Abstract
Purpose :
Mesenchymal stem cells from corneal stroma (CSSC) suppress scar formation and induce regeneration of transparent tissue in wounded corneas. We recently observed that a fraction of small extracellular vesicles with properties of exosomes (Exo) isolated from conditioned CSSC culture medium have similar regenerative properties. This study investigated the mechanism by which CSSC Exo prevent scarring and induce regeneration of transparent stroma tissue.
Methods :
Exo were isolated from conditioned CSSC medium by repeated sedimentation. RNA was isolated using Qiagen miRNeasy kits. RNA content was determined by RNAseq and Nanostring array. Corneas of C57B/L6 mice were wounded by debridement with Algerbrush II as previously described(PMID28257425)and treated topically with Exo embedded in a fibrin gel immediately after wounding. Inflammation was assessed by ELISA for myeloperoxidase at day 1, and by TaqMan qPCR at day 3 for Cd80, uPAR, Cxcl7, Lcn2. Fibrotic scarring was determined by OCT and qPCR for Col3a1, Acta2, Tnc at day 14.
Results :
Exo treatment of wounded mouse cornea reduced visible scarring and suppressed early inflammation in wounded corneas and blocked upregulation of fibrotic gene expression at day 14 compared to controls treated with Exo from HEK 293. RNAseq showed presence of mRNA, lncRNA and miRNA in the EXOs, with a number of RNA species upregulated compared to Exo from HEK 293 cells. Incubation of EXO with HCEC or with corneal fibroblasts found that Exo rapidly fused with corneal cells and transferred miRNA into them. Knockdown of Alix protein in CSSC reduced the amount of miRNA in EXO by >85% but did not change the amount of miRNA in the cells. EXO with reduced miRNA failed to block scarring and inflammation in wounded corneas.
Conclusions :
These results support the idea that microRNA is essential in the ability of CSSC to prevent corneal scarring and to induce regeneration of transparent corneal tissue after wounding. Delivery of the miRNA to the damaged tissue appears to be require the presence of exosomes loaded with miRNA.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.