Abstract
Purpose :
Corneal blindness due to scarring is treated with cornea transplantation. However, there exists a global challenge of donor material shortage. Preclinical and clinical studies have shown that cell therapies using corneal stromal stem cells (CSSC) suppress scar formation. However, not every CSSC batches achieve stromal regenerative effects. MicroRNA-29 is a “master fibro-regulatory miRNA”, with pivotal roles in regulating tissue fibrosis. Our group identified an upregulated expression of hsa-miR-29a in extracellular vesicles (EV) produced by human CSSC with good healing effects. This work examined miR-29a expression among CSSC batches, and the anti-scarring potency of cells was assessed with a mouse model of corneal injury. We aimed to design a quantitative tool to screen CSSC with healing potency for clinical applications.
Methods :
EV fractions from conditioned media of human CSSC cultures (from n=13 donors) were harvested for small RNAs using miRNeasy protocol (Qiagen). The abundance of miR-29a and 16 (housekeeping EV-miRNA) was analyzed with Taqman RT-PCR assay (Thermo Fisher). Corneal stromal wound was induced by Algerbrush burring in Swiss-webster mice (n=18) and treated with human CSSC (5x104 cells) in fibrin gel. Corneas were examined by Spectral Domain OCT every 4 days and harvested at day 14 for scar evaluation and fibrosis marker expression analysis by qPCR and immunohistochemistry.
Results :
The normalized levels of miR-29a expression were upregulated in EV fractions of HC540 and 641 and were low in HC572 and 618 (P<0.05, Mann-Whitney U test). On injured mouse corneas, topical treatment using HC540 and 641 (high miR-29a levels) nearly prevented corneal scarring, maintained normal corneal thickness, and lower expression levels of fibrosis genes (mouse collagen III, fibronectin, α-smooth muscle actin, and Thy-1). In contrast, treatment with HC618 and 572 (low miR-29a levels) resulted in thicker corneas with intense scarring, and upregulated fibrosis gene expression, similar to wound controls.
Conclusions :
We demonstrated that EV-miR-29a expression could distinguish CSSC with anti-scarring quality, providing a tool to select suitable CSSC for clinical applications.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.