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Originally published In Press as doi:10.1167/iovs.07-1263 on April 25, 2008
 (Investigative Ophthalmology and Visual Science. 2008;49:3395-3401.)
© 2008 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.07-1263
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Targeted Cornea Limbal Stem/Progenitor Cell Transfection in an Organ Culture Model

Bojun Zhao, Sarah L. Allinson, Aihua Ma, Adam J. Bentley, Francis L. Martin, and Nigel J. Fullwood

From Biomedical Sciences, Department of Biological Sciences, Lancaster University, Lancaster, United Kingdom.

PURPOSE. To optimize a nonviral gene transfection system targeting the corneal limbal stem/progenitor cells.

METHODS. A plasmid containing LacZ gene coding for β-galactosidase (β-gal) was transfected into human corneal epithelial cells (HCECs) and multilineage progenitor cells (MLPCs) with different transfection reagents, to determine the optimal transfection reagent. In an ex vivo study, the bovine corneal epithelium and limbal stem/progenitor cells were transfected with a microinjection system with a 36-gauge needle that delivered plasmid/transfection reagent (Lipofectamine 2000; Invitrogen, Carlsbad, CA) complexes. The transfected corneoscleral discs were cultured in an air-interface culture system. The expression of β-gal was determined with an X-gal staining assay, and images were acquired with light microscopy and transmission electron microscopy. The expression of cytokeratin K5/14 and K3/K12 in corneal and limbal epithelium was determined by immunohistochemistry.

RESULTS. The highest percentages of β-gal expression in HCECs and MLPCs were achieved when the transfection reagent Lipofectamine 2000 was used. Corneal epithelial and limbal basal cells were successfully transfected with the reporter gene by targeted microinjection of plasmid/liposomal complexes. The location of the bovine limbal stem/progenitor cells was confirmed by positive K5/K14 labeling and negative K3/12 labeling.

CONCLUSIONS. Targeted microinjection of plasmid/liposomal complexes resulted in limbal stem/progenitor cell transfection. This technique has potential for the short-term treatment of corneal diseases.






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