Ex Vivo Expansion Of Human Limbal Epithelial Stem Cells And Primary Identification Of Cultured Cells

Objective:Severe ocular surface diseases can compromise the ocular surface and cause catastrophic visual loss,and human limbal allograft transplantation or limbal autograft transplantation can not be accepted extensively in clinic because of its defects.With the development of tissue engineering technologies,transplantation of ex vivo cultured limbal epithelial stem cells has been an advanced treatment.The aim of this study was to expand human limbal epithelial stem cells ex vivo under the confocal miscroscope's guidance,which lay the foundation for fabricating ex vivo cultured cell sheets for transplantation.Methods:Ten eyes of ten patients were observed with the Heidelberg Retina TomographyⅢRostock Cornea Module(HRT3-RCM) examination to elucidate the structure of the human corneoscleraI limbus and to correlate limbal epithelial dimensions.The written informed consent was obtained from any subjects before initiation of any examination.According to the analysis of the images of limbal epithelia,the limbal tissues provided by Eye Bank was cut into suitable explants for culture.This study was conducted to expand limbal epithelial stem cells ex vivo on denuded amniotic membrane by limbal explant culture.The phenotypes of primary cultured cells were evaluated by morphology and immunofluorescent staining with antibodies for proposed keratinocyte stem cell markers(p63,cytokeratin19) and differentiation markers(keratin 3,involucrin).Results:Human limbal palisade morphology was imaged clearly on laser scanning in vivo confocal microscopy and many hyperreflective cells were observed in palisade basal cells.The "cell islands" phenomenon was also observed in the basement membrane by laser scanning in vivo confocal microscopy.The oblique sections of limbus show that many papilla-like epithelial columns were present under the superficial limbal epithelia.In this study,the successful epithelial cell growth rates from these tissues were 68.62±16.94%and the time to observation of initial cell growth was 5.83±2.04 day,which depended on the tissue freshness.The phenotypes of cultured cells,ranging from putative stem cells to superficial differentiated cells, are well maintained by limbal explant culture.Conclusion:Under the confocal miscroscope's guidance,human limbal epithelial stem cells have been successfully expanded ex vivo on denuded amniotic membrane by limbal explant culture.