Targeted cornea limbal stem/progenitor cell transfection in an organ culture model.
OBJECTIVE
To optimize a nonviral gene transfection system targeting the corneal limbal stem/progenitor cells.


METHODS
A plasmid containing LacZ gene coding for beta-galactosidase (beta-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 beta-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 beta-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.
Zhao
Bojun
B
Department of Biological Sciences, Biomedical Sciences, Lancaster University, Lancaster, United Kingdom.
Allinson
Sarah L
SL
Ma
Aihua
A
Bentley
Adam J
AJ
Martin
Francis L
FL
Fullwood
Nigel J
NJ
D000818:Animals; D002417:Cattle; D019070:Cell Lineage; D019573:Epithelium, Corneal / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D018390:Gene Targeting / Q000379:methods; D017930:Genes, Reporter; D006801:Humans; D007633:Keratins / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology*; D008055:Lipids; D009924:Organ Culture Techniques; D010957:Plasmids; D013234:Stem Cells / Q000378:metabolism*; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics*
D008055:Lipids; C086724:Lipofectamine; D007633:Keratins; D001616:beta-Galactosidase