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Originally published In Press as doi:10.1167/iovs.09-3503 on May 6, 2009
 (Investigative Ophthalmology and Visual Science. 2009;50:4640-4645.)
© 2009 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.09-3503
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N-Cadherin in the Maintenance of Human Corneal Limbal Epithelial Progenitor Cells In Vitro

Kazunari Higa,1,2 Shigeto Shimmura,1,2 Hideyuki Miyashita,2 Naoko Kato,2 Yoko Ogawa,2 Tetsuya Kawakita,1,2 Jun Shimazaki,1,2 and Kazuo Tsubota1,2

1From the Cornea Center, Tokyo Dental College, Chiba Japan; and the 2Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan.

PURPOSE. To demonstrate the role of N-cadherin (N-cad) in maintaining the progenitor status of primary human limbal epithelial cells in vitro.

METHODS. Immunohistochemistry and immunoelectron microscopy against N-cad was performed in human limbal tissue. The expression of N-cad, cytokeratin (K) 12, and K14 was also observed in primary cultured limbal epithelial cell colonies in 3T3 feeder cells, which also express N-cad. Laser microdissection of individual colonies was performed to separate central cells from peripheral cells to compare secondary colony formation. Finally, an artificial small interfering RNA (siRNA) expression vector was used to downregulate N-cad in 3T3 cells (N-cadlow 3T3) to observe changes in colony formation and cultivated epithelial sheet phenotype.

RESULTS. N-cad was expressed in clusters of basal limbal epithelial cells. Limbal epithelial cell colonies cocultured with N-cad+ 3T3 feeder cells showed N-cad expression along the edge of each colony. When individual colonies were divided into peripheral and central sections by laser microdissection, peripheral cells had a significantly higher secondary colony-forming efficiency than did central cells. Furthermore, colonies using N-cadlow 3T3 cells were significantly smaller than mock-transfected cells. Then a duplex-feeder model with two layers of either 3T3 or N-cadlow 3T3 cells was used to produce stratified epithelial sheets. Only N-cad+ 3T3 cells produced epithelial sheets with basal K15/ suprabasal K12 expression observed in limbal tissue.

CONCLUSIONS. N-cad plays a pivotal role in the maintenance of the progenitor phenotype in cultured limbal epithelial cells.






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