Magnetic separation and characterization of keratinocyte stem cells from human gingiva

Background and Objective: Although keratinocyte stem cells play a key role in tissue homeostasis, wound healing and neoplasia, they remain difficult to identify and characterize. The purpose of this study was to isolate and characterize an oral keratinocyte stem-cell population. Material and Methods: Oral human keratinocytes obtained from keratinized oral mucosa were magnetically separated using alpha(6)beta(4) integrin and a proliferation-related marker, CD71. The isolated cell fractions were analyzed for cell size, cell cycle stage (using flow cytometry) and colony-forming ability. The expression of stem cell markers p63 and cytokeratin 19 and of differentiation markers cytokeratin 10 and involucrin was checked using immunocytochemical analysis. Results: The stem cell alpha(6)beta(pos)(4) CD71(neg) fraction had the smallest cell size compared with alpha(6)beta(pos)(4) CD71(pos) and alpha(6)beta 4(neg) fractions [ 780.7 +/- 141.5 ( pixels), 1422.9 +/- 264.6 ( pixels) and 3844.4 +/- 220.1 (pixels) respectively, p < 0.01; analysis of variance ( ANOVA)]. Also, the alpha(4)beta(pos)(4)CD71(neg) subpopulation consistently had the highest colony- forming ability among the three cell fractions ( 126.2 +/- 21.7 vs. 32.8 +/- 4.5 vs. 12.4 +/- 2.1 compared with alpha(6)beta(pos)(4) CD71(pos) and alpha(6)beta(neg)(4) subpopulations, respectively, p < 0.01; ANOVA). Moreover, the alpha(6)beta(pos)(4) CD71(neg) fraction contained more quiescent cells and fewer actively cycling cells than the alpha(6)beta(pos)(4) CD71(pos) cell fraction. The candidate stem cells were positive for cytokeratin 19 and p63 keratinocyte stem cell markers, while differentiation markers such as cytokeratin 10 or involucrin were absent. Conclusion: The human gingival alpha(6)beta(pos)(4) CD71(neg) cell fraction, separated by a magnetic system, demonstrated several characteristics of gingival keratinocyte stem cells. It is also suggested that a magnetic system may be an important tool in acquiring oral keratinocyte stem cells for research.