1. Identification of HUMSCs and HUMSC-exosomes
Results of flow cytometry analysis confirmed the presence of positive expressions of typical MSC makers CD105, CD90, CD73, while the surface markers of hematopoietic cells such as CD34, CD11b, CD19, CD45 and HLA-DR were fairly weak to detect compared with the isotype control (Fig. 1a). In addition, according to inverted microscopic observation, the morphology of the cells was regular long spindle with directional arrangement, and presented a typical spindle shape, which grew as whirlpool or cluster (Fig. 1b).
The classical structure of the isolated exosome, including “rim of a cup” and double-layer membrane morphology, were observed by TEM (Fig. 1c). NTA results demonstrated that the diameters of the particles were around 50–150 nm (Fig. 1d). The identity of these particles was further confirmed as exosomes by Western blot, which showed the presence of widely expressed exosomal markers, including CD9, CD63, CD81 (Fig. 1e). Therefore, results above confirmed that the EVs we extracted were indeed exosomes.
Morphological observation and identification of HUMSCs and HUMSC-exosomes. a Flow cytometry analysis of surface markers in HUMSCs. b Light morphology image of HUMSCs. c Morphology of HUMSC-exosomes under TEM. Scale bar, 100 nm. d Peak size of HUMSC-exosomes was around 80 nm as showed by NTA. e HUMSC-exosomes were positive for CD9, CD81 and CD63 as indicated by Western blot.
2. Topical application of HUMSC-exosomes accelerates corneal wound healing in a rat model
To investigate the influences of HUMSC-exosomes on mechanical corneal epithelium defect model, 5 µL of 1 µg/µL exosome suspensions or PBS were topically dropped on defect eye four times a day. The change of corneal defect area was monitored using the corneal fluorescein staining every 12 hours. HUMSC-exosomes treatment significantly decreased the percentages of residual defect compared to the PBS-treated group at 48 hours. (Fig. 2a-b). The injured corneas treated with HUMSC-exosomes regained more regular arrangement and compact structure than those treated with PBS through assessing corneal tissues microstructure by H&E staining. In addition, corneal epithelium detachment and inflammatory cells infiltration occurred in the PBS group (Fig. 2c).
The effect of HUMSC-exosomes on corneal epithelial wound healing in vivo. a, b Fluorescein-stained images of defect corneas, before and after treatment with HUMSC-exosomes or PBS. Wounds treated with HUMSC-exosomes had healed significantly more than control. c H&E staining showed the histologic appearance of the cornea in exosome-group mice and PBS-group mice at 72 h. Data are expressed as the means ± SD. *P < 0.05.
3. HUMSC-exosomes promote the proliferation and migration of HCECs in vitro
To demonstrate the uptake of exosomes, CFSE-labelled HCECs were co-cultured with Dil-labelled exosomes and then visualized with laser scanning confocal microscope. Localization results showed that exosomes derived from HUMSCs had been taken up by HCECs with the dye distributing within in the cell (Fig. 3a). In addition, the fusion process was also observed by TEM (Fig. 3b).
In order to evaluate whether HUMSC-exosomes stimulates HCECs migration, the effect on wound closure rates were investigated. The disparity of the remaining area during scratch wound assays confirmed the promigratory effects of HUMSC-exosomes with a dose-related trend after 18 hours incubation (Fig. 3c-d). Considering corneal healing is a dynamic interwoven process composed of cell proliferation, migration and adhesion, and the proliferation ability is the basis. We further investigated whether HUMSC-exosomes could enhance the proliferation-promoting behavior of HCECs in vitro. The CCK-8 assay showed that the proliferation of HCECs after incubating with exosomes was significantly improved in a dose-dependent manner (Fig. 3e). And the EdU assays for visualization of proliferating cells also demonstrated that HUMSC-exosomes treatment increased the percentage of proliferating cells compared to controls (Fig. 3f-g).
