Experimental transplantation of corneal epithelium-like cells induced by Pax6 gene transfection of mouse embryonic stem cells.
OBJECTIVE
Corneal epithelial stem cells are deficient in cases of limbal disorders, leading to conjunctival epithelial ingrowth, vascularization, and eventually visual disturbance. We introduced the eye development-associated transcription factor pax6 to embryonic stem (ES) cells and tested whether pax6-transfected cells resembling purified corneal epithelial cells were applicable as a cell source for corneal transplantation.


METHODS
pax6 cDNA with green fluorescence protein was electrotransfected to ES cells and the cells were cultured with G418 for 14 days. They were characterized by reverse transcription-polymerase chain reaction and immunohistochemistry. The cells were transplanted onto experimentally damaged mouse corneas. Histologic reconstitution of the corneal epithelium was assessed.


RESULTS
pax6-transfected cells formed a monolayer of epithelium-like cells in vitro. They expressed cytokeratin12, a specific keratin of corneal epithelial cells, E-cadherin, and CD44, which are important adhesion molecules of corneal epithelial cells on the cell membrane. They accumulated to make a colony that gave a staining pattern of reticular configuration for cytokeratin 12, E-cadherin, and CD44. When the cells were transplanted onto damaged cornea, they have been kept alive on the cornea.


CONCLUSIONS
The purified corneal epithelium-like cells derived from ES cells transfected with pax6 gene adapted to the injured cornea and were kept alive on it. These results suggested application of ES cell-derived corneal epithelial cells for treating corneal injuries.
Department of Ophthalmology, St Marianna University School of Medicine, Kawasaki, Kanagawa, Japan.
Hiroki
H
Ueno
Manae S
MS
Kurokawa
Maki
M
Kayama
Ryusuke
R
Homma
Yuta
Y
Kumagai
Chieko
C
Masuda
Erika
E
Takada
Kazuo
K
Tsubota
Satoki
S
Ueno
Noboru
N
Suzuki
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D015820:Cadherins / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D003316:Corneal Diseases / Q000378:metabolism / Q000473:pathology / Q000601:surgery*; D018274:Electroporation; D053595:Embryonic Stem Cells / Q000378:metabolism*; D019573:Epithelium, Corneal / Q000378:metabolism / Q000473:pathology / Q000637:transplantation*; D005136:Eye Proteins / Q000235:genetics*; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D005822:Genetic Vectors; D018398:Homeodomain Proteins / Q000235:genetics*; D018960:Hyaluronan Receptors / Q000378:metabolism; D053551:Keratin-12 / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D033581:Stem Cell Transplantation; D014162:Transfection
D015415:Biomarkers; D015820:Cadherins; C476399:Cd44 protein, mouse; D005136:Eye Proteins; D018398:Homeodomain Proteins; D018960:Hyaluronan Receptors; D053551:Keratin-12; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D012333:RNA, Messenger; D012097:Repressor Proteins