Stem cell-like keratinocytes are responsible for the high regenerative potential of the skin. For clinical applications using keratinocytes in artificial skin constructs, it is suitable to work with serum-free medium under defined conditions. This is also true for the preceding expansion of the stem cell-like keratinocyte population. Therefore, we analyzed the effect of a serum-free medium on the population distribution in comparison to an established serum-containing standard medium for keratinocyte culture. We quantified the freshly isolated as well as cultured primary human keratinocytes by their expression of the β1 integrin (CD29) in combination with the expression of the α6 integrin (CD49f) and the transferrin receptor (CD71) by flow cytometric methods. We were able to show that cultivation with serum-free medium induces a switch of the cell population to higher expression of the β1 integrin. In addition, the proportion of the α6bri/ CD71dim-expressing keratinocyte cell population was enhanced about 35.4 ± 6.56% after cultivation with serum-free medium. Culture in serum-containing medium increased this proportion of the keratinocyte cell population only about 17.3 ± 8.06%, when compared to the α6bri/ CD71dim-expressing keratinocyte cell population measured directly after isolation. Our data show that the applied culture conditions already have an enormous impact on the development of a stem cell-like phenotype of keratinocytes. This work demonstrates that the serum-free medium significantly increases the proportion of β1bri6bri/CD71dim-expressing keratinocytes. In conclusion, these findings implicate new applications in keratinocyte stem cell research and regenerative medicine.

1.
Barrandon, Y., H. Green (1987) Three clonal types of keratinocyte with different capacities for multiplication. Proc Natl Acad Sci USA 84: 2302–2306.
2.
Breitkreutz, D., A. Bohnert, E. Herzmann, P.E. Bowden, P. Boukamp, N.E. Fusenig (1984) Differentiation specific functions in cultured and transplanted mouse keratinocytes: environmental influences on ultrastructure and keratin expression. Differentiation 26: 154–169.
3.
Fuchs, E., J.A. Segre (2000) Stem cells: a new lease on life. Cell 100: 143–155.
4.
Jensen, U.B., S. Lowell, F.M. Watt (1999) The spatial relationship between stem cells and their progeny in the basal layer of human epidermis: a new view based on whole-mount labelling and lineage analysis. Development 126: 2409–2418.
5.
Jones, P.H., S. Harper, F.M. Watt (1995) Stem cell patterning and fate in human epidermis. Cell 80: 83–93.
6.
Jones, P.H., F.M. Watt (1993) Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression. Cell 73: 713–724.
7.
Kaur, P., A. Li (2000) Adhesive properties of human basal epidermal cells: an analysis of keratinocyte stem cells, transit amplifying cells, and postmitotic differentiating cells. J Invest Dermatol 114: 413–420.
8.
Lavker, R.M., K.H. Kaidbey (1982) Redistribution of melanosomal complexes within keratinocytes following UV-A irradiation: a possible mechanism for cutaneous darkening in man. Arch Dermatol Res 272: 215–228.
9.
Lavker, R.M., T.T. Sun (2000) Epidermal stem cells: properties, markers, and location. Proc Natl Acad Sci USA 97: 13473–13475.
10.
Li, A., P. Kaur (2005) FACS enrichment of human keratinocyte stem cells. Methods Mol Biol 289: 87–96.
11.
Li, A., P.J. Simmons, P. Kaur (1998) Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype. Proc Natl Acad Sci USA 95: 3902–3907.
12.
Morris, R.J., C.S. Potten (1994) Slowly cycling (label-retaining) epidermal cells behave like clonogenic stem cells in vitro. Cell Prolif 27: 279–289.
13.
Ohyama, M., A. Terunuma, C.L. Tock, M.F. Radonovich, C.A. Pise-Masison, S.B. Hopping, J.N. Brady, M.C. Udey, J.C. Vogel (2006) Characterization and isolation of stem cell-enriched human hair follicle bulge cells. J Clin Invest 116: 249–260.
14.
Pittenger, M.F., A.M. Mackay, S.C. Beck, R.K. Jaiswal, R. Douglas, J.D. Mosca, M.A. Moorman, D.W. Simonetti, S. Craig, D.R. Marshak (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284: 143–147.
15.
Potten, C.S., J.C. Bullock (1983) Cell kinetic studies in the epidermis of the mouse. I. Changes in labeling index with time after tritiated thymidine administration. Experientia 39: 1125–1129.
16.
Potten, C.S., J.A. O’Shea, C.L. Farrell, K. Rex, C. Booth (2001) The effects of repeated doses of keratinocyte growth factor on cell proliferation in the cellular hierarchy of the crypts of the murine small intestine. Cell Growth Differ 12: 265–275.
17.
Rheinwald, J.G., H. Green (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6: 331–343.
18.
Schaefer, B.M., H.J. Stark, N.E. Fusenig, R.F. Todd, 3rd, M.D. Kramer (1995) Differential expression of urokinase-type plasminogen activator (uPA), its receptor (uPA-R), and inhibitor type-2 (PAI-2) during differentiation of keratinocytes in an organotypic coculture system. Exp Cell Res 220: 415–423.
19.
Sonnenberg, A., J. Calafat, H. Janssen, H. Daams, L.M. van der Raaij-Helmer, R. Falcioni, S.J. Kennel, J.D. Aplin, J. Baker, M. Loizidou, et al. (1991) Integrin alpha 6/beta 4 complex is located in hemidesmosomes, suggesting a major role in epidermal cell-basement membrane adhesion. J Cell Biol 113: 907–917.
20.
Stark, H.J., M. Baur, D. Breitkreutz, N. Mirancea, N.E. Fusenig (1999) Organotypic keratinocyte cocultures in defined medium with regular epidermal morphogenesis and differentiation. J Invest Dermatol 112: 681–691.
21.
Tani, H., R.J. Morris, P. Kaur (2000) Enrichment for murine keratinocyte stem cells based on cell surface phenotype. Proc Natl Acad Sci USA 97: 10960–10965.
22.
Taylor, G., M.S. Lehrer, P.J. Jensen, T.T. Sun, R.M. Lavker (2000) Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell 102: 451–461.
23.
van Rossum, M.M., M.E. Franssen, W.A. Cloin, G.J. van de Bosch, J.B. Boezeman, J. Schalkwijk, P.C. van de Kerkhof, P.E. van Erp (2004) Functional characterization of beta1-integrin-positive epidermal cell populations. Acta Derm Venereol 84: 265–270.
24.
Watt, F.M. (1998) Epidermal stem cells: markers, patterning and the control of stem cell fate. Philos Trans R Soc Lond B Biol Sci 353: 831–837.
25.
Wu, Y.J., L.M. Parker, N.E. Binder, M.A. Beckett, J.H. Sinard, C.T. Griffiths, J.G. Rheinwald (1982) The mesothelial keratins: a new family of cytoskeletal proteins identified in cultured mesothelial cells and nonkeratinizing epithelia. Cell 31: 693–703.
26.
Zellmer, S., D. Reissig (2002) Isolation, cultivation, and differentiation of normal human epidermal keratinocytes in serum-free medium. Methods Mol Biol 188: 179–184.
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