The effect of HUMSC-exosomes on HCECs proliferation and migration in vitro. a Fluorescence images of CFSE-labelled HCECs (green) incubated with Dil-labelled HUMSC-exosomes (red). Nuclei were stained with DAPI (blue). b TEM of HCECs incubated with HUMSC-exosomes. c, d Representative images from in vitro scratch wound healing assays demonstrating that cell migrate into the cell-free region is significantly accelerated in the presence of HUMSC-exosomes when compared to controls. e CCK-8 assay showed increased proliferation of HCECs incubated with HUMSC-exosomes after 48 hours. f, g The proliferating HCECs was detected by EdU incorporation. The cells were treated with HUMSC-exosomes or blank control. Blue: nuclear staining (Hoechst33342); Red: EdU staining. h, i The expression of phospho-PI3K, phospho-Akt and cyclin D1 were detected through Western blot. Data are expressed as the means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.
4. HUMSC-exosomes promote HCECs proliferation and migration though PI3K/Akt pathway
To further investigate the potential mechanism of HUMSC-exosomes regulated proliferation and migration in HCECs, full-length transcriptome sequencing was used to detect the mRNA expression levels of related genes. 240 differentially expressed genes (DEG) were identified (Fold Change ≥ 2 and P value < 0.05), including 104 up-regulated DEGs and 136 down-regulated DEGs (Fig. 4a). Then, we interpreted the potential biological functions of DEGs from the gene function and signaling pathway through KEGG enrichment analysis, and revealed that the PI3K/Akt signaling pathways had a significant difference between before and after HUMSC-exosome treated HCECs (Fig. 4b).
Previous studies demonstrated that PI3K/Akt pathway involved deeply in the modulation of the process of corneal epithelial wound healing .Therefore, the PI3K/Akt signaling pathways involved in HCECs proliferation and migration process after HUMSC-exosomes treatment were explored. The activation of the PI3K/Akt pathway in HCECs following HUMSC-exosome stimulation was verified by treating the cells with HUMSC-exosomes or PBS for 24 hours and assessing the phosphorylated protein levels (Fig. 3h). In addition, since the PI3K/Akt pathway is a strong activator of cyclin D1 which plays a critical role in regulating cell cycle from G1 phase to S phase , we reasoned that exosomes may exert its effects on proliferation through modulating the cell phase transition. As expected, the protein levels of cyclin D1 were also increased after HUMSC-exosomes treatment. (Fig. 3h).
Transcriptome and pathway analysis of HUMSC-exosomes treatment. a Volcano Plot of DEGs between HUMSC-exosomes treated and untreated HCECs. Dots in green stands for down-regulated DEGs and red dots mean up-regulated DEGs, black dots are non-significant DEGs. b The KEGG annotation results of the DEGs were classified according to the pathway types in KEGG. DEG, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes
5. HUMSC-exosomes promote HCECs proliferation and migration through miR-21
Exosomes regulate a large number of physiological activities via exosomal miRNAs . As miRNAs are abundant in HUMSC-derived exosomes, we hypothesized that HUMSC-exosomes promote the healing of corneal epithelial defect mainly through miRNAs. The downloaded dataset was used to determine the content of various miRNAs in HUMSC-exosomes . Among the several miRNAs selectively enriched in HUMSC-exosomes, we focused on the most abundant one, miR-21(Fig. 5a). The downstream targets were predicted by Target Scan, mirBase and miRDB database, then imputed DAVID online to conduct GO analysis. The results showed that miR-21 was involved in the regulation of various molecular function, containing calcium ion binding, peptidase inhibitor activity, growth factor activity etc. (Fig. 5b). Among them, the phosphatase activity may involve in the regulation of PI3K/Akt.
In order to assess whether the exosome-mediated miR-21 transfer plays a role in HCECs proliferation and migration, a subsequent knockdown experiment was conducted. HUMSCs were transfected with miR-21 inhibitors (at final concentration of 100 nM) or NC, and the culture supernatants were collected subsequently for isolating the exosomes. Then, HCECs were incubated with the same concentration of miR-21 contained or miR-21 knockdown HUMSC-exosomes for migration and CCK-8 analysis. Results showed that the up-regulation of migration (Fig. 6a, d), as well as proliferation (Fig. 6e) induced by HUMSC-exosomes were partially negated by miR-21 knockdown.
