Abstract
Purpose :
Previous work has demonstrated that the extracellular matrix (ECM) plays an integral role in regulating cell behavior, with noticeable differences observed between cells cultured in 2D and 3D environments. Our lab has utilized a variety of in vitro models to study the phenotypic response of corneal keratocytes to biophysical and biochemical stimuli from the ECM. However, much remains to be explored regarding the transcriptional changes and signaling pathways responsible for the cells’ ability to sense and respond to these cues.
Methods :
Primary rabbit corneal keratocytes were seeded on monomeric collagen-coated glass substrates (2D-CM) and on top of (2DF) or embedded within (3DF) fibrillar collagen gels. All samples were cultured in serum-free media to maintain a quiescent keratocyte phenotype. For each condition, total RNA was collected for three experimental replicates and sent to Novogene for bulk RNA-sequencing. Pairwise comparisons were made to determine the number of significant differentially expressed genes (DEG) based on a |log2(FoldChange)|>1 and Padj<0.05. Additional functional analysis was performed to identify cellular components, biological processes, and signaling pathways enriched for DEGs.
Results :
Cells from each ECM condition exhibited unique transcriptional profiles based on principal component and differential expression analyses. Overall, there were 795 significant DEGs between 2D-CM and 2DF, 1153 between 2D-CM and 3DF, and 605 between 2DF and 3DF. When comparing cells in fibrillar collagen conditions to those on monomeric collagen, genes characteristic of quiescent keratocytes were generally upregulated (e.g. ALDH1A1, LUM). This was also true for the transition from a 2D fibrillar matrix to 3D (e.g. KERA). In contrast, genes associated with activated keratocytes had higher expression in the 2D-CM condition (e.g. FN1, TNC, ITGA5, ACTA2). Functional analysis revealed that pathways enriched within our differential gene sets included focal adhesions, motor proteins, ECM-receptor interactions, and protein digestion and absorption.
Conclusions :
These data demonstrate that keratocytes cultured in 3D fibrillar collagen matrices obtain a transcriptional profile that better reflects the quiescent nature of these cells in vivo. This highlights the importance of using models that mimic the native ECM environment when studying keratocyte behavior in physiological and pathological conditions.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.