Treatment of Rabbit Bullous Keratopathy with Precursors Derived from Cultured Human Corneal Endothelium

Abstract

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. 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. Cultured HCE formed primary and secondary sphere colonies. The spheres expressed alpha-smooth muscle actin and nestin, and progeny expressed alpha-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. 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.