Document Detail


N-cadherin in the maintenance of human corneal limbal epithelial progenitor cells in vitro.
MedLine Citation:
PMID:  19420343     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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-cad(low) 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-cad(low) 3T3 cells were significantly smaller than mock-transfected cells. Then a duplex-feeder model with two layers of either 3T3 or N-cad(low) 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.
Authors:
Kazunari Higa; Shigeto Shimmura; Hideyuki Miyashita; Naoko Kato; Yoko Ogawa; Tetsuya Kawakita; Jun Shimazaki; Kazuo Tsubota
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2009-05-06
Journal Detail:
Title:  Investigative ophthalmology & visual science     Volume:  50     ISSN:  1552-5783     ISO Abbreviation:  Invest. Ophthalmol. Vis. Sci.     Publication Date:  2009 Oct 
Date Detail:
Created Date:  2009-09-24     Completed Date:  2009-10-02     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  7703701     Medline TA:  Invest Ophthalmol Vis Sci     Country:  United States    
Other Details:
Languages:  eng     Pagination:  4640-5     Citation Subset:  IM    
Affiliation:
Cornea Center, Tokyo Dental College, Chiba, Japan.
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MeSH Terms
Descriptor/Qualifier:
3T3 Cells
Animals
Antigens, CD / physiology*
Blotting, Western
Cadherins / physiology*
Cells, Cultured
Coculture Techniques
Down-Regulation
Epithelium, Corneal / cytology*,  metabolism
Genetic Vectors
Humans
Immunohistochemistry
Keratin-12 / metabolism
Keratin-14 / metabolism
Limbus Corneae / cytology*,  metabolism
Mice
Microscopy, Immunoelectron
Phenotype
RNA, Small Interfering / genetics
Stem Cells / cytology*,  metabolism
Transfection
Chemical
Reg. No./Substance:
0/Antigens, CD; 0/CDH2 protein, human; 0/Cadherins; 0/Keratin-12; 0/Keratin-14; 0/RNA, Small Interfering

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