A Role for Topographic Cues in the Organization of Collagenous Matrix by Corneal Fibroblasts and Stem Cells

Dimitrios Karamichos,James D Zieske,Martha L Funderburgh,Yiqin Du,James L Funderburgh,Jian Wu,Audrey E K Hutcheon,Fu-Shin Yu

doi:10.1371/journal.pone.0086260

Abstract

Human corneal fibroblasts (HCF) and corneal stromal stem cells (CSSC) each secrete and organize a thick stroma-like extracellular matrix in response to different substrata, but neither cell type organizes matrix on tissue-culture polystyrene. This study compared cell differentiation and extracellular matrix secreted by these two cell types when they were cultured on identical substrata, polycarbonate Transwell filters. After 4 weeks in culture, both cell types upregulated expression of genes marking differentiated keratocytes (KERA, CHST6, AQP1, B3GNT7). Absolute expression levels of these genes and secretion of keratan sulfate proteoglycans were significantly greater in CSSC than HCF. Both cultures produced extensive extracellular matrix of aligned collagen fibrils types I and V, exhibiting cornea-like lamellar structure. Unlike HCF, CSSC produced little matrix in the presence of serum. Construct thickness and collagen organization was enhanced by TGF-ß3. Scanning electron microscopic examination of the polycarbonate membrane revealed shallow parallel grooves with spacing of 200–300 nm, similar to the topography of aligned nanofiber substratum which we previously showed to induce matrix organization by CSSC. These results demonstrate that both corneal fibroblasts and stromal stem cells respond to a specific pattern of topographical cues by secreting highly organized extracellular matrix typical of corneal stroma. The data also suggest that the potential for matrix secretion and organization may not be directly related to the expression of molecular markers used to identify differentiated keratocytes.

Highlights

The cornea forms the optical portal for light into the eye [1] and its integrity is critical for vision
We report that corneal stromal stem cells (CSSC), like Human corneal fibroblasts (HCF), exhibit the ability to express and organize an extracellular matrix (ECM) resembling that of the human corneal stroma when cultured on Transwell filter substrata
After 4 weeks in culture, expression of 6 genes marking keratocyte differentiation was compared between the two cell types using Quantitative Reverse Transcription PCR (qPCR)

Summary

Introduction

The cornea forms the optical portal for light into the eye [1] and its integrity is critical for vision. The cornea derives its shape from the stroma, which contributes to as much as 90% of the total corneal structure, with the epithelial and endothelial layers comprising the remaining 10%. This highly ordered tissue has long been a subject of interest among scientists, as maintaining optical transparency of this dense connective tissue is essential for vision. Once the cornea is wounded or trauma occurs, the cornea may become permanently scarred, resulting in loss of visual acuity. The primary treatment used to correct stromal scarring is corneal transplantation

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