Oral Keratinocyte Stem Cells and 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.

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, Oct3/4, CD117 was checked by immunofluorescence. The ability of α₆β₄ ^pos CD71^neg 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 CD71^pos and α₆β₄ ^neg. Our α₆β₄ ^pos CD71^neg stem cell fraction was positive for Oct ¾, CD44H and cytokeratin 19 while Nanog, Nestin and CD117 expression was absent. At the same time the other two cell fractions α₆β₄ ^pos CD71^pos 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 CD71^neg 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.