Magnetic separation and characterization of keratinocyte stem cells from human gingiva.
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.


METHODS
Oral human keratinocytes obtained from keratinized oral mucosa were magnetically separated using α(6) β(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 CD71(neg) fraction had the smallest cell size compared with CD71(pos) and 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 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 CD71(pos) and subpopulations, respectively, p < 0.01; ANOVA). Moreover, the CD71(neg) fraction contained more quiescent cells and fewer actively cycling cells than the 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.


CONCLUSIONS
The human gingival 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.
Calenic
B
B
Department of Oral Health Oral Maxillofacial Surgery Research Center for Odontology, Nippon Dental University, Tokyo, Japan.
Ishkitiev
N
N
Yaegaki
K
K
Imai
T
T
Kumazawa
Y
Y
Nasu
M
M
Hirata
T
T
D053687:Adult Stem Cells / Q000166:cytology* / Q000378:metabolism; D000704:Analysis of Variance; D015703:Antigens, CD; D002453:Cell Cycle; D002454:Cell Differentiation; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D048429:Cell Size; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay / Q000379:methods; D005434:Flow Cytometry; D005881:Gingiva / Q000166:cytology*; D006801:Humans; D039503:Integrin alpha6; D039663:Integrin beta4; D053539:Keratin-19 / Q000096:biosynthesis; D015603:Keratinocytes / Q000166:cytology* / Q000378:metabolism; D008280:Magnetics; D008565:Membrane Proteins / Q000096:biosynthesis; D008863:Microspheres; D011990:Receptors, Transferrin
D015703:Antigens, CD; C068126:CD71 antigen; C081876:CKAP4 protein, human; D039503:Integrin alpha6; D039663:Integrin beta4; D053539:Keratin-19; D008565:Membrane Proteins; D011990:Receptors, Transferrin