Abstract
Purpose :
Transparent cornea is paramount for vision. Corneal scarring is a top leading cause of global blindness. The conventional corneal transplantation has been challenging due to the scarcity of donor tissue. Alternative corneal stromal stem cell (CSSC) therapy was reported to successfully prevent the scarring development. The paracrine action of CSSC, mediated by extracellular vesicles (EVs), was shown to block corneal fibrosis and stimulate native tissue regeneration. This study investigated the role of anti-fibrotic transforming growth factor (TGF) β3 transported in CSSC-EVs in the scarless regeneration of mouse corneas after injury
Methods :
Primary human CSSCs isolated from donor limbus were co-cultured with lipopolysaccharide-treated pro-inflammatory M1 mouse macrophages (RAW264.7) or conditioned media concentrates. The time course cellular and EV expression of hTGFβ1, 2 and 3 were examined by immunofluorescence and qPCR, respectively. Naïve CSSC or hTGFβ3 knockdown CSSCs were applied to mouse corneas after wounding by Algerbrush burring Corneal clarity (scar development), corneal thickness and the expression of inflammatory and fibrosis genes on mouse corneas were examined.
Results :
hTGFβ3 was upregulated in CSSCs after stimulation with M1 RAW cells, in both co-culture and paracrine conditions. More importantly, EVs derived from the stimulated CSSCs contained increasing levels of hTGFβ3 mRNA transcripts. After CSSC application to injured mouse corneas, hTGFβ3 was significantly upregulated, in conjunction with the inhibition of mouse fibrotic genes. At day 14 post-injury, the treated corneas remained clear and the expression of fibronectin, hyaluronan synthase 2, SPARC, tenascin C, collagen 3a1, and α-smooth muscle actin were significantly reduced. CSSCs with TGFβ3 knockdown by specific siRNAs lost these therapeutic effects.
Conclusions :
Our study revealed the paracrine action of human CSSCs on corneal scar prevention through EV-mediated delivery of TGFβ3 mRNA transcripts. The elevated TGFb3 expression in injured corneas could redirect the wound healing process to a reduced scarring or scar-free tissue regeneration, resulting in clear corneas.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.