Stromal matrix directs corneal fibroblasts to re-express keratocan after injury and transplantation

**Background:** The corneal stroma is populated by quiescent keratocytes that express keratocan, a proteoglycan essential for transparency. After injury, keratocytes undergo apoptosis, and activated fibroblasts and myofibroblasts repair the tissue but do not express keratocan. It is unknown whether these transformed cells can revert to a keratocyte phenotype once healing resolves. This study investigates the cellular dynamics of corneal wound healing and the role of the extracellular matrix (ECM) in regulating fibroblast-to-keratocyte conversion.

**Methods:** The authors created a novel triple-transgenic mouse strain (I-KeramTmG) that expresses eGFP only in keratocan-expressing cells after doxycycline induction, enabling cell-lineage tracing. Two strategies were used: (1) full-thickness corneal incisions (1 mm) were made in I-KeramTmG mice, and scars were analyzed at 1, 2, and 3 months post-injury using fluorescence microscopy and second-harmonic generation (SHG) imaging; (2) in vitro-expanded corneal fibroblasts (passage 1-3) or tenocytes from I-KeramTmG mice were injected into the stroma of wild-type C57BL/6 mice, and eGFP expression was induced with doxycycline for 1 week. Keratocan mRNA levels were measured by qPCR in cells cultured on plastic vs. a matrix with stiffness similar to normal stroma (30-60 kPa).

**Key Results:** After injury, all cells in scars at 1 and 3 months expressed eGFP, indicating a keratocyte lineage. However, at 1 and 2 months post-injury, no eGFP-expressing cells were found in maturing scars, suggesting that keratocytes are absent during early healing. At 3 months, a mixed population of eGFP-positive and -negative cells was present, with disorganized collagen fibrils on SHG imaging. In vitro, corneal fibroblasts lost keratocan expression after one passage on plastic with serum, but keratocan mRNA was significantly higher when cells were cultured on a matrix with normal stiffness (P=0.0003). When early-passage (P1) fibroblasts were transplanted into normal stroma, a significant proportion re-expressed eGFP (no significant difference between eGFP and tdTomato pixel counts), whereas passage 3 cells showed significantly fewer eGFP-expressing cells (P<0.05). Tenocytes, which express keratocan in vivo, failed to re-express it after corneal transplantation.

**Clinical Implications:** This study demonstrates that corneal fibroblasts retain phenotypic plasticity and can revert to keratocytes when placed in a healthy stromal niche, but this ability diminishes with prolonged culture (passage 3). The ECM, particularly matrix stiffness, plays a critical role in regulating keratocan expression. These findings suggest that therapies aimed at normalizing the ECM (e.g., reducing stiffness, promoting matrix reorganization) could facilitate fibroblast-to-keratocyte conversion and reduce scarring. The transplantation technique also offers a platform for cell-based therapies, such as delivering genetically modified cells that can be induced to express therapeutic factors. Future studies should explore mechanotransduction pathways and the role of matrix disorganization in limiting fibroblast reversal.