Characterization of oral keratinocyte stem cells and prospects of its differentiation to oral epithelial equivalents

Objective: Although oral keratinocyte stem cells play a key role in tissue homeostasis, wound healing, and neoplasia, they remain difficult to identify and characterize. The specific aim of the present study is to characterize an oral keratinocyte stem-cell population separated using a magnetic technique. Material and Methods: Oral human keratinocytes obtained from keratinized oral mucosa were magnetically separated using a proliferation-related marker, CD71 and α6β4 integrin. The expression of different stem cell markers: CD44H, Nestin, Nanog, Oct 3/4, CD117 was checked by immunofluorescence. The ability of α6β4 pos CD71neg fraction to form oral epithelial equivalents was also assayed. Results: Three different oral keratinocyte subpopulations were obtained following magnetic separation: α6β4 pos CD71 neg , α6β4 pos CD71pos and α6β4 neg. Our α6β4 pos CD71neg stem cell fraction was positive for Oct 3/4, CD44H and cytokeratin 19 while Nanog, Nestin and CD117 expression was absent. At the same time, the other two cell fractions α6β4 pos CD71pos and α6β4 neg were negative for all stem cell markers. Also, α6β4 pos CD71 neg fraction was able to regenerate a well stratified and organized oral epithelial equivalent. The distribution of cytokeratin 19 and involucrin in the oral epithelial equivalent reflected the in vivo situation in oral gingival epithelium. Conclusions: The human gingival α6β4 pos CD71neg fraction was strongly positive for a panel of stem cell markers and could form oral epithelial equivalent. It is also suggested that a magnetic system may be an important tool in acquiring oral keratinocyte stem cells for research.