TY - JOUR

T1 - Targeted cornea limbal stem/progenitor cell transfection in an organ culture model

AU - Zhao, Bojun

AU - Allinson, Sarah L.

AU - Ma, Aihua

AU - Bentley, Adam J.

AU - Martin, Francis L.

AU - Fullwood, Nigel J.

PY - 2008/8

Y1 - 2008/8

N2 - PURPOSE. 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.

AB - PURPOSE. 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.

KW - EPITHELIAL PROGENITOR CELLS

KW - STEM-CELLS

KW - GENE-THERAPY

KW - INFRARED MICROSPECTROSCOPY

KW - IN-VIVO

KW - EXPRESSION

KW - DELIVERY

KW - IDENTIFICATION

KW - RENEWAL

KW - VECTOR

UR - http://www.scopus.com/inward/record.url?scp=49049122068&partnerID=8YFLogxK

U2 - 10.1167/iovs.07-1263

DO - 10.1167/iovs.07-1263

M3 - Journal article

VL - 49

SP - 3395

EP - 3401

JO - Investigative Ophthalmology and Visual Science

JF - Investigative Ophthalmology and Visual Science

SN - 0146-0404

IS - 8

ER -