Abstract
Purpose :
Preclinical studies have shown success using cultivated human corneal stromal keratocytes (CSK) and their progenitors (corneal stromal stem cells [CSSC]) to prevent corneal scarring and regenerate transparent corneal stroma. However, there is no study comparing the treatment efficacy of CSK versus CSSC. This work investigated if both cell types have comparable corneal regenerative effects in a mouse corneal injury model.
Methods :
Human CSSC and CSK were isolated from same donor corneas (n=3) and cultured following reported protocols. Cell phenotypes were characterized by marker expression, doubling time and collagen deposition. In a mouse model of anterior stromal injury created by Algerbrush burring, CSSC were topically applied in fibrin gel immediately after injury whereas CSK were intrastromally injected after epithelial healing. Corneas were examined weekly with anterior segment optical coherent tomography and harvested at 14 days post-treatment for scar analysis, fibrosis gene expression (Col3a1, aSMA, fibronectin [FN], and tenascin C [TNC])by qPCR and immunostaining.
Results :
Cultivated human CSSC were proliferative and expressed stem cell markers (Pax6, ABCG2, nestin and CXCR4), and lumican but negative for keratocan and ALDH3A1. Growth-arrested CSK became quiescence and expressed ALDH3A1, keratocan, and lumican. Using ELISA, CSSC produced significantly less pro-collagen I than quiescent CSK, and both had minimal collagen III expression when compared to stromal fibroblasts. When applied on mouse corneal wounds, both cell types reduced corneal scarring in a dosage-dependent manner and downregulated inflammatory (iNOS, MCP1) and fibrosis markers (Col3a1, aSMA, FN, and TNC), when compared to wound controls. Comparing between CSSC and CSK, fibrosis gene expression was generally lower in CSK group than those treated with CSSC, particularly significant for MCP1, FN and TNC (P<0.05, Mann-Whitney U test). The lowest effective dosage for CSSC was 50x103 cells and CSK was 20x103 cells on mouse corneas.
Conclusions :
Treatments with CSSC and CSK were effective to reduce corneal fibrosis and scarring. CSK injection showed a greater scar inhibition than topical CSSC. This could be due to a better retention of functional CSK inside stroma whereas CSSC could be partly removed by blinking. In addtion, batch-to-batch variability and differentiation of CSSC could affect the treatment outcomes.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.