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purpose. To isolate precursor cells derived from rabbit corneal endothelium (CE) and to use them for the treatment of CE deficiency in a rabbit model.

methods. A sphere-forming assay was performed to isolate precursor cells from rabbit CE. Immunocytochemistry was used to examine marker expressions of neural and mesenchymal cells in the sphere colonies and their progenies. The pump function of the CE sheet was evaluated by measurement of the potential difference and short circuit current. Precursors obtained from rabbit CE by a sphere-forming assay were injected into the anterior chamber of the eye, after which an eye-down (ie, CE up) position was maintained for 24 hours to allow attachment by gravitation (sphere eye-down group). The sphere eye-down and control groups, observed for 28 days after surgery, underwent histologic and fluorescence microscopic examinations.

results. Rabbit CE formed primary and secondary sphere colonies. The progeny expressed α-smooth muscle actin, nestin, and neural markers and showed a CE-like hexagonal shape and adequate transport activity. Mean corneal thickness in the sphere eye-down group was significantly less than in the other control groups 3, 7, 14, 21, and 28 days (P< 0.05) after surgery. CE-like hexagonal cells were detected on Descemet’s membrane, and corneal edema was substantially suppressed. DiI-labeled cells were spread over the rear corneal surface in the sphere eye-down group only.

conclusions. Precursors from rabbit CE were isolated by a sphere-forming assay. Rabbit CE-derived sphere therapy is an effective treatment in a rabbit CE deficiency model.