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Corneal endothelial dysfunction remains a major indication for corneal transplantation. Both corneal endothelial cells and stromal cells originate from the neural crest, but have distinct phenotypes and function in the adult cornea. We previously reported that stem cells isolated from the adult corneal stroma [cornea-derived precursors (COPs)] show characteristics of multipotent neural crest-derived stem cells. In this study, we report the induction of functional tissue-engineered corneal endothelium (TECE) from mouse and human COPs. TECE was engineered from Wnt1-Cre/Floxed EGFP mouse COPs in a medium containing retinoic acid and glycogen synthase kinase (GSK) 3β inhibitor (activator of Wnt/β-catenin signaling). The expression levels of major markers characterizing corneal endothelial function (Atp1a1, Slc4a4, Car2, Col4a2, Col8a2, and Cdh2) were significantly upregulated. Both retinoic acid and GSK 3β inhibitor upregulated the expression of Pitx2, a homeobox gene involved in the development of the anterior segment of the eye. GSK 3β inhibitor increased Atp1a1 expression and Na,K-ATPase pump activity of TECE, which was significantly higher than COPs or control 3T3 cells, and 2.6-fold higher than cultured mouse corneal endothelial cells. Mouse TECE transplanted into rabbit corneas maintained transparency and corneal thickness, whereas control corneas without TECE showed marked edema and increased corneal thickness. Furthermore, we successfully induced TECE from human COPs, and human TECE transplanted into rabbit corneas also maintained corneal transparency and thickness. This protocol enables efficient production of corneal endothelium from corneal stromal stem cells by direct induction, which may lead to a novel stem cell therapy for corneal endothelial dysfunction.

Cultivated limbal epithelial transplantation (CLET) is a recently developedsurgical method to reconstruct the ocular surface in eyes with limbal dysfunction. The concept of the CLET is to produce a limbal epithelial sheet containing progenitor cells of the corneal epithelium, and then to transplant to the ocular surface following excision of cicatricial tissues. The method was first reported by Pellegrini et al in 1997, followed by several other investigators including our group. [1][2][3][4][5][6][7][8][9][10][11][12] In our previous report in 2002, the short-term outcomes of CLET were comparable with conventional limbal epithelial transplantation. 7 We found that the method did not produce excellent outcomes in patients with severe cicatricial ocular surface disorders such as Stevens-Johnson syndrome (SJS) and chemical/thermal burns of the cornea. 7 This may be due to the fact that many of the patients had risk factors for poor epithelialization such as decreased tear production, lid abnormalities, persistent inflammation. [13][14][15] It is not clear whether the relatively poor outcomes of our previous report can be improved by the modification of the epithelial sheet preparation. There are a variety of factors that may influence the quality of the epithelial sheet, which include; source of cells, preparation of the cells, types of substrates used, cultivation medium, the use of feeder cells, and culture conditions. Our group has made some modification of the cultivation/preparation methods including the use of feeder cells, cell suspension technique and air-lifting of the cell sheet.In order to investigate whether the modification resulted in improvement in surgical outcomes, we retrospectively studied the mid-term outcomes of CLET. The mid-term outcomes of CLET for chronic cicatricial ocular surface disorders were compared with the different cultivation methods. Also, other factors were analyzed to see if they influenced the results. METHODS PatientsTwenty nine eyes of 27 patients that had CLET between June 1999 and November 2003 were retrospectively analyzed in this study. Patients consisted of 16 males and 11 females, with a mean age of 50.2 + 20.7 years (15-82 years). All eyes had total limbal dysfunction. Conjunctivalization was confirmed preoperatively by impression cytology in 10 of 27 eyes. Mean corrected visual acuity was counting fingers, and two-third of the eyes had less than 20/2000. Schirmer`s test was performed in 21 eyes and decreased tear secretion (Schirmer`s value < 5 mm) was noted in 12 eyes. As for preoperative complications, glaucoma, macular degeneration, and lid deformity were noted in 4, 1, and 3 eyes, respectively.

A better midterm clinical outcome was achieved with COMET of a substrate-free cell sheet than with COMET of AM as a substrate for treating severe stem cell deficiency.

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