Ex vivo transduction of corneal epithelial progenitor cells using a retroviral vector.
OBJECTIVE
To transduce corneal epithelial progenitor cells with a reporter gene using a retroviral vector and follow their progeny in vitro and in vivo.


METHODS
Using a lacZ-producing retroviral vector, rabbit keratolimbal explants were transduced ex vivo, autografted onto their original sites, and assessed for lacZ-producing cells in the cornea throughout a 6-month period. Four autografts served as control samples, having received no vector. Experimental and control rabbits were euthanized and corneas with scleral rims harvested, weekly for 4 weeks and then monthly for 6 months. The corneas were first stained with 5-bromo-4-chloro-3-indolyl galactopyranoside (X-gal) as wholemounts and then sectioned for histology and immunohistochemistry to examine lacZ-positive cell outgrowth. Three additional transduced explants were observed in culture. These explants were transferred to new culture dishes every week for 9 weeks. The previously occupied culture dish was stained for lacZ to detect transduced epithelial cells, and the number of lacZ-positive cells was quantitated.


RESULTS
LacZ-positive cells were found in the corneas of 18 of 20 eyes in which virally transduced keratolimbal autografts had been implanted. The cells were epithelial in nature, originated from the limbus, and were found in colonies throughout the epithelial layer of the cornea. The appearance of lacZ-positive cells in four of five corneas harvested after 6 months showed long-term transgene expression consistent with transduction of corneal epithelial stem cells. In vitro, the number of lacZ-positive cells migrating from the keratolimbal autografts decreased rapidly during the first 4 weeks and then remained stable through week 9.


CONCLUSIONS
This study shows that a retroviral vector can effectively transduce corneal epithelial progenitor cells, shown by the long-term appearance of transduced cells on the cornea in vivo and the stable production of lacZ-positive cells in vitro. The appearance and disappearance of labeled cells is consistent with the initial transduction of stem cells and transient amplifying cells.
Bradshaw
J J
JJ
Department of Ophthalmology, University of Minnesota Medical School, Minneapolis, USA.
Obritsch
W F
WF
Cho
B J
BJ
Gregerson
D S
DS
Holland
E J
EJ
D000818:Animals; D017690:Cell Transplantation; D019573:Epithelium, Corneal / Q000201:enzymology* / Q000637:transplantation / Q000821:virology; D005697:Galactosides / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D007124:Immunoenzyme Techniques; D007211:Indoles / Q000378:metabolism; D007763:Lac Operon / Q000235:genetics*; D008979:Moloney murine leukemia virus / Q000235:genetics*; D011817:Rabbits; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000201:enzymology* / Q000821:virology; D014182:Transplantation, Autologous; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D005697:Galactosides; D007211:Indoles; D001616:beta-Galactosidase; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside