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Tatsuya Mimura, Seiichi Yokoo, Makoto Araie, Shiro Amano, Satoru Yamagami; Treatment of Rabbit Bullous Keratopathy with Precursors Derived from Cultured Human Corneal Endothelium. Invest. Ophthalmol. Vis. Sci. 2005;46(10):3637-3644. doi: 10.1167/iovs.05-0462.
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purpose. To establish a method for the mass production of human corneal endothelium (HCE) precursors and the therapeutic application of these cells in a rabbit CE-deficiency model.
methods. A sphere-forming assay was performed to produce precursors from cultured HCE. Various marker expressions were examined in the sphere colonies, and their progenies by immunocytochemistry and reverse transcription–polymerase chain reaction (RT-PCR). The transport activity of the sphere-derived cell sheet was evaluated by the Ussing chamber system. DiI-labeled precursors obtained from cultured HCE were injected the anterior chamber of the eye in a rabbit CE-deficiency model, and the eye-down position was maintained for 24 hours for attachment to Descemet’s membrane (sphere eye-down group). The sphere eye-down and control groups, observed for 28 days after surgery, underwent histologic and fluorescence microscopic examinations.
results. Cultured HCE formed primary and secondary sphere colonies. The spheres expressed α-smooth muscle actin and nestin, and progeny expressed α-smooth muscle actin, confirmed by RT-PCR. The progeny showed an HCE-like hexagonal shape, were confluent, and had adequate transport activity. Mean corneal thickness in the sphere eye-down group was significantly less than in the other control groups 14, 21, and 28 days (P < 0.006) after surgery. The HCE-like hexagonal cells detected on the Descemet’s membrane are DiI-positive in the sphere eye-down group.
conclusions. The findings demonstrate that culture of HCE can promote mass production of HCE precursors, determined by sphere-forming assay. Injection of precursors derived from cultured HCE into the anterior chamber is an effective treatment strategy for CE deficiency in a rabbit model.
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