Keratocyte Gene Expression Shaped by ECM Dimensionality: Evidence for Enhanced Quiescence in 3D Culture

In addition to soluble biochemical factors, biophysical cues from the extracellular matrix (ECM) can play an integral role in regulating cell behavior. Previous work has demonstrated that varying ECM dimensionality (3D vs. 2D) and structure (fibrillar vs. non-fibrillar) can produce distinct cell phenotypes; however, the transcriptional changes underlying how cells sense and respond to these different environments are not well understood. Here, we used bulk RNA sequencing to compare the gene expression profiles of primary rabbit corneal keratocytes cultured on collagen-coated substrates and on top of or embedded within fibrillar collagen matrices under serum-free conditions. Differential expression and functional analyses revealed distinct transcriptional profiles across the culture conditions and identified differentially expressed genes and enriched signaling pathways primarily related to the ECM, cell-matrix interactions, cell mechanics, and proliferation. Cells cultured in 3D fibrillar conditions exhibited gene expression patterns that better reflected the normal in vivo keratocyte phenotype, including broad suppression of proliferation and ECM synthesis-related genes. In contrast, cells cultured on 2D substrates showed higher expression of genes associated with an activated phenotype. Overall, these findings demonstrate the strong influence of biophysical cues from the ECM on keratocyte gene expression and highlight the importance of selecting physiologically relevant in vitro models for studies of corneal cell biology, wound healing, and regenerative therapies.