Mithun Santra, Elizabeth Rubin, Syeda R Ali, Moira L Geary, Christine Chandran, Julia T Coelho, Ella J Dewald, Xi Cheng, Martha L Funderburgh, Deepinder K Dhaliwal, Xin D Gao, Vishal Jhanji, Gary H F Yam
M1-primed hCSSC-Exo containing protein-coding TGFB3 mRNA significantly inhibited corneal opacity and promoted scar-free healing in injured mouse corneas. These findings support the anti-fibrotic and regenerative potential of hCSSC-Exo, which can be developed as a cell-free therapeutic strategy for corneal fibrosis and scarring.
INTRODUCTION: Human corneal stromal stem cells (hCSSCs), which exhibit mesenchymal stem cell (MSC) features, prevent fibrotic scarring and promote regeneration of injured stromal tissue, thereby restoring corneal clarity in mice. These therapeutic effects rely on hCSSCs' ability to respond to early inflammatory cues, suppress chronic neutrophil infiltration, and inhibit fibrosis. In this study, we examined the responses of hCSSCs to transient pro-inflammatory M1 stimulation and characterized the exosomes (Exo) they secreted.
METHODS: We evaluated the expression of full-length protein-coding human TGFB3 mRNA molecules in purified Exo, and investigated Exo uptake, induction of human transforming growth factor β3 (hTGFβ3) protein expression, and the effects of different hTGFβ3/β1 ratios on myofibroblast generation from human corneal stromal keratocytes (CSKs) and stromal fibroblasts (SFs). The anti-scarring effect of hCSSC-Exo enriched in TGFB3 mRNA was assessed in a mouse model of acute corneal stromal injury.
RESULTS: Our results showed that hCSSC-Exo contained TGFB3 mRNA transcripts, and their abundance increased following pro-inflammatory M1 stimulation. Exo uptake enhanced hTGFβ3 protein expression in hCSKs, and a higher hTGFβ3/β1 ratio in hCSKs and hSFs suppressed expression of α-smooth muscle actin and TGFβ1-induced myofibroblast differentiation. In vivo, topical treatment with M1-primed hCSSC-Exo reduced corneal opacity compared with sham-treated controls. Notably, hCSSC-Exo enriched with TGFB3 mRNA demonstrated greater scar-reducing and wound-healing efficacy.
CONCLUSIONS: M1-primed hCSSC-Exo containing protein-coding TGFB3 mRNA significantly inhibited corneal opacity and promoted scar-free healing in injured mouse corneas. These findings support the anti-fibrotic and regenerative potential of hCSSC-Exo, which can be developed as a cell-free therapeutic strategy for corneal fibrosis and scarring.