Phenotypic characterization of human corneal epithelial cells expanded ex vivo from limbal explant and single cell cultures

Abstract

Cultivated human corneal epithelial cells have been successfully used for corneal reconstruction. Explant and single cell systems are currently used for human corneal epithelial cultivation. This study was conducted to characterize the phenotypes of human corneal epithelial cells expanded ex vivo by these two culture systems with regard to their growth potential, morphology and antigen expression patterns. Human corneal epithelial cells were expanded by limbal explant culture or limbal single cell suspension culture on a mitomycin C treated 3T3 fibroblast feeder layer. The phenotypes of primary cultured cells were evaluated by morphology and immunohistochemical staining with antibodies for proposed keratinocyte stem cell markers (p63, EGFR, K19 and integrin β1) and differentiation markers (K3, involucrin and gap junction protein connexin 43). BrdU labeling was performed to identify the label-retaining cells. Human corneal epithelial cells were grown from limbal tissues preserved as long as 16 days by both culture systems. The growth rate depended on the tissue freshness, the time from death to preservation and the time from death to culture, but not on the donor age. Cell growth was observed in 96.2% (n = 43) of single cell suspension cultures and in 90.8% (n = 213) of explant cultures. The cell expansion was confluent in 10-14 days in single cell suspension cultures and 14-21 days in explant cultures. The cell morphology in single cell suspension culture was smaller, more compact and uniform than that in explant culture. Immunostaining showed a greater number of the small cells expressing p63, EGFR, K19 and integrin β1, while more larger cells stained positively for K3, involucrin and connexin 43 in both culture systems. BrdU-label retaining cells were identified in 2.3 ± 0.7% of explant cultures and 3.73 ± 1.5% of single cell cultures chased for 21 days. In conclusion, the limbal rims are a great treasure for ex vivo expansion of human corneal epithelial cells. The phenotypes of corneal epithelial cells, ranging from basal cells to superficial differentiated cells, are well maintained in both culture systems. Slow-cycling BrdU-label retaining cells, that are characteristic of stem cells, were identified in the cultures.

Factors Influencing Outcomes in Cultivated Limbal Epithelial Transplantation for Chronic Cicatricial Ocular Surface Disorders

“…[1][2][3][4][5][6][7][8][9][10][11][12][17][18][19][20][21][22][23][24][25][26] Although short-term outcomes appeared promising, only a few mid-term or long-term results have been reported. Validity of CLET for acute phase of chemical burns and SJS was reported, 6 however, the efficacy of the method for chronic cicatricial keratoconjunctivitis seems to be less dramatic.…”

“…The cell-suspension culture system produced more desmosomal junctions and smaller intercellular spaces between the basal epithelial cells compared with explant culture, although both culture systems produced healthy, corneal phenotype cell layers. 21,23 Air-lifting was also shown to promote epithelial stratification and tight junction formation. 39 Ang et al demonstrated by using cultivated conjunctival epithelium that air-lifting caused reduced proliferating potential and it may be disadvantageous for long-term transplantation outcome.…”

Age-Dependent Depletion of Human Skin-Derived Progenitor Cells

“…: conception and design, financial support, data analysis and interpretation, manuscript writing, final approval of manuscript. expanded by limbal culture, differentiation potential of mesenchymal stromal cells from bone marrow, or the number and phenotype of pluripotent hepatic progenitors [13][14][15][16]. Although some of the observed discrepancies will undoubtedly be accounted for by tissue-specific differences [17], illdefined cell populations of origin and unreliable assays used for phenotypic readout have also contributed to blur the general picture [18].…”

Amnios et problèmes de surface oculaire

“…Certains des marqueurs proposés pour les cellules souches pourraient ne pas leur être exclusifs : ils pourraient, en effet, marquer également les cellules amplificatrices transitoires. Les cellules souches de la cornée seraient négatives pour les connexines 43 et 50 (Cx43, Cx50), protéines des jonctions gap, et positives pour la kéra-tine 19 [5,6], pour le facteur de transcription p63 [7] et pour ABCG2 [8].…”