerase chain reaction analysis of top 10 uMSC-Exo-abundant miRNAs in uMSC-Exo-treated fibroblasts (upper). UEFS-treated fibroblasts served as control. Expression level of pre-miRNAs in the indicated groups (right). Data are presented as mean ± SD; ∗∗, p < .01; ∗∗∗, p < .001. (F): Gene Ontology analysis of the TargetScan-predicted mRNA targets for the 10 most abundantly expressed miRNAs in uMSC-derived exosomes. The red dash-highlighted term, TGF-β receptor pathway, highly correlated with SMA expression and myofibroblasts formation. (G): The miRNA-mRNA interacting network showing the predicted targets for the top 10 abundant miRNAs in uMSC-derived exosomes. Target genes predicted to be targeted by more than 2 of the 10 miRNAs are shown. The yellow dots represents target mRNA, and the red arrow, miRNA. Abbreviations: Exo, exosome; hsa, Homo sapiens; miR, microRNA; miRNA, microRNA; pre-miR, before microRNA; SMA, smooth muscle actin; TGF-β, transforming growth factor-β; UEFS, umbilical cord-derived mesenchymal stem cell exosome-free supernatant; uMSC, umbilical cord-derived mesenchymal stem cell; uMSC-Exo, umbilical cord-derived mesenchymal stem cell-derived exosomes.

Figure 5.

uMSC-Exo-specific microRNAs target the TGF-β/SMAD2 pathway to suppress myofibroblast formation. (A): The effect of uMSC-Exo-specific miRNAs on TGF-β-stimulated SMA expression. The miRNAs were overexpressed using agomirs. Data are presented as mean ± SD; n = 3; ∗∗, p < .01. (B): A list of predicted binding sites of uMSC-Exo-specific miRNAs and their targets. (C): Luciferase reporter assay showing exosomal miR-21, miR-23a, miR-125b, and miR-145 regulates the target gene reporters’ luciferase activities. Data are presented as mean ± SD; n = 4; ∗∗, p < .01; ∗∗∗, p < .001. (D): Western blot analysis showing the effect of exosomal miR-21, miR-23a, miR-125b, and miR-145 on the protein expression of SMA, SMAD2, and p-SMAD2 in each treatment group. (E): SMAD2 reporter analysis showing the luciferase level of SMAD2-binding sequence-contained luciferase reporters under the indicated treatment. Data are presented as mean ± SD; n = 4; ∗∗, p < .01. (F): Collagen gel contraction by overexpressing exosomal miR-21, miR-23a, miR-125b, miR-145, and scramble negative agomir. Contraction inhibitor (1 M BDM) was used in each 48-well plate. Left: Representative images are shown. Right: The gel diameter was measured and is presented as the fold change of diameter compared with BDM. Data are presented as mean ± SD; n = 3; ∗∗, p < .01. Abbreviations: BDM, 2,3-butanedione monoxime; Exo, exosome; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hsa, Homo sapiens; NC, negative control; miR, microRNA; miRNA, microRNA; Mock, phosphate-buffered saline group; p-SMAD2, phosphorylated SMAD2; SMA, α-smooth muscle actin; TGF-β, transforming growth factor-β; uMSC, umbilical cord-derived mesenchymal stem cell; 3′-UTR, 3′-untranslated region.

Figure 6.

Inhibition of uMSC-Exo-specific miRNAs abolished the ability of uMSC-Exos to suppress TGF-β/SMAD2 activation in vitro. (A): Schematic showing the procedure of preparing Antago-uMSC-Exos. (B): Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) showing the miRNA level of fibroblasts treated with modified uMSC-Exos. The inhibitory efficiency of Antago-uMSC-Exos is shown compared with NC-uMSC-Exos. Equal amounts of phosphate-buffered saline (PBS) were used as exosome control (Mock). Data are presented as mean ± SD; n = 3; ∗∗, p < .01. (C): The effect of modified exosomes on SMA expression level assessed by both Western blot (upper) and qRT-PCR (lower). Equal amounts of PBS served as negative control. Data are presented as mean ± SD; n = 3; ∗∗, p < .01. (D): Flow cytometry analysis showed the percentage of p-SMAD2-negative and SMA-negative cells for the indicated groups; the bar region was standardized using the isotype antibody incubated cells as controls. (E): Reporter assay showing the effect of modified uMSC-Exo on TGFB2, TGFBR2, and SMAD2 3′UTR reporters’ luciferase activities. Data are presented as mean ± SD; n = 4; ∗∗, p < .01. (F): SMAD2 reporter analysis showing the luciferase level of SMAD2-binding sequence-contained luciferase reporter under the modified uMSC-Exo treatment. Data are presented as mean ± SD; n = 4; ∗∗, p < .01. Abbreviations: Antago-uMSC-Exos, antagomir-contained uMSE-Exos; DMEM, Dulbecco’s modified Eagle’s medium; Exo, exosome; miR, microRNA; Mock, phosphate-buffered saline group; NC, treating cells with same amount of phosphate-buffered saline used in NC-uMSC-Exos and Antago-uMSC-Exos; NC-uMSC-Exos, scramble antagomir-contained uMSC-Exos; NS, N.S., not significant; p-SMAD2, phosphorylated SMAD2; TGF-β, transforming growth-β; uMSC, umbilical-derived mesenchymal stem cell; uMSC-Exos, umbilical cord-derived mesenchymal stem cell-derived exosomes; 3′-UTR, 3′-untranslated region.

Figure 7.

uMSC-Exo-specific miRNAs play essential roles in the myofibroblast-suppressing function of uMSC-Exos in vivo. (A): Fluorescence in situ hybridization assay showing the existence and abundance of miR-145, miR-125b, miR-21, miR-23a (green) and p-SMAD2 (red) in mouse skin wound model treated with uMSC-Exos or phosphate-buffered saline (PBS) control. The nucleus was counterstained with 4′,6-diamidino-2-phenylindole. Scale bars = 200 μm. (B): Representative images of immunohistochemistry showing SMA and p-SMAD2 expression in normal and wound skin tissue. Wounded mice were treated with either NC-uMSC-Exo or Antago-uMSC-Exo. Equal amounts of PBS served as negative control. Scale bars = 500 μm. Abbreviations: Antago-uMSC-Exo, antagomir contained uMSC-Exo; Blank, no treatment; miR, microRNA; Mock, treatment using equal amounts of phosphate-buffered saline as exosome control; N, normal region; NC-uMSC-Exo, scramble antagomir contained uMSC-Exo; p-SMAD2, phosphorylated SMAD2; SMA, α-smooth muscle actin; uMSC-Exo, umbilical cord-derived mesenchymal stem cell-derived exosome; W, wound region.