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Delta Np63 protein is typically detected in human corneal epithelial cells with high proliferative capacity including, limbal epithelial stem cells (SCs) and probably also transient amplifying cells (TACs). AM supports Delta Np63 protein expression in HLECs and maintains a higher resistance against phorbol ester-induced differentiation, indicating that characteristic signs of limbal epithelial progenitor cells may be preserved during ex vivo expansion on AM.

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Aim: To analyse the correlation of p63 expression and cell cycle kinetics of human limbal epithelial cells (HLECs) expanded on amniotic membrane (AM) or plastic. Methods: Primary HLECs were cultured either on cryopreserved intact AM or plastic dishes for 2 weeks. Cells were labelled with 5 μM 5-bromo-2′-deoxyuridine (BrdU) for 3 days, followed by an interval of either 7 or 14 days in BrdU-free medium. The expression of p63 and BrdU labelling was detected by immunocytochemistry. Results: More cells on AM (56%) than on plastic (24%) retained their BrdU label after the 7-day interval (p < 0.001). The difference was even more pronounced after 14 days (20 and 2%, p < 0.001). All BrdU-labelled cells were also p63 positive. 2.5-fold more cells on AM (54%) than on plastic (21%) were BrdU positive/p63 positive after 7 days (p < 0.001). It increased to a 9-fold difference after 14 days (p < 0.001). The BrdU label was lost more quickly than the p63 expression during the observation period, indicating that p63 expression was not confined to stem cells but existed also in transient amplifying cells. Conclusions: The combination of p63 expression and BrdU label retention is a better criterion to characterize stemness than either marker alone. AM as a culture substrate preserves stemness better than plastic.