To further study the potential involvement of miR-21, HCECs were transiently transfected with miR-21 mimics or NC. Proliferation of HCECs following transfection with miR-21 mimics or NC was assessed using CCK-8 and EdU assay. As shown in Fig. 7c, transient transfection of miRNA mimic is efficient. miR-21 mimics transfection significantly promoted the proliferation of HCECs compared with the NC group (Fig. 6b, f, g). In addition, the ability of HCECs transfected with miR-21 mimics to regain monolayer integrity was raise compared with NC-transfected cells (Fig. 6c, h).
miR-21 involved in the process of HUMSC-exosomes promote cell proliferation and migration. a, d HCECs were treated with miR-21KD HUMSC-exosomes or miR-21 contained HUMSC-exosomes for 18h. The scratch assay showed the healing of the miR-21KD HUMSC-exosomes treated group was slower than the miR-21 contained HUMSC-exosomes treated group. b, f The proliferation of HCECs was detected by EdU incorporation after transfected with miR-21 mimics (at final concentration of 50 nM). Blue: nuclear staining (Hoechst33342); Red: EdU staining. e The CCK-8 assay showed the proliferation of the miR-21 KD HUMSC-exosomes treated group was lower than the miR-21 contained HUMSC-exosomes treated group after 18 hours. c, h The scratch assay showed significantly faster wound closure in HCECs incubated with miR-21 mimics than NC after 18 hours. g The CCK-8 assay showed the proliferation of the miR-21 mimics group was higher than control group after 48 hours. Data are expressed as the means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001. KD, knockdown
6. miR-21 regulates cell proliferation and migration by targeting PTEN
Unsurprisingly, we found that the effects of miR-21 on the PI3K/Akt phosphorylation and cyclin D1 protein expression were stimulative (Fig. 7g). miRNAs can exert their functions by interacting with the 3' untranslated region (3' UTR) or protein coding sequence of target mRNAs. According to the miRbase database, PTEN might be the potential downstream of miR-21 (Fig. 7a). To confirm whether PTEN is a target of miR-21 in HCECs, we further measured the expression of PTEN in HCECs transfected independently with miR-21 mimics or inhibitors and their corresponding NC to verify the interaction between the miR-21 and PTEN by qRT–PCR and Western blot. Once transfected with miR-21 mimics, the protein levels of PTEN were significantly reduced in HCECs (Fig. 7f), and the difference was also detected in transcription level (Fig. 7d). Meanwhile, the inhibition of miR-21 resulted in opposite effect on PTEN expression (Fig. 7f). These results suggested that miR-21 regulate PTEN within HCECs via post-transcriptional modify. Reduced mRNA and protein expression levels of PTEN were identified within HCECs after treated with HUMSC-exosomes (Fig. 7b, e), whereas PI3K/Akt phosphorylation and cyclin D1 expression were increased (Fig. 3h). PTEN down-regulated the PI3K/Akt pathway, which was important for proliferation and migration (Fig. 7h).
Taken together, our data indicate that exosomal miR-21 promotes proliferation by activating PTEN/PI3K/Akt signaling pathway, which might play a critical role to enhance corneal epithelial wound healing.
Exosomal miR-21 regulate HCECs proliferation and migration by activating PI3K/Akt pathway through targeting PTEN. a The binding site between miR-21 and PTEN mRNA. b, e, f HUMSC-exosomes treatment decreased the RNA and protein levels of PTEN in HCECs. c The expression level of miR-21 in HCECs. d, g, h The expression of PTEN changed with miR-21 variation. i, j The expression level of PTEN, phospho-PI3K, phospho-Akt and cyclin D1 after transfected with miR-21 mimics were detected by Western blot. k, l The expression of phospho-PI3K, phospho-Akt and cyclin D1 after overexpression of PTEN. Data are expressed as the means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.
7. Schematic diagram
Schematic diagram describes the mechanism of HUMSC-exosomes in corneal epithelial defect.
We first determined the positive effects of exosomes derived from HUMSCs in promoting corneal epithelial wound healing, then determined the main molecules PI3K/Akt in the wound healing process and the fact that miR-21 was the most abundantly contained in exosomes through bioinformatics analysis. Finally, we anchored PTEN as the downstream target of miR-21, which was the key link between miR-21 and related protein, and determined that PTEN/PI3K/Akt were involved in cell proliferation and migration.