Establishment of Novel Limbus-Derived, Highly Proliferative ABCG2⁺/ABCB5⁺ Limbal Epithelial Stem Cell Cultures

Homeostasis and regeneration of corneal epithelia are sustained by limbal epithelial stem cells (LESCs); thus, an LESC deficiency is a major cause of blindness worldwide. Despite the generally promising results of cultivated LESC transplantation, it has been limited by variations in long-term success rates, the use of xenogeneic and undefined culture components, and a scarcity of donor tissues. In this study, we identified the culture conditions required to expand LESCs in vitro and established human limbus-derived highly proliferative ABCG2⁺/ABCB5⁺ double-positive LESCs. These LESCs exhibited the LESC marker profile and differentiated into corneal epithelial cells. In addition, cultured LESCs expressed high levels of the stem cell markers Sox2, Oct4, c-Myc, and Klf4, had high telomerase activity, and had stable, normal genomes. These results suggest that our novel cultivation protocol affects the phenotype and differentiation capacity of LESCs. From the limbus, which contains a heterogenous cell population, we have derived highly proliferative ABCG2⁺/ABCB5⁺ double-positive cells with the ability to differentiate into corneal epithelial cells. This study opens a new avenue for investigation of the molecular mechanism of LESC maintenance and expansion in vitro and may impact the treatment of corneal disease, particularly corneal blindness due to an LESC deficiency.