Reconstruction of Highly Proliferative Auto-Tissue-Engineered Lamellar Cornea Enhanced by Embryonic Stem Cell

To increase the epithelial proliferation of an auto-tissue-engineered lamellar cornea, 3.0×10 6 corneal epithelial cells (CECs) were combined with 3×10 5 mouse embryonic stem cells (ESCs) pretransfected with the HSV-tk gene (CECs+ESCs-TK group), and 3.3×10 6 corneal epithelial cells (CECs group) were seeded between the acellular porcine corneal stroma and the amniotic membrane using the centrifugal cell seeding method. After 4 days of perfusion culture (treatment with ganciclovir starting on day 2), a thicker corneal epithelium (four to five layers) formed in the CECs+ESCs-TK group compared with that observed in the CECs group (two to three layers). More stem/progenitor cell (K3 −, p63+, ABCG2+, and integrin-β1+) and proliferation phenotypes (Ki67+) were measured in the CECs+ESCs-TK group compared with the CECs group using immunofluorescence staining, real-time quantitative reverse transcription polymerase chain reaction, and flow cytometry. Consistent with these findings, the colony-forming efficiency and cellular doubling time were significantly different between the CECs+ESCs-TK group (16.18%±3.98%, 28.45±2.03 h) and CECs group (11.96%±2.60%, 36.3±1.15 h). In a rabbit lamellar transplantation model, the CECs+ESCs-TK group had better epithelial barrier functions and wound healing abilities compared with the CECs group. Furthermore, ESCs-TK could be completely and safely removed by ganciclovir. Thus, the ESCS-TK coculture system could serve as a potential strategy for corneal tissue engineering.