Corneal epithelial stem cells residing within the annular limbal crypts regulate adult tissue homeostasis. Autologous limbal grafts and tissue engineered corneal epithelial cell sheets have been widely used in the treatment of various ocular surface defects. In case of bilateral limbal defects, pluripotent stem cell (PSC) derived corneal epithelial cells are now being explored as an alternative to allogeneic limbal grafts. We report here an efficient method to generate complex three dimensional corneal organoids from human PSCs. The eye field primordial (EFP) clusters that emerged from differentiating PSCs developed into whole eye ball-like, self-organized, three dimensional, miniature structures consisting of retinal primordia (RP), corneal primordia (CP), primitive eye lid-like outer covering and ciliary margin zone-like adnexal tissues in a step-wise maturation process within 15 weeks. These minicorneal organoids recapitulate the early developmental events in vitro and displayed similar anatomical features and marker expression profiles as that of adult corneal tissues and offers an alternative tissue source for regenerating different layers of the cornea and eliminates the need for complicated cell enrichment procedures.
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