Role of Mesenchymal Stem Cell-Derived Conditioned Medium in Modulating the Benzalkonium Chloride-Induced Cytotoxic Effects in Cultured Corneal Epithelial Cells In Vitro.

PURPOSE: Benzalkonium chloride (BAK) is a common preservative in ophthalmic formulations that causes cytotoxic damage to the corneal epithelial cells. This study aims to explore the role of mesenchymal stem cell (MSC)-derived conditioned medium in modulating the BAK-induced cytotoxic effects in cultured human corneal epithelial cells (HCECs) as a cell-free therapeutic agent. METHODS: The in vitro cultured HCECs derived from a HCE cell line were treated with BAK (0.001% and 0.005%, diluted in DMEM/F12, v/v) for 15 min, washed with 1xPBS, and allowed to recover for 24 h in human bone marrow MSC-derived conditioned medium (MSC-CM: undiluted (100%) and diluted (50%, v/v)). On the other hand, HCECs were co-incubated with BAK (0.005%, v/v) and MSC-CM (100% and 50%, v/v) for 24 h. The HCEC-derived conditioned medium (HCE-CM) was used as an optimal control for MSC-CM, whereas HCECs cultured in DMEM/F12 were used as a control. The DMEM/F12 was used as the base medium for the culture of HCECs and preparation of HCE- and MSC-CM. The role of MSC-CM in modulating the metabolic activity, cell death, epithelial repair, and proliferation, in BAK-treated HCECs was evaluated using MTT assay, Propidium iodide staining, scratch assay, and Ki-67 staining, respectively. RESULTS: Compared to the control, recovery of BAK-treated (0.001% and 0.005%, for 15 min) HCECs in MSC-CM showed significantly reduced cell death with enhanced metabolic activity, epithelial repair, and proliferation. However, in comparison with HCE-CM, the beneficial effects of MSC-CM were predominantly observed at lower BAK concentration (0.001%, for 15 min). Whereas the co-incubation of BAK (0.005%) and MSC-CM for a longer duration (24 h) was marginally beneficial. CONCLUSIONS: Our results suggest that the MSC-CM is effective in modulating the BAK-induced cell death, retardation of metabolic activity and proliferation in cultured HCECs, particularly at lower concentration (0.001%) and shorter exposure (15 min) of BAK.

Bone marrow mesenchymal stem cell-derived extracellular vesicles promote corneal epithelial repair and suppress apoptosis via modulation of Caspase-3 in vitro.

Corneal injuries are the major cause of blindness and visual impairment. Available treatments are limited by their efficacy and side effects. Mesenchymal stem cell-derived extracellular vesicles are presumed as functional equivalents and potential candidates for cell-free therapy. This study reports isolation and characterization of extracellular vesicles from human bone marrow mesenchymal stem cells and evaluates their role in mediating epithelial repair and apoptosis in cultured corneal epithelial cells through scratch assay, PCR, immunofluorescence, and flow cytometry in vitro. The isolated extracellular vesicles were spherical, < 150 nm in diameter, and characterized as CD9+, CD63+, CD81+, TSG101+, and Calnexin-. Further, these vesicles promoted corneal epithelial repair by enhancing proliferation and suppressed apoptosis by regulating the expression of BAD, P53, BCL-2, and cleaved CASPASE-3. Thus, our results suggest that BM-MSC-EVs might have the potential to be used for the treatment of injury-induced corneal epithelial defects. Clinical translation of this work would require further investigations.

Mesenchymal stem cell-based adjunctive therapy for Pseudomonas aeruginosa-induced keratitis: A proof-of-concept in-vitro study.

PURPOSE: Pseudomonas aeruginosa-induced keratitis is one of the most severe and challenging forms of corneal infection, owing to its associated intense inflammatory reactions leading to corneal necrosis and dense corneal scar with loss of vision. Since mesenchymal stem cells (MSCs) are reported to possess antimicrobial and immunomodulatory properties, they can be tested as an adjuvant treatment along with the antibiotics which are the current standard of care. This study aims to investigate the anti-bacterial and immunomodulatory roles of human bone marrow MSC-derived conditioned medium (MSC-CM) in P. aeruginosa-infected human corneal epithelial cells (HCECs) in vitro. METHODS: The effect of MSC-CM on the growth of clinical isolates of P. aeruginosa was evaluated by colony-forming unit assay. The expression of inflammatory cytokines (IL-6 and TNF-α) and an antimicrobial peptide (Lipocalin 2) in lipopolysaccharide-treated MSCs and HCECs was analyzed through ELISA. Corneal epithelial repair following infection with P. aeruginosa was studied through scratch assay. RESULTS: Compared to control (P. aeruginosa (5*105) incubated in DMEM (1 ml) at 37 °C for 16 h), MSC-CM significantly: i) inhibits the growth of P. aeruginosa (159*109 vs. 104*109 CFU/ml), ii) accelerates corneal epithelial repair following infection with P. aeruginosa (9% vs. 24% closure of the wounded area after 12 h of infection), and iii) downregulates the lipopolysaccharide-induced expression of IL-6, TNF-α and Lipocalin 2 in HCECs. A combination of MSC-CM with an antibiotic, Ciprofloxacin moderately regulated the expression of IL-6, TNF-α, and Lipocalin 2. CONCLUSION: MSC-CM holds promise as an adjunctive therapeutic approach for P. aeruginosa-induced corneal epithelial damage.