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Xiaoqing Q Guo, Audrey E K Hutcheon, James D Zieske; Human Corneal Endothelial Development on 3D Constructs. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2048.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal endothelium is one of the three major cell layers in the cornea playing a critical role in maintaining corneal clarity. Many current investigations focus on generating an endothelium in vitro for transplantation and for the development of corneal equivalents. Our lab has developed a cell-based 3-dimensional (3D) corneal stromal construct consisting of human corneal fibroblasts (HCF) and their self-assembled matrix, which mimics stromal development. Our present study is to test if human corneal endothelial cells (HCEn) can develop into mature endothelium on our 3D construct.
HCF were grown on Transwells in EMEM with 10% FBS and 0.5mM Vitamin C (VitC) for 4 weeks to make our 3D constructs. An endothelial cell line (HCEn) generated in the laboratory of Dr. May Griffith was seeded on top of the constructs and cultivated for another 5 days. Cultures were examined for morphology and ultrastructure using light and transmission electron microscopy. Indirect immunofluorescence microscopy (IF) also was used to examine tight junction formation (ZO-1), maturation (ALDH1A1), basement membrane formation (Laminin), cell proliferation (Ki67) and cell death (caspase-3).
The HCEn formed a monolayer on the constructs. IF showed that ZO-1 was present in cell-cell junctions in the endothelial layer, indicating the formation of tight-junctions. ALDH1A1 was positive in the cytoplasm of both endothelial and stromal cells, and Laminin was present in the endothelial cells, as well as in the interface between HCEn and construct. In addition, minimum numbers of HCEn cells were labeled with Ki67 or Caspase-3.
The detection of ZO-1 and ALDH1A1 in HCEn cells indicates that HCEn cells are forming a mature monolayer. The localization of Laminin suggests that the cells are beginning to assemble a basement membrane as early as five days after the addition of the endothelial cells. The HCEn also shared the in vivo characteristics of minimal proliferative and apoptotic activity. These data suggest that HCEn cells appear to develop in vivo like characteristics in our model, and that the 3D construct is a good substrate to study corneal endothelial cell development.
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