Three-dimensional construction of tissue-engineered cornea in vitro

Abstract

Objective To develop a corneal replacement for penetrating keratoplasty (PKP). A three-dimentional, tissue-engineered cornea is constructed by using human embryonic stem cells (hESCs) derived corneal endothelial-like cells and corneal epithelial-like cells as seed cells, and acellular porcine cornea matrix (APCM) as scaffold. Methods Experimental study. To obtain APCM, sterile fresh porcine cornea was decellularized with a 0.5% SDS solution for 24 hours at 4 ℃ and then washed 8 times with PBS over 24 hours to remove SDS. To obtain corneal endothelial-like cells derived from hESCs, periocular mesenchymal precursors (POMPs) were developed first by using a transwell co-culture system of hESCs with human corneal stromal cells, and then culturing in a lens epithelial cell-conditioned medium for 14 days. The isolated corneal endothelial-like cells were expanded, labeled and seeded onto APCM lamellas to construct the corneal endothelial-like cell sheets. Pump functions of the cell sheets were measured by the corneal endothelium transplantation of rabbit corneal endothelium dysfunction models in vivo. Corneal epithelial-like cells derived from hESCs were cultured with an LSC-conditioned medium, and then seeded on APCM to construct corneal epithelial-like cell sheet for limbal stem cells deficiency model transplantation. Animals were followed up with a weekly slit lamp microscope exam and HE staining was performed after surgery. Results The induced cells expressed corneal endothelial cells related markers such as Na+/K + ATPase alpha1, ZO-1, N-cadherin and vimentin. The constructed corneal endothelial-like cell sheets improved the corneal transparency of rabbit cornea endothelium in dysfunction models. Corneal epithelial-like cells formed 4 to 5 layers of cells on APCM and possessed the potential to repair a damaged ocular surface in vivo. Conclusion The construct derived from hESCs induced cells and APCM possessed the similar function and structure of a natural cornea, and might be used for PKP in future. Key words: Cornea; Embryonic stem cells; Embryonic induction; Tissue engineering; Tissue transplantation