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Increased expression of p63 protein and sonic hedgehog signalling molecule in buccal epithelial holoclones.

Construction of many tissues and organs de novo requires the use of external epithelial cell sources. In the present study we optimized the isolation, expansion and characterization of porcine oral epithelial cells from buccal tissue (Buccal Epithelial Cells, BECs). Additionally, we tested whether key markers (cytokeratin 14 (ck14), p63 protein and sonic hedgehog molecule (shh)) expression profiles are correlated with three buccal epithelial clone types. Two digestion methods of BECs isolation (Method 1, M1 (collagenase IV/ dispase and accutase) and Method 2, M2 (collagenase IV/ dispase and trypsin/EDTA)) were compared. Cells obtained by more effective method were further cultured to the 3rd passage and analysed. Holoclone-, meroclone- and paraclone-like colonies were identified based on BECs morphology. Immunofluorescent stainings were performed to compare selected markers for the indicated buccal clone types. Comparative analysis demonstrated the advantage of isolation using M1 over M2. Cells from the 3rd passage exhibited average 92.73%±2.27% presence of ck14. Real-time PCR confirmed expression of tested genes (cytokeratin 8 (ck8), ck14, integrin β and p63). The highest level of ck14, shh and p63 was observed for holoclones. The comparable ck14 expression was observed in the mero- and paraclones. Meroclones expressed significantly lower levels of shh compared to paraclones. The weakest p63 expression was observed in the paraclone-like cells. It was demonstrated that holoclones are the richest in shh (+) and p63 (+) stem cells and these cells should appear to be a promising alternative for obtaining epithelial cells for tissue engineering purposes.