Keratocytes in the corneal stroma express keratan sulfate-containing proteoglycans including cornea-specific keratocan. On plastic dishes, human, bovine, and rabbit keratocytes lose their characteristic dendritic morphology and keratocan expression when cultured in serum-containing media. Herein, we demonstrated that murine keratocytes also acquired a fibroblastic shape and lost keratocan expression after first passage when cultured on plastic in the presence of serum. In contrast, cells expanded on human amniotic membrane (AM) stromal matrix maintained a three-dimensional dendritic morphology and expressed keratocan mRNA and protein for at least 8 passages before senescence. When keratocytes were cultured on AM, the promoter activity of transforming growth factor (TGF)-β2 and TGF-β receptor II was down-regulated as compared with that on plastic. Furthermore, cells on AM continuously retained Smad 2 and Smad 4 in the cytoplasm and did not express α-smooth muscle actin, even when 10 ng/ml TGF-β1 was added in a serum-free medium for up to 5 days. In parallel to such down-regulation of TGF-β signaling, keratocan promoter-driven ECFP expression was observed in cells cultured either on AM in the presence of serum or on plastic containing serum treated with a neutralizing antibody to TGF-β. Collectively, these results indicate that down-regulation of Smad-mediated TGF-β signaling is an important mechanism for cultured keratocytes to maintain a normal phenotype while continuously expanded in a serum-containing medium. This strategy of suppressing TGF-β signaling, achieved by AM stromal matrix in part via suppression of TGF-β gene transcription, can be used to expand keratocytes in culture without the use of AM in the future.
This work was supported by a research grant from TissueTech, Inc. and in part by an unrestricted grant from the Ocular Surface Research & Education Foundation (Miami, FL) and National Institutes of Health Grants EY06819 (to S. C. G. T.) and EY12486 (to C.-Y. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
