Generating minicorneal organoids from human induced pluripotent stem cells

Abstract

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.

Improved Ocular Tissue Models and Eye-On-A-Chip Technologies Will Facilitate Ophthalmic Drug Development

“…22 Nevertheless, these cornea organoids recapitulate the structures of the human cornea in that they are semitransparent, produce corneal epithelium, have stroma characterized by organized collagen fibrils, contain a basement reminiscent of Descemet's membrane, and have a corneal endothelium. 23 The identification of these cell types by morphological analysis is corroborated by gene expression analyses; corneal epithelium from these organoids expresses KRT3 and KRT14; corneal endothelium expresses COL8A1, F11R, and S100A4; and both epithelium and endothelium show low expression of the cornea precursor marker KRT12. 23 In addition, stromal keratocyte markers CD34 and KERA are readily detectable in the stroma of these organoids.…”

“…23 The identification of these cell types by morphological analysis is corroborated by gene expression analyses; corneal epithelium from these organoids expresses KRT3 and KRT14; corneal endothelium expresses COL8A1, F11R, and S100A4; and both epithelium and endothelium show low expression of the cornea precursor marker KRT12. 23 In addition, stromal keratocyte markers CD34 and KERA are readily detectable in the stroma of these organoids. 23 These advances raise the intriguing possibility that cornea organoids could be used for the study of human diseases with unclear etiology, such as Fuchs' dystrophy or for the production of corneal cells that could be used for cell therapy approaches to corneal disease.…”

“…23 In addition, stromal keratocyte markers CD34 and KERA are readily detectable in the stroma of these organoids. 23 These advances raise the intriguing possibility that cornea organoids could be used for the study of human diseases with unclear etiology, such as Fuchs' dystrophy or for the production of corneal cells that could be used for cell therapy approaches to corneal disease. 22,23 Corneal tissue chip platforms that could be used for preclinical evaluations of therapeutic drugs are also in development.…”

“…Two independent groups have pioneered organoid technology to develop miniature corneas that capture the morphology of the developing tissue. 22,23 Current efforts coax iPSCs to an anterior neural commitment using Matrigel Ò scaffolds, retinoic acid exposure, and inhibition of Wnt and Notch signaling pathways, but the low efficiency of producing cornea organoids using this methodology suggests that much more refinement of the differentiation protocol is needed. 22 Nevertheless, these cornea organoids recapitulate the structures of the human cornea in that they are semitransparent, produce corneal epithelium, have stroma characterized by organized collagen fibrils, contain a basement reminiscent of Descemet's membrane, and have a corneal endothelium.…”

“…23 These advances raise the intriguing possibility that cornea organoids could be used for the study of human diseases with unclear etiology, such as Fuchs' dystrophy or for the production of corneal cells that could be used for cell therapy approaches to corneal disease. 22,23 Corneal tissue chip platforms that could be used for preclinical evaluations of therapeutic drugs are also in development. 24 Dr. Dan Huh is working on an eye-on-a-chip that mimics blinking by incorporating a hydrogel ''eyelid.''…”