Cultivation and characterization of limbal epithelial stem cells in rabbits.
OBJECTIVE
In the last decades, strong evidence emerged regarding the presence of stem cells located at the corneal limbus. Our objective was to find a way to isolate and cultivate rabbit corneal stem cells in vitro, into an epithelial tissue.


METHODS
Two in vitro systems were developed to culture rabbit corneal stem cells: (1) limbal biopsies used as explants and cultivated on fresh denuded amniotic membrane and (2) a monolayer culture obtained by enzymatic treatment of the corneal biopsies. Genetic characterization (PCR) was performed. Specific triggers were used to induce differentiation of corneal stem cells.


RESULTS
At four weeks, 16 explant samples out of 18 cultures showed good expansion, ranging from 1 cm to 2 cm. Genetic characterization showed similar expression of genetic stem markers for corneal stem cells and placental stem cells, previously characterized (stem cell factor, Oct3/4, Vimentin, Nestin and Neurofilament). Corneal stem cells showed high Rhodamine efflux and were effective progenitors for neuronal, myocardial, osteogenic and endothelial lineage.


CONCLUSIONS
In one month, it was possible to grow enough epithelial tissue with preserved proliferative state to allow transplantation on the cornea.
Ophthalmology Clinic, Cluj-Napoca, Romania; iovidius@yahoo.com.
Ovidiu
O
Samoilă
Olga
O
Soriţău
Lăcrămioara
L
Totu
Sergiu
S
Şuşman
Carmen Mihaela
CM
Mihu
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002469:Cell Separation; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D019573:Epithelium, Corneal / Q000166:cytology*; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D008297:Male; D008858:Microscopy, Phase-Contrast; D011817:Rabbits; D020112:Rhodamine 123 / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism
D020112:Rhodamine 123