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

Abstract

PurposeMesenchymal 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.MethodsCorneal 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.ResultscMSC 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).ConclusionsHuman cMSC exosomes can accelerate corneal epithelial wound healing, and thus, may provide a therapeutic approach for ocular surface injuries.

Increased cell size, structural complexity and migration of cancer cells acquiring fibroblast organelles by cell-projection pumping

“…It is noteworthy that although CPP may be more akin to intercellular exchange via TNT than via exosomes, that we are unable to find any reports of altered cell morphology following uptake of material via CPP, while there are several separate reports of altered cell morphology following exosome uptake [14][15][16]. Similarly, while it has been long known that exosomes from a variety of sources can increase migration of several cell types [17][18][19][20][21], there is less evidence for a similar effect for cellular contents transferred via TNT. Our observation of increased SAOS-2 migration following fibroblast cytoplasmic CPP, is consistent with a recent report of increased cancer cell migration after uptake of mitochondria via TNT from macrophages [22].…”

Potential hydrodynamic cytoplasmic transfer between mammalian cells: Cell-projection pumping

“…For example, exosome uptake alters cell morphology (14,15,42), but we find no literature of TNT mediating this effect. Similarly, while exosomes from a variety of sources can increase migration of several cell types (16,(43)(44)(45)(46), there is less evidence for a similar effect for cellular contents transferred via TNT (17). Seemingly different effects of cytoplasmic transfer by CPP and TNT, underscore the distinction between the two processes.…”

Reproducible Derivation and Expansion of Corneal Mesenchymal Stromal Cells for Therapeutic Applications

“…Our in vitro experiments demonstrated enhanced corneal wound healing with factors secreted by cMSCs, consistent with previous studies. 49 Moreover, the secretome woundhealing properties were maintained through P5 without diminished effect. Future studies might compare the wound-healing efficacy of cMSCs to that of the secretome.…”