Wound-Healing Effects of Mesenchymal Stromal Cell Secretome in the Cornea and the Role of Exosomes
Seungwon An, Khandaker N. Anwar, Mohammad Javad Ashraf, Hyungjo Lee, Rebecca Jung, Raghuram Koganti, Mahmood Ghassemi, Ali R. Djalilian
Abstract
Mesenchymal stromal/stem cells (MSCs) and their secreted factors have been shown to have immunomodulatory and regenerative effects. In this study, we investigated human bone-marrow-derived MSC secretome (MSC-S) for the treatment of corneal epithelial wounds. Specifically, we evaluated the role of MSC extracellular vesicles (EV)/exosomes in mediating the wound-healing effects of the MSC-S. In vitro studies using human corneal epithelial cells showed that MSC-CM increased cell proliferation in HCEC and HCLE cells, while EV-depleted MSC-CM showed lower cell proliferation in both cell lines compared to the MSC-CM group. In vitro and in vivo experiments revealed that 1X MSC-S consistently promoted wound healing more effectively than 0.5X MSC-S, and MSC-CM promoted wound healing in a dose-dependent manner, while exosome deprivation delayed wound healing. We further evaluated the incubation period of MSC-CM on corneal wound healing and showed that MSC-S collected for 72 h is more effective than MSC-S collected for 48 h. Finally, we evaluated the stability of MSC-S under different storage conditions and found that after one cycle of freeze-thawing, MSC-S is stable at 4 °C for up to 4 weeks. Collectively, we identified the following: (i) MSC-EV/Exo as the active ingredient in MSC-S that mediates the wound-healing effects in the corneal epithelium, providing a measure to optimize its dosing for a potential clinical product; (ii) Treatment with EV/Exo-containing MSC-S resulted in an improved corneal barrier and decreased corneal haze/edema relative to EV/Exo-depleted MSC-S; (iii) The stability of MSC-CM for up to 4 weeks showed that the regular storage condition did not significantly impact its stability and therapeutic functions.