Abstract
Purpose :
Human Corneal Stromal Stem Cells (CSSC) have been shown to promote regeneration of transparent stromal tissue and suppress inflammation in wounded mouse corneas. Small extracellular vesicles (sEV) secreted by these CSSC have been implicated to play a crucial role in this process. It is important to identify those CSSC that display high regenerative capacity. This study investigated the capacity of CSSC and their sEVs to suppress inflammation and fibrosis using in vitro assays.
Methods :
Conditioned media of primary human CSSC were collected for sEV isolation and assay analysis. Anti-inflammatory response was evaluated using a RAW 264.7 cell assay, in which osteoclast differentiation was induced by receptor activator of nuclear factor kappa-Β ligand (RANKL) and Concanavalin A. Cell morphology and mRNA level of matrix metalloproteinase 9 (MMP-9), Tartrate resistant acid phosphatase (ACP5), and Cathepsin K (CTSK) was quantified by qRT-PCR. Anti-fibrotic activity was assessed by a differentiation protocol in which primary human corneal fibroblasts differentiated into myofibroblasts after TGF-β1 treatment and were assayed for pro-collagen I alpha-1 (pro-COLIa1) level via ELISA and the protein expression of myofibroblast markers, α-smooth muscle actin (αSMA) and fibronectin extra domain-A (EDA-FN) by immunohistochemistry.
Results :
Conditioned media and sEVs from primary CSSCs were capable of significantly reducing gene expression of MMP-9 (5.37 folds and 2.07 folds, respectively, p<0.05), CTSK (2.1 and 3 folds, p<0.05), and ACP5 (2.2 and 2.1 folds, p<0.05) compared to the positive control in the RAW 264.7 cell assay. CSSC conditioned media potency correlates with their sEVs’ potency to prevent monocyte-to-osteoclast differentiation. In anti-fibrotic assays, sEVs from CSSC reduced pro-COLIa1 level by 40.9% relative to the positive control and lowered αSMA-positive cells by 32% (p<0.001). Among these different CSSC derived from multiple donors, one distinct donor CSSC produced sEVs that repeatedly demonstrate a higher anti-inflammatory and anti-fibrotic capacity than other CSSC (p<0.001).
Conclusions :
The anti-fibrotic activity of sEV could be quantified using in vitro assays. There is donor variation among CSSCs and their sEVs in respect to their anti-inflammatory and anti-fibrotic capacity. The factors didact the activities of sEV remain to be elucidated.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.