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Deshea L. Harris, Biagio Saitta, Nancy C. Joyce; In Vitro Tests of Whether UCB MSCs Could Help Repair Damaged Corneal Endothelium. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6467.
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To test whether human umbilical cord blood mesenchymal stem cells (UCB MSCs) could be used to repair damaged corneal endothelium.
UCB MSCs were isolated, characterized, and grown in basal medium. Donor human corneas were obtained from National Disease Research Interchange, Philadelphia, PA. In some cases, human corneal endothelial cells (HCEC) were cultured according to published protocols. In others, HCEC on ex vivo corneas were damaged by mechanical scraping or by crushing with a capsule polisher. MSCs (5x105 of GFP-labeled) were applied to the wound in the presence of MSC basal medium, HCEC medium containing growth factors plus 8% FBS or the same medium containing only 8% FBS, basal medium used for growing a lens epithelial cell line (ATCC), medium conditioned using lens epithelial cells (LCM), rabbit aqueous humor (AH) diluted with lens basal medium, or LCM plus AH. Identification, attachment and morphology of MSCs were followed by confocal microscopy for GFP and DAPI. Cell-cell junctions were visualized by immunostaining for ZO1. The ability of MSCs to attach to different substrates was also tested in culture by seeding cells on tissue culture plastic, FNC coating mix, and HCEC-derived matrix in the presence of the above culture media. Cell-cell associations were observed by immunostaining for ZO1 and N-cadherin.
In ex vivo corneas, UCB MSCs did not adhere to intact endothelium or to Descemet’s membrane denuded of HCEC by scraping, but did adhere to areas of damaged endothelium under all medium conditions. LCM tended to increase elongation of MSCs. LCM alone and LCM + AH supported close cell-cell association resulting in increased ZO1 plaque formation at MSC-MSC and MSC-HCEC cell-cell boundaries. In culture, MSCs attached most efficiently to FNC-coated plates under all medium conditions. Of the media tested, LCM had the greatest effect on morphology by inducing cell elongation. This medium also promoted greater MSC-MSC association, as determined by morphology and localization of both ZO1 and N-cadherin at cell-cell borders, indicating the formation of junctional complexes.
UCB MSCs attach to areas of damaged HCEC in ex vivo corneal wounds. In culture, MSCs appeared to form cell-cell associations similar to those found in the corneal endothelial monolayer, particularly when grown in the presence of LCM. This preliminary in vitro data strongly suggests that UCB MSCs could be used to repair damaged corneal endothelium.
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