Effect of Human Corneal Mesenchymal Stromal Cell-derived Exosomes on Corneal Epithelial Wound Healing

Abstract

Purpose: Mesenchymal stromal cells (MSCs) have been used therapeutically to modulate inflammation and promote repair. Extracellular vesicles, including exosomes, have been identified as one of the important mediators. This study investigated the effect of human corneal MSC-derived exosomes on corneal epithelial wound healing.

Methods: Corneal MSCs (cMSCs) were isolated from human cadaver corneas. The secretome was collected after 72 hours and exosomes were isolated using differential ultracentrifugation. Morphology and size of exosomes were examined by electron microscopy and dynamic light scattering. Expression of CD9, CD63, and CD81 by cMSC exosomes was evaluated by western blotting. Cellular uptake of exosomes was studied using calcein-stained exosomes. The effect of exosome on wound healing was measured in vitro using a scratch assay and in vivo after 2-mm epithelial debridement wounds in mice.

Results: cMSC exosomes were morphologically round and main population ranged between 40 and 100 nm in diameter. They expressed CD9, CD63, and CD81, and did not express GM130, Calnexin, and Cytochrome-C. Stained cMSC exosomes were successfully taken up by human cMSCs, human corneal epithelial cells (HCECs), and human macrophages in vitro and by corneal epithelium in vivo. In scratch assay, after 16 hours, cMSC exosome treated HCECs had 30.1% ± 14% remaining wound area compared to 72.9% ± 8% in control (P < 0.005). In vivo, after 72 hours, cMSC exosome-treated corneas had 77.5% ± 3% corneal wound healing compared to 41.6% ± 7% in the control group (P < 0.05).

Conclusions: Human cMSC exosomes can accelerate corneal epithelial wound healing, and thus, may provide a therapeutic approach for ocular surface injuries.

Figures

Figure 1

Characterization of exosomes. (A, B) DLS analysis, showing round particles with a size distribution of 40 to 280 nm. Most of the particles fall in the range defined for exosomes (40–100 nm). (C) Transmission electron microscopy of isolated exosomes showing a round morphology with a central depression, which is characteristic for exosomes. (D, E) Western blot results showing the presence of positive exosome markers CD9, CD63, CD81, and HSP70, as well as the absence or underrepresentation of negative exosome markers GM130, Calnexin, and Cytochrome-C, in isolated exosomes (Exo). cMSC whole cell lysate was used as control.

Figure 2

Uptake of cMSC derived exosomes by different type of cells in vitro. Cultured cells were incubated with calcein (green) labeled cMSC derived exosomes for 4 hours. Following incubation, fluorescent exosomes were taken up by human cMSCs (A), HCECs (B), and human macrophages (C).

Figure 3

Uptake of cMSC derived exosomes by cornea epithelium in vivo. Mice corneas (after linear scratches) were topically treated with green labeled cMSC derived exosomes for 15 minutes under anesthesia. After 4 hours, imaging of the whole mount of the cornea showed wide distribution of exosomes throughout the mouse corneal epithelium (A). Z-stack imaging of the cornea whole mount showed penetration of the exosomes through the epithelium (B).

Figure 4

The effect of cMSC derived exosomes on epithelial wound healing and proliferation. (A, B) Scratch assay on HCECs showed significantly greater wound closure in cells incubated with cMSC derived exosomes after 16 hours. (C) MTT assay showed increased proliferation of HCECs incubated with cMSC derived exosomes after 16 hours. There appears to be a slight dose dependent effect with increasing concentration of exosomes. *Statistically significant, P

Figure 5

The effect of cMSC derived exosomes on corneal epithelial wound healing in vivo. Fluorescein-stained images of 2-mm wounded mice corneas, before and 24 hours after treatment with either cMSC derived exosomes or vehicle control. Exosome treated wounds had healed significantly more than control (n = 6). *Statistically significant, P