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To examine the epithelial–mesenchymal transition (EMT) that is induced on the human corneal stroma, two- and three-dimensional (2D and 3D) cultures of human corneal stroma fibroblasts (HCSFs) were used. In this study, HCSF 2D monolayers and 3D spheroids were characterized by (1) scanning electron microscopy (SEM), (2) trans-endothelial electrical resistance (TEER) measurements and fluorescein isothiocyanate (FITC)-dextran permeability, (3) cellular metabolic measurements, (4) the physical properties of 3D HCSF spheroids, and (5) the extracellular matrix (ECM) molecule gene expressions, including collagen (<i>COL</i>) <i>1</i>, <i>4 and 6</i>, and fibronectin (<i>FN</i>), a tissue inhibitor of metalloproteinase (<i>TIMP</i>) <i>1–4</i>, matrix metalloproteinase (<i>MMP</i>) <i>2</i>, <i>3</i>, <i>9 and 14</i>, and several endoplasmic reticulum (ER) stress-related factors. In the 2D HCSFs, TGF-β2 concentration-dependently generated (1) a considerable increase in ECM deposits revealed by SEM, (2) an increase in TEER values and a decrease in FITC-dextran permeability, (3) increases in both mitochondrial and glycolytic functions, and a substantial upregulation of <i>COL1</i>, <i>COL4</i>, <i>FN</i>, <i>αSMA</i>, <i>TIMP1</i>, TIMP, and most ER stress-related genes and the downregulation of <i>COL6</i> and <i>MMP3</i>. In the case of 3D spheroids, TGF-β2 induced the downsizing and stiffening of 3D spheroids and the upregulation of <i>COL6</i>, <i>MMP14</i>, and most ER stress-related genes. These findings suggest that TGF-β2 significantly induced a number of EMT-associated biological events including planar proliferation, cellular metabolic functions, and the production of ECM molecules in the 2D cultured HCSF cells, but these effects were significantly less pronounced in the case of 3D HCSF spheroids.