Abstract
Purpose :
To isolate extracellular vesicles (EVs) from human conjunctival mesenchymal stromal cells (Conj-MSCs) and analyze their effect on conjunctival epithelium.
Methods :
Conjunctival stromal cells isolated from cadaveric donor tissues were cultured and analyzed to determine whether they could be defined MSC (n=5). Expression of MSC markers was analyzed by flow cytometry. Cells were cultured in adipogenic, osteogenic, and chondrocyte differentiation media, and stained with Oil Red, Von Kossa, and Alcian Blue, respectively, to determine multipotent capacity. Cells were grown at 70% confluence, secretome was collected after 48h with EVs-depleted FBS-supplemented medium and EVs were isolated by differential ultracentrifugation. EV morphology was evaluated by atomic force microscopy, size distribution analyzed by dynamic light scattering, and EVs were individually characterized by nanoflow cytometry. To analyze the effect on oxidative stress, the human conjunctival epithelial cell line IM-HConEpiC was exposed to 50 µg/ml Conj-MSCs-derived EVs for 3h (n=3), then loaded with 2′,7′-Dichlorofluorescin diacetate (30 min, 37°C) and finally exposed to 200 µM H2O2 to induce oxidative stress. Fluorescence was measured to quantify reactive oxygen species (ROS). Cell viability was determined by alamarBlue assay. Data were shown as mean ± standard deviation. One-way ANOVA was done to analyze differences.
Results :
Cultured stromal cells fulfilled the criteria of MSCs: they adhered to plastic; expressed CD90 (99.95±0.03% positive cells), CD105 (99.04±1.43%), CD73 (99.99±0.19%), CD44 (99.93±0.05%), and lacked CD34, CD11b, CD19, CD45 and HLA-DR (0.82±0.91%); and differentiated in vitro into different lineages. Conj-MSCs EVs were morphologically round. Main EV subpopulations were <300nm, but larger differential subpopulations were also observed. EVs expressed CD9, CD63, CD81, and CD147 markers. H2O2 increased ROS by 1.61+0.31-fold (p=0.0061) compared to untreated cells set as 1, and Conj-MSC EVs decreased it to 0.42±0.46 (p<0.0001), in similar levels than adipose tissue-MSC-derived EVs (0.65±0.50; p=0.0002) and 250 µM ascorbic acid (0.50±0.59; p<0.0001), used as control. EVs did not affect cell viability.
Conclusions :
Conj-MSC-derived EVs show no toxicity and promising antioxidant activity on conjunctival epithelial cells, warranting further research to determine their potential therapeutic effect.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.