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Jian Wu, Yiqin Du, Jelena Rnjak-Kovacina, David Kaplan, James Funderburgh; Corneal Stromal Bioequivalents Secreted on Patterned Silk Substrata by Corneal Fibroblasts and Stem Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4690.
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© ARVO (1962-2015); The Authors (2016-present)
Engineering of corneal stromal tissue is an important goal in the development of a functional corneal bioequivalent. In this study, we conducted a comparison study to assess the applicability of human corneal stromal stem cells (hCSSCs) versus human corneal fibroblasts (hCFs) in the generation of human corneal stromal tissue employing a combination of surface guidance and growth factor supplementation.
Surface patterned silk films were prepared by solution casting, followed by surface-derivatization with Arg-Gly-Asp (RGD)-peptide. hCSSCs (passage 4) isolated from limbal stroma and hCFs (passage 6) from central stroma were seeded on the silk film and cultured in serum-free media supplemented with FGF2, TGFβ3, and ascorbate-2-phosphate. After 9 weeks of culture, extracellular matrixes (ECM) deposited by hCSSCs and hCFs on the substrates were evaluated by transmission electron microscopy, wholemount immunohistochemistry, and Western blotting of culture media. Gene expression was examined by quantitative RT-PCR (qPCR).
Immunohistochemistry demonstrated that hCSSCs deposited fibrous ECM abundant in type-I collagen on patterned silk films with RGD surface modification. The hCSSC construct was 90~100 μm in thickness and featured stratified multilayered collagen-fibril lamellae with orthogonal orientation, morphologically close to that of native human corneal stromal tissue. In contrast, ECM secreted by hCFs was only 20~30 μm thick and contained less abundant type-I collagen than that from hCSSCs. Like scar tissue of human corneal stroma, ECM from hCFs lacked orderly organization. Gene expression profiles showed that hCSSCs up-regulated mRNA for several tissue-specific markers of keratocytes (KERA, ALDH, B3GNT7, CHST6,) much more substantially than hCFs. Western blotting revealed that hCSSCs secreted proteoglycans including lumican and keratocan modified with keratan sulfate. In contrast, these cornea-specific ECM components were hardly detected in hCF-secreted ECM.
These observations demonstrated that hCSSCs could be a powerful tool to develop an artificial bio-equivalent of human corneal stromal tissue by stem cell tissue engineering strategy. hCFs would not be an appropriate cell line for corneal repair and regeneration, and might rather be responsible for scar tissue formation in the human cornea.
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