Effect of Stem Cell-Derived Extracellular Vesicles on Damaged Human Corneal Endothelial Cells

Abstract

Purpose. Human corneal endothelial cells (HCECs) are essential to visual function; however, since they have limited proliferative capacity in vivo, they are prone to corneal endothelial dysfunction. At present, the only treatment is a corneal transplantation from donor cadavers. Also, due to a global shortage of donor corneas, it is important to find alternative strategies. Recent studies highlight that stem cell–derived extracellular vesicles (EVs) play a relevant role in stem cell-induced regeneration by reprogramming injured cells and inducing proregenerative pathways. The aim of this work is to evaluate whether EVs derived from mesenchymal stem cells (MSC-EVs) are able to promote regeneration of damaged HCECs. Methods. We isolated HCECs from discarded corneas in patients undergoing corneal transplantation or enucleation ( N = 23 patients). Bone marrow mesenchymal stem cells (MSCs) were obtained from Lonza, cultured, and characterized. MSC-EVs were obtained from supernatants of MSCs. In order to establish a valid in vitro damage model to test the regenerative potential of EVs on HCECs, we evaluated the proliferation rate and the apoptosis after exposing the cells to serum-deprived medium at different concentrations for 24 hours. We then evaluated the HCEC migration through a wound healing assay. Results. In the selected serum deprivation damage conditions, the treatment with different doses of MSC-EVs resulted in a significantly higher proliferation rate of HCECs at all the tested concentrations of EVs ( 5 ‐ 20 × 10 3 MSC-EV/cell). MSC-EVs/cell induced a significant decrease in number of total apoptotic cells after 24 hours of serum deprivation. Finally, the wound healing assay showed a significantly faster repair of the wound after HCEC treatment with MSC-EVs. Conclusions. Results highlight the already well-known proregenerative potential of MSC-EVs in a totally new biological model, the endothelium of the cornea. MSC-EVs, indeed, induced proliferation and survival of HCECs, promoting the migration of HCECs in vitro.

Mesenchymal Stem Cell-Derived Extracellular Vesicles Protect Human Corneal Endothelial Cells from Endoplasmic Reticulum Stress-Mediated Apoptosis

“…HCECs were isolated and characterized as previously described [ 20 ]. In brief, we isolated HCECs from discarded corneas of patients undergoing corneal transplantation or enucleation (N = 23 patients; Supplementary Table S1 ).…”

“…When tunicamycin was applied to HCECs, the expression levels of the main ER stress-related proteins GRP78 [ 26 ] and C/EBP homologous protein (CHOP) [ 27 ] were significantly increased in a dose-dependent manner ( Figure 3 ). In parallel, we used the serum deprivation model of HCEC damage already set for previous experiments [ 20 ]. In this model, ER stress related genes were upregulated in a comparable manner to the tunicamycin treatment, confirming nutrient deprivation as an inducer of ER stress.…”

“…Human corneal endothelial cells (HCECs) were isolated and characterized as previously described [ 20 ]. The study was approved by the Intercompany Ethics Committee of A.O.U.…”

“…From each patient, we isolated a cell line of HCECs, each cell line was kept in culture, and all of the experiments were performed between passage 2 and 4. The cells were characterized by immunofluorescence using phalloidin antibody (ThermoFisher Scientific, Waltham, MA, USA) and DAPI for nuclear staining, for the expression of HCEC main marker Na+K+ATPase, ZO-1, and by presence of the surface marker CD166, as described [ 20 ].…”

“…Our group recently reported the proangiogenic effect of MSC-EVs in an in vitro model of corneal endothelium damage due to serum deprivation. MSC-EVs induced the proliferation and survival of damaged human corneal endothelial cells (HCECs), and promoted their wound closure [ 20 ].